Self-adhesive powder capable of sealing dura mater and promoting the repair of traumatic brain injury, and preparation method and use thereof

Abstract

The present invention provides a self-adhesive powder that takes into account both dura mater closure and promotion of traumatic brain injury repair, which is prepared from the following raw materials in weight ratio: 1-3 parts of azobenzene-functionalized hyaluronic acid (mAZo-HA), 1-3 parts of cyclodextrin-functionalized hyaluronic acid (CD-HA), and 1-3 parts of type I collagen. The present invention also provides a preparation method and use of the composition. The present invention is a self-assembling powder adhesive composed of a highly bioactive biomimetic matrix (HA and Col modified with cyclodextrin and azobenzene, respectively). In vivo experiments have shown that it has good hemostatic properties, can effectively prevent cerebrospinal fluid leakage, and can also improve angiogenesis, nerve regeneration and functional recovery after traumatic brain injury. It is easy to prepare and use, and provides convenient conditions for the clinical treatment of tissue regeneration and trauma repair.

Description

Technical Field

[0001] The present invention relates to a composition for treating traumatic brain injury. Background Art

[0002] Traumatic brain injury (TBI) caused by war, traffic, and disease is a global public health problem, with more than 10 million people hospitalized or dying from TBI globally each year. For severe TBI, in order to prevent cerebrospinal fluid (CSF) leakage and reduce the risk of intracranial infection, craniotomy is usually used to remove hematomas and decompress, with the focus on repairing dural defects. In addition, due to the lack of an appropriate matrix bridge, the activated astrocytes and dysplastic gliotic scars formed in the area around the brain injury make it extremely difficult for brain tissue regeneration and nerve recovery after TBI. Many preclinical studies have used hydrogels as scaffolds for TBI repair because hydrogels can mimic the viscoelastic properties of brain tissue and promote the repair of damaged brain tissue by filling the lesion cavity.

[0003] Self-gelling powder adhesives are a new type of biomaterial that can rapidly absorb moisture upon contact with wet tissues to form hydrogels, thereby achieving strong adhesion to wet tissues. This material has broad application prospects in the biomedical field, especially in situations where rapid hemostasis and wound healing are required. For example, there are literature reports on a polyethylenimine / polyacrylic acid / quaternized chitosan (PEI / PAA / QCS) powder adhesive hemostatic material and wound dressing (Xin Peng, Ultrafast Self-Gelling and Wet Adhesive Powder for Acute Hemostasis and Wound Healing, ADVANCED FUNCTIONAL MATERIALS, 17 June 2021). It can rapidly absorb a large amount of blood, concentrate clotting factors, and in-situ form a viscous hydrogel within 4 seconds after hydration, forming a pressure-resistant physical barrier, thus achieving rapid and effective hemostasis. In addition, the self-setting powder adhesive can penetrate into interconnected pores or fill rough surfaces, showing immediate sealing and regeneration properties. On the one hand, the adhesive powder is filled into the brain defect cavity and assembled into a gel by rapid water absorption, serving as a biomimetic extracellular matrix (ECM) material to provide mechanical support and cell recruitment in the cavity. On the other hand, the powder adhesive covers the tissue surface through mechanical interlocking to establish a strong adhesive interface, serving as a physical barrier for adhering and sealing dural defects. Compared with traditional adhesives, self-gelling powder adhesives are easy to prepare and can quickly convert the powder into a viscous hydrogel after hydration, which helps to fill damaged cavities with irregular shapes, reduce acute bleeding, and establish a stable and durable physical barrier on the tissue surface. The favorable microporosity of the powder adhesive can enhance cell migration and vascular infiltration during tissue repair, which is beneficial for the realization of nerve regeneration.

[0004] The disadvantages of traditional hydrogels for traumatic brain injury repair are still obvious. For example, the small specific surface area and poor water absorption affect the sealing performance and hemostasis efficiency. The nanoscale porosity limits the transport of nutrients and restricts the regenerative capacity.

[0005] In addition, although powder adhesives can solve some of the disadvantages of hydrogels, there are also some problems. They are easily dispersed during body fluid flushing, and their relatively weak mechanical strength and poor tissue adhesion cannot effectively prevent cerebrospinal fluid leakage, and it is difficult to form a complete dural closure. In addition, the lack of bioactivity and the mismatch of the brain ECM components and structure further limit the regeneration of brain tissue, and the endogenous repair is limited. Therefore, it is necessary to rationally design self-setting powder adhesives with improved sealing and regeneration capabilities to address these challenges.

