A silk fibroin / hyaluronic acid interpenetrating network self-adapting hydrogel scaffold

Abstract

The application discloses a silk fibroin / hyaluronic acid interpenetrating network self-adaptive hydrogel support and a preparation method thereof, and belongs to the technical field of medical supplies. The SF / HA interpenetrating network biomimetic hydrogel is developed by using an ice crystal template method in combination with a freeze-drying technology, a microtubule array biomimetic microstructure and a compressive modulus distribution changing in a gradient manner along a radial direction are constructed, and nerve tissue extracellular matrix is fully simulated and nerve axon directional growth is induced. Through a 'biological marker precise response, dynamic coordination bond dissociation and penetration-diffusion mechanism', biological active substances are sequentially delivered, and a regenerative microenvironment is precisely and dynamically regulated. The intelligent biomimetic hydrogel with the optimal comprehensive performance is screened out, and is expected to be prepared in an engineering mode.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical supplies, and particularly relates to a silk fibroin / hyaluronic acid interpenetrating network self-adaptive hydrogel scaffold and a preparation method thereof. BACKGROUND

[0002] Peripheral nerve injury (PNI) often leads to motor dysfunction and even paralysis, usually resulting from traffic accidents, natural disasters, wars, disease complications, and extreme sports accidents. The large patient population and low postoperative excellent recovery rate seriously endanger human health and social and economic development. Although peripheral nerves can spontaneously repair after injury, the limited regenerative capacity of nerve tissue and the harsh requirements for the microenvironment that allows growth have severely limited the rehabilitation effect, especially for long peripheral nerve defects (>5 cm). At present, for nerve transection and short nerve defects (2-4 cm), the nerve can be directly anastomosed by "end-to-end". However, for long nerve defects (>5 cm), tension-free suture cannot be performed clinically, and autologous nerve transplantation is still mainly relied on. However, autologous transplantation has defects such as limited donor, need for secondary surgery, mismatch between donor and recipient size, and formation of neuroma, which hinder the application of this method.

[0003] The construction of nerve repair scaffolds provides an effective method for long nerve defect repair. Currently, clinical commercial scaffolds are mainly divided into two categories. The first category is a hollow conduit of biological materials (usually <4 cm in commercial specifications), such as Neurotube and NeuraGen, which bridges the nerve defect ends to create a locally closed regeneration environment, reducing external inflammatory substance infiltration and also providing an extension channel for regenerating axons. However, due to the lack of three-dimensional structure inside the hollow conduit and insufficient mechanical properties, it cannot effectively guide long nerve regeneration. The second category is a decellularized nerve scaffold (usually <6 cm in commercial specifications), such as Avance and "Shenqiao", which removes the cells and myelin sheath components in allogeneic / xenogeneic nerves, leaving the microtubular array structure of the extracellular matrix in the nerve tissue. This type of product has a three-dimensional structure to a certain extent, guiding nerve regeneration, but the three-dimensional structure of the scaffold collapses easily during decellularization, the inherent microstructure of the nerve changes, and there is a risk of cross-infection and potential immune rejection; in addition, the production raw material is derived from amputated limbs, which also seriously limits large-scale production and application. In summary, the two types of products currently on the market lack nerve induction activity and cannot precisely regulate the nerve regeneration process, and cannot meet the repair needs of long segment nerve defects in the clinic.

[0004] The application constructs a microtubule array biomimetic microstructure by ice crystal template method-freeze drying technology combined with electrospinning technology, provides physical clues for improving optimal directional extension of nerve axons, constructs a physical modulus gradient distribution to meet different requirements of mechanical support and mechanics of proximal and distal ends of nerve regeneration, realizes long-term directional chemotaxis and guidance of nerve scaffolds to axon growth, adjusts the microenvironment through different types of stimulus response functional groups to realize precise dynamic regulation of key links of nerve regeneration. The defects of the above commercialized scaffolds are effectively overcome, and it becomes a potential scheme for repairing long peripheral nerve defects in the clinic. SUMMARY

[0005] The application aims to provide a silk fibroin / hyaluronic acid interpenetrating network adaptive hydrogel scaffold and a preparation method thereof. The adaptive hydrogel has mechanical properties matching natural nerve tissue, meets the modulus dynamic evolution requirement required for proximal to distal repair, precisely regulates the nerve regeneration microenvironment, is finally used for efficient repair of long-distance peripheral nerve defects, and is expected to become an engineered preparation.

[0006] To achieve the above application purposes, the application adopts the following technical solutions:

[0007] A silk fibroin / hyaluronic acid interpenetrating network adaptive hydrogel scaffold and a preparation method thereof. Specifically, a biomimetic multi-channel micro-pore structure is constructed by ice crystal template method-freeze drying technology, and the three-dimensional structure of the nerve conduit is realized by regulating the ice crystal size, orientation, freeze-drying time and temperature gradient and other parameters. Secondly, the composition ratio, concentration and surface modification functional groups of the HA / SF base are changed to realize the gradient change of the compression modulus of the hydrogel scaffold in the radial direction in the range of 1-10 kPa. The HA grafted diselenium segment can quickly respond to the biological marker (ROS) of nerve regeneration repair in the early stage of repair, so as to release the anti-inflammatory factor IAA to regulate the inflammatory microenvironment; in addition, the dynamic metal coordination bond of magnesium ion obtained after the modification of Pam on the HA molecular chain can be dissociated as needed to release Mg 2+ ; and the growth factor@polymer nanoparticles formed by physical action self-assembly realize long-term sustainable release behavior through the penetration-diffusion mechanism, and comprehensively promote myelin regeneration. The types of the above stimulus response segments, the types of biologically active substances, the components of the polymer carrier, the size and the like are systematically regulated, and finally a series of biomimetic hydrogel scaffolds meeting the phased requirements of tissue repair are obtained.

[0008] The preparation method of the hydrogel scaffold comprises the following steps:

[0009] (1) Anhydrous sodium carbonate is added to boiling deionized water, stirred and mixed, silk is added for full degumming and then washed, dried in an oven, and dry degummed silk is obtained. The dry degummed silk is added to a lithium bromide solution and stirred and dissolved. The solution is incubated in an oven, and the silk fibroin (SF) solution is obtained after dialysis in deionized water, centrifugal treatment, and removal of the precipitate;

[0010] (2) Hyaluronic acid (HA) powder is dissolved in deionized water, stirred and mixed at room temperature, and a hyaluronic acid solution is obtained;

[0011] (3) Selenocystamine hydrochloride, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) are added to the hyaluronic acid solution to obtain solution S1. 3-indoleacetic acid (IAA), EDC, and NHS are added to solution S1 to obtain solution S2;

[0012] (4) Pamidronate disodium hydrate (Pam), EDC, and NHS are added to solution S2 to obtain solution S3. Magnesium chloride is added to solution S3 to obtain solution S4;

[0013] (5) Polyvinyl alcohol (PVA) is dissolved in deionized water, stirred and mixed, and nerve growth factor (NGF) is added to obtain solution S5;

[0014] (6) Polycaprolactone (PCL) is dissolved in dichloromethane to obtain solution S6. Solution S6 is mixed with S5, ultrasonically emulsified to obtain emulsion S7. The emulsion S7 is mixed with the PVA solution, ultrasonically emulsified, stirred at room temperature to volatilize dichloromethane, and then centrifuged to obtain nerve growth factor-coated nanoparticles (NGF@PCLNPs);

[0015] (7) The NGF@PCLNPs in step (6) are added to solution S4 to obtain solution S8;

[0016] (8) The silk fibroin solution and solution S8 are mixed at room temperature, stirred and mixed, and bubbles are removed to obtain mixed solution S9;

[0017] (9) S9 is injected into a customized mold, one end of the mold is slowly immersed in liquid nitrogen for freezing, and after complete freezing, the mold is placed in a freeze dryer for freeze-drying to preliminarily obtain an aerogel scaffold. The aerogel scaffold is soaked in an ice bath crosslinking agent solution for crosslinking to obtain a hydrogel scaffold;

[0018] (10) PCL is dissolved in an organic solvent to obtain a polymer macromolecular solution, and electrospinning treatment is performed to obtain an oriented electrospun membrane;

[0019] (11) wrapping the electrospun membrane obtained in step (10) outside the hydrogel scaffold obtained in step (9) to obtain a final hydrogel scaffold.

[0020] Preferably, the mixing ratio of anhydrous sodium carbonate and deionized water in step (1) is 1.6-2.5 g / L; the mixing ratio of silk and deionized water is 3-6 g / L; the mixing ratio of dry degummed silk and lithium bromide solution is 0.15-0.35 g / ml; the incubation time is 4-8 h; the dialysis time is 24-72 h; and the centrifugation condition is 9000 r / min, 4℃, 40 min.

[0021] Preferably, the mass concentration of the hyaluronic acid solution in step (2) is 1-3%, and the stirring time is 24-48 h.

