Cell culture and characterization
New Zealand rabbits weighing approximately 0.4–0.5 kg (regardless of gander) were selected as the source of primary BMSCs. The ends of the epiphyses were cut, the middle bone marrow was then extracted and centrifuged at 1000 rpm for 5 min. The supernatant was removed, and 2 mL of L-DMEM (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA) was added for resuspension. The cells were evenly divided into six-well plates and incubated at 37 °C in a 5% incubator. Suitable rabbit BMSCs were selected and made into single-cell suspensions. BMSCs were identified using flow cytometry with the cell surface markers Rabbit Anti-CD34/FITC (1:100, Bioss, China), Rabbit Anti-CD44/FITC (1:100, Bioss, China), and Rabbit Anti-CD45/FITC (1:100, Bioss, China). As described in the literature, suitable cells were selected for induction of chondrogenic, osteogenic, and adipogenic differentiation and identified by Alisin Blue (Sigma, USA), Alizarin Red (Sigma, USA), and Oil Red O (Sigma, USA) staining respectively after 21 days.
Exosomes preparation and identification
BMSCs cultured to P3 generation were transferred to 6-well plates, and cell supernatants were collected daily when the cell density grew to 80% (approximately 5 × 105 cells/well). BMSCs-exosomes in the supernatant were extracted according to the instructions of the VEX Exosome Isolation Reagent kit (Vazyme, China), and the concentration of exosomes was measured using the BCA protein quantification kit (Beyotime, China). The structure of the exosomes was observed using transmission electron microscopy (TEM; Titan, FEL, USA), the size distribution and concentration of the exosomes were determined by nanoparticle tracking characterization system (Zatasizer Nano ZS90, Malvern Panalytical), and the surface-specific proteins Calnexin (Bioss, China), CD9 (Bioss, China), CD81 (Bioss, China), and TSG101 (Bioss, China) were detected by Western Blot.
BMSCs-exosomes labeling
First, 500 µl BMSCs-exosomes (50 µg/ml) were resuspended in 0.5 ml Diluent C (Solarbio, China) and then mixed with 4 µl PKH26 (Solarbio, China). The solution was incubated at room temperature and avoided light for 5 min. Then, 2 ml of 0.5% BSA (Beyotime, China) was used to end the staining. The mixture was centrifuged at 10,0000 g for 70 min to remove the residual dye, followed by resuspension of the precipitate in 200 µl phosphate buffered saline (PBS).
GelMA preparation and pore size analysis
A 0.25% initiator standard solution (EFL, China) was prepared according to the kit instructions. 0.5 g of GelMA-30 or GelMA-60 solid (EFL, China) and 1 mL of initiator standard solution were added into the tube and heated in a water bath to dissolve completely. The GelMA solution was aspirated using a sterile syringe and filtered through a 0.22 μm sterile filter in an ultraclean table. The filtered sterile GelMA solution was irradiated with a 405 nm Ultraviolet (UV) light source for 30 s to solidify. The gelatinized samples were frozen at − 80 °C for 2 h, then dried in a vacuum dryer for 24 h. The samples were then sprayed with gold and placed on a scanning electron microscope (SEM; Gemini 2, Sigma, USA) irradiation stage for photography. Image J software was used to measure the average diameter of each hole and calculate the hole size based on the SEM photographs.
GelMA-exosomes distribution and release curve analysis
The 5% GelMA-30 was prepared according to the method mentioned above. BMSCs-exosomes were marked with PKH-26. An appropriate amount of BMSCs-exosomes was mixed with 5% GelMA-30 solution (the final concentration of exosomes was 50 µg/ml) and irradiated with UV light for 30 s to allow sufficient gelation. Subsequently, the distribution of BMSCs-exosomes in GelMA was observed by confocal microscopy. GelMA-exosomes was placed in 1 ml PBS. The supernatant was gathered daily and quantified after 7 days.
Establishment of rabbit TBJ injury model
All the animal experiments were approved by the Ethics Committee of Soochow University (Approval No.: SUDA20230619A02) and all animal manipulations were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Soochow University. Additionally, the study was conducted in compliance with the ARRIVE guidelines.
A total of 72 three-month-old rabbits (weight 2.7 ± 0.2 kg) were studied. The rabbits were continuously anaesthetised using isoflurane inhalation anaesthesia. Successful anaesthesia was indicated by the disappearance of the corneal reflex. Surgical preparation of the left lower limb of the rabbit was treated. A longitudinal incision was made on the posterior lateral aspect of the lower leg using a blade to expose the flexor digitorum longus and tibia. Along the walk of the tendon, the flexor digitorum longus tendon was severed distally and the severed end was secured with suture. A 2.0 Kirschner wire (Pfizer, USA) was used to drill a hole in the lower 1/3 of the tibia vertically when fixed with a suture anchor (2.0 Mini Quickanchor Plus, AO, China) (n = 36). A 4.0 Kirschner wire (Pfizer, USA) was used to drill when secured with an interference screw (5 × 12 mm Milagro BR Interference Screw, AO, China) (n = 36) (Fig. 3). A 50 µg/ml GelMA-exosome mixture was prepared by encapsulating the exosomes in GelMA, which was subsequently injected into the TBJ site using a 1 ml syringe. Then, the experiment was divided equally into 3 groups according to the implants: (1) control group (only inject 1 ml PBS at the injury site), (2) GelMA group (only inject 1 ml GelMA at the injury site), (3) GelMA-exosomes group (inject 1 ml GelMA-exosomes at the injury site). The wound was then closed layer by layer and dressed with aseptic gauze. All animals resumed normal diets after the operation and were given ceftiofur sodium for anti-infection treatment the next day. The experimental animals were euthanised by intravenous pentobarbital. At 1 week postoperatively, qRT-PCR assays were performed to detect inflammatory factors at the TBJ and Edu assays were used to detect cell proliferation. HE staining, SO-FG staining, immunohistochemical analysis, Micro-CT analysis of surface bone volume changes, and biomechanical tests were used to detect the strength of tendon-bone healing at 3 and 6 weeks postoperatively.