[0006] Application No. 202110993194.X, Invention Title: Near-infrared Responsive Hyaluronic Acid Hydrogel for Articular Cartilage Repair and Its Preparation Method, discloses a near-infrared responsive hyaluronic acid hydrogel for articular cartilage repair and its preparation method. The hydrogel is formed by cross-linking azobenzene derivative-modified hyaluronic acid and cyclodextrin derivative-modified hyaluronic acid with a mass ratio of 1:1. The azobenzene derivative is tetra-o-methoxy-substituted azobenzene modified with a long alkyl chain, and the cyclodextrin derivative is cyclodextrin modified with hexamethylenediamine. Dissolve the azobenzene derivative-modified hyaluronic acid and cyclodextrin derivative-modified hyaluronic acid in proportion, mix the two solutions evenly, and let it stand or sonicate to form a gel. The hydrogel has the self-healing property and injectability of a non-covalently cross-linked hydrogel, and can undergo a partial gel-sol transition under the stimulation of near-infrared light, resulting in a decrease in the cross-linking network density on the gel surface to form a hydration layer. The formation of this near-infrared light-responsive mechanics and hydration layer makes it potentially significant in the field of cartilage repair. This patent prepares a gel from azobenzene-functionalized hyaluronic acid and cyclodextrin-functionalized hyaluronic acid for forming a hydration layer by light stimulation to increase joint lubrication, and the administration method is intra-articular injection.

[0007] There is no literature report on using azobenzene-functionalized hyaluronic acid and cyclodextrin-functionalized hyaluronic acid for the treatment of traumatic brain injury. Summary of the Invention

[0008] The technical solution of the present invention is to provide a composition for the treatment of traumatic brain injury, its preparation method and use.

[0009] The present invention provides a composition for the treatment of traumatic brain injury, which is prepared from the following raw materials in the following weight ratios:

[0010] Azobenzene-functionalized hyaluronic acid (mAZo-HA) 1-3 parts, cyclodextrin-functionalized hyaluronic acid (CD-HA) 1-3 parts, type I collagen 1-3 parts.

[0011] Further preferably, it is prepared from the following raw materials in the following weight ratios:

[0012] Azobenzene-functionalized hyaluronic acid (mAZo-HA) 1 part, cyclodextrin-functionalized hyaluronic acid (CD-HA) 1 part, type I collagen 1 part.

[0013] The composition of the present invention dissolves azobenzene-functionalized hyaluronic acid and cyclodextrin-functionalized hyaluronic acid in a type I collagen solution, adjusts the pH to 7-9 in an ice bath, incubates in a 37°C incubator, and after assembling into a gel, lyophilizes and pulverizes to obtain a powder adhesive.

[0014] Further preferably, the pH value is 7.4.

[0015] The composition of the present invention is obtained by separately pulverizing powders to internal sizes of 50 - 100 μm and 200 - 300 μm, and then compounding the powders of the two sizes. The weight ratio of the formulation is: 1 - 3:1.

[0016] The weight ratio of the powders of the two sizes is: 1:1.

[0017] The present invention also provides a method for preparing the composition for treating traumatic brain injury, which comprises the following steps:

[0018] a. Weigh the raw materials according to the respective weight ratios;

[0019] b. Dissolve azobenzene-functionalized hyaluronic acid and cyclodextrin-functionalized hyaluronic acid in type I collagen solution, adjust the pH to 7 - 9 in an ice bath, place it in an incubator at 37 °C for incubation. After assembling into a gel, freeze-dry and pulverize to obtain a powder adhesive.

[0020] Among them, the pH value is 7.4; the pulverizing method is: pulverize to internal sizes of 50 - 100 μm and 200 - 300 μm, and then compound the powders of the two sizes. The weight ratio of the formulation is: 1 - 3:1.

[0021] Among them, the particle size is obtained by means of a sieve.

[0022] The present invention provides the use of the composition in the preparation of an external medicine for treating traumatic brain injury.

[0023] The use of the composition in the preparation of a self-adhesive powder with both dura mater occlusion and promotion of traumatic brain injury repair.

