Preparation of Ag-pIOPNs nanoparticles
To prepare Ag-pIOPNs nanoparticles, 3 g of IOPNs powder (XFJ119-12227-89-3102138, Nanjing Xianfeng Nanomaterials Technology Co., Ltd.) was dispersed in a dopamine solution (2 g/L) (dopamine hydrochloride (DA-HCl, 99%, BP468, CAS: 62-31-7, Aladdin) at 60 °C for 12 h. The suspension was then centrifuged and washed with deionized water to remove residual dopamine. After drying, the resulting polydopamine-modified IOPNs were labeled as pIOPNs. Subsequently, a silver ammonia solution (0.06 mol/L) was prepared by dropping an ammonia solution (NH3·H2O, 105423, CAS: 1336-21-6, Sigma-Aldrich) into a silver nitrate solution (0.17 mol L− 1, AgNO3, 209139, Sigma-Aldrich). Next, 0.7 g of pIOPNs powder was added to the prepared silver ammonia solution (40 mL) and stirred continuously for 2 h. Finally, Ag-modified pIOPN nanoparticles were obtained by centrifugation, washing, and drying.
Analysis and characterization of materials
The morphology and elemental distribution of the samples were examined using a scanning electron microscope (SEM, S-4800, Hitachi, purchased from Shanghai Fulai Optical Technology Co., Ltd.), TEM (Hitachi H-7650, purchased from Shanghai Baihe Instrument Technology Co., Ltd.), and energy dispersive spectrometer (EDS, Bruker QUANTAX EDS). The powder morphology was observed using a TEM (FEI, USA). The chemical structure of the samples was analyzed using Fourier-transform infrared spectroscopy (FT-IR, 912A0770, Thermo Fisher, USA). The size distribution of the nanoparticles and the zeta potential were measured using a nanoparticle tracking analyzer (Zeta View_Particle Metrix, purchased from Dachang Huajia Scientific Instruments) (all sample solutions were measured at pH 7.0) [66, 67]. The thermal stability of the samples was determined using a thermogravimetric analyzer (TGA-601, Nanjing Huicheng Instruments and Meters Co., Ltd.) under a nitrogen atmosphere at a heating rate of 20 °C/min. The compressive performance of the composite material scaffold was tested using a universal mechanical testing machine (CMTS5205, MTS, USA) at a constant deformation rate of 2.5 mm/min until the specimen height decreased by 40%. The compressive modulus was calculated from the slope of the initial linear region of the stress-strain curve. The release of ions from the scaffold in deionized water was quantitatively analyzed using an inductively coupled plasma optical emission spectrometer (ICP-OES, Spectro Blue Sop, purchased from Germany Hua Pu General Distribution).
3D printing and printability testing of hydrogels
Hydrogels were printed using the extrusion-based 3D printer (AXO A3) from Axolotl BIOSYSTEMS. Prior to printing, the hydrogel precursor was loaded into a 22 g syringe and injected into a 5 ml syringe. Printing parameters were set as follows: both the syringe and nozzle temperature were set to 31 °C, while the printing speed and pressure were adjusted accordingly. The hydrogel was sequentially printed onto a platform maintained at 4 °C, forming 1, 2, 5, and 10 layers. Subsequently, the printed hydrogel was immersed in a 1% calcium chloride solution for 2 min to allow for curing. Morphological observations were made on the cured hydrogel. Optimal process parameters were selected for the preparation of 3D-printed scaffolds.
Preparation of hybrid gel
Collagen (EFL-GEL-001, Suzhou Intelligent Manufacturing Research Institute), sodium alginate (EFL-Alg-300 K, Suzhou Intelligent Manufacturing Research Institute), or dopamine-modified sodium alginate (C6H7O6Na, molecular weight: 216.12) along with self-prepared Ag-pIOPNs were dissolved and dispersed. A solution of 0.25% (w/v) collagen and 0.1% (w/v) sodium alginate in PBS (pH 7.4) was stirred in a water bath at 70 °C for 30 min to obtain pre-fabricated water gelatin with the acronym GA. Subsequently, the GA pre-fabricated water gel was immersed in a 100mM calcium chloride solution (C4901, Sigma-Aldrich) for 48 h to solidify and form GA hydrogel [68]. In accordance with a cited reference [69], dopamine-modified sodium alginate (Alg-DA) was formed under the catalysis of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide), which was then mixed with the collagen solution to prepare GAD hydrogel. Ag-pIOPNs were sonicated in water using an ultrasonic cleaner (US-22D, China Scientific Instruments (Beijing) Co., Ltd.) to form an ion-crosslinked primary network, subsequently loaded into the GAD hydrogel to prepare GAD/Ag-pIO hydrogel [70].
