A tissue adhesive, its preparation method and application

Abstract

This invention provides a tissue adhesive, its preparation method, and its application. The preparation method specifically includes a one-pot reaction method, in which biological proteins, reducing sugars, polyphenolic unit substances, and metal chloride salts are mixed and heated to react, resulting in a protein-reducing sugar tissue adhesive with a polymeric network structure. After low-temperature incubation, dense and ordered hydrogen bonds are reconstructed within the network structure to obtain the tissue adhesive. The tissue adhesive provided by this invention exhibits excellent adhesive properties due to the synergistic effect of chain reinforcement caused by Maillard chemical reactions and physical forces induced by the one-pot cooking method. This allows it to match the modulus and electroactivity of natural soft tissue; it also possesses injectability, self-healing ability, and faster and stronger adhesion properties. It can be effectively injected into nerve injury sites to achieve incompressible nerve hemostasis and seamless filling of lesion cavities, promoting nerve healing and regeneration.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a tissue adhesive, its preparation method, and its application. Background Technology

[0002] Tissue adhesives, as a new generation of medical materials that can replace medical sutures and staples, are considered an ideal biomedical material for wound management and wound healing due to their ease of use and the fact that they generally also have the functions of sealing the wound site and preventing body fluid leakage, mechanical support, hemostasis, and matching the microenvironment of the injury site.

[0003] However, existing clinical tissue adhesives suffer from drawbacks such as limited variety, certain toxicity, complex and slow preparation, weak adhesion, and lack of functionality. For example, fibrin glue, commonly used in clinical practice, has low adhesive strength and potential immunogenicity; albumin is generally polymerized and cross-linked using toxic aldehyde cross-linking agents (such as glutaraldehyde); cyanoacrylate adhesives polymerize quickly, but their wet-surface adhesion strength is significantly reduced, and their in vivo degradation toxicity limits their further application. Although some novel adhesive hydrogels have partially solved the toxicity problem, their weak wet-surface adhesion strength, poor hemostatic properties, and complex preparation procedures greatly limit their synthesis efficiency and application scope. For example, the polymer-based hydrogel adhesives described in Chinese invention patents CN113209358A and CN113512133A have complex preparation processes, weak wet surface adhesion, and poor mechanical properties due to their inherent instability. This limits their application in areas requiring high intrinsic sealing strength, such as emergency hemostasis and incompressible injuries (e.g., arterial bleeding, bleeding from major tissue defects, nerve bleeding). Furthermore, existing tissue adhesives are functionally limited, serving simply as dressings or hemostatic agents, and generally lack electrical conductivity.

[0004] Based on the technical problems existing in the prior art, the present invention provides a tissue adhesive that can be prepared by a simple one-pot cooking method with heating. It can be prepared rapidly within minutes, has adjustable mechanical properties and adhesive characteristics, and also has rapid hemostatic properties. In addition, it has the characteristic of forming reversible micro-batteries and thus having good electrical conductivity, so it can be used in the fields of emergency hemostasis and regenerative medicine. Summary of the Invention

[0005] In view of this, in order to solve the above problems, the present invention provides a tissue adhesive, its preparation method and application, which aims to solve the problem that the tissue adhesives prepared by the prior art have only one function, simply serving as dressings or hemostatic agents, and generally do not have conductive properties.

[0006] To achieve the above objectives, the present invention provides a method comprising: providing a functionalized protein; modifying or blending the protein with a polyphenol-containing unit substance or a small molecule containing a phenolic hydroxyl group to obtain the functionalized protein; providing a protein-reducing sugar tissue adhesive; mixing the functionalized protein, reducing polysaccharide or derivative, and hygroscopic salt, and then subjecting the mixture to a Maillard chemical reaction and ion complexation reaction under heating conditions via a one-pot cooking method to obtain a three-dimensional network structure, thereby preparing the protein-reducing sugar tissue adhesive; preparing the tissue adhesive; and incubating the protein-reducing sugar tissue adhesive to reform dense and ordered hydrogen bonds in the network structure to obtain the tissue adhesive.

[0007] Preferably, the functionalized protein can be added to the protein chain by physical or chemical modification methods using the polyphenol-containing unit substance or the small molecule containing phenolic hydroxyl groups.

[0008] Preferably, the physical modification method includes blending the protein with the polyphenol-containing unit substance or the small molecule containing phenolic hydroxyl groups and then incubating it through interactions such as hydrogen bonding and hydrophobic interactions; preferably, the incubation temperature is 4–30°C.

[0009] Preferably, the chemical modification method includes preparing the substance containing polyphenol units or small molecules containing phenolic hydroxyl groups through esterification or condensation reactions.

[0010] Preferably, the polyphenol unit substance or small molecule containing phenolic hydroxyl groups includes at least one of dopamine, tannic acid, epigallocatechin, gallic acid and its derivatives.

[0011] Preferably, the protein includes at least one of collagen, gelatin, silk fibroin, bovine serum albumin, soy protein, or macromolecular polypeptide.

[0012] Preferably, the reducing polysaccharide has oxidizing properties, including polysaccharides containing oxidizing functional groups, that is, the reducing polysaccharide itself has oxidizing properties.

[0013] Preferably, the reducing polysaccharide is one or a combination of several of glucose, fructose, galactose, lactose, maltose, etc., but is not limited thereto.

[0014] Preferably, the reduced polysaccharide can be obtained by oxidation of a non-reducing polysaccharide.

