Conductive silk fibroin loaded methacrylated gelatin hydrogel and preparation method thereof

Abstract

The invention relates to conductive silk fibroin loaded methacrylated gelatin hydrogel and a preparation method thereof, and belongs to the technical field of biomedical materials. The preparation method comprises the following steps: S1, adding silk fibroin fibers into a dopamine hydrochloride solution, stirring, standing, then adjusting the pH value to 8-9, and carrying out a heating reaction to obtain polydopamine grafted silk fibroin fibers; s2, sequentially dipping the polydopamine grafted silk fibroin fibers in an ammonium persulfate solution and a pyrrole solution to obtain conductive silk fibroin fibers; and S3, adding the conductive silk fibroin fibers into the methacrylated gelatin solution, carrying out ultraviolet light cross-linking curing, and then soaking the conductive silk fibroin fibers in a glycerol solution to obtain the conductive silk fibroin loaded methacrylated gelatin hydrogel. The method not only can ensure the structural integrity and conductive continuity of a conductive network in the hydrogel, but also breaks through the dependence of in-situ polymerization on the permeability of a matrix.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and particularly relates to a methacrylated gelatin hydrogel loaded with conductive silk fibroin and a preparation method thereof. BACKGROUND

[0002] As a novel hydrogel with electrical conductivity, conductive hydrogel shows significant potential in the field of biomedicine, especially in the direction of electroactive tissue regeneration, bioelectronic interface and wound healing monitoring. Its advantages lie in providing a three-dimensional scaffold microenvironment required for cell growth and regulating cell behavior through electrical signals, thereby more effectively promoting tissue function reconstruction. In the construction strategy of conductive polymer hydrogel, the introduction of conductive fillers such as carbon nanotubes and metal nanowires can improve electrical conductivity, but such fillers have potential biological toxicity and dispersion problems that are difficult to overcome. Although conductive polymer polypyrrole (PPy) has electrical conductivity and biological safety, it has inherent defects such as brittleness and difficulty in processing, and existing technologies mostly rely on physically doped PPy particles, which often leads to uneven distribution of fillers, easy loss of fillers, and insufficient mechanical properties due to weak interfacial bonding between fillers and matrix, seriously affecting the long-term electrical conductivity stability of the material. Therefore, it is an urgent need to develop a composite hydrogel scaffold with excellent biocompatibility, high electrical conductivity and matching mechanical properties of tissues.

[0003] Invention patent CN 109575318 A discloses a dopamine-mediated polypyrrole conductive hydrogel and a preparation method thereof. Although the dopamine-mediated polypyrrole conductive hydrogel can improve electrical conductivity, pyrrole polymerization needs to be carried out at a low temperature of 0-10℃ for 18-36h, which has the problems of high energy consumption and long cycle time, and the use of immersion method can lead to preferential polymerization of pyrrole monomers on the surface of the hydrogel, making it difficult to ensure the formation of a continuous and homogeneous conductive network inside.

[0004] Invention patent CN 114796603 A discloses a chitosan / xanthan gum interpenetrating network conductive hydrogel and a preparation method thereof. Although the chitosan / xanthan gum interpenetrating network conductive hydrogel scaffold has good biocompatibility, PEDOT-HA nanoparticles are prone to agglomeration, resulting in low electrical conductivity (only about 4.1×10 -5 S / m), and insufficient mechanical strength in the target application field. SUMMARY

[0005] To solve the above technical problems, the application provides a methacrylated gelatin hydrogel loaded with conductive silk fibroin and a preparation method thereof. Through dopamine-assisted interfacial polymerization, polypyrrole (PPy) is firmly combined with the surface of silk fibroin fiber (SFF) by means of the synergistic effect of covalent and non-covalent, to form conductive silk fibroin fiber; and then the conductive silk fibroin fiber is dispersed in a methacrylated gelatin (GelMA) matrix, so that three-dimensional uniform distribution can be achieved, the mechanical properties of the scaffold can be enhanced, and the electrical signal can be effectively transmitted. The covalent grafting strategy also fully utilizes the inherent high biocompatibility, biodegradability and low immunogenicity of silk fibroin fiber, and avoids the potential cytotoxicity risk caused by physical doping filler leaching. The technology not only ensures the structural integrity and electrical continuity of the conductive network in the hydrogel, but also breaks through the dependence of in-situ polymerization on the permeability of the matrix.