[0022] Preferably, in step (3), the mass ratio of selenocystamine hydrochloride to hyaluronic acid is 5-12%, the mass ratio of EDC to HA is 5-12%, the mass ratio of NHS to HA is 2-8%, the reaction temperature is 20-25℃, the reaction time is 3-6 h, the mass ratio of IAA to HA in the S1 solution is 8-15%, the mass ratio of EDC to HA is 8-15%, the mass ratio of NHS to HA is 2-8%, the reaction temperature is 20-25℃, and the reaction time is 0.5-2 h.

[0023] Preferably, in step (4), the mass ratio of Pam to HA is 10-25%, the mass ratio of EDC to HA is 10-25%, the mass ratio of NHS to HA is 5-15%, the reaction temperature is 20-25℃, the reaction time is 3-8 h, pH=8.5, the concentration of magnesium chloride is 25-75 mM, the reaction temperature is 20-25℃, and the reaction time is 20-40 min.

[0024] Preferably, in step (5), the mass concentration of the PVA solution is 0.1-2%, the reaction temperature is 60-90℃, and the stirring time is 8-12 h.

[0025] Preferably, in step (6), the PCL solution concentration is 5%-10% W / V, the ultrasonic power is 500 W, the ultrasonic time is 10-20 min, the emulsion S7 and the PVA solution ultrasonic power is 400-900 W, the ultrasonic time is 5-40 min, the room temperature stirring time is 2-6 h, and the centrifugation condition is 12000 r / min, 20-60 min.

[0026] Preferably, in step (7), the mass ratio of NGF@PCLNPs to HA in solution S8 is 5-15%.

[0027] Preferably, in step (8), the mass ratio of silk fibroin to hyaluronic acid in the mixed solution is 1 / 9, 2 / 8, 5 / 5, 8 / 2, or 9 / 1.

[0028] Preferably, in step (9), the mold is immersed in liquid nitrogen at a rate of 0-3 mm / min, the freezing time is 1-3 h, the freeze-drying time is 24-48 h, the mass ratio of crosslinking agent EDC / NHS is 2-1, and the mass concentration of the crosslinking agent solution is 1-2%.

[0029] Preferably, in step (10), the organic solvent is one or two of chloroform, dichloromethane, dimethylformamide, and methanol; the mass concentration of the obtained polymer solution is 10-15%, the electrospinning voltage is 10-20kV, the temperature is 20-50℃, the humidity is 40-60%, the receiving distance is 10-20cm, and the roller speed is 1000-2000rpm.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The SF / HA hydrogel scaffold prepared by this invention has mechanical properties that match those of natural nerves. The integrated design of elastic modulus gradient distribution and microtube array biomimetic microstructure along the nerve regeneration direction provides physical clues for improving the directional extension of nerve regeneration, meets the evolving needs of long-distance nerve regeneration for different mechanical support and mechanical requirements at the proximal and distal ends, and realizes long-term directional chemotaxis and guidance for nerve growth.

[0032] (2) The hydrogel scaffold prepared in this invention enables the orderly delivery of bioactive substances and dynamically regulates the microenvironment for peripheral nerve regeneration. Based on biomarker response, dynamic coordination bond dissociation and repolymerization, and the permeation-diffusion mechanism of self-assembled nanoparticles, this invention releases inflammation inhibitors, metal ions, and growth factors on demand, matching the three microenvironments of "inflammation control, axonal growth, and myelin formation" in the nerve regeneration and repair process. This invention comprehensively improves the repair efficiency of long-distance peripheral nerve defects by precisely regulating the microenvironment for nerve tissue regeneration. Attached Figure Description

[0033] Figure 1 Prepared for Example 7 Se IAA / Pam Mg / P The morphology, mechanical properties, swelling, and water contact angle characterization of the three SF / HA ratios used in the NGF@H composite hydrogel scaffold are shown in the following figures: A. Scanning electron microscope images of radial cross-sections of hydrogels with different ratios; B. Scanning electron microscope images of cross-sections of hydrogels with different ratios; C, D. Quantitative analysis of pore size and porosity of hydrogels with different ratios; E, F, G. Compression curves, quantitative analysis of compressive modulus, and quantitative analysis of compressive strength of hydrogels with different ratios; H. Swelling curves of hydrogels with different ratios; I, J, K. Compression curves, quantitative analysis of compressive modulus, and quantitative analysis of compressive strength of hydrogels with different ratios; L. Quantitative analysis of water contact angle of hydrogels with different ratios.

[0034] Figure 2 Characterization figures for the morphology, degradation performance, and mechanical properties of the hydrogel scaffolds prepared in Examples 4, 5, 6, and 7: A. Scanning electron microscopy morphology and orientation of cross-sections of the uniform random scaffold (UR-SF / HA), uniformly oriented scaffold (UO-SF / HA), gradient random scaffold (GR-SF / HA), and gradient oriented scaffold (GO-SF / HA); B. Degradation curves of the UR-SF / HA, UO-SF / HA, GR-SF / HA, and GO-SF / HA scaffolds; C. Schematic diagram of degradation matching nerve regeneration with uniform and gradient scaffolds; D. Vertical compression, parallel compression, vertical stretching, and parallel stretching curves of the four groups of scaffolds.

[0035] Figure 3 This is a diagram illustrating the modification mechanism of linking anti-inflammatory factors, metal ions, and nerve growth factors in Example 7.

[0036] Figure 4 The verification characterization diagrams for the successful connection of anti-inflammatory factors, metal ions, and nerve growth factors in Example 7 are as follows: A. Infrared test results of HA, HA+Se2, and HA+Se2+IAA; B. X-ray photoelectron spectroscopy of HA+Se2; C. Release curve of anti-inflammatory factor IAA in response to reactive oxygen species; D. Release curve of anti-inflammatory factor IAA under different concentrations of reactive oxygen species; E. HA+Pam+Mg 2+ X-ray photoelectron spectroscopy analysis data; infrared test results for F.HA and HA+Pam; G.HA and HA+Pam+Mg 2+ Mechanical property test results; H. Mg at different concentrations 2+ I. Release curve of nanoparticles; J. Particle size distribution curve of nanoparticles; K. Potential distribution curve of nanoparticles; L. Release curve of nerve growth factor.

[0037] Figure 5 Basic characterization of the hydrogel scaffolds prepared for 7 and the comparative example: A. Scanning electron microscopy images of cross sections; B. Quantitative analysis of pore size and porosity; C-ray photoelectron spectroscopy data; D. Quantitative analysis of water contact angle; E. Swelling curves; F. Degradation curves; G. Compression curves; H. Quantitative analysis of compressive modulus and strength; I. Tensile curves; J. Quantitative analysis of tensile modulus and strength; K. Release curve of anti-inflammatory factor IAA; L. Release curve of metal ions; M. Release curve of nerve growth factor; N. In vitro response to elimination of reactive oxygen species.

[0038] Figure 6 Prepared for Example 7 Se IAA / Pam Mg / PCharacterization of electrospun outer membrane in NGF@H composite hydrogel scaffold: A. SEM image of oriented spinning membrane; B. Orientation degree analysis of oriented spinning membrane; C. Fiber diameter distribution image of oriented spinning membrane; D. Stretching schematic diagram of oriented spinning membrane along the vertical and parallel directions; E. Stretching curve diagram of oriented spinning membrane along the vertical and parallel directions; F. Quantitative analysis of tensile modulus and strength of oriented spinning membrane along the vertical and parallel directions.

[0039] Figure 7 The prepared Se IAA / Pam Mg / P Biocompatibility performance characterization diagram of NGF@H composite hydrogel scaffold: five groups of different scaffolds after 24h co-cultured with Schwann cells under the data picture of live and dead cell immunofluorescence staining. DETAILED DESCRIPTION

[0040] In order to make the content of the application more convenient to understand, the technical solutions of the application will be further described below in combination with specific embodiments, but the application is not limited to this.

[0041] The application discloses a silk fibroin / hyaluronic acid interpenetrating network self-adaptive hydrogel scaffold and a preparation method thereof.

