A medical adhesive based on mussel mucoprotein and its preparation method and use

Abstract

The present application relates to a kind of medical adhesive of mussel mucin base and its preparation method and purposes, the medical adhesive of mussel mucin base of the present application is made of component A and crosslinking accelerator;Component A includes mussel mucin complex and functional regulator, and mussel mucin complex includes mussel mucin and anionic functional material;The mass ratio of mussel mucin, anionic functional material and functional regulator in component A is 3:(50~2.5):(0.2~1);The mass ratio of crosslinking accelerator and mussel mucin complex in component A is 1:(200~800).The medical adhesive of mussel mucin base of the present application has multi-scene high adaptability, function integration and active repair are combined to realize "adhesion-repair" integration;Meanwhile, it has excellent biocompatibility and safety, process feasibility and stability.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a mussel adhesive protein-based medical adhesive, its preparation method, and its uses. Background Technology

[0002] Medical adhesives, as important alternatives or auxiliary biomedical materials to traditional sutures and hemostatic materials, have irreplaceable application value in clinical fields such as surgical closure, trauma emergency repair, and tissue regeneration induction. Currently, medical adhesives used clinically are mainly divided into two categories: synthetic and natural. Synthetic medical adhesives have the core advantages of fast curing rate and high initial bond strength, and are widely used in scenarios such as rapid closure of superficial wounds. However, due to the material's brittleness and insufficient toughness, when used in dynamically stressed tissues or moving parts such as joints, the heart, and blood vessels, the adhesive interface is prone to cracking and detachment due to tissue deformation, making long-term stable adhesion impossible. Furthermore, because these adhesives lack bioactive sites, they can only achieve physical adhesion and fixation, and cannot participate in the tissue repair process, making it difficult to meet the clinical needs of damaged tissue regeneration. Natural medical adhesives possess good biocompatibility, biodegradability, and weak hemostatic function, and have certain advantages in soft tissue filling and bonding. However, natural medical adhesives rely on the cross-linking of protein molecules in a dry environment. In wet environments such as wound bleeding and body fluid infiltration, the bonding interface is easily washed away by body fluid, resulting in a significant decrease in adhesive strength and thus causing adhesive failure. On the other hand, due to their low mechanical strength, they are difficult to meet the bonding and fixation requirements of tissues with high mechanical loads such as cartilage, bone, and tendons, and are not suitable for hard tissue repair scenarios. To address the technical bottlenecks of traditional medical adhesives, the development of novel bioadhesive materials has become a research hotspot in this field. Among them, mussel adhesive protein (MAP) is considered an ideal substrate for constructing a new generation of medical adhesives due to its unique structural and performance advantages. The core adhesion mechanism of MAP originates from the catechol groups rich in its molecular structure. These groups can achieve strong adhesion to various substrates (including biological tissues) in an aqueous environment through multiple mechanisms such as hydrogen bonding, hydrophobic interaction, metal coordination bonds, and oxidative crosslinking. At the same time, MAP has excellent biocompatibility, biodegradability, and potential cell proliferation and anti-inflammatory activities, which can effectively avoid the toxicity problems and biological inertness defects of traditional materials. Although mussel adhesive protein has significant application potential, the research and development of existing mussel adhesive protein-based medical adhesives still face three key technical problems that urgently need to be overcome: (1) Limitations in scene adaptability: Existing studies mostly use a single crosslinking mechanism (such as simple enzymatic oxidative crosslinking, single metal ion coordination crosslinking, or physical crosslinking), resulting in fixed adhesive properties of the adhesive, which cannot flexibly adapt to the needs of different clinical scenarios. (2) Insufficient functional integration: The core design of existing mussel adhesives focuses on optimizing adhesive strength and degradation rate, lacking multi-dimensional consideration of actual clinical needs. On the one hand, it fails to achieve responsive regulation of the tissue microenvironment (such as the acidic pH of the inflamed site, specific enzymes at the wound site, and changes in body temperature); on the other hand, it fails to effectively integrate active repair functions (such as osteogenic induction, angiogenesis, anti-inflammatory and antibacterial effects). Therefore, it is difficult to achieve the integration of "adhesion-repair-regulation". (3) Defects in process and stability: The catechol groups in mussel adhesive molecules are chemically active and are prone to spontaneous oxidation and inactivation under normal temperature and aerobic conditions, leading to a decrease in the adhesive's adhesion performance and seriously affecting the product's storage stability. At the same time, the current crosslinking preparation process of MAP adhesives requires strict control of reaction conditions, making large-scale production difficult and hindering clinical translation and widespread application. In summary, there is an urgent need in this field to develop a mussel adhesive protein-based medical adhesive that is flexible and adaptable to different scenarios, has excellent wet adhesion strength, good biocompatibility, controllable degradation rate and active repair function, and is stable in preparation process, convenient to store and easy to use, so as to meet the comprehensive needs of different clinical scenarios and promote the technological upgrading of medical adhesive materials. Summary of the Invention

[0003] In response to the current technical challenges faced by the industry, this invention develops a mussel adhesive protein-based medical adhesive, its preparation method, and its applications.

[0004] On the one hand, the present invention provides a mussel adhesive protein-based medical adhesive, which is composed of component A and a crosslinking accelerator; Component A includes a mussel adhesive protein complex and a functional regulator; the mussel adhesive protein complex includes mussel adhesive protein and anionic functional materials. The mass ratio of mussel adhesive protein, anionic functional material, and functional regulator in component A is 3:(50~2.5):(0.2~1); The functional regulator is selected from one or more combinations of temperature-sensitive components, enzyme-sensitive components, bioactive factors, and bioactive inorganic fillers; The mass ratio of the cross-linking promoter to the mussel adhesive protein complex in component A is 1:(200~800).

[0005] Furthermore, in the mussel adhesive protein-based medical adhesive of the present invention, the crosslinking accelerator is selected from any one of oxidants, polyvalent metal ion compounds, and polyethylene glycol derivatives.

[0006] Furthermore, in the mussel adhesive protein-based medical adhesive of the present invention, the oxidant is an oxidant capable of oxidizing catechol groups, selected from any one of hydrogen peroxide, sodium periodate, and persulfate.

[0007] Furthermore, the polyvalent metal ion compound is selected from any one of soluble calcium salts, ferric salts, zinc salts, and magnesium salts.

[0008] Furthermore, the polyethylene glycol derivative is selected from any one of multi-arm polyethylene glycol-acrylate (e.g., 4-arm-PEG-Acrylate, 2-arm-PEG-acrylate), multi-arm polyethylene glycol-maleimide (e.g., 4-arm-PEG-Maleimide), and multi-arm polyethylene glycol-succinimide glutarate (e.g., 4-arm-PEG-SG, 8-arm-PEG-SG).

[0009] Furthermore, in the mussel adhesive protein-based medical adhesive of the present invention, the temperature-sensitive component is selected from one or more combinations of poly(N-isopropylacrylamide)-grafted hyaluronic acid (HA-g-PNIPAM), polyethylene glycol-poly(N-isopropylacrylamide) double-grafted hyaluronic acid, poly(N-isopropylacrylamide) and its copolymers, poloxamer, and hydroxypropyl methylcellulose.

[0010] Furthermore, in the mussel adhesive protein-based medical adhesive of the present invention, the enzyme-sensitive component is selected from one or more combinations of matrix metalloproteinases.

[0011] Furthermore, the matrix metalloproteinases include, but are not limited to, matrix metalloproteinase-3 (MMP-3) and matrix metalloproteinase-2 (MMP-2).

[0012] Furthermore, in the mussel adhesive protein-based medical adhesive of the present invention, the bioactive factor is selected from one or more combinations of vascular endothelial growth factor (VEGF), bone morphogenetic protein-2 (BMP-2), and platelet-derived growth factor (PDGF).

[0013] Furthermore, in the mussel adhesive protein-based medical adhesive of the present invention, the bioactive inorganic filler is selected from one or more combinations of hydroxyapatite and β-tricalcium phosphate.

[0014] Furthermore, in the mussel adhesive protein-based medical adhesive of the present invention, the anionic functional material is one or more combinations of polyanionic materials, amphiphilic polyelectrolyte materials, and biomaterials with anionic groups.

[0015] Furthermore, the anionic functional material is selected from any one or more combinations of hyaluronic acid, alginate, chondroitin sulfate, and cholesterol-modified hyaluronic acid.

[0016] Furthermore, the polyanionic material includes, but is not limited to, sodium hyaluronate and sodium alginate.

[0017] Furthermore, in the mussel adhesive protein-based medical adhesive of the present invention, the mussel adhesive protein can be selected from naturally extracted or genetically engineered recombinant mussel adhesive protein.

[0018] Furthermore, the naturally extracted mussel adhesive protein refers to the adhesive protein components isolated from the mussel foot, including but not limited to Mfp-1, Mfp-2, Mfp-3, Mfp-4, Mfp-5, and Mfp-6; the genetically engineered recombinant mussel adhesive protein includes but is not limited to recombinant proteins with the amino acid sequence of natural mussel adhesive proteins, fusion proteins constructed from two or more of the above-mentioned mussel adhesive proteins, and activity-enhanced fusion proteins constructed by further fusing functional peptides derived from the extracellular matrix.

