Silk fibroin-hyaluronic acid temperature-sensitive bioactive hydrogel stent, preparation method thereof and application of silk fibroin-hyaluronic acid temperature-sensitive bioactive hydrogel stent in TBI treatment

Abstract

The invention discloses a silk fibroin-hyaluronic acid temperature-sensitive bioactive hydrogel scaffold, a preparation method thereof and application of the silk fibroin-hyaluronic acid temperature-sensitive bioactive hydrogel scaffold in TBI treatment, hyaluronic acid, silk fibroin and N-isopropylacrylamide are used as raw materials to construct temperature-sensitive hydrogel, and bone marrow mesenchymal stem cells (BMSCs) and paeoniflorin (PF) are loaded to form a treatment unit. Hyaluronic acid is one of components of an extracellular matrix (ECM), has high affinity to cells, and can promote proliferation of stem cells. Silk fibroin has high biocompatibility and biodegradability and good mechanical strength and ductility / toughness, and can promote tissue repair and stem cell attachment and proliferation. N-isopropylacrylamide endows the hydrogel with the characteristic of in-situ temperature-sensitive gelling. Therefore, the bioactive hydrogel stent has the advantages of components, combines the BMSCs and the PF, overcomes the obstacle that the medicine is limited by BBB to be transferred into the brain, and is implanted into a brain injury part after a TBI surgical operation to be used for repairing and treating nerve injury.

Description

Technical Field

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

[0002] Traumatic brain injury (TBI) is an injury caused by external force acting on the brain. It is one of the most common injuries in emergency work, with a mortality rate of 10.8%. Various pathological mechanisms constitute primary and secondary injuries of TBI, causing temporary or permanent sensory and motor function loss, cognitive dysfunction, etc. Its pathological factors are also important risk factors for various neurodegenerative diseases and stroke. Therefore, the treatment of secondary injuries of TBI is of great significance. The inflammatory response lasts for several hours to several months after the primary injury, damaging neurons, 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 cause dysfunction, leading to neuronal apoptosis. Therefore, regeneration and repair of damaged neurons, anti-inflammatory, and inhibition of oxidative stress response are the core strategies for treating secondary injuries of TBI.

[0003] Mesenchymal stem cells (MSCs) have received extensive attention for their tissue repair and immunomodulatory potential. Transplantation of MSCs in the brain injury area increases the expression of brain-derived neurotrophic factor, VEGF, EGF, BDNF, NT-3, GDNF and synaptic proteins, promotes axon regeneration, and repairs damaged nerves. MSCs can increase the proliferation and differentiation of neural stem cells and repair neural networks. The mechanism of action involves the synergistic effects of chemokines, growth factors and adhesion molecules. However, there are still some problems with stem cell transplantation therapy: local injection methods, such as factors such as cerebrospinal fluid, cause stem cells to be unable to reside in the site of injury, and the local cell quantity is insufficient, affecting the therapeutic effect. Therefore, direct implantation in the site of injury and the creation of a local microenvironment that is conducive to stem cell retention, survival and proliferation have become one of the important research contents to improve the efficacy of stem cell therapy.

[0004] Paeoniflorin (PF) is the main active ingredient of the traditional Chinese medicine red peony root and white peony root, and has significant therapeutic effects in cardiovascular and nervous system diseases. Studies have shown 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 ROS production and upregulates antioxidant function to accelerate ROS clearance to reduce mitochondrial ROS levels, alleviate mitochondrial damage under oxidative stress, and thus inhibit oxidative stress. Due to the barrier function of the BBB, PF is a water-soluble small molecule, and its brain transport is very limited after intravenous injection.

[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 research on 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 therapy. Hydrogel scaffolds with three-dimensional network structures are one of the most promising dosage forms for tissue engineering due to their good biodegradability and microstructures similar to the extracellular matrix environment. Hydrogels have a 3D topological structure that fits the central nervous system, simulates the natural extracellular matrix environment (Extracellularmatrix, ECM), and adapts to the brain's biochemical environment (water content and pH value) and biophysical environment (viscoelasticity and porosity).