[0042] (1) anhydrous sodium carbonate is added to boiling deionized water, stirred and uniformly mixed, silk is added for full degumming, washed, placed in an oven for drying, dry degummed silk is obtained, the dry silk is added to a lithium bromide solution for stirring and dissolving, the dissolving solution is placed in an oven for incubation, the incubated silk solution is dialyzed in deionized water, after dialysis, centrifugal treatment is performed to remove the precipitate, and a silk fibroin (SF) solution is obtained;

[0043] (2) hyaluronic acid (HA) powder is dissolved in deionized water, stirred uniformly at room temperature, and a hyaluronic acid solution is obtained;

[0044] (3) selenocystamine hydrochloride, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are added to the hyaluronic acid solution for reaction, a solution S1 is obtained, 3-indoleacetic acid (IAA), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are added to the S1 solution for reaction, and a solution S2 is obtained;

[0045] (4) pamidronate disodium hydrate (Pam), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are added to the S2 solution for reaction, a solution S3 is obtained, magnesium chloride is added to the S3 solution for reaction, and a solution S4 is obtained;

[0046] (5) Dissolve polyvinyl alcohol (PVA) in deionized water, stir well, and then add nerve growth factor (NGF) to obtain solution S5;

[0047] (6) Polycaprolactone (PCL) was dissolved in dichloromethane to obtain solution S6. Solution S6 was mixed with S5 and ultrasonically emulsified to obtain primary emulsion S7. Emulsion S7 was mixed with PVA solution, ultrasonically emulsified, and then stirred at room temperature to evaporate dichloromethane. After centrifugation, nanoparticles encapsulating nerve growth factor (NGF@PCLNPs) were obtained.

[0048] (7) Add the NGF@PCLNPs from step (8) to solution S4 to obtain solution S8;

[0049] (8) Mix the silk fibroin solution and solution S8 at room temperature, stir evenly, remove air bubbles, and obtain mixed solution S9;

[0050] (9) Inject S9 into a custom mold, slowly immerse one end of the mold in liquid nitrogen to freeze, and after it is completely frozen, put the mold into a freeze dryer to freeze dry, and initially obtain an aerogel scaffold; soak the aerogel scaffold in an ice bath crosslinking agent solution to crosslink, and obtain a hydrogel scaffold.

[0051] (10) Dissolve PCL in an organic solvent to obtain a polymer solution, and perform electrospinning treatment to obtain an oriented electrospinned film.

[0052] (11) Wrap the electrospun membrane obtained in step (10) around the hydrogel scaffold obtained in step (9) to obtain the final hydrogel scaffold.

[0053] In this invention, silkworm silk is first degummed to prepare a silk fibroin solution. More preferably, the mixing ratio of anhydrous sodium carbonate and deionized water is 1.6 g / L; the mixing ratio of silkworm silk and deionized water is 3 g / L; the mixing ratio of degummed dry silkworm silk and lithium bromide solution is 0.25 g / ml; the incubation time is 5-6 h; the dialysis time is 48-72 h; and the centrifugation conditions are 9000 r / min, 4℃, and 40 min.

[0054] The hyaluronic acid is stirred to form a solution at room temperature, more preferably, the stirring time is 24-36 h, and the mass concentration of the obtained hyaluronic acid solution is 1%-2%. After obtaining the hyaluronic acid solution, selenium cystamine hydrochloride, EDC and NHS are added during the stirring process to fully react to obtain solution S1. After obtaining solution S1, IAA, EDC and NHS are added during the stirring process to fully react to obtain solution S2. More preferably, the mass ratio of selenium cystamine hydrochloride to hyaluronic acid is 5-12%, the mass ratio of EDC to HA in this step is 5-12%, the mass ratio of NHS to HA is 2-8%, the reaction temperature is 20-25°C, and the reaction time is 3-6 h; the mass ratio of IAA to HA is 8-15%, the mass ratio of EDC to HA in this step is 8-15%, the mass ratio of NHS to HA is 2-8%, the reaction temperature is 20-25°C, and the reaction time is 0.5-2 h.

[0055] After obtaining solution S2, Pam, EDC and NHS are added during the stirring process to fully react to obtain solution S3, and magnesium chloride is added to fully stir and react to obtain solution S4. More preferably, the mass ratio of Pam to HA is 10-25%, the mass ratio of EDC to HA is 10-25%, the mass ratio of NHS to HA is 5-15%, the reaction temperature is 20-25°C, the reaction time is 3-8 h, and the pH is 8.5. The concentration of magnesium chloride is 25-75 mM, the reaction temperature is 20-25°C, and the reaction time is 20-40 min.

[0056] PVA is dissolved in deionized water to obtain PVA solutions with mass concentrations of 0.1% and 2%, respectively, the reaction temperature is 60-90°C, and the reaction time is 8-12 h. NGF is added to the PVA solution with a mass concentration of 2% to obtain solution S5. PCL is dissolved in dichloromethane to obtain solution S6, S6 and S5 are mixed and ultrasonically emulsified to obtain primary emulsion S7, S7 is subjected to secondary emulsification with a PVA solution with a mass concentration of 0.1%, dichloromethane is volatilized at room temperature after the emulsification is completed, and centrifugal treatment is performed to obtain NGF-coated nanoparticles (NGF@PCLNPs). The nanoparticles are added to solution S4 to disperse uniformly to obtain solution S8. More preferably, the mass ratio of NGF to PVA is 1*10 -4 ~2*10 -4 ; the mass concentration of the PCL solution is 5%; the ultrasonic power is 500 W during the primary emulsification, and the ultrasonic time is 10-20 min; the ultrasonic power is 400-900 W during the secondary emulsification, the ultrasonic time is 10-20 min, the stirring time at room temperature is 2-6 h, the centrifugal conditions are 12000 r / min and 20-60 min, and the mass ratio of NGF@PCLNPs to HA in solution S8 is 5-15%.

[0057] After obtaining the solution S8, it is mixed with the silk fibroin solution at room temperature, stirred uniformly, and bubbles are removed to obtain a mixed solution S9; the S9 is injected into a customized mold, one end of the mold is slowly immersed in liquid nitrogen for freezing, and after complete freezing, the mold is placed in a freeze dryer for freeze-drying to preliminarily obtain an aerogel scaffold; the aerogel scaffold is soaked in an ice bath crosslinking agent solution for crosslinking to obtain a hydrogel scaffold. More preferably, the mass ratio of silk fibroin and hyaluronic acid in the mixed solution S9 is 1 / 9, 2 / 8, 5 / 5, 8 / 2, or 9 / 1; the speed of immersing the mold in liquid nitrogen is 0-3 mm / min, the freezing time is 1-3 h, and the freeze-drying time is 24-48 h. The crosslinking agent is EDC and NHS, the mass ratio of EDC / NHS is = 2-1, and the mass concentration of the crosslinking agent solution is 1-2%.

[0058] PCL is dissolved in chloroform to obtain a polymer macromolecular solution, and then electrospinning treatment is performed to obtain an oriented electrospun film; the electrospun film is wrapped outside the hydrogel scaffold to obtain a final hydrogel scaffold. More preferably, the mass concentration of the obtained polymer macromolecular solution is 10-15%; the voltage for electrospinning is 10-20 kV, the temperature is 20-50°C, the humidity is 40-60%, the receiving distance is 10-20 cm, and the roller rotation speed is 1000-2000 rpm.

[0059] In order to further understand the present application, the self-adaptive and sequential response hydrogel scaffold for dynamically regulating the microenvironment of peripheral nerve regeneration and the preparation method thereof provided by the present application are described below in conjunction with examples, and the protection scope of the present application is not limited by the following examples.

[0060] Example 1

[0061] (1) 6.4 g of anhydrous sodium carbonate is added to 4 L of boiling deionized water, stirred and mixed, 12 g of silk is added and washed after being fully degummed, and then dried in an oven to obtain degummed dry silk. The dry silk is then added to a lithium bromide solution and stirred to dissolve. The dissolved solution is incubated in an oven for 6 h. The incubated silk solution is dialyzed in deionized water for 48 h, and then centrifuged at 9000 r / min and 4°C for 40 min to remove the precipitate, thereby obtaining a silk fibroin (SF) solution;

[0062] (2) Hyaluronic acid (HA) powder is dissolved in deionized water and stirred uniformly at room temperature to obtain a 1% mass concentration hyaluronic acid solution;

[0063] (3) selenium cystamine hydrochloride, EDC, NHS are added into the hyaluronic acid solution, and the reaction is stirred at room temperature for 3h, wherein the mass ratio of selenium cystamine hydrochloride, EDC, NHS to HA is 10%, 10%, and 5%, to obtain solution S1, IAA, EDC and NHS are added into S1 solution, and the reaction is stirred at room temperature for 0.5h, wherein the mass ratio of IAA, EDC, NHS to HA is 10%, 10%, and 5%, to obtain solution S2;

[0064] (4) Pam, EDC and NHS are added into S2 solution, and the reaction is stirred at room temperature for 4h, and the pH is adjusted to 8.5, wherein the mass ratio of Pam, EDC, NHS to HA is 20%, 10%, and 10%, to obtain solution S3, and magnesium chloride is added into S3 solution, and the concentration is 50Mm, and the reaction is stirred at room temperature for 20min, to obtain solution S4;

[0065] (5) polyvinyl alcohol (PVA) and deionized water are stirred at 90℃ for 8h to completely dissolve, and nerve growth factor (NGF) is added to obtain solution S5;