[0019] On the other hand, the present invention provides a method for preparing a mussel adhesive based on mussel adhesive protein, wherein the mussel adhesive based on mussel adhesive protein is any of the mussel adhesive based on mussel adhesive protein described above; the preparation method includes the following steps: Step 1) Dissolve mussel adhesive protein in solvent A to obtain a mussel adhesive protein solution with a mass fraction of 0.1~8wt%; the solvent A is a phosphate buffer with a pH range of 5.5~7; Step 2): Dissolve the anionic functional material and the functional regulator in solvent A to prepare anionic functional material solution and functional regulator solution, respectively; add the anionic functional material solution to the mussel adhesive protein solution to obtain mussel adhesive protein complex; then add the functional regulator solution to the mussel adhesive protein complex; stir at 150-200 rpm for 20-30 min; and then aseptically process to obtain component A. In component A, the mass ratio of mussel adhesive protein, anionic functional material, and functional regulator is 3:(50~2.5):(0.2~1). Step 3): Dissolve the crosslinking accelerator in solvent B and adjust the pH of the system to 7.0-7.4 to obtain a crosslinking accelerator solution; the solvent B is phosphate buffer (PBS) with a pH range of 7.0-7.4. The mass ratio of the cross-linking promoter to the mussel adhesive protein complex is 1:(200~800); Step 4): After sterilizing the components A and crosslinking accelerator solutions, dispense them into sterile sealed containers under aseptic conditions and store them at low temperature and away from light. The mussel adhesive protein-based medical adhesive described above is obtained by mixing component A and the crosslinking accelerator solution at a volume ratio of 1:1, and then immediately applied to the target site.

[0020] Furthermore, the low temperature in step 4) is 2~10℃.

[0021] In another aspect, the present invention provides a use of a mussel adhesive protein-based medical adhesive, wherein the mussel adhesive protein-based medical adhesive is any of the mussel adhesive protein-based medical adhesives described above, or a mussel adhesive protein-based medical adhesive prepared by any of the preparation methods described above; the mussel adhesive protein-based medical adhesive is used for closing superficial skin wounds, repairing moist wounds, sealing mucous membranes, and bonding bone / cartilage defects.

[0022] Furthermore, the mussel adhesive protein-based medical adhesive is used for bonding bone or cartilage defects, and preferably the mass ratio of mussel adhesive protein, anionic functional material and functional regulator in component A is 3:(10~15):(0.8~1).

[0023] Furthermore, the functional regulator is preferably one or a combination of bioactive factors and bioactive inorganic fillers.

[0024] Furthermore, the mass ratio of the cross-linking promoter to the mussel adhesive protein complex in component A is 1:(300~500).

[0025] Furthermore, the crosslinking accelerator is preferably selected from any one of metal ion compounds and polyethylene glycol derivatives.

[0026] The beneficial effects of this invention are: The mussel adhesive protein-based medical adhesive of this invention is suitable for tissue bonding needs under various physiological conditions, and can form a stable and reliable adhesive interface at wet, dynamic, and highly challenging tissue interfaces. Through a two-stage "complex-crosslinking" design, this invention significantly improves the adhesive strength and shear resistance while maintaining good biocompatibility, exhibiting both broad tissue adaptability and reliable mechanical stability, effectively expanding its clinical application range. The mussel adhesive protein-based medical adhesive of this invention possesses high adaptability to multiple scenarios, integrates functions, and combines active repair to achieve integrated "adhesion-repair"; simultaneously, it exhibits excellent biocompatibility and safety, process feasibility, and stability.

[0027] The mussel adhesive-based medical adhesive of this invention significantly expands its biological functions and effectively improves the material's mechanical properties and environmental adaptability by introducing specific functional regulators. Specifically, the added thermosensitive component undergoes a phase transition near body temperature, endowing the adhesive with in-situ gelation capabilities and optimizing the operating window and tissue adhesion; the added bioactive factors enable the adhesive to possess multiple biological functions, including wound closure, hemostasis, anti-inflammation, and tissue repair; and the added bioactive inorganic filler not only enhances the material's adhesive strength and shear resistance but also slowly releases calcium and silicon ions, promoting tissue mineralization and cellular response.

[0028] The various functional modulators in the mussel adhesive protein-based medical adhesive of this invention, while performing their specific biological functions, can also penetrate and enhance the integrity of the mussel adhesive protein complex network structure through secondary interactions such as hydrophobic interactions, hydrogen bonds, or ionic cross-linking, thereby effectively overcoming the shortcomings of insufficient mechanical strength in traditional mussel adhesive protein-based adhesive materials. Therefore, this invention provides a multifunctional medical adhesive that can be customized according to actual clinical needs, possessing both structural support and bioactivity, significantly improving its applicability and reliability in complex physiological environments, and meeting the needs of various clinical application scenarios such as superficial skin wound closure and moist wound repair. Attached Figure Description

[0029] Figure 1 This is a schematic diagram illustrating the healing-promoting effects of Examples 3-4 and Comparative Examples 1-2 of the present invention in a rat model of full-thickness skin defects with chronic wounds. Detailed Implementation

[0030] To provide a clearer understanding of the technical features, objectives, and effects of the present invention, specific embodiments are now described in detail with reference to the accompanying drawings. The described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the detailed embodiments, conventional conditions or conditions provided by the manufacturer shall apply.

[0031] In the following description, when referring to the accompanying drawings, the same numbers in different drawings denote the same or similar elements unless otherwise indicated. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this disclosure. The terminology used in this disclosure is for the purpose of describing particular embodiments only and is not intended to be limiting of this disclosure. Example 1

[0032] This invention discloses a mussel adhesive protein-based medical adhesive for closing superficial skin wounds. The preparation method of the mussel adhesive protein-based medical adhesive includes the following steps: Step 1) Dissolve Mfp-5 mussel adhesive protein in PBS solution with a pH of 6.0 to prepare a 5 wt% mussel adhesive protein solution; Step 2) Dissolve sodium alginate and poly(N-isopropylacrylamide) separately in PBS solution with a pH of 6.5 to prepare sodium alginate solution and poly(N-isopropylacrylamide) solution respectively; Step 3) Mix the sodium alginate solution obtained in Step 2) and the mussel adhesive protein solution obtained in Step 1) evenly to obtain a mussel adhesive protein complex. Then, add the poly(N-isopropylacrylamide) solution to the mussel adhesive protein complex and stir at 200 rpm for 20 min. After sterilization, obtain component A, wherein the mass ratio of Mfp-5 type mussel adhesive protein to sodium alginate and poly(N-isopropylacrylamide) is 3:30:0.5. Step 4) Dissolve ferric chloride hexahydrate in PBS solution with pH 7.4, and adjust the pH of the system to 7.0~7.4 using a pH adjuster to obtain a cross-linking promoter solution; wherein, the mass ratio of ferric chloride hexahydrate to mussel adhesive protein complex is 1:720; Step 5) Sterilize component A and crosslinking accelerator solution separately, and dispense them into sterile sealed containers under aseptic conditions. Store at a low temperature of 2~10℃ away from light. In use, component A and the crosslinking accelerator solution are mixed at a volume ratio of 1:1 to obtain the mussel adhesive protein-based medical adhesive of Example 1, and then applied to the target site immediately.

[0033] The mussel adhesive-based medical adhesive of this embodiment 1 is composed of component A and ferric chloride hexahydrate; component A includes mussel adhesive protein complex and poly(N-isopropylacrylamide), the mussel adhesive protein complex includes Mfp-5 type mussel adhesive protein and sodium alginate; the mass ratio of mussel adhesive protein, sodium alginate and poly(N-isopropylacrylamide) in component A is 3:30:0.5; the mass ratio of ferric chloride hexahydrate to mussel adhesive protein complex is 1:720.

[0034] Example 2 This invention discloses a mussel adhesive protein-based medical adhesive for closing superficial skin wounds. The preparation method of the mussel adhesive protein-based medical adhesive includes the following steps: Step 1) Dissolve Mfp-5 mussel adhesive protein in PBS solution with pH 6 to prepare a 3wt% mussel adhesive protein solution; Step 2) Dissolve sodium hyaluronate, poly(N-isopropylacrylamide)-grafted hyaluronic acid, and hydroxypropyl methylcellulose in PBS solution at pH 7 to prepare sodium hyaluronate solution and poly(N-isopropylacrylamide)-grafted hyaluronic acid and hydroxypropyl methylcellulose solution, respectively. Step 3) Mix the sodium hyaluronate solution obtained in Step 2) and the mussel adhesive protein solution obtained in Step 1) evenly to obtain a mussel adhesive protein complex. Then, add the poly(N-isopropylacrylamide) grafted hyaluronic acid and hydroxypropyl methylcellulose solution to the mussel adhesive protein complex and stir at 200 rpm for 20 min. After sterilization, obtain component A, wherein the mass ratio of Mfp-5 type mussel adhesive protein to sodium hyaluronate and poly(N-isopropylacrylamide) grafted hyaluronic acid and hydroxypropyl methylcellulose is 3:45:0.5:0.5. Step 4) Dissolve calcium chloride in PBS solution with pH 7.4, and adjust the pH of the system to 7.0~7.4 using a pH adjuster. After sterile treatment, obtain the cross-linking promoter solution; wherein the mass ratio of calcium chloride to mussel adhesive protein complex is 1:300. Step 5) After sterilizing the A component and the crosslinking accelerator solution, dispense them into sterile sealed containers under aseptic conditions and store them at 2~10℃ in the dark. In use, component A and the crosslinking accelerator solution are mixed at a volume ratio of 1:1 to obtain the mussel adhesive protein-based medical adhesive of Example 2, and then applied to the target site immediately.

