Synthesis of double-bonded hyaluronic acid (HAMA)

According to literature [20], weigh 1.8 g of HA with a molecular weight of 100,000, fully dissolve it in 90 mL of deionized water, then add 60 mL of N,N-dimethylformamide (DMF) to it and mix well, then slowly add 2.88 mL of methacrylic anhydride water drop by drop, and react for 30 minutes. Then, a 1 mol/L sodium hydroxide aqueous solution was prepared, and the pH of the reaction solution was adjusted to 8–9 until the pH of the reaction solution was stable and no longer changed with time. After 24 hours of reaction, 0.9 g of sodium chloride was added and continued to react for half an hour. After the reaction was completed, the reaction solution was added to 360 mL of absolute ethanol solution, stirred to obtain a white precipitate, which was collected by centrifugation and dissolved in deionized water, dialyzed through a MWCO7000 dialysis bag for three days, and then freeze-dried to obtain double-bond modified hyaluronic acid (HAMA) [21] (Figure 1A).

Dopamine-modified double bond hyaluronic acid (HAC)

According to the literature [22], HAMA requires first conversion to the corresponding tetrabutylammonium salt (HAMA-TBA). Weigh 1.0 g of HAMA and completely dissolve it in 200 mL of deionized water, add 3.0 g of strong acidic cation exchange resin to react overnight, and then adjust the pH of the solution to 7.02–7.06 with tetrabutylammonium hydroxide (TBA-OH) to obtain an aqueous solution of HAMA-TBA, which is freeze-dried for later use. 1 mmol of freeze-dried HAMA-TBA was completely dissolved in 27 mL of deionized water, then 63 mL of DMF, 2.5 mmol of 1-hydroxybenzotriazole hydrate (HOBt), 2.5 mmol of N,N-diisopropylcarbodiimide (DIC) were added, and stirred evenly for 1 hour under the protection of nitrogen, then 2.5 mmol of dopamine hydrochloride, 0.5 mmol of 4-dimethylaminopyridine (DMAP) and 2.5 mmol of N,N-diisopropylethylamine (DIPEA) were added. The reaction was carried out under nitrogen protection at room temperature for 24 hours, and then the solution of the reaction was dialyzed using a dialysis bag with a molecular weight cut-off of 7,000 Da, in concentrated hydrochloric acid adjusted sodium chloride solution (1 M, pH = 5) for 1 day, followed by 4 days in deionized water. The dialyzed solution was lyophilized to obtain dopamine-modified double bonded hyaluronic acid (HAC). The route map for HAC synthesis (Figure 1B), the structure of the obtained HAC product was identified using ¹H-NMR, and the degree of substitution on it was calculated.

Preparation of amidated regenerated silk fibroin (SFMA)

Degumming: Weigh 30 g of chopped silkworm cocoons and boil them in a Na₂CO₃ aqueous solution with a concentration of 0.05 M, wash them with deionized water, and repeat the steps three times to remove the sericin remaining in the silk. Then, the degummed SF was completely dried in a constant temperature oven at 60°C to obtain SF fiber for later use.

Preparation of pure regenerated silk fibroin samples (RSF): Take 5 g of degummed SF fiber, fully dissolve it in 25 mL of 9.3 M LiBr aqueous solution in a water bath at 60°C, and put the dissolved aqueous solution into a dialysis bag (MWCO7000), dialyze it in deionized water for 3 days. After the dialysis is completed, centrifuge to remove insoluble impurities, and freeze dry the supernatant to obtain a pure regenerated SF powder sample with high purity.

Preparation of amidated regenerated silk fibroin (SFMA) [21]: According to the literature method [23], weigh 5 g of degummed SF fiber, place it into a 50 mL beaker, then add 25 mL of 9.3 M LiBr aqueous solution, stir in a constant temperature water bath at 60°C until the SF is completely dissolved, and then slowly add 2 mL of GMA to it to obtain a mixture with a GMA concentration of 604 mM. Under the condition of 300 rpm, stirring was continued for 3 hours to allow GMA and SF to completely react. The solution after the completion of the reaction was put into a dialysis bag (MWCO7000) and dialyzed in deionized water for 4 days. Finally, the dialyzed solution was centrifuged to remove impurities, and the supernatant was freeze-dried to obtain an SFMA powder sample (Figure 1C).