[0066] (6) PCL is dissolved in dichloromethane to obtain a solution S6 with a PCL concentration of 5% W / V, solution S6 is mixed with S5, ultrasonic emulsification is performed at 500W ultrasonic power for 12min to obtain a primary emulsion S7, the emulsion S7 is mixed with the PVA solution, ultrasonic emulsification is performed at 500W ultrasonic power for 15min, and then dichloromethane is volatilized at room temperature for 6h, and centrifugation is performed at 12000r / min for 40min to obtain nerve growth factor wrapped nanoparticles (NGF@PCLNPs);

[0067] (7) NGF@PCLNPs are added into solution S4 and stirred uniformly, wherein the mass ratio of NGF@PCLNPs to HA is 10%, to obtain solution S8;

[0068] (8) a silk fibroin solution and solution S8 are mixed at room temperature, wherein the mass ratio of SF / HA is 1 / 9, and stirring is uniformly performed to remove bubbles, to obtain a mixed solution S9;

[0069] (9) the mixed solution S9 is injected into a customized mold, one end of the mold is immersed into liquid nitrogen at a speed of 1mm / min for 1h, the mold is placed into a freeze dryer for freeze-drying for 48h, an aerogel scaffold is preliminarily obtained, and the aerogel scaffold is soaked in an ice bath crosslinking agent solution for crosslinking to obtain a hydrogel scaffold. The crosslinking agent is EDC and NHS, the mass ratio of EDC / NHS is =2, and the mass concentration of the crosslinking agent solution is 1%;

[0070] (10) PCL was dissolved in chloroform to obtain a 10% mass fraction of polymer solution, and the solution was subjected to electrospinning treatment, with a voltage of 15 kV, a temperature of 30°C, a humidity of 50%, a receiving distance of 15 cm, and a drum rotation speed of 1500 rpm, to obtain an oriented electrospun film;

[0071] (11) The electrospun film obtained in step (10) was wrapped outside the hydrogel scaffold obtained in step (9) to obtain a final hydrogel scaffold.

[0072] Example 2

[0073] (1) 6.4 g of anhydrous sodium carbonate was added to 4 L of boiling deionized water, stirred and mixed, 12 g of silk was added and fully degummed, washed, and dried in an oven to obtain degummed dry silk. The dry silk was then added to a lithium bromide solution and stirred to dissolve. The dissolved solution was incubated in an oven for 6 h. The incubated silk solution was dialyzed in deionized water for 48 h, and then centrifuged at 9000 r / min at 4°C for 40 min to remove the precipitate, to obtain a silk fibroin (SF) solution;

[0074] (2) Hyaluronic acid (HA) powder was dissolved in deionized water and stirred uniformly at room temperature to obtain a 1% mass concentration of hyaluronic acid solution;

[0075] (3) Selenocystamine hydrochloride, EDC, and NHS were added to the hyaluronic acid solution and stirred at room temperature for 3 h, with a mass ratio of selenocystamine hydrochloride, EDC, and NHS to HA of 10%, 10%, and 5%, respectively, to obtain solution S1. IAA, EDC, and NHS were added to solution S1 and stirred at room temperature for 0.5 h, with a mass ratio of IAA, EDC, and NHS to HA of 10%, 10%, and 5%, respectively, to obtain solution S2;

[0076] (4) Pam, EDC, and NHS were added to solution S2 and stirred at room temperature for 4 h, with a pH of 8.5, and a mass ratio of Pam, EDC, and NHS to HA of 20%, 10%, and 10%, respectively, to obtain solution S3. Magnesium chloride was added to solution S3 to a concentration of 50 Mm and stirred at room temperature for 20 min to obtain solution S4;

[0077] (5) Polyvinyl alcohol (PVA) and deionized water were stirred at 90°C for 8 h to completely dissolve, and nerve growth factor (NGF) was added to obtain solution S5;

[0078] (6) PCL was dissolved in dichloromethane to obtain a solution S6 with a PCL concentration of 5% W / V, the solution S6 was mixed with S5, and the primary emulsion S7 was obtained by ultrasonic emulsification at a power of 500 W for 12 min, the emulsion S7 was mixed with the PVA solution by stirring, and the emulsion was ultrasonic emulsified at a power of 500 W for 15 min, and then dichloromethane was volatilized at room temperature for 6 h, and the emulsion was centrifuged at 12000 r / min for 40 min to obtain the NGF@PCLNPs;

[0079] (7) The NGF@PCLNPs were added to the solution S4 and stirred uniformly, wherein the mass ratio of NGF@PCLNPs to HA was 10%, to obtain the solution S8;

[0080] (8) The solution S8 was mixed with the solution of silk fibroin at room temperature, wherein the mass ratio of SF to HA was 2 / 8, and the mixture was stirred uniformly to remove bubbles to obtain the mixed solution S9;

[0081] (9) The mixed solution S9 was injected into a customized mold, one end of the mold was immersed in liquid nitrogen at a speed of 1 mm / min for 1 h, and the mold was placed in a freeze dryer for freeze-drying for 48 h to obtain a preliminary aerogel scaffold, and the aerogel scaffold was immersed in an ice bath crosslinking agent solution for crosslinking to obtain a hydrogel scaffold. The crosslinking agent was EDC and NHS, the mass ratio of EDC / NHS was 2, and the mass concentration of the crosslinking agent solution was 1%;

[0082] (10) PCL was dissolved in chloroform to obtain a polymer solution with a mass fraction of 10%, and the solution was subjected to electrospinning treatment, the voltage was 15 kV, the temperature was 30°C, the humidity was 50%, the receiving distance was 15 cm, and the drum rotation speed was 1500 rpm to obtain an oriented electrospun film;

[0083] (11) The electrospun film obtained in step (10) was wrapped outside the hydrogel scaffold obtained in step (9) to obtain the final hydrogel scaffold.

[0084] Example 3

[0085] (1) 6.4 g of anhydrous sodium carbonate was added to 4 L of boiling deionized water, stirred and mixed, 12 g of silk was added and fully degummed, then washed and dried in an oven to obtain dried degummed silk, and then the dried degummed silk was added to a lithium bromide solution and stirred to dissolve, the dissolved solution was incubated in an oven for 6 h, and then the silk fibroin solution after incubation was dialyzed in deionized water for 48 h, and then centrifuged at 9000 r / min and 4°C for 40 min to remove the precipitate to obtain a silk fibroin (SF) solution;

[0086] (2) Hyaluronic acid (HA) powder was dissolved in deionized water and stirred uniformly at room temperature to obtain a 1% mass concentration hyaluronic acid solution;

[0087] (3)Selenium cystamine hydrochloride, EDC, NHS are added into the hyaluronic acid solution, and stirred at room temperature for 3h, wherein the mass ratio of selenium cystamine hydrochloride, EDC, NHS to HA is 10%, 10%, 5%, to obtain solution S1, IAA, EDC and NHS are added into S1 solution, and stirred at room temperature for 0.5h, wherein the mass ratio of IAA, EDC, NHS to HA is 10%, 10%, 5%, to obtain solution S2;

[0088] (4)Pam, EDC and NHS are added into S2 solution, and stirred at room temperature for 4h, and the pH is adjusted to 8.5, wherein the mass ratio of Pam, EDC, NHS to HA is 20%, 10%, 10%, to obtain solution S3, and magnesium chloride is added into S3 solution, and the concentration is 50Mm, and stirred at room temperature for 20min, to obtain solution S4;

[0089] (5)Polyvinyl alcohol (PVA) and deionized water are stirred at 90℃ for 8h to completely dissolve, and nerve growth factor (NGF) is added to obtain solution S5;

[0090] (6)PCL is dissolved in dichloromethane to obtain a solution S6 with a PCL concentration of 5% W / V, solution S6 is mixed with S5, ultrasonic emulsification is performed at 500W ultrasonic power for 12min to obtain a primary emulsion S7, the emulsion S7 is mixed with the PVA solution, ultrasonic emulsification is performed at 500W ultrasonic power for 15min, and then dichloromethane is volatilized at room temperature for 6h, and centrifugation is performed at 12000r / min for 40min to obtain nerve growth factor wrapped nanoparticles (NGF@PCLNPs);

[0091] (7)NGF@PCLNPs are added into solution S4 and stirred uniformly, wherein the mass ratio of NGF@PCLNPs to HA is 10%, to obtain solution S8;

[0092] (8)The solution of silk fibroin and solution S8 are mixed at room temperature, wherein the mass ratio of SF / HA is 8 / 2, and stirred uniformly to remove bubbles, to obtain mixed solution S9;

[0093] (9)The mixed solution S9 is injected into a customized mold, one end of the mold is immersed into liquid nitrogen at a speed of 1mm / min for 1h, the mold is placed into a freeze dryer for freeze-drying for 48h, to preliminarily obtain an aerogel scaffold, and the aerogel scaffold is soaked in an ice bath crosslinking agent solution for crosslinking, to obtain a hydrogel scaffold. The crosslinking agent is EDC and NHS, the mass ratio of EDC / NHS is =2, and the mass concentration of the crosslinking agent solution is 1%;

[0094] (10) PCL was dissolved in chloroform to obtain a 10% mass fraction polymer solution, and the solution was subjected to electrospinning treatment at a voltage of 15 kV, a temperature of 30°C, a humidity of 50%, a receiving distance of 15 cm, and a drum rotation speed of 1500 rpm to obtain an oriented electrospinning film;

[0095] (11) The electrospinning film obtained in step (10) was wrapped outside the hydrogel scaffold obtained in step (9) to obtain a final hydrogel scaffold.

