Adhesion-therapeutic synergistic tissue adhesive and method of making and use thereof

Abstract

The application discloses an adhesion-treatment synergic integrated tissue adhesive and a preparation method and application thereof, and belongs to the technical field of medical adhesives. The adhesion-treatment synergic integrated tissue adhesive comprises: (1) 100 parts by mass of an adhesion precursor, wherein the adhesion precursor is a mixture of a silk fibroin-polyphenol composite hydrogel and urea; and (2) 0.1-100 parts by mass of a medically acceptable metal oxide particle, wherein the metal oxide particle has a hollow mesoporous shell structure and is loaded with a tissue repair promoting component. The adhesion-treatment synergic integrated tissue adhesive not only has excellent adhesion strength, mechanical bearing capacity and a suitable curing window time, can be adapted to the repair of complex tissue damage sites, but also has biological activity and good compatibility for promoting tissue healing, so that the adhesion fixation, the repair and regeneration of the tissue are effectively realized.

Description

Technical Field

[0001] This application belongs to the field of medical adhesive technology, specifically an adhesion-therapeutic integrated tissue adhesive, its preparation method, and its application. Background Technology

[0002] Tissue adhesives are indispensable materials in surgical and wound repair procedures, including tissue closure, defect repair, and implant fixation. Their performance directly affects healing quality and recovery progress. However, current mainstream tissue adhesives generally have significant limitations, failing to meet the clinical requirements for integrated "strong adhesion, biocompatibility, and active repair." For example, polymethyl methacrylate (PMMA) adhesives exhibit significant exothermic curing and residual monomers are cytotoxic; cyanoacrylate (CA) adhesives cure too quickly, have a narrow operating window, and are difficult to adapt to precise application to complex wounds; while inorganic adhesives such as calcium phosphate cement (CPC) have low bonding strength and insufficient mechanical properties, making them prone to failure in dynamic or load-bearing areas. More importantly, these mainstream adhesives primarily function as mechanical fixation, lacking the ability to actively promote tissue regeneration, and their long-term retention may interfere with the healing microenvironment. Therefore, developing adhesive materials that combine strong adhesion, good biocompatibility, and active tissue regeneration promotion has become an urgent technical need in the fields of surgery and regenerative medicine. Summary of the Invention

[0003] This application discloses an adhesion-therapeutic integrated tissue adhesive, its preparation method, and its application, effectively solving the technical problem of poor clinical performance of existing mainstream tissue adhesives.

[0004] To achieve the above objectives, the technical solution provided in this application is as follows:

[0005] A first aspect of this application provides an adhesion-therapeutic synergistic integrated tissue adhesive, the components of which comprise:

[0006] (1) 100 parts by weight of the adhesion precursor, wherein the adhesion precursor is a mixture of silk fibroin-polyphenol composite hydrogel and urea;

[0007] (2) 0.1 to 100 parts by weight of medically acceptable metal oxide particles, wherein the metal oxide particles have a hollow mesoporous shell structure and are loaded with tissue-repairing components.

[0008] According to a preferred disclosure of the first aspect, the metal oxide is selected from zinc oxide, magnesium oxide, copper oxide, cuprous oxide, cobalt oxide, manganese oxide, manganese dioxide, and combinations thereof.

[0009] According to the preferred disclosure of the first aspect, the silk fibroin-polyphenol composite hydrogel comprises silk fibroin-tannic acid composite hydrogel, silk fibroin-dopamine composite hydrogel, silk fibroin-tea polyphenol composite hydrogel, silk fibroin-caffeic acid composite hydrogel and silk fibroin-chlorogenic acid composite hydrogel.

[0010] According to the preferred disclosure of the first aspect, the tissue repair promoting ingredient is selected from 8DSS peptide, isoflavones, collagen peptide, abapapeptide, teriparatide, amorphous calcium phosphate, demineralized bone matrix, hydroxyapatite, bone morphogenetic protein, NGF nerve growth factor, BDNF brain-derived neurotrophic factor, EGF epidermal growth factor, extracellular matrix, and VEGF vascular endothelial growth factor.

[0011] According to the preferred disclosure of the first aspect, the loading amount of the tissue repair promoting component is 0.1% to 15%.

[0012] The second aspect of this application also discloses a method for preparing the adhesion-therapeutic integrated tissue adhesive described in this application, comprising:

[0013] Adhesion precursors and metal oxide particles with hollow mesoporous shell structures were prepared separately.

[0014] After loading the metal oxide particles with tissue-repairing components, the adhesion precursor is mixed with the metal oxide particles loaded with tissue-repairing components to form an integrated adhesion-treatment tissue adhesive.

