A silk fibroin-hyaluronic acid thermosensitive bioactive hydrogel scaffold, its preparation method and application in the treatment of TBI

Abstract

The present invention discloses a silk fibroin - hyaluronic acid thermosensitive bioactive hydrogel scaffold, its preparation method and application in the treatment of TBI. A thermosensitive hydrogel is constructed using hyaluronic acid, silk fibroin, and N - isopropylacrylamide as raw materials, and loaded with bone marrow mesenchymal stem cells (BMSCs) and paeoniflorin (PF) to form a treatment unit. Hyaluronic acid is one of the components of the extracellular matrix (ECM), has a high affinity for cells, and can promote the proliferation of stem cells. Silk fibroin has high biocompatibility, biodegradability, good mechanical strength and ductility / toughness, and can promote tissue repair and the attachment and proliferation of stem cells. N - isopropylacrylamide endows the hydrogel with the property of in situ thermosensitive gelation. Therefore, the bioactive hydrogel scaffold of the present invention combines the advantages of its components, composites BMSCs and PF, overcomes the obstacle of drug transport into the brain restricted by the BBB, and is implanted into the brain injury site after TBI surgery for the treatment of nerve injury repair.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue engineering, and particularly to a silk fibroin-hyaluronic acid bioactive hydrogel scaffold, a preparation method thereof, and an application thereof in the treatment of TBI. Background Art

[0002] Traumatic brain injury (TBI) is an injury caused by external force acting on the brain and is one of the most common traumas in emergency work, with a fatality rate of 10.8%. A variety of pathological mechanisms constitute the primary and secondary injuries of TBI, causing temporary or permanent sensory and motor function deficits, cognitive dysfunction, etc. Its pathological factors are also important risk factors for a variety of neurodegenerative diseases and stroke. Therefore, the treatment of TBI secondary injury is of great significance. The inflammatory response persists for several hours to several months after the primary injury, damaging nerve cells, glial cells, and blood vessels, and is related to programmed cell death. At the same time, oxidative stress plays a key role in the pathological process of TBI. After TBI occurs, high concentrations of reactive oxygen species (ROS) cause mitochondrial membrane lipid peroxidation, destroy the membrane structure and lead to dysfunction, resulting in neuronal apoptosis. Therefore, the regeneration and repair of damaged neurons, anti-inflammation, and inhibition of oxidative stress response are the core strategies for treating TBI secondary injury.

[0003] The tissue repair and immunomodulatory potential of mesenchymal stem cells (MSCs) have received extensive attention. Transplanting MSCs in the brain injury area increases the expression of brain-derived neurotrophic factor, VEGF, EGF, BDNF, NT-3, GDNF, and synaptic proteins, promotes axonal regeneration, and repairs damaged nerves. MSCs can increase the proliferation and differentiation of neural stem cells, repair neural networks, and the mechanism of action involves the synergistic action of chemokines, growth factors, and adhesion molecules. However, there are still some problems in stem cell transplantation therapy: the local injection method, such as cerebrospinal fluid and other factors, causes the stem cells to be unable to stay in the injury site, and the insufficient local cell amount affects the treatment effect. Therefore, directly implanting at the injury site and simultaneously creating a local microenvironment conducive to the retention, survival, and proliferation of stem cells has become one of the important research contents for improving the treatment effect of stem cells.

[0004] Paeoniflorin (PF) is the main active ingredient of traditional Chinese medicines Radix Paeoniae Rubra and Radix Paeoniae Alba, and has significant curative effects in cardiovascular diseases and neurological diseases. Research shows that PF inhibits inflammation and apoptosis by targeting intracellular cascade signals, such as the NF-κB pathway, STAT3 pathway, KLF4 factor, etc. At the same time, it reduces the production of ROS and upregulates the antioxidant function to accelerate the clearance of ROS, thereby reducing the mitochondrial ROS level and alleviating mitochondrial damage under oxidative stress, thus inhibiting oxidative stress. Due to the barrier effect of the BBB, PF is a water-soluble small molecule, and its transport to the brain after intravenous injection is very limited.

[0005] The concept of tissue engineering was proposed in the 1980s. Biocompatible and biodegradable polymer materials are used to load bioactive units (such as stem cells / extracellular vesicles) for the research of damaged tissue repair and regenerative medicine. Tissue engineering technology has greatly overcome the problems of low cell retention rate and reduced survival ability affected by the microenvironment in stem cell transplantation. Hydrogel scaffolds with a three-dimensional network structure are one of the most promising dosage forms in tissue engineering due to their good biodegradability and microscopic structure similar to the extracellular matrix environment. Hydrogels have a 3D topological structure that fits the central nervous system, simulating the natural extracellular matrix environment (ECM), and are adapted to the brain biochemical environment (water content and pH value) and biophysical environment (viscoelasticity and porosity).