[0096] Example 4

[0097] (1) 6.4 g of anhydrous sodium carbonate was added to 4 L of boiling deionized water, stirred and mixed, 12 g of silk was added and fully degummed, then washed and dried in an oven to obtain degummed dry silk. The dry silk was then added to a lithium bromide solution and stirred to dissolve. The dissolved solution was incubated in an oven for 6 h. The incubated silk solution was dialyzed in deionized water for 48 h, and then centrifuged at 9000 r / min at 4°C for 40 min to remove the precipitate, obtaining a silk fibroin (SF) solution;

[0098] (2) Hyaluronic acid (HA) powder was dissolved in deionized water and stirred uniformly at room temperature to obtain a 1% mass concentration hyaluronic acid solution;

[0099] (3) Selenocystamine hydrochloride, EDC, and NHS were added to the hyaluronic acid solution and stirred at room temperature for 3 h, wherein the mass ratio of selenocystamine hydrochloride, EDC, and NHS to HA was 10%, 10%, and 5%, respectively, to obtain solution S1. IAA, EDC, and NHS were added to solution S1 and stirred at room temperature for 0.5 h, wherein the mass ratio of IAA, EDC, and NHS to HA was 10%, 10%, and 5%, respectively, to obtain solution S2;

[0100] (4) Pam, EDC, and NHS were added to solution S2 and stirred at room temperature for 4 h, and the pH was adjusted to 8.5, wherein the mass ratio of Pam, EDC, and NHS to HA was 20%, 10%, and 10%, respectively. Magnesium chloride was added to solution S3 to a concentration of 50 Mm and stirred at room temperature for 20 min to obtain solution S4;

[0101] (5) Polyvinyl alcohol (PVA) and deionized water were completely dissolved by stirring at 90°C for 8 h, and nerve growth factor (NGF) was added to obtain solution S5;

[0102] (6) PCL was dissolved in dichloromethane to obtain a solution S6 with a PCL concentration of 5% W / V, the solution S6 was mixed with S5, and the primary emulsion S7 was obtained by ultrasonic emulsification at 500 W for 12 min, the emulsion S7 was mixed with the PVA solution by stirring, and the emulsion was ultrasonic emulsified at 500 W for 15 min, and then dichloromethane was volatilized at room temperature for 6 h, and the nanoparticles (NGF@PCLNPs) were obtained by centrifugation at 12000 r / min for 40 min;

[0103] (7) The NGF@PCLNPs were added to the solution S4 and stirred uniformly, wherein the mass ratio of NGF@PCLNPs to HA was 10%, to obtain a solution S8;

[0104] (8) The silk fibroin solution and the solution S8 were mixed at room temperature, wherein the mass ratio of SF to HA was 5 / 5, and the mixture was stirred uniformly to remove bubbles to obtain a mixed solution S9;

[0105] (9) The mixed solution S9 was injected into a customized mold, one end of the mold was immersed in liquid nitrogen at a speed of 1 mm / min for 1 h, the mold was placed in a freeze dryer for freeze-drying for 48 h, and a preliminary aerogel scaffold was obtained, the aerogel scaffold was soaked in an ice bath crosslinking agent solution for crosslinking, and a hydrogel scaffold was obtained. The crosslinking agent is EDC and NHS, the mass ratio of EDC / NHS is = 2, and the mass concentration of the crosslinking agent solution is 1%;

[0106] (10) PCL was dissolved in chloroform to obtain a polymer solution with a mass fraction of 10%, the solution was subjected to electrospinning treatment, the voltage was 15 kV, the temperature was 30°C, the humidity was 50%, the receiving distance was 15 cm, and the drum rotation speed was 1500 rpm, to obtain an oriented electrospun film;

[0107] (11) The electrospun film obtained in step (10) was wrapped outside the hydrogel scaffold obtained in step (9) to obtain the final hydrogel scaffold.

[0108] Example 5

[0109] (1) 6.4 g of anhydrous sodium carbonate was added to 4 L of boiling deionized water, stirred and mixed, 12 g of silk was added and fully degummed, then washed and dried in an oven to obtain dried degummed silk, the dried degummed silk was then added to a lithium bromide solution and stirred to dissolve, the dissolved solution was incubated in an oven for 6 h, and the incubated silk solution was dialyzed in deionized water for 48 h, then centrifuged at 9000 r / min at 4°C for 40 min to remove the precipitate, and a silk fibroin (SF) solution was obtained;

[0110] (2) Hyaluronic acid (HA) powder was dissolved in deionized water and stirred uniformly at room temperature to obtain a 1% mass concentration hyaluronic acid solution;

[0111] (3) Add selenocystamine hydrochloride, EDC and NHS to the hyaluronic acid solution and stir for reaction at room temperature for 3 h, wherein the mass ratio of selenocystamine hydrochloride, EDC and NHS to HA is 10%, 10% and 5%, to obtain solution S1, and then add IAA, EDC and NHS to solution S1 and stir for reaction at room temperature for 0.5 h, wherein the mass ratio of IAA, EDC and NHS to HA is 10%, 10% and 5%, to obtain solution S2;

[0112] (4) Add Pam, EDC and NHS to solution S2 and stir for reaction at room temperature for 4 h, and adjust pH to 8.5, wherein the mass ratio of Pam, EDC and NHS to HA is 20%, 10% and 10%, to obtain solution S3, and then add magnesium chloride to solution S3 to a concentration of 50 mM and stir for reaction at room temperature for 20 min, to obtain solution S4;

[0113] (5) Dissolve polyvinyl alcohol (PVA) and deionized water at 90°C for 8 h to obtain a complete solution, and then add nerve growth factor (NGF) to obtain solution S5;

[0114] (6) Dissolve PCL in dichloromethane to obtain a solution S6 with a PCL concentration of 5% W / V, mix solution S6 with S5, and ultrasonically emulsify at a power of 500 W for 12 min to obtain a primary emulsion S7, mix emulsion S7 with the PVA solution, ultrasonically emulsify at a power of 500 W for 15 min, then stir at room temperature for 6 h to volatilize dichloromethane, and centrifuge at 12000 r / min for 40 min to obtain nerve growth factor-coated nanoparticles (NGF@PCLNPs);

[0115] (7) Add NGF@PCLNPs to solution S4 and stir uniformly, wherein the mass ratio of NGF@PCLNPs to HA is 10%, to obtain solution S8;

[0116] (8) Mix the silk fibroin solution and solution S8 at room temperature, wherein the mass ratio of SF to HA is 5 / 5, stir uniformly, and remove air bubbles to obtain a mixed solution S9;

[0117] (9) Inject the mixed solution S9 into a mold, directly immerse the mold in liquid nitrogen for freezing for 1 h, place the mold in a freeze dryer for freeze-drying for 48 h, preliminarily obtain an aerogel scaffold, immerse the aerogel scaffold in an ice bath crosslinking agent solution for crosslinking, and obtain a hydrogel scaffold. The crosslinking agent is EDC and NHS, the mass ratio of EDC / NHS is =2, and the mass concentration of the crosslinking agent solution is 1%;

[0118] (10) PCL was dissolved in chloroform to obtain a 10% mass fraction of polymer solution, and the solution was subjected to electrospinning treatment, with a voltage of 15 kV, a temperature of 30°C, a humidity of 50%, a receiving distance of 15 cm, and a drum rotation speed of 1500 rpm, to obtain an oriented electrospun film;

[0119] (11) The electrospun film obtained in step (10) was wrapped outside the hydrogel scaffold obtained in step (9) to obtain a final hydrogel scaffold.