Rheological analysis
Rheological measurements were conducted using an Anton Paar MCT 302 rheometer (Anton Paar, Austria) at 37 °C. To evaluate shear-thinning behavior, the bioink was placed between parallel plates and subjected to increasing shear rates (0.01 to 200 s-1). To assess the viscosity of the bioink prior to printing, the bioink underwent low shear at 1 s-1 for 600 s. Subsequently, the bioink was solidified into gel sheets with a diameter of 10 mm and a height of 1 mm. Scans were performed under fixed strain and frequency conditions (1% strain and 5 rad s-1) for 300 s to evaluate the storage modulus and loss modulus [71]. Each experiment was conducted in triplicate.
Swelling ratio and degradation rate
Circular disc-shaped samples with a diameter of 8 mm and a height of 3 mm were prepared using bioink solutions of varying concentrations to calculate the swelling ratio. The samples were immersed in PBS at 37 °C for 12 h and weighed as M1, then lyophilized for 12 h and weighed as M2. The swelling ratio (Qs) was calculated using the equation Qs = M1/M2. Degradation experiments were conducted by placing the samples in a 24-well plate containing collagenase I (1148089, Merck, Germany) at a concentration of 0.5 mg/mL. The samples were removed at different time points, lyophilized, and weighed as mass (Wr). The initial sample mass was denoted as W0, and the degradation rate (Qd) was calculated as Qd = Wr/W0 [72]. Each experiment was performed in triplicate.
Isolation, cultivation, and identification of primary cells
BMSCs were collected from the tibia and femur of 7–8 week-old C57BL/6 mice (213, Beijing Vital River Laboratory Animal Technology Co., Ltd.) weighing 16–20 g. The bone marrow was washed with Dulbecco’s Modified Eagle Medium (DMEM)/F12 (DF-041, Sigma Aldrich, Shanghai, China) to obtain a mixed cell suspension. The suspension was centrifuged at 800 g for 5 min to obtain a pellet containing BMSCs, which was resuspended in DMEM/F12 and cultured at 37 °C with 5% CO2 [73]. The culture medium was replaced every 2–3 days.
Differentiation of BMSCs into adipocytes was induced using adipogenic induction medium (IMDM supplemented with 10% fetal bovine serum (FBS) (12103 C, Sigma Aldrich, Shanghai, China), 10 µg/mL insulin (Y0001717, Sigma Aldrich, Shanghai, China), 1 µM dexamethasone (D1756, Sigma Aldrich, Shanghai, China), 0.5 mM phosphodiesterase (PDE) inhibitor (IBMX) (I5879, Sigma Aldrich, Shanghai, China), and 0.1 mM indomethacin (I7378, Sigma Aldrich, Shanghai, China)). Osteogenic differentiation of BMSCs was induced using osteogenic induction medium (IMDM supplemented with 10% FBS, 5 µg/mL insulin, 0.1 µM dexamethasone, 0.2 mM vitamin C (PHR1008, Sigma Aldrich, Shanghai, China), and 10 mM β-glycerophosphate (G5422, Sigma Aldrich, Shanghai, China)). ARS staining was used to assess calcium deposition at week 3 as an indicator of osteogenic differentiation, while Oil Red O staining was used to evaluate lipid droplet formation as an indicator of adipogenic differentiation at week 3. Cell morphology and growth were observed using an inverted microscope. Cell passaging was performed every 3–5 days when the cell confluence reached 80% [73,74,75].