Preparation of hyaluronic acid/silk fibroin hydrogel (HASF) by rapid photo-curing

Dissolve methacrylated and dopamine-modified HA and methacrylated SF protein in deionized water, stir and mix well, and change the addition amount of SFMA (0%, 1%, 2%, 4%). By controlling the content of SF in the hydrogel, hyaluronic acid/silk fibroin hydrogel (HASF) gels with different degrees of crosslinking can be obtained. According to the feed ratio of hydrogel, it is used to formulate various HASF hydrogels (Table 1).

A series of hydrogel samples (HAC-gel, HASF-gel-1, HASF-gel-2, and HASF-gel-4) with varying degrees of crosslinking were prepared by adjusting the SFMA content.

Four groups of hydrogel photo-cured precursor fluids were prepared according to different feed ratios. LAP photo-initiator at a concentration of 0.001 g/mL was added to each group of solutions. After mixing evenly, precursor solutions of HAC-gel, HASF-gel-1, HASF-gel-2, and HASF-gel-4 were individually loaded into circular silicone molds with a diameter of 15 mm and a height of 4 mm. Photocuring was subsequently performed using a UV lamp (365 nm wavelength) operating at 18 W power output. The irradiation process was conducted for 30 seconds under controlled UV intensity set to 40 mW/cm².

Nuclear magnetic resonance hydrogen spectroscopy (¹H-NMR)

¹H-NMR (Bruker AVANCE III 500MHz, Switzerland) was used to measure the ¹H-NMR spectra of HA before and after modification and SF before and after modification. Samples of HA, HAMA, HAC, RSF, and RSFMA were dissolved in D₂O at room temperature at a concentration of 10 mg/mL, and were characterized by ¹H nuclear magnetic resonance spectroscopy. MestReNova software was used to accurately calibrate our measurement data and calculate the degree of modification of different samples based on their corresponding integrated areas. The degree of modification of methacrylic anhydride (D₁) and dopamine (D₂) on the HA molecule is calculated according to Equations 1 and 2, respectively.

Among them, I_{6.10 ppm}, I_{5.63 ppm}, and I_{1.91 ppm} are the integrated areas of the signal peaks with chemical shifts at 6.10 ppm, 5.63 ppm, and 1.91 ppm.

Among them, I_{6.5 ppm~7.0 ppm} is the area of the signal peak with chemical shifts of 6.5 ppm to 7.0 ppm, I_{3.0 ppm~4.0 ppm} is the area of the signal peak with chemical shifts of 3.0 ppm to 4.0 ppm, and the degree of substitution of vinyl groups on the SF molecule is calculated according to Equation 3.

where D represents the degree of amidation on the SF molecule, and I₀ represents the integrated area of lysine in SF without GMA modification. I represent the integrated area of lysine in SFMA modified by GMA.

UV-Vis spectral

Prepare HAC and HAMA samples into aqueous solutions with a concentration of 1–2 mg/mL, pour them into 2/3 of the cuvette, and use a UV-Vis-NIR spectrometer (LAMBDA 750, PerkinElmer, USA) to perform absorption spectrum detection on the HAMA and HAC samples. Using deionized water as a control, scan the range of 100–600 nm, and measure the absorption peak.

Molecular weight determination

Gel permeation chromatography (GPC, Waters GPC 1515, USA) was used to measure the absolute molecular weight of HA before and after modification. Unmodified HA (Mn = 100,000), double-bonded modified HAMA and dopamine-modified double-bonded HAC were respectively prepared into aqueous solutions with a concentration of 3–6 mg/mL, and then the respective prepared solutions were passed through a chromatographic column to measure the absolute molecular weight.

Rheological properties

Rheological experiments were performed on the hydrogel samples using a rotary rheometer (Anton-Paar MCR 302, Austria), and they were analyzed. In the oscillation time sweep experiment, the changes of storage modulus (G') and loss modulus (G'') with time were measured at a strain amplitude of 1% and a speed of 6.28 rad/s. The curing light source uses a 365 nm wavelength ultraviolet lamp with a power of 18 W, and the intensity of the ultraviolet lamp is set to 40.