[0120] Example 6

[0121] (1) 6.4 g of anhydrous sodium carbonate was added to 4 L of boiling deionized water, stirred and mixed, 12 g of silk was added and washed after being fully degummed, and then dried in an oven to obtain degummed dry silk. The dry silk was then added to a lithium bromide solution and stirred to dissolve. The dissolved solution was incubated in an oven for 6 h. The incubated silk solution was dialyzed in deionized water for 48 h, and then centrifuged at 9000 r / min at 4°C for 40 min to remove the precipitate, to obtain a silk fibroin (SF) solution;

[0122] (2) Hyaluronic acid (HA) powder was dissolved in deionized water and stirred uniformly at room temperature to obtain a 1% mass concentration of hyaluronic acid solution;

[0123] (3) Selenocystamine hydrochloride, EDC, and NHS were added to the hyaluronic acid solution and stirred at room temperature for 3 h, with a mass ratio of selenocystamine hydrochloride, EDC, and NHS to HA of 10%, 10%, and 5%, respectively, to obtain solution S1. IAA, EDC, and NHS were added to solution S1 and stirred at room temperature for 0.5 h, with a mass ratio of IAA, EDC, and NHS to HA of 10%, 10%, and 5%, respectively, to obtain solution S2;

[0124] (4) Pam, EDC, and NHS were added to solution S2 and stirred at room temperature for 4 h, with a pH of 8.5, and a mass ratio of Pam, EDC, and NHS to HA of 20%, 10%, and 10%, respectively, to obtain solution S3. Magnesium chloride was added to solution S3 to a concentration of 50 Mm and stirred at room temperature for 20 min to obtain solution S4;

[0125] (5) Polyvinyl alcohol (PVA) and deionized water were stirred at 90°C for 8 h to completely dissolve, and nerve growth factor (NGF) was added to obtain solution S5;

[0126] (6) PCL was dissolved in dichloromethane to obtain a solution S6 with a PCL concentration of 5% W / V, the solution S6 was mixed with S5, and the primary emulsion S7 was obtained by ultrasonic emulsification at a power of 500 W for 12 min, the emulsion S7 was mixed with the PVA solution by stirring, and the emulsion was ultrasonic emulsified at a power of 500 W for 15 min, and then dichloromethane was volatilized at room temperature for 6 h, and the emulsion was centrifuged at 12000 r / min for 40 min to obtain the NGF@PCLNPs;

[0127] (7) The NGF@PCLNPs were added to the solution S4 and stirred uniformly, wherein the mass ratio of NGF@PCLNPs to HA was 10%, to obtain a solution S8;

[0128] (8) The silk fibroin solution and the solution S8 were mixed at room temperature, wherein the mass ratio of SF to HA was 2 / 8, 5 / 5, and 8 / 2, respectively, and the mixture was stirred uniformly to remove bubbles, to obtain three mixed solutions;

[0129] (9) The three mixed solutions were sequentially injected into a mold, the mold was directly soaked in liquid nitrogen for 1 h, and the mold was placed in a freeze dryer for freeze-drying for 48 h to preliminarily obtain an aerogel scaffold, and the aerogel scaffold was soaked in an ice bath crosslinking agent solution for crosslinking to obtain a hydrogel scaffold. The crosslinking agent was EDC and NHS, the mass ratio of EDC / NHS was 2, and the mass concentration of the crosslinking agent solution was 1%;

[0130] (10) PCL was dissolved in chloroform to obtain a polymer solution with a mass fraction of 10%, and the solution was subjected to electrospinning treatment, the voltage was 15 kV, the temperature was 30°C, the humidity was 50%, the receiving distance was 15 cm, and the drum rotation speed was 1500 rpm to obtain an oriented electrospun film;

[0131] (11) The electrospun film obtained in step (10) was wrapped outside the hydrogel scaffold obtained in step (9) to obtain a final hydrogel scaffold.

[0132] Example 7

[0133] (1) 6.4 g of anhydrous sodium carbonate was added to 4 L of boiling deionized water, and stirred to mix, 12 g of silk was added and fully degummed, then washed and dried in an oven to obtain degummed dry silk, and then the dry silk was added to a lithium bromide solution and stirred to dissolve, and the dissolved solution was incubated in an oven for 6 h, and then the silk solution after incubation was dialyzed in deionized water for 48 h, and then centrifuged at 9000 r / min and 4°C for 40 min to remove the precipitate, to obtain a silk fibroin (SF) solution;

[0134] (2) Hyaluronic acid (HA) powder was dissolved in deionized water and stirred uniformly at room temperature to obtain a 1% mass concentration hyaluronic acid solution;

[0135] (3) Add selenocystamine hydrochloride, EDC and NHS to the hyaluronic acid solution and stir to react at room temperature for 3h, wherein the mass ratio of selenocystamine hydrochloride, EDC and NHS to HA is 10%, 10% and 5%, to obtain solution S1, and then add IAA, EDC and NHS to solution S1 and stir to react at room temperature for 0.5h, wherein the mass ratio of IAA, EDC and NHS to HA is 10%, 10% and 5%, to obtain solution S2;

[0136] (4) Add Pam, EDC and NHS to solution S2 and stir to react at room temperature for 4h, and adjust pH to 8.5, wherein the mass ratio of Pam, EDC and NHS to HA is 20%, 10% and 10%, to obtain solution S3, and then add magnesium chloride to solution S3 to a concentration of 50Mm and stir to react at room temperature for 20min, to obtain solution S4;

[0137] (5) Stir polyvinyl alcohol (PVA) and deionized water at 90°C for 8h to completely dissolve, and then add nerve growth factor (NGF) to obtain solution S5;

[0138] (6) Dissolve PCL in dichloromethane to obtain a solution S6 with a PCL concentration of 5% W / V, mix solution S6 with solution S5, ultrasonically emulsify at a power of 500W for 12min to obtain a primary emulsion S7, stir and mix emulsion S7 with the PVA solution, ultrasonically emulsify at a power of 500W for 15min, then stir at room temperature for 6h to volatilize dichloromethane, and centrifuge at 12000r / min for 40min to obtain nerve growth factor-coated nanoparticles (NGF@PCLNPs);

[0139] (7) Add NGF@PCLNPs to solution S4 and stir uniformly, wherein the mass ratio of NGF@PCLNPs to HA is 10%, to obtain solution S8;

[0140] (8) Mix the silk fibroin solution and solution S8 at room temperature, wherein the mass ratio of SF to HA is 2 / 8, 5 / 5 and 8 / 2 respectively, stir uniformly and remove bubbles to obtain three mixed solutions;

[0141] (9) Inject the three mixed solutions into a customized mold in turn, immerse one end of the mold into liquid nitrogen at a speed of 1mm / min for 1h, and then place the mold into a freeze dryer for freeze-drying for 48h to preliminarily obtain an aerogel scaffold, and then immerse the aerogel scaffold in an ice bath crosslinking agent solution for crosslinking to obtain a hydrogel scaffold. The crosslinking agent is EDC and NHS, the mass ratio of EDC / NHS is =2, and the mass concentration of the crosslinking agent solution is 1%;

[0142] (10) PCL was dissolved in chloroform to obtain a 10% mass fraction of polymer solution, and the solution was subjected to electrospinning treatment, with a voltage of 15 kV, a temperature of 30°C, a humidity of 50%, a receiving distance of 15 cm, and a drum rotation speed of 1500 rpm, to obtain an oriented electrospun film;

[0143] (11) The electrospun film obtained in step (10) was wrapped outside the hydrogel scaffold obtained in step (9) to obtain a final hydrogel scaffold.

[0144] Example 8

[0145] (1) 6.4 g of anhydrous sodium carbonate was added to 4 L of boiling deionized water, stirred and mixed, 12 g of silk was added and fully degummed, then washed and dried in an oven to obtain degummed dry silk. The dry silk was then added to a lithium bromide solution and stirred to dissolve. The dissolved solution was incubated in an oven for 6 h. The incubated silk solution was dialyzed in deionized water for 48 h, and then centrifuged at 9000 r / min at 4°C for 40 min to remove the precipitate, to obtain a silk fibroin (SF) solution;

[0146] (2) Hyaluronic acid (HA) powder was dissolved in deionized water and stirred uniformly at room temperature to obtain a 1% mass concentration of hyaluronic acid solution;

[0147] (3) Selenocystamine hydrochloride, EDC, and NHS were added to the hyaluronic acid solution and stirred at room temperature for 3 h, with a mass ratio of selenocystamine hydrochloride, EDC, and NHS to HA of 15%, 10%, and 5%, respectively, to obtain solution S1. IAA, EDC, and NHS were added to solution S1 and stirred at room temperature for 0.5 h, with a mass ratio of IAA, EDC, and NHS to HA of 15%, 10%, and 5%, respectively, to obtain solution S2;

[0148] (4) Pam, EDC, and NHS were added to solution S2 and stirred at room temperature for 4 h, with a pH of 8.5, and a mass ratio of Pam, EDC, and NHS to HA of 25%, 10%, and 10%, respectively, to obtain solution S3. Magnesium chloride was added to solution S3 to a concentration of 75 Mm and stirred at room temperature for 20 min to obtain solution S4;

[0149] (5) Polyvinyl alcohol (PVA) and deionized water were completely dissolved by stirring at 90°C for 8 h, and nerve growth factor (NGF) was added to obtain solution S5;