[0024] The self-adhesive powder of the present invention is a special material, which can quickly form an adhesion when contacting with wet tissues and has the ability of rapid gelation. This powder is usually composed of polymers with strong physical interactions, which can absorb interfacial water in a very short time and form a physically crosslinked hydrogel in situ.

[0025] Under humid conditions, the powder of the present invention induces gelation through physical or chemical interactions to form an adhesive. However, when exposed to an aqueous environment for a long time, the gel may decompose due to loss of bonding strength and lose its adhesive properties. The supramolecular strategy based on host-guest interactions introduces a reversible bonding method, providing a potential solution for promoting adhesion and regulating cohesion. Since the dura mater is composed of highly oriented collagen tissues, the use of type I collagen (Col) in the present invention is particularly suitable as a 3D scaffold material because it can self-assemble into a fibrous gel and has excellent bioactivity. In addition, hyaluronic acid (HA) is a major component of the extracellular matrix (ECM) of nerve cells involved in nerve development and function regulation, containing many highly reactive functional groups. This promotes various chemical modifications to form a variety of functional derivatives, making it an ideal choice for mimicking the ECM microenvironment in nerve tissue engineering.

[0026] In view of the problems of intracranial infection caused by cerebrospinal fluid leakage and tissue regeneration and repair at the defect site during severe traumatic brain injury (TBI), the present invention proposes a solution - by implanting a bridging scaffold in the brain defect cavity to establish an induced microenvironment for nerve growth, and then sealing or repairing the dura mater defect, the treatment effect of TBI can be improved.

[0027] The present invention can improve the treatment effect of TBI by implanting a bridging scaffold in the brain defect cavity to establish an induced microenvironment for nerve growth, and then sealing or repairing the dura mater defect. The present invention has developed a self-assembling powder adhesive composed of a highly bioactive biomimetic matrix (HA and Col modified with cyclodextrin and azobenzene respectively). The powder adhesive has self-gelling, rapid hemostasis properties and bioactivity. It can rapidly absorb the exuded blood and undergo various supramolecular interactions such as collagen self-assembly, host-guest interaction, and cyclodextrin / aromatic residue interaction within seconds, immediately forming an adhesion barrier at the defect site to firmly seal the bleeding site. It can not only form a stable physical barrier against blood pressure but also create a favorable microenvironment to promote tissue regeneration in the traumatic brain injury cavity. After in-depth exploration, this powder adhesive has shown good hemostasis performance in a live bleeding model, effectively preventing CSF leakage in live dura mater sealing experiments on rats and rabbits, and at the same time can also improve angiogenesis, nerve regeneration and functional recovery after traumatic brain injury. This easily prepared and convenient-to-use powder adhesive shows the potential for in vivo tissue repair, will inspire the design of new biomaterials, and provide convenient conditions for the clinical treatment of tissue regeneration and wound repair. Description of the Drawings

[0028] Figure 1: (A) Schematic diagram and optical photograph of obtaining three different-sized powders by crushing and sieving; (B) Void map of powders of different sizes after self-gelation, with FITC-labeled collagen showing green and TRITC-labeled dextran used as a simulated solution showing red; (C) Statistical chart of particle size distribution of different powders; (D) Intrinsic size distribution of powders after self-gelation; (E) Porosity statistics of powders after gelation; (F) Rheological data of powders after self-gelation;

[0029] Figure 2 1H NMR spectra (A) 1H NMR spectrum of CD-HA; (B) 1H NMR spectrum of mAzo-HA;

[0030] Figure 3 Performance characterization of the collagen-based powder adhesive of the present invention, including SEM images (A) and particle size distribution diagrams (B) of CCA-P; (C) Process record of rapid gelation of CCA-P powder upon contact with water; (D) Stability of CCA-P powder in solution after gelation; (E) Shape adaptability and injectability of powders after self-gelation;

[0031] Figure 4 Adhesive and sealing properties of the powder adhesive of the present invention, including (A) Schematic diagram of adhesion measurement after self-gel powder forms a gel; (B) Stress-strain curve of adhesion force test; (C) Statistical chart of adhesion force test; (D) Display diagram of tissue adhesion performance of self-gelation to form a gel; (E) Picture of small intestine occlusion performance of self-gelation powder to form a gel; (F) Self-gelation powder forms a gel for dural sealing after TBI injury to prevent cerebrospinal fluid leakage; (G) Schematic diagram of burst pressure test and (H) Quantification data of burst pressure performance;

