A methacrylated gelatin hydrogel loaded with electrically conductive silk fibroin and a method of making the same

Abstract

The present application relates to a kind of methyl methacrylated gelatin hydrogel of electrically conductive silk fibroin and its preparation method, belong to biological medical material technical field.The preparation method of the present application includes the following steps: S1, to dopamine hydrochloride solution is added silk fibroin fiber, after stirring, then adjust pH to 8-9 and carry out heating reaction, obtain polydopamine silk fibroin fiber;S2, polydopamine silk fibroin fiber is immersed in ammonium persulfate solution and pyrrole solution in turn, obtain electrically conductive silk fibroin fiber;S3, electrically conductive silk fibroin fiber is added to methyl methacrylated gelatin solution, after ultraviolet light crosslinking solidification, it is immersed in glycerol solution, obtain methyl methacrylated gelatin hydrogel of electrically conductive silk fibroin loading.The method can not only ensure the structural integrity and electrically conductive continuity of electrically conductive network in hydrogel, also break through the dependence of in-situ polymerization on matrix permeability.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and particularly relates to a methacrylamide gel hydrogel loaded with conductive silk fibroin and its preparation method. Background Technology

[0002] Conductive hydrogels, as a novel type of hydrogel with both conductivity and chemical conductivity, have shown significant potential in the biomedical field, particularly in electroactive tissue regeneration, bioelectronic interfaces, and wound healing monitoring. Their advantages lie in providing the 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 strategies of conductive polymer hydrogels, the introduction of conductive fillers such as carbon nanotubes and metal nanowires can improve conductivity; however, these fillers have potential biotoxicity and dispersion problems are difficult to overcome. While the conductive polymer polypyrrole (PPy) possesses conductivity and biosafety, it suffers from inherent defects such as brittleness and difficulty in processing. Furthermore, current technologies often rely on physical doping of PPy particles, which often leads to uneven filler distribution, easy loss, and insufficient mechanical properties due to weak interfacial bonding between the filler and the matrix, severely affecting the long-term conductive stability of the material. Therefore, the development of composite hydrogel scaffolds with excellent biocompatibility, high conductivity, and tissue mechanical properties is urgently needed.

[0003] Invention patent CN 109575318 A discloses a dopamine-mediated polypyrrole conductive hydrogel and its preparation method. Although the dopamine-mediated polypyrrole conductive hydrogel can improve the conductivity, the pyrrole polymerization needs to be carried out at a low temperature of 0℃-10℃ for 18h-36h, which has the problems of high energy consumption and long cycle. Moreover, the immersion method used tends to cause the pyrrole monomer to preferentially polymerize 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 conductive hydrogel based on a chitosan / xanthan gum interpenetrating network and its preparation method. Although the chitosan / xanthan gum interpenetrating network conductive hydrogel scaffold has good biocompatibility, the PEDOT-HA nanoparticles are prone to aggregation, resulting in a low conductivity (only about 4.1 × 10⁻⁶). -5 (S / m), and its mechanical strength performance in the target application area is insufficient. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a methacrylamide gelatin hydrogel loaded with conductive silk fibroin and its preparation method. Through dopamine-assisted interfacial polymerization, polypyrrole (PPy) is firmly bonded to the surface of silk fibroin fibers (SFF) via covalent and non-covalent synergistic effects, forming conductive silk fibroin fibers. These fibers are then dispersed in a methacrylamide gelatin (GelMA) matrix, achieving a three-dimensional uniform distribution that enhances the mechanical properties of the scaffold and effectively transmits electrical signals. This covalent grafting strategy also fully utilizes the inherent high biocompatibility, biodegradability, and low immunogenicity of silk fibroin fibers, avoiding the potential cytotoxic risks associated with the leaching of physically doped fillers. This technology not only ensures the structural integrity and conductive continuity of the conductive network within the hydrogel but also overcomes the dependence of in-situ polymerization on matrix permeability.