Fourier transform infrared spectroscopy (FT-IR)

The changes in the molecular structure of amidated SF were analyzed by infrared spectroscopy (Nicolet iS50, Thermo Fisher, USA). The SFMA powder modified with GMA and the unmodified RSF powder were characterized in the range of 4,000–400 cm^–1.

Microscopic morphology of the hydrogels

The internal microstructure of the lyophilized hydrogel samples was observed using a benchtop scanning electron microscope (SEM, TESCAN VEGA3, Czech). The freeze-dried hydrogel samples HAC-gel, HASF-gel-1, HASF-gel-2, and HASF-gel-4 were brittle broken with liquid nitrogen, adhered to a standard cross-section table with conductive glue, and then the surface was sprayed with gold. The scanning voltage was set to 15 kV, and the magnification was adjusted to 50 times and 100 times to observe the internal morphology of the hydrogel.

Mechanical properties of hydrogels

The compression properties of the prepared hydrogels were measured using a high and low temperature dual column tester (Instron 5966, America). Precursors of HAC-gel, HASF-gel-1, HASF-gel-2, and HASF-gel-4 were placed in a silica gel orifice plate, and cylindrical samples with a diameter of about 15 mm and a height of about 4 mm were photocured to measure the compression properties of the hydrogels. The speed was set at 2 mm/min, and the compression shape variable was 80% until the hydrogel was broken and the experiment was stopped. Each group of samples was tested 3 times and the average value was taken.

Shear adhesion of hydrogel

Referring to the ASTM standard (F2255-05 method), the shear adhesion strength of the hydrogel was tested using a high and low temperature double column testing machine (Instron5966, USA) [24]. First, the fresh pig casings were cut into rectangular strips with a length of about 7 cm × 2 cm. Secondly, the precursor liquids of HAC-gel, HASF-gel-1, HASF-gel-2, and HASF-gel-4 hydrogels were prepared, and the precursor liquids were evenly coated on the pig casings. One end was evenly coated and covered with another strip in the opposite direction. The coverage range of the prepared gel precursor liquid was about 4 cm². Then, 365 nm ultraviolet light was irradiated for 30 seconds. After completing the bonding, the length b (cm) and the width l (cm) of the bonded part were measured. and record relevant data. Place the prepared specimen in the film fixture of a high-low-temperature double column testing machine, and stretch it at a speed of 5 mm/min with the long axis of the specimen as the force direction until the adhesive joint breaks, and obtain the maximum load of shear failure of the specimen—F (N). The shear adhesion strength τ (MPa) of the samples can be calculated from Equation 4, and the experiment is repeated three times for each group of samples.

Swelling property

Hydrogels have the function of absorbing blood oozing from wounds, keeping wounds moist and accelerating healing. Therefore, it is necessary to evaluate their swelling characteristics. The swelling behavior of the prepared hydrogels was evaluated using a weighing method. Four groups of lyophilized hydrogel samples HAC-gel, HASF-gel-1, HASF-gel-2, and HASF-gel-4, were weighed respectively, the initial weight was recorded as W₀ (g), and the samples were immersed in 1× PBS solution at room temperature. After a fixed time, the swollen hydrogel was taken out, dried on a clean filter paper, weighed (W_t, g), and the swelling rate of the hydrogel was calculated by a formula.

The calculation formula for hydrogel swelling ratio is shown in Equation 5:

Degradation property

In vitro degradation experiments of HASF hydrogel were carried out with 1× PBS and 300 U/mL hyaluronidase solution. The dried HASF hydrogel column (diameter 15 mm, thickness 4 mm) was weighed. Place each component hydrogel in a 12-well plate, add 2 mL of PBS containing 300 U/mL to each well until it is completely immersed, and then place it in a constant temperature air bath at 37°C, and replace the PBS every two days. Take samples, freeze-dry, and weigh regularly to calculate the weight loss percentage of the initial hydrogel sample. The degradation rate calculation formula is as follows:

where W₀ (g) is the initial dry weight of the HASF hydrogel, and W_d (g) is the dry weight of the hydrogel measured after a specific time.