Bone marrow-derived macrophages (BMDMs): Bone marrow progenitor cells were collected from the femur and tibia of 7–8 week-old C57BL/6 mice weighing 16–20 g. The progenitor cells were differentiated in RPMI-1640 (11875093, Thermofisher, USA) supplemented with 10% FBS (12103 C, Sigma Aldrich, Shanghai, China), 1% L-glutamine (G7513, Sigma Aldrich, Shanghai, China), 1% penicillin/streptomycin (15140148, Thermofisher, USA), and 15% L929 fibroblast-conditioned medium for 7 days. After differentiation, BMDMs were washed with PBS and resuspended in 5 ml RPMI-1640. Cell scraping was performed, followed by centrifugation at 200 × g for 5 min. The BMDMs were then seeded and stimulated in R10/5 medium (RPMI-1640 supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 5% LCCM) [76,77,78]. BMDMs were treated with 500 ng/ml of LPS for three hours [79]. Subsequently, cell viability, levels of inflammatory factors, and oxidative stress were assessed. Macrophage-conditioned medium post LPS treatment was used to culture BMSCs, and mitochondrial signaling was analyzed after treating with 10 µM CCCP for 2 h [80].
Cell viability assessment
After 3D bioprinting, live and dead cells were identified using Calcein AM (ThermoFisher, USA, Product No. C3099) and Propidium Iodide (PI) (innibio, USA, Product No. DN1005-010). Cells were incubated in a medium containing 1 µM Calcein AM at 37 ℃ for 30 min, followed by three washes with PBS. Subsequently, cells were incubated in a medium containing 1 µM PI for 10 min, followed by three washes with PBS. Images were captured using a confocal microscope (Carl Zeiss AG, Germany, Model 880), with each image representing a different field of view. Three independent samples were tested. Cell viability was calculated using ImageJ software (Version 1.8.0) [81].
Additionally, cell viability was assessed according to the manufacturer’s instructions for the CCK-8 assay kit (Beyotime, Shanghai, China, Product No. C0041). After 48 h of incubation, cell viability was measured using the CCK-8 method. CCK-8 detection solution (10 µL) was added at each measurement, followed by 4 h of incubation in a CO2 incubator. Absorbance at 450 nm was then measured using an enzyme-linked immunosorbent assay (ELISA) reader to calculate cell viability [82].
Antioxidant activity determination
DPPH radical was used to assess the antioxidant activity of the scaffolds. Firstly, 4 mg of DPPH (Aldrich, Product No. 300267) was dissolved in 100 ml of methanol solution (Aldrich, Shanghai, China, Product No. 439193). Next, cylindrical scaffolds with dimensions of 10 mm (height) × 10 mm (diameter) were immersed in 4 ml of DPPH solution and subjected to antioxidant reaction at 37 °C. Absorbance at 516 nm was measured to evaluate the antioxidant activity by calculating the DPPH clearance efficiency [83].
Detection of total ROS and mitochondrial ROS
The assessment of ROS was conducted using the green fluorescent dye dichlorodihydrofluorescein diacetate (DCF) (HY-D0940, Abcam, UK). This dye has the ability to enter cells and interact with ROS molecules, forming a fluorescent compound called dichlorodihydrofluorescein (DCFDA). In brief, a stock solution of DCFDA (10 mM) was prepared in methanol and then diluted with culture medium to obtain a working solution of 100 µM. Cells were cultured overnight in a six-well plate with coverslips. On the following day, the cells were treated with H2O2 (200 µM) for 24 h. After treatment, coverslips were washed with ice-cold Hank’s Balanced Salt Solution (HBSS) (H8264, Sigma Aldrich, Shanghai, China) and incubated with 100 µM DCFDA at 37 °C for 30 min. Following washing, coverslips were mounted onto slides. Fluorescence intensity and ratio of positively stained cells were observed and analyzed using a multiphoton confocal microscope (A1R, Nikon, USA) with a ×100 objective, and ImageJ software was employed for data analysis at specific excitation and emission wavelengths.