[0006] Silk fibroin (SF) is a natural biomaterial derived from silk. It has high biocompatibility, biodegradability, and physical strength. It can promote cell attachment and survival for tissue repair and recovery. SF-based hydrogels have been shown to support neuronal growth for central nervous system tissue repair. Hyaluronic acid (HA) is a linear polysaccharide and the main component of 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 neural tissue engineering. HA can specifically bind to the stem cell surface receptor CD44 to promote the attachment 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 human body temperature. It 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 TBI treatment. They are adaptable to the central biophysical environment and have good safety. However, it is difficult to study bioactive hydrogel scaffolds that meet both functionality and biocompatibility. Therefore, it is of research value and application potential to prepare a bioactive hydrogel scaffold with central adaptability and loaded with BMSCs and PF to promote TBI nerve regeneration and repair. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides a silk fibroin-hyaluronic acid thermosensitive bioactive hydrogel scaffold, a preparation method thereof, and an application thereof in the treatment of TBI. The present invention utilizes hyaluronic acid as one of the components of the extracellular matrix (ECM), which has a high affinity for cells and can promote the proliferation of stem cells. Silk fibroin has high biocompatibility and 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 has the advantages of both components, compounding BMSCs and PF, overcoming the obstacle of drug transport into the brain restricted by BBB, and being implanted in the brain injury site after TBI surgery for nerve injury repair treatment, such as improvement of sensory motor function, improvement of cognitive memory function, and 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, wherein the thermosensitive hydrogel is constructed using hyaluronic acid, silk fibroin and N-isopropylacrylamide as raw materials, wherein bone marrow mesenchymal stem cells and paeoniflorin are loaded in the thermosensitive hydrogel; the thermosensitive bioactive hydrogel has a three-dimensional porous network structure inside. 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.

[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 method for preparing the silk fibroin-hyaluronic acid thermosensitive hydrogel scaffold, comprising the following steps:

[0013] S1, connecting N-isopropylacrylamide and hyaluronic acid through redox reaction and amide formation reaction to obtain N-isopropylacrylamide-hyaluronic acid (NIPAM-HA);

[0014] S2, grafting silk fibroin onto hyaluronic acid in N-isopropylacrylamide-hyaluronic acid through an amide reaction to obtain a hydrogel NIPAM@HA-SF precursor;

[0015] S3. Dissolve PF using hydrogel NIPAM@HA-SF precursor, sterilize, and resuspend BMSCs

[0016] In some embodiments of the present invention, in step S1, after the N-isopropylacrylamide aqueous solution is deoxygenated in an ice bath, a potassium persulfate solution and a 2-mercaptoethylamine hydrochloride (AET) solution are added, mixed and reacted, and then a hyaluronic acid solution is added. Under the catalysis of EDC / NHS, N-isopropylacrylamide-hyaluronic acid is obtained by reaction.

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

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

[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, 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.

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

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

[0025] The above technical solution of the present invention has the following advantages compared with the prior art:

[0026] The present invention utilizes hyaluronic acid, which is one of the components of the extracellular matrix (ECM), and silk fibroin, which has high affinity for cells, has high biocompatibility and biodegradability, good mechanical strength and ductility / toughness, and can promote tissue repair and cell attachment and survival. Therefore, the bioscaffold of the present invention has the advantages of both components. It combines BMSCs and PF to improve the immune microenvironment of the lesion site, inhibit inflammation and oxidative stress damage by regulating mitochondrial autophagy, inhibit cell 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 more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0028] Figure 1 is a 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 to 200 rad / s);

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

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

[0032] Figure 5 The Morris water maze test of mice in each group in Example 4 of the present invention (29-34 days after TBI surgery): A, statistical graph of escape latency in the positioning cruising stage; B: statistical graph of the number of platform crossings in the space exploration stage; C, swimming trajectory graph in the space exploration stage; D: swimming heat map in the space 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 air stepping 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 score of each group of mice 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 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 tissue of each group of mice (35 days after traumatic brain injury). B is the percentage of DCX and MAP-2 positive expression in the brain tissue of each group of mice (n=3). DETAILED DESCRIPTION

[0036] The present invention is 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 implement it, but the embodiments are not intended to limit the present invention.

[0037] Example 1 Preparation and formulation optimization of thermosensitive hydrogel scaffold

[0038] The thermosensitive polymer NIPAM was connected to HA by redox reaction and amide formation reaction. Weigh an appropriate amount of NIPAM (1.224 g), dissolve it in deionized water, and deoxygenate it with nitrogen in an ice bath for 1 hour. KPS (0.0296 g) and AET (0.0248 g) were dissolved in deionized water and put into the reaction. After 4 hours, HA (0.256 g) dissolved in advance was added, EDC (0.200 g) / NHS (0.100 g) was catalyzed, and N 2 The reaction was kept at low temperature for 24 hours to obtain NIPAM-HA.