[0006] The first objective of this invention is to provide a method for preparing a methacrylamide gelatin hydrogel loaded with conductive silk fibroin, comprising the following steps:

[0007] S1. Add silk fibroin fiber to the hydrochloric acid dopamine solution, stir and let stand, then adjust the pH to 8-9 and heat to react, to obtain polydopamine-linked silk fibroin fiber.

[0008] S2. The polydopamine fibroin fiber described in S1 is sequentially immersed in ammonium persulfate solution and pyrrole solution to obtain conductive fibroin fiber.

[0009] S3. The conductive silk fibroin fibers described in S2 are added to a methacrylamide gelatin solution, crosslinked and cured under ultraviolet light, and then immersed in a glycerol solution to obtain the methacrylamide gelatin hydrogel loaded with conductive silk fibroin; the methacrylamide gelatin solution is prepared by dissolving methacrylamide gelatin and a photoinitiator in a phosphate buffer solution.

[0010] In one embodiment of the present invention, 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 the key interface engineering molecule, which forms a polydopamine transition layer on the surface of silk fibroin fibers through oxidative self-polymerization, thereby initiating the directional grafting polymerization of pyrrole; in addition, by grafting polydopamine onto the surface of silk fibroin fibers through chemical methods, subsequent conductive-mechanical dual-function synergy can be achieved;

[0011] The mass ratio of dopamine hydrochloride to silk fibroin fiber in the dopamine hydrochloride solution is (2-2.5):1.

[0012] In one embodiment of the present invention, in S1, the temperature of the heating reaction is 65°C-75°C and the time is 2.5h-3.5h.

[0013] In one embodiment of the invention, after S1, an ultrasonic cleaning step is further included to wash away loosely bonded dopamine agglomerates on the surface.

[0014] In one embodiment of the present invention, during S1, the alkaline environment during the grafting of polydopamine onto the surface of silk fibroin fibers not only promotes the oxidation of the catechol groups in the dopamine molecules to quinone structures by dissolved oxygen in the solution, but also further promotes the polymerization reaction of the active quinone intermediates generated after oxidation. Simultaneously, this alkaline environment synergistically works 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.

[0015] 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. Using ammonium persulfate as an oxidant benefits from its good water solubility (58.2 mg / 100 mL water), and the decomposition products do not affect the properties of silk, thus meeting application requirements. In contrast, although potassium persulfate has a smaller impact on silk fibroin fibers, its low solubility (only 1.75 mg / 100 mL water) is not conducive to its full effect. While ferric chloride is widely used in pyrrole polymerization, its strong acidity (pH≈2) can damage silk fibroin fibers, and the residual Fe ions may also affect biocompatibility. Furthermore, ammonium persulfate, as an oxidant, can provide a sufficiently high oxidation potential (higher than the pyrrole oxidation potential) when preparing a polypyrrole (PPy) coating on the surface of silk fibroin fibers, thereby triggering the oxidative polymerization reaction of pyrrole monomers.

[0016] The soaking time in ammonium persulfate solution is 30-120 minutes, so that the ammonium persulfate can fully penetrate into the silk fibroin fibers and the surface.

[0017] In one embodiment of the present invention, in S2, the mass fraction of the pyrrole solution is 1%-5%;

[0018] The immersion time in the pyrrole solution is 12h-30h; the pyrrole monomer can achieve in-situ uniform polymerization by contacting and adsorbing ammonium persulfate on the surface of silk fibroin fibers, thereby forming a tightly bonded polypyrrole (PPy) coating on the surface of silk fibroin fibers.

[0019] In one embodiment of the present invention, in S3, the concentration of the conductive silk fibroin fiber in the solution is 1 mg / mL-3 mg / mL.

[0020] In one embodiment of the present invention, in S3, the photoinitiator is Irgacure 2959;

[0021] The concentration of methacrylamide gelatin in the methacrylamide gelatin solution is 9% w / v-11% w / v, and the concentration of photoinitiator is 1.2% w / v-1.8% w / v.

