Synthesis of NB and HA-NB

Methyl 4-(4-(hydroxymethyl)−2-methoxy-5-nitrophenoxy)butanoate (mNB) was synthesized as follows17: 4-hydroxy-3-methoxybenzaldehyde (vaniline) (8.90 g, 58.5 mmol, 1.06 eq.), methyl 4-bromobutanoate (9.89 g, 55.0 mmol, 1.0 eq.), and potassium carbonate (10.2 g, 73.8 mmol, 1.34 eq.) were dissolved in N, N-Dimethylformamide (DMF) (40 mL). The mixture was stirred at ambient temperature for 16 h, after which time the resulting solution was poured into chilled water (200 mL) and allowed to precipitate for 15 min at 0 °C. The solid was filtered off, washed with water, redissolved in dichloromethane, and dried over magnesium sulfate. The solvent was removed under reduced pressure to yield a white solid (methyl 4-(4-formyl-2-methoxyphenoxy)butanoate, 12.2 g, 48.4 mmol, 88 %). Methyl 4-(4-formyl-2-methoxyphenoxy)butanoate (9.4 g, 37.3 mmol, 1 eq.) was added slowly to a pre-cooled (−2 °C) solution of nitric acid (70%, 140 mL) and stirred at −2 °C for 3 h. It is important to note that—depending on the temperature of the nitration reaction—ipso substitution of the formyl moiety occurs. The resulting solution was poured into chilled water (500 mL) and allowed to precipitate for 15 min at 0 °C (note: as saponification of the product can occur under these conditions, the precipitation time should be kept as short as possible). The product was filtered, washed with water, and dissolved in dichloromethane. The organic layer was dried over magnesium sulfate. The solvent was removed under reduced pressure to yield a slightly yellow powder (methyl 4-(4-formyl-2-methoxy-5-nitrophenoxy)butanoate, 7.7 g, 25.9 mmol, 69%). Sodium borohydride (1.50 g, 39.7 mmol, 1.5 eq.) was slowly added at 0 °C to a solution of methyl -(4-formyl-2-methoxy-5-nitrophenoxy)butanoate (7.7 g, 25.9 mmol, 1.0 eq.) in EtOH/THF 1:1 v/v (100 mL). After 3 h, all solvents were removed in vacuo and the residue was suspended in water (50 mL) and dichloromethane (50 mL). The aqueous layer was extracted two times with dichloromethane (2 × 50 mL) and the combined organic layers were dried over magnesium sulfate. The solvent was removed under reduced pressure. In order to increase the overall yield and to remove partially saponified products, methanol (100 mL) and tosylic acid (50 mg) were added to the residue. The solution was stirred at room temperature overnight. The solvent was removed in vacuo and the residue was suspended in water (50 mL) and dichloromethane (50 mL). The aqueous layer was extracted two times with dichloromethane (2 × 50 mL) and the combined organic layers were dried over magnesium sulfate. The solvent was removed under reduced pressure to yield a yellow solid as a raw product, which was purified by column chromatography on silica gel using hexane/ethyl acetate = 1:1 (Rf = 0.6) and finally 5.22 g (4.81 mmol, 75%) of a slightly yellow powder mNB were obtained.

Then mNB (0.5 g, 1.8 mmol) and ethylenediamine (1.1 mL, 2 mmol, Sigma-Aldrich) were dissolved in methanol. The mixture was refluxed overnight until the starting individual components were undetectable by thin layer chromatography. After the reaction was complete, the solvent was evaporated under vacuum. The crude precipitate was dissolved in methanol and re-precipitated three times using ethyl acetate. The filter cake was then dried for 12 h at 30 °C under vacuum until NB appeared as light yellow powder (0.4 g, 1.2 mmol, 66.7%). HA-NB was synthesized according to a previous report17. Briefly, HA (408 mg, 1 mmol of disaccharide unit, Dongyuan Biotech, Zhenjiang) was dissolved in 50 mL deionized water at room temperature and NB (224 mg, 0.69 mmol) was added followed by HOBt (153 mg, 1 mmol, Sigma-Aldrich). The pH of the mixed solution was adjusted to pH 4.5, after which 1-(3-Dimethylaminopropyl)–3-ethylcarbodimide hydrochloride (200 mg, 1.04, Sigma-Aldrich) was added to the mixture and stirred for 48 h at room temperature. The solution was loaded into dialysis tubing (Molecular Weight (MW) cutoff 3500, Spectrum®) and dialyzed against diluted HCl (pH 3.5) containing 0.1 M NaCl for 2 days, then dialyzed against deionized water for a further 2 days. The solution was lyophilized and HA-NB was obtained in powder form. The substitution degree of nitrobenzyl groups (3% of HA disaccharide units) was verified by 1H-nuclear magnetic resonance (NMR).