[0032] Figure 5 Biological functions of the powder adhesive of the present invention, including (A) Schematic diagram of cell compatibility and FDA / PI staining; (B) MTT test of cell viability; (C) Schematic diagram of cell protection by powder forming a gel during cell injection and live cell membrane staining; (D) Evaluation of cell viability before and after injection; (E) Blood compatibility and schematic diagram and picture display; (F) Statistical data of blood compatibility;

[0033] Figure 6 Test results of CCA-P of the present invention promoting endogenous NSC recruitment and vascularization after traumatic brain injury (Nestin staining (A) and Tuj-1 staining (B) of the Sham group, TBI group, CCA-G group and CCA-P group at 10 days; (C) Masson staining of the Sham group, TBI group, CCA-G group and CCA-P group at 10 days; (D) CD31 / α-SMA staining and CD31 / VEGF staining of the Sham group, TBI group, CCA-G group and CCA-P group at 30 days (E)). Detailed implementation mode

[0034] Example 1 Preparation of the composition powder adhesive for treating traumatic brain injury of the present invention

[0035] 1. Synthesis of azobenzene-functionalized hyaluronic acid (mAZo-HA): Through amidation reaction, amino-functionalized azobenzene was grafted onto hyaluronic acid.

[0036] 2. Synthesis of cyclodextrin-functionalized hyaluronic acid (CD-HA): First, prepare hexanediamine-modified cyclodextrin (CD-HDA), and then graft amino-functionalized cyclodextrin onto hyaluronic acid through amidation reaction.

[0037] 3. Dissolve mAZo-HA and CD-HA at a concentration of 2% respectively, and dissolve the type I collagen (Col) solution at 14 mg / ml. Mix the three according to a volume ratio of 1:1:1, adjust the pH to 7.4 in an ice bath at 4°C, then place it in an incubator at 37°C for incubation. After assembling into a gel, perform freeze-drying treatment. Then place it in a cryogenic grinder and grind it at a speed of 10,000 revolutions per minute for 10 minutes to obtain the powder adhesive.

[0038] Among them, azobenzene-functionalized hyaluronic acid and cyclodextrin-functionalized hyaluronic acid are prepared according to the method disclosed in patent application No. 202110993194.X.

[0039] Example 2 Preparation of the composition powder adhesive for treating traumatic brain injury of the present invention

[0040] The powders with internal sizes of 50 - 100 μm and 200 - 300 μm prepared in Example 1 were physically mixed according to a mass ratio of 1:1, and shaken thoroughly to make them evenly mixed.

[0041] The beneficial effects of the present invention are demonstrated by the following specific experiments.

[0042] Test Example 1 Screening test for the ratio of different internal sizes of the powder adhesive of the present invention

[0043] In order to deeply explore the effect of powder size on the material, the powder size was screened more carefully. Through the method of sieve, three sizes of powders with sizes of 50 - 100 μm, 120 - 180 μm and 200 - 300 μm were selected, corresponding to large, medium and small sizes.

[0044] Experimental process: First, the freeze-dried material was crushed by a crusher to obtain a powdery material. To deeply explore the effect of powder size on the hydrogelation of the material, the powder size was more carefully screened. By means of a sieve, three sizes of powder with sizes of 50 - 100 μm, 120 - 180 μm, and 200 - 300 μm were selected, corresponding to large, medium, and small sizes. Specifically, after the powder was crushed by a pulverizer, it was first passed through a sieve with a pore size of 50 μm, and the powder larger than 50 μm was sieved out. Then, this part of the powder was passed through a sieve with a pore size of 100 μm, and the powder with a pore size between 50 μm and 100 μm was selected. The remaining powder was first passed through a sieve with a pore size of 120 μm, and the powder larger than 120 μm was sieved out. Then, this part of the powder was passed through a sieve with a pore size of 180 μm, and the powder with a pore size between 120 μm and 180 μm was selected. Finally, the remaining powder was passed through a sieve with a pore size of 200 μm, and the powder larger than 200 μm was sieved out. Then, this part of the powder was passed through a sieve with a pore size of 300 μm, and the powder with a pore size between 200 μm and 300 μm was selected. As Figure 1 shown in A, C.