For the evaluation of superoxide compounds (SOX) present in the mitochondria, the red dye mito-SOX (M36008, Thermo Fisher, USA) was utilized. THP-P cells (2 × 104) were cultured overnight in a six-well plate with coverslips. On the next day, the cells were treated with H2O2 (200 µM) for 24 h. Post-treatment, coverslips were washed with ice-cold 1×PBS and incubated at 37 °C for 30 min with a working solution of 2 µM mito-SOX. Following a final wash with 1×PBS, coverslips were mounted onto slides. Fluorescence intensity and ratio of positively stained cells were observed and analyzed using a multiphoton confocal microscope (A1R, Nikon, USA) with a ×100 objective. ImageJ software was then used for data analysis at specific excitation and emission wavelengths [84].
Flow cytometry analysis
To identify surface markers of BMSCs, we used the following antibodies: CD44 (ab25064, 1:500, Abcam, USA), CD90 (ab24904, 1:50, Abcam, USA), CD45 (ab210225, 1:500, Abcam, USA), and CD34 (ab23830, 1:500, Abcam, USA). Flow cytometry analysis was performed using an Attune NxT flow cytometer (Thermo Fisher Scientific Inc.) [85]. For the identification of surface markers of BMDMs, we used the F4/80 antibody (ab237332, 1:500, Abcam, USA). Flow cytometry analysis was similarly conducted using the Attune NxT flow cytometer (Thermo Fisher Scientific Inc.) [76]. Cell nuclei were stained using an APC-conjugated anti-F4/80 antibody (1:100, ab105080, Abcam, USA) and Sytox Green (R37109, 1:100, Bioscience, USA). An APC-conjugated mouse IgG2a kappa antibody (ab154434, 1:100, Abcam, USA) was used as an isotype control. All antibodies were incubated on ice and protected from light for 15 min. After incubation, the cells were centrifuged and washed with 500 µL of staining buffer. Flow cytometry was used to identify surface markers of BMDMs, with undifferentiated BMs serving as a control [76,77,78]. To measure ROS levels, we employed the DCFH2-DA probe (HY-D0940, Abcam, UK, 1 µM, 30 min). Cells were collected, and fluorescent intensity was measured using the Attune NxT flow cytometer (Thermo Fisher Scientific Inc.) [86].
Extraction of macrophage mitochondria
The supernatant from macrophage lysis was centrifuged at 10,000 xg for 10 min at 4 °C to obtain the mitochondrial pellet. The mitochondrial pellet was then resuspended in mitochondrial isolation buffer (MIB) containing 70 mM sucrose, 210 mM mannitol, 5 mM HEPES, 1 mM EGTA, and 0.5% (w/v) fatty acid-free BSA at pH 7.2. Subsequently, the resuspended mitochondria were layered on a 15% Percoll gradient (15% Percoll, 10% sucrose in 2.5 M, 75% MIB) and centrifuged at 21,000 xg for 10 min at 4 °C. Finally, the pellet was washed with MIB at 13,000 xg for 10 min at 4 °C [87].
Mitochondrial transfer experiment
In order to observe mitochondrial transfer, macrophages were incubated in a pre-warmed staining solution containing a MitoTracker Red CMXRos probe (50 nM) for 30 min and then washed three times with neutral PBS to remove the unbound probe. Co-cultivation with labeled BMSCs (using Mitotracker Green) was then performed, and after 24 h of cultivation, images were taken using confocal microscopy. The average fluorescence intensity of received mitochondria in BMSCs was analyzed using FlowJo software.
TSNE clustering analysis and cell annotation
To reduce the dimensionality of the scRNA-Seq dataset, we applied Principal Component Analysis (PCA) to the top 2000 highly variable genes with maximum variance. The first 14 principal components were selected for downstream analysis using the Elbowplot function in the Seurat package. To determine the major cell subgroups, we utilized the FindClusters function in Seurat with the default resolution value (resolution = 0.2). Subsequently, we employed the TSNE algorithm to reduce the dimensionality of the scRNA-Seq sequencing data nonlinearly. Lastly, known cell lineage-specific marker genes were utilized, and cell annotation was performed using the online website CellMarker [88].