[0006] Silk fibroin (SF) is a natural biomaterial derived from silk, with high biocompatibility, biodegradability, and physical strength, and can promote cell adhesion and survival for tissue repair and recovery. SF-based hydrogels have been proven to support neuron growth for central nerve tissue repair. Hyaluronic acids (HA) is a linear polysaccharide and a major component of the cell ECM. Due to its biocompatibility and regulatory role in inflammation and glial scar formation, HA has become one of the most suitable materials for nerve tissue engineering. HA can specifically bind to the stem cell surface receptor CD44, promoting the adhesion and proliferation of stem cells. N-isopropylacrylamide (NIPAM) is a typical temperature-responsive polymer with a lower critical phase transition temperature of 32 °C, which is close to the human body temperature and can be used as an ideal material for preparing temperature-responsive hydrogels.

[0007] Hydrogel scaffolds based on natural biomaterials are used for brain implantation and have the characteristics of adapting to the central biophysical environment and good safety in the treatment of TBI. However, it is difficult to study bioactive hydrogel scaffolds that simultaneously meet functionality and biocompatibility. Therefore, it is of research value and application potential to prepare a bioactive hydrogel scaffold with central adaptability that simultaneously loads BMSCs and PF to promote nerve regeneration and repair in TBI. Summary of the Invention

[0008] To solve the above technical problems, the present invention provides a silk fibroin-hyaluronic acid thermosensitive bioactive hydrogel scaffold, its preparation method, and its application in the treatment of TBI. The present invention utilizes that hyaluronic acid is one of the components of the extracellular matrix (ECM), has a high affinity for cells, and can promote the proliferation of stem cells. Silk fibroin has high biocompatibility, biodegradability, good mechanical strength, and ductility / toughness, and can promote tissue repair and cell attachment and survival. Therefore, the bioactive hydrogel scaffold of the present invention combines the advantages of the two components, composites BMSCs and PF, overcomes the obstacle of drug transport into the brain restricted by the BBB, and is implanted into the brain injury site after TBI surgery for nerve injury repair treatment, such as the improvement of sensory and motor functions, the improvement of cognitive and memory functions, and the regeneration and repair of damaged nerves.

[0009] The first object of the present invention is to provide a silk fibroin-hyaluronic acid thermosensitive bioactive hydrogel scaffold. A thermosensitive hydrogel is constructed from hyaluronic acid, silk fibroin, and N-isopropylacrylamide, and the thermosensitive hydrogel is loaded with bone marrow mesenchymal stem cells and paeoniflorin; the interior of the thermosensitive bioactive hydrogel presents a three-dimensional porous network structure. The present invention utilizes the porous network structure to provide space for cell extension and proliferation, and can also load drugs.

[0010] In some embodiments of the present invention, the loading amount of the bone marrow mesenchymal stem cells is 1×10 5 -1×10 6 cells in every 20 μL of the 4% (mass fraction) silk fibroin-hyaluronic acid thermosensitive bioactive hydrogel scaffold.

[0011] In some embodiments of the present invention, the loading amount of paeoniflorin is 400-500 μM.

[0012] The second object of the present invention is to provide a preparation method of the silk fibroin-hyaluronic acid thermosensitive hydrogel scaffold, comprising the following steps:

[0013] S1. Connect N-isopropylacrylamide and hyaluronic acid through a redox reaction and an amidation reaction to obtain N-isopropylacrylamide-hyaluronic acid (NIPAM-HA);

[0014] S2. Graft silk fibroin onto hyaluronic acid in N - isopropylacrylamide - hyaluronic acid through amidation reaction to obtain the hydrogel NIPAM@HA - SF precursor;

[0015] S3. Dissolve PF using the hydrogel NIPAM@HA - SF precursor, after sterilization, resuspend BMSCs

[0016] In some embodiments of the present invention, in step S1, after deoxygenating the N - isopropylacrylamide aqueous solution in an ice bath, add potassium persulfate solution and 2 - mercaptoethylamine hydrochloride (AET) solution, mix and react, and then add hyaluronic acid solution. Under the catalysis of EDC / NHS, react to obtain N - isopropylacrylamide - hyaluronic acid.