Cell viability

The biocompatibility was evaluated by indirect contact method. Indirect contact method: L929 cells were seeded in a 24-well plate (5 × 10⁴ cells) for 24 hours, and then treated with different concentrations of hydrogel soaking solution (hydrogel:DMEM, mass ratio 1:5) to treat for 24 hours. Add 10 μL of CCK-8 solution to each well. After incubation for 2 hours, the absorbance was measured with a 450 nm microplate reader. Take A_s and A_c as the absorbance of the experimental and control groups, and A_b as the OD value of the blank group. The calculation formula is shown in Equation 7.

where A_s is OD_{experimental hole}, A_c is OD_{control hole}, and A_b is OD_{blank hole}.

For more biocompatibility experiments, please refer to the literature [25] of our group.

Animals

SD rats were approved by the Ethics Committee of Zhejiang Provincial People’s Hospital (Ethics Permission No. 20230527175018933362). Diabetic male SD rats (250–300 g, n = 6) from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd. were employed in this study. All animal care and experimental protocols were approved by the Animal Experimental Center of Zhejiang University of Technology and conducted in accordance with ethical guidelines. Following a one-week acclimatization period, the rats were anesthetized via intraperitoneal injection of pentobarbital sodium [26] (1%, 45 mg/kg), and their dorsal fur was shaved. Three circular wounds (1.2 cm in diameter) were created on the dorsal surface of each rat, with each wound receiving one of three treatments: HASF hydrogel precursor, Tegaderm (3M) dressing, or PBS. Wound healing was assessed at predefined time points, and wound size was quantified using ImageJ software. The day of surgery was designated as Day 0, and wounds were photographed and healing progress recorded on Days 1, 3, 5, and 10. On Day 7, the rats were humanely euthanized, and the wound tissue was harvested and fixed in 4% paraformaldehyde for paraffin embedding. Hematoxylin and eosin (H&E) and Masson’s trichrome staining were performed by Xavier Biological Co., Ltd. 1% pentobarbital sodium was injected intraperitoneally at a dosage of 45 mg/kg body weight. After injection, the depth of anesthesia was monitored to ensure loss of consciousness and analgesia. Once adequately anesthetized, tissues or organs were collected according to the experimental protocol. All procedures were performed by trained personnel to ensure consistency and animal welfare. Following sample collection, euthanasia was performed by administering an additional dose of 1% pentobarbital sodium via intraperitoneal injection to ensure humane death without pain. The euthanasia of SD rats was performed via cervical dislocation, in accordance with standard protocols.

Hemostasis experiment

Hepatic hemorrhage models were established in 9 SD rats and its hemostatic effect in vivo was evaluated. First, anesthetics (1% sodium pentobarbital 45 mg/kg) were injected intraperitoneally, fixed on the operating table, and the abdomen of the rat was cut open to expose the liver. A 15 mm long and 5 mm deep incision was made on the liver of each mouse with a scalpel, and HASF hydrogel and commercially available gelatin sponge were immediately injected. The control group was not treated. Use a filter paper sheet to suck blood for 150 seconds. The change in weight before and after filtration with filter paper is the amount of wound bleeding (mg). The same method was used to repeat the measurements on different SD rats, and the average value was taken.

Construction and treatment of skin wound models

After one week of adaptive culture, SD rats (n = 6) were anesthetized by intraperitoneal injection with sodium pentobarbital (1% sodium pentobarbital 45 mg/kg) and their backs were depilated. We will construct and treat the wound model in accordance with the experimental procedures delineated in the aforementioned Animals section. HASF hydrogel precursor solution was applied to the wounds of rats and photocured to form gels. The wounds were treated with Tegaderm (3M) dressing and the wounds were treated with PBS. The wounds were observed at specific time points and photographed with a camera. The wound size was evaluated with ImageJ. The day of surgery was recorded as Day 1. Each treated wound was photographed on Days 1, 3, 5 and 10, and its healing status was recorded.