Single-cell sequencing analysis
The scRNA-Seq data were processed using the Seurat package (version 3.1) in R software with standard downstream processing. The processing steps involved excluding cells with fewer than 200 detected genes and genes detected in less than 3 cells. Additionally, cells with mitochondrial proportions exceeding 10% were restricted. The data were then normalized using the LogNormalize method [89].
Next, clustering of cells was performed using the FindClusters function, and visualization was conducted using the RunUMAP function. Specific marker genes for cell clusters were identified using the FindMarkers function in the Seurat package. To determine differentially expressed genes (DEGs) specific to a particular cluster, the Wilcoxon rank-sum test was applied to compare cells within that cluster against all other cells. A Bonferroni-corrected p-value of less than 0.05 was used as the cutoff for identifying DEGs with statistical significance. Known cell lineage-specific marker genes were used, and cell markers were annotated using the online website CellMarker [90].
RT-qPCR
Total RNA was extracted from tissues or cells using Trizol reagent (15596026, Invitrogen, USA). The concentration and purity of total RNA at 260/280 nm were determined using NanoDrop LITE (ND-LITE-PR, Thermo Scientific™, Germany). The extracted total RNA was then reverse transcribed into cDNA using the PrimeScript RT reagent Kit with gDNA Eraser (RR047Q, TaKaRa, Japan). Subsequently, the SYBR Green PCR Master Mix reagent (4364344, Applied Biosystems, USA) and ABI PRISM 7500 Sequence Detection System (Applied Biosystems) were used to perform RT-qPCR for each gene. The primers for each gene were synthesized by TaKaRa (Table S1), with GAPDH serving as the internal reference gene. The relative expression levels of each gene were analyzed using the 2−ΔΔCt method, where △△Ct = (average Ct value of target gene in the experimental group – average Ct value of reference gene in the experimental group) – (average Ct value of target gene in the control group – average Ct value of reference gene in the control group) [91,92,93]. All RT-qPCR detections were performed in triplicate.
Western blot
Total protein from tissues or cells was extracted using highly efficient RIPA lysis buffer (C0481, Sigma-Aldrich, USA) containing 1% protease inhibitor (ST019-5 mg, Beyotime, Shanghai, China) and 1% phosphatase inhibitor. After 15 min of lysis at 4℃, the samples were centrifuged at 13,000 g for 15 min, and the supernatant was collected. The protein concentration of each sample was determined using the BCA assay kit (23227, TH&Ermo, USA). The protein samples were then quantified based on different concentrations and mixed with a 5x loading buffer (P0015, Beyotime, China). The proteins were separated by polyacrylamide gel electrophoresis and transferred onto a PVDF membrane (IPVH00010, Millipore, Billerica, MA, USA). The membrane was blocked with 5% BSA at room temperature for 1 h, followed by incubation with the following primary antibodies overnight: OPN (ab228748, 45 kDa, 1:2000, Abcam, UK), Runx2 (ab264077, 57 kDa, 1:1000, Abcam, UK), ALP (ab229126, 39 kDa, 1:1000, Abcam, UK), OCN (ab93876, 11 kDa, 1:1000, Abcam, UK). The membrane was then washed three times for 5 min each with TBST and incubated with the appropriate diluted secondary antibodies: HRP-conjugated goat anti-rabbit IgG (1:2000, ab205718, Abcam, UK) or goat anti-mouse IgG (1:2000, ab6789, Abcam, UK) at room temperature for 1.5 h. After incubation, the membrane was washed three times for 5 min each with TBST and developed using the chemiluminescent substrate (NCI4106, Pierce, Rockford, IL, USA). Protein quantification analysis was performed using ImageJ software by calculating the grayscale ratio of each protein to the internal control GAPDH (ab8245, 36 kDa, 1:1000, Abcam, UK) [92]. Each experiment was repeated three times.