[0022] In one embodiment of the present invention, in S3, the wavelength of the ultraviolet crosslinking curing is 365 nm and the time is 10 min to 30 min.

[0023] In one embodiment of the present invention, in S3, the glycerol solution is obtained by mixing glycerol and water in a volume ratio of (6.5-7.5):3;

[0024] Soaking time in glycerol solution is 12h-36h.

[0025] A second objective of this invention is to provide a methacrylamide gelatin hydrogel loaded with conductive silk fibroin prepared by the method.

[0026] The technical solution of the present invention has the following advantages compared with the prior art:

[0027] The hydrogel described in this invention combines rigid conductive silk fibroin fibers (PPy-PDA-SFF) with a flexible methacrylamide gelatin (GelMA) network. The synergistic effect of covalent and non-covalent forces strengthens the interfacial bonding, significantly improving the mechanical strength and toughness of the hydrogel. This effectively overcomes the mechanical defects caused by weak interfacial bonding in physically doped systems, making its mechanical properties more suitable for the needs of biological tissues. Simultaneously, PPy is firmly grafted onto the silk fibroin fiber framework through covalent and hydrogen bonds, avoiding the problems of conductive filler aggregation and uneven distribution caused by physical doping. This ensures the uniformity and long-term stability of the conductive network. In addition, biodegradable 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 silk fibroin fibers. Its quinone groups react with the amino groups of silk fibroin fibers to form imine bonds, and the hydrophobic water area combines with the hydrophobic region of the β-sheet of silk fibroin (alanine / glycine enrichment region). PPy is firmly grafted with PDA through π-π stacking and hydrogen bonding, further solving the problem of uneven distribution of PPy particles in traditional physical doping.

[0028] The preparation method described in this invention first utilizes polydopamine (PDA) to firmly adhere to the surface of silk fibroin fibers through the synergistic effect of covalent and non-covalent interactions, forming an activation layer. Subsequently, in the presence of ammonium persulfate, conductive polymer polypyrrole (PPy) is polymerized in situ on this activation layer to obtain conductive silk fibroin fibers. Then, using a physical blending method, the conductive silk fibroin fibers, a photoinitiator, and a methacrylamide gelatin (GelMA) solution are uniformly mixed, and crosslinked and cured under ultraviolet light to obtain a methacrylamide gelatin hydrogel scaffold loaded with conductive silk fibroin.

[0029] The preparation method described in this invention allows for the adjustment of the pore size, mechanical properties, and degradation cycle of a three-dimensional porous hydrogel by controlling the content of conductive silk fibroin fibers. The resulting hydrogel not only exhibits excellent mechanical properties, conductivity, biocompatibility, and safety, but also supports the adhesion and proliferation of various cells. Therefore, it can be used for the repair and regeneration of electrically active tissues such as skeletal muscle, showing broad application prospects in the fields of biomedical materials and regenerative medicine. Furthermore, this method is simple to operate, operates under mild conditions, allows for rapid gelation of the solution, and requires no organic solvents or toxic cross-linking agents, while also possessing good biocompatibility and degradability. Attached Figure Description

[0030] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0031] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0032] Figure 1 The tabletop scanning electron microscope images (scale bar is 10 μm) of SFF, PDA-SFF and PPy-PDA-SFF in Test Example 1 of the present invention.

[0033] Figure 2 The infrared spectra of SFF, PDA-SFF and PPy-PDA-SFF in Test Example 2 of this invention are shown below.

[0034] Figure 3 The conductivity of methacrylamide gelatin hydrogels prepared with different amounts of conductive silk fibroin fibers in Test Example 4 of this invention is measured.

[0035] Figure 4 Stress-strain curves of methacrylamide gelatin hydrogels prepared with different amounts of conductive silk fibroin fibers in Test Example 4 of this invention.

[0036] Figure 5Tabletop scanning electron microscope images (scale bar is 100 μm) of methacrylamide gelatin hydrogels prepared from conductive silk fibroin fibers with different addition amounts in Test Example 4 of the present invention.