[0150] (6) PCL was dissolved in dichloromethane to obtain a solution S6 with a PCL concentration of 5% W / V, the solution S6 was mixed with S5, and the primary emulsion S7 was obtained by ultrasonic emulsification at a power of 500 W for 12 min, the emulsion S7 was mixed with the PVA solution by stirring, and the emulsion was ultrasonic emulsified at a power of 500 W for 15 min, and then dichloromethane was volatilized at room temperature for 6 h, and the nanoparticles (NGF@PCLNPs) were obtained by centrifugation at 12000 r / min for 40 min;

[0151] (7) The NGF@PCLNPs were added to the solution S4 and stirred uniformly, wherein the mass ratio of NGF@PCLNPs to HA was 15%, to obtain a solution S8;

[0152] (8) The silk fibroin solution and the solution S8 were mixed at room temperature, wherein the mass ratios of SF to HA were 2 / 8, 5 / 5, and 8 / 2, respectively, and the mixture was stirred uniformly to remove bubbles, to obtain three mixed solutions;

[0153] (9) The three mixed solutions were sequentially injected into a customized mold, one end of the mold was immersed in liquid nitrogen at a speed of 1 mm / min for 2 h, the mold was placed in a freeze dryer for freeze-drying for 36 h, and a preliminary aerogel scaffold was obtained, the aerogel scaffold was immersed in an ice bath crosslinking agent solution for crosslinking, and a hydrogel scaffold was obtained. The crosslinking agent was EDC and NHS, the mass ratio of EDC / NHS was 2, and the mass concentration of the crosslinking agent solution was 1%;

[0154] (10) PCL was dissolved in chloroform to obtain a polymer solution with a mass fraction of 10%, the solution was subjected to electrospinning treatment, the voltage was 15 kV, the temperature was 30°C, the humidity was 50%, the receiving distance was 15 cm, and the drum rotation speed was 1500 rpm, to obtain an oriented electrospun film;

[0155] (11) The electrospun film obtained in step (10) was wrapped outside the hydrogel scaffold obtained in step (9) to obtain a final hydrogel scaffold.

[0156] Comparative Example 1

[0157] (1) 6.4 g of anhydrous sodium carbonate was added to 4 L of boiling deionized water, stirred and mixed, 12 g of silk was added and fully degummed, then washed and dried in an oven to obtain dried degummed silk, the dried degummed silk was then added to a lithium bromide solution and stirred to dissolve, the dissolved silk solution was incubated in an oven for 6 h, and then dialyzed in deionized water for 48 h, and then centrifuged at 9000 r / min at 4°C for 40 min to remove the precipitate, to obtain a silk fibroin (SF) solution;

[0158] (2) Dissolve hyaluronic acid (HA) powder in deionized water, stir uniformly at room temperature, and obtain a 1% mass concentration of hyaluronic acid solution;

[0159] (3) Add IAA, EDC and NHS to the S1 solution, stir and react at room temperature for 0.5 h, wherein the mass ratio of IAA, EDC, NHS to HA is 10%, 10%, 5%, and obtain a solution S2;

[0160] (4) Add Pam, EDC and NHS to the S2 solution, stir and react at room temperature for 4 h, adjust pH = 8.5, wherein the mass ratio of Pam, EDC, NHS to HA is 20%, 10%, 10%, and obtain a solution S3. Add magnesium chloride to the S3 solution, the concentration is 50 Mm, stir and react at room temperature for 20 min, and obtain a solution S4;

[0161] (5) Add NGF to the solution S4 and stir uniformly, wherein the concentration of NGF is 800 ng / mL, and obtain a solution S8;

[0162] (6) Mix the silk fibroin solution and the solution S8 at room temperature, wherein the mass ratio of SF / HA is 2 / 8, 5 / 5, 8 / 2 respectively, stir uniformly, and remove bubbles, and obtain three mixed solutions;

[0163] (7) Inject the three mixed solutions into a customized mold in turn, immerse one end of the mold into liquid nitrogen at a speed of 1 mm / min for 1 h, and place the mold into a freeze dryer for freeze-drying for 48 h, and preliminarily obtain an aerogel scaffold. Crosslink the aerogel scaffold in an ice bath crosslinking agent solution to obtain a hydrogel scaffold. The crosslinking agent is EDC and NHS, the mass ratio of EDC / NHS is = 2, and the mass concentration of the crosslinking agent solution is 1%;

[0164] (8) Dissolve PCL in chloroform to obtain a 10% mass fraction of polymer solution, and perform electrospinning treatment on the solution, wherein the voltage is 15 kV, the temperature is 30°C, the humidity is 50%, the receiving distance is 15 cm, and the drum rotation speed is 1500 rpm, and obtain an oriented electrospun film;

[0165] (9) Wrap the electrospun film obtained in step (8) outside the hydrogel scaffold obtained in step (7) to obtain the final hydrogel scaffold.

[0166] Comparative Example 2

[0167] (1) 6.4 g of anhydrous sodium carbonate was added to 4 L of boiling deionized water, stirred and mixed, 12 g of silk was added and degummed, washed and dried in an oven, to obtain degummed dry silk, then the dry silk was added to a lithium bromide solution and stirred to dissolve, the dissolved solution was incubated in an oven for 6 h, the incubated silk solution was dialyzed in deionized water for 48 h, after dialysis, the precipitate was removed by centrifugation at 9000 r / min, 4°C for 40 min, to obtain a silk fibroin (SF) solution;

[0168] (2) Hyaluronic acid (HA) powder was dissolved in deionized water, stirred uniformly at room temperature to obtain a 1% mass concentration of hyaluronic acid solution;

[0169] (3) Selenocystamine hydrochloride, EDC and NHS were added to the hyaluronic acid solution and stirred at room temperature for 3 h, wherein the mass ratio of selenocystamine hydrochloride, EDC and NHS to HA was 10%, 10% and 5%, respectively, to obtain solution S1, IAA, EDC and NHS were added to solution S1 and stirred at room temperature for 0.5 h, wherein the mass ratio of IAA, EDC and NHS to HA was 10%, 10% and 5%, respectively, to obtain solution S2;

[0170] (4) Pam, EDC and NHS were added to solution S2 and stirred at room temperature for 4 h, the pH was adjusted to 8.5, wherein the mass ratio of Pam, EDC and NHS to HA was 20%, 10% and 10%, respectively, to obtain solution S3, magnesium chloride was added to solution S3 to a concentration of 50 mM and stirred at room temperature for 20 min to obtain solution S4;

[0171] (5) NGF was added to solution S4 and stirred uniformly, wherein the concentration of NGF was 800 ng / mL, to obtain solution S8;

[0172] (6) The silk fibroin solution and solution S8 were mixed uniformly at room temperature, wherein the mass ratio of SF / HA was 2 / 8, 5 / 5 and 8 / 2, respectively, and stirred uniformly to remove bubbles, to obtain three mixed solutions;

[0173] (7) The three mixed solutions were sequentially injected into a customized mold, one end of the mold was immersed in liquid nitrogen at a speed of 1 mm / min for 1 h, the mold was placed in a freeze dryer for freeze-drying for 48 h, to preliminarily obtain an aerogel scaffold, the aerogel scaffold was soaked in an ice bath crosslinking agent solution for crosslinking, to obtain a hydrogel scaffold. The crosslinking agent was EDC and NHS, the mass ratio of EDC / NHS was 2, and the mass concentration of the crosslinking agent solution was 1%;

[0174] (8) PCL was dissolved in chloroform to obtain a 10% polymer solution, which was subjected to electrospinning treatment at a voltage of 15 kV, a temperature of 30°C, a humidity of 50%, a receiving distance of 15 cm, and a roller speed of 1500 rpm to obtain an oriented electrospinning film;

[0175] (9) The electrospinning film obtained in step (8) was wrapped outside the hydrogel scaffold obtained in step (7) to obtain a final hydrogel scaffold.

[0176] Through Figure 1 Further analysis of the data of the three ratios of SF / HA used in the composite hydrogel scaffold in Example 7 shows that the scaffold has a microtubular array biomimetic microstructure, realizing three-dimensional multi-channel micro-pore structure biomimicry. The scaffold has good hydrophilicity, and the modulus of the three ratio scaffolds can be dynamically regulated between 1-10 kPa, matching the natural nerve tissue. As can be seen from Table 1, the three ratios of SF / HA have obvious differences in mechanical properties, the higher the SF content, the better the mechanical properties, and the slower the degradation rate. After a large number of experimental condition adjustments, the freezing time is finally determined to be 1 h, and the freeze-drying time is 48 h.