[0035] The mussel adhesive-based medical adhesive of this embodiment 2 is composed of component A and calcium chloride; component A includes mussel adhesive protein complex and poly(N-isopropylacrylamide)-grafted hyaluronic acid and hydroxypropyl methylcellulose. The mussel adhesive protein complex includes Mfp-5 type mussel adhesive protein and sodium hyaluronate; the mass ratio of mussel adhesive protein, sodium hyaluronate and poly(N-isopropylacrylamide)-grafted hyaluronic acid and hydroxypropyl methylcellulose in component A is 3:45:0.5:0.5; the mass ratio of calcium chloride to mussel adhesive protein complex is 1:300.

[0036] Example 3 This invention discloses a mussel adhesive protein-based medical adhesive for the repair of moist chronic wounds. The preparation method of the mussel adhesive protein-based medical adhesive includes the following steps: Step 1) Dissolve Mfp-3 type mussel adhesive protein in PBS solution with a pH of 5.5 to prepare a mussel adhesive protein solution with a mass fraction of 0.1 wt%. Step 2) Dissolve sodium alginate, sodium hyaluronate, vascular endothelial growth factor (VEGF), and matrix metalloproteinase-3 (MMP-3) in PBS solution at pH 5.5 to prepare a mixed solution of sodium alginate and sodium hyaluronate and a mixed solution of VEGF and MMP-3. Step 3) Mix the sodium alginate and sodium hyaluronate mixed solution obtained in Step 2) with the mussel adhesive protein solution obtained in Step 1) to obtain a mussel adhesive protein complex. Then add the VEGF and MMP-3 mixed solution to the mussel adhesive protein complex and stir at 150 rpm for 20 min. After sterilization, obtain component A. The mass ratio of Mfp-3 type mussel adhesive protein to sodium alginate, sodium hyaluronate, VEGF, and MMP-3 is 3:25:25:0.1:0.1. Step 4) Dissolve sodium periodate in PBS buffer solution with pH 7.0, and adjust the pH of the system to 7.0~7.4 using a pH adjuster. After sterile treatment, obtain the cross-linking promoter solution; wherein the mass ratio of sodium periodate to mussel adhesive protein complex is 1:800. Step 5) After sterilizing the components A and crosslinking accelerator solutions, dispense them into sterile sealed containers under aseptic conditions and store them at 2~10℃ in the dark. In use, component A and the crosslinking accelerator solution are mixed at a volume ratio of 1:1 to obtain the mussel adhesive protein-based medical adhesive of Example 3, and then applied to the target site immediately.

[0037] The mussel adhesive-based medical adhesive of this embodiment 3 is composed of component A and sodium periodate; component A includes mussel adhesive protein complex, vascular endothelial growth factor (VEGF), and matrix metalloproteinase-3 (MMP-3); the mussel adhesive protein complex includes Mfp-3 type mussel adhesive protein, sodium alginate, and sodium hyaluronate; the mass ratio of Mfp-3 type mussel adhesive protein to sodium alginate, sodium hyaluronate, VEGF, and MMP-3 in component A is 3:25:25:0.1:0.1; the mass ratio of sodium periodate to mussel adhesive protein complex is 1:800.

[0038] Example 4 This invention discloses a mussel adhesive protein-based medical adhesive for the repair of moist chronic wounds. The preparation method of the mussel adhesive protein-based medical adhesive includes the following steps: Step 1) Dissolve Mfp-3 mussel adhesive protein in PBS solution with a pH of 7.0 to prepare a mussel adhesive protein solution with a mass fraction of 8 wt%. Step 2) Dissolve chondroitin sulfate, platelet-derived growth factor (PDGF), and polyethylene glycol-poly(N-isopropylacrylamide) double-grafted hyaluronic acid in PBS solution at pH 7.0 to prepare chondroitin sulfate solution and PDGF and polyethylene glycol-poly(N-isopropylacrylamide) double-grafted hyaluronic acid mixed solution. Step 3) Mix the chondroitin sulfate solution obtained in Step 2) and the mussel adhesive protein solution obtained in Step 1) evenly to obtain a mussel adhesive protein complex. Then, add the mixed solution of PDGF and polyethylene glycol-poly(N-isopropylacrylamide) double-grafted hyaluronic acid to the mussel adhesive protein complex and stir at 200 rpm for 30 min. After sterilization, obtain component A, wherein the mass ratio of Mfp-3 type mussel adhesive protein to chondroitin sulfate, PDGF, and polyethylene glycol-poly(N-isopropylacrylamide) double-grafted hyaluronic acid is 3:2.5:0.6:0.4. Step 4) Dissolve 2-arm polyethylene glycol-acrylate (2-arm-PEG-acrylate) in PBS buffer solution with pH 7.4, and adjust the pH of the system to 7.0~7.4 using a pH adjuster. After sterile treatment, obtain the cross-linking promoter solution; wherein, the mass ratio of 2-arm-PEG-acrylate to mussel adhesive protein complex is 1:200. Step 5) After sterilizing the components A and crosslinking accelerator solutions, dispense them into sterile sealed containers under aseptic conditions and store them at 2~10℃ in the dark. In use, component A and the crosslinking accelerator solution are mixed at a volume ratio of 1:1 to obtain the mussel adhesive protein-based medical adhesive of Example 4, and then applied to the target site immediately.

[0039] The mussel adhesive-based medical adhesive of Example 4 is composed of component A and 2-arm polyethylene glycol-acrylate (2-arm-PEG-acrylate). Component A includes mussel adhesive protein complex, platelet-derived growth factor (PDGF), and polyethylene glycol-poly(N-isopropylacrylamide) double-grafted hyaluronic acid. The mussel adhesive protein complex includes Mfp-3 type mussel adhesive protein and chondroitin sulfate. The mass ratio of Mfp-3 type mussel adhesive protein to chondroitin sulfate, platelet-derived growth factor (PDGF), and polyethylene glycol-poly(N-isopropylacrylamide) double-grafted hyaluronic acid in component A is 3:2.5:0.6:0.4. The mass ratio of 2-arm-PEG-acrylate to mussel adhesive protein complex is 1:200.

[0040] Example 5 This invention discloses a mussel adhesive protein-based medical adhesive for mucosal occlusion. The preparation method of the mussel adhesive protein-based medical adhesive includes the following steps: Step 1) Dissolve Mfp-361 mussel adhesive protein in PBS solution with a pH of 6.5 to prepare a 6 wt% mussel adhesive protein solution; Step 2) Chondroitin sulfate, cholesterol-modified hyaluronic acid, matrix metalloproteinase-2 (MMP-2), and vascular endothelial growth factor (VEGF) were dissolved in PBS solution at pH 7.0 to prepare chondroitin sulfate and cholesterol-modified hyaluronic acid mixed solution and MMP-2 and VEGF mixed solution, respectively. Step 3) Mix the chondroitin sulfate and cholesterol-modified hyaluronic acid mixed solution obtained in Step 2) with the mussel adhesive protein solution obtained in Step 1) to obtain a mussel adhesive protein complex. Then add the MMP-2 and VEGF mixed solution to the mussel adhesive protein complex and stir at 200 rpm for 30 min. After sterilization, obtain component A, in which the mass ratio of Mfp-361 mussel adhesive protein to chondroitin sulfate, cholesterol-modified hyaluronic acid, MMP-2 and VEGF is 3:1:3:0.5:0.5. Step 4) Dissolve 8-arm-PEG-SG in PBS buffer at pH 7.4, and adjust the pH of the system to 7.0~7.4 using a pH adjuster. After sterile treatment, obtain the cross-linking promoter solution; wherein, the mass ratio of 8-arm-PEG-SG to mussel adhesive protein complex is 1:350. Step 5) After sterilizing the components A and crosslinking accelerator solutions, dispense them into sterile sealed containers under aseptic conditions and store them at 2~10℃ in the dark. In use, component A and the crosslinking accelerator solution are mixed at a volume ratio of 1:1 to obtain the mussel adhesive protein-based medical adhesive of Example 5, and then applied to the target site immediately.

[0041] The mussel adhesive-based medical adhesive of Example 5 is composed of component A and 8-arm polyethylene glycol-succinimide glutarate (8-arm-PEG-SG). Component A includes a mussel adhesive protein complex, matrix metalloproteinase-2 (MMP-2), and vascular endothelial growth factor (VEGF). The mussel adhesive protein complex includes Mfp-361 mussel adhesive protein, chondroitin sulfate, and cholesterol-modified hyaluronic acid. The mass ratio of Mfp-361 mussel adhesive protein to chondroitin sulfate, cholesterol-modified hyaluronic acid, matrix metalloproteinase-2, and vascular endothelial growth factor (VEGF) in component A is 3:1:3:0.5:0.5. The mass ratio of 8-arm-PEG-SG to the mussel adhesive protein complex is 1:350.