[0006] A first object of the application is to provide a preparation method of a methacrylated gelatin hydrogel loaded with conductive silk fibroin, comprising the following steps: S1, adding silk fibroin fibers to a dopamine hydrochloride solution, stirring, then adjusting the pH to 8-9 and performing a heating reaction to obtain polydopamine-coated silk fibroin fibers; S2, immersing the polydopamine-coated silk fibroin fibers obtained in S1 in an ammonium persulfate solution and a pyrrole solution in sequence to obtain conductive silk fibroin fibers; S3, adding the conductive silk fibroin fibers obtained in S2 to a methacrylated gelatin solution, cross-linking and curing under ultraviolet light, and then immersing in a glycerol solution to obtain the methacrylated gelatin hydrogel loaded with conductive silk fibroin; the methacrylated gelatin solution is prepared by dissolving methacrylated gelatin and a photoinitiator in a phosphate buffer solution.

[0007] In an embodiment of the application, in S1, the concentration of the dopamine hydrochloride solution is 0.01 mol / L-0.06 mol / L, and the pH is 8-9; dopamine is used as a key interfacial engineering molecule, which is oxidized and self-polymerized on the surface of the silk fibroin fibers to form a polydopamine transition layer, and then initiates the directional graft polymerization of pyrrole; in addition, the surface of the silk fibroin fibers is grafted with polydopamine through a chemical method, so that subsequent conductive-mechanical dual-function synergy can be achieved. The mass ratio of dopamine hydrochloride to silk fibroin in the dopamine hydrochloride solution is (2-2.5):1.

[0008] In an embodiment of the application, in S1, the heating reaction is performed at a temperature of 65℃-75℃ for 2.5h-3.5h.

[0009] In an embodiment of the application, after S1, a step of ultrasonic cleaning is further included to wash away the large agglomerated particles of dopamine that are not firmly combined on the surface.

[0010] In one embodiment of the present invention, in step S1, during the grafting of polydopamine onto the surface of the silk fibroin fibers, the alkaline environment not only promotes the oxidation of the catechol groups in the dopamine molecules into quinone structures by dissolved oxygen in the solution, but also further promotes the polymerization of the reactive quinone intermediates generated after oxidation. Simultaneously, this alkaline environment synergizes with the subsequent water bath heating to ensure the efficient and rapid formation and deposition of the polydopamine coating on the surface of the silk fibroin fibers.

[0011] In one embodiment of the present invention, in S2, the concentration of the ammonium persulfate solution is 0.05 mol / L-1.5 mol / L; ammonium persulfate is used as an oxidant, thanks to its good water solubility (solubility is 58.2 mg / 100 mL water), and the decomposition products will not affect the performance of silk, and can meet the application requirements. In contrast, although potassium persulfate has less effect on silk fibroin fibers, its solubility is low (only 1.75 mg / 100 mL water), which is not conducive to full play of its role; and although ferric chloride is widely used in pyrrole polymerization, it is highly acidic (pH ≈ 2), which will damage silk fibroin fibers, and the residual Fe ions may also affect biocompatibility. In addition, ammonium persulfate is used as an oxidant, and when a polypyrrole (PPy) coating is subsequently prepared on the surface of the silk fibroin fiber, it can provide a sufficiently high oxidation potential (higher than the pyrrole oxidation potential), thereby triggering the oxidative polymerization reaction of the pyrrole monomer; The immersion time in the ammonium persulfate solution is 30 minutes to 120 minutes, so that the ammonium persulfate can fully penetrate between and on the surface of the silk fibroin fibers.

[0012] In one embodiment of the present invention, in S2, the mass fraction of the pyrrole solution is 1%-5%; The immersion time in the pyrrole solution is 12h-30h; the pyrrole monomer can achieve in-situ uniform polymerization by contacting and adsorbing on the ammonium persulfate on the surface of the silk fibroin fiber, thereby forming a tightly bound polypyrrole (PPy) coating on the surface of the silk fibroin fiber.

[0013] In one embodiment of the present invention, in S3, the concentration of the conductive silk fibroin fibers in the solution is 1 mg / mL-3 mg / mL.

[0014] In one embodiment of the present invention, in S3, the photoinitiator is Irgacure 2959; The concentration of methacrylated gelatin in the methacrylated gelatin solution is 9% w / v-11% w / v, and the concentration of the photoinitiator is 1.2% w / v-1.8% w / v.