Synthesis of GelMA

Type A gelatin (Sigma-Aldrich) was dissolved in PBS at 50 °C to make a 10% w/v homogeneous solution. Then, a 0.1 mL methacrylic anhydride (MA) (Sigma-Aldrich) per gram of gelatin was added to the gelatin solution at a rate of 0.5 mL/min, with continuous stirring. The mixture was allowed to react at 50 °C for 3 h. The GelMA solution was dialyzed against deionized water using 8–14 kDa cutoff dialysis tubing (VWR Scientific USA) for 6 days at 50 °C to remove unreacted MA and any byproducts. The GelMA solution was frozen overnight at −80 °C, then lyophilized and stored at −20 °C until further use31,32. The substitution degree of MA was verified by 1H-NMR.

Synthesis of the photo-initiator

Dimethyl phenylphosphonite (Ourchem) was reacted with 2,4,6-trimethylbenzoyl chloride (Sigma-Aldrich) via a Michaelis–Arbuzov reaction. At room temperature and under argon gas, 3.2 g (0.018 mol) of 2,4,6-trimethylbenzoyl chloride was added dropwise to an equimolar amount of continuously stirred dimethyl phenylphosphonite (3.0 g). The reaction mixture was stirred for 18 h whereupon a fourfold excess of lithium bromide (Aladdin, 6.1 g) in 100 mL of 2-butanone (Sinopharm Chemical Reagent) was added to the reaction mixture from the previous step, which was then heated to 50 °C. Ten minutes later, a solid precipitate had formed. The mixture was cooled to ambient temperature, allowed to rest for 4 h, and then filtered. The filtrate was washed and filtered three times with 2-butanone to remove unreacted lithium bromide and excess solvent was removed by vacuum33.

Precursor preparation of the hydrogels

For precursors of GelMA/HA-NB/LAP hydrogel, the freeze-dried GelMA foams and HA-NB foams were dissolved in PBS solution at 40 °C, then the photo-initiator LAP was added. The final precursor is composed of 5% GelMA, 1.25% HA-NB, and 0.1% LAP for low-concentration matrices, and 10% GelMA, 2.5% HA-NB, and 0.2% LAP for high-concentration matrices. For precursors of GelMA/HA-NB hydrogels, the freeze-dried GelMA foams and HA-NB foams were dissolved in PBS solution at 40 °C to a final concentration of 5% GelMA and 1.25% HA-NB. For precursors of GelMA/LAP hydrogels, the freeze-dried GelMA foams were dissolved in PBS solution at 40 °C and then added to the photo-initiator LAP to a final concentration of 5% GelMA and 0.1% LAP.

Adhesion mechanism study

XPS (Kratos AXIS Ultra DLD) was used to characterize the surface composition of the sausage skin membranes; skins were treated with non-photo illuminated HA-NB and also with photo-illuminated HA-NB, using an Al Kα source (1486.6 eV). A detailed scan for Nitrogen was carried out with a step of 0.1 eV. The Carbon 1s peak (284.6 eV) was used for calibration.

SEM analysis

Cryo-SEM imaging was performed on a FEI Helios NanoLab 600i Analytical Field Emission Scanning Electron Microscope fitted with low-temperature sample carrier Quaroum PP3000T, to examine the gross morphology hydrogels. The samples were loaded on the cryo-specimen holder and cryo-fixed in slush nitrogen (−210 °C), then quickly transferred to the cryo-stage in the frozen state. The sample was transferred into the cryo-stage (Quorum Technologies, PP300T, East Sussex, UK), a chamber attached to the microscope (FEI, Helios Nanolab 600i, Hillsboro, USA). Once the sample was inside the chamber, a fracture of the sample was made to get a fresh clean surface to be examined. The temperature of the sample was raised by heating the holder to −90 °C for 30 min, in order to increase the contrast and sublimate free-water in the solid-state lakes, followed by a temperature decrease to −180 °C, to stabilize the sample. The surface of the frozen preparation was then coated with platinum (10 mA, 30 s) to prevent charging of the sample and to obtain a good relation between signal and noise. The coated sample was thereafter transferred into the microscope chamber where it was analyzed at a temperature range of −180 °C. To evaluate the integration of the tissue and hydrogels, the samples were first fixed with 2.5% glutaraldehyde in phosphate buffer (0.1 M, pH 7.0) for >4 h, then washed three times in phosphate buffer for 15 min each. The samples were dehydrated by a graded series of ethanol extractions (30%, 50%, 70%, 80%, 90%, 95%, and 100%) for 15–20 min at each step and the dehydrated samples were coated with gold-palladium in a Hitachi E-1010 ion sputter for 4–5 min before viewing. Then the samples were observed under a SEM (Hitachi TM-1010, Japan).