[0042] Example 6 This invention discloses a mussel adhesive protein-based medical adhesive for mucosal occlusion. The preparation method of the mussel adhesive protein-based medical adhesive includes the following steps: Step 1) Dissolve Mfp-361 mussel adhesive protein in PBS solution with a pH of 5.8 to prepare a 3wt% mussel adhesive protein solution; Step 2) Dissolve sodium hyaluronate and hyaluronic acid grafted poly(N-isopropylacrylamide) in PBS solution with pH 5.8 to prepare sodium hyaluronate and hyaluronic acid grafted poly(N-isopropylacrylamide) solution respectively. Step 3) Mix the sodium hyaluronate solution obtained in Step 2) and the mussel adhesive protein solution obtained in Step 1) evenly to obtain a mussel adhesive protein complex. Then, add the hyaluronic acid grafted poly(N-isopropylacrylamide) solution to the mussel adhesive protein complex and stir at 200 rpm for 30 min. After sterilization, obtain component A, in which the mass ratio of Mfp-361 mussel adhesive protein to sodium hyaluronate and hyaluronic acid grafted poly(N-isopropylacrylamide) is 3:40:1. Step 4) Dissolve hydrogen peroxide in PBS buffer at pH 7.4, and adjust the pH of the system to 7.0-7.4 using a pH adjuster. After sterile treatment, obtain a cross-linking promoter solution; wherein the mass ratio of hydrogen peroxide to mussel adhesive protein complex is 1:500. Step 5) After sterilizing the components A and crosslinking accelerator solutions, dispense them into sterile sealed containers under aseptic conditions and store them at 2~10℃ in the dark. In use, component A and the crosslinking accelerator solution are mixed at a volume ratio of 1:1 to obtain the mussel adhesive protein-based medical adhesive of Example 6, and then applied to the target site immediately.

[0043] The mussel adhesive-based medical adhesive of Example 6 is composed of component A and hydrogen peroxide; component A includes mussel adhesive protein complex and hyaluronic acid grafted poly(N-isopropylacrylamide), the mussel adhesive protein complex includes Mfp-361 type mussel adhesive protein and sodium hyaluronate; the mass ratio of Mfp-361 type mussel adhesive protein to sodium hyaluronate and hyaluronic acid grafted poly(N-isopropylacrylamide) in component A is 3:40:1; the mass ratio of hydrogen peroxide to mussel adhesive protein complex is 1:500.

[0044] Example 7 This invention discloses a mussel adhesive protein-based medical adhesive for bone defect repair. The preparation method of the mussel adhesive protein-based medical adhesive includes the following steps: [The invention relates to a mussel adhesive protein-based medical adhesive, which is not directly related to the preceding text and can be omitted.] Step 1) Dissolve Mfp-1-RGD mussel adhesive protein in PBS solution with a pH of 6.5 to prepare a mussel adhesive protein solution with a mass fraction of 8 wt%. Step 2) Cholesterol monosubstituted hyaluronic acid, bone morphogenetic protein-2 (BMP-2) and hydroxyapatite were dissolved in PBS solution at pH 6.5 to prepare cholesterol monosubstituted hyaluronic acid solution and BMP-2 and hydroxyapatite mixture solution respectively. Step 3) Mix the cholesterol monosubstituted hyaluronic acid solution obtained in Step 2) and the mussel adhesive protein solution obtained in Step 1) evenly to obtain the mussel adhesive protein complex. Then add the BMP-2 and hydroxyapatite mixture to the mussel adhesive protein complex and stir at 200 rpm for 25 min. After sterilization, obtain component A, in which the mass ratio of Mfp-1-RGD type mussel adhesive protein to cholesterol monosubstituted hyaluronic acid, BMP-2 and hydroxyapatite is 3:10:0.5:0.5. Step 4) Dissolve magnesium chloride in PBS solution with pH 7.0, and adjust the pH of the system to 7.0~7.4 using a pH adjuster. After sterile treatment, obtain the cross-linking promoter solution; wherein the mass ratio of magnesium chloride to mussel adhesive protein complex is 1:300. Step 5) After sterilizing the components A and crosslinking accelerator solutions, dispense them into sterile sealed containers under aseptic conditions and store them at 2~10℃ in the dark. In use, component A and the crosslinking accelerator solution are mixed at a volume ratio of 1:1 to obtain the mussel adhesive protein-based medical adhesive of Example 7, and then applied to the target site immediately.

[0045] The mussel adhesive-based medical adhesive of this embodiment 7 is composed of component A and magnesium chloride; component A includes mussel adhesive protein complex, bone morphogenetic protein-2 (BMP-2) and hydroxyapatite, the mussel adhesive protein complex includes Mfp-1-RGD type mussel adhesive protein and cholesterol monosubstituted hyaluronic acid; the mass ratio of Mfp-1-RGD type mussel adhesive protein to cholesterol monosubstituted hyaluronic acid, bone morphogenetic protein-2 (BMP-2) and hydroxyapatite in component A is 3:10:0.5:0.5; the mass ratio of magnesium chloride to mussel adhesive protein complex is 1:300.

[0046] Example 8 This invention discloses a mussel adhesive protein-based medical adhesive for bone defect repair. The preparation method of the mussel adhesive protein-based medical adhesive includes the following steps: Step 1) Dissolve Mfp-1-RGD mussel adhesive protein in PBS solution with a pH of 5.5 to prepare a mussel adhesive protein solution with a mass fraction of 8 wt%. Step 2) Dissolve cholesterol monosubstituted hyaluronic acid, BMP-2, and β-tricalcium phosphate in PBS solution with pH 6.5 to prepare cholesterol monosubstituted hyaluronic acid solution and BMP-2 and β-tricalcium phosphate solution, respectively. Step 3) Mix the cholesterol monosubstituted hyaluronic acid solution obtained in Step 2) and the mussel adhesive protein solution obtained in Step 1) evenly to obtain the mussel adhesive protein complex. Then add BMP-2 and β-tricalcium phosphate solution to the mussel adhesive protein complex and stir at 200 rpm for 25 min. After sterilization, obtain component A, in which the mass ratio of Mfp-1-RGD type mussel adhesive protein to cholesterol monosubstituted hyaluronic acid, BMP-2 and β-tricalcium phosphate is 3:15:0.5:0.3. Step 4) Dissolve sodium persulfate in PBS buffer solution with pH 7.0, and adjust the pH of the system to 7.0~7.4 using a pH adjuster. After sterile treatment, obtain the cross-linking promoter solution; wherein, the mass ratio of sodium persulfate to mussel adhesive protein complex is 1:500. Step 5) After sterilizing the components A and crosslinking accelerator solutions, dispense them into sterile sealed containers under aseptic conditions and store them at 2~10℃ in the dark. In use, component A and the crosslinking accelerator solution are mixed at a volume ratio of 1:1 to obtain the mussel adhesive protein-based medical adhesive of Example 8, and then applied to the target site immediately.

[0047] The mussel adhesive-based medical adhesive of this embodiment 8 is composed of component A and sodium persulfate; component A includes mussel adhesive protein complex, bone morphogenetic protein-2 (BMP-2) and β-tricalcium phosphate, the mussel adhesive protein complex includes Mfp-1-RGD type mussel adhesive protein and cholesterol monosubstituted hyaluronic acid; the mass ratio of Mfp-1-RGD type mussel adhesive protein to cholesterol monosubstituted hyaluronic acid, bone morphogenetic protein-2 (BMP-2) and β-tricalcium phosphate in component A is 3:15:0.5:0.3; the mass ratio of sodium persulfate to mussel adhesive protein complex is 1:500.

[0048] Example 9 This invention discloses a mussel adhesive protein-based medical adhesive for repairing cartilage defects. The preparation method of the mussel adhesive protein-based medical adhesive includes the following steps: Step 1) Dissolve Mfp-151 mussel adhesive protein in PBS solution with a pH of 7.0 to prepare a 1 wt% mussel adhesive protein solution; Step 2) Dissolve sodium alginate and bone morphogenetic protein-2 (BMP-2) in PBS solution with pH 7.0 to prepare sodium alginate solution and BMP-2 solution, respectively. Step 3) Mix the sodium alginate solution obtained in Step 2) and the mussel adhesive protein solution obtained in Step 1) evenly to obtain the mussel adhesive protein complex. Then add the BMP-2 solution to the mussel adhesive protein complex and stir at 200 rpm for 25 min. After sterilization, obtain component A, in which the mass ratio of Mfp-151 mussel adhesive protein to sodium alginate and BMP-2 is 3:12:0.8. Step 4) Dissolve 4-arm-PEG-maleimide in PBS buffer solution with pH 7.0, and adjust the pH of the system to 7.0~7.4 using a pH adjuster. After sterile treatment, obtain the cross-linking promoter solution; wherein, the mass ratio of 4-arm-PEG-maleimide to mussel adhesive protein complex is 1:400. Step 5) After sterilizing the components A and crosslinking accelerator solutions, dispense them into sterile sealed containers under aseptic conditions and store them at 2~10℃ in the dark. In use, component A and the crosslinking accelerator solution are mixed at a volume ratio of 1:1 to obtain the mussel adhesive protein-based medical adhesive of Example 9, and then applied to the target site immediately.

[0049] The mussel adhesive-based medical adhesive of Example 9 is composed of component A and 4-arm-PEG-maleimide; component A includes mussel adhesive protein complex and bone morphogenetic protein-2 (BMP-2), the mussel adhesive protein complex includes Mfp-151 mussel adhesive protein and sodium alginate; the mass ratio of Mfp-151 mussel adhesive protein to sodium alginate and bone morphogenetic protein-2 (BMP-2) in component A is 3:12:0.8; the mass ratio of 4-arm-PEG-maleimide to mussel adhesive protein complex is 1:400.