Immunofluorescence
Cells or tissues were washed with ice-cold PBS and then fixed with 4% paraformaldehyde (P885233, Macklin, USA) for 15 to 30 min. Subsequently, they were treated with 0.1% Triton (L885651, Macklin, USA) for 15 min. After two PBS washes, samples were incubated with PBS solution containing 15% FBS at 5 °C for 4 min overnight. For staining, cells or tissues were covered with antibodies against TNF-α (ab237353, Alexa Fluor® 488, Abcam, UK; 1:100)/iNOS (ab209027, Alexa Fluor® 647, Abcam, UK; 1:100), CD68 (ab201844, Alexa Fluor® 488, Abcam, UK; 1:250)/iNOS (ab209027, Alexa Fluor® 647, Abcam, UK; 1:100), or CD86 (ab275741, Alexa Fluor® 488, Abcam, UK; 1:100)/CD163 (ab313666, Alexa Fluor® 647, Abcam, UK; 1:100). The samples were then incubated overnight at 4 °C. DAPI staining (D1306, Thermo Fisher, USA) was performed, and observations were made using a fluorescence microscope (Zeiss Observer Z1, Germany). The fluorescent intensity was measured in selected areas, and image processing and quantification were performed using ImageJ software to determine the number of positive cells [94].
ATP measurement
Cellular ATP levels were measured using an ATP assay kit (BC0300, Solarbio, Beijing, China). Cells were lysed by centrifugation to separate cell pellets from culture supernatants. Cell pellets were resuspended in 300 µL of hot double-distilled water, homogenized by heat in a water bath at 95 °C, and further heated in a boiling water bath for 10 min. Next, 30 µL of the sample was mixed with reagents one, two, three, and distilled water following the kit’s instructions. After thorough mixing and a 30-minute incubation at 37 °C, 50 µL of reagent four was added, mixed well, and centrifuged at 4000 rpm for 5 min. The supernatant (300 µL) was collected, mixed with 500 µL of reagent five, incubated at room temperature for 2 min, followed by the addition of 500 µL of reagent six and a 5-minute incubation at room temperature. The absorbance was measured at 636 nm with a path length of 0.5 cm using a UV spectrophotometer (DU720, Beckman, USA) [95].
Bioenergetic analysis
Bioenergetic measurements were performed using the Seahorse Bioscience XFe24 Flux Analyzer (Agilent Technologies) following the manufacturer’s instructions. Briefly, cells were seeded at a density of 5 × 104 cells per well on a collagen-coated 24-well Seahorse XFe plate overnight. The oxygen consumption rate (OCR) was measured using a basal assay medium containing 1 mM pyruvate, 10 mM glucose, and 2 mM glutamine (25030081, Thermo Fisher, USA). Subsequently, samples were treated with 1 µM oligomycin (ab141829, Abcam, UK), 2 µM FCCP (ab120081, Abcam, UK), and 0.5 µM rotenone/antimycin A (ab52922, Abcam, UK) sequentially. Finally, the measured values were normalized to cell number per well and analyzed using Seahorse Wave software [96].
JC-1 staining
Cells were seeded at a density of 2 × 104 in 35 mm culture dishes and incubated overnight. The following day, cells were treated for 24 h in different experimental groups. Afterward, cells were washed three times with 1×PBS and co-incubated with JC-1 dye (T3168, Thermo Fisher, USA) for 20 min at 37 °C. After washing, cells were mounted on glass slides and observed under a fluorescence microscope. ImageJ software was used for image analysis, statistical analysis, and quantification of the percentage of positive cells [97].
ELISA
Cell or tissue samples to be tested were collected and lysed or centrifuged to obtain supernatants, following the instructions provided with the assay kits. IL-1β ELISA kit (ab197742, Abcam, UK), IL-6 ELISA kit (ab100712, Abcam, UK), and TNF-α ELISA kit (ab208348, Abcam, UK) were used. First, the antigen used was diluted to an appropriate concentration in coating buffer, and 5% bovine serum (F8318, MSK, Wuhan, China) was added to seal the enzyme reaction wells at 37 °C for 40 min. Diluted samples were added to the enzyme reaction wells, followed by the addition of the enzyme conjugate and substrate solution. The reaction was stopped by adding 50µL of stop solution to each well within 20 min. The plate was read at 450 nm using an enzyme reader (Bio-Rad, USA), and a standard curve was plotted. Finally, data analysis was performed [98, 99].