SR of the hydrogels

The GelMA/HA-NB/LAP (n = 3), GelMA/HA-NB (n = 3), and GelMA/LAP hydrogels (n = 3) were incubated in PBS at 37 °C for 24 h, then lightly blotted dry and weighed (Ws). Hydrogels were then freeze-dried and weighed to determine the dry weight (Wd). The SR of the swollen gel was calculated according to the following equation34.

$${\mathrm{SR}} = \frac{{({\mathrm{Ws}} - {\mathrm{Wd}})}}{{{\mathrm{Wd}}}}$$

Rheological studies

Rheological properties of hydrogels were analyzed following a reported method17. In brief, dynamic rheology experiments were performed using a HAAKE MARS III photo-rheometer with parallel-plate (P20 TiL, 20-mm diameter) geometry and OmniCure Series 2000 (365 nm: 30 mW•cm−2) at 37 °C. Time-sweep oscillatory tests of GelMA/HA-NB/LAP (n = 3), GelMA/HA-NB (n = 3), and GelMA/LAP hydrogels (n = 3) were performed at a 10% strain, 1 Hz frequency, and a 0.5 mm gap (CD mode). Strain sweeps were performed on the pregel solution to verify the linear response. The gel point was determined at the time when the torsion modulus (G’) surpassed the loss modulus (G”).

Burst pressure test

Burst pressure testing was performed using a published method35. Briefly, a piece of 4 × 4 cm porcine sausage skin membrane was cut and cleaned to remove any excess fat. The membrane was fixed to the measurement device linked to a syringe pump filled with PBS solution. A 2 mm incision was made on the sausage skin membrane surface and the membrane surface was kept wet. Then, 500 μL precursor solutions were injected onto the incision, after which the hydrogels formed in situ on the puncture site after UV illumination. The thickness of the hydrogels was ~4.4 mm and burst pressure was measured after gel formation. Peak pressure before pressure loss was considered the burst pressure. All measurements were repeated three times. Fibrin Glue (Shanghai RAAS Blood Products, Co., Ltd, Shanghai, China), CA (Beijing Compont Medical Devices, Co., Ltd, Beijing, China), and SurgifloTM (Ethicon, Inc., Somerville, NJ) were tested using the same parameters and conditions.

Wound-closure tests

The adhesion strengths of the GelMA/HA-NB/LAP hydrogels and of Fibrin Glue, CA, and SurgifloTM were tested using the modified ASTM F2458–05 standard, a standard test for the determination of tissue/sealant material adhesive strength15. Porcine skin was prepared from fresh porcine skin pieces obtained from a local slaughterhouse. The skin sample dimensions were 30 mm × 10 mm. Tissues were pre-wet by immersion in PBS before testing, then fixed onto two glass slides (25 mm × 50 mm) using the ParafilmTM. The tissue was cut in the middle with a straight edge razor to simulate wounding, hydrogel solutions (1 mL) were injected onto the desired adhesion zone (20 × 20 mm), and crosslinked by UV. The two glass slides were placed into an Instron mechanical tester (Instron-5543 with a 1 kN sensor) for adhesion strength test by tensile loading with a strain rate of 1 mm/min. Maximum adhesive strength of each sample was obtained at the point of tearing. All measurements were repeated three times.

Peeling adhesion test

The peeling adhesion test was performed according to reported methods10. The sausage skin membrane was bonded to a rigid polyethylene terephthalate (PET) film with CA glue. One end of the PET film was kept open, in order to limit deformation at the crack tip. Then, hydrogel solutions (1 mL) were injected on the membrane surface (40 × 15 mm) and a hydrogel polymerized by UV irradiation. Finally, another PET film was bonded to the hydrogel (40 × 15 mm) with CA, forming a bilayer with an edge crack. This experimental set-up was used, because the sausage membranes are not transparent to UV and the hydrogel could not form if these membranes were used on both sides. An Instron machine (Instron-5543 with a 1 kN sensor) was used to apply unidirectional tension, while recording the force and the extension. The loading rate was kept constant at 1 mm/min. All measurements were repeated three times.