[0037] Figure 6 Line graph showing the swelling rate of methacrylamide gelatin hydrogels prepared with different amounts of conductive silk fibroin fibers in Test Example 4 of this invention as a function of time.

[0038] Figure 7 Line graph showing the change of the residual mass percentage of methacrylamide gelatin hydrogels prepared with different amounts of conductive silk fibroin fibers in Test Example 4 of the present invention over time.

[0039] Figure 8 Cell proliferation staining images of myocytes cultured from methacrylamide gelatin hydrogels prepared with different amounts of conductive silk fibroin fibers in Test Example 5 of this invention (scale bar is 100 μm).

[0040] Figure 9 The results of CCK-8 cell proliferation of myocytes cultured from methacrylamide gelatin hydrogels prepared with different amounts of conductive silk fibroin fibers in Test Example 5 of this invention are shown. Detailed Implementation

[0041] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0042] In this invention, unless otherwise stated, the preparation of methacrylamide gelatin (GelMA) used in the embodiments of this invention includes the following steps: dissolving 10g of gelatin powder, swelling at room temperature for 1h, then stirring in a constant temperature water bath at 60℃ until completely dissolved; subsequently, adding 8mL of methacrylic anhydride dropwise to the gelatin solution at a rate of 1mL / min, and stirring at high speed at 50℃ for 2h; after stirring, adding 200mL of deionized water at 50℃ to dilute the reaction solution, and dialysis the solution for 6 days at room temperature; after dialysis, centrifuging the solution at 4000rpm for 8min, and freeze-drying the supernatant to obtain methacrylamide gelatin (GelMA).

[0043] Example 1

[0044] The methacrylamide gel loaded with conductive silk fibroin and its preparation method in this embodiment specifically include the following steps:

[0045] S1. Preparation of polydopamine splicing fibroin fibers

[0046] S11. Preparation of silk fibroin fiber: The silk was boiled in a 0.05wt% Na2CO3 aqueous solution for 30 minutes, washed thoroughly with deionized water, and repeated three times. Then it was dried in a 60℃ oven for 24 hours to obtain silk fibroin fiber (SFF).

[0047] S12. Preparation of dopamine hydrochloride solution: Weigh 2.285g of dopamine hydrochloride and dissolve it completely in hydrochloric acid buffer to prepare a dopamine hydrochloride solution with a concentration of 0.05mol / L and a pH of 8.5.

[0048] S13. Preparation of polydopamine-based fibroin fiber: Add 1g of fibroin fiber to 30mL of dopamine hydrochloride solution, stir at room temperature for 10h and let stand for 12h; discard 15mL of solution, add 15mL of deionized water, add 0.07g of sodium hydroxide, heat at 70℃ for 3h, and ultrasonically clean for 3min to obtain polydopamine-based fibroin fiber (PDA-SFF).

[0049] S2. Preparation of conductive silk fibroin fibers

[0050] First, polydopamine-bonded silk fibroin fibers are immersed in a 1 mol / L ammonium persulfate solution for 90 minutes to allow the oxidant to fully penetrate between and onto the surface of the silk fibroin fibers. Then, they are immersed in a 3% pyrrole solution for 24 hours. The pyrrole monomers contact the oxidant adsorbed on the silk fibroin fibers, achieving in-situ uniform polymerization and forming a tightly bonded PPy coating. After drying, conductive silk fibroin fibers (PPy-PDA-SFF) are obtained.

[0051] S3. Preparation of methacrylamide gelatin hydrogel loaded with conductive silk fibroin

[0052] S31. Preparation of methacrylamide gelatin solution: Methacrylamide gelatin (GelMA) was added to the photoinitiator Irgacure 2959 and dissolved in a phosphate buffer solution to obtain a methacrylamide gelatin solution; wherein, the concentration of GelMA in the methacrylamide gelatin solution was 10% (w / v) and the concentration of the photoinitiator was 1.5% (w / v).