Wound histochemical staining

Rats were modeled and therapeutic interventions were given respectively according to the above method. On the 7th day, the rats were sacrificed, and the skin at the wound site was fixed in 4% paraformaldehyde, and paraffin sections were prepared for subsequent H&E staining, and the skin tissue was also stained with Masson. Both H&E staining and Masson staining were entrusted to Seville Biotech Co., Ltd.

Rapid photocuring preparation of HASF-gel

The photo-initiator LAP was added to the precursor liquid of the hydrogel, and the hydrogel sample HASF was successfully prepared under ultraviolet light irradiation. Hydrogel samples HAC-gel, HASF-gel-1, HASF-gel-2, and HASF-gel-4 with different crosslinking degrees were obtained by changing the content of SFMA. As the SFMA content increases, the transparency of the hydrogel decreases due to the increase in the degree of crosslinking of the HASF hydrogel and the changes in the internal structure of the hydrogel (Figure 1K).

The curing times of four groups of hydrogels were measured by the tilt method. The light curing times of the four groups of samples were 10 s, 12 s, 13 s, and 18 s, respectively, and all hydrogel groups were quickly cured and formed in tens of seconds. Shorter ultraviolet light irradiation can reduce toxic effects on cells and biological tissues.

In a word, all hydrogels rapidly gelled under the action of the initiator and UV light, with gelation times ranging from 10 to 20 s.

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Figure 1. Character of hydrogel. A) Synthesis of HAMA. B) Synthesis of HAC. C) Synthesis of SFMA. D) & E) ¹H-NMR spectrum of HAMA, HAC and SFMA. F) FT-IR spectra of pure RSF and GMA-modified SFMA. G) UV-Vis spectra of HAMA and HAC. H) HA, HAMA and HAC molecular weight distribution. I) Rheological properties of hydrogels with different SFMA contents. J) SEM image of microporous morphology of sections of different hydrogels. K) Preparation process of HASF hydrogel. HA: hyaluronic acid; HAC: dopamine-modified double bonded hyaluronic acid; HAMA: double-bond modified hyaluronic acid; HASF: hyaluronic acid/silk fibroin hydrogel; RSF: regenerated silk fibroin; SF: silk fibroin; SFMA: amidated regenerated silk fibroin; TBA: tetrabutylammonium

Characterization of HAC and SFMA precursors

To further confirm the successful modification of the SF molecule with GMA, we performed ¹H-NMR (Figure 1E) analysis on the modified SFMA. From the results, it can be seen that after GMA modification, two new NMR signal peaks appeared at two chemical shifts of 6.02 ppm and 5.60 ppm, respectively. They were caused by the vibration of two hydrogen nuclei in C=CH₂. An obvious methyl (CH₃) signal peak appeared at the chemical position of 1.8 ppm, and the vibration signal of the proton in N–CH₂ appeared at 3.30–3.70 ppm. In addition, it can be found from the spectrum that compared with pure SF, the signal peak intensity of lysine on the SF molecule modified with GMA is weaker. This may be due to the reaction of GMA with the lysine on the SF molecule, resulting in the decrease in the content of lysine, and the spectrum shows that the signal peak intensity of lysine becomes weaker. At the same time, the GMA modification degree was calculated to be 34.8% through quantitative analysis of nuclear magnetic resonance spectra. Combined with FT-IR characterization results, it was confirmed that GMA could effectively amidate SF.

Comparing the nuclear magnetic spectra of HA and HAMA (Figure 1D), it was observed that three new signal peaks appeared on the HAMA spectrum, one partial peak at about 1.84 ppm, and two new nuclear magnetic peaks at 6.1 ppm and 5.63 ppm. These three peaks are all characteristic peaks of MA, which are signal peaks of methyl hydrogen of MA and two hydrogen atoms on the double bond carbon. The integrated area of MA hydrogen was compared with the integrated area of HA methyl hydrogen. The modification degree of MA on HA was calculated to be 47%. Comparing the nuclear magnetic spectra of HAMA and HAC, it can be found that a new peak at 6.5–7.0 ppm has been added to the spectra of HAC. These three chemical shift peaks correspond to the chemical shift values of hydrogen on the three carbons at positions 2, 3 and 4 on the dopamine benzene ring, respectively, indicating that dopamine hydrochloride has been successfully modified onto methacrylic acid liver modified HA. The peak of 3.0–4.0 ppm indicates the signal peak presented by hydrogen atoms on the two six-membered ring skeletons of HA. The degree of substitution of dopamine calculated from the peak area is about 34.6%, which is close to that reported in the document [22].