[0017] In some embodiments of the present invention, the concentration of the N - isopropylacrylamide aqueous solution is 1.35 - 135 mmol / L;

[0018] The reaction time with potassium persulfate and 2 - mercaptoethylamine is 3 - 5 h;

[0019] The concentration of the hyaluronic acid solution is 0.032 - 0.512 g / mL.

[0020] In some embodiments of the present invention, the concentration of the potassium persulfate solution is 0.0135 - 0.216 mol / L;

[0021] The concentration of the AET solution with potassium persulfate and AET is 0.027 - 0.432 mol / L.

[0022] In some embodiments of the present invention, in step S2, mix N - isopropylacrylamide - hyaluronic acid and silk fibroin, react under the catalysis of EDC / NHS, and then dialyze the reaction solution and freeze - dry to obtain the NIPAM@HA - SF.

[0023] The third object of the present invention is to provide a nerve regeneration material, including the silk fibroin - hyaluronic acid thermosensitive hydrogel scaffold.

[0024] The fourth object of the present invention is to provide the application of the silk fibroin - hyaluronic acid thermosensitive hydrogel scaffold in traumatic brain injury repair materials.

[0025] The above - mentioned technical solutions of the present invention have the following advantages compared with the prior art:

[0026] The present invention utilizes the fact that hyaluronic acid is one of the components of the extracellular matrix (ECM), and silk fibroin, which has a high affinity for cells, has high biocompatibility, biodegradability, good mechanical strength, and ductility / toughness, and can promote tissue repair and cell attachment and survival. Therefore, the biological scaffold of the present invention combines the advantages of the two components. Its combination with BMSCs and PF can improve the immune microenvironment at the lesion site, inhibit inflammation and oxidative stress damage by regulating mitophagy, inhibit apoptosis, promote the regeneration of damaged axons and myelin sheaths, and induce nerve regeneration and repair, ultimately promoting the recovery of sensory, motor, and cognitive functions in TBI mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to make the content of the present invention easier to be clearly understood, the following further describes the present invention in detail according to the specific embodiments of the present invention in combination with the drawings, wherein,

[0028] Figure 1 is the scanning electron microscope image of the hydrogel obtained in Example 1 of the present invention;

[0029] Figure 2 is the elastic modulus diagram of the hydrogel in Example 2 of the present invention (platform temperature 37 °C, strain 1%, dynamic oscillation frequency range 1 - 200 rad / s);

[0030] Figure 3 is the statistical chart of the mass retention rate of the hydrogel in Example 2 of the present invention;

[0031] Figure 4 is the 3D growth image of BMSCs in the hydrogel in Example 2 of the present invention;

[0032] Figure 5 is the Morris water maze test of each group of mice in Example 4 of the present invention (29 - 34 days after TBI): A. Statistical chart of escape latency in the positioning cruise stage; B: Statistical chart of the number of times of crossing the platform in the spatial exploration stage; C: Swimming trajectory diagram in the spatial exploration stage; D: Swimming heat map in the spatial exploration stage ( *P <0.05, compared with the control group; **P <0.01, compared with the control group; ***P <0.001, compared with the control group);

[0033] Figure 6 is the result of the stepping stone test of each group of mice in Example 5 of the present invention (*** P <0.001, compared with the control group; ****P <0.0001, compared with the control group);

[0034] Figure 7is the mNSS scores of mice in each group in Example 6 of the present invention ( **P <0.01, compared with the control group; ****P <0.0001, compared with the control group);

[0035] Figure 8 are the results of neuron migration and regeneration in Example 7 of the present invention. A is the positive expression of DCX and MAP-2 in the brain tissues of mice in each group (35 days after traumatic brain injury). B is the percentage of positive expression of DCX and MAP-2 in the brain tissues of mice in each group (n = 3). Specific embodiments

[0036] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the exemplified embodiments are not intended to limit the present invention.

[0037] Example 1 Preparation and prescription optimization of temperature-sensitive hydrogel scaffolds

[0038] Connect the thermosensitive polymer NIPAM with HA by redox reaction and amidation reaction. Weigh an appropriate amount of NIPAM (1.224 g), dissolve it in deionized water, and remove oxygen with nitrogen under ice bath conditions for 1 h. KPS (0.0296 g) and AET (0.0248 g) are respectively dissolved in deionized water and added to the reaction. After 4 h, add the pre-dissolved HA (0.256 g), catalyzed by EDC (0.200 g) / NHS (0.100 g), and protect it at low temperature for 24 h to obtain NIPAM-HA. 2 Protect the low-temperature reaction for 24 h to obtain NIPAM-HA.