[0015] Preferably, the non-reducing polysaccharide includes hyaluronic acid, starch, dextran, cyclodextrin, and chitosan, but is not limited to these.

[0016] Preferably, the hygroscopic salt includes a metal chloride salt.

[0017] Preferably, the mass ratio of the polyphenolized functional protein, the reducing sugar and its derivatives, the metal chloride salt and the solvent is (10-100):(10-500):(30-1200):(100-3000).

[0018] Preferably, the chloride metal salt includes one or a combination of calcium chloride, magnesium chloride, ferric chloride, zinc chloride and lithium chloride, but is not limited thereto.

[0019] Preferably, the added metal chloride salt is 20-30% by mass. Unless otherwise specified, the amount of metal chloride salt added in this invention refers to the mass percentage.

[0020] Preferably, the chloride metal salt comprises a mixture of calcium chloride and lithium chloride.

[0021] Preferably, the mass ratio of calcium chloride to lithium chloride is (1:10) to 1; more preferably, the mass ratio of calcium chloride to lithium chloride is 5 to 1.

[0022] Preferably, the solvent includes, but is not limited to, deionized water or an acetic acid solution with a mass fraction of 1%.

[0023] Preferably, in the one-pot cooking method, the heating temperature is 40–200°C.

[0024] Based on the problems existing in the prior art, the present invention uses the above preparation method to provide a tissue adhesive containing an ion-quinone redox couple, thereby having stable conductivity; based on this function, combined with electrical stimulation therapy, it can promote neuronal differentiation and axon growth in vivo, enhance endogenous neurogenesis and promote functional recovery in rats with spinal cord injury.

[0025] Furthermore, the tissue adhesive possesses wet surface adhesion (overlap shear strength ≥ 80 kPa) and tunable electrical conductivity (~1 × 10⁻⁶ kPa). -3 The product features good shape adaptability, biocompatibility, and biodegradability, and preferably, a wet surface adhesion strength greater than 80 kPa, enabling its application in integrated repair of nerve transections. Specifically, this includes emergency hemostasis, incompressible nerve hemostasis and repair, sealing connection, and defect filling.

[0026] Furthermore, the tissue adhesive has adjustable mechanical properties that can match the mechanical properties and electrical conductivity of the natural spinal cord.

[0027] Based on the aforementioned technical solution, a one-pot high-temperature cooking synthesis process is used to rapidly prepare a strong and adhesive protein-reducing sugar-based natural polymer hydrogel tissue adhesive. This process utilizes polyphenol-functionalized biological proteins, reducing sugars, and mixed metal chlorides to induce Maillard reactions and ion complexation reactions in the network. The hydrogel adhesive exhibits rapid hemostasis and electrical conductivity, making it suitable for emergency hemostasis and the repair of incompressible nerves. First, the protein is modified or blended using polyphenolic unit substances or small molecules containing phenolic hydroxyl groups to obtain functionalized proteins. Then, the functionalized proteins are blended with reducing sugars or derivatives and metal chlorides, and Maillard reactions and ion complexation reactions occur under heating conditions to rapidly prepare the protein-reducing sugar tissue adhesive. Finally, the resulting adhesive is incubated overnight at 4°C to allow the network to reform dense and ordered hydrogen bonds, yielding the final tissue adhesive. Specifically, taking catecholized collagen, oxidized starch, and a mixed calcium chloride-lithium chloride metal salt as an example, using the aforementioned one-pot cooking strategy, catecholized collagen, oxidized starch, and a mixed ion (CaCl2 and LiCl) solution were rapidly synthesized (~25s) via a low-high temperature heating one-pot method to produce a solution with an elastic modulus (2~3kPa) and electroactivity (~1×10⁻⁶) matching that of natural soft tissue. -3 This tissue adhesive exhibits a shear strength of ≥80 kPa (S / cm). It is also injectable, self-healing, and demonstrates faster and stronger wet-surface adhesion (shear adhesion strength ≥80 kPa). The enhanced adhesion is attributed to the synergistic effect of chain reinforcement caused by Maillard chemical reactions during high-temperature cooking and physical forces. Based on these characteristics, the tissue adhesive provided by this invention can be effectively injected into nerve injury sites to achieve incompressible nerve hemostasis and seamless filling of lesion cavities. Furthermore, it incorporates high-mobility Li... + The formation of the quinone redox couple enables continuous and stable electrical signal transmission.

[0028] Furthermore, verification through combined electrical stimulation therapy revealed that the injectable, functional, and conductive tissue adhesive provided by this invention can effectively modulate the local microenvironment of spinal cord injury, reduce fibrotic scar formation, promote neurogenesis, promote myelin regeneration, and enhance axonal regeneration, enabling its application in the field of regenerative medicine. In particular, this invention proposes a multifunctional tissue adhesive that can integrate hemostasis, non-invasive connection, and filling of defect lesions in spinal cord injury, while promoting spinal cord regeneration, thus providing a new integrated repair concept.

[0029] In summary, the strong and functional hydrogel adhesive synthesized using the one-pot cooking strategy of this invention has excellent hemostatic and sealing effects, and is a promising medical tissue adhesive for rapid hemostasis, wound sealing, and damage repair.