[0050] Example 10 This invention discloses a mussel adhesive protein-based medical adhesive for repairing cartilage defects. The preparation method of the mussel adhesive protein-based medical adhesive includes the following steps: Step 1) Dissolve Mfp-151 mussel adhesive protein in PBS solution with a pH of 5.5 to prepare a mussel adhesive protein solution with a mass fraction of 0.5 wt%. Step 2) Dissolve sodium hyaluronate and hydroxypropyl methylcellulose in PBS solution with pH 6.0 to prepare sodium hyaluronate solution and hydroxypropyl methylcellulose solution, respectively; Step 3) Mix the sodium hyaluronate solution obtained in Step 2) and the mussel adhesive protein solution obtained in Step 1) evenly to obtain a mussel adhesive protein complex. Then add the hydroxypropyl methylcellulose solution to the mussel adhesive protein complex and stir at 200 rpm for 25 min. After sterilization, obtain component A, in which the mass ratio of Mfp-151 mussel adhesive protein to sodium hyaluronate and hydroxypropyl methylcellulose is 3:15:0.9. Step 4) Dissolve magnesium chloride in PBS buffer solution with pH 7.0, and adjust the pH of the system to 7.0~7.4 using a pH adjuster. After sterile treatment, obtain the cross-linking promoter solution; wherein the mass ratio of magnesium chloride to mussel adhesive protein complex is 1:450. Step 5) After sterilizing the components A and crosslinking accelerator solutions, dispense them into sterile sealed containers under aseptic conditions and store them at 2~10℃ in the dark. In use, component A and the crosslinking accelerator solution are mixed at a volume ratio of 1:1 to obtain the mussel adhesive protein-based medical adhesive of Example 10, and then applied to the target site immediately.

[0051] The mussel adhesive-based medical adhesive of this embodiment 10 is composed of component A and magnesium chloride; component A includes mussel adhesive protein complex and hydroxypropyl methylcellulose, the mussel adhesive protein complex includes Mfp-151 type mussel adhesive protein and sodium hyaluronate; the mass ratio of Mfp-151 type mussel adhesive protein to sodium hyaluronate and hydroxypropyl methylcellulose in component A is 3:15:0.9; the mass ratio of magnesium chloride to mussel adhesive protein complex is 1:450.

[0052] Comparative Example 1 The only difference between Comparative Example 1 and Example 1 is that in step 3), the mass ratio of Mfp-5 mussel adhesive protein to sodium alginate and poly(N-isopropylacrylamide) is 3:1:0.5, resulting in a medical adhesive. All other aspects are the same as in Example 1.

[0053] Comparative Example 2 The only difference between Comparative Example 2 and Example 1 is that in step 3), the mass ratio of Mfp-5 mussel adhesive protein to sodium alginate and poly(N-isopropylacrylamide) is 3:60:0.5, resulting in a medical adhesive. All other aspects are the same as in Example 1.

[0054] Comparative Example 3 The only difference between Comparative Example 3 and Example 1 is that in step 3), the mass ratio of Mfp-5 mussel adhesive protein to sodium alginate and poly(N-isopropylacrylamide) is 3:30:2, resulting in a medical adhesive. All other aspects are the same as in Example 1.

[0055] Comparative Example 4 The only difference between Comparative Example 4 and Example 1 is that no functional component was prepared in Comparative Example 4, and a medical adhesive was finally obtained. All other aspects are the same as in Example 1.

[0056] Comparative Example 5 The only difference between Comparative Example 5 and Example 1 is that no crosslinking accelerator was prepared in Comparative Example 5, and a medical adhesive was finally obtained. All other aspects are the same as in Example 1.

[0057] Comparative Example 6 The only difference between Comparative Example 6 and Example 1 is that in step 4), the mass ratio of ferric chloride hexahydrate to mussel adhesive protein complex is 1:1000, and a medical adhesive is finally obtained. All other aspects are the same as in Example 1.

[0058] Comparative Example 7 The only difference between Comparative Example 7 and Example 1 is that in step 4), the mass ratio of ferric chloride hexahydrate to mussel adhesive protein complex is 1:50, resulting in a medical adhesive. All other aspects are the same as in Example 1.

[0059] Comparative Example 8 In Comparative Example 8, a cross-linking promoter was added before preparing the mussel adhesive protein complex. The specific preparation steps are as follows: Step 1) Dissolve Mfp-5 type mussel adhesive protein in PBS solution with pH 6 to prepare a 5 wt% mussel adhesive protein solution; Step 2) Dissolve ferric chloride hexahydrate in a Tris-HCl solution with a pH of 7.4, and adjust the pH of the system to 7.0~7.4 using a pH adjuster. After sterile treatment, obtain a cross-linking accelerator solution. Step 3) Mix the ferric chloride hexahydrate solution obtained in Step 2) and the mussel adhesive protein solution obtained in Step 1) at a mass ratio of 11:720 until homogeneous; Step 4) Dissolve sodium alginate and poly(N-isopropylacrylamide) separately in PBS solution with a pH of 6.5 to prepare sodium alginate solution and poly(N-isopropylacrylamide) solution, respectively. Step 5) Mix the sodium alginate solution obtained in Step 4) and the mussel adhesive protein / ferric chloride hexahydrate mixed solution obtained in Step 3) evenly, then add the poly(N-isopropylacrylamide) solution and stir at 200 rpm for 20 min. After sterilization, obtain component A, wherein the mass ratio of Mfp-5 type mussel adhesive protein to sodium alginate and poly(N-isopropylacrylamide) is 3:30:0.5. After sterilization, obtain the medical adhesive of Comparative Example 8, and store it under sterile sealing at low temperature.

[0060] When using, simply apply the above-mentioned mussel adhesive protein-based medical adhesive to the target area.

[0061] The medical adhesives prepared in Examples 1-10 and Comparative Examples 1-8 were subjected to adhesion strength testing, in vitro cytotoxicity testing, functional testing, osteogenic performance evaluation, and chondrogenic performance evaluation; the specific testing procedures are as follows: 1. Adhesion strength test The adhesion properties of different samples were evaluated using the lap shear-tension test, following the ASTM F2256 standard. Different biological tissue substrates were selected for testing based on the application scenarios of the test examples and comparative examples, as detailed below: (1) Skin base: Fresh pig skin was taken, the subcutaneous fat tissue was peeled off, and it was cut into rectangles of 2.5cm×5cm. The dermal layer side was used as the adhesion surface during the test. The test samples were Examples 1-4, Comparative Examples 1-8 and commercial fibrin glue (Hualan Biological Engineering Co., Ltd., trade name: Kangpuxin, specification: 1.0ml / set).

[0062] (2) Mucosal substrate: Fresh porcine gastric mucosa was taken, the muscle layer tissue was removed, and it was cut into rectangles of 2.5cm × 5cm. During the test, the mucosal layer side was used as the adhesion surface. The test samples were from Examples 5 and 6.

[0063] (3) Bone base: Take fresh pig ribs, remove the periosteum, grind them flat, and cut them into 2.5cm×2.5cm blocks; the test samples are from Examples 7 and 8.

[0064] (4) Cartilage base: Take fresh pig knee joint cartilage, remove the subchondral bone tissue, and cut it into 2.5cm×2.5cm blocks; the test samples are from Examples 9~10.

[0065] The specific testing method is as follows: The test adhesive sample was evenly applied to the adhesion area of ​​the substrate, with the application size controlled at 2.5cm × 2.5cm.

[0066] Quickly cover the coating area with another substrate of the same material, ensuring the two substrates overlap parallel to each other and the bonding areas are strictly aligned. Place a weight over the overlap for 30 seconds, then remove the weight and allow the sample to stand at 25°C for 5 minutes to allow the adhesive sample to fully cure.

[0067] (3) The cured test sample is symmetrically clamped in the upper and lower clamps of the universal testing machine, ensuring that the clamps hold the uncoated parts at both ends of the substrate and that the long axis of the sample is parallel to the tensile direction. Set the tensile rate to 5 mm / min and start the test until the bonding interface of the sample is damaged.

[0068] Record the maximum tensile force (in N) during the test. Calculate the lap shear adhesion strength of the sample using the following formula:

[0069] (4) Five parallel samples were prepared for each group of test samples, and the test results were expressed as mean ± standard deviation. See Table 1 for specific results.

[0070] 2. In vitro cytotoxicity test (1) The in vitro cytotoxicity of the samples was detected by the CCK-8 colorimetric method according to ISO 10993-5 standard. Mouse fibroblasts (L929) were used and cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The culture conditions were 37°C, 5% CO2 and saturated humidity in an incubator.

[0071] (2) Under aseptic conditions, the medical adhesives prepared in Examples 1-10 and Comparative Examples 1-8 were immersed in DMEM medium containing 10% fetal bovine serum according to ISO 10993-12 standard, with an extraction ratio of 0.2 g / mL. The extraction container was placed in a constant temperature shaking incubator and incubated at 37°C for 24 hours. After incubation, the supernatant was collected to obtain a 100% concentration extract.

[0072] (3) Take L929 cells in the logarithmic growth phase and use 1×10 4 Cells were seeded at a density of 100 μL per well in 96-well plates and incubated for 24 hours to allow the cells to adhere completely.

[0073] (4) Discard the original culture medium in the wells. Add 100 μL of the 100% sample extract prepared above to each well in the experimental group. The following control group was also established: Negative control group: Add 100 μL of fresh complete culture medium (DMEM containing 10% FBS) to each well.

[0074] Blank control group: wells containing no cells and only 100 μL of complete culture medium were set up.

[0075] Positive control group: Add 100 μL of complete culture medium containing 5% DMSO (or other positive substance recommended by ISO 10993-5) to each well. (5) After culturing the plate for another 24 hours, the supernatant in the wells was aspirated, and 10 μL CCK-8 solution was added to each well. The plate was then incubated in the incubator for another 2 hours. After the incubation was completed, the absorbance (OD) value of each well at a wavelength of 450 nm was measured using an ELISA reader.