qRT-PCR analysis
At one week after operation, animals of the corresponding group were sacrificed by air embolization introduced from the ear vein. The specimens from the TBJ were collected. 1 ml Trizol (Thermo Fisher, China) was added to the tissue and grinded. Subsequently, chloroform (Servicebio, China), isopropanol (Servicebio, China), and ethanol (Servicebio, China) were added in the appropriate proportions to extract the RNA. The primers were added according to the kit instructions (RiboBio, China) (Table 1), and performed the reverse transcription reaction at 42 °C for 60 min and 70 °C for 10 min. The cDNA concentration was measured after the reverse transcription. The reverse transcription products were amplified by adding the appropriate primers according to the kit instructions in a fluorescence PCR machine. The reaction program was set as follows: pre-denaturation at 95 °C for 10 min, denaturation at 95 °C for 2 s, annealing at 60 °C for 2 s, and extension at 70 °C for 10 s. A total of 40 cycles were performed. Melting curve analysis was performed immediately after the end of the cycle. CT values were obtained, and gene expression ploidy differences were calculated.
Cell proliferation analysis
Three days after surgery, 1 ml Edu staining solution (RiboBio, China) was injected subcutaneously. The corresponding rabbits were sacrificed after 4 days and specimens were collected from the TBJ. The specimens were collected for optimal cutting temperature compound (OCT, abcam) embedding to produce frozen sections, and 5 μm tissue sections were prepared. Staining was carried out according to the kit instructions (RiboBio, China), and 50 µl of anti-fluorescence quenching sealer was added to each specimen after staining was completed to seal the coverslips. Fluorescence microscopy (Nikon, China) was used for imaging and analysis.
Histological and immunohistochemical analysis
The specimens were fixed in 4% paraformaldehyde (Servicebio, China) for 24 h and decalcified in 15% EDTA (Servicebio, China) for 1 week. The fixed specimens were placed in tissue embedding boxes, and the serial numbers of the specimens were marked using an oil-based pen. The specimens were dehydrated in a gradient using different concentrations of ethanol, followed by wax immersion, embedding, and sectioning, with the thickness of the sections controlled at 3 μm. The sections were dehydrated in gradient alcohol at room temperature and stained for HE (Servicebio, China) and SO-FG (Servicebio, China). The corresponding anti-ACAN antibody (1:1000, Servicebio, China) and anti-Col II antibody (1:1000, Servicebio, China) were diluted at the corresponding ratios, and immunohistochemical staining was performed. The sections were stained and then air-dried. The sections were placed under a microscope for observation and image analysis.
Biomechanical testing
Specimens were collected 6 weeks after surgery and placed at – 80 °C for storage to prevent tissue damage. Samples were thawed at room temperature for more than 6 h before testing. The tendon tissue was fixed using a blood-repellent band to increase friction. Then, the specimen was fixed with clamps. The initial position of the tensiometer was adjusted so that the tester displays around 0 N. Then, the tensiometer was set to zero, and the tendon was stretched at a 5 mm/minute rate until the tendon-bone connection was separated. Measurements were taken to calculate the maximum tensile force and strength, where the maximum tensile force was the maximum rupture force, and strength was calculated by dividing the maximum tensile force by the contact area of the tendon-bone interface. The contact area for the interference screw group was calculated as follows: With a bone tunnel radius of 2 mm, the tendon-to-bone contact length is approximately one third of the tunnel’s circumference, the depth is based on the cortical thickness of the tibia in rabbits, with a mean value of about 1 mm. Thus, the contact area is 1/3 × π × 22 × 1 = 4π/3 mm2. For the suture anchor group, the bone tunnel radius was 1 mm. The tendon was anchored at the tunnel’s opening, making the contact area roughly the same as the area of the circular opening: π × 12 = π mm2.
Micro-CT scanning
The specimens were collected 3 and 6 weeks postoperatively and placed in 4% paraformaldehyde for 24 h of fixation. The specimens were then placed on a machine foam box and scanned after setting the scanning parameters. The X-ray scans were algorithmically reconstructed into tomograms using the NRecon Reconstruction software; the scanned tomograms were reconstructed in 3D using the CTVox software. CTan software was used to analyze the quality and density of new bone tissue within the bone tunnel and changes in bone quality around the bone tunnel.
Statistical analysis
This study used ImageJ software for image processing, SPSS 25.0 software for data analysis, and GraphPad Prism 8.0 software for plotting. Data between two groups were analyzed using an unpaired t-test, and differences between multiple groups were analyzed using ANOVA. T-tests were used to determine statistical differences between groups. Differences were considered statistically significant at P < 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
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- Source: https://www.nature.com/articles/s41598-024-79787-0