[0030] The beneficial technical effects obtained by this invention are as follows:

[0031] 1. The tissue adhesive provided by the present invention exhibits excellent adhesive properties due to the synergistic effect of chain reinforcement and physical forces induced by Maillard chemical reaction through a one-pot cooking method. This results in a modulus (~2 kPa) and electroactivity (~1 × 10⁻⁶) matching those of natural soft tissue. -3 It also features injectability, self-healing ability, and faster and stronger wet surface adhesion (shear adhesion strength ≥80KPa), which can be effectively injected into the nerve injury site to achieve incompressible nerve hemostasis and seamless filling of the lesion cavity.

[0032] 2. The tissue adhesive provided by the technical solution of the present invention includes Li with high migration rate. + - Quinone redox couples can ensure the continuous and stable transmission of electrical signals by tissue adhesives. Combined with electrical stimulation therapy, injectable functional conductive tissue adhesives can effectively regulate the local microenvironment of spinal cord injury, promote neuronal differentiation and axon growth in vivo, enhance endogenous neurogenesis and promote functional recovery in rats with spinal cord injury, thereby reducing fibrotic scar formation, promoting neurogenesis, promoting myelin regeneration and enhancing axon regeneration.

[0033] 3. The tissue adhesive provided by the technical solution of this invention is a multifunctional tissue adhesive, which proposes an integrated repair concept, including the ability to achieve hemostasis, non-destructive connection and filling of defect lesions in spinal cord injury in an integrated manner, and promote spinal cord injury regeneration and other repair functions.

[0034] 4. The tissue adhesive provided by the technical solution of this invention is a strong and multifunctional hydrogel tissue adhesive synthesized using a one-pot cooking strategy. It has good hemostatic and sealing effects and is a medical tissue adhesive with broad application prospects for rapid hemostasis, wound sealing and damage repair.

[0035] 5. The tissue adhesive provided by the technical solution of the present invention has wet surface adhesion, and the wet surface adhesion strength can reach more than 80 kPa. It can seamlessly connect completely severed spinal cords and has shape adaptability, biocompatibility and biodegradability. It has adjustable mechanical properties and can match the mechanical properties and conductivity of natural spinal cords.

[0036] 6. The tissue adhesive provided by the technical solution of this invention contains hygroscopic salts and intrinsic hemostatic properties, which can quickly absorb interfacial water and hemostatic properties, thereby achieving rapid and incompressible nerve hemostasis. Attached Figure Description

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0038] Figure 1 This is a schematic diagram simulating the reaction of synthesizing protein-reducing sugar tissue binder using the one-pot cooking method of this invention;

[0039] Figure 2 In Examples 1, 5, and Comparative Examples 1-2 of this invention, the gelation time was monitored by a rheometer when different salt ion contents were added during the preparation of catecholized collagen-oxidized starch tissue adhesive.

[0040] Figure 3 This is a statistical chart showing the gelation time when different salt ion contents were added during the preparation of catecholized collagen-oxidized starch tissue adhesive in Examples 1, 5, and Comparative Examples 1-2 of the present invention.

[0041] Figure 4 This is a statistical analysis of the modulus of materials obtained when different salt ion contents were added during the preparation of catecholized collagen-oxidized starch tissue binders in Examples 1, 5, and Comparative Examples 1-2 of the present invention.

[0042] Figure 5 This is a SEM micrograph of the catecholized collagen-oxidized starch tissue adhesive prepared in Example 1 of the present invention;

[0043] Figure 6 The circular dichroism chromatograms before and after heating are shown in Example 1 of this invention for the preparation of catecholized collagen-oxidized starch tissue binder.

[0044] Figure 7 This is a statistical graph of the shear adhesion strength when different salt ion contents are added during the preparation of the catecholized collagen-oxidized starch tissue adhesive in Example 1 of the present invention.

[0045] Figure 8 These are the CV curves of different salt ion contents added during the preparation of the catecholized collagen-oxidized starch tissue adhesive in Example 1 of this invention;

[0046] Figure 9 The catecholized collagen-oxidized starch tissue adhesive prepared in Example 1 of this invention, combined with electrical stimulation therapy, is used for motor behavioral BBB evaluation at different recovery stages after spinal cord injury repair.

[0047] Figure 10The results are histological staining of the catecholized collagen-oxidized starch tissue adhesive prepared in Example 1 of this invention, combined with electrical stimulation therapy, after 8 weeks of recovery following spinal cord injury repair. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some embodiments of this application, but not all embodiments.

[0049] This invention provides a tissue adhesive that uses a one-pot cooking method to react polyphenol-functionalized biological proteins, reducing sugars, and metal chlorides under heating conditions to obtain a functional protein-reducing sugar tissue adhesive with a polymer network structure. After low-temperature incubation, dense and ordered hydrogen bonds are reconstructed in the network structure to obtain the tissue adhesive.

[0050] See Figure 1 The diagram shows a reaction simulation of the one-pot cooking method for synthesizing protein-reducing sugar tissue adhesive according to the present invention. As can be seen from the diagram, polyphenol-functionalized protein, reducing polysaccharide, and hygroscopic salt are added to the reaction vessel for reaction. First, under heating conditions, Maillard chemical reaction and ion complexation reaction are carried out through a one-pot cooking method to obtain a three-dimensional network structure and prepare protein-reducing sugar tissue adhesive. Then, it is incubated to allow dense and ordered hydrogen bonds to reform in the three-dimensional network structure of the protein-reducing sugar tissue adhesive, thus obtaining the tissue adhesive.