[0039] SF was grafted onto HA by amide formation reaction. NIPAM-HA and SF (0.256 g) were mixed and catalyzed by EDC (0.400 g) / NHS (0.200 g). 2 The reaction was protected at low temperature for 24 hours. Dialysis was performed for 3 days, and the product was collected, stored at -80°C overnight, and freeze-dried in a vacuum freeze dryer for 3 days to obtain the freeze-dried product of NIPAM@HA-SF.

[0040] Through the preliminary prescription screening, the three factors with the greatest impact on the elastic modulus and gelation time were found. A three-factor three-level star point screening design was performed to further screen and optimize the hydrogel prescription. The elastic modulus and gelation time were used as the evaluation indicators to prepare the corresponding hydrogels and compare them. Based on the elastic modulus of brain tissue and the ideal gelation time, the optimal prescription was selected as 1.35mmol NIPAM dosage, SF / HA=1, and 4% mass fraction.

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

[0042]

[0043] Example 2 Characterization of Thermosensitive Hydrogel Scaffold

[0044] ① Observation of the microscopic morphology of the hydrogel: The prefabricated hydrogel precursor of group number 13 obtained in Example 1 was dispensed into a silica gel mold, 2 mL per well, and then the silica gel mold was placed in a 37°C constant temperature box until the gel was completely formed, kept at -80°C overnight, freeze-dried using a vacuum freeze dryer, and exposed by liquid nitrogen fracture, gold spraying (to increase surface conductivity), and finally the internal microscopic morphology of the hydrogel was observed using a scanning electron microscope (SEM). The results are shown in Figure 1 The results showed that the hydrogel had a three-dimensional porous network structure. The porous network structure could provide space for cells to expand and proliferate, and provide conditions for the exchange of oxygen, nutrients and metabolites.

[0045] ② Determination of elastic modulus: Add the prefabricated hydrogel precursor of group number 13 obtained in Example 1 into a silicone mold, control the liquid level at 2 cm, and place it in a 37°C constant temperature box. After the gel is formed, take out the sample and place it on the rheometer platform. First, perform a strain scan to determine the linear viscoelastic range of the hydrogel. Set the strain range to 1% to 1000% and the oscillation frequency to 6.2832 rad / s. Then perform a frequency scan, set the platform temperature to 37°C, and measure the elastic modulus of the hydrogel. The results are shown in Figure 2 The results showed that the elastic modulus of the optimal prescription was consistent with the elastic modulus of brain tissue and could be used for intracranial use.

[0046] ③ Degradation performance: After the gel obtained in step ① is formed, the mass of the hydrogel is weighed and recorded as W 0 Then, the hydrogel was placed in artificial cerebral-spinal fluid (ACSF) at 37°C and taken out on days 1, 3, 7, 14, 21, 28, and 35, and the surrounding water was dried with filter paper. The mass of the hydrogel was recorded again as W. n , calculate the hydrogel mass retention rate, the formula is as follows:

[0047]

[0048] Three parallel samples were taken from each sample 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 almost completely degraded after 35 days.

[0049] ④ Biocompatibility: The hydrogel obtained in step ① was co-incubated with BMSCs, and the survival of BMSCs was detected using the Calcein / PI cell activity and cytotoxicity detection kit. The specific steps are as follows: BMSCs with good growth status were taken, digested and centrifuged, and the supernatant was discarded. The BMSCs were resuspended in MSCs complete medium and counted. 1×10 5 The cells were inoculated into the confocal dish, and the sterilized hydrogel obtained in step ① was added and placed at 37°C and 5% CO. 2 Culture in a cell culture incubator. After 1, 3, and 7 days of culture, discard the supernatant, wash with PBS, add 1 mL of CalceinAM / PI detection working solution, and incubate at 37°C in the dark for 40 minutes. 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 showed that after 7 days of culture, BMSCs could attach and stretch on the hydrogel surface, and the cells grew well, indicating that the hydrogel had good biocompatibility.

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

[0051] (I) Material preparation: The specific preparation steps of PF@Gel, BMSCs@Gel, and PF+BMSCs@Gel are as follows (Gel here 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%, and after sterilization, take 20 μL to dissolve PF, wherein the amount of PF used is 0.5 mg.

[0053] Preparation steps of BMSCs@Gel material: prepare a 4% gel precursor, sterilize and take 20 μL to resuspend BMSCs, where the amount of BMSCs is 1×10 6 indivual.

[0054] Preparation steps of PF+BMSCs@Gel material: prepare a Gel precursor with a mass fraction of 4%, take 20 μL of it after sterilization to dissolve PF, and resuspend BMSCs, where the amount of PF is 0.5 mg and the amount of BMSCs is 1×10 6 indivual.