[0076]

[0077] Cytotoxicity is classified according to ISO 10993-5:2009 standard: Grade 0 (No cytotoxicity): Cell viability ≥ 90% Grade 1 (mild cytotoxicity): Cell viability is 70-89%. Grade 2 (Moderate cytotoxicity): Cell viability is 50-69%. Grade 3 (Severe cytotoxicity): Cell viability <50% Five replicates were set up for each group, and the experimental results are expressed as mean ± standard deviation (Mean ± SD). Specific test results are shown in Table 1.

[0078] Table 1. Results of adhesion strength and in vitro cytotoxicity tests in Examples 1-10 and Comparative Examples 1-8

[0079] As shown in Table 1, considering the combined results of adhesion strength and cell viability tests, Examples 1-10 all exhibited higher adhesion strength (33.7 kPa) than commercial fibrin glue on different tissue substrates, and the cell viability was ≥90%, meeting the ISO 10993-5 standard requirement for non-cytotoxicity. Comparative Examples 1-8, using Example 1 as a baseline, revealed the influence of each component and process on performance through variable adjustments: Too low anionic functional material (sodium alginate) addition (Comparative Example 1, 57.6 ± 11.8 kPa) or too high an addition (Comparative Example 2, 49.3 ± 13.2 kPa) resulted in a significant decrease in adhesion strength compared to Example 1; Too high an addition of functional regulating polymer (N-isopropylacrylamide) (Comparative Example 3, 69.4 ± 10.9 kPa) or... The absence of functional modifiers (Comparative Example 4, 61.7 ± 11.2 kPa) also reduced adhesive performance; the absence of crosslinking accelerator (ferric chloride hexahydrate) (Comparative Example 5, 18.9 ± 13.7 kPa), the excessively low amount (Comparative Example 6, 40.3 ± 10.5 kPa), the excessively high amount (Comparative Example 7, 45.7 ± 14.1 kPa), and the improper timing of addition (Comparative Example 8, 22.5 ± 12.3 kPa) all resulted in adhesive strength lower than in Example 1. These data indicate that the amount of anionic functional material, functional modifiers, and crosslinking accelerators, as well as the timing of the crosslinking accelerator addition, have a significant impact on the adhesive performance of mussel adhesive protein-based medical adhesives.

[0080] Regarding cell viability, the cell viability rates of Comparative Example 5 (no crosslinking promoter), Comparative Example 7 (excessive crosslinking promoter dosage), and Comparative Example 8 (improper timing of crosslinking promoter addition) were 87.9%, 80.8%, and 88.7%, respectively, all below 90%, while the cell viability rates of Examples 1-10 were all above 90%. This data comparison illustrates that the dosage and timing of crosslinking promoter addition are key factors affecting the biocompatibility of materials. Excessive dosage can lead to residual free metal ions, directly causing cytotoxicity; insufficient dosage (such as in Comparative Example 5) may result in the material failing to form a stable crosslinking network, leading to component leaching and indirectly affecting cell activity; improper timing of addition (such as in Comparative Example 8) may result in uneven crosslinking, localized toxicity or leaching, and a decrease in cell viability.

[0081] Adhesion strength and in vitro cell viability experiments demonstrate that the formulation and preparation process used in the examples achieve a balance between adhesive performance and biosafety. While maintaining excellent biosafety, it also exhibits significantly improved wet tissue adhesion performance, making it suitable for further development and application in the field of medical adhesives.

[0082] 3. Functional testing 3.1 Cell Scratch Assay The effects of Examples 1-2 and Comparative Examples 1-8 on the migration ability of human skin fibroblasts (HSF) were evaluated using a cell scratch assay to verify their promoting effect on the healing of superficial skin wounds. The specific methods are as follows: (1) Human skin fibroblasts (HSF) in the logarithmic growth phase were seeded at a density of 5 × 10⁶ cells per well. 5 One cell was seeded into a 6-well plate, and 2 mL of DMEM medium containing 10% fetal bovine serum was added. The cells were cultured at 37°C in a 5% CO2 incubator until the cell confluence reached more than 90%.

[0083] (2) Using a 200μL sterile pipette tip, make a straight line with uniform width perpendicular to the bottom of the well plate in the center of the cell monolayer in each well. After making the line, discard the culture medium and rinse the well plate three times with sterile PBS buffer to remove floating cells and cell debris.

[0084] (3) Dilute the samples (Examples 1-2 and Comparative Examples 1-8) to 100 μg / mL with sterile PBS, and prepare working solutions using DMEM low-serum medium containing 1% fetal bovine serum. The following groups were set up for the experiment: Experimental group: Add 2 mL of 1% FBS medium containing the sample (100 μg / mL) to each well; Blank control group: Add 2 mL of culture medium containing 1% FBS to each well; Solvent control group: Add 2 mL of 1% FBS medium containing the same volume of PBS as the experimental group to each well; Positive control group: Add 2 mL of culture medium containing 10% FBS to each well.

[0085] Each group has 3 parallel holes.

[0086] (4) Place the culture plate in the incubator and continue to incubate. At 0h, 24h and 48h of scratch, observe and photograph the scratch area under the same field of view using an inverted microscope and record the changes in scratch width.

[0087] (5) Use image analysis software to measure the scratch width at each time point, and calculate the scratch healing rate using the following formula:

[0088] Experimental results are expressed as mean ± standard deviation (Mean ± SD), and the results are shown in Table 2.

[0089] Table 2. Results of cell scratch assays for Examples 1-2 and Comparative Examples 1-8

[0090] The cell scratch assay results (see Table 2) showed that the cell migration rates of Examples 1 and 2 were 58.7% and 62.5% at 24 h, respectively, and reached 89.3% and 92.1% at 48 h, respectively, both significantly higher than those of Comparative Examples 1-8. Examples 1-2 demonstrated a significant migration-promoting advantage as early as 24 h, indicating their ability to rapidly activate fibroblast migration and maintain a high migration rate even after 48 h. This suggests that Examples 1-2 have a significant advantage in promoting fibroblast migration and possess the potential to promote the healing of superficial skin wounds.

[0091] In comparison, the 24-hour migration rates of Comparative Examples 1-8 were 28.9%-42.3%, and the 48-hour migration rates were 60.3%-75.8%, indicating a significant decrease in cell migration-promoting performance compared to Examples 1-2. Specifically, Comparative Example 7 had the lowest 24-hour migration rates (28.9%) and 48-hour migration rates (60.3%), indicating that the residual cytotoxicity caused by excessive crosslinking promoters had the most significant inhibitory effect on migration activity. The 24-hour migration rates of Comparative Examples 5 and 6 were 32.6% and 35.8%, respectively, and the 48-hour migration rates were 65.9% and 68.7%, respectively, indicating that the absence or insufficiency of crosslinking promoters may lead to material structural instability, thereby affecting cell migration. The 24-hour migration rates of Comparative Examples 1-2 and Comparative Examples 3-4 were 38.5%-42.3%, and the 48-hour migration rates were 71.2%-75.8%, indicating that deviations in the dosage of anionic materials and functional regulators may indirectly affect the cell migration environment by disrupting the network structure. The above results indicate that, under the component ratio and preparation process specified in this invention, the adhesive can simultaneously provide an excellent microenvironment for rapid activation (24h) and continuous support (48h) of cell migration, while any deviation from the key parameters will lead to a significant decrease in the migration-promoting performance.

[0092] 3.2 Animal experiments on chronic wound repair Using a rat model of chronic wounds with full-thickness skin defects on the back, the repair capabilities of samples from Examples 3-4 and Comparative Examples 1-8 in a moist environment were evaluated. The specific methods are as follows: (1) SPF-grade SD rats (weighing 250-300g) were selected. After acclimatization for one week, the rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (40mg / kg). The back was prepared, and the hair was removed with 8% sodium sulfide solution. The surgical area was disinfected with povidone-iodine. Using a sterile scalpel and punch, circular full-thickness skin defects with a diameter of 1.5cm and a depth of 0.5cm were prepared on both sides of the spine on the back of the rat. The full-thickness skin and subcutaneous fascia were removed to expose the muscle fascia. For three consecutive days after the operation, dexamethasone sodium phosphate injection (1mg / kg / d) was injected intraperitoneally to inhibit the inflammatory response and granulation tissue growth, simulating the clinical state of chronic and refractory wounds.

[0093] (2) The rats were randomly divided into 12 groups of 6 rats each. The specific grouping and treatment were as follows: Experimental group (Examples 3-4): The adhesives prepared in Examples 3-4 were immediately and evenly applied to the wound surface, with the application thickness controlled at 0.3 mm to ensure complete coverage of the wound surface.

[0094] Comparative groups (Comparative Examples 1-8): The adhesives prepared in Comparative Examples 1-8 were immediately and evenly applied to the wound surface, with the application thickness controlled at 0.3 mm to ensure complete coverage of the wound surface.

[0095] Blank control group: The wound was covered with sterile gauze only, and no sample was applied.

[0096] Positive control group: Commercially available recombinant human epidermal growth factor gel (yeast) (Guilin Huanowei Gene Pharmaceutical Co., Ltd., Yifu, 10g: (50,000 IU (100μg)) was applied to the wound as a reference for promoting healing.

[0097] After applying the sample or control material to all wounds, they were covered with sterile gauze and secured with medical tape. To simulate the moist environment of clinical chronic wounds and avoid mechanical damage to newly formed tissue from frequent changes, the first gauze change was 48 hours post-surgery. Thereafter, the gauze was changed daily and the wound was observed. However, the sample layer (the hardened gel) was only reapplied when it broke or detached, avoiding forced removal.