[0015] According to a preferred disclosure of the second aspect, the method for loading the metal oxide particles with tissue-repairing components comprises:

[0016] The metal oxide particles with hollow mesoporous shell structure are vacuum treated to remove air, then injected into a tissue repair-promoting component solution and ultrasonically vibrated in an ice bath to obtain the final product.

[0017] The third aspect of this application also discloses the application of the adhesion-therapeutic integrated tissue adhesive described in this application, specifically the application of the adhesion-therapeutic integrated tissue adhesive in the preparation of therapeutic drugs or implantable devices for tissue defects, infections, inflammation, etc.

[0018] The fourth aspect of this application also discloses a method for using the adhesion-therapy synergistic integrated tissue adhesive described in this application, comprising:

[0019] After the adhesive-therapeutic synergistic tissue adhesive is applied to the tissue defect site, it is cured by water immersion for at least 24 hours to form a stable adhesive support structure.

[0020] Compared with the prior art, the advantages or beneficial effects of this application include at least:

[0021] This application involves the directional introduction of medically acceptable metal oxide particles with a hollow mesoporous shell structure and loaded with tissue-repair-promoting components into an adhesion precursor composed of silk fibroin-polyphenol composite hydrogel and urea. Firstly, upon contact with an aqueous medium, the metal oxide particles can dissociate at a controllable rate, releasing metal ions that synergistically interact with the adhesion precursor. This not only drives the adhesion precursor to complete dynamic equilibrium and ordered network reconstruction within 24 hours, forming a dense, high-level network solidification structure with enhanced cohesion, thus endowing the solidified structure with excellent adhesion strength and mechanical load-bearing capacity, but also ensures that the dissociation rate is compatible with the solidification process, providing sufficient window time for clinical operation and precisely adapting to the repair needs of complex tissue damage sites. Secondly, the aforementioned medical-grade metal oxide particles with a hollow mesoporous shell structure exhibit mild (low exothermic) exothermic properties during the solidification process. Its properties can avoid damage to surrounding tissues caused by excessively high local temperatures, thereby creating a physiological microenvironment suitable for tissue healing. Furthermore, the particles have excellent biocompatibility and component compatibility, enabling them to form a good fit with adhesion precursors and body tissues, ultimately achieving an integrated synergistic effect of "adhesion fixation and tissue repair," which efficiently accelerates tissue repair and regeneration. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 Transmission electron microscope images of HMn and HMn@8DSS provided for this application;

[0024] Figure 2 Strain scanning rheology diagram of STU-10wt%HMn@8DSS before curing provided for this application;

[0025] Figure 3 Comparison of scanning electron microscope morphology of STU-10wt%HMn@8DSS before and after underwater immersion curing provided for this application;

[0026] Figure 4 A comparison diagram of the protein secondary structure content of STU adhesion precursor and STU-10wt%HMn@8DSS after curing, provided in this application;

[0027] Figure 5Comparison of the overlap shear strength of underwater bonded bone fragments containing different mass concentrations of HMn@8DSS, provided for this application;

[0028] Figure 6 Comparison of the overlap shear strength of the STU adhesion precursor, tissue adhesive with different metal oxide particles, tissue adhesive with different tissue repair-promoting components, and adhesion-treatment synergistic integrated tissue adhesive loaded with different tissue repair-promoting components provided in this application.

[0029] Figure 7 This is a representative plate coating result showing the antibacterial activity of the adhesion-therapeutic synergistic integrated tissue adhesive prepared by combining STU adhesion precursor with different tissue repair-promoting components against Staphylococcus aureus in this application.

[0030] Figure 8 Immunofluorescence staining results of TNF-α, an M1 polarization marker, on the integrated tissue adhesive prepared by combining STU adhesion precursor with different tissue repair-promoting components according to this application;

[0031] Figure 9 Immunofluorescence staining results of IL-10, an M2 polarization marker, on the integrated tissue adhesive prepared by combining STU adhesion precursor with different tissue repair-promoting components according to this application;

[0032] Figure 10 Tube formation assay results of human umbilical vein endothelial cells (HUVECs) after treatment with STU adhesion precursor, STU-10wt%HCu, STU-10wt%VEGF and STU-10wt%HCu@VEGF groups respectively;

[0033] Figure 11 Alizarin Red staining results of MC3T3-E1 cells induced by STU adhesion precursor, STU-10wt%HMn, STU-10wt%8DSS and STU-10wt%HMn@8DSS groups provided in this application;

[0034] Figure 12 Postoperative Micro-CT imaging results for each treatment group provided in this application;

[0035] Figure 13 Postoperative HE-stained histological sections of each treatment group provided in this application;

[0036] Figure 14 Representative photographs of STU adhesion precursor, STU-10wt%HCa, STU-10wt%EGF, and STU-10wt%HCa@EGF used in this application on the postoperative healing of diabetic wounds. Detailed Implementation

[0037] 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 only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments described in this application without creative effort are within the scope of protection of this application.

