Precursor synthesis
Gelatin methacrylate (GelMA) was prepared according to Khademhosseini et al.35 with small modifications. Gel (20.00 g) was added to Dulbecco’s phosphate-buffered saline (DPBS, 200 mL) at 50 °C, and the mixture was stirred magnetically until complete dissolution. Methacrylic anhydride (MA, 16.00 mL) was added and stirring was continued for 3 h. More PBS (600 mL) was added and the solution was dialyzed against distilled water (40 °C, 2 h, membrane cut-off 12–14 kDa). Then, the solution was frozen (−80 °C, 24 h) and freeze-dried for 3 days (Boyikang Experimental Instrument Co. Ltd., Beijing, China). The yield of GelMA (white powder): 16.27 g. Analogously, HA (2.00 g) was added to phosphate-buffered saline (PBS, 200 mL) at 50 °C, and the mixture was stirred magnetically until complete dissolution. MA anhydride (2.00 mL) was added and stirring was continued for 24 h at 4 °C while maintaining the pH between 8 and 10 with the addition of sodium hydroxide (5 M). In addition to more PBS, dialysis, and freeze-drying were done as described above. The yield of HAMA (white powder): 1.84 g.
Cartilage (~200 g) was harvested from the ribs of an α-1, 3-Gal gene-knockout pig. The ribs were provided by Yifan Dai’s lab from Nanjing Medical University (Nanjing, China). Soft tissues and blood were removed carefully, the cartilage was washed repeatedly with PBS (500 mL, 3×) and cut into small pieces (~1 g each). The cartilage was further treated with 10 mM Tris-HCl (500 mL) at 45 °C for 24 h. The supernatant was removed and replaced by 0.25% (m/v) Trypsin (500 mL). The mixture was left to stand for 24 h at 37 °C. The supernatant was removed again and replaced by a protease inhibitor (500 mL, room temperature). The supernatant was refreshed once (after 1 h). The cartilage pieces were washed with PBS (500 mL, 3×), frozen (−20 °C) for 4 h, and ground with the automatic freezing grinding machine (Retsch, Haan, Germany), using a frequency of 20 sec−1, grinding cycle 3× (15 min each). Decellularized cartridge particles sized <40 μm were sieved out and stored at −80 °C. The animal experiment included in this part was in line with this ethical requirement, as approved by the Ethics Committee of Wenzhou Institute of the University of Chinese Academy of Sciences (WIUCAS 20033115 and 20200331).
Preparation of the hybrid hydrogels
Materials (eight formulations) were prepared according to Table 2. Note that the GelMA content is either 10 or 15% (m/v); content of HAMA is 1% (m/v) invariably, and content of DCM is 0, 3, 6, or 12% (m/v). Note, furthermore, that materials 1–4 (containing 10% GelMA) are designated Series A, and materials 5–8 (containing 15% GelMA) are designated Series B.
To 1.00 g of each formulation, photo-initiator [5 mg, 0.5% (m/v); 2-hydroxy-4′-(2-hydroxyethoxy)-2-methyl acetone (Igracur 2959)] was added in the dark. A Teflon mold containing six holes (8.0 mm diameter, 1.0 mm depth) was used. First, the cavities were filled with mixtures 1 and 5, and irradiated with UV light (365 nm, 18 mW/cm2, 2 min), thus yielding three samples per composition. This was repeated for compositions (2 + 3), (4 + 6), and (7 + 8) thus yielding 8 × 3 = 24 samples.
Spectroscopic analysis by NMR and FTIR
Nuclear magnetic resonance (NMR) spectra (1H, 500 MHz) of GelMA and HAMA (i.e., the precursors of the cross-linking reaction) were run on a Bruker Avance Neo Spectrometer Durham, USA). D2O was used as the solvent, chemical shifts were referenced against residual solvent signals. Special notice was given to the resonances appearing in the range 5.2–5.7 ppm, as these stem from the vinylic protons of the tethered methacrylate groups. Fourier-transform infrared (FTIR) spectra in the frequency range 500–4000 cm−1 were recorded with a Fourier infrared spectrometer instrument (Bruker, Tensor II, Bremen, Germany). Samples were at room temperature. Data were analyzed with OriginPro 8.5. The results of NMR and FTIR were listed in Supplementary materials.
Verification of cartilage decellularization
The matrix architecture of pristine and decellularized cartilage was studied with scanning electron microscopy (SEM; Hitachi SU 8010, Tokyo, Japan). The specimens were frozen (−80 °C), freeze-dried during 48 h (Boyikang Experimental Instrument Co. Ltd., Beijing, China), cracked, and sputter-coated with Pt, using a Leica EM ACE600 instrument (Leica GmbH, Wetzlar, Germany).