Infrared spectrum analysis of SFMA (Figure 1F) was carried out using FT-IR to initially judge the modification effect of GMA on SF. The GMA and SF-related peaks were identified from the spectra, and the relationship between the two was confirmed. The spectra of RSF and GMA modified SFMA samples both showed that the peaks of amide type I, type II and type III were located at 1,639, 1,517 and 1,234 cm^–1, respectively. From the spectrum, we can find that SFMA has a new peak with a weak band at a wave number of 1,295 cm^–1, which may be related to the stretching vibration of the alcoholic hydroxyl group (–CHOH) generated after the ring-opening reaction between the epoxy group on GMA and the SF molecule. In addition, weak peaks appeared at wavenumbers of approximately 950 cm^–1 and 1,120 cm^–1, representing the stretching vibration of the methacrylate vinyl group (–C= CH₂) in GMA. This peak is not obvious in the spectrum, probably because it is covered by the peak positions of other structures. In addition, since the molecular weight of GMA is much smaller than SF, the GMA functional groups can only be slightly detected or hidden.

Generally speaking, in the UV-visible spectrum, the catechol group has a typical absorption peak at 280 nm (assigned to the π–π* absorption band). By absorbance measurement of HAMA and HAC solutions (Figure 1G), the single absorption peak of HAC appears at 280 nm, and no obvious absorption peak is observed in HAMA, indicating that the dopamine catechol structure has been successfully grafted to HAMA, and there are no other peaks at wavelengths greater than 300 nm, indicating that the catechol conjugated structure has not been oxidized.

GPC testing was performed on the synthesized HAC and HAMA, and the results showed that (Figure 1H), the weight-average molecular weight Mw of HAC is 2.4 × 10⁵ g/mol, the number-average molecular weight Mn is 1.46 × 10⁵ g/mol, and the molecular weight distribution coefficient (PDI) is 1.656. The weight-average molecular weight Mw of HAMA is 1.8 × 10⁵ g/mol. The number-average molecular weight Mn is 1.1 × 10⁵ g/mol and the molecular weight distribution coefficient (PDI) is 1.653. The weight-average molecular weight Mw of raw material HA-10W is 1 × 10⁴ g/mol. The number average molecular weight Mn is 1.4 × 10⁵ g/mol, and the distribution coefficient (PDI) is 1.45. As shown in Figure 1H, the molecular weight of the modified HA becomes larger.

The crosslinking and the structure of hydrogels

The changes in storage modulus G' and loss modulus G'' of copolymer hydrogels with different content ratios were studied by rheological methods (Figure 1I). When the elastic modulus is greater than the viscosity modulus, the hydrogel is in a gel state and is first scanned for time under ultraviolet radiation from a rheometer. All hydrogel precursors are stabilized for 25 s before turning on the ultraviolet light source to irradiate. All solutions have stable G' and G' before ultraviolet light irradiation. When the ultraviolet lamp is turned on, G' increases rapidly as the illumination time increases, then exceeds G'', and finally reaches a plateau within 300 s. We found that the addition of SFMA had a certain impact on the gel point time (the G' and G' data curves crossed). When the addition of SFMA was 4%, the longest gel point time was about 22 s, indicating that the hydrogel precursor requires more time to crosslink.

By controlling the SFMA content, the degree of crosslinking in the hydrogels could be adjusted, resulting in porous network structures with different crosslinking densities. Mechanical, swelling, and degradation tests confirmed that the properties of the hydrogels were directly influenced by their crosslinking density.