[0015] In one embodiment of the present application, in S3, the wavelength of the ultraviolet light cross-linking curing is 365 nm, and the time is 10 min-30 min.

[0016] In one embodiment of the present application, in S3, the glycerol solution is obtained by mixing glycerol and water in a volume ratio of (6.5-7.5):3. The time of soaking in the glycerol solution is 12 h-36 h.

[0017] A second object of the present application is to provide a methacrylated gelatin hydrogel loaded with conductive silk fibroin prepared by the method.

[0018] The technical solution of the present application has the following advantages compared with the prior art: The hydrogel of the present application combines rigid conductive silk fibroin fibers (PPy-PDA-SFF) with flexible methacrylated gelatin (GelMA) networks, and the interface bonding is strengthened by the synergistic effect of covalent bonds and non-covalent forces, which significantly improves the mechanical strength and toughness of the hydrogel, effectively overcomes the mechanical defects caused by weak interface bonding in the physical doping system, and makes the mechanical properties of the hydrogel more suitable for the needs of biological tissues; at the same time, PPy is firmly grafted on the silk fibroin fiber skeleton through covalent bonds and hydrogen bonds, avoiding the problem of agglomeration and uneven distribution of conductive fillers caused by physical doping, ensuring the uniformity and long-term stability of the conductive network; in addition, degradable polypyrrole (PPy) and silk fibroin fibers are selected as conductive components to avoid the risk of heavy metal or carbon nanotube residues; furthermore, polydopamine (PDA) forms a covalent-non-covalent synergistic interface on the surface of the silk fibroin fiber, the quinone group reacts with the amino group of the silk fibroin fiber to form an imine bond, the hydrophobic domain combines with the silk beta-sheet hydrophobic region (alanine / glycine-rich region), and PPy is firmly grafted with PDA through π-π stacking and hydrogen bonding, further solving the problem of uneven distribution of traditional physical doping PPy particles.

[0019] The preparation method of the present application first uses polydopamine (PDA) to firmly adhere to the surface of the silk fibroin fiber through the synergistic effect of covalent bonds and non-covalent interactions to form an activation layer; then, in the presence of ammonium persulfate, conductive polymer polypyrrole (PPy) is in-situ polymerized on the activation layer to obtain conductive silk fibroin fibers. Subsequently, the conductive silk fibroin fibers, a photoinitiator, and a methacrylated gelatin (GelMA) solution are uniformly mixed by physical blending, and a methacrylated gelatin hydrogel scaffold loaded with conductive silk fibroin is obtained by ultraviolet light cross-linking curing.

[0020] The preparation method can adjust the pore size, mechanical property and degradation period of the three-dimensional porous hydrogel by controlling the content of the conductive silk fibroin fiber; the hydrogel formed thereby not only exhibits excellent mechanical property, conductivity, biocompatibility and safety, but also can support adhesion and proliferation of various cells, and thus can be used for repairing and regenerating electrically active tissues such as skeletal muscles, and has a wide application prospect in the fields of biomedical materials and regenerative medicine. In addition, the method is simple in operation, mild in conditions, and the solution can be quickly gelled without organic solvents and toxic cross-linking agents, and has good biocompatibility and degradability. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to make the content of the present application more easily and clearly understood, the present application will be further described in detail below according to specific embodiments of the present application and in conjunction with the drawings, in which: In order to make the content of the present application more easily and clearly understood, the present application will be further described in detail below according to specific embodiments of the present application and in conjunction with the drawings, in which: Figure 1 It is a table scanning electron microscope image (the scale is 10 μm) of SFF, PDA-SFF and PPy-PDA-SFF in Test Example 1 of the present application; Figure 2 It is an infrared spectrum of SFF, PDA-SFF and PPy-PDA-SFF in Test Example 2 of the present application; Figure 3 It is the conductivity of the methacrylated gelatin hydrogel prepared by different adding amounts of conductive silk fibroin fibers in Test Example 4 of the present application; Figure 4 It is a stress-strain curve of the methacrylated gelatin hydrogel prepared by different adding amounts of conductive silk fibroin fibers in Test Example 4 of the present application; Figure 5 It is a table scanning electron microscope image (the scale is 100 μm) of the methacrylated gelatin hydrogel prepared by different adding amounts of conductive silk fibroin fibers in Test Example 4 of the present application; Figure 6 It is a broken line graph of the swelling rate of the methacrylated gelatin hydrogel prepared by different adding amounts of conductive silk fibroin fibers in Test Example 4 of the present application; Figure 7 It is a broken line graph of the residual mass percentage of the methacrylated gelatin hydrogel prepared by different adding amounts of conductive silk fibroin fibers in Test Example 4 of the present application; Figure 8 It is a cell proliferation staining image (the scale is 100 μm) of the myoblast cells cultured by the methacrylated gelatin hydrogel prepared by different adding amounts of conductive silk fibroin fibers in Test Example 5 of the present application; Figure 9 Cell proliferation CCK-8 results of myoblast cells cultured in the methacrylated gelatin hydrogels prepared from the electrically conductive silk fibroin fibers with different addition amounts in Example 5 of the present application. DETAILED DESCRIPTION

