Ethics Approval
Bone marrow samples from healthy volunteers complied with the protocols by The Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (No. S347). Protocols of animal experiments were approved by The Institutional Animal Care and Use Committee at Tongji Medical College, Huazhong University of Science and Technology (No. S3553). The study was performed in accordance with the Declaration of Helsinki.
Chemicals and Materials
gelatin methacryloyl (EFL-GM-30, Engineering For Life, China), folic acid (Aladdin, China), carboxymethyl cellulose sodium (CMCNa, Aladdin, China), poly(ethylene glycol) diacrylate (PEGDA, Aladdin, China; 400Mw), N-hydroxysuccinimide (NHS, Macklin, China), dimethylsulfoxide (DMSO, Aladdin, China), Tartrazine (Aladdin, China), methacrylic anhydride (MA, Macklin, China), N-(3Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, Aladdin, China), ethylenediamine-β-cyclodextrin (EDA-β-CD, Zhiyuan Biotechnology, China), hyaluronic acid methacryloyl (HAMA, 150 kDa, Engineering For Life, China), China), recombinant human connective tissue growth factor (CTGF, PeproTech, USA), and recombinant human transforming growth factor-b3 (TGF-β3, PeproTech, USA), lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate (LAP, Engineering For Life, China). fetal bovine serum (Gibco, USA), 1% penicillin‒streptomycin (15140122, Thermo Fisher), DMEM/F12 (Gibco, USA), 1×PBS pH 7.4 (10010023, Thermo Fisher), Trypsin-EDTA (25200056, Thermo Fisher).
Isolation and identification of hMSCs and goat MSCs
Bone marrow samples were obtained from the iliac crest of healthy volunteers. Goat bone marrow samples were obtained from the femoral marrow cavity. hMSCs were isolated from human and goat bone marrow using density gradient centrifugation and cultured in expansion medium (DMEM/F12 containing 1% penicillin-streptomycin and 15% fetal bovine serum). The multilineage differentiation potential of MSCs was evaluated by staining with Oil Red O and Alizarin Red after induction in the corresponding differentiation media (Cyagen).
Synthesis of GelMA-FA
The interaction between GelMA (DS: 30%) and FA occurs in an EDC/NHS/DMSO solution. Briefly, FA (30 mg) and GelMA (2.5g) was completely dissolved in DMSO (5 mL) on magnetic stirrer (37°C, 2 hours). Then, EDC (30mg) and NHS (50mg) were added and thoroughly mixed (37°C, 12 h) and dialyzed for 5 days. Then freeze-dry for 3 days and collect GelMA-FA. The FA motif peak in the 1H NMR spectrum (δ = 1.79 and δ = 2.57) indicates the conjugation of the FA group to GelMA. In addition, the relatively weak peak at 7.16 ppm in the complex can be attributed to the resonance of aromatic protons of GelMA and FA. By using the reported FA extinction coefficient of 6230 M− 1 cm− 1 and monitoring the absorbance of GelMA-FA at 350 nm, the DS of FA binding to GelMA was calculated to be 21.5% according to the following equation:
DS of FA \(\:=\frac{Mole\:of\:FA\:conjugated\:to\:GelMA}{Total\:mole\:of\:lysine\:present\:in\:the\:GelMA}\times\:100\%\)(1)
Synthesis of β-cyclodextrin (β-CD)-modified hyaluronic acid methacryloyl (HAMA)
The linkage between HAMA and EDA-β-CD occurred within EDC/NHS/HAMA in phosphate buffer solution (PBS). Briefly, 25 mg of NHS and 50 mg of EDC were fully dissolved in a solution of 25 mg of HAMA in 7.5 mL of 0.1 mol PBS (room temperature, 30 min). Then, 73 mg of β-CD-EDA in 10 mL of PBS was added with sufficient mixing. After the mixture was stirred and dialyzed for 5 days at room temperature. Then freeze-dry for 3 days and collect HAMA-β-CD. The β-CD motif peak in the 1H NMR spectrum (H1 at δ = 5.0 ppm) indicates the conjugation of the β-CD group to HAMA. DS was calculated to be 15.2% according to Eq. (2). In this equation, A2ppm is the area of the characteristic peak at 2 ppm chemical shift for HAMA in the 1H NMR spectrum, and A5ppm is the area of the characteristic peak at 5 ppm chemical shift for β-CD in the 1H NMR spectrum.