[0039] Graft SF onto HA by amidation reaction. Mix NIPAM-HA and SF (0.256 g), catalyzed by EDC (0.400 g) / NHS (0.200 g), and protect it at low temperature for 24 h. Dialyze for 3 days, collect the product, freeze it at -80°C overnight, and lyophilize it with a vacuum freeze dryer for 3 days to obtain the NIPAM@HA-SF lyophilized product. 2 Protect the low-temperature reaction for 24 h. Dialyze for 3 days, collect the product, freeze it at -80°C overnight, and lyophilize it with a vacuum freeze dryer for 3 days to obtain the NIPAM@HA-SF lyophilized product.

[0040] Through preliminary prescription screening, 3 factors that have the greatest influence on the elastic modulus and gelation time are found, and a three-factor three-level central composite design is carried out on them to further screen and optimize the hydrogel prescription. Taking the elastic modulus and gelation time as the investigation indexes, the corresponding hydrogels are prepared and compared. Considering the elastic modulus of the brain tissue and the ideal gelation time, the optimal prescription is selected as the NIPA dosage of 1.35 mmol, SF / HA = 1, and the mass fraction is 4%.

[0041] Table 1. Central composite screening of elastic modulus and gelation time of NIPAM@HA-SF

[0042] Group Number Factor 1 Factor 2 Factor 3 Result 1 Result 2 Std Run A: NIPAM B: SF / HA C: wt% Elastic Modulus Gelation Time mmol 1 % Pa h 11 1 68.175 1 6 3823.5 12 9 4 68.175 0.6 6 187.7 0.5 3 5 1.35 1.4 1 276.9 60 10 6 68.175 0.6 3 2440.8 2.25 5 7 1.35 0.6 5 2638.9 0.25 1 11 1.35 0.6 1 306.2 2.25 7 12 1.35 1.4 5 1791.1 0.25 13 13 1.35 1 4 733.9 0.33

[0043] Example 2. Characterization of thermosensitive hydrogel scaffolds

[0044] ① Observation of the microstructure of the hydrogel: The prefabricated hydrogel precursor with the group number 13 obtained in Example 1 was dispensed into a silicone mold, 2 mL per well. Subsequently, the silicone mold was placed in an incubator at 37 °C until the gel was completely formed, stored at -80 °C overnight, freeze-dried using a vacuum freeze dryer, fractured in liquid nitrogen to expose the cross-section, and sputter-coated (to increase surface conductivity). Finally, the internal microstructure of the hydrogel was observed using a scanning electron microscope (SEM). The results are shown in Figure 1 . The results showed that the hydrogel presented a three-dimensional porous network structure inside. The porous network structure can provide space for cell extension and proliferation, and conditions for the exchange of oxygen, nutrients, and metabolites.

[0045] ② Measurement of the elastic modulus: The prefabricated hydrogel precursor with the group number 13 obtained in Example 1 was added to a silicone mold, and the liquid level was controlled at 2 cm. Then it was placed in an incubator at 37 °C. After the gel was formed, the sample was taken out and placed on the rheometer platform. First, a strain sweep was performed to determine the linear viscoelastic region of the hydrogel. The strain range was set from 1% to 1000%, and the oscillation frequency was 6.2832 rad / s. Subsequently, a frequency sweep was performed, and the platform temperature was set at 37 °C to measure the elastic modulus of the hydrogel. The results are shown in Figure 2 . The results showed that the elastic modulus of the optimal formulation was in line with that of brain tissue and could be used for intracranial use.

[0046] ③ Degradation performance: After the gel obtained in step ① was formed, the mass of the hydrogel was weighed and recorded as W 0 , and then it was soaked in artificial cerebrospinal fluid (ACSF) at 37 °C. On the 1st, 3rd, 7th, 14th, 21st, 28th, and 35th days, the samples were taken out respectively, and the surrounding moisture was blotted dry with filter paper. The mass of the hydrogel was recorded again as W n . The mass retention rate of the hydrogel was calculated using the following formula:

[0047] ,

[0048] Three parallel samples were taken for each specimen for measurement and statistical analysis and comparison. The experimental results are shown in Figure 3 . The results showed that the hydrogel had good biodegradability and was basically completely degraded after 35 days.