[0053] S32. Preparation of methacrylamide gelatin hydrogel loaded with conductive silk fibroin: Conductive silk fibroin fibers were brittled with liquid nitrogen, sieved, and then added to a methacrylamide gelatin solution and mixed evenly (the concentrations of conductive silk fibroin fibers in the solution were 0 mg / mL, 1 mg / mL, 2 mg / mL, and 3 mg / mL, respectively). After crosslinking and curing under ultraviolet light at a wavelength of 365 nm for 15 min, the gel was immersed in a glycerol solution (oil-water volume ratio of 7:3) for 24 h to obtain a methacrylamide gelatin hydrogel loaded with conductive silk fibroin.

[0054] Test Example 1

[0055] Based on Example 1, silk fibroin fibers (SFF), polydopamine-bonded silk fibroin fibers (PDA-SFF), and conductive silk fibroin fibers (PPy-PDA-SFF) were bonded onto the electron microscope stage and subjected to gold sputtering in a vacuum environment for 120 seconds. The cross-sectional morphology was observed using a tabletop scanning electron microscope, and the results are as follows: Figure 1 As shown. From Figure 1 It can be seen that the SFF surface is relatively smooth, while the PDA-SFF surface exhibits an uneven coating layer, which is an amorphous aggregate formed by the random polymerization of PDA. The PPy-PDA-SFF surface displays a rougher granular and fibrous network structure, which is the random growth structure during PPy polymerization. This preliminarily proves that polydopamine and polypyrrole have been successfully grafted onto silk fibroin fibers.

[0056] Test Example 2

[0057] Based on Example 1, samples of silk fibroin fiber (SFF), polydopamine-bonded silk fibroin fiber (PDA-SFF), and conductive silk fibroin fiber (PPy-PDA-SFF) were cut and ground into tiny particles, which were then filled into a total internal reflection test platform at 500 cm⁻¹. -1 -4000cm -1 The scan was performed within the wavenumber range at a scan rate of 80 spectra / second, yielding... Figure 2 The infrared spectrum shown is from... Figure 2 It can be seen that at 1622cm -1 Imine C=N stretching occurs at 1170 cm⁻¹. -1 3280cm -1 3070cm -1 The absorption peaks at 1172 cm⁻¹ correspond to the aromatic CH bending vibration and CN stretching vibration peaks, as well as the -OH stretching vibration and symmetrical NH stretching vibration peaks, indicating successful PDA grafting; simultaneously, at 1172 cm⁻¹... -1 CN appears at [location] + Stretching vibration, a characteristic of PPy doped states, 1550 cm⁻¹. -1 The presence of C=C and the symmetrical contraction of the conjugated double bond of the pyrrole ring indicates that PPy grafting was successful.

[0058] Test Example 3

[0059] Use a multimeter to measure the resistance of conductive silk fibroin fibers and calculate the conductivity. 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 (m²). 2 The conductivity of the conductive silk fibroin fiber was measured to be 9.91 × 10⁻⁶. -3 S / m.

[0060] Test Example 4

[0061] Based on Example 1, the conductivity, compressive mechanics, and other properties of methacrylamide gelatin hydrogels prepared with different amounts of conductive silk fibroin fibers (mg / mL) were tested.

[0062] (1) Conductivity (S / m): The hydrogel was sandwiched between two copper electrode plates with a diameter of 1 cm, and the resistance was measured with a multimeter. Each sample was tested three times, and the conductivity was calculated according to the formula: σ = L / (R × A), where: L is the conductor length (m), R is the resistance (Ω), and A is the cross-sectional area (m²). 2 );

[0063] (2) Maximum compressive strength (kPa) and maximum compressive strain (%): The hydrogel was fabricated into a cylindrical structure with a bottom diameter of 14 mm and a height of 12 mm. The compressibility of these materials was evaluated using a universal testing machine at a compression speed of 2 mm / min until the hydrogel broke. The load displacement obtained from the test was converted into stress and strain according to the formula to obtain the maximum compressive strength and maximum compressive strain. The stress calculation formula is σ=F / S, where: σ is the compressive stress (Pa), F is the compressive load applied to the hydrogel (N), and S is the cross-sectional area (m²). 2 The strain calculation formula is ε=(L0-L) / L0; where: ε is the compressive strain, L0 is the original height (m), and L is the actual height of the sample when the maximum compressive deformation is reached (m).