[0038] In the following description of this application, the term "and / or" is used to describe the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, B existing alone, and A and B existing simultaneously. Here, A and B can be singular or plural; the symbol " / " means "or".

[0039] In the following description of this application, the term "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions mean any combination of such items, including any combination of single or multiple items. For example, "at least one of A, B or C" or "at least one of A, B and C" can mean any one of A, B, and C, or A+B, or A+C, or B+C, or A+B+C, where A, B, and C can be single or multiple.

[0040] In the following description of this application, the sequence of numbers does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be specifically determined by its function and internal logic, and does not constitute any limitation on the execution process of this embodiment.

[0041] In the following description of this application, the numerical range should be understood to also specifically disclose each intermediate value between the upper and lower limits of the range. Any intermediate value within a stated range, as well as any other stated value or each smaller range between intermediate values ​​within a stated range, are also included in this embodiment, and the upper and lower limits of the smaller ranges may be independently included or excluded from the range.

[0042] Unless otherwise stated, the technical / scientific terms used in this application have the meanings commonly understood by one of ordinary skill in the art. While this application describes only preferred materials and methods, similar or equivalent methods and materials may be used in specific embodiments or test cases. All references to this application are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this application shall prevail.

[0043] To address the problem of poor clinical performance of existing tissue adhesives, the first aspect of this application provides an adhesion-therapeutic integrated tissue adhesive, the components of which include: (1) 100 parts by weight of an adhesion precursor, wherein the adhesion precursor is a mixture of silk fibroin-polyphenol composite hydrogel and urea; and (2) 0.1 to 100 parts by weight of medically acceptable metal oxide particles, wherein the metal oxide particles have a hollow mesoporous shell structure and are loaded with tissue repair-promoting components.

[0044] It should be noted that the adhesion precursor in this application embodiment can be water-conductively driven to reconstruct and solidify its network structure. Specifically, when the adhesion-therapeutic integrated tissue adhesive comes into contact with an aqueous medium, the silk fibroin peptide chains in the components undergo directional self-assembly of their β-sheet secondary structures. Through the orderly cross-linking of hydrogen bonds between molecular chains, the adhesion precursor is transformed from a porous, loose network into a denser, higher-order structure, thus solidifying through a physical self-assembly process. The medically acceptable metal oxide particles not only meet the biocompatibility and component compatibility requirements of medical implant materials, but also enable the controlled dissociation of metal ions upon contact with an aqueous medium, thereby driving the adhesion precursor to complete network reconstruction and solidification and exert a synergistic effect on tissue repair.

[0045] This application embodiment introduces medically acceptable metal oxide particles with a hollow mesoporous shell structure into an adhesion precursor composed of silk fibroin-polyphenol composite hydrogel and urea. Firstly, these particles can dissociate into specific metal ions at a controllable rate upon contact with the aqueous medium. These metal ions synergistically interact with the adhesion precursor, driving it to complete a dynamically balanced, ordered network reconstruction within 24 hours, forming a dense, high-level network solidification structure with enhanced cohesion. This imparts excellent adhesion strength and mechanical load-bearing capacity to the solidified structure. Furthermore, the dissociation rate is adapted to the solidification process, providing sufficient window time for clinical operation and precisely addressing the repair needs of complex tissue injuries. Secondly, the aforementioned medical-grade metal oxide particles with a hollow mesoporous shell structure exhibit mild (low exothermic) exothermic properties during the solidification reaction. Its properties can avoid the problem of excessive local temperature causing damage to surrounding tissues, thereby creating a physiological microenvironment suitable for tissue healing; moreover, the particles have excellent biocompatibility and component compatibility, and can form a good fit with adhesion precursors and body tissues, ultimately achieving an integrated synergistic effect of "adhesion fixation and tissue repair", which can efficiently accelerate tissue repair and regeneration.

[0046] In possible examples of disclosure, the metal oxide described in this application is selected from zinc oxide (ZnO), magnesium oxide (MgO), copper oxide (CuO), cuprous oxide (Cu2O), cobalt oxide (Co3O4), manganese oxide (MnO), manganese dioxide (MnO2), and combinations thereof. In this application, MnO2 is selected as a representative example for illustrative purposes. First, the hollow mesoporous shell structure of MnO2 is easily controlled precisely through existing mature preparation processes. Its key structural parameters, such as shell pore size, hollow cavity size, and specific surface area, can be flexibly matched to the curing requirements of the adhesion precursor in this application, facilitating clear verification of the core technical logic of "controllable dissociation of metal ions - network reconstruction curing - synergistic tissue repair" in this application. Second, MnO2 itself has excellent biological activity, which can not only provide a suitable microenvironment for tissue cell proliferation and differentiation, but also has certain anti-inflammatory and antioxidant properties, which can more intuitively reflect the integrated technical advantage of "adhesion fixation and tissue repair" in this application. However, this does not constitute any limitation on the scope of protection of this application. Other metal oxides with the same hollow mesoporous shell structure, meeting medical acceptability and achieving the same technical effect are all within the scope of protection of this application, and will not be listed one by one in this application.