Animal experiments
BALB/c male mice aged 7–8 weeks and weighing 16–20 g were obtained from the Experimental Animal Research Center at our institution. All animal studies were conducted in accordance with our institution’s “Guidelines for the Care and Use of Laboratory Animals.” The mice were housed at a temperature of 23 ± 1 °C and a relative humidity of 55 ± 5%, with a 12-hour light/dark cycle and free access to food and water. To acclimatize to the environment, the mice were kept under these conditions for 2 to 3 days prior to the experiments [100].
Establishment of a mouse model for femoral fractures
A mouse model for femoral fractures was established using 7-8-week-old male BALB/c mice weighing 16–20 g. The mice were anesthetized with chloral hydrate (50 mg/kg, C8383, Sigma-Aldrich, USA). The leg was cleaned with 10% iodine solution, and a longitudinal incision was made on the skin to expose the femur after separating the muscles. Subsequently, a three-point bending device was used to induce a fracture near the proximal end of the femur, followed by fixation with a 30-gauge intramedullary needle. The incision was closed using 5 − 0 absorbable sutures, and another fracture was induced using the three-point bending device. After anesthesia recovery, the mice were allowed unrestricted movement without any weight-bearing restrictions. Fractures that were not located in the midshaft or severe comminuted fractures were excluded from the study. Prior to fracture induction, sustained-release buprenorphine (3.25 mg/kg) was administered as an analgesic and repeated every 72 h for a total of 7 days [101].
For the group receiving scaffold treatment for fractures, a printed gel scaffold was placed in the fracture marrow cavity and fixed using a 30-gauge needle after the fracture. Animal experimental groups were as follows:
Normal group: Normal mice without any intervention.
Model group: Mice with fractures treated using natural healing after fixation and suturing.
GA group: Mice with fractures treated using a 3D-printed GA scaffold at the fracture site.
GAD/Ag-pIO group: Mice with fractures treated using a 3D-printed GAD/Ag-pIO scaffold at the fracture site.
GAD/Ag-pIO + mito group: Mice with fractures treated using a 3D-printed GAD/Ag-pIO scaffold loaded with extracted macrophage mitochondria (mito) at the fracture site.
Immunohistochemistry and immunofluorescence observations were conducted on randomly selected six mice from each group on the 3rd, 7th, and 14th days after fracture to observe changes in the inflammatory process during the healing process. Micro-CT was used to observe the growth of newly formed bone at the fracture site on the 14th and 35th days after fracture. On the 35th day after complete fracture healing, bone tissue was stained with H&E, and the mechanical strength of the healing bone was evaluated using a four-point bending mechanical test. Six mice from each group were sampled [101, 102].
Micro-CT analysis
The femoral bone tissue region was scanned using a micro-CT system (mCT-40, Scanco Medical, Switzerland) to investigate the growth characteristics of the femoral bone tissue. The scanning parameters were set as follows: power current: 385µA, power voltage: 65 kV, pixel size: 9 μm, filter: AI 1.0 mm, rotation step: 0.4°. Image reconstruction was performed using Bruker’s NRecon software, followed by data analysis using the CTAn program. Two specific volumes of interest were created at a height of 0.5 mm above the growth plate of the femoral head and at a height of 0.25 mm. The region of interest (ROI) beneath the articular cartilage within these volumes was manually defined, and a constant threshold (50–255) was applied for binarization using trabecular bone. The micro-CT parameters analyzed were as follows: (a) trabecular bone volume fraction (bone volume/total tissue volume, BV/TV): the ratio of bone surface area to tissue volume; (b) trabecular bone thickness (Tb.Th): the average thickness of trabeculae, used to describe trabecular structure changes; (c) trabecular bone number (Tb.N): the number of intersections between bone and non-bone tissues per unit length; (d) bone density (bone mineral density, BMD): the distribution of bone mass and density within the skeleton [103, 104].
