Materials
Amine-terminated G5 PAMAM was purchased from Weihai Chenyuan Molecular New Material Co., Ltd. (Shandong, China). 4-(Bromomethyl)phenylboronic acid (PBA), gelatin, N-hydroxysuccinimide (NHS), NaIO4, and ethylene glycol were purchased from Aladdin (Shanghai, China). GelMA (EFL-GM-60) and lithium pheny1-2,4,6-trimethybenzoylphosphinate (LAP) were purchased from Suzhou Intelligent Manufacturing Research Institute (Suzhou, China). Dulbecco’s modified Eagle’s medium, Ham’s F-12 medium (DMEM/F12), and fetal bovine serum (FBS) were purchased from Gibco (Carlsbad, CA).
Synthesis and characterization of G5-PBA-dendrimer nanoplatforms (GPNPs)
As mentioned previously, PBA-functionalized dendritic macromolecules were synthesized via the following method, in which phenylboronic acid (PBA) was grafted onto the surface of dendritic macromolecules through a substitution reaction under heating conditions [37]. First, amine-terminated G5 dendritic macromolecules and 4-(bromomethyl)phenylboronic acid were dissolved in a 40 mL anhydrous methanol beaker at a molar ratio of 1:256, followed by thorough stirring at 70 °C for 24 h. The reaction product was transferred into a dialysis bag (MWCO = 3500 Da) and extensively dialyzed against anhydrous methanol and deionized water to remove unreacted substances, impurities, and solvents, with the dialysis solution replaced every 3 h. The purified product was freeze-dried to obtain para-PBA-modified dendritic macromolecules. The fully purified product was characterized using 1 H nuclear magnetic resonance (1 H NMR) spectroscopy, and the average number of PBA molecules grafted per dendritic macromolecule was calculated based on the integral areas corresponding to the characteristic peaks of PBA and the dendritic macromolecules.
Preparation of SOX9@GPNPs, SIRT1@GPNPs, and SOX9/SIRT1@GPNPs
The preparation of SOX9@GPNPs, SIRT1@GPNPs, and SOX9/SIRT1@GPNPs was conducted separately. For SOX9@GPNPs preparation, GPNPs were mixed with SOX9 expression plasmid (0.8 µg) at N/P ratios (0, 1, 2, 4, 8, 16) in 100 µl solution according to a previously reported method for N/P ratio calculation [37]. The corresponding GPNP masses were 0, 0.18 µg, 0.36 µg, 0.72 µg, 1.44 µg, and 2.88 µg. The mixtures were vortexed thoroughly for 3 min and incubated at 25 °C for 30 min. For agarose gel electrophoresis, samples with different N/P ratios and DNA marker were loaded onto a 1% agarose gel. Electrophoresis was performed in 1×TAE buffer at 130 V for 30 min, and band patterns were visualized using a gel imaging system. Subsequently, SOX9@GPNPs were diluted with PBS to 1 ml for hydrodynamic size and zeta potential measurements using a Zetasizer Nano-ZS (Malvern, UK) at room temperature. Parameters including size distribution, average particle size, and PDI were recorded. For SIRT1@GPNPs preparation, SIRT1 protein was mixed with GPNPs at W/W ratios (0, 2, 4, 8) in 100 µl solution, corresponding to SIRT1 masses of 0, 2.88 µg, 5.76 µg, and 11.52 µg (with fixed GPNP mass of 1.44 µg based on the optimal N/P ratio for SOX9@GPNPs). The mixtures were vortexed for 3 min, incubated at 25 °C for 30 min, and then diluted to 1 ml with PBS for hydrodynamic size and zeta potential measurements using the same protocol as SOX9@GPNPs. For SOX9/SIRT1@GPNPs preparation, SOX9 plasmid (0.8 µg) was first complexed with GPNPs (1.44 µg) at N/P = 8, followed by the addition of SIRT1 protein (2.88 µg). Other steps followed the protocols for SOX9@GPNPs and SIRT1@GPNPs. The final complexes were diluted to 1 ml with PBS for hydrodynamic size analysis. The particle size stability of GPNPs, SOX9@GPNPs, and SIRT1/SOX9@GPNPs was monitored at 0, 12, 24, and 36 h during PBS storage. Additionally, the morphology of SIRT1/SOX9@GPNPs was characterized by TEM (JEOL JEM 2100 F).