[0055] (ii) Fifty male C57 mice (20 ± 2 g) were prepared and divided into five groups: sham operation group (n = 10), control group (no drug, n = 10), PF@Gel group (n = 10), BMSCs@Gel group (n = 10), and PF+BMSCs@Gel group (n = 10). The TBI model was established using the CCI instrument. First, the anesthetized mice were fixed on a stereotaxic frame. The head skin was incised along the midline of the head and 1 cm was cut longitudinally to peel off the periosteum. A 3 mm diameter circular hole was opened with a bone drill 1 mm behind the fontanelle and 2 mm to the right of the coronal suture. A needle with a diameter of 2.5 mm was selected, and the operating parameters of the instrument were set to 5 m / s, 1.0 mm depth, and 150 ms. The mouse brain was impacted according to the set parameters. After the operation, 20 μL of drug-containing hydrogel precursor was injected into the injury site according to the group, and the wound was disinfected and sutured. The sham operation group was only given bone window treatment, while the control group was only given modeling without drug treatment.

[0056] Example 4 Evaluation of cognitive memory function in TBI mice

[0057] Morris water maze (MWM) assessment was performed 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 180 cm) filled with a mixture of white pigment and water was evenly divided into four quadrants. Then, each mouse (n=10 per group) was forced to swim in the pool to find a hidden platform. Spatial learning studies were performed from day 29 to day 33, and escape latencies were collected. Subsequently, the platform was removed from the pool, and a spatial memory study was performed on day 34. Quadrant residence time (swimming time spent in the platform quadrant) and the number of site crossings were collected in the spatial memory study. The experimental results are shown in Figure 5 ,Depend on Figure 5 It can be seen that PF+BMSCs@Gel can improve the cognitive memory function of TBI mice.

[0058] Example 5 Evaluation of motor function in TBI mice after treatment

[0059] Each group of TBI mice obtained in Example 3 was randomly placed on a specific grid, and the number of times the forelimbs of the mice with traumatic brain injury stepped on the ground and the total number of steps taken by the forelimbs during movement were observed and recorded. The ratio of the number of steps to the total number of steps was calculated, and the larger the value, the more severe the neurological damage of the TBI mice. The mice were evaluated and statistically analyzed for 1-35 days of behavior. The results showed that ( Figure 6 ): Compared with the control group, the missing rate 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 function scores after treatment of TBI mice

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

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

[0063] Immunofluorescence staining was performed on the brain tissue of each group of mice obtained in Example 3 to evaluate the expression of proteins related to neural 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 increased significantly, indicating that ,neuronal migration and regeneration were significantly improved.

[0064] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled 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 implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the 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. The thermosensitive hydrogel is loaded with bone marrow mesenchymal stem cells and paeoniflorin. The thermosensitive bioactive hydrogel has a three-dimensional porous network structure.

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. A method for preparing a silk fibroin-hyaluronic acid thermosensitive bioactive hydrogel scaffold as claimed in any one of claims 1 to 3, characterized in that: The following steps are involved: 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. Dissolve PF using the hydrogel NIPAM@HA-SF precursor obtained in step S2, sterilize, and resuspend BMSCs to obtain the silk fibroin-hyaluronic acid thermosensitive bioactive hydrogel scaffold.

5. The preparation method according to claim 4, characterized in that: In step S1, after deoxygenating the N-isopropylacrylamide aqueous solution in an ice bath, a potassium persulfate solution and a 2-mercaptoethylamine solution are added, mixed and reacted, and then a hyaluronic acid solution is added, and N-isopropylacrylamide-hyaluronic acid is obtained by reaction under the catalysis of EDC / NHS.

6. The preparation method according to claim 5, characterized in that: The concentration of the N-isopropylacrylamide aqueous solution is 1.35-135 mol / L; 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.

7. The preparation method according to claim 5, characterized in that: The concentration of potassium persulfate solution is 0.0135-0.216 mol / L; The concentration of the AET solution is 0.027-0.432 mol / L.

8. The preparation method according to claim 4, 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.

9. A nerve regeneration and repair preparation, characterized in that: The invention comprises the silk fibroin-hyaluronic acid thermosensitive hydrogel scaffold as described in any one of claims 1 to 3.

10. Use of the silk fibroin-hyaluronic acid thermosensitive bioactive hydrogel scaffold according to any one of claims 1 to 3 or the nerve regeneration repair preparation according to claim 9 in a traumatic brain injury repair preparation.

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