[0045] As Figure 1 shown in B, the powder was labeled with FITC fluorescence (green), and a TRITC-labeled dextran solution (red) was selected as the permeation solution to explore a series of physicochemical property changes of powders with different sizes during the processes of water absorption and swelling and self-adhesion to form a gel. The results showed that: The internal size of the self-adhesive powder formed by large-size powder (named CCA-P-H) was 230 ± 120 μm, the internal size of the self-adhesive powder formed by medium-size powder (named CCA-P-M) was 190 ± 115 μm, and the internal size of the self-adhesive powder formed by small-size powder (named CCA-P-L) was 100 ± 65 μm.

[0046] It should be noted that, as Figure 1 shown in D, the internal size of the self-adhesive powder (CCA-P-H+L) formed by large-size and small-size powders in a 1:1 ratio was more uniform, being 75 ± 35 μm. The porous material with a size of 100 μm was beneficial to the ingrowth and migration of nerve cells and promoted the repair of craniocerebral injuries.

[0047] It was further found that a complementary combination of powder sizes would obtain a more uniform internal size. The porosity test results showed that the CCA-P-H+L group had a smaller porosity, accounting for about 10% of the entire self-adhesive gel, and this lower porosity often brought more stable gel strength ( Figure 1 E).

[0048] It was also found through rheological tests that the self-adhesive powder formed by large-sized powders has a lower modulus, while the CCA-P-H+L composed of large-sized and small-sized powders has a higher modulus, enabling more effective packing. In addition, it has a shear-thinning property with a modulus of about 300 Pa, which is very suitable for brain applications. It not only facilitates the growth of nerve cells but also has a certain skeletal support effect. Figure 1 F). Therefore, in subsequent experiments, the powder formed by large-sized and small-sized powders in a 1:1 ratio was selected as the self-adhesive powder for further characterization. At a 1:1 ratio, the best match between large-sized and small-sized powders can be achieved.

[0049] Experimental Example 2 Screening Test for the Optimal Proportion of Raw Materials in the Composition for Treating Traumatic Brain Injury of the Present Invention

[0050] The cyclodextrin in CD-HA can not only undergo host-guest interactions with mAzo-HA but also interact with the aromatic residues in collagen. The grafting degree of cyclodextrin is about 18% Figure 2 A), and the grafting degree of azobenzene is 9% Figure 2 B); considering that another part of the cyclodextrin needs to react with collagen, the ratio of mAzo-HA:CD-HA is determined to be 1:1, and the concentration of collagen can be between 0.5 and 2. That is, mAzo-HA:CD-HA:Col = 2:2:1 - 1:1:2, and the optimal ratio is 1:1:1.

[0051] Experimental Example 3 Performance Characterization of the Collagen-Based Powder Adhesive of the Present Invention

[0052] First, after freeze-drying and grinding, Col / CD-HA / Azo-HA powder (abbreviation: CCA-P) and Col powder (abbreviation: C-P) were obtained respectively. Scanning electron microscope images showed that the powder was in the form of curly irregular flakes Figure 3 A), and the powder size was about 200 μm Figure 3 B).

[0053] Observations under an optical microscope showed that CCA-P quickly swelled and connected into a whole within 10 seconds, as Figure 3 C shows.

[0054] To prove the self-gelling ability, the powder was hydrated, stained and immersed in PBS solution. As Figure 3 D shows, a red dispersion appeared in the supernatant of C-P, while CCA-P formed a more stable hydrogel. The hydrogel scaffold with shape plasticity and ductility can better adapt to the irregular wound shape. After adding an appropriate amount of water or PBS, the CCA powder can quickly fill the mold, form a large amount of stretchable hydrogel, and has certain injectability. Figure 3E)

[0055] This is due to the topological entanglement and physical interactions between two polymer chains, thus forming a cohesive bulk hydrogel.

[0056] Test Example 4 Bonding and Sealing Properties of Powder Adhesives

[0057] The bioactive adhesive CCA-P based on supramolecular interactions has excellent tissue adhesiveness and burst pressure, indicating its great potential for biological functions.