Intracellular delivery of nanocomplexes
The prepared SOX9@GPNPs, SIRT1@GPNPs, and SOX9/SIRT1@GPNPs were diluted to 500 µl using DMEM/F12 complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin for subsequent cell culture and incubated in a 37 °C, 5% CO₂ humidified incubator. For cytotoxicity testing, NPCs were seeded into 96-well plates to ensure uniform distribution. Groups included SOX9@GPNPs at varying N/P ratios (0, 1, 2, 4, 8, 16) and SIRT1@GPNPs at varying W/W ratios (0, 2, 4, 8). The complexes were added to designated wells and cultured for 24 h. After incubation, 100 µL CCK-8 reagent (dissolved in sterile DMEM/F12 medium, Beyotime, China) was added to each well and incubated in the dark for 1 h. Absorbance at 450 nm was measured using a full-wavelength microplate reader. To evaluate GPNPs’ plasmid DNA delivery efficiency, GPNPs were mixed with pcDNA3.1-Sox9-ZsGreen1 at N/P ratios of 0, 1, 2, 4, 8, and 16. When NPCs reached 80% confluency, the mixtures were added to the culture medium. After 6 h incubation, the medium was replaced, and cells were cultured for an additional 18 h. Green fluorescence expression was observed under an inverted microscope. Successful delivery by SOX9@GPNPs was confirmed by detecting Sox9, Col2a1 and Acan gene expression (NC group: Sox9 none-expressed; SOX9@GPNPs: Sox9 expressed). Total RNA was extracted using TRIzol reagent (Invitrogen, American), quantified with NanoDrop 2000 spectrophotometers (Thermo Fisher Scientific, American), reverse-transcribed into cDNA, and amplified following manufacturer protocols. Primer sequences (Sangon Biotech, Shanghai, China) are detailed in Table S1. Gene expression levels were normalized to GAPDH. For protein delivery assessment, BSA-FITC (green fluorescent) was used as a model protein. Groups included control, GPNPs, BSA-FITC, and BSA-FITC@GPNPs (WBSA-FITC: WGPNPs = 1.5:1; 2.16 µg BSA-FITC, 1.44 µg GPNPs). After 6 h treatment, NPCs were fixed with 4% paraformaldehyde, stained for cytoskeleton (37 °C, dark, 1 h), and nuclei (DAPI). Fluorescence distribution was observed under an inverted microscope. To evaluate intracellular ROS levels before and after SIRT1 delivery, a DCFH-DA probe-based fluorescence assay was performed. Groups included CTRL (Control no other treatment), PBS/LPS (PBS and LPS co-treatment), FREE SIRT1/LPS (SIRT1 protein and LPS co-treatment), and SIRT1@GPNPs/LPS (SIRT1@GPNPs and LPS co-treatment). The concentration of LPS was 1 µg/mL. NPCs were seeded in 24-well plates, cultured to 80–90% confluency in DMEM/F12 medium, and treated according to groups for 24 h. After washing with serum-free medium, DCFH-DA working solution (1:1000 dilution in PBS, Beyotime, China) was added and incubated (37 °C, dark, 20 min). Nuclei were stained with Hoechst 33,342 (Beyotime, China) for 10 min. Fluorescence was observed under a fluorescence microscope. Sequential delivery was monitored by delivering BSA-FITC and pcDNA3.1-Sox9-DsRed plasmid (2.16 µg BSA-FITC, 1.44 µg GPNPs, 0.8 µg plasmid) into cells. Fluorescence distribution was observed at 6, 12, 24, and 48 h post-delivery. For follow-up experiments (antioxidant, mitochondrial function, ECM synthesis/catabolism, and phenotypic assays), groups included: (1) Normal medium (CTRL); (2) PBS + 1 µg/mL LPS (PBS group); (3) SOX9@GPNPs + 1 µg/mL LPS (SOX9@GPNPs group); (4) SIRT1@GPNPs + 1 µg/mL LPS (SIRT1@GPNPs group); (5) SIRT1/SOX9@GPNPs + 1 µg/mL LPS (SIRT1/SOX9@GPNPs group). NPCs were seeded in 24-well plates, treated with group-specific solutions for 6 h after reaching 80% confluency, washed twice to remove residual medium, and incubated in fresh complete medium post-treatment.