[0022] The present application will be further described below in conjunction with the drawings and specific examples so that those skilled in the art can better understand the present application and implement it. However, the examples are not intended to limit the present application.

[0023] In the present application, unless otherwise specified, the preparation of the methacrylated gelatin (GelMA) used in the examples of the present application includes the following steps: dissolving 10 g of gelatin powder, swelling at room temperature for 1 h, then stirring in a constant temperature water bath at 60℃ until completely dissolved; then adding 8 mL of methacrylic anhydride dropwise into the gelatin solution at a rate of 1 mL / min, and stirring at high speed for 2 h at 50℃; after the stirring is completed, adding 200 mL of deionized water at 50℃ to dilute the reaction solution, and performing dialysis treatment on the solution for 6 days at room temperature; after the dialysis is completed, centrifuging the solution at a speed of 4000 rpm for 8 min, freezing and drying the supernatant to obtain the methacrylated gelatin (GelMA).

[0024] Example 1

[0025] The electrically conductive silk fibroin-loaded methacrylated gelatin hydrogel of the present embodiment and the preparation method thereof specifically include the following steps: S1, Preparation of polydopamine-coated silk fibroin fibers S11, Preparation of silk fibroin fibers: silk is placed in a 0.05wt% Na2CO3 aqueous solution and boiled for 30 min, washed thoroughly with deionized water, repeated three times, and then placed in a 60℃ oven for drying for 24 h to obtain silk fibroin fibers (SFF); S12, Preparation of dopamine hydrochloride solution: 2.285 g of dopamine hydrochloride is weighed and dissolved in a hydrochloric acid buffer to prepare a dopamine hydrochloride solution with a concentration of 0.05 mol / L and a pH of 8.5; S13, Preparation of polydopamine-coated silk fibroin fibers: 1 g of silk fibroin is added to 30 mL of dopamine hydrochloride solution, stirred at room temperature for 10 h, and then left to stand for 12 h; 15 mL of the solution is poured out, 15 mL of deionized water is added, 0.07 g of sodium hydroxide is added, heated at 70℃ for 3 h, and ultrasonically cleaned for 3 min to obtain polydopamine-coated silk fibroin fibers (PDA-SFF); S2, Preparation of electrically conductive silk fibroin fibers First, the polydopamine interfaced silk fibroin fiber is immersed in 1 mol / L ammonium persulfate solution for 90 min, so that the oxidant fully penetrates into the silk fibroin fibers and the surface, and then immersed in 3% pyrrole solution for 24 h, and the pyrrole monomer is adsorbed on the oxidant on the surface of the silk fibroin fiber, realizing in-situ uniform polymerization, forming a tightly combined PPy coating, and after drying, the conductive silk fibroin fiber (PPy-PDA-SFF) is obtained; S3, Preparation of methacrylated gelatin hydrogel loaded with conductive silk fibroin S31, Preparation of methacrylated gelatin solution: methacrylated gelatin (GelMA) is added to a photoinitiator Irgacure 2959 dissolved in a phosphate buffer solution to obtain a methacrylated gelatin solution; wherein the concentration of GelMA in the methacrylated gelatin solution is 10% (w / v), and the concentration of the photoinitiator is 1.5% (w / v); S32, Preparation of methacrylated gelatin hydrogel loaded with conductive silk fibroin: the conductive silk fibroin fiber is broken by liquid nitrogen, sieved, and then added to the methacrylated gelatin solution and mixed uniformly (the concentration of the conductive silk fibroin fiber in the solution is 0 mg / mL, 1 mg / mL, 2 mg / mL, and 3 mg / mL, respectively), and then cross-linked and cured under ultraviolet light of 365 nm for 15 min, and then soaked in a glycerol solution (the oil-water volume ratio is 7:3) for 24 h to obtain the methacrylated gelatin hydrogel loaded with conductive silk fibroin.