The internal structure of hydrogels plays a key role in the performance of materials. Therefore, the internal microstructures of four groups of hydrogel samples HAC-gel, HASF-gel-1, HASF-gel-2, and HASF-gel-4 were observed through a benchtop scanning electron microscope (Figure 1J). It can be seen from the figure that all components of hydrogels have interconnected and uniform porous microstructures, and the internal cross-linked structure of hydrogels remains intact after freeze-drying. The pores of HAC/SFMA composite gels of different components penetrate through to form a unique 3D porous structure, which is conducive to the transport of oxygen and nutrients, and also creates conditions for cell growth. In addition, with the increase of SFMA concentration, the pore size of the hydrogel slightly increases, which is due to the increase in cross-linking degree of HASF hydrogel and changes in the internal structure of the hydrogel. This unique loose porous structure provides an ideal growth environment for cells. These pores not only facilitate tight adhesion between cells but also promote their migration and distribution. At the same time, this material can maintain a moist microenvironment, which is crucial for the absorption of exudate from the wound. Due to the existence of these holes, new tissue can adapt to the surrounding environment more quickly, thus accelerating the process of wound healing. In addition, the porosity of this structure also allows liquids and nutrients to flow inside the material, further enhancing the support and nourishment of damaged areas [27, 28].

Mechanical and adhesive properties of hydrogels

Effective hemostatic materials should have sufficient mechanical strength to protect wounds and promote tissue healing. We conducted mechanical compression tests on hydrogel samples HAC-gel, HASF-gel-1, HASF-gel-2, and HASF-gel-4, and measured the compressive strain (%) on the X-axis and the compressive stress on the Y-axis (MPa), where HAC is a hydrogel without SFMA added, and the compressive strengths of the four groups of hydrogels are 0.22 MPa, 0.25 MPa, 0.33 MPa, and 0.44 MPa, respectively, indicating that with the increase of SFMA content, the compressive strength of the hydrogel is improved to a certain extent, and the degree of crosslinking of the hydrogel increases (Figure 2A). In addition, the four groups of hydrogel samples fractured at compressive strains of 42.85%, 50.28%, 49.51%, and 50.71%, respectively, indicating that changes in SFMA content can improve the toughness of the hydrogel to a certain extent, and the adjustable compressive strength of the hydrogel can be better applied to various tissues.

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Figure 2. Property of hydrogel. A) Compression performance of hydrogel samples. B) Schematic diagram and photos of hydrogel lap shear test. C) Shear tensile curves of hydrogels with different SFMA contents on wet pig casings. D) Shear adhesion strength of hydrogels with different SFMA contents on pig casing substrate. E)–G) Schematic diagram of hydrogel adhesion to human finger joints, pig skin surface and other different substrates. H) Hydrogel adhesion mechanism. HAC: dopamine-modified double bonded hyaluronic acid; HASF: hyaluronic acid/silk fibroin hydrogel

The good tissue adhesion of hydrogels can promote wound sealing to stop bleeding, create a physical barrier to prevent bacterial invasion, promote cell growth and differentiation, and provide a moist environment to accelerate wound healing. To this end, we investigated the adhesion properties of the prepared hydrogels. We used pig casings as skin tissue to study the adhesion strength of the hydrogel through lap shear tests (Figure 2B). As shown in Figures 2C and 2D, the prepared hydrogel HASF-gel-2 has a maximum adhesion strength of 160.3 kPa, while the adhesion strengths of HAC-gel, HASF-gel-1, and HASF-gel-4 are 71.96 kPa, 117.35 kPa, and 136.51 kPa, respectively. Adding a suitable amount of SFMA can further improve the adhesion strength of the hydrogel, and the reduction of the adhesion performance of HASF-gel-4 may be related to the crosslinking density of the hydrogel. Comparison found that the adhesive strength of the prepared HASF hydrogel was much higher than that of the marketed fibrin glue (2–40 kPa). Figures 2E and 2G show that the prepared hydrogel HASF-gel-2 not only has good adhesion to human finger joints, but also has excellent adhesion flexibility to fresh pig skin and will not separate when stretched, bent, rolled and twisted. In addition, we also found that the prepared hydrogel HASF-gel-2 can also be easily cured in situ and adhered to various materials, as shown in Figure 2F. All of the good adhesion properties of the hydrogel are attributed to the dopamine catechol structure grafted on HA and some functional groups, such as amino groups on SF that interact with the tissue interface to produce adhesion (Figure 2H). This adhesion effect allows the hydrogel to maintain good adhesion properties even in a humid environment.