Extraction and culture of NPCs
NPCs were isolated from the tail vertebrae of 6-week-old male Sprague–Dawley rats. Under sterile conditions, the nucleus pulposus tissue was removed from the IVDs and incubated at 37 °C for 4 h in a 0.25% type II collagenase solution (Yuanye, Shanghai, China). Following incubation, the digested mixture was collected, centrifuged to separate the cells, and the supernatant was discarded. The resulting cell pellet was then resuspended in DMEM/F12 medium enriched with 10% FBS. Once the cells had attached, the culture medium was refreshed every two days, and cell morphology was routinely inspected.
Biocompatibility evaluation of SOX9@GPNPs and SIRT1@GPNPs
The in vitro cytotoxicity was assessed using the CCK8 assay. Approximately 6,000 cells were seeded in a 96-well plate and incubated overnight for attachment. Grouping was based on the aforementioned culture systems, and each group had three replicates. The next day, NPCs were treated with different formulations for 24 h and then incubated with 100 µl CCK8 solution for an additional 1 h. Absorbance at 450 nm was measured using a spectrophotometer (Invitrogen, USA).
Mitochondrial membrane potential detection
The mitochondrial membrane potential was assessed using the JC-1 dye, a sensitive indicator for mitochondrial membrane potential, as per the manufacturer’s instructions (Beyotime, China). The functional mechanism of JC-1 relies on its aggregation state in response to the membrane potential: it forms red fluorescent J-aggregates at high potentials and exists as green fluorescent monomers at low potentials. Prior to staining, NPCs were washed with PBS, then treated with pre-warmed JC-1 dye (1:200) and the nuclear stain Hoechst 33,342, respectively, at 37 °C for 30 min. Following staining, cells were washed three times with PBS to remove unbound dye. Finally, the stained cells were visualized under an inverted microscope to assess the intensity and distribution of red and green fluorescence, and fluorescence intensity was analyzed using ImageJ software.
MitoSOX red detection
To measure mitochondrial ROS (mtROS), we employed MitoSOX Red, a fluorescent probe specifically designed to detect superoxide in mitochondria (MCE), following the manufacturer’s protocol. For staining, a working solution of MitoSOX Red at 10 µM was prepared. Following the removal of the culture medium, a freshly prepared MitoSOX Red working solution was added to cover all cells. The cells were subsequently incubated in darkness at 37 °C and 5% CO2 for 30 min, and then stained with Hoechst 33,342 under the same environmental conditions. After the incubation period, the cells were carefully washed three times with PBS to eliminate any unbound dye. The cells were then examined using an inverted microscope, and the analysis was performed using ImageJ software.
Microscopic observation of mitochondria by TEM
For the microscopic observation of mitochondria by transmission electron microscopy (TEM) (JEOL JEM F200,Japan), NPCs were collected after stimulation and centrifuged. The culture medium was discarded, and the cells were fixed with pre-cooled electron microscopy fixative at room temperature for 2 h. The cell pellet was then processed further: it was washed three times with 0.1 M phosphate buffer (pH 7.2), refixed with 1% osmium tetroxide, and washed again three times with the same phosphate buffer. Afterward, the samples underwent dehydration in a graded series of alcohols, infiltration, and embedding in resin. The resin blocks were sliced into ultrathin Sect. (50 nm) for staining. Ultimately, the sections were observed under a transmission electron microscope.
Flow detection
The collected cells were resuspended in PBS and centrifuged at 300 g for 5 min. After discarding the supernatant, the cells were resuspended in binding buffer and adjusted to a concentration of 1 × 106 cells/ml. Annexin-V/PI staining was conducted in darkness. Flow cytometry analysis was performed using a flow cytometer (Merck Millipore, Germany). Subsequently, FlowJo software was utilized to determine the proportions of cells in different states and to calculate the apoptosis rate for each group.
Western blot
Proteins were extracted from NPCs under various conditions using RIPA buffer with protease inhibitors (Thermo Fisher, USA). Protein concentration was measured with a BCA Assay Kit (Beyotime, China). After denaturing the proteins at temperatures exceeding 95 °C, equal volumes were loaded onto a 10% SDS-PAGE gel for electrophoresis. Proteins were then transferred to a PVDF membrane (Millipore, USA). The membrane was blocked for 2 h and incubated with a diluted primary antibody (1:1,000) at 4 °C overnight. The next day, the membrane was washed with TBST, incubated with a diluted secondary antibody (1:1,000) for 2 h, and washed again. The membrane was scanned, and band intensity was analyzed using ImageJ to assess protein expression. Antibodies used are listed in Table S2.