[0098] (3) On postoperative days 0, 3, 7, and 14, photographs of the wound were taken using a digital camera at a fixed distance and under the same lighting conditions. The wound area was measured using image analysis software. The wound healing rate was calculated using the following formula: .

[0099] All data are expressed as mean ± standard deviation (Mean ± SD). Statistical analysis was performed, with one-way ANOVA used for comparisons among multiple groups and repeated measures ANOVA used for comparisons of different time points within the same group. The results are shown in Table 3.

[0100] Table 3. Results of chronic wound healing rate tests in Examples 3-4 and Comparative Examples 1-8

[0101] As shown in Table 3 and Figure 1The experimental results of chronic wound repair presented show that the healing rates of Examples 3 and 4 at each time point were significantly better than those of the control group and the comparative group, and showed a continuous and efficient growth trend. Three days post-surgery, the healing rates of Examples 3 and 4 were 28.9% and 32.6%, respectively, higher than those of the positive control group (18.5%), the blank control group (8.9%), and the comparative group (10.8%~18.7%), indicating that they could quickly initiate the repair process in the early stage of wound healing. Seven days post-surgery, the healing rates of Examples 3 and 4 reached 65.7% and 70.5%, respectively, while the blank control group was only 32.6% and the comparative group (35.9%~48.9%), showing that they maintained a strong healing-promoting effect during the proliferation phase. Fourteen days post-surgery, the healing rates of Examples 3 and 4 further increased to 91.2% and 94.8%, significantly better than those of the positive control group (84.5%), the blank control group (62.3%), and the comparative group (65.7%~78.5%), further confirming the advantages of Examples 3 and 4 in promoting the healing of chronic wounds. The adhesives prepared in Examples 3-4 can exert a stable healing-promoting effect throughout the inflammatory, proliferative, and remodeling phases, and have long-lasting healing properties, making them suitable for the clinical treatment needs of chronic wounds.

[0102] 3.3 Evaluation of mucosal sealing stability The mucosal sealing stability and sealing effect of Examples 5-6 and Comparative Examples 1-8 under simulated bodily fluid flushing conditions were evaluated through in vitro simulated flushing tests and liquid leakage tests. Specific test methods are as follows: 3.3.1 Erosion Resistance Test (1) Take porcine small intestinal mucosal tissue, peel off the submucosa and muscle layer, cut it into 2cm×2cm pieces, rinse it with physiological saline, spread it flat in a sterile culture dish, and evenly spread the test sample (Examples 5~6 and Comparative Examples 1~8) on the surface of porcine small intestinal mucosal tissue. The spreading area is controlled at 1cm×1cm and the thickness is 0.5mm. Place the spread sample tissue in a 37℃ constant temperature incubator and let it stand for 3min to fully solidify. (2) Fix the solidified mucosal tissue in an in vitro simulated flushing device, adjust the flushing angle to 45°, the flushing distance to 5cm, use 37℃ physiological saline as flushing fluid, adjust the flow rate to 5mL / min, and flush continuously for 60min. Set up 5 parallel samples for each group. (3) During the rinsing process, observe and record any detachment, cracking, or edge lifting of the test sample. After rinsing, remove the mucosal tissue, absorb the surface moisture with filter paper, weigh the remaining test sample, and calculate the erosion resistance rate using the following formula:

[0103] The results are shown in Table 4.

[0104] 3.3.2 Liquid Leakage Rate Test (1) Take double-layered porcine small intestinal mucosa tissue, simulate the clinical mucosal damage state, fix the tissue between the two cavities of the customized leakage test device, and prepare a 1cm×1cm damaged area in the central region; (2) Each test sample (Examples 5-6 and Comparative Examples 1-7) was evenly applied to the damaged area, completely covering the window, with a coating thickness of 0.5 mm. The device was placed at 37°C and left to stand for 3 minutes to fully cure.

[0105] (3) Slowly add 10 mL of physiological saline (37℃) to the upper chamber of the device and record the addition time as T0. Place a pre-weighed sterile centrifuge tube in the lower chamber of the device to collect the leakage liquid. After 60 min, remove the centrifuge tube, weigh the collected leakage liquid, and calculate the liquid leakage rate according to the following formula:

[0106] All data are expressed as mean ± standard deviation (Mean ± SD), with 5 replicates per test group. One-way ANOVA was used for comparisons among multiple groups, and the results are shown in Table 4.

[0107] Table 4. Test results of erosion resistance and liquid leakage rate for Examples 5-6 and Comparative Examples 1-8

[0108] Note: During the rinsing process, it was observed that the surfaces of the samples in Examples 5 and 6 were intact, without any peeling, cracks or edge lifting; Comparative Examples 1 to 8 all showed varying degrees of peeling, cracks or edge lifting, with Comparative Examples 5, 7 and 8 being particularly severe.

[0109] The results of the mucosal sealing stability test (see Table 4) show that the erosion resistance of Examples 5-6 is ≥87%, the liquid leakage rate is ≤2.5%, and no detachment, cracking, or edge lifting is observed during the 60-minute erosion process. In contrast, the erosion resistance of all comparative examples is below 72%, the liquid permeability is above 11%, and all show varying degrees of physical structural damage. This indicates that the adhesive prepared in the embodiments of the present invention has significantly better sealing stability and erosion resistance than the comparative examples under simulated body fluid erosion conditions, and can meet the basic performance requirements as a mucosal sealing material.

[0110] 3.4 Evaluation of osteogenic performance Using alkaline phosphatase (ALP) activity as the evaluation index, the osteogenic properties of the adhesives prepared in Examples 7-8 were verified by comparing them with Comparative Examples 1-8. The specific methods are as follows: (1) Prepare three samples of each adhesive sample prepared in Examples 7-8 and Comparative Examples 1-8. Immerse each sample in α-MEM medium containing 10% fetal bovine serum at a ratio of 0.1 g / mL and extract in a constant temperature shaking incubator at 37℃ for 24 h. After extraction, collect the supernatant and filter it through a 0.22 μm filter membrane to remove bacteria, thus obtaining a 100% concentration extract for later use.

[0111] (2) Take rat osteoblasts in the logarithmic growth phase and adjust the cell density to 1×10⁻⁶. 4 Cells were seeded into 96-well plates, with 100 μL of α-MEM medium containing 10% fetal bovine serum added to each well. The plates were then incubated at 37°C in a 5% CO2 incubator for 24 h until cell attachment was achieved. The following experimental groups were set up, with 5 replicates per group: Experimental group: Discard the original culture medium and add 100 μL of each sample extract (100% concentration) to each well.

[0112] Blank control group: Add 100 μL of fresh α-MEM medium (containing 10% fetal bovine serum) to each well.

[0113] Negative control group: 100 μL of negative control RM-C (Shenzhen Boseng Technology, RM-C, specification 30×100×0.5mm (5 tablets / pack)) extract with no osteogenic induction was added to each well.

[0114] Positive control group: 100 μL of osteogenic induction medium (α-MEM medium containing 10% fetal bovine serum, 10 mM β-glycerophosphate sodium, 50 μg / mL ascorbic acid and 100 nM dexamethasone) was added to each well.

[0115] (3) Place the culture plate in an incubator and continue culturing. Change the corresponding culture medium or extraction solution every 48 hours to keep the culture conditions constant. After culturing for 72 hours, follow the instructions of the ALP detection kit. Take 50 μL of cell lysis supernatant and add it to a new 96-well plate. Add 50 μL of reaction substrate solution (PNPP) and incubate at 37°C in the dark for 15-30 minutes. Add 50 μL of reaction stop solution (3M NaOH) and use a microplate reader to measure the absorbance (OD) value at a wavelength of 405 nm. (4) Simultaneously, 25 μL of cell lysis supernatant was taken and the total protein concentration (OD) of each well was determined using a BCA protein quantification kit. 562 The relative ALP activity is calculated using the following formula:

[0116] Results are expressed as mean ± standard deviation and statistical analysis was performed. One-way ANOVA was used for comparisons among multiple groups, and t-test was used for comparisons between two groups. Normalization was performed with the blank control group as the baseline (set as 100%). The results are shown in Table 5.

[0117] Table 5. Results of alkaline phosphatase (ALP) activity assays in Examples 7-8 and Comparative Examples 1-8

[0118] Table 5 shows the osteogenic performance evaluation results. The relative ALP activities of Examples 7 and 8 (1.86±0.12 and 1.92±0.10, respectively) were significantly higher than those of the blank control group (0.32±0.04) and the negative control group (0.34±0.05), and comparable to those of the positive control group (2.05±0.14), demonstrating their good potential for promoting osteogenic differentiation. The ALP activities of Comparative Examples 1-8 were significantly lower than those of Examples 7-8, with ALP activities of Comparative Examples 5-8 ranging from 0.63 to 0.79, and ALP activities of Comparative Examples 1-4 ranging from 0.87 to 1.02. This indicates that the amounts of crosslinking promoters, anionic functional materials, and functional regulators all have a certain impact on osteogenic differentiation, with crosslinking promoters being more significant. The above data show that, under the component ratios and preparation processes specified in this invention, the adhesive materials prepared in Examples 7-8 can provide a suitable microenvironment for osteoblast differentiation and have a significant promoting effect on osteoblast differentiation.