DS of β-CD \(\:=\frac{\text{A}5\text{p}\text{p}\text{m}}{\text{A}2\text{p}\text{p}\text{m}}\times\:\frac{3}{7}\times\:100\%\)(2)
Preparation of the GelMA, HG, and SilMA Hydrogels
For the GelMA hydrogels, 8% w/t GelMA was dissolved in PBS. For the HG hydrogel, 4% w/t GelMA-FA and 4% w/t HAMA-β-CD were dissolved in PBS. For the SilMA hydrogels, 8% w/t SilMA was dissolved in PBS. For all the hydrogels, 0.5% w/t LAP was added and dissolved at 37°C. Then, the solution was exposed to 20 mW cm− 2 405 nm UV radiation (30 s).
Cell migration
To evaluate cell migration, hMSCs were seeded into a 24-well plate. When the cells reached 70–80% confluence, use a 200 µL pipette tip to create a straight scratch in the center of the well. The liquid in the wells was aspirated, and 8% GelMA, 8% SilMA, and HG hydrogels were cast into the 24-well plate. To eliminate the influence of cell proliferation, serum-free medium was used to replace complete medium. The wound healing process was observed at 0, 6, 12, and 24 hours using a bright field microscope, and the scratch area was measured with Image J.
Hydrogel Swelling Tests
GelMA and HG hydrogels (2 mm thick, Φ 8 mm) were immersed in PBS (37°C). The weight of the hydrogels (W0) was subsequently measured. The weights of the hydrogels (W12, W24, W48, and W72) were measured at different swelling durations (12, 24, 48, and 72 hours). The swelling rate was calculated according to the following equation:
swelling ratio\(\:=\frac{{W}_{n}-{W}_{0}}{{W}_{0}}\times\:100\%\)(3)
Degradation of Hydrogels In Vitro
Circular samples (2 mm thick, Φ 8 mm) of GelMA and HG hydrogels were incubated in PBS (37°C). The degradation rate was calculated by thoroughly washing the hydrogel in deionized water, freeze-drying and weighing, and finally applying the following formula:
Degradation ratio\(\:=\frac{{W}_{o,dry}-{W}_{t,dry}}{{W}_{o,dry}}\times\:100\%\)(4)
where Wo,dry represents the initial dry weight of the sample, while Wt,dry denotes the dry weight at each incubation time point.
In Vitro Release of Folic Acid
The in vitro release of FA from GelMA and HG hydrogels was evaluated by immersing HG hydrogel samples (2 mm thick, Φ 8 mm) in PBS (2mL, 37°C). extract 100 µL of PBS for absorbance measurement, then add another 100 µL of PBS and return it to the incubator. Use NanoDrop to measure the absorbance of the sample at 288 nm, and calculate the concentration based on the FA standard curve dissolved in the same medium.
Mechanical Properties of Hydrogels
Mechanical property tests were conducted using the EUT2000 electromechanical testing machine (Shenzhen Sansi Testing Co., Ltd., China) with reference to GB/T 1041–2008 standard at room temperature. The crosshead speed was set at 10 mm min− 1, and the tests involved compression of cylindrical samples with a height of 8 mm and a diameter of 10 mm.
Differentiation of NPO and AFO through 3D culture
For NPO differentiation, hMSCs were passaged to P3, and on day − 2, wash the cells twice with 1×PBS. Subsequently, incubate with trypsin-EDTA at 37°C and 5% CO2 for 1 minute. Then, collect the cells and resuspend at a concentration of 1×106 cells ml− 1 in expansion medium. Next, add 200µL of the cell suspension to each well of a 96-well ultra-low attachment round-bottom microplate (7007, Corning). On day 0, encapsulate cell spheroids in GelMA hydrogel (10% GelMA and 0.25% w/t LAP) or HG hydrogel (4% w/t GelMA-FA, 4 wt% HAMA-β-CD and 0.25% w/t LAP) using a 1 mL Luer-Lok syringe (605–002601, Winner, China) and a female Luer-threaded coupler. Dissolve the HG hydrogel in 1×PBS and mix with the cell spheroids in the syringe, followed by deposition into a 12-well plate and exposed to UV irradiation (20s, 405 nm, 20 mW cm− 2). Culture the cell spheroids in DMEM/F12 containing 10% fetal bovine serum, 1% ITS+ (41400045, Thermo Fisher), 0.1% sodium pyruvate (11360070, Thermo Fisher), 1% NEAA (11140050, Thermo Fisher), 1% penicillin/streptomycin, 10 nM dexamethasone (D1756, Sigma), 40 mg/ml L-proline (P0380, Sigma), 50 mg/ml ascorbic acid-2-phosphate (A8960, Sigma), TGF-β3 (10 ng/ml) and FA (30 µm) for a 21-day differentiation period. For AF organoid differentiation, the HG hydrogel was replaced with 8% SilMA. CTGF (100 ng/ml) was added for further AFO differentiation.