Immunofluorescence staining
Roughly 10,000 NPCs were plated in a 24-well dish and left to adhere overnight. The next day, various stimuli were introduced. NPCs were immobilized with 4% paraformaldehyde at ambient temperature and subsequently blocked with an immunostaining solution (Beyotime, China) incorporating Triton X-100 for an hour. Cells were then incubated with diluted primary antibodies (1:200; targeting COL II, KRT-19, NLRP3, Caspase-1, GSDMD) at 4 °C overnight. The following day, after three PBST washes, cells were exposed to diluted fluorescent secondary antibodies for two hours. Observations were conducted using an inverted microscope, and fluorescence strength was analyzed quantification via imageJ software.
Alcian blue staining
Alcian Blue forms a blue insoluble complex with glycosaminoglycans, indicating positive staining under a microscope. After fixing the cell samples with 4% paraformaldehyde, they were washed three times with PBS. Alcian blue staining was then performed by adding 500 µl of Alcian staining solution. After staining, samples were thoroughly washed with PBS to remove unbound dye. Finally, the staining results were observed under a microscope.
RNA sequencing and differential bioinformatic analyses
To explore the impact of LPS and SIRT1/SOX9@GPNPs (SSGP) on gene expression patterns, RNA sequencing analysis was carried out. This involved two study groups: the LPS group and the SSGP group, each with three biological replicates. RNA was isolated from the treated cells with TRIzol reagent (Invitrogen, Carlsbad, CA), and its quality and integrity were evaluated. The sequencing of RNA libraries was performed on the Illumina platform by Genedenovo Biotechnology Co., Ltd (Guangzhou, China). DESeq2 was utilized for differential gene expression analysis, where genes with an adjusted p-value less than 0.05 and an absolute log2 fold change greater than 1 were considered significantly differentially expressed.
Preparation of oxidised hyaluronic acid
First, 1.5 g of hyaluronic acid was placed in a beaker, and 150 mL of deionized water was added. The mixture was stirred at room temperature using a magnetic stirrer until the hyaluronic acid was completely dissolved. Subsequently, to oxidize the hyaluronic acid, 802 mg of sodium periodate (NaIO₄) was added to the solution as an oxidizing agent, followed by thorough stirring for 2 h. After the reaction, 200 µL of ethylene glycol was added to terminate the process. The resulting product was transferred into a dialysis bag, sealed, and dialyzed in deionized water. The deionized water was replaced every 3 h over 48 h to thoroughly remove small-molecule impurities. After dialysis, the purified product was freeze-dried to obtain OHA. The prepared OHA sample was sealed in a 50 mL centrifuge tube and stored in a 4 °C refrigerator for subsequent experiments.
Preparation and characterization of composite hydrogels
First, the previously prepared SOX9@GPNPs, SIRT1@GPNPs, and SOX9/SIRT1@GPNPs were crosslinked with OHA, respectively, and stirred until fully dissolved. Subsequently, gelatin methacryloyl (GelMA) with a substitution degree of 60% and the photoinitiator lithium phenyl-2,4,6-trimethyl-benzoyl phosphinate (LAP) were added to the above solution for further crosslinking. After stirring until complete dissolution, the hydrogel prepolymer was obtained. The final concentrations were 5% for GelMA, 1% for OHA, and 0.05% for LAP. Each milliliter of the prepolymer contained 0.8 µg of SOX9-expressing plasmid, 1.44 µg of GPNPs, and 2.88 µg of SIRT1 protein. The precursor solution was photo-crosslinked for 1 min using a blue light source with a wavelength of 405 nm, yielding the composite hydrogels SOX9@GPNPs@G-HA, SIRT1@GPNPs@G-HA, and SOX9/SIRT1@GPNPs@G-HA. These hydrogels were then freeze-dried and stored at 4 °C. The freeze-dried samples were cut into slices approximately 2 mm thick. The dried hydrogel slices were adhered to SEM sample stubs using conductive adhesive and sputter-coated with gold using an ion sputter coater (coating parameters: 20 mA current, 45 s). Finally, the microstructures of the hydrogels were observed using scanning electron microscopy (SEM). To investigate the in vitro degradation of the hydrogels under physiological conditions, newly prepared hydrogel samples (500 µL) were incubated in PBS at 37 °C with a rotation speed of 100 rpm. The samples were weighed (Wt) on the 7th, 14th, 21st, and 28th days. Subsequently, the degradation of the hydrogels was computed using the following formula: weight remaining (%) = Wt/W0 × 100%. To test the release of SOX9-expressing plasmid and SIRT1 protein, 500 µL SIRT1/SOX9@GPNPs@G-HA hydrogel was prepared. Hydrogels were put into 2 mL PBS and then placed in a constant temperature shaker (37 °C, 80 cycles/min). At desired time points (1 d, 3 d, 5 d, 7 d, 14 d and 28 d), the whole medium was taken out, and an equal amount of fresh medium was added. The supernatant was evaluated by nanodrop (Thermo Fisher, American) and SIRT1 ELISA (Solarbio, China).