The Quant-iTTM PicoGreen® dsDNA assay kit (Invitrogen, Carlsbad CA, USA) was used to quantify residual DNA in the DCM material. Analyses of DCM (10 mg) and untreated cartilage (10 mg) were done in triplo. The materials were added to 0.1% Triton-X 100 (1 mL) and left to stand for 10 min. The supernatant was removed and replaced by TE buffer (200 mM Tris-HCl, 20 mM EDTA, pH 7.5), present in the kit. Then, PicoGreen detection reagent was added, following the instructions of the supplier. The absorbance was read at 520 nm using a microplate reader (ThermoFisher, Varioskan LUX, USA) for all six samples (three for DCM and three for pristine cartilage). Whether or not the genetic material was eliminated completely, and the cartilage matrix was retained was assessed further by hematoxylin & eosin (H&E) staining, Masson staining, Safranin O staining, and Alcian blue staining. DCM and cartilage specimens were placed in tissue fixative for 24 h, dehydrated (ethanol series 70% → 80% → 90% → 95% → 100%) and permeated in xylene. The completely infiltrated tissues were embedded in paraffin (6 h) and sliced (8 μm thickness) on a microtome. Staining was done with H&E solution and Masson pine trichrome dye solution. Dyed slices were studied by fluorescence microscopy.
Swelling tests
Each of the 24 samples (see above) was weighed (W0), and subsequently placed in a 5-mL Eppendorf tube containing 1 mL of PBS; the temperature was maintained at 37 °C. The samples were taken out at different time points (6, 12, 24, 36, 48, and 60 h), dried superficially with a filter paper, and weighed (Wt). Expansion rates were calculated according to the formula: Pt = (Wt – W0)/W0 × 100%.
Compression tests
A second Teflon mold was used to generate cylindrical material samples with the dimensions: diameter 5.0 mm, height 5.0 mm. Formulations were according to Table 2, and irradiation was done as described above. It was decided not to equilibrate the specimens in PBS or any other aqueous medium, as materials 1–8 already have high water contents, which are on par with the water content of cartilage (see also Table 2). It is well known that up to 80% of cartilage is water36. Then, the samples were compressed using an electronic universal material testing machine (Instron 5944, Norwood, MA, USA), using a crosshair speed of 2 mm/min. Stress-strain curves were measured and Young’s modulus was abstracted as the slope of the curve in the 5–10% strain region37. Experiments were done fivefold per composition.
Degradation tests
Samples had the geometry as described in Swelling tests. Materials were immersed in PBS (24 h, 37 °C); dried superficially, and weighed (W0). Then, samples were placed in 0.5 mg/mL collagenase type II and kept at 37 °C. Samples were taken out, dried, weighed (Wt), and put back at 2, 4, 6, and 8 h. Degradation was quantified using the equation: Rt = Wt/W0 × 100%. Experiments were done in triplo.
Scanning electron microscopy (SEM) analysis—microstructure
Samples of the eight materials were according to the preparation of the hybrid hydrogels (diameter 8.0 mm and height 1.0 mm). First, the specimens were frozen (−80 °C) and then freeze-dried during 48 h (Boyikang Experimental Instrument Co. Ltd., Beijing, China). Samples were cracked and the fracture surfaces were examined using SEM (Hitachi SU 8010, Tokyo, Japan). Prior to the measurements, samples were sputter-coated with Pt, using a Leica EM ACE600 instrument (Leica GmbH, Wetzlar, Germany).
Cell cultures
Dental Pulp Stem Cells (DPSCs, Stem Cell Bank, Chinese Academy of Sciences, Beijing) were cultured in the α-minimum eagle’s medium (α-MEM) with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin. The cells were cultured in a 37 °C incubator with a 5% volume fraction of CO2, and the cell culture medium was replaced every 2–3 days. These cells were used in our in vitro experiments.
Cytotoxicity tests
Formulations 1–8 (see Table 2) were added to a 24-well plate (three wells per composition). The plate was then irradiated with UV light (365 nm, 18 mW/cm2, 2 min), which leads to photo cross-linking at the bottom of each well. Alcohol (75%) was pipetted into each well to fill it for ~50% and left for 2 h. Then the wells were washed with PBS (3×, 30 min standing for each wash step). DPSCs (5 × 103) were added to each well, and the plate was placed in a 37 °C incubator. Quantitative-iTTm PicoGreen® dsDNA assay kit was used to detect the proliferation activity of DPSCs seeded onto each of the hybrid hydrogel materials. At 3, 7, and 14th days after the cells were seeded, the DNA content was determined by measuring the UV absorbance (520 nm) with a microplate reader.