[0047] In possible public examples, the silk fibroin-polyphenol composite hydrogel described in this application is preferably a silk fibroin-tannic acid composite hydrogel, a silk fibroin-dopamine composite hydrogel, a silk fibroin-tea polyphenol composite hydrogel, a silk fibroin-caffeic acid composite hydrogel, and a silk fibroin-chlorogenic acid composite hydrogel. The silk fibroin-tannic acid composite hydrogel is chosen as a representative example in this application because its preparation process is simple and easy to scale up for verification. However, this does not constitute any limitation on the scope of protection of this application. Any other silk fibroin-polyphenol composite hydrogel that achieves the technical effects of this application falls within the scope of protection of this application, and will not be listed individually here.

[0048] In possible public examples, the tissue repair-promoting components described in this application include 8DSS peptides, isoflavones, collagen peptides, abapapeptide, teriparatide, amorphous calcium phosphate, demineralized bone matrix, hydroxyapatite, bone morphogenetic protein, NGF nerve growth factor, BDNF brain-derived neurotrophic factor, EGF epidermal growth factor, extracellular matrix, and VEGF vascular endothelial growth factor. These tissue repair-promoting components, as functional active ingredients, synergistically accelerate tissue repair and regeneration, further enhancing the integrated technical advantages of "adhesion fixation and tissue repair." In this application, the 8DSS peptide (a peptide containing eight repeating aspartic-serine-serine sequences) is selected as a representative example for illustrative description. First, the structure and function of the 8DSS peptide possess the typical characteristics of tissue repair-promoting components, which can fully verify the feasibility of the technical solution of this application. Second, the 8DSS peptide has good compatibility with the metal oxide particles with hollow mesoporous shell structure described in this application, which can clearly verify that this application can synergistically accelerate tissue repair and regeneration through the composite system of "metal oxide particles-tissue repair-promoting components". However, this does not constitute any limitation on the scope of protection of this application. Other tissue repair-promoting components that can achieve the same technical effect are all within the scope of protection of this application, and will not be listed one by one in this application.

[0049] In possible public examples, the loading amount of the tissue repair promoting component described in this application is preferably 0.1% to 15%. The embodiments of this application use loading amounts of 5%, 10%, and 15% for illustrative purposes because these three ratios respectively cover the lower, middle, and upper limits of the aforementioned preferred range, enabling comprehensive and intuitive verification of the technical effects of the system within this range. This facilitates understanding by those skilled in the art and does not constitute a limitation on the scope of protection of this application. Any other ratio falling within the 0.1% to 15% range is within the scope of protection of this application, and will not be listed individually here.

[0050] In possible disclosed examples, the adhesion precursor described in this application is preferably a solution or a powder. Specifically, when the adhesion precursor is in solution form, it can be formed by mixing SF solution and TA solution and stirring at room temperature until an ST composite hydrogel is formed, followed by mixing the ST composite hydrogel with urea powder and stirring at room temperature until a homogeneous viscous liquid of the STU adhesion precursor is formed for direct use. When the adhesion precursor is in powder form, the formed viscous liquid of the STU precursor can be processed into a powder for later use. For example, after freeze-drying the viscous liquid of the STU adhesion precursor, the freeze-dried block is sequentially ground and sieved to obtain a homogeneous STUSTU precursor powder.

[0051] It should be noted that this application does not impose any special limitations on the concentration and mixing ratio of SF solution and TA solution, with the aim of preparing ST composite hydrogel. The example described in this application uses a 10wt% SF solution and 10wt% TA solution mixed at a volume ratio of 1:4 to prepare an ST composite hydrogel, because the materials at this concentration ratio produce an ST composite hydrogel with a uniform network structure, while simultaneously exhibiting excellent adhesion, mechanical support, and biocompatibility, facilitating the verification of the core technical logic of this application. However, this does not constitute a limitation on the concentration and mixing ratio of SF solution and TA solution in this application; other concentrations and mixing ratios that achieve the same or similar technical effects are acceptable, and will not be listed individually in this application.