NPCs seeding and culturing on hydrogel surfaces (2D Culture)
First, the prepared hydrogel materials underwent strict sterilization. Subsequently, 300 µL of sterilized hydrogel solution was evenly inoculated into each well of a 24-well plate and photocrosslinked for 1 min using a 405 nm blue light source. After complete hydrogel solidification, NPCs were added to the plate at a density of 2 × 10⁴ cells/well, and the plate was gently shaken repeatedly to ensure uniform cell distribution on the hydrogel surface. After seeding, the 24-well plate was placed in a constant-temperature cell culture incubator for incubation. The experimental groups included the G-HA group, GPNPs@G-HA group, SOX9@GPNPs@G-HA group, SIRT1@GPNPs@G-HA group, and SIRT1/SOX9@GPNPs@G-HA group. For cytotoxicity testing, the CCK-8 assay was performed on days 1, 3, and 5 post-seeding according to the manufacturer’s instructions. During measurement, 100 µL of CCK-8 reagent (dissolved in sterile DMEM/F12 medium) was added to each well, followed by 1 h of incubation in the dark. Finally, the absorbance of each well was measured at 450 nm using a full-wavelength microplate reader. For live/dead staining, cytotoxicity was assessed on days 3 and 5 post-seeding using the live/dead staining method as per the manufacturer’s protocol. After the culture period, the medium was gently aspirated to avoid damaging the hydrogel and cells, and the wells were washed three times with PBS. Calcein AM/PI detection working solution (Beyotime, China) was then added, and the cells were incubated at 37 °C in the dark for 30 min. After three additional PBS washes, the samples were observed under a fluorescence microscope. For cytoskeleton staining, cell morphology was evaluated on days 3 and 5 post-seeding using cytoskeleton staining according to the manufacturer’s instructions. After aspirating the medium and washing with PBS three times, the cells were fixed with 4% paraformaldehyde at room temperature for 40 min, washed three times with PBS, and then treated with blocking solution containing Triton-X-100 (Beyotime, China) for 30 min to enhance cell membrane permeability. Rhodamine-labeled phalloidin working solution (prepared by diluting phalloidin stock solution 1:200 with PBS, Yuanye, China) was used to stain the cytoskeleton, followed by 40 min of incubation at 37 °C in the dark. After discarding the medium and washing with PBS three times, the nuclei were labeled with DAPI (Beyotime, China) for 5 min. The cells were washed again with PBS and observed under a fluorescence microscope. For EdU staining, NPC proliferation on the hydrogel surface was assessed on day 3 post-seeding using the EdU staining method. According to the manufacturer’s instructions, NPCs were stained with EdU working solution (Beyotime, China) and DAPI, followed by fluorescence microscopy observation.
NPCs encapsulation and culturing within hydrogels (3D Culture)
First, the prepared composite hydrogel materials were subjected to sterile processing. Subsequently, NPCs were digested and collected from culture dishes, then mixed with the hydrogel solution. The cell concentration was adjusted to 2 × 10⁶ cells/mL, and the mixture was gently homogenized to avoid bubble formation or cell aggregation. The uniformly mixed cell-hydrogel suspension was dispensed at 50 µL/well, photocrosslinked for 1 min using a 405 nm blue light source, and then transferred to a 24-well plate pre-filled with complete medium. The plate was incubated in a constant-temperature cell culture incubator. During cultivation, the medium was replaced every other day to ensure nutrient sufficiency, and cell growth was monitored under an optical microscope. Subsequent experimental groups included the G-HA group, GPNPs@G-HA group, SIRT1/SOX9@GPNPs@G-HA group, 2× group (SIRT1/SOX9@GPNPs concentration doubled compared to the original hydrogel preparation, with other components unchanged), and 3× group (SIRT1/SOX9@GPNPs concentration tripled compared to the original hydrogel preparation, with other components unchanged).