Cellular morphology
Live-dead cell staining and cytoskeleton staining were performed at 1, 3, 7, and 14 days after inoculation. The AO and EB solutions (1:1) in the Live-Dead staining kits were mixed in the dark, following instructions of the supplier. Each well was washed three times with PBS, incubated with AO/EB working solution, observed by fluorescence microscopy, and photographed. Specimens which were used to study the DPSC cytoskeletons were washed with PBS (3×), permeated with 0.1% Triton-X 100, stained with rhodamine and DAPI, observed by fluorescence microscopy, and photographed.
Cell Differentiation
The main markers of chondrogenic and hypertrophic differentiation include ACAN, Sox9, Coll2, Col2α1, ALP, and Col 10A1. The change of expression level of these specific markers is an important indicator of chondrogenic differentiation. DPSCs were cultured for 7, 14, or 21 days. Total RNA was extracted by Trizol and reverse transcribed into cDNA. These marker genes were subjected to a real-time polymerase chain reaction (RT-PCR) using SYBR Green Master Mix. The primer sequences are listed in Table 3. Real-time PCR was performed at 95 °C for 15 min, and then it was denatured at 95 °C for 10 s, extended at 60 °C for 15 s, and annealed at 72 °C for 15 s, and was cycled 40 times. The melting curve is prepared from 75 °C to 95 °C with a temperature increase of 1 °C every 20 s. The GAPDH gene is used as an internal reference gene and normalized expression level, and the ΔΔCt method is used to calculate the relative expression level of the gene. In addition, the ability of the material to induce stem cell chondrogenic differentiation was further verified by Safranin O and Alcian blue staining, as presented in the supplementary material.
Qualitative characterization of chondrogenic differentiation
Safranin O and Alcian blue staining was performed at 1, 7, and 14 days after incubation. The samples were washed with PBS (3×) and fixed with 4% neutral formaldehyde, then dyed according to the instructions. After dyeing, washed with PBS (3×) and observed by microscopy and photographed. The results were listed in Supplementary materials.
Animal experiments
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The animal experimental plan of this study was approved by the Ethical Committee of Wenzhou Institute, University of Chinese Academy of Sciences (WIUCAS20033115 and 20200331). Adult male Sprague-Dawley rats (20, weight ~260 g) from Wenzhou Medical University were used to study the utility of the hydrogels (see Table 2) 5 (15%GelMA/1%HAMA), 6 (15%GelMA/1%HAMA/3%DCM), 7 (15%GelMA/ 1%HAMA/6% DCM), and 8 (15%GelMA/1%HAMA/12%DCM) in the rat knee cartilage repair model as described by Cao Tong et al.38 with some modifications. Series B were first photo-polymerized into circular disks with a diameter of 8.0 mm and a height of 2.0 mm as described above. Then, cylindrical specimens with a diameter of 2.0 mm were cut with a cork drill. Knee cartilage defects with a cylindrical shape (diameter 2.0 mm and depth of 2.0 mm) were created in the right hind leg knee of each animal. Animals were anesthetized with 10% chloral hydrate (4 μL/g). Animals were housed under normal conditions, in the Vivarium facilities of Wenzhou Institute, University of Chinese Academy of Sciences with appropriate feeding and water ad libitum. Specimens 5, 6, 7, or 8 were carefully inserted into the defects (each material in four animals); four animals were used as sham controls. Surgical wounds were carefully closed and penicillin was injected to prevent inflammation39. The animals were sacrificed (chloral hydrate, 8 μL/g) after 9 weeks. Specimens and surrounding tissues were excised, photographed, and stored in 10% neutral formalin at 4 °C until histopathological analysis.
International cartilage repair society (ICRS) analyses
At predetermined time points, samples from each group were graded blindly by two independent observers in terms of cartilage repair according to the international cartilage repair society (ICRS) scoring system (Table 4)40,41.
Histopathology
The samples were fixed overnight in 10% neutral buffered formalin. Then, the samples were decalcified (EDTA 0.1 g/mL, PBS 2 L, Sodium hydroxide 11 mg/mL, pH 7.2, decalcification for 1 month, the supernatant was refreshed every 2 days), sealed with paraffin and sliced using a microtome (Histotome, Leica RM2265, Wetzlar, Germany) to obtain the tissue thickness of 5 µm. The slices were mounted onto microscopic glass and stained with H&E or Safranin O Fast Green following the standard procedures as stated in the instructions. Safranin O Fast Green allowed us to distinguish bone (blue or green) from cartilage (red). The dyed slices were studied in detail with light microscopy and fluorescence microscopy; extensive photography was used to document the results.
Statistical analysis
Statistics were performed with GraphPad Prism 5 software (GraphPad Software, San Diego, California). Group sizes are specified for each data set, and data were presented as mean ± standard deviation. Statistical significance and p levels are indicated as *p < 0.05, **p < 0.01, and ***p < 0.001.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