[0064] (3) Pore size (μm): The hydrogel was frozen and fractured by liquid nitrogen and then freeze-dried to retain the original pore structure. The cross-sectional morphology was observed using a desktop electron scanning microscope. Five regions were selected from the scanning electron microscope images of each sample, and 10 relatively uniform pores were selected from each region. The diameter of the pores was measured using the line tool of ImageJ software. Each region was repeated 3 times and the average value was taken.

[0065] (4) Porosity (%): The hydrogel was frozen and fractured by liquid nitrogen and then freeze-dried to retain the original pore structure. The cross-sectional morphology was observed using a desktop electron scanning microscope. Five regions were selected from the scanning electron microscope images of each sample. The scale was set according to the hydrogel electron microscope images. The circular snapping tool of ImageJ software was used to calculate the proportion of the area of ​​each pore to the area of ​​the selected region to obtain the porosity. Each region was repeated 3 times and the average value was taken.

[0066] (5) Swelling rate (%): The hydrogel was made into a cylinder with a bottom diameter of 14 mm and a height of 12 mm. After freeze-drying, its weight was weighed. Then, it was soaked in PBS solution (pH=7.4) to absorb water and swell. It was placed in a 37℃ water bath shaker and shaken at a speed of 100 rpm. After a period of time, it was taken out, the surface moisture was gently wiped off, the wet weight was weighed, and the swelling rate of the hydrogel was calculated. The swelling rate was calculated by formula SR=(M2-M1) / M1×100%, where: SR is the swelling rate, M1 is the weight of the hydrogel after freeze-drying (g), and M2 is the wet weight of the hydrogel after absorbing water and swelling (g).

[0067] (6) Residual mass percentage (%): The hydrogel was freeze-dried and placed in a phosphate buffer solution with a solid-liquid ratio of 1:50 (pH=7.4). The degradation system was placed in a constant temperature water bath at 37℃ and shaken at 100 rpm. The samples were taken out and freeze-dried at 1, 2, 3, 4, 6 and 8 weeks. The mass of the dried gel before degradation and the mass after degradation were weighed and the residual mass percentage of the hydrogel was calculated. The residual mass percentage was calculated as MR=M2 / M1×100%, where: MR is the residual mass percentage, M2 is the mass after degradation (g), and M1 is the initial mass (g).

[0068] Figures 3-7 The relevant test results are shown in Table 1:

[0069] Table 1

[0070]

[0071] Figure 3 As shown in Table 1, the conductivity of the hydrogel gradually increases with the increase of conductive silk fibroin fiber content. The conductivity range of native human skeletal muscle is 8.0 × 10⁻⁶. -4 -4.5×10 -3 The conductivity of the implant should be within 0.1-10 times that of the muscle tissue (i.e., the difference is ±1 order of magnitude) to effectively reconstruct the physiology of the damaged tissue. Within this range, the higher the conductivity, the more beneficial it is to the electrophysiological functional adaptability and cell behavior regulation, thereby promoting skeletal muscle repair. Hydrogels with conductive silk fibroin fiber content of 2 mg / mL and 3 mg / mL have suitable conductivity and the potential to promote skeletal muscle repair.