[0051] The tissue adhesive obtained by adopting the above technical solution has wet surface adhesion, shape adaptability, biocompatibility and biodegradability, which enables it to be applied to the integrated repair of nerve transection, specifically including emergency hemostasis, hemostasis and repair of incompressible nerves, sealing connection and defect filling.

[0052] Furthermore, the tissue adhesive obtained using the above-mentioned technical solution is conductive, which, when combined with electrical stimulation therapy, can promote neuronal differentiation and axonal growth in vivo, enhance endogenous neurogenesis, and promote functional recovery in rats with spinal cord injury.

[0053] In particular, the tissue adhesive prepared by the present invention through a simple one-pot cooking method can be rapidly prepared within minutes (as fast as 25 seconds), has adjustable mechanical and adhesive properties, rapid hemostatic properties, and the ability to form micro-batteries, thus exhibiting good electrical conductivity. Therefore, it can be used in the fields of emergency hemostasis and regenerative medicine.

[0054] In some preferred embodiments, the present invention also provides a method for preparing the above-mentioned tissue adhesive, comprising: providing a functionalized protein; modifying or blending the protein with a polyphenol-containing unit substance or a small molecule containing a phenolic hydroxyl group to obtain a functionalized protein; providing a reducing polysaccharide or derivative; blending the functionalized protein, the reducing polysaccharide or derivative, and a metal chloride salt, and then subjecting the mixture to a Maillard chemical reaction and an ion complexation reaction under heating conditions via a one-pot cooking method to obtain a three-dimensional network structure, thereby preparing the protein-reducing sugar tissue adhesive; preparing the tissue adhesive; and incubating the protein-reducing sugar tissue adhesive to reform dense and ordered hydrogen bonds in the network structure to obtain the tissue adhesive.

[0055] In some preferred embodiments, the physical modification method includes incubating the protein with the polyphenol-containing unit or the small molecule containing phenolic hydroxyl groups. The incubation temperature is 4–30°C.

[0056] In some preferred embodiments, the chemical modification method includes preparing the substance by esterification or condensation reaction of the polyphenol-containing unit substance or the small molecule containing phenolic hydroxyl groups.

[0057] In some preferred embodiments, the polyphenol unit substance or small molecule containing phenolic hydroxyl groups includes at least one of dopamine, tannic acid, epigallocatechin, gallic acid and its derivatives.

[0058] In some preferred embodiments, the protein includes at least one of collagen, gelatin, silk fibroin, bovine serum albumin, soy protein, or macromolecular polypeptide.

[0059] In some preferred embodiments, the oxidized polysaccharide includes one or more of sugars that are oxidizing in themselves, such as glucose, fructose, galactose, lactose, maltose, etc.

[0060] In some preferred embodiments, the reduced polysaccharide can also be obtained by oxidation of the non-reduced polysaccharide;

[0061] In some preferred embodiments, the non-oxidized polysaccharide includes, but is not limited to, hyaluronic acid, starch, dextran, cyclodextrin, chitosan, etc.

[0062] In some preferred embodiments, the chloride metal salt includes one or a combination of calcium chloride, magnesium chloride, ferric chloride, zinc chloride, and lithium chloride.

[0063] It should be noted that, unless otherwise specified, the tissue adhesives synthesized in this invention with different formulations refer to tissue adhesives or conductive tissue adhesives.

[0064] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0065] Example 1

[0066] This embodiment provides a tissue adhesive. Taking catecholized collagen, oxidized starch, and a mixed metal chloride salt of calcium chloride and lithium chloride as an example, the feasibility of the technical solution of the present invention is explained in detail by using the above-mentioned one-pot cooking method to rapidly gel the catecholized collagen, oxidized starch, and mixed ion (CaCl2 and LiCl) solution through a low-temperature and high-temperature heating one-pot method (gelation time ~25s).

[0067] Specifically, the preparation method includes the following steps:

[0068] S1. Provides catecholated functional proteins;

[0069] Weigh 500 mg of collagen and dissolve it in 50 mL of 0.5% trifluoroacetic acid. Stir and dissolve at room temperature for about 2-3 hours until completely dissolved. Then add 0.5 g of dopamine hydrochloride to the collagen solution and stir and dissolve under sealed conditions. Adjust the pH of the mixed solution to 5.5 by adding 1 mol / L sodium hydroxide solution dropwise. Next, weigh 0.85 g of N-hydroxysuccinimide (NHS) and dissolve it in 15 mL of deionized water. Adjust the pH of the mixed solution to 5.5 by adding 1 mol / L sodium hydroxide solution dropwise. Similarly, weigh 1.15 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and dissolve it in 15 mL of deionized water. Adjust the pH of the mixed solution to 5.5 by adding 10% trifluoroacetic acid solution dropwise.

[0070] The prepared NHS and EDC solutions were added to the mixed solution sequentially, with an interval of 15 min between additions. Finally, the entire reaction system was placed in a sealed container and reacted at room temperature for 20 h. After 20 h, the reaction solution was transferred to a dialysis bag and dialyzed in 4 mM hydrochloric acid solution for three days, with the dialysate changed once a day. The purified solution was then frozen at -80 degrees Celsius and freeze-dried using a freeze dryer to obtain the catecholized functional protein, which was ready for use.