[0052] It should be noted that this application does not have a specific limitation on the mixing ratio of the ST composite hydrogel and urea powder. The goal is to achieve the regulation of the ST composite hydrogel network structure and the formation of a modified hydrogel with target properties (e.g., adhesion, injectability, biocompatibility) through the introduction of urea. Those skilled in the art can flexibly adjust the ratio according to the performance requirements of actual application scenarios. In this application, the embodiment is described exemplarily with a urea powder to dried silk fibroin protein mass ratio of 2:3 because the hydrogel formed under this ratio has excellent injectability and can solidify in contact with a water-containing environment to form an adhesive structure with good adhesion and support. This is easy for those skilled in the art to understand, but it does not constitute any limitation on the scope of protection of this application. Any other ratio that can achieve the same or similar technical effects falls within the scope of protection of this application, and will not be listed one by one.

[0053] It should be noted that this application does not specifically limit the specific strategies and parameters for processing the viscous liquid STU adhesion precursor into STU adhesion precursor powder, as long as a uniform STU adhesion precursor powder can be prepared. Specifically, when the STU adhesion precursor is in solution form, the liquid method is used, that is, the adhesion precursor solution is directly mixed with metal oxide particles loaded with tissue repair-promoting components; when the adhesion precursor is in powder form, the powder method is used, that is, the adhesion precursor powder is first physically dry-mixed with metal oxide particles loaded with tissue repair-promoting components, and then activated by adding water or an aqueous solution before use.

[0054] In a second aspect, embodiments of this application also provide a method for preparing the aforementioned adhesion-therapeutic integrated tissue adhesive, which includes the following steps:

[0055] Adhesion precursors and metal oxide particles with hollow mesoporous shell structures were prepared separately.

[0056] After loading the metal oxide particles with tissue-repairing components, the adhesion precursor is mixed with the metal oxide particles loaded with tissue-repairing components to form an integrated adhesion-treatment tissue adhesive.

[0057] It should be noted that this application does not particularly limit the specific preparation method of metal oxide particles with hollow mesoporous shell structure, and they can be prepared by mature processes known to those skilled in the art. In the embodiments of this application, manganese dioxide particles with hollow mesoporous shell structure are preferably prepared by the following process, specifically including:

[0058] Ethanol, water, and ammonia were mixed and stirred at 50°C. An ethanol solution of tetraethyl orthosilicate was then added dropwise, and stirring continued at 50°C for 2 hours. The precipitate was then collected by centrifugation and washed with ethanol and water. The product was dispersed in water, potassium permanganate (KMnO4) was added, and the mixture was sonicated for 1 hour and stirred overnight. The precipitate was then centrifuged and washed, dispersed in sodium carbonate (Na2CO3) solution, etched at 60°C for 12 hours, centrifuged, and washed to obtain manganese dioxide particles with a hollow mesoporous shell structure.

[0059] This application embodiment also provides a method for loading tissue repair-promoting components onto the metal oxide particles, preferably comprising:

[0060] After removing air from the metal oxide particles with hollow mesoporous shell structure under vacuum, they are injected into a tissue repair-promoting component solution and ultrasonically vibrated in an ice bath to obtain the final product.

[0061] It should be noted that this application does not have any special limitations on the concentration ratio of the tissue repair promoting component solution. Conventional concentration ratios and solvents can be used. For example, the 250 mg / mL 8DSS solution is selected for illustrative description in this application because this concentration is within the conventional effective concentration range of tissue repair promoting components in tissue repair-related experiments and applications. It has good universality and representativeness and can clearly and stably present the core effect of the technical solution protected by this application. However, this does not constitute any limitation on the scope of protection of this application. Any other ratios are within the scope of protection of this application, and this application will not list them one by one.

[0062] In a third aspect, embodiments of this application also provide typical applications of the adhesion-therapeutic synergistic integrated tissue adhesive described in this application, specifically, using the adhesion-therapeutic synergistic integrated tissue adhesive to prepare therapeutic drugs or implantable devices for tissue defects, infections, and inflammation.

[0063] In a fourth aspect, embodiments of this application also provide a method for using the adhesion-therapy synergistic integrated tissue adhesive described in this application, comprising:

[0064] After the adhesive-therapeutic synergistic tissue adhesive is applied to the tissue defect site, it is cured by water immersion for at least 24 hours to form a stable adhesive support structure.

[0065] It should be noted that this application uses water immersion to cure the material for at least 24 hours, which can achieve curing based on the human physiological environment, thus improving the biosafety and clinical applicability of the material. At the same time, the aqueous environment can promote the slow release of tissue repair-promoting components, ensuring that they continue to play a role in promoting tissue repair during the curing process and subsequent healing stages, thereby achieving the simultaneous implementation of "curing and bonding" and "synergistic treatment".

[0066] The technical solution of this application will be further described below with reference to specific embodiments.