Hydrogel frozen sections and Immunofluorescence staining
After 7 days of 3D culture, the cell-hydrogel composite samples were fixed in 4% paraformaldehyde. Following fixation, the samples underwent sucrose gradient dehydration, were embedded in OCT (Biosharp, China), and flash-frozen in liquid nitrogen to prepare frozen specimens. Frozen sections were then cut at a thickness of 10 μm. The sections were subjected to antigen retrieval using citrate sodium buffer (Beyotime, China) and exposed to hydrogen peroxide solution for 10 min to eliminate endogenous peroxidase activity. Subsequently, the sections were sealed with blocking agent and incubated overnight at 4 °C with diluted primary antibodies. The next day, after washing three times with PBST, fluorescent secondary antibodies were added for staining. Finally, the sections were observed under a fluorescence microscope.
In vivo release efficiency of SIRT1/SOX9@GPNPs
For Cy3 labeling of GPNPs, GPNPs were dissolved in PBS, and Cy3 (Cy3 to GPNPs molar ratio of 3:1) was added to the solution. The mixture was reacted at room temperature under light-protected conditions for 24 h. The reaction product was transferred into a dialysis bag and thoroughly dialyzed against PBS and deionized water, with the dialysis solution replaced every 3 h over a 24-hour period. After dialysis, the product was freeze-dried, weighed, labeled as GPNPs-Cy3, and stored in a light-protected − 20 °C freezer. To investigate the in vivo release kinetics of the composite hydrogel, 10-week-old male Sprague-Dawley rats were randomly divided into two groups. One group received direct injection of SIRT1/SOX9@GPNPs-Cy3 into the caudal vertebrae, while the other group was injected with SIRT1/SOX9@GPNPs-Cy3@G-HA composite hydrogel. Fluorescence intensity changes were monitored using an IVIS imaging system at postoperative days 0, 7, and 14.
Surgical establishment of the rat model of IVDD
All procedures were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Soochow University (SUDA20240415A02). Thirty adult male rats, weighing about 300 g and aged between 8 and 10 weeks, were obtained from the Experimental Animal Center at Soochow University. The rats were randomly divided into four experimental groups: Sham, Defect, G-HA, and SIRT1/SOX9@GPNPs@G-HA. The concentration of hydrogel was 2×. In the Sham group, no puncture surgery was performed. In the Defect group, puncture surgery was carried out, but no therapeutic agents were injected; only 10 µl of PBS was injected. In the G-HA group, puncture was performed and 10 µl of G-HA hydrogel was injected. Finally, in the SIRT1/SOX9@GPNPs@G-HA group, puncture was conducted and 10 µl of SIRT1/SOX9@GPNPs@G-HA hydrogel was injected. Specifically, prior to surgery, the workbench and rat coccyges were sterilized. To minimize the interference of adjacent degenerated segments, a 20-G needle was used to puncture the central NP tissue of the 7–8th (Co7–8) and 9–10th (Co9–10) coccygeal segments to a depth of 5 mm, rotated 360°, and held in place for 30 s. Then, PBS and different compositions of hydrogels were injected at the puncture sites. Post-surgery, the coccygeal area was sterilized again, and rats were transferred to a constant temperature and ventilated environment for recovery.
Radiological imaging assessment
Radiological imaging of the rats was conducted at 4 and 8 weeks post-surgery, with the animals in an anesthetized and supine state. Normalization of the DHI was achieved using X-Ray images processed via Image J software. Additionally, T2-weighted MRI scans of the intervertebral discs were acquired using a 1.5T MRI scanner (Magnetom Essenza, Siemens Medical Solution, Erlangen, Germany) and subsequently analyzed using Image J to determine white signal intensity.
Histological and Immunofluorescence assessment of in vivo therapeutic effects
At 4 and 8 weeks, coccygeal samples from the SD rats were harvested and preserved in formalin for 24 h. Following fixation, the samples underwent a 30-day decalcification process in 10% EDTA. The NP tissue was then extracted, embedded in paraffin, and sliced into 5 μm sections. These sections were subjected to H&E and Safranin O/Fast Green (SO/FG) staining techniques to visualize the disc structure and the distribution of matrix components. Subsequently, the degree of disc degeneration was evaluated and graded based on established histological grading criteria.