[0071] S2. Preparation of oxidizing reducing sugars

[0072] Weigh 6.4 g of starch and dissolve it in 100 mL of dry tetrahydrofuran. Stir thoroughly at room temperature. After about 1 hour, add 1.5 g of p-benzaldehyde to the starch solution and stir to dissolve under sealed, light-protected conditions. Then add 0.08 g of DMPA (2,2-dimethylolpropionic acid). Similarly, weigh 2 g of DCC (dicyclohexylcarbodiimide) and add it to the above solution. After purging with nitrogen, place the entire reaction system in a sealed container and react at room temperature for 18 hours. After 18 hours, transfer the reaction solution to a beaker and wash three times each with anhydrous diethyl ether and distilled water. Dry the purified starch using a rotary evaporator to obtain a white powder. Dry and seal the powder for later use. This is the oxidized reducing sugar (oxidized starch).

[0073] S3. Preparation of protein-reducing sugar conductive tissue adhesive:

[0074] Dissolve catechin-modified collagen in deionized water and sonicate to fully dissolve the catechin-modified collagen, obtaining a catechin-modified collagen solution with a concentration of 50 mg / mL, which is then set aside for use. Subsequently, pour 2 mL of the catechin-modified collagen solution into a 10 mL beaker. Weigh 400 mg of oxidized starch, 500 mg of calcium chloride, and 100 mg of lithium chloride and add them to the 10 mL beaker in sequence. Place the beaker on a heating platform, adjust the temperature to 80 °C, and start the stirrer to stir rapidly.

[0075] Then, slowly add 1 mL of 0.1 mol / L sodium bicarbonate solution to the beaker and stir rapidly until the starch is completely dissolved to obtain a viscous solution. The solution is then ultrasonically centrifuged to remove air bubbles. Finally, the solution is frozen overnight to obtain a catecholated collagen-reduced starch conductive tissue adhesive with a gelation time of approximately 25 seconds.

[0076] The mass percentage content of calcium chloride and lithium chloride (the mass ratio of the two is always maintained at 5:1) is 20%.

[0077] The tested catecholated collagen-reduced starch conductive tissue adhesive had a modulus of approximately 2 kPa, a shear strength of approximately 80 kPa, and a conductivity of approximately 9.88 × 10⁻⁶ kPa. -4 S / cm. It should be noted that the modulus in this article refers to the elastic modulus, which is obtained by time-scan testing using a rheometer (Malvin). The moduli mentioned in other embodiments and comparative examples are all elastic moduli.

[0078] See Figure 5 The figure shows the SEM microstructure of the catecholized collagen-oxidized starch tissue adhesive with a metal chloride ion content of 20% in this embodiment. As can be seen from the figure, the molecular chain network in the network is tightly and orderly connected with uniform pore size, indicating that the material has an orderly cross-linked network similar to nerve fibers. At the same time, the orderly structure with the same direction further enhances the strength of the material.

[0079] Furthermore, the protein-reducing sugar conductive tissue adhesive obtained from S3 was used for the repair of complete transection spinal cord injuries, and the results were tested. The testing steps included:

[0080] Female rats were anesthetized, and a 3cm incision was made in their backs. The muscles on the dorsal side of the spine were dissected to expose the bilateral lamina at T9-11. The lamina were then pried open to expose the spinal cord. One side of the dura mater was carefully lifted with microforceps, and a 2mm section of the spinal cord was quickly removed at T10 using microscissors. Hemostasis was then performed to establish a complete transverse spinal cord injury model. Rats with spinal cord injury were treated with this protein-reducing sugar conductive tissue adhesive combined with electrical stimulation. After nearly two months of treatment, motor function scores and histological staining showed that the group treated with the protein-reducing sugar conductive tissue adhesive combined with electrical stimulation had the best recovery of motor function.

[0081] Example 2

[0082] Catecholized collagen was prepared according to the method in Example 1, and after freeze-drying, it was sealed and stored in a light-proof container for later use.

[0083] The catechin-modified collagen was dissolved in deionized water and then sonicated to fully dissolve it, resulting in a catechin-modified collagen solution with a concentration of 50 mg / mL, which was then set aside for use.

[0084] Then, 2 mL of catechin-modified collagen solution was poured into a 10 mL beaker. 600 mg of maltose, 500 mg of calcium chloride, and 100 mg of lithium chloride were weighed and added to the 10 mL beaker in sequence. The beaker was placed on a heating platform, the temperature was adjusted to 80 °C, and the stir bar was turned on for rapid stirring.

[0085] Then slowly add 1 mL of 0.1 mol / L sodium bicarbonate solution to the beaker and stir rapidly until the maltose is completely dissolved, resulting in a viscous solution. Then, centrifuge the solution using ultrasound to remove air bubbles.

[0086] Finally, the above solution was frozen overnight to obtain the catecholated collagen-maltose conductive tissue adhesive. The gelation time was approximately 200 s. Various tests and demonstration experiments showed that the modulus of this tissue adhesive was approximately 1.5 kPa, the shear strength was approximately 100 kPa, and the conductivity was approximately 1 × 10⁻⁶. -3 S / cm.

[0087] Example 3

[0088] Weigh 4000 mg of gelatin and dissolve it in 50 mL of 0.5% trifluoroacetic acid. Stir and dissolve at room temperature. It will completely dissolve in about 0.5-1 hour to obtain a gelatin solution.