[0177] Table 1 Influence of key experimental conditions on the mechanical properties of the scaffold

[0178]

[0179]

[0180] Through Figure 2 Characterization of the morphology, degradation performance, and mechanical characteristics of the scaffolds of Examples 4, 5, 6, and 7 shows that when there is no heat conduction during preparation, the pore structure inside the scaffold does not have a directional arrangement, but is a random pore structure. Through degradation testing, it can be seen that the gradient scaffold will have a gradient degradation process compared to the uniform scaffold, the proximal scaffold degrades faster to provide space for regenerating nerve fibers, and the distal scaffold can better match the regeneration process of the nerve from the proximal to the distal end by providing mechanical support and inducing the migration and growth of various cells of the nerve and the matrix. Through compression and tensile testing of the four groups of scaffolds in the two directions perpendicular and parallel to the radial pores, it can be seen that: first, the scaffolds with oriented pore structures have better mechanical properties than the scaffolds without random pores; second, the tensile properties parallel to the oriented pore structure direction are better than the mechanical properties perpendicular to the direction; finally, we can also see that the gradient scaffold is a continuous and hierarchical structure, and the vertical compression of the gradient scaffold is a smooth curve, the different gradients are well connected, forming a whole scaffold, while the parallel compression curve will have fluctuations when the modulus changes, which can also be seen from the hierarchical fracture of the vertical tensile.

[0181] Figure 3 , 4 The successful synthesis of three types of bioactive factors was verified using infrared spectroscopy, elemental analysis, X-ray photoelectron spectroscopy, and transmission electron microscopy. Based on the successful linking of bioactive substances... Figure 5 Characterization was performed using Example 7 and a comparative example lacking precise response from biomarkers and sustained release of growth factors. It was observed that the hydrogel scaffold prepared in Example 7 firstly released anti-inflammatory factors in the first two weeks through intelligent ROS response, and secondly released metal ions on demand through dynamic coordination bond dissociation and repolymerization, while growth factor polymer nanoparticles were released sustainably. Figure 6 As can be seen, the external electrospun membrane of this adaptive hydrogel scaffold has a high degree of orientation, exhibiting significantly different mechanical properties in the parallel and perpendicular directions of the fibers, which helps to enhance the directional guiding effect of the scaffold.

[0182] By culturing Schwann cells and dorsal root ganglia on a scaffold, from Figure 7 As can be seen, the scaffolds exhibit good biocompatibility. Table 1 quantifies cell viability, with cell viability on different scaffolds exceeding 97%. The growth and migration of nerve axons were quantified by the extension length of the dorsal root ganglion on the scaffold. Table 2 shows that the scaffolds with random apertures in Examples 5 and 6 lacked directional induction of nerve axons, with an average nerve axon length of only 500–600 μm after 48 hours. In contrast, Examples 7 and Comparative Example 1, with their directional microchannels, provided physical clues for optimal directional extension of nerve axons, achieving an average nerve axon length of 1000–1300 μm after 48 hours. This demonstrates the directional chemotaxis and guidance effect of the scaffolds on axon growth.

[0183] Table 2. Statistics on the bioactivity and neurite growth length of the scaffold.

[0184]

[0185] The above embodiments are provided only to help understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from its principles, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a silk fibroin / hyaluronic acid interpenetrating network self-adaptable hydrogel scaffold, characterized by: Comprise the following steps: (1) Anhydrous sodium carbonate is added to boiling deionized water, stirred and mixed, and silk is added for full degumming and then washed and dried to obtain dry silk; The dry silk is added to a lithium bromide solution and stirred to dissolve, the solution is incubated, dialyzed with deionized water, and centrifuged to obtain a silk fibroin solution; (2) Hyaluronic acid powder is dissolved in deionized water and stirred uniformly at room temperature to obtain a hyaluronic acid solution; (3) adding selenocystamine hydrochloride to the hyaluronic acid solution, 1 - ethyl- (2-ethoxy- 1 -hydroxyethyl) carbodiimide hydrochloride and 3 - dimethylaminopropyl) carbodiimide hydrochloride and N - hydroxysuccinimide, obtaining a solution S1 ; - indoleacetic acid, 3 - indoleacetic acid, 1 - ethyl-( 3 - dimethylaminopropyl) carbodiimide hydrochloride and N - hydroxysuccinimide, to obtain solution S2; (4) to solution S2 is added pamidronic acid disodium hydrate, 1 - ethyl- (1-methyl- 1H-imidazol-2-yl) carbodiimide hydrochloride and 3 - dimethylaminopropyl) carbodiimide hydrochloride and N - hydroxysuccinimide, to obtain solution S3; to solution S3 is added magnesium chloride, to obtain solution S4; (5) Polyvinyl alcohol is dissolved in deionized water and stirred uniformly, and nerve growth factor is added to obtain solution S5; (6) Polycaprolactone is dissolved in dichloromethane to obtain solution S6; solution S6 is mixed with solution S5, ultrasonic emulsified to obtain emulsion S7; emulsion S7 is mixed with a polyvinyl alcohol solution, ultrasonic emulsified, and dichloromethane is volatilized at room temperature after stirring, and centrifuged to obtain nerve growth factor-coated nanoparticles NGF@PCL NPs; (7) NGF@PCL NPs are added to solution S4 to obtain solution S8; (8) The silk fibroin solution and solution S8 are mixed at room temperature, stirred uniformly, and bubbles are removed to obtain solution S9; (9) Solution S9 is injected into a mold, one end of the mold is slowly immersed in liquid nitrogen for freezing, and after complete freezing, the freeze-dried product is obtained, cross-linked in an ice bath to obtain a hydrogel scaffold; (10) Polycaprolactone is dissolved in an organic solvent to obtain a polymer solution, and electrospinning treatment is performed to obtain an oriented electrospun membrane; (11) The electrospun membrane is wrapped outside the hydrogel scaffold to obtain the silk fibroin / hyaluronic acid interpenetrating network self-adapting hydrogel scaffold.

2. The method of claim 1, wherein: In step (2), the mass concentration of the hyaluronic acid solution is 1-10%, and the stirring time is 10-50 h.

3. The method of claim 1, wherein: In step (3), the mass ratio of selenocystamine hydrochloride to hyaluronic acid in solution S1 is 5-20%, the mass ratio of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride to hyaluronic acid is 5-20%, the mass ratio of N-hydroxysuccinimide to hyaluronic acid is 2-10%, the reaction temperature is 15-40℃, and the reaction time is 2-8 h; the mass ratio of 3-indoleacetic acid to hyaluronic acid added in solution S1 is 1-20%, the mass ratio of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride to hyaluronic acid is 1-20%, the mass ratio of N-hydroxysuccinimide to hyaluronic acid is 1-10%, the reaction temperature is 15-40℃, and the reaction time is 0.5-4 h.

4. The method of claim 1, wherein: In step (4), the mass ratio of pamidronate disodium hydrate to hyaluronic acid is 5-40%, the mass ratio of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride to hyaluronic acid is 5-40%, the mass ratio of N-hydroxysuccinimide to hyaluronic acid is 1-20%, the reaction temperature is 15-40℃, the reaction time is 2-10 h, and the pH is adjusted to 8.5; the concentration of magnesium chloride in solution S4 is 20-80 mM, the reaction temperature is 15-40℃, and the reaction time is 10-60 min.

5. The method of claim 1, wherein: The mass concentration of the polyvinyl alcohol solution in step (5) is 0.1-5%, the reaction temperature is 40-100 DEG C, and the reaction time is 5-12 h; the concentration of the polycaprolactone solution in the solution S6 of step (6) is 1-20% w / v , the ultrasonic power is 400-900 W, the ultrasonic time is 5-30 min; the second ultrasonic power is 400-900 W, the ultrasonic time is 5-40 min; the stirring time at room temperature is 2-6 h; and the centrifugal condition is 12000 r / min, 20-60 min.

6. The method of claim 1, wherein: Step (7) the mass ratio of NGF@PCL NPs to hyaluronic acid in solution S8 is 5-20%.

7. The method of claim 1, wherein: Step (8) the mass ratio of silk fibroin to hyaluronic acid in solution S9 is 0.1-1.

8. The method of claim 1, wherein: The mold immersion liquid nitrogen speed in step (9) is 1-10 mm / min, the freezing time is 1-5 h, the freeze-drying time is 12-72 h, the crosslinking agent is 1 -ethyl-( 3 -dimethylaminopropyl) carbodiimide hydrochloride and N -hydroxy succinimide, the mass ratio of EDC / NHS is 3-1, and the mass concentration of the crosslinking agent solution is 1-10%.

9. The method of claim 1, wherein: In step (10), the organic solvent is at least one of chloroform, dichloromethane, dimethylformamide and methanol; the mass concentration of the polymer solution is 5-25%; the voltage for electrospinning is 5-25 kV, the temperature is 10-60 ℃, the humidity is 30-70%, the receiving distance is 5-25 cm, and the rotating speed of the drum is 800-2500 rpm.

10. A silk fibroin / hyaluronic acid interpenetrating network self-adapting hydrogel scaffold prepared by the method of any one of claims 1-9.

Images