[0072] Figure 4As shown in Table 1, the compressive stress of different groups of hydrogels is approximately 46 kPa-83 kPa, the local compressive stress of skeletal muscle at rest is approximately 10 kPa-50 kPa, and the peak load during daily activities (such as walking) is ≤80 kPa. The strength range of the hydrogel of this invention is within this range, which can effectively bear physiological loads and prevent structural collapse after implantation. Within the above range, increasing the content of conductive silk fibroin fibers can enhance the mechanical properties of the hydrogel. This is because the GelMA molecular chains (containing amino / carboxyl groups) form hydrogen bonds with the polar groups (-OH, -NH2) on the surface of the silk fibroin fibers, and the hydrophobicity of PPy generates van der Waals forces with the hydrophobic regions of GelMA, 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 excessive silk fibroin fibers will hinder the photocrosslinking of GelMA molecular chains, reducing the crosslinking density and thus leading to a decrease in mechanical properties. In addition, within the range required for skeletal muscle repair, the hydrogel with a conductive silk fibroin fiber content of 2 mg / mL has the best mechanical properties.

[0073] Figure 5 As shown in Table 1, the pore size and porosity of the hydrogel increase with the increase of conductive silk fibroin fiber content. This is because the conductive silk fibroin fibers are embedded in the gaps of 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. This provides space and pathways for cell migration, blood vessel ingrowth, and the removal of metabolic waste. The ideal pore size range for hydrogels that meet the needs of cell migration and infiltration is 50μm-120μm, while the capillary network construction requires 30μm-100μm (skeletal muscle cell diameter is 50μm-80μm, Schwann cell long axis is 20μm-40μm, and endothelial cell lumen is 15μm-50μm). The diffusion of metabolic waste requires a porosity >60%. The pore size and porosity of the hydrogel of this invention are within a suitable range and can be adjusted according to the conductive silk fibroin fiber content. Hydrogels with conductive silk fibroin fiber content of 2mg / mL and 3mg / mL have more suitable pore size and porosity.

[0074] Figure 6 As shown in Table 1, the swelling rate of the hydrogel slightly increases with the increase of conductive silk fibroin fiber content. This is because the conductive silk fibroin fiber adsorbs GelMA molecular chains, reducing its effective concentration for photocrosslinking, thus decreasing the crosslinking density and leading to an increase in pore size and porosity, thereby increasing the swelling rate of the hydrogel. The hydrogel scaffold used for skeletal muscle repair is suitable for low to medium swelling (50%-200%), and the swelling rate range of the four groups of hydrogels is 110%-125%, which is beneficial for the repair of load-bearing tissues and can avoid compression of blood vessels and nerves.

[0075] Figure 7As can be seen from Table 1, the degradation process of this hydrogel exhibits phased characteristics; in the early stage (0-2 weeks), it can maintain 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, making room for new muscle fibers; in the late stage (>6 weeks), most of it degrades, which helps to avoid long-term foreign body reactions. Increased conductive silk fibroin fiber content in hydrogels leads to increased hydrogel degradation rate. At week 8, the remaining mass percentages of GelMA hydrogels and hydrogels with conductive silk fibroin fiber contents of 1 mg / mL, 2 mg / mL, and 3 mg / mL were 70.92±2.83%, 63.05±2.57%, 50.14±2.02%, and 41.77±2.05%, respectively. In vitro degradation rates are typically 1.3-1.5 times slower than in vivo (due to the lack of enzyme / cell action). The degradation rate at week 8 should reach 50%-80% (corresponding to the new muscle fibers taking over the mechanical load and the scaffold gradually withdrawing). This indicates that the degradation of hydrogels with conductive silk fibroin fiber contents of 2 mg / mL and 3 mg / mL satisfies the triple requirements of mechanical support, porosity conduction, and degradation timing.

[0076] In summary, when the amount of conductive silk fibroin fiber added is 2 mg / mL, the prepared methacrylamide gelatin hydrogel can balance mechanical properties and electrical conductivity, while also having a suitable degradation rate and meeting the conditions required to support cell growth.

[0077] Test Example 5

[0078] Based on Example 1, the cell compatibility of methacrylamide gelatin hydrogels prepared from the control group and different amounts of conductive silk fibroin fibers (mg / mL) was characterized. The test steps are as follows:

[0079] According to GB / T 16886.5-2016 "Biological Evaluation of Medical Devices - Part 5: In Vitro Cytotoxicity Tests", the hydrogel was first sterilized with ethanol gradient. Then, under sterile conditions at 37°C, it was immersed in serum-free high-glucose DMEM medium (extraction ratio 0.1 mg / mL) for 24 ± 2 h at 37°C. After that, 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin were added to prepare the extraction medium for subsequent cytotoxicity assessment.