[0089] Subsequently, 2g of dopamine hydrochloride was added to the gelatin solution, and the mixture was stirred and dissolved under sealed conditions. The pH of the mixed solution was adjusted to 5.5 by adding 1mol / L sodium hydroxide solution dropwise. Then, 3.4g of N-hydroxysuccinimide (NHS) was weighed and dissolved in 15mL of deionized water, and the pH of the mixed solution was adjusted to 5.5 by adding 1mol / L sodium hydroxide solution dropwise. Similarly, 4.6g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) was weighed and dissolved in 15mL of deionized water, and the pH of the mixed solution was adjusted to 5.5 by adding 10% trifluoroacetic acid solution dropwise. The prepared NHS and EDC solutions were added to the mixed solution sequentially, with an interval of 15 minutes between additions. Finally, the entire reaction system was placed in a sealed container and reacted at room temperature for 20 hours. After 20 hours, the reaction solution was transferred to a dialysis bag and dialyzed in 4 mM hydrochloric acid solution for three days, with the dialysate changed daily. The purified solution was then frozen at -80 degrees Celsius and freeze-dried using a freeze dryer to obtain the functionalized protein (catecholized gelatin), which was ready for use.

[0090] Dissolve the catechin-modified gelatin in deionized water, and then sonicate to fully dissolve the catechin-modified gelatin to obtain a catechin-modified gelatin solution with a concentration of 80 mg / mL, which is then ready for use.

[0091] Then, 2 mL of catechinized gelatin solution was poured into a 10 mL beaker. 500 mg of fructose, 500 mg of calcium chloride, and 100 mg of lithium chloride were weighed and added to the 10 mL beaker in sequence. The beaker was placed on a heating platform, the temperature was adjusted to 80 °C, and the stir bar was turned on for rapid stirring.

[0092] Then, slowly add 1 mL of 0.1 mol / L sodium bicarbonate solution to the beaker and stir rapidly until the fructose is completely dissolved to obtain a viscous solution. The solution is then ultrasonically centrifuged to remove air bubbles. Finally, the above solution is frozen overnight to obtain a catecholized gelatin-fructose conductive tissue adhesive with a gelling time of approximately 160 s.

[0093] Various tests and demonstration experiments revealed that the modulus of this tissue adhesive was approximately 2 kPa, the shear strength was approximately 100 kPa, and the conductivity was approximately 1 × 10⁻⁶ kPa. -3 S / cm.

[0094] Example 4

[0095] Oxidized starch was prepared according to the method in Example 1, washed, dried, sealed and stored in the dark for later use.

[0096] Dissolve silk fibroin in deionized water, and then sonicate to fully dissolve the silk fibroin, obtaining a silk fibroin solution with a concentration of 100 mg / mL, which is then ready for use.

[0097] Then, 2 mL of silk fibroin solution was poured into a 10 mL beaker, and 1 mL of 0.1 g / mL tannic acid (TA) solution was added to the silk fibroin stock solution. 400 mg of aldehyde-modified oxidized starch, 500 mg of calcium chloride and 100 mg of lithium chloride were weighed and added to the 10 mL beaker in sequence. The beaker was placed on a heating table, the temperature was adjusted to 80 °C, and the stir bar was turned on for rapid stirring.

[0098] Then, slowly add 1 mL of 0.1 mol / L sodium bicarbonate solution to the beaker and stir rapidly until the fructose is completely dissolved to obtain a viscous solution. The solution is then ultrasonically centrifuged to remove air bubbles. Finally, the above solution is frozen overnight to obtain the silk fibroin-tannin-reduced starch conductive tissue adhesive, with a gelation time of approximately 20 seconds.

[0099] Various tests and demonstration experiments revealed that the modulus of this tissue adhesive was approximately 3 kPa, the shear strength was approximately 120 kPa, and the conductivity was approximately 1 × 10⁻⁶. -3 S / cm.

[0100] Example 5

[0101] The preparation method of this embodiment is the same as that of Example 1, except that the content of the metal chloride salt is 30% (that is, the total content of calcium chloride and lithium chloride, and the mass ratio of the two is always kept at 5:1). All other steps are the same as those of Example 1.

[0102] Tests showed that the shear adhesion strength was approximately 100 kPa and the conductivity was approximately 1 × 10⁻⁶. -3 S / cm.

[0103] Comparative Example 1

[0104] The preparation method is the same as in Example 1, except that the content of the metal chloride salt is reduced from 20% to 10% (that is, the total content of calcium chloride and lithium chloride, and the mass ratio of the two is always maintained at 5:1), and the other steps are the same as in Example 1.

[0105] The protein-reducing sugar conductive tissue adhesive prepared in this comparative example has a gelation time of approximately 50 s, a modulus of approximately 0.5-1 kPa, an overlap shear strength of approximately 50 kPa, and a conductivity of approximately 5 × 10⁻⁶. -4 S / cm. The results showed that when the ion content was reduced from 20% to 10%, the adhesion strength, modulus and conductivity decreased significantly, with the adhesion strength and modulus decreasing by about half and the conductivity decreasing by an order of magnitude.

[0106] Comparative Example 2

[0107] The preparation method is the same as in Example 1, except that no metal chloride salt is added, that is, the content of metal chloride salt is 0%, and the other steps are the same as in Example 1.

[0108] The hydrogel adhesive prepared in this comparative example showed a significant decrease in adhesion strength, with a shear adhesion strength of 20 kPa and a conductivity of less than 1 × 10⁻⁶ kPa. -6 With a conductivity of S / cm, it has almost no electrical conductivity.