[0026] Test Example 1

[0027] Based on Example 1, the silk fibroin fiber (SFF), the polydopamine interfaced silk fibroin fiber (PDA-SFF), and the conductive silk fibroin fiber (PPy-PDA-SFF) are pasted on the electron microscope sample table, and the sample table is subjected to 120 s of gold spraying treatment in a vacuum environment, and the cross-sectional morphology is observed by a desktop scanning electron microscope, and the results are shown in Figure 1 It can be seen from Figure 1 that the surface of the SFF is relatively smooth, the surface of the PDA-SFF appears uneven coating, which is the amorphous aggregation formed by the random polymerization of PDA, and the surface of the PPy-PDA-SFF presents a more rough granular and fibrous network structure, which is the random growth structure during PPy polymerization. It is preliminarily proved that the polydopamine and polypyrrole are successfully grafted on the silk fibroin fiber.

[0028] Test Example 2

[0029] Based on Example 1, the samples of the silk fibroin fiber (SFF), the polydopamine interfaced silk fibroin fiber (PDA-SFF), and the conductive silk fibroin fiber (PPy-PDA-SFF) are cut and ground into tiny particles, which are respectively filled into a total reflection test platform, and the total reflection loss of the particles is measured at a wavelength of 500 cm-1 -4000cm -1 The scanning was carried out within the wave number range of 100 nm at a scanning rate of 80 spectra / s. Figure 2 The infrared spectrum shown in Figure 2 is as follows. Figure 2 It can be seen that at 1622cm -1 Imine C=N stretching appears at 1170 cm -1 , 3280cm -1 , 3070cm -1 The absorption peaks at 1172 cm-1 are the aromatic CH bending vibration and CN stretching vibration peaks, -OH stretching vibration and symmetrical NH stretching vibration, indicating that PDA was successfully grafted. -1 CN appears + Stretching vibration, which is the characteristic of PPy doping state, 1550cm -1 The appearance of C=C and the symmetrical contraction of the conjugated double bonds of the pyrrole ring indicate that PPy was successfully grafted.

[0030] Test Example 3

[0031] The resistance of the conductive silk protein fiber was measured with a multimeter, and the conductivity was calculated. The conductivity calculation formula is: σ = L / (R × A), where L is the conductor length (m), R is the resistance (Ω), and A is the cross-sectional area (m). 2 ); The conductivity of the conductive silk protein fiber was measured to be 9.91×10 -3 S / m.

[0032] Test Example 4

[0033] Based on Example 1, the conductivity, compression mechanics, and other tests were performed on methacrylated gelatin hydrogels prepared with different amounts of conductive silk fibroin fibers (mg / mL). (1) Conductivity (S / m): The hydrogel was sandwiched between two copper electrodes with a diameter of 1 cm. The resistance value was measured with a multimeter. Each sample was tested three times and the conductivity was calculated according to the formula; the conductivity calculation formula is σ = L / (R × A) , where L is the conductor length (m), R is the resistance (Ω), and A is the cross-sectional area (m). 2 ); (2) Maximum compressive strength (kPa) and maximum compressive strain (%): The hydrogel was made into a cylindrical structure with a bottom diameter of 14 mm and a height of 12 mm, and a universal material testing machine was used to evaluate the compression performance of the material, with a compression speed of 2 mm / min, compression to the breaking of the hydrogel, and the load displacement obtained by the test was converted into stress and strain according to the formula to obtain the maximum compression strength and the maximum compression strain; the stress calculation formula is σ=F / S, wherein: σ is the compression stress (Pa), F is the compression load applied on the hydrogel (N), S is the cross-sectional area (m 2 ); the strain calculation formula is ε= (L0-L) / L0; in the formula: ε is the compression strain, L0 is the original height (m), and L is the actual height of the sample when the maximum compression deformation is reached (m); (3) Pore size (μm): The hydrogel was frozen and broken with liquid nitrogen and freeze-dried to retain the original pore structure, and the cross-sectional morphology was observed by a table electron scanning microscope. Five regions were selected on the scanning electron microscope photos of each sample, 10 pores in each region were selected, the diameter of the pores was measured using the straight line tool of Image J software, and the average value was obtained by repeating 3 times in each region; (4) Porosity (%): The hydrogel was frozen and broken with liquid nitrogen and freeze-dried to retain the original pore structure, and the cross-sectional morphology was observed by a table electron scanning microscope. Five regions were selected on the scanning electron microscope photos of each sample, the scale was set according to the hydrogel electron microscope photos, the circular extraction tool of Image J software was used to calculate the proportion of the area of each pore to the area of the selected region to obtain the porosity, and the average value was obtained by repeating 3 times in each region; (5) Swelling rate (%): The hydrogel was made into a cylinder with a bottom diameter of 14 mm and a height of 12 mm, and after freeze-drying, its weight was measured, then it was soaked in PBS solution (pH=7.4) to absorb water and swell, and was placed in a 37℃ water bath oscillator with a shaking speed of 100 rpm. It was taken out at intervals, the surface water was wiped off gently, the wet weight was measured, and the swelling rate of the hydrogel was calculated; the swelling rate calculation formula is SR=(M2-M1) / M1x100%, wherein: SR is the swelling rate, M1 is the weight of the freeze-dried hydrogel (g), and M2 is the wet weight of the water-swollen hydrogel (g); (6) Residual mass percentage (%): freeze-dry the hydrogel, place it in a phosphate buffer solution (pH = 7.4) with a solid-liquid ratio of 1:50, and place the degradation system in a 37℃ constant temperature water bath at a speed of 100 rpm; at 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, and 8 weeks, respectively, take out the sample for freeze-drying treatment, weigh the mass of the dried gel before degradation and the mass after degradation, and calculate the residual mass percentage of the hydrogel; the residual mass percentage calculation formula is MR = M2 / M1 x 100%, wherein: MR is the residual mass percentage, M2 is the mass after degradation (g), and M1 is the initial mass (g).