Four-point bending mechanical testing
Mechanical testing was conducted on the second day following animal euthanasia at ambient temperature. The contralateral femur was compared to an internal control group. A four-point bending device equipped with a 50 N load cell sensor (Hounsfield Test Equipment Ltd, UK) was employed to assess femoral healing. During the tests, loading was applied along the anteroposterior axis, with an inner span of 8 mm and an outer span of 20 mm. The femur’s long axis was maintained perpendicular to the blades. The QMAT Professional software (Tinius Olsen Inc., Horsham, PA, USA) was used to measure and analyze ultimate load (UL), elastic modulus (E-modulus), and failure energy. These biomechanical properties were expressed as percentages relative to the values obtained from the intact contralateral skeleton [105].
Pathology tissue/cell staining
Hematoxylin and Eosin (H&E) Staining: Tissue samples for examination are obtained and subjected to fixation. Paraffin-embedded sections are placed in xylene to remove the wax. Then, they are dehydrated in sequential baths of 100% ethanol, 95% ethanol, and 70% ethanol. Subsequently, the sections can be either embedded or rinsed with water. The prepared sections are stained in hematoxylin staining solution (H8070, Solarbio, Beijing, China) for approximately 5–10 min at room temperature. Afterward, the slides are rinsed with distilled water and dehydrated in 95% ethanol. The sections are then stained with an eosin staining solution (G1100, Solarbio, Beijing, China) for 5–10 min. Finally, standard procedures for dehydration, clearing, and slide coverslipping are followed [106]. ImageJ software is utilized for image processing and quantitative analysis of the newly formed bone area [102, 107].
ALP Staining: ALP detection is performed using an ALP staining kit (CTCC-JD002, Puhui Biotech, Wuxi, China). ALP staining solution is prepared according to the instructions provided. In each 10 mL of the color development buffer, 33 µL of BCIP solution and 66 µL of NBT solution are added. After thorough mixing, an appropriate amount of staining solution is added to each well to cover the cells. The plate is incubated at room temperature in the dark for 30 min. If no blue staining is observed within 30 min, incubation can be continued overnight. After discarding the staining solution, the plate is washed twice with distilled water to stop the reaction. Following the removal of the wash solution, images are captured, and ALP activity is quantitatively analyzed by measuring the optical density [107].
ARS Staining: ARS staining kit (C01383, Yagebio, Shanghai, China) is used for detection. Alizarin Red forms a visible orange-red complex with calcium ions, which is used to identify the presence of calcium nodules. The specific steps are as follows: add an appropriate amount of ARS staining solution to cover the cells and incubate at room temperature for 5 min, during which orange-red clusters can be observed. Discard the staining solution, and wash the plate three times with distilled water to stop the reaction. Images are captured, and quantitative analysis is performed by measuring absorbance values [107].
Immunohistochemistry staining
The tissue or cells to be tested were fixed and embedded. The embedded tissue was then sectioned and subjected to dewaxing treatment to remove the paraffin, making it hydrophilic for subsequent immunostaining procedures. The dewaxed tissue sections were treated with SOD1 protein antibody (ab308181, Abcam, UK, 1:100) and IL-1β protein antibody (ab315084, Abcam, UK, 1:500). The sections were processed with Anti-Rabbit-HRP secondary antibody (12–348, Sigma Aldrich, Shanghai, China, 1:1000). DAPI staining (ab64238, Abcam, USA) was used to visualize the sites where the secondary antibody bound to the primary antibodies. The stained tissue sections were then dewaxed and coverslipped. The sections were observed under a microscope, and Image J software was used for image processing and quantitative analysis of the positive efficiency [108, 109].
Statistical analysis
Statistical analysis was performed using ImageJ, SPSS 25, and GraphPad Prism 8 software. All data were processed using GraphPad Prism 8.0 and presented as mean ± standard deviation (Mean ± SD) for continuous variables. An unpaired t-test was used to compare the two groups, and one-way analysis of variance (ANOVA) was employed for multiple group comparisons. Levene’s test was used to test for homogeneity of variance. In the case of homogeneity, Dunnett’s t-test and LSD-t-test were used for pairwise comparisons. In the presence of heterogeneity of variance, Dunnett’s T3 test was employed. Pearson’s correlation analysis was used to determine the correlation between genes and the content of immune cells. A p-value of < 0.05 was considered statistically significant for group comparisons [110].
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