[0109] Comparative Example 3

[0110] The preparation method is the same as in Example 1, except that unmodified collagen is used instead of catecholized collagen.

[0111] The overall properties of the hydrogel tissue adhesive prepared in this comparative example were significantly reduced, with a gelation time of approximately 40 s, a modulus of approximately 1.6 kPa, a shear strength of approximately 40 kPa, and a conductivity of approximately 8 × 10⁻⁶. -4 S / cm.

[0112] Comparative Example 4

[0113] The preparation method is the same as in Example 1, except that non-reduced starch is used instead of reduced starch.

[0114] The hydrogel tissue adhesive prepared in this comparative example has a gelation time of approximately 25 s, a modulus of approximately 2 kPa, a shear strength reduction of approximately 45 kPa, and a conductivity of approximately 1 × 10⁻⁶. -3 S / cm.

[0115] See Figure 2 In the preparation of tissue adhesives according to the methods described in Example 1 (col / C-S 20% group), Example 5 (col / CS 30% group), Comparative Example 1 (col group), and Comparative Example 2 (col / CS 10% group), when different concentrations of metal chloride salt ions were added (20%, 30%, 0%, and 10%, respectively, the same below), the hydrogel forming time accelerated with the increase of ion content. This result indicates that salt ions participate in the construction of the hydrogel network. 2+ It will undergo a complexation reaction with the carboxyl groups on collagen, Li + It will interact with the quinone groups on collagen to form lithium bonds, thereby forming a cross-linked network.

[0116] See Figure 3The figure shows a comparison of the gelation time of different amounts of metal chloride salt particles added during the preparation of tissue adhesives for Example 1 (col / C-S 20%), Example 5 (col / CS 30%), Comparative Example 1 (col group), and Comparative Example 2 (col / CS 10%). As can be seen from the figure, when the mass percentage content of metal chloride salt ions is 20-30%, gelation occurs within 10 seconds. When the salt ion content is too low or not added, the gelation time is significantly prolonged, exceeding 50 seconds.

[0117] See Figure 4 During the preparation of tissue adhesives according to the methods described in Example 1 (col / C-S 20% group), Example 5 (col / CS 30% group), Comparative Example 1 (col group), and Comparative Example 2 (col / CS 10% group), the modulus of the hydrogel after gelation changed significantly when different amounts of metal chloride salt ions were added. As the content of metal chloride salts increased, the modulus also increased. The main reason is as mentioned above: salt ions participate in the construction of the hydrogel network. As the salt ion content increases, the complexation effect in the hydrogel increases, the ionic bond content increases significantly, thereby enhancing the strength of the hydrogel and thus increasing the modulus.

[0118] See Figure 6 The circular dichroism chromatograms of the tissue adhesive prepared according to the method described in Example 1 before and after heating demonstrate that the secondary structure of collagen is well preserved before and after heating. The activity of collagen affects the strength of the interaction between hydrogel and cells, thereby affecting cell adhesion. The heating process often affects the activity of collagen. Therefore, we proved through circular dichroism chromatogram that the activity of collagen did not change significantly before and after heating. This result proves that the three-dimensional structure of collagen remains intact before and after heating, thus maintaining stable biological activity.

[0119] See Figure 7 The tissue adhesives prepared according to the methods described in Example 1 (col / C-S 20% group), Example 5 (col / CS 30% group), Comparative Example 1 (col group), and Comparative Example 2 (col / CS 10% group) showed that the hydrogel adhesion strength increased with the increase of ion content when different amounts of metal chloride salt ions were added during the preparation process. This result is because the introduction of a large number of ions firstly makes the matrix more uniformly dispersed, and secondly, more reversible ion interactions are formed in the network, which enhances the connection with the interface. At the same time, the enhancement of the hydrogel network leads to a significant increase in the hydrogel adhesion strength.

[0120] See Figure 8Cyclic voltammetry curves of hydrogel adhesives prepared with different ion contents were measured using an electrochemical workstation for Example 1 (col / C-S 20%), Example 5 (col / CS 30%), and Comparative Example 2 (col / CS 10%). The results showed that the current through the hydrogel increased with the increase of metal salt ion content. This is because the increase of salt content significantly increases the ion concentration, thereby increasing conductivity.

[0121] See Figure 9 The catecholized collagen-oxidized starch tissue adhesive prepared in Example 1, combined with electrical stimulation therapy, was used to evaluate motor behavior (BBB) ​​at different recovery stages after spinal cord injury repair. Rat spinal cord injury models were established in four groups: control group (Control), electrical stimulation group (ES), material group (Col / CS), and electrical stimulation + material group (ES-Col / CS). After 8 weeks of treatment with different regimens, the rats' behavior was scored. The results showed that using the hydrogel prepared in Example 1 as a tissue adhesive for transplantation combined with percutaneous electrical stimulation significantly enhanced the motor ability of rats after spinal cord injury, significantly exceeding that of the control group, the transplantation alone, and the electrical stimulation therapy strategy group. The aforementioned treatment regimens refer to the use of tissue engineering strategies to treat complete transection spinal cord injuries. The first group was the control group, where no treatment was given after creating the injury; the second group was the electrical stimulation group, where no material was transplanted, and percutaneous electrical stimulation was performed on the injured rats; the third group involved transplanting material to the injury site without electrical stimulation; and the fourth group involved transplanting material while simultaneously receiving electrical stimulation.