[0034] Figures 3-7 and Table 1 shows the relevant test results: Table 1

[0035] Figure 3 And from Table 1, it can be seen that as the content of conductive silk fibroin fibers increases, the conductivity of the hydrogel gradually increases. The electrical conductivity of human native skeletal muscle ranges from 8.0 x 10 -4 -4.5 x 10 -3 S / m, and the conductivity of the implant should be in the range of 0.1-10 times that of muscle tissue (i.e. a difference of ±1 order of magnitude), which can effectively reconstruct the physiology of damaged tissue, and a higher conductivity is more conducive to electrical physiological function adaptability and cell behavior regulation, thereby promoting skeletal muscle repair. The hydrogel with a content of 2 mg / mL and 3 mg / mL of conductive silk fibroin fibers has a suitable conductivity and potential to promote skeletal muscle repair.

[0036] Figure 4 And from Table 1, it can be seen that the compressive stress of the hydrogel in different groups is about 46 kPa-83 kPa, the local compressive stress of skeletal muscle in a resting state is about 10 kPa-50 kPa, and the peak load of daily activities (such as walking) is ≤80 kPa. The strength range of the hydrogel of the present application is within this interval, which can effectively bear physiological load and avoid structural collapse after implantation. Within the above range, increasing the content of conductive silk fibroin fibers can enhance the mechanical properties of the hydrogel, because the amine group / carboxyl group of the GelMA molecular chain forms a hydrogen bond with the polar group (-OH, -NH2) on the surface of the silk fibroin fiber, and the hydrophobicity of PPy and the hydrophobic region of GelMA produce van der Waals force, further enhancing the interfacial bonding force. When the content of conductive silk fibroin fibers reaches 3 mg / mL, the mechanical properties of the hydrogel decrease, because too much silk fibroin fiber will hinder the photo-crosslinking of the GelMA molecular chain, reducing the crosslinking density and thus leading to a decrease in mechanical properties; in addition, within the above required range for skeletal muscle repair, the hydrogel with a content of 2 mg / mL of conductive silk fibroin fibers has the best mechanical properties.

[0037] Figure 5 As can be seen from Table 1, as the conductive silk fibroin fiber content increases, the pore size and porosity of the hydrogel increase. This is because the conductive silk fibroin fibers are embedded in the gaps between the GelMA network, occupying the original pore space, causing the network to break and form micropores, resulting in an increase in the pore size and porosity of the hydrogel, thereby providing space and pathways for cell migration, blood vessel ingrowth, and metabolic waste discharge. The ideal hydrogel pore size range for cell migration and infiltration is 50μm-120μm, while the capillary network construction requires 30μm-100μm (skeletal muscle cells have a diameter of 50μm-80μm, the long axis of Schwann cells is 20μm-40μm, and the lumen of endothelial cells is 15μm-50μm). The diffusion of metabolic waste requires a porosity of >60%. The pore size and porosity of the hydrogel of the present invention are within the appropriate range and can be controlled according to the conductive silk fibroin fiber content. Hydrogels with conductive silk fibroin fiber contents of 2mg / mL and 3mg / mL have more suitable pore sizes and porosities.