[0080] According to the standard GB / T 16886.5-2017, this extract medium was used for myoblast culture tests: cells were seeded in 96-well plates (1×10³ cells / well). After the cells were fully adhered, the medium was replaced with the extract medium (experimental group). A control group (complete medium without added materials) and a blank group (containing only CCK-8 working solution) were also set up. After 1, 3, and 5 days of culture, the medium was discarded, and 100 μL of live / dead cell staining reagent (Calcein-AM / PI) diluted 1000 times with PBS was added to each well. The cells were incubated at room temperature in the dark for 30 min. After incubation, the staining solution was aspirated, and unbound staining solution was removed by washing with PBS. Cells were observed using an inverted fluorescence microscope, and images were captured and collected within the same field of view. Figure 8 ).

[0081] Cells were seeded in 96-well plates (1×10³ cells / well). After complete cell adhesion, the medium was replaced with extraction medium (experimental group). A control group (complete medium without added materials) and a blank group (containing only CCK-8 working solution) were also set up. After 1, 3, and 5 days of culture, the medium was discarded, and 100 μL of DMEM basal medium containing 10% CCK-8 was added to each well. The plates were incubated for 1 hour. After incubation, 90 μL of medium was transferred from each well to a new 96-well plate, and the absorbance at 450 nm was measured using a microplate reader. The absorbance was calculated using the formula: OD value = OD experimental group - OD blank group, where 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. Statistical analysis was then performed. Figure 9 ).

[0082] from Figures 8-9 It can be seen that the methacrylamide gelatin hydrogels prepared with different amounts of conductive silk fibroin fibers showed no cytotoxicity. Among them, the hydrogels with conductive silk fibroin fiber contents of 2 mg / mL and 3 mg / mL significantly promoted myoblast proliferation within 5 days. Increased number of viable cells, enhanced metabolic activity, and increased production of colored products led to an increase in the OD value. Therefore, the OD value is positively correlated with the number of viable cells and can indirectly reflect cell proliferation.

[0083] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing a methacrylamide gelatin hydrogel loaded with conductive silk fibroin, characterized in that, Includes the following steps: S1. Add silk fibroin fiber to the hydrochloric acid dopamine solution, stir and let stand, then adjust the pH to 8-9 and heat to react to obtain polydopamine-linked silk fibroin fiber. S2. The polydopamine fibroin fiber described in S1 is sequentially immersed in ammonium persulfate solution and pyrrole solution to obtain conductive fibroin fiber. S3. The conductive silk fibroin fibers described in S2 are added to a methacrylamide gelatin solution, crosslinked and cured under ultraviolet light, and then immersed in a glycerol solution to obtain the methacrylamide gelatin hydrogel loaded with conductive silk fibroin; the methacrylamide gelatin solution is prepared by dissolving methacrylamide gelatin and a photoinitiator in a phosphate buffer solution; the concentration of the conductive silk fibroin fibers in the solution is 1 mg / mL-3 mg / mL.

2. The method for preparing methacrylamide 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 methacrylamide gelatin hydrogel loaded with conductive silk fibroin according to claim 1, characterized in that, In S1, the heating reaction is carried out at a temperature of 65°C-75°C for a duration of 2.5h-3.5h.

4. The method for preparing methacrylamide 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 to 1.5 mol / L; The immersion time in ammonium persulfate solution is 30-120 minutes.

5. The method for preparing methacrylamide 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 soaking time in pyrrole solution is 12h-30h.

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

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

8. The method for preparing methacrylamide 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; Soaking time in glycerol solution is 12h-36h.

9. Methacrylamide gelatin hydrogel loaded with conductive silk fibroin prepared by the method according to any one of claims 1-8.

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