[0119] 3.5 Evaluation of cartilage performance The chondrogenic properties of the adhesives prepared in Examples 9 and 10 were verified by comparing them with those of Comparative Examples 1-8, using the expression level of type II collagen (COL2A1) as the evaluation index. The specific methods are as follows: (1) The adhesive samples prepared in Examples 9-10 and Comparative Examples 1-8 were divided into groups of 3. Each group was prepared into 3 samples. Each sample was immersed in DMEM / F12 medium containing 10% fetal bovine serum at a ratio of 0.1 g / mL and extracted in a constant temperature shaking incubator at 37°C for 24 h. After extraction, the supernatant was collected and filtered through a 0.22 μm filter membrane to remove bacteria, thus obtaining a 100% concentration extract.

[0120] (2) Take New Zealand rabbit chondrocytes in the logarithmic growth phase and adjust the cell density to 1×10⁻⁶. 4 Cells were seeded in 96-well plates, with 100 μL of DMEM / F12 medium containing 10% fetal bovine serum added to each well. The plates were then incubated at 37°C with 5% CO2 for 24 hours until cell attachment was achieved. The experiment was conducted in the following groups, with 5 replicates per group: Experimental group: Discard the original culture medium and add 100 μL of each sample extract (100% concentration) to each well.

[0121] Blank control group: Add 100 μL of fresh DMEM / F12 medium (containing 10% fetal bovine serum) to each well.

[0122] Negative control group: 100 μL of negative control RM-C (Shenzhen Boseng Technology, RM-C, specification 30×100×0.5mm (5 tablets / pack)) extract with no chondrogenic induction was added to each well.

[0123] Positive control group: 100 μL of chondrogenesis induction medium (DMEM / F12 medium containing 10% fetal bovine serum, 10 ng / mL TGF-β1, 100 nM dexamethasone, 50 μg / mL ascorbic acid and 1% ITS) was added to each well.

[0124] (3) Place the culture plate in an incubator and continue culturing. Replace the corresponding culture medium or extract every 48 hours to keep the culture conditions constant. Perform the test after 72 hours of culture.

[0125] (4) After culture, the FITC fluorescence channel was detected using flow cytometry (excitation wavelength 488 nm, emission wavelength 525 nm). 1 × 10⁻⁶ cells were collected from each tube. 4 We analyzed the percentage of COL2A1-positive cells and the mean fluorescence intensity of individual cells. Results are expressed as the percentage of COL2A1-positive cells (%) and the relative mean fluorescence intensity.

[0126]

[0127] All data are expressed as mean ± standard deviation (Mean ± SD). Statistical analysis was performed, with one-way ANOVA used for comparisons among multiple groups and t-tests used for comparisons between two groups. The results are shown in Table 6.

[0128] Table 6. Percentage (%) of COL2A1 positive cells and relative mean fluorescence intensity of Examples 9-10 and Comparative Examples 1-8

[0129] The chondrogenic performance evaluation results (see Table 6) show that the percentage of COL2A1 positive cells in Examples 9 and 10 (78.3% and 72.5%) and the relative average fluorescence intensity (165.2% and 152.3%) were significantly higher than those in the blank control group (18.6%, 100%) and the negative control group (17.8%, 98.2%), and comparable to those in the positive control group (85.4%, 180.6%), demonstrating that they have good potential to promote chondrocyte differentiation.

[0130] The COL2A1 expression levels in Comparative Examples 1-8 were significantly lower than those in Examples 9-10. Specifically, the percentage of COL2A1-positive cells in Comparative Examples 5-8 was 31.2%-38.7%, with a relative average fluorescence intensity of 80.5%-91.2%, indicating that the absence or improper dosage of the crosslinking promoter had the most significant inhibitory effect on chondrogenic differentiation. The percentage of COL2A1-positive cells in Comparative Examples 1-4 was 42.1%-47.9%, with a relative average fluorescence intensity of 98.5%-108.7%, indicating that deviations in the dosage of anionic functional materials or functional regulators also weaken chondrogenic performance, but the impact is less than that related to the crosslinking promoter. These data demonstrate that, under the component ratios and preparation processes defined in this invention, the adhesive materials prepared in Examples 9-10 can provide a suitable microenvironment for chondrocyte differentiation.

[0131] This invention has been described through the specific embodiments described above. Those skilled in the art should understand that various modifications and equivalent substitutions can be made to this invention without departing from its scope. Parts not described in detail in this specification are well-known to those skilled in the art. Furthermore, various modifications can be made to this invention for specific situations or circumstances without departing from its scope. Therefore, this invention is not limited to the specific embodiments disclosed, but should include all embodiments falling within the scope of the claims.

Claims

1. A mussel adhesive protein-based medical adhesive, characterized in that, The mussel adhesive protein-based medical adhesive is composed of component A and a cross-linking accelerator; Component A includes a mussel adhesive protein complex and a functional regulator, wherein the mussel adhesive protein complex includes mussel adhesive protein and anionic functional materials. The mass ratio of mussel adhesive protein, anionic functional material, and functional regulator in component A is 3:(50~2.5):(0.2~1); The functional regulator is selected from any one or more combinations of temperature-sensitive components, enzyme-sensitive components, bioactive factors, and bioactive inorganic fillers. The mass ratio of the cross-linking promoter to the mussel adhesive protein complex in component A is 1:(200~800).

2. The mussel adhesive protein-based medical adhesive according to claim 1, characterized in that, The crosslinking accelerator is selected from any one of oxidants, polyvalent metal ion compounds, and polyethylene glycol derivatives.

3. The mussel adhesive protein-based medical adhesive according to claim 2, characterized in that, The oxidant is selected from any one of hydrogen peroxide, sodium periodate, and persulfate; The metal ion compound is selected from any one of soluble calcium salts, ferric salts, zinc salts, and magnesium salts; The polyethylene glycol derivative is selected from any one of multi-arm polyethylene glycol-acrylate, multi-arm polyethylene glycol-maleimide, and multi-arm polyethylene glycol-succinimide glutarate.

4. The mussel adhesive protein-based medical adhesive according to claim 1, characterized in that, The temperature-sensitive component is selected from one or more combinations of poly(N-isopropylacrylamide) grafted hyaluronic acid, polyethylene glycol-poly(N-isopropylacrylamide) double-grafted hyaluronic acid, poly(N-isopropylacrylamide) and its copolymers, poloxamer, and hydroxypropyl methylcellulose. The enzyme-sensitive component is selected from one or more combinations of matrix metalloproteinases; The bioactive factor is selected from one or more combinations of vascular endothelial growth factor, bone morphogenetic protein-2, and platelet-derived growth factor. The bioactive inorganic filler is selected from one or more combinations of hydroxyapatite and β-tricalcium phosphate.

5. The mussel adhesive protein-based medical adhesive according to claim 1, characterized in that, The anionic functional material is one or more of the following: polyanionic materials, amphiphilic polyelectrolyte materials, and biomaterials with anionic groups.

6. The mussel adhesive protein-based medical adhesive according to claim 5, characterized in that, The anionic functional material is selected from any one or more combinations of hyaluronic acid, alginate, chondroitin sulfate, and cholesterol-modified hyaluronic acid.

7. The mussel adhesive protein-based medical adhesive according to claim 1, characterized in that, The mussel adhesive protein can be selected from naturally extracted mussel adhesive protein or genetically engineered recombinant mussel adhesive protein.

8. A method for preparing a mussel adhesive protein-based medical adhesive, characterized in that, The mussel adhesive protein-based medical adhesive is the mussel adhesive protein-based medical adhesive according to any one of claims 1 to 7; the preparation method includes the following steps: Step 1): Dissolve mussel adhesive protein in solvent A to obtain a mussel adhesive protein solution with a mass fraction of 0.1~8wt%; the solvent A is a phosphate buffer with a pH of 5.5~7; Step 2): Dissolve the anionic functional material and the functional regulator in solvent A to prepare anionic functional material solution and functional regulator solution, respectively; add the anionic functional material solution to the mussel adhesive protein solution to obtain mussel adhesive protein complex; then add the functional regulator solution to the mussel adhesive protein complex; stir at 150-200 rpm for 20-30 min; and then aseptically process to obtain component A. In component A, the mass ratio of mussel adhesive protein, anionic functional material, and functional regulator is 3:(50~2.5):(0.2~1). Step 3): Dissolve the crosslinking accelerator in solvent B and adjust the pH of the system to 7.0-7.4 to obtain a crosslinking accelerator solution; the solvent B is a phosphate buffer solution with a pH of 7.0-7.4; The mass ratio of the cross-linking promoter to the mussel adhesive protein complex is 1:(200~800); Step 4): After sterilizing the components A and crosslinking accelerator solutions, dispense them into sterile sealed containers under aseptic conditions and store them at low temperature and away from light. The mussel adhesive protein-based medical adhesive according to any one of claims 1 to 7 is obtained by mixing component A and the crosslinking accelerator solution at a volume ratio of 1:1 and applying it immediately to the target site.

9. The use of the mussel adhesive protein-based medical adhesive according to any one of claims 1 to 7 in medical products for closing superficial skin wounds, repairing moist wounds, sealing mucous membranes, and bonding bone / cartilage defects.

10. The use of the mussel adhesive protein-based medical adhesive according to claim 9, characterized in that, The mussel adhesive protein-based medical adhesive is used for bonding bone or cartilage defects; In the mussel adhesive protein-based medical adhesive, the mass ratio of mussel adhesive protein, anionic functional material, and functional regulator in component A is 3:(10~15):(0.8~1); The functional regulator is one or more combinations of bioactive factors and bioactive inorganic fillers; The mass ratio of the cross-linking promoter to the mussel adhesive protein complex in component A is 1:(300~500); The crosslinking accelerator is selected from any one of metal ion compounds and polyethylene glycol derivatives.

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