Rheological Testing of bioinks
The rheological properties of HG hydrogels, with and without 4% PEGDA, were studied using a rheometer (HAAKE MARS, Thermo Scientific) before UV irradiation. The temperature sweep test involves placing an appropriate amount of ink onto a rheometer, cooling it to 4°C to form a gel state, and recording the viscosity within the temperature range of 4 ~ 50°C at a heating rate of 2°C/min (σ = 20Pa, f = 1Hz). For the sweep test under 405 nm 20 mW cm− 2 light irradiation. The solution of HG and SilMA hydrogel was placed on the rheometer at 30 ºC, which operated at 10 Hz and 1% strain. Turn on the light source at the 60 s and maintain it until the storage modulus reaches a stable state.
Fabricatioin of NP and AF scaffolds
The bioink solution was based on HG and SilMA hydrogels. We added 4% w/t PEGDA and 1% w/t CMCNa to ensure subsequent 3D printing, after which the solution was dissolved in PBS. Subsequently, 0.25% w/t LAP and 0.05% w/t tartrazine was added to the bioink as photoabsorbers. A 3D printer (AUTOCERA-R, TenDimensions Technology, China) was used to print NP and AF scaffolds. First, the bioink was prepared and placed in an ink cartridge. Materialise Magics 22 was used to design the 3D model, which was output as a StereoLithography file. Subsequently, the model was programmatically sliced in the Z direction. The predesigned printing parameters (layer thickness and curing time) and the 3D model were input into the 3D printer. Printing was carried out by controlling the transmission of images to the projector and the movement of the build platform. During this process, the ink was exposed to 405 nm 20 mW cm− 2 light. The printing conditions included a curing time of 20 s and a layer thickness of 25 µm. The obtained scaffold was named the NP or AF scaffold.
Assembly of 3D printed IVDO
On day 0 of the differentiation protocol, the cell spheroids suspended in HG and SilMA hydrogels were respectively deposited into NP and AF scaffolds using a 1 mL Luer-Lok syringe, followed by UV (20s, 405 nm, 20 mW cm− 2) irradiation. Subsequently, according to the differentiation protocol, a 21-day induced differentiation culture was conducted in 12-well plates to form 3D-printed NPO and AFO. On day 21, the NPO was removed from the well plate and placed in the center of the AFO, followed by a 7-day culture in differentiation medium without FA and CTGF to allow for their fusion. On day 28, an integrated IVDO was formed and collected for subsequent experiments.
Scanning electron microscopy (SEM)
Assembled NP and AF scaffolds were prepared and allowed to reach swelling equilibrium in PBS at 37°C. Subsequently, freeze-dry the samples under vacuum conditions at -80°C. Observe the dried hydrogels using SEM (TESCAN MIRA LMS, TESCAN, Czech Republic).
Cell proliferation assay
The CCK-8 assay kit was used to conduct a cell proliferation experiment to verify the in vitro cell compatibility of the hydrogel. hBMSCs resuspended in hydrogels were seeded into a 24-well plate (15,000 cells/well), with three replicates per group. The CCK-8 assay was performed after 1, 3, 7, and 12 days of 3D culture. The specific experimental steps are as follows: Prepare a cell culture medium containing 10% CCK-8 solution (HYCEZMBIO, HYCCK8), remove the culture medium from the wells and wash with PBS, add 400µL of 10% CCK-8 culture medium to each well, and incubate at 37°C for 2.5 hours. Transfer the liquid from the 24-well plate to a 96-well plate, and measure the absorbance at 450 nm for each well using the VICTOR Nivo® multimode plate reader (PerkinElmer, Waltham, USA).
Cell viability assay
Cells were embedded in hydrogels and photopolymerized. Cell culture medium was added immediately and incubated in a CO2 incubator. Cell viability was assessed using the Calcein-AM/PI Cell Viability/Cytotoxicity Assay Kit (Beyotime, China). The cells were stained on the first, third, seventh, and fourteenth days after bioprinting. The stained cells were observed after they had incubated for 30 minutes. ImageJ software was used for image analysis, and cell viability was calculated based on the percentage of live cells.
RNA sequencing
The experiment included a control group and a folate group. Total RNA was extracted from the aforementioned hBMSCs using TRIzol reagent, and the quality and integrity of the samples were assessed. After quantifying the RNA samples, an RNA sequencing library was prepared. BGI Genomics (Shenzhen, China) collaborated in processing the raw data, and the human reference genome was mapped using STRA software. The cutoff values for the fold change and p value were set at 0.05 and 2, respectively.
qRT–PCR
Use GelMA lysis buffer (EFL, China), Collagenase Ⅱ (Biofroxx, 2275GR001), and Hyaluronidase (Solarbio, H8030) to decompose the hydrogel and extracellular matrix. Organoids can be isolated through further centrifugation, making them suitable for subsequent RNA/cDNA extraction. Cellular RNA was lysed and purified using TRIzol (Invitrogen, 15596026, USA), followed by reverse transcription using a cDNA synthesis kit (Vazyme, Nanjing, R312-01). And use Bio-Rad fluorescence quantitative PCR system for amplification. Target genes were quantified by normalizing their expression to that of 18sRNA. Table S1 displays the primer sequences used.