[0049] ④ Biocompatibility: The hydrogel obtained in step ① was co-incubated with BMSCs, and the viability of BMSCs was detected using the Calcein / PI Cell Viability and Cytotoxicity Detection Kit. The specific steps are as follows: Take BMSCs in good growth state, digest, centrifuge, discard the supernatant, resuspend with complete medium for MSCs and count. Inoculate 1×10 5 cells into a confocal dish, and add the sterilized hydrogel obtained in step ①. Place it in an incubator at 37 °C with 5% CO 2 for culture. After culturing for 1, 3, and 7 days, aspirate the supernatant, wash with PBS, add 1 mL of Calcein AM / PI detection working solution, and incubate at 37 °C in the dark for 40 min. Observe the staining effect with a laser confocal microscope. Live cells show green fluorescence and dead cells show red fluorescence. The experimental results are shown in Figure 4 . The results show that after culturing for 7 days, BMSCs can adhere and spread on the surface of the hydrogel, and the cell growth state is good, indicating that the hydrogel has good biocompatibility.

[0050] Example 3 Establishment of a TBI animal model and transplantation of a composite scaffold

[0051] (I) Material preparation: The specific preparation steps of PF@Gel, BMSCs@Gel, and PF + BMSCs@Gel are as follows (here Gel refers to NIPAM@HA-SF obtained in Example 1):

[0052] Preparation steps of PF@Gel material: Prepare a Gel precursor with a mass fraction of 4%. After sterilization, take 20 μL to dissolve PF, and the dosage of PF is 0.5 mg.

[0053] Preparation steps of BMSCs@Gel material: Prepare a Gel precursor with a mass fraction of 4%. After sterilization, take 20 μL to resuspend BMSCs, and the dosage of BMSCs is 1×10 6 cells.

[0054] Preparation steps of PF + BMSCs@Gel material: Prepare a Gel precursor with a mass fraction of 4%. After sterilization, take 20 μL to dissolve PF and resuspend BMSCs. The dosage of PF is 0.5 mg and the dosage of BMSCs is 1×10 6 cells.

[0055] (2) Prepare 50 male C57 mice (20±2 g) and divide them into 5 groups evenly: sham operation group (n = 10), control group (not administered drugs, n = 10), PF@Gel group (n = 10), BMSCs@Gel group (n = 10), PF + BMSCs@Gel group (n = 10). The TBI model was established using a CCI instrument. First, fix the anesthetized mice on a stereotaxic frame. Incise the head skin along the midline of the head and longitudinally cut 1 cm to strip the periosteum. Use a dental drill to make a round hole with a diameter of 3 mm at 1 mm behind the bregma and 2 mm to the right of the coronal suture. Select a needle with a diameter of 2.5 mm, and set the operating parameters of the instrument to 5 m / s, 1.0 mm depth, and 150 ms. Impact the mouse brain according to the set parameters. After the operation, inject 20 μL of the drug-loaded hydrogel precursor into the injury site, and disinfect and suture the wound. Among them, the sham operation group only underwent craniotomy, and the control group only underwent modeling without drug treatment.

[0056] Example 4 Evaluation of Cognitive Memory Function of TBI Mice

[0057] The Morris water maze (MWM) was evaluated 21 - 27 days after brain injury to explore the cognitive memory function of each group of mice obtained in Example 3. Briefly, a cylindrical pool (inner diameter of 180 cm) filled with a mixture of white pigment and water was evenly divided into four quadrants. Then, each mouse (n = 10 in each group) was forced to swim in the pool to find a hidden platform. Spatial learning studies were conducted from the 29th day to the 33rd day, and the escape latency was collected. Subsequently, the platform was removed from the pool, and a spatial memory study was conducted on the 34th day. The quadrant residence time (swimming time spent in the platform quadrant) and the number of platform crossings were collected during the spatial memory study. The experimental results are shown in Figure 5 , and it can be seen from Figure 5 that PF + BMSCs@Gel can improve the cognitive memory function of TBI mice.

[0058] Example 5 Evaluation of Motor Function of TBI Mice after Treatment

[0059] Randomly place each group of TBI mice obtained in Example 3 on a specific grid, observe and record the number of times the forelimbs touch the ground and the total number of steps taken by the forelimbs during the movement of the traumatic brain injury mice. Calculate the ratio of the number of times of touching the ground to the total number of steps. The larger the value, the more severe the nerve injury of the TBI mice. Behavioral evaluation and statistical analysis of the mice were carried out for 1 - 35 d. The results showed ( Figure 6 ) that compared with the control group, the rate of missing steps in the PF + BMSCs@Gel group was significantly reduced, indicating that PF + BMSCs@Gel can improve the sensorimotor function of mice with brain trauma.