[0058] We selected the above-mentioned fast self-gelling powder CCA-P for subsequent experiments. First, the adhesion properties of these two powder hydrogels to porcine skin were evaluated by lap shear tests ( Figure 4 A). Specifically, the powder was sprinkled on one side of the sheet of porcine skin, then it absorbed water and swelled to gel, and the two pieces of porcine skin were closely attached together. The adhesion strength of CCA-P based on supramolecular interactions was 10 kPa, much higher than that of C-P (5 kPa, Figure 4 B, C). This is because the reversible bonds in the double cross-linked network can effectively improve the adhesion force by regulating the cohesion of CCA-P. After in-situ adhesion to tissues, it was found that CCA-P could withstand bending and deformation, which further demonstrated the excellent adhesion ability of CCA-P ( Figure 3 D).

[0059] Next, we adhered CCA-P to damaged porcine intestines to evaluate its tissue sealing ability ( Figure 4 E). Based on supramolecular interactions, CCA-P quickly formed a gel at the gaps of porcine intestines and diffused into the matrix through rich physical interactions and entanglement of polymer chains, quickly plugging the gaps and forming a stable physical barrier, so that the intestine filled with PBS no longer leaked. CCA-P was further placed on the surface of the ruptured dura mater of rabbits to evaluate the immediate leakage-stopping ability. By simulating the overflow rate of normal cerebrospinal fluid (CSF), it was found that the defect surface sealed by CCA-P could effectively prevent liquid leakage ( Figure 4 F). The burst test showed that the burst pressure of CCA-P (150 mmHg) was much higher than that of C-P (35 mmHg), and this anti-liquid erosion ability was sufficient to resist the dynamic changes of CSF pressure and form an effective seal for the brain tissue ( Figure 4 G).

[0060] Test Example 5 Biological Functions of Powder Adhesives (Biocompatibility, Cell Protection and Hemostatic Properties)

[0061] The bioactive adhesive CCA-P based on supramolecular interactions has excellent biocompatibility and blood compatibility and can achieve the protection of cells.

[0062] In cell experiments, we co-cultured CCA-P with NIH 3T3 cells in high-glucose DMEM medium at 37 °C to evaluate its biocompatibility. As Figure 5 shown in A and B, the cells in the CCA-P group proliferated significantly on the third day, similar to the control group (without hydrogel), indicating that CCA-P has good biocompatibility. In addition, due to the supramolecular interaction endowing CCA-P with shear-thinning properties, the hydrogel can also provide protection when cells pass through the needle. We evaluated the cell viability of CCA-P mixed with cells before and after injection and observed that the cell viability of the control group (only PBS) decreased by 10%, while the cell viability of CCA-P did not change significantly ( Figure 5 C, D). Therefore, CCA-P can be used as a cell carrier to protect cells from mechanical damage.

[0063] Mixed brain injury is often accompanied by subdural hematoma and subarachnoid hemorrhage, so rapid hemostasis must be achieved during acute bleeding. CCA-P can form a hydrogel in situ at the injury site by sucking blood and is an ideal hemostatic material. Hemolysis refers to the rupture of a large number of red blood cells after contact with the material, and the hemolysis rate can be used to characterize the blood compatibility of blood-contact materials. The hemolysis test showed that there was no significant difference in the hemolysis rate among CCA-P, C-P, and the negative control (0.9% NaCl), all of which were less than 5% (safe level), which proved that CCA-P has good blood compatibility ( Figure 5 E, F).

[0064] Test Example 6 CCA-P Promotes the Recruitment and Vascularization of Endogenous NSCs after Traumatic Brain Injury

[0065] Given the ability of CCA-P to gel in situ to fill the lesion cavity, as well as its good bioactivity and functions (tissue adhesion, hemostasis, and wound sealing), it becomes an ideal nerve scaffold for treating traumatic brain injury. Different from the nanoscale pores of the bulk gel, the pores of the microgel formed by the powder are micron-scale, which greatly improves the cell migration ability and vascular infiltration ability.