Western blot analysis
Cells were washed twice with pre-cooled PBS, and then were lysed using RIPA lysis buffer containing 1% PMSF (Solarbio). The lysates were then centrifuged (12,000 × g, 15 min). The lysed protein concentration was measured using the BCA method. SDS‒PAGE 5× loading buffer (Beyotime) was added and boiled (95–100°C, 5 min) to denature the protein. The proteins were separated via SDS‒PAGE (120 V, 90 minutes) based on their molecular weight. The loading amount of protein antigen was 30 µg, and the thickness of the glass plate gap was selected to be 1.5 mm. After electrophoresis, the PVDF membrane (Millipore) were used to transfer the proteins, with a current of 300 mA for 60 minutes. Use skim milk powder (5% w/t) to block the membrane (1 hours) with low-speed shaking and was then washed with TBST. After the membrane washing was completed the primary antibody, which had been diluted in advance, was immediately added and incubated (4°C, 8 hours). Incubate with HRP-conjugated secondary antibody at room temperature for 1.5 hours. The antibodies used can be found in Supplementary Table S2. Visualize protein bands using ECL reagent (Affinity) and analyze them with ImageJ (NIH, USA). The experiment was repeated three times.
Animal preparation and in vivo test
Under anesthesia, a lateral approach between the lumbar muscle and peritoneum was used to simulate clinical intervertebral disc (IVD) excision surgery, during which a circular defect approximately 17 mm in diameter was created to ensure the implantation of IVDOs. IVDOs were implanted into the excised discs, along with hMSC-seeded IVD scaffolds or no implantation as a control. An untreated disc was used as the intact control. Euthanasia was performed on all animals six weeks later.
CT and MRI Examination
Prior to euthanasia, lateral CT images and T1 and T2 magnetic resonance images, including axial, sagittal, and coronal planes, were acquired. Three blinded evaluators quantitatively analyzed the IDD using the Pfirrmann grading system based on the signal intensity and uniformity of the nucleus pulposus (NP) in sagittal and axial T2 MR images, as well as the boundary distinction between the NP and AF, grading the lesions from 1 to 5. The Pfirrmann grade for each IVD was based on the median. The disc height index (DHI) was calculated by dividing the disc height by the height of adjacent vertebral bodies to measure the intervertebral disc height. The posttreatment intervertebral disc DHI was normalized against the unaffected portion within the same spine to account for interanimal differences.
Histological evaluation
Organoid and goat intervertebral disc samples were collected, fixed, decalcified and dehydrated. The dehydrated tissue was then embedded in paraffin, sectioned into 4 µm slices, and subjected to safranin O/fast green staining, Masson staining, and H&E staining.
Immunofluorescence Assessment
Fix the cells or tissues with 4% paraformaldehyde (Biosharp, China), wash three times with Tris-buffered saline for 10 minutes each. Then incubate in Tris-buffered saline containing 0.3% Triton X-100 (w/v) and 5% normal donkey serum (w/v) for 1 hour to block nonspecific reactions. Subsequently, incubate the sections overnight at 4°C or for 3 hours at room temperature with the antibodies listed in Table S2. After washing three times with Tris-buffered saline for 10 minutes each, incubate the cells at 22–28°C with FITC, CY3, and CY5-conjugated secondary antibodies (Thermo Fisher) for 1 hour. Prior to observation under a fluorescence microscope (Olympus), staining was performed using DAPI (Thermo Fisher).
Immunohistochemistry Assessment
For IHC, tissue sections were deparaffinized and rehydrated. Sections were incubated for 1 hour at room temperature using COL-1, COL-2, ACAN as primary antibodies. And were then incubated with biotin-labeled secondary antibodies for 1 hour at room temperature. Finally, DAB peroxidase substrate kit (AR1000, Boster) was used for staining.
Quantification and statistical analysis
Data are expressed as mean ± SD of three independent experiments. Comparisons between multiple groups were performed using One-way analysis of variance (ANOVA). The significance of differences between two groups was assessed using the Student's t-test. P < 0.05 is considered statistically significant, P > 0.05 is considered not significant (ns) (* P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). n = 3 independent experiments per group.