In vitro lap shear test

The lap shear test was performed according to a previous study15. The shear resistance of the GelMA/HA-NB/LAP hydrogels (n = 4), GelMA/HA-NB hydrogels (n = 4), and GelMA/LAP hydrogels (n = 4) was tested according to the modified ASTM F2255-05 standard for lap shear strength property of tissue adhesives. The sausage skin membrane was bonded to glass slides with CA glue. Then, hydrogel solutions (200 μL) were injected on the membrane surface (10 mm × 15 mm) and a hydrogel polymerized by UV irradiation. Finally, another glass slide was bonded to the hydrogel (10 mm × 15 mm) with CA. The two glass slides were placed into an Instron mechanical tester for shear testing by tensile loading with a strain rate of 1 mm/min. The sealant shear strength was determined at the point of detachment.

Compression test

The compressive stress–strain measurements were performed using a tensile-compressive tester (Instron-5543 with a 1 kN sensor). In compression-crack test, compressive samples were prepared in the molds for compression tests (10 mm in diameter and 5 mm in depth). Prior to the test, hydrogels were incubated in PBS at 37 °C for 4 h. The compressive strain rate was 5 mm/min and strain level was up to 75% of the original height. The compressive moduli were the approximate linear fitting values of the stress–strain curves in the strain range of 15–25%. All measurements were repeated three times.

Cell encapsulation and proliferation assay

L929 fibroblast cells (L929, Cell bank of the Chinese Academy of Science), at a density of 4 × 106 cells/mL were suspended in the sterile polymer precursor solution GelMA/HA-NB/LAP to evaluate the cytotoxicity of hydrogels. The cell-containing precursor solution (25 μL) was irradiated (30 mW/cm2, 3 min) to produce a cell-laden hydrogel and cultured in Dulbecco’s modified Eagle’s media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C and 5% CO 2 for 1, 3, and 5 days. Cell viability was determined using the live/dead cytotoxicity kit (Dojindo, Japan). Encapsulated cells were imaged under a fluorescence microscope (X71; Olympus). The proliferation of L929 cells was assessed by the Counting Kit-8 (CCK-8) method. Briefly, L929 cells were seeded into 96-well plates with a density of 1000 cells/100 μL/well and incubated for 24 h at 37 °C in a 5% CO 2 humidified incubator to obtain a monolayer of cells. Cell medium was replaced with hydrogel extracts and further incubated for 1, 3, and 5 days. The sample solution was removed and CCK-8 reagent was added to each well and incubated for 2 h at 37 °C. The absorbance was measured using a microplate reader (SpectraMax 190, USA) at 450 nm. For each sample, five independent cultures were prepared and proliferation assays were repeated three times for each culture.

Cell attachment test

C3H cells (C3H10T1/2, Cell bank of the Chinese Academy of Science) (5 × 104 cells/hydrogel) were seeded onto the GelMA/HA-NB/LAP hydrogel coated onto coverslips. Cells were stained with phalloidin (Cytoskeleton, Inc.) and DAPI (4′,6-diamidino- 2-phenylindole, Beyotime Institute of Biotechnology, Inc., Jiangsu, China) 1, 3, and 5 days following seeding. Before staining, cells were fixed in 4% (v/v) paraformaldehyde for 20 min, permeabilized in 0.1% (w/v) Triton X-100 (Sigma-Aldrich) for 5 min, and then blocked with 1% bovine serum albumin (Sigma-Aldrich) for 30 min. Actin filaments were stained in 200 × 10−9 M phalloidin for 45 min and nuclei were stained in 14.3 × 10−6 M DAPI for 5 min. Stained cells were then imaged under a confocal microscope with a ×40 water objective (BX-FV1000, Olympus).

In vivo degradation of hydrogels

Male rats (~ 250 g) were used in the in vivo degradation studies. All animals were treated according to the standard guidelines approved by the Zhejiang University Ethics Committee (ZJU20170969). A 1 cm incision in the mediodorsal skin of was made and a lateral subcutaneous pocket prepared. Hydrogel samples (n = 20; 10 × 3 mm cylinders) were implanted under sterile conditions. At designated time intervals (days 7, 14, 28, and 56), the rats were sacrificed and the samples were processed for histological analyses and biodegradation studies.