[0067] Example 1

[0068] This example provides an experimental preparation of the adhesion-therapeutic integrated tissue adhesive STU-HMn@8DSS, specifically prepared through the following steps:

[0069] Preparation of S1-Adhesion Precursor

[0070] S101: Add 10g of tannic acid (TA) powder to 90g of deionized water and stir to dissolve for 1 hour to form a 10wt% TA solution;

[0071] S102: 120g of natural silkworm cocoons were soaked in 10L of 0.02M Na2CO3 solution and boiled for 1 hour. The cocoons were removed, the liquid was discarded, and the soaking and boiling process was repeated three times to obtain degummed silk fibroin fibers. The silk fibroin fibers were washed five times with deionized water and then dried in an oven at 45℃ for 48 hours. The dried silk fibroin fibers were then dissolved in 100mL of 9.3M LiBr solution at 60℃ to form a silk fibroin (SF) solution. The silk fibroin (SF) solution was dialyzed with deionized water for three days (dialysis bag molecular weight 1.4×10⁻⁶). 4 Da), and 15wt% PEG solution were reverse dialyzed for 24h to obtain 10wt% SF solution, wherein the PEG solution was prepared by 300g of 2×10⁻⁶ ppm PEG solution. 4 Da's PEG powder was added to 1700g of deionized water and stirred for 2 hours to form a solution.

[0072] S103: The SF solution and the TA solution are mixed and stirred at a volume ratio of 1:4 to form an ST adhesion hydrogel, and the ST adhesion hydrogel is mixed and stirred with urea powder to form a uniform STU precursor viscous liquid, wherein the mass ratio of urea powder to dried silk protein is 2:3.

[0073] Preparation of S2-HMn manganese dioxide particles with hollow mesoporous shell structure

[0074] 60 mL of ethanol, 20 mL of water, and 3 mL of ammonia were stirred and mixed at 50 °C for 5 minutes. Then, 5 mL of tetraethyl orthosilicate and 20 mL of ethanol mixture were added dropwise, and stirring was continued at 50 °C for 2 hours. The precipitate was collected by centrifugation and washed with ethanol and water. The product was then dispersed in 100 mL of water, and 2 g of potassium permanganate (KMnO4) was added. The mixture was sonicated for 1 hour and stirred overnight. The precipitate was then centrifuged and washed. The precipitate was then dispersed in 0.2 M sodium carbonate (Na2CO3) solution and etched at 60 °C for 12 hours. After centrifugation and washing, manganese dioxide particles (HMn) with a hollow mesoporous shell structure and a diameter of approximately 150 nm were finally obtained.

[0075] Preparation of S3-loaded 8DSS peptide-containing manganese dioxide particles HMn@8DSS with hollow mesoporous shell structure

[0076] 1000 mg of HMn was placed in a vacuum for 10 min to remove air completely, then 1 mL of 250 mg / mL 8DSS solution was injected, and the solution was sonicated in an ice bath for 30 min. After centrifugation, HMn@8DSS was obtained.

[0077] Preparation of S4-Adhesion-Therapy Synergistic Integrated Tissue Adhesive STU-10wt%HMn@8DSS

[0078] The viscous liquid of the STU adhesion precursor prepared above is thoroughly mixed with HMn@8DSS at a mass ratio of 100:10 to prepare the fluid dynamic adhesion-therapeutic synergistic integrated tissue adhesive STU-10wt%HMn@8DSS.

[0079] To illustrate the performance of the adhesion-therapeutic integrated tissue adhesive prepared in Example 1 above, this application also provides Comparative Examples 1-2.

[0080] Comparative Example 1

[0081] This comparative example provides a viscous liquid of the STU adhesion precursor without the therapeutic component, specifically derived from Example 1.

[0082] Comparative Example 2

[0083] This comparative example provides an STU-10wt% 8DSS adhesion-therapeutic synergistic integrated tissue adhesive, which differs from Example 1 only in that:

[0084] The viscous liquid of the STU adhesion precursor prepared above was thoroughly mixed with 8DSS peptide at a mass ratio of 100:10 to prepare STU-10wt%8DSS, an integrated tissue adhesive for adhesion-treatment synergy.

[0085] Test Example 1

[0086] 1.1 Morphological characterization of HMn and HMn@8DSS

[0087] The morphology of the prepared HMn and HMn@8DSS was characterized by transmission electron microscopy, and the results were as follows: Figure 1 As shown.

[0088] according to Figure 1 As can be seen, both HMn and HMn@8DSS prepared in this application have uniform spherical structures, with a lighter color in the central region and a darker color at the edges, indicating that both HMn and HMn@8DSS have hollow shell structures; at the same time, the BET results show the presence of obvious hysteresis loops, proving that the prepared HMn has a mesoporous structure.

[0089] 1.2 Characterization of mechanical properties before curing

[0090] The strain scanning test of the prepared STU-10wt%HMn@8DSS was performed using a rheometer to monitor the changes in its storage modulus (G') and loss modulus (G'') with strain. The results are as follows: Figure 2 As shown.