[0066] Next, we conducted animal experiments to establish a TBI injury model in the brains of male rats. Specifically, first, an approximately 8-mm defect was drilled in the rat brain using a mold, then the material was filled, sutured, and samples were taken after 30 days. We first explored the recruitment of endogenous neural stem cells (NSCs) in the cavity. We performed immunofluorescence staining for nestin (a neural stem cell marker) on day 10 and found that there were only a few nestin-positive cells in the TBI group (without any treatment), while the unground bulk gel (CCA-G) led to an increase in positive signals due to its good biological activity, indicating that endogenous NSCs were recruited. Notably, the CCA-P group could effectively promote the recruitment of endogenous NSCs, which was closely related to the fact that the pore space of CCA-P was suitable for cell growth ( Figure 6 A). In addition to being able to effectively recruit endogenous neural stem cells in the injury cavity, promoting the differentiation of neural stem cells into neurons is also equally important. New neurons can replace the lost neurons and reconstruct neural circuits. βⅢ-Tubulin (Tuj-1, a maker of early neurons) staining showed that more cells in the CCA-P group expressed Tuj-1 on day 14. In addition, the high expression of NeuN (neuronal nuclear marker) and ChAT (choline acetyltransferase marker) in the CCA-P group also indicated that CCA-P promoted the regeneration and maturation of neurons ( Figure 6 B). Nissl staining showed that the number of Nissl bodies in the TBI group was significantly reduced, while the density of Nissl bodies in the CCA-P group was higher than that in the TBI group and the CCA-G group, indicating that CCA-P could reduce neuronal damage and showed higher neuronal viability ( Figure 6 C).

[0067] After traumatic brain injury, blood vessels rupture and necrosis occur. CCA-P can effectively stop bleeding, and further angiogenesis and vascular remodeling are the keys to the late repair of brain injury. We studied the angiogenesis situation on day 30 after the implantation of CCA-P. First, CD31 (a vascular endothelial marker) and α-SMA (a smooth muscle actin marker) were used to detect the integrity of the vascular structure at the time of injury. Immunofluorescence and quantitative statistics showed that almost no α-SMA was expressed in the TBI group, indicating the absence of blood vessels after brain injury, while the addition of CCA-G promoted the expression of α-SMA. In the CCA-P group, cells at the injury site showed a high level of α-SMA, indicating that CCA-P could promote angiogenesis after brain injury ( Figure 6 D). Since the vascular endothelial growth factor marker VEGF can induce the proliferation of endothelial cells, we further detected the expression of VEGF. As Figure 6As shown in E, the expression of vascular endothelial growth factor in the CCA-P group was significantly higher than that in the other two groups (the TBI group and the CCA-P group), which was similar to the previous results. In summary, CCA-P can promote the recruitment of endogenous stem cells and vascular infiltration, which is crucial for brain tissue repair and neural circuit remodeling.

Claims

1. A composition for treating traumatic brain injury, characterized in that: It is prepared from the following raw materials in weight ratio: 1 part of azobenzene functionalized hyaluronic acid (mAZo-HA), 1 part of cyclodextrin functionalized hyaluronic acid (CD-HA), 1 part of type I collagen; The composition is prepared by dissolving azobenzene-functionalized hyaluronic acid and cyclodextrin-functionalized hyaluronic acid in a type I collagen solution, adjusting the pH to 7-9 in an ice bath, incubating in a 37°C incubator, and lyophilizing and pulverizing to obtain a powder adhesive after assembling into a gel; The pulverizing method is: pulverizing the powders to inner sizes of 50-100um and 200-300um respectively, and then compounding the powders of the two sizes in a weight ratio of 1-3:

1.

2. The composition for treating traumatic brain injury according to claim 1, characterized in that: The pH value is 7.

4.

3. The composition for treating traumatic brain injury according to claim 1, characterized in that: The weight ratio of the two sizes of powder is 1:

1.

4. A method for preparing the composition for treating traumatic brain injury according to any one of claims 1 to 3, characterized in that: It includes the following steps: a. Weigh the raw materials of each weight ratio; b. Dissolve azobenzene-functionalized hyaluronic acid and cyclodextrin-functionalized hyaluronic acid in type I collagen solution, adjust the pH to 7-9 in an ice bath, incubate in a 37°C incubator, and after assembling into a gel, freeze-dry and grind to obtain a powder adhesive.

5. The method for preparing the composition for treating traumatic brain injury according to claim 4, characterized in that: The pH value is 7.4; the pulverizing method is: pulverizing to inner sizes of 50-100um and 200-300um, and then compounding the powders of the two sizes, with the weight ratio of 1-3:

1.

6. Use of the composition according to any one of claims 1 to 3 in the preparation of an external-use medicament for treating traumatic brain injury.

7. Use of the composition according to any one of claims 1 to 3 in the preparation of a self-adhesive powder having the effects of both dura mater occlusion and promoting the repair of traumatic brain injury.

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