[0038] Figure 6 As can be seen in Table 1, the hydrogel swelling rate increases slightly with increasing conductive silk fibroin fiber content. This is because the conductive silk fibroin fibers adsorb GelMA molecular chains, reducing their effective concentration for photocrosslinking and lowering the crosslinking density, which in turn increases the pore size and porosity, and increases the hydrogel swelling rate. The hydrogel scaffolds used for skeletal muscle repair have suitable low to medium swelling rates (50%-200%). The swelling rates of the four hydrogel groups ranged from 110% to 125%, which facilitates the repair of load-bearing tissues and avoids compression of blood vessels and nerves.

[0039] Figure 7 As can be seen from Table 1, the degradation process of the hydrogel presents a staged characteristic; in the early stage (0-2 weeks), it can maintain a high structural integrity with a degradation rate of less than 20%, which can support cell migration and proliferation; in the middle stage (2-6 weeks), it gradually degrades to make room for new muscle fibers; in the late stage (>6 weeks), most of it is degraded, which helps to avoid long-term foreign body reactions. As the content of conductive silk protein fibers in the hydrogel increased, the degradation rate of the hydrogel increased. The remaining mass percentages of GelMA hydrogel and hydrogels with conductive silk protein fiber contents of 1 mg / mL, 2 mg / mL, and 3 mg / mL at week 8 were 70.92±2.83%, 63.05±2.57%, 50.14±2.02%, and 41.77±2.05%, respectively. The degradation rate in vitro is usually 1.3-1.5 times slower than that in vivo (due to the lack of enzyme / cell action). The degradation rate should reach 50%-80% at week 8 (corresponding to the newborn muscle fibers taking over the mechanical load and the scaffold gradually withdrawing), indicating that the degradation of hydrogels with conductive silk protein fiber contents of 2 mg / mL and 3 mg / mL met the triple requirements of mechanical support-pore conduction-degradation timing.

[0040] In summary, when the addition amount of the conductive silk fibroin fiber is 2 mg / mL, the prepared methacrylated gelatin hydrogel can balance the mechanical property and the electrical conductivity, has a suitable degradation rate, and can meet the required conditions for supporting cell growth.

[0041] Test Example 5

[0042] Based on Example 1, the cell compatibility of the methacrylated gelatin hydrogel prepared by adding different amounts of conductive silk fibroin fiber (mg / mL) in the control group was characterized, and the test steps were as follows: According to the standard of GB / T 16886.5-2016 "GB / T 16886.5-2016 Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Test", the hydrogel was first subjected to ethanol gradient sterilization, and then immersed in serum-free high-glucose DMEM medium (extraction ratio 0.1 mg / mL) at 37°C in a sterile environment. After 24±2h of extraction at 37°C, 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin were added to prepare the extraction medium for subsequent cytotoxicity evaluation.

[0043] According to the standard of GB / T 16886.5-2017, the extraction medium was used for the culture test of myoblast cells: cells were inoculated in a 96-well plate (1×10³ cells / well), and after the cells were completely adhered, the extraction medium was replaced (experimental group). At the same time, a control group (complete medium without adding materials) and a blank group (containing only CCK-8 working solution) were set up; after 1, 3, and 5 days of culture, the culture medium was discarded, 100μL of Calcein-AM / PI diluted 1000 times with PBS was added to each well, and incubated at room temperature for 30min in the dark; after incubation, the dye solution was aspirated, and PBS was used to wash and remove the unbound dye solution. An inverted fluorescence microscope was used to observe and record the cells in the same field of view and collect images. Figure 8 ).