[0091] according to Figure 2 It can be seen that the STU-10wt%HMn@8DSS before curing exhibits a loss modulus (G'') greater than the storage modulus (G'), indicating that the STU-10wt%HMn@8DSS before curing is a viscous fluid with good flowability and injectability.

[0092] 1.3 Morphological characterization after curing

[0093] STU-10wt%HMn@8DSS was immersed in an underwater environment for 24 hours for curing, followed by freeze-drying and gold sputtering. Its microstructure was then observed using a scanning electron microscope. The results were as follows: Figure 3 As shown.

[0094] according to Figure 3 It can be seen that the structure of STU-10wt%HMn@8DSS before curing is relatively loose with large pores; after curing for 24 hours, the structure becomes dense and the pore size also decreases, indicating that the network structure of STU-10wt%HMn@8DSS has undergone reorganization after being soaked in water.

[0095] 1.4 Secondary Structure Analysis

[0096] The STU adhesion precursor and STU-10wt%HMn@8DSS prepared above were immersed in an underwater environment for 24 hours for curing. The protein secondary structure content was then analyzed by circular dichroism (CD) chromatography. The results were as follows: Figure 4 As shown.

[0097] according to Figure 4 It can be seen that, compared with STU adhesive, STU-10wt%HMn@8DSS has a significantly increased β-sheet content, confirming that HMn@8DSS effectively drives the transformation of protein secondary structure.

[0098] Test Example 2

[0099] 2.1 Overlap Shear Strength Test

[0100] This test case validates the addition amount of HMn@8DSS. Specifically, HMn@8DSS equivalent to 0 wt%, 0.1 wt%, 5 wt%, 10 wt%, 20 wt%, 40 wt%, 80 wt%, and 100 wt% of its mass was added to the viscous liquid of the STU adhesion precursor to prepare an adhesion-therapeutic synergistic tissue adhesive STU-x·wt%HMn@8DSS. Following the ASTM F2255 standard lap shear strength test method, tissue adhesives with different HMn@8DSS concentrations (x·wt%HMn@8DSS / STU, x=0, 0.1, 1, 5, 10, 20, 40, 60, 80, and 100) were coated between two stainless steel sheets (adhesion area 2.5 cm²). After curing underwater for 24 hours, the lap shear strength was tested using a universal testing machine. The results were... Figure 5 As shown.

[0101] according to Figure 5 It can be seen that as the concentration of HMn@8DSS increases, the adhesion strength of the tissue adhesive increases significantly, reaching the maximum value at a concentration of 80wt%, and the adhesion strength decreases slightly at a concentration of 100wt%.

[0102] Test Example 3

[0103] To verify the universality of the adhesion-therapeutic integrated tissue adhesive of this application, Examples 2-3 and Comparative Examples 3-6 are also provided.

[0104] Example 2

[0105] A viscous liquid containing HCu@VEGF, a copper oxide-loaded vascular endothelial growth factor (VEGF), and STU adhesion precursor was prepared according to the method in Example 1. The two were thoroughly mixed at a mass ratio of 100:10 to prepare a fluid dynamic adhesion-therapeutic synergistic integrated tissue adhesive STU-10wt%HCu@VEGF.

[0106] Example 3

[0107] A viscous liquid containing HCa@EGF and STU adhesion precursor loaded with calcium oxide was prepared according to the method in Example 1. The two were thoroughly mixed at a mass ratio of 100:10 to prepare a fluid dynamic adhesion-therapeutic synergistic tissue adhesive STU-10wt%HCa@EGF.

[0108] Comparative Example 3

[0109] This comparative example provides STU-HCu, specifically derived from Example 2.

[0110] Comparative Example 4

[0111] This comparative example provides STU-HCa, specifically derived from Example 3.

[0112] Comparative Example 5

[0113] This comparative example provides an STU-10wt%VEGF adhesion-therapeutic synergistic integrated tissue adhesive, which differs from Example 1 only in that:

[0114] The viscous liquid of the STU adhesion precursor prepared above was thoroughly mixed with VEGF at a mass ratio of 100:10 to prepare the adhesion-treatment synergistic integrated tissue adhesive STU-10wt%VEGF.

[0115] Comparative Example 6

[0116] This comparative example provides an STU-10wt%EGF adhesion-therapeutic synergistic integrated tissue adhesive, which differs from Example 1 only in that:

[0117] The viscous liquid of the STU adhesion precursor prepared above was thoroughly mixed with EGF at a mass ratio of 100:10 to prepare the adhesion-treatment synergistic integrated tissue adhesive STU-10wt%EGF.

[0118] According to the ASTM F2255-24 standard method for testing lap shear strength, the tissue adhesives prepared in Examples 1-3 and the products prepared in Comparative Examples 1-6 were respectively coated between two stainless steel sheets (bonding area 2.5 cm²). After curing in an underwater environment for 24 hours, the lap shear strength was tested using a universal testing machine. The results were as follows: Figure 6 As shown.