In vivo biocompatibility

Male rats (~ 250 g) were used for the in vivo biocompatibility studies. All animals were treated according to the standard guidelines approved by the Zhejiang University Ethics Committee (ZJU20170969). A 1 cm incision was made in the rat mesodorsal epidermis and a small lateral subcutaneous pocket was prepared. Matrix hydrogel (n = 8), CA (n = 8), and Fibrin glue (n = 8) were implanted into the dorsal subcutaneous pockets under sterile conditions. At designated time intervals (days 7 and 14), the rats were killed and the samples were processed for histological analyses. The degree of inflammation was assessed by three expert histopathologists, under blinded experimental conditions.

In vitro hemostasis experiments on pig livers

In vitro experiments were carried out on fresh pig livers purchased from the market to study hemostatic performance under wet and dynamic conditions. First, a 10 mm incision was pierced in pig liver and a perfusion tube was inserted with a 20 mL/min blood flow volume to mimic the heavy bleeding. The hydrogel was applied to the bleeding site and crosslinked by 365 nm UV light. The Fibrin Glue was used as control group.

In vivo hemostasis on rabbit’s livers and arteries

To study the hemostatic properties of hydrogels in vivo, the liver lobes and femoral artery of male New Zealand white rabbits (2.5–3.0 kg, n = 22), and the heart and carotid artery of male BA-MA Mini-pigs (20–25 kg, n = 5) were used as models. All animals were treated according to guidelines approved by the Zhejiang University Ethics Committee (ZJU20170969). For liver hemostasis, a large (3 cm) incision was made in the liver using surgical scissors. The hydrogel was injected onto the incision, followed by illumination with 365 nm UV light for 3~ 5 s. During the surgery, the blood was carefully collected with filter papers at time point of 10 min. The total amount of the blood loss was determined by weighing the papers and recorded10. For femoral artery hemostasis, the femoral artery of rabbit was peeled from the surrounding tissues and an incision (2 mm) created by scalpel. Hemostatic forceps were then used to clamp the blood vessels. Then the hydrogel was applied to the incision site and illuminated by UV light for 3~ 5 s. After 30 s for gelation, hemostatic forceps clamped in the proximal vascular was removed to observe whether the bleeding was stopped or not. Then, the distal part of the artery was clipped to observe whether the vessel was free or not.

Hemostatic experiments on pig carotid arteries and hearts

For hemostasis of penetrated cardiac injuries, after general anesthesia, a 6 mm inner diameter needle was used to pierce the ventriculus sinister of pig hearts (n = 7). For the experimental group, the defects and surrounding tissue were rapidly covered with hydrogel and irradiated by UV (n = 4). For the control group, the wound and surrounding tissue were first covered with Fibrin Glue, then with SurgifloTM, and finally with the hydrogel, followed by UV fixation (n = 3). The electrocardiograph was recorded using noninvasive telemetry for large animals (Emka Technologies, France).

The carotid artery hemostasis of pigs was carried out in a similar way to the femoral artery hemostasis of rabbit, the difference being that the incision (4–5 mm) was created by needle puncture followed by full scalpel incision. Blood flow volume was tested before and after surgery using the MFV-3200 electromagnetic flow meter (Nihon Kohden, Japan). The comparative experiments with Fibrin Glue and SurgifloTM were made using carotid artery hemostasis. After these operations, one pig was sacrificed for cardiac wall section histopathology and SEM analysis, and other pigs were allowed to recover.

All animals were treated according to guidelines approved by the Zhejiang University Ethics Committee (ZJU20170969).

Myocardial enzyme test

The levels of AST, CK, and LDH in the blood were analyzed by automatic biochemical analyzer (HITACHI 7020, Tokyo, Japan). The levels of BNP, cTn-T, and CK-MB were measured by ELISA kit (Nanjing Jiancheng Bioengineering Institute).

Histological evaluation

After 2 weeks recovery, the pigs were sacrificed. The heart and carotid artery were surgically removed and the samples were processed for histological analyses.

Statistical analysis

All data are presented as the mean ± SD. Differences between the values were evaluated using one-way analysis of variance (ANOVA; Tukey’s post-hoc test), except in vivo biocompatibility experiment (two-way ANOVA; Tukey’s post-hoc test). Data are presented as means with 95% confidence interval. p < 0.05 was considered statistically significant.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.