[0122] See Figure 10 This study analyzed the histological staining results of the catecholized collagen-oxidized starch tissue adhesive prepared in Example 1 combined with electrical stimulation therapy after 8 weeks of recovery following spinal cord injury repair. Sections and pathological staining of neonatal spinal cord tissue from rats in different groups after 2 months of treatment were performed. H&E staining results showed that the hydrogel prepared in Example 1, when transplanted as a tissue adhesive and combined with percutaneous electrical stimulation, significantly enhanced the growth of neonatal spinal cord tissue, reduced cavity area, and increased blood vessel content. In contrast, the control group, the transplanted tissue alone, and the electrical stimulation treatment group had significantly larger cavities and significantly fewer blood vessels in the neonatal spinal cord. This demonstrates that the transplantation of hydrogel tissue adhesive combined with percutaneous electrical stimulation can effectively promote the recovery of spinal cord injury in rats, thereby promoting the recovery of the rats' motor function.

[0123] In addition, the inventors of this case also conducted experiments with other raw materials, process operations, and process conditions described in this specification, referring to the aforementioned embodiments, and obtained relatively ideal results in all cases.

[0124] It should be understood that the technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made to the technical solutions of the present invention without departing from the spirit and scope of the claims are within the scope of protection of the present invention.

Claims

1. A method for preparing a tissue adhesive, comprising: Provide functionalized proteins; use polyphenol-containing substances or small molecules containing phenolic hydroxyl groups to chemically modify or blend proteins to obtain functionalized proteins; A protein-reducing sugar tissue binder is provided; the functionalized protein, reducing sugar, and hygroscopic salt are mixed and then subjected to Maillard chemical reaction and ion complexation reaction under heating conditions through a one-pot cooking method to form a three-dimensional network structure, thereby preparing the protein-reducing sugar tissue binder. Preparation of tissue adhesives; The protein-reducing sugar tissue adhesive is incubated and then remodeled to form dense and ordered hydrogen bonds and secondary structures in the network structure to obtain the tissue adhesive. The hygroscopic salts include metal chloride salts; The mass ratio of the functionalized protein, the reducing sugar, the metal chloride, and the solvent is (10~100):(10~500):(30~1200):(100~3000); The metal chloride salt includes a mixture of calcium chloride and lithium chloride; The reducing sugar includes one or a combination of glucose, fructose, galactose, lactose, and maltose; Alternatively, the reducing sugar is obtained by oxidation of a non-reducing polysaccharide; The non-reducing polysaccharides include hyaluronic acid, starch, dextran, cyclodextrin, and chitosan.

2. The method for preparing the tissue adhesive according to claim 1, characterized in that, The tissue adhesive comprises an ion-quinone redox couple.

3. The method for preparing the tissue adhesive according to claim 1, characterized in that, The tissue adhesive has an overlap shear strength ≥80 kPa and a conductivity of approximately 1 × 10⁻⁶ kPa. -3 S / cm.

4. The method for preparing the tissue adhesive according to claim 1, characterized in that, The functionalized protein is modified onto the protein chain by means of polyphenol-containing unit substances or small molecules containing phenolic hydroxyl groups through physical or chemical modification methods.

5. The method for preparing the tissue adhesive according to claim 4, characterized in that, The physical modification method includes blending the protein with the polyphenol-containing unit substance or the small molecule containing phenolic hydroxyl groups and then incubating it through hydrogen bonding and / or hydrophobic interactions.

6. The method for preparing tissue adhesive according to claim 4, wherein the chemical modification method comprises preparing the substance containing polyphenol units or small molecules containing phenolic hydroxyl groups through esterification or condensation reactions.

7. The method for preparing the tissue adhesive according to claim 1, characterized in that, The polyphenol unit substance or small molecule containing phenolic hydroxyl groups includes at least one of dopamine, tannic acid, epigallocatechin, gallic acid and its derivatives.

8. The method for preparing the tissue adhesive according to claim 4, characterized in that, The protein includes at least one of collagen, gelatin, silk fibroin, bovine serum albumin, soy protein, or macromolecular polypeptide.

9. The method for preparing the tissue adhesive according to claim 1, characterized in that, In the one-pot cooking method, the heating temperature is 40~200℃.

10. The method for preparing the tissue adhesive according to claim 1, characterized in that, In the one-pot cooking method, the reaction time is 25–300 seconds.

11. The method for preparing the tissue adhesive according to claim 1, characterized in that, The mass ratio of calcium chloride to lithium chloride is 5 to 1.

12. The method for preparing the tissue adhesive according to claim 1, characterized in that, The solvent is deionized water or a 1% acetic acid solution.

13. A tissue adhesive, prepared by the method according to any one of claims 1-12; the tissue adhesive having wet surface adhesion, adjustable electrical conductivity, shape adaptability, biocompatibility, and biodegradability; in, The tissue adhesive has an overlap shear strength ≥80 kPa and a conductivity of approximately 1 × 10⁻⁶ kPa. -3 S / cm.

14. The application of the tissue adhesive as described in claim 13 in the preparation of an integrated repair material for nerve transection; wherein the integrated repair of the nerve transection includes emergency hemostasis, incompressible nerve hemostasis and repair, sealing connection, defect filling, and electrical conductivity to promote neurogenesis and axonal growth.

15. The use of the tissue adhesive as described in claim 13 in the preparation of a repair material for seamlessly connecting a completely severed spinal cord.

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