All hydrogels exhibited strong adhesion, and the addition of SFMA improved their adhesive properties. The hydrogels adhered well to various surfaces and were capable of adapting to shape changes without breaking or peeling, showing promise for use as wound dressings.

Swelling properties

In a liquid environment, hydrogels will swell to varying degrees, and a suitable swelling rate is the key to maintaining their good structure. In order to investigate the swelling behavior of the prepared hydrogels, hydrogel swelling tests were carried out on four groups of hydrogel samples of HAC-gel, HASF-gel-1, HASF-gel-2, and HASF-gel-4 in 1× PBS solution. The swelling behavior and equilibrium swelling of each group of hydrogel samples. As shown in Figures 3A and 3B, all hydrogels showed the ability to absorb water rapidly in the first 1 hour, and could reach an equilibrium swelling state within 4 hours. A stable swelling rate could be maintained for a period of time. This shows that the hydrogel has relatively good size stability, and good swelling properties are conducive to the adsorption of wound exudate by the hydrogel wound dressing. HAC-gel has the highest swelling ratio of 2,100%, and the HASF-gel-4 hydrogel sample has the lowest swelling ratio of 1,288%. The swelling ratio of HASF hydrogel decreases with the increase of SFMA concentration. The addition of SFMA results in the formation of hydrogels with a higher degree of crosslinking, so the equilibrium swelling ratio of the hydrogels is lower.

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Figure 3. Biocompatibility properties of hydrogel. A) Swelling behavior of HASF hydrogels in PBS buffer. B) Equilibrium swelling ratio of HASF hydrogels in PBS buffer. C) Degradation behavior of HASF hydrogel. D) OD and cell viability of HASF hydrogel after 24 h of L929 cell culture. HAC: dopamine-modified double bonded hyaluronic acid; HASF: hyaluronic acid/silk fibroin hydrogel

Degradation property

The degradation behavior of materials plays a crucial role in many applications of tissue engineering. It not only affects the stability and long-term performance of biological scaffolds or tissue substitutes, but also affects the treatment effect and patient satisfaction. Under ideal conditions, the degradation rate of hydrogel should maintain a quick-fitting relationship with the growth of tissue and skin. To this end, we explored the in vitro degradation behavior of the prepared hydrogel. In vitro degradation curves of the hydrogels obtained by placing hydrogel samples of HAC-gel, HASF-gel-1, HASF-gel-2, and HASF-gel-4 in PBS solution containing hyaluronidase for 2 weeks (Figure 3C). It can be seen from the figure that the degradation rates of HAC-gel, HASF-gel-1, HASF-gel-2, and HASF-gel-4 hydrogels decrease in turn over time. All hydrogel groups could degrade by 80% within two weeks, and HASF-gel-4 hydrogel degrades relatively slowly than the pure HAC-gel group. This is attributed to the relatively high degree of crosslinking of HASF-gel-4 hydrogel. The degradation rate of hydrogel can be changed by adjusting the content of SFMA.

Cell viability

The biocompatibility of HAC-gel, HASF-gel-1, HASF-gel-2, and HASF-gel-4 hydrogel samples was characterized by the CCK-8 kit [25] (Figure 3D). WST-8 in the kit was reduced to a highly water-soluble yellow formazan product by the dehydrogenase in the cells under the action of the electron carrier. The amount of formazan produced is proportional to the number of living cells. Therefore, this characteristic can be used to directly conduct cell proliferation and toxicity analysis. The optimal measurement time can be selected for multiple determinations. Compared with the MTT method, the linear range is wider and the sensitivity is higher.

The cell viability value of the control group was 1, and the cell viability values of HAC-gel, HASF-gel-1, HASF-gel-2, and HASF-gel-4 hydrogels were 1.015, 1.085, 1.136, and 1.263, respectively, which were higher than the control group, indicating that the prepared hydrogel group has good biocompatibility, and the addition of appropriate amount of silk protein is conducive to cell proliferation and provides a good growth environment for cell culture.

Biocompatibility tests indicated that all hydrogels had good biocompatibility, with the hydrogel groups promoting cell proliferation compared to the control group.