[0060] Example 6 Neurological deficit functional score after treatment of TBI mice

[0061] In addition, the modified neurological severity score (mNSS) was calculated to explore the repair of neurological injury function in the five groups of mice obtained in Example 3 at 1-35 days after TBI. The experimental results are shown in Figure 7 , compared with the control group, the mNSS score of the PF+BMSCs@Gel group was significantly reduced, indicating that PF+BMSCs@Gel could improve the sensorimotor function of mice with traumatic brain injury.

[0062] Example 7 Evaluation of endogenous neural stem cell regeneration and differentiation in TBI mice

[0063] Immunofluorescence staining was performed on the brain tissues of each group of mice obtained in Example 3 to evaluate the expression of proteins related to nerve migration and regeneration (DCX, MAP-2). The experimental results are shown in Figure 8 , the results showed that the positive expression of DCX in the PF+BMSCs@Gel group was significantly increased, indicating that neuron migration and regeneration were significantly improved.

[0064] Obviously, the above examples are only for illustration and are not intended to limit the implementation. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementations here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A silk fibroin-hyaluronic acid thermosensitive bioactive hydrogel scaffold, characterized in that: A thermosensitive hydrogel is constructed using hyaluronic acid, silk fibroin and N-isopropylacrylamide as raw materials, wherein the thermosensitive hydrogel is loaded with bone marrow mesenchymal stem cells and paeoniflorin; the thermosensitive bioactive hydrogel has a three-dimensional porous network structure; the silk fibroin-hyaluronic acid thermosensitive bioactive hydrogel scaffold is prepared by the following method: S1, connecting N-isopropylacrylamide and hyaluronic acid through redox reaction and amide formation reaction to obtain N-isopropylacrylamide-hyaluronic acid; S2, grafting silk fibroin onto hyaluronic acid in N-isopropylacrylamide-hyaluronic acid through an amide reaction to obtain a hydrogel NIPAM@HA-SF precursor; S3, using the hydrogel NIPAM@HA-SF precursor obtained in step S2 to dissolve PF, sterilize, and resuspend BMSCs to obtain the silk fibroin-hyaluronic acid thermosensitive bioactive hydrogel scaffold; After deoxygenating the N-isopropylacrylamide aqueous solution in an ice bath, potassium persulfate solution and 2-mercaptoethylamine solution are added, mixed and reacted, and then hyaluronic acid solution is added, and N-isopropylacrylamide-hyaluronic acid is obtained by reaction under the catalysis of EDC / NHS; The concentration of the N-isopropylacrylamide aqueous solution is 1.35-135 mmol / L; The concentration of the 2-mercaptoethylamine solution is 0.027-0.432 mol / L.

2. The silk fibroin-hyaluronic acid thermosensitive bioactive hydrogel scaffold according to claim 1, characterized in that: The loading amount of the bone marrow mesenchymal stem cells is 1×10 5 -1×10 6 cells.

3. The silk fibroin-hyaluronic acid thermosensitive bioactive hydrogel scaffold according to claim 1, characterized in that: The loading amount of paeoniflorin is 400-500 μM.

4. The silk fibroin-hyaluronic acid thermosensitive bioactive hydrogel scaffold according to claim 1, characterized in that: The reaction time with potassium persulfate and 2-mercaptoethylamine is 3 to 5 hours; The concentration of the hyaluronic acid solution is 0.032-0.512 g / mL.

5. The silk fibroin-hyaluronic acid thermosensitive bioactive hydrogel scaffold according to claim 1, characterized in that: The concentration of potassium persulfate solution is 0.0135-0.216 mol / L.

6. The silk fibroin-hyaluronic acid thermosensitive bioactive hydrogel scaffold according to claim 1, characterized in that: In step S2, N-isopropylacrylamide-hyaluronic acid and silk fibroin are mixed and reacted under the catalysis of EDC / NHS, and then the reaction solution is dialyzed and freeze-dried to obtain the NIPAM@HA-SF.

7. A nerve regeneration and repair preparation, characterized in that: It comprises the silk fibroin-hyaluronic acid thermosensitive hydrogel scaffold as described in any one of claims 1 to 6.

8. Use of the silk fibroin-hyaluronic acid thermosensitive bioactive hydrogel scaffold according to any one of claims 1 to 6 or the nerve regeneration repair preparation according to claim 7 in a traumatic brain injury repair preparation.

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