[0119] according to Figure 6 It can be seen that the tissue adhesive with only added tissue repair components does not show a significant increase in adhesion performance compared to the STU adhesion precursor, while the addition of metal oxide particles carrying tissue repair components shows a significant improvement compared to metal oxide particles alone. This indicates that there is a new synergistic effect between metal oxide particles and tissue repair components in enhancing adhesion strength.

[0120] Test Example 4

[0121] 4.1 In vitro antibacterial performance evaluation

[0122] The STU-10wt%HMn@8DSS, STU-10wt%HCu@VEGF, and STU-10wt%HCa@EGF prepared in Examples 1-3 above were used to co-culture Staphylococcus aureus, specifically as follows:

[0123] Add 200 μL of bacterial suspension (approximately 1 × 10⁻⁶) to each well of a sterile 48-well plate. 8 Add sterilized adhesion complex sample (CFU) to each well, ensuring a concentration of 0.5 mg / mL and complete immersion of the sample in the bacterial solution. Set up a blank control group (bacterial solution). Place the culture plate in a 37°C incubator and co-culture for 4 or 6 hours. Each group has 3 replicates, and the experiment is repeated 3 times as independent biological replicates. After co-culture, thoroughly mix the bacterial solution in each well by pipetting, and transfer 100 μL of the bacterial solution to an EP tube containing 900 μL of sterile PBS, vortex to mix, and obtain 10... - ¹Dilution. After repeating the process three times, take 50 μL of each diluted solution and spread it evenly on a solid culture medium plate. Incubate the plates upright for 20 min to allow the bacterial suspension to absorb the solution, then invert them and incubate at 37°C for 18–24 h. Results are as follows. Figure 7 As shown.

[0124] according to Figure 7 It can be seen that, compared with the blank group, STU-10wt%HMn@8DSS, STU-10wt%HCu@VEGF and STU-10wt%HCa@EGF all have effective bactericidal properties against Staphylococcus aureus.

[0125] 4.2 In vitro anti-inflammatory performance evaluation

[0126] This experiment was divided into a control group, an LPS-induced control group, and STU-10wt%HMn@8DSS, STU-10wt%HCu@VEGF, and STU-10wt%HCa@EGF groups. Specifically, Raw264.7 macrophages were used at a rate of 1x102... 6Raw264.7 macrophages were seeded at a density of cells / mL into 6-well plates. After 24 hours of seeding, except for the control group, each group was treated with 100 ng / mL LPS to induce the polarization of Raw264.7 macrophages into M1 macrophages for 24 hours. Simultaneously, each experimental group was treated with different material extracts. After induction, the culture medium was aspirated, 2 mL of PBS was added to each well, cells were collected, and then centrifuged at 5000 rpm for 5 min. After centrifugation, 10 μL of TNF-α fluorescently labeled antibody was added to label M1 macrophages, and incubated at 4°C for 30 min. After staining, cells were collected by centrifugation, the supernatant was removed, and then 250 μL of PBS and 250 μL of Fixation Buffer were added to resuspend the cells, and incubated at room temperature in the dark for 60 min. After incubation, 1 mL of diluted transmembrane working solution was added, and then centrifuged and the supernatant was removed. After centrifugation, 500 μL of transmembrane working solution and 10 μL of IL-10 fluorescently labeled antibody were added to label M2 macrophages, mixed well, and incubated at room temperature in the dark for 30 min. Then 1 mL of PBS was added, centrifuged, the supernatant was removed, and finally 500 μL of PBS was added to resuspend the cells. After the intervention treatment, immunofluorescence staining analysis of TNF-α and IL-10 was performed using a high-content cell imaging analysis system.

[0127] The protein expression levels of TNF-α, a marker of M1 macrophages, and IL-10, a marker of M2 macrophages, were measured by immunofluorescence staining to assess the effects of STU-10wt%HMn@8DSS, STU-10wt%HCu@VEGF, and STU-10wt%HCa@EGF on Raw264.7 macrophage polarization. The results are shown in Figures 8 to 9. Figure 9 As shown.

[0128] according to Figure 8 It was observed that LPS stimulation of Raw264.7 macrophages significantly enhanced the fluorescence intensity of TNF-α, a marker on the surface of M1 macrophages. After intervention with different extracts, the fluorescence intensity of TNF-α was significantly lower than that of the LPS group, indicating a significant inhibition of M1 macrophage polarization.

[0129] As shown in Figure 9, while LPS induced M1 macrophages, STU-10wt%HMn@8DSS, STU-10wt%HCu@VEGF, and STU-10wt%HCa@EGF all enhanced the fluorescence intensity of