[0044] Cells were inoculated in a 96-well plate (1×10³ cells / well), and after the cells were completely adhered, the extraction medium was replaced (experimental group); at the same time, a control group (complete medium without adding materials) and a blank group (containing only CCK-8 working solution) were set up; after 1, 3, and 5 days of culture, the culture medium was discarded, 100μL of DMEM basic medium containing 10% CCK-8 was added to each well, and incubated in the incubator for 1h; after incubation, 90μL was aspirated from each well to a new 96-well plate, the absorbance at 450nm wavelength was detected by a microplate reader, and the absorbance was calculated; the absorbance calculation formula is OD value=OD experimental group-OD blank group, wherein: OD value is the absorbance value, OD experimental group is the absorbance value of the experimental group, and OD blank group is the absorbance value of the blank group; and statistical analysis was performed Figure 9).

[0045] From Figures 8-9 It can be seen that the methylacrylated gelatin hydrogels prepared by different amounts of conductive silk fibroin fibers have no cytotoxicity; among them, the hydrogels with conductive silk fibroin fiber content of 2 mg / mL and 3 mg / mL can significantly promote the proliferation of myoblasts within 5 days. The number of living cells increases, the metabolic activity is enhanced, and the colored product generated increases, resulting in an increase in OD value, so the OD value is positively correlated with the number of living cells, which can indirectly reflect the cell proliferation situation.

[0046] Obviously, the above examples are only examples for clearly illustrating, and are not limitation to the embodiments. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, it is not necessary and also impossible to enumerate all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A method for preparing a methacrylated gelatin hydrogel loaded with conductive silk fibroin, characterized in that: The following steps are involved: S1. Adding silk fibroin fibers to a dopamine hydrochloride solution, stirring and then allowing to stand, then adjusting the pH to 8-9 and heating to react, to obtain polydopamine-linked silk fibroin fibers; S2. Sequentially immersing the polydopamine-connected silk fibroin fiber described in S1 in an ammonium persulfate solution and a pyrrole solution to obtain a conductive silk fibroin fiber; S3. Add the conductive silk fibroin fiber described in S2 to a methacrylated gelatin solution, crosslink and cure with ultraviolet light, and then soak in a glycerol solution to obtain the methacrylated gelatin hydrogel loaded with conductive silk fibroin; the methacrylated gelatin solution is prepared by dissolving methacrylated gelatin and a photoinitiator in a phosphate buffer solution.

2. The method for preparing the methacrylated gelatin hydrogel loaded with conductive silk fibroin according to claim 1, characterized in that: In S1, the concentration of the dopamine hydrochloride solution is 0.01 mol / L-0.06 mol / L, and the pH is 8-9; The mass ratio of dopamine hydrochloride to silk fibroin fiber in the dopamine hydrochloride solution is (2-2.5):

1.

3. The method for preparing the methacrylated gelatin hydrogel loaded with conductive silk fibroin according to claim 1, characterized in that: In S1, the heating reaction temperature is 65°C-75°C, and the time is 2.5h-3.5h.

4. The method for preparing the methacrylated gelatin hydrogel loaded with conductive silk fibroin according to claim 1, characterized in that: In S2, the concentration of the ammonium persulfate solution is 0.05 mol / L-1.5 mol / L; The immersion time in the ammonium persulfate solution is 30 min-120 min.

5. The method for preparing the methacrylated gelatin hydrogel loaded with conductive silk fibroin according to claim 1, characterized in that: In S2, the mass fraction of the pyrrole solution is 1%-5%; The immersion time in the pyrrole solution is 12h-30h.

6. The method for preparing the methacrylated gelatin hydrogel loaded with conductive silk fibroin according to claim 1, characterized in that: In S3, the concentration of the conductive silk fibroin fibers in the solution is 1 mg / mL-3 mg / mL.

7. The method for preparing the methacrylated gelatin hydrogel loaded with conductive silk fibroin according to claim 1, characterized in that: In S3, the photoinitiator is Irgacure 2959; The concentration of methacrylated gelatin in the methacrylated gelatin solution is 9% w / v-11% w / v, and the concentration of the photoinitiator is 1.2% w / v-1.8% w / v.

8. The method for preparing the methacrylated gelatin hydrogel loaded with conductive silk fibroin according to claim 1, characterized in that: In S3, the wavelength of the ultraviolet light cross-linking curing is 365 nm, and the time is 10 min-30 min.

9. The method for preparing the methacrylated gelatin hydrogel loaded with conductive silk fibroin according to claim 1, characterized in that: In S3, the glycerol solution is obtained by mixing glycerol and water in a volume ratio of (6.5-7.5):3; The soaking time in the glycerol solution is 12h-36h. 10 . The methacrylated gelatin hydrogel loaded with conductive silk fibroin prepared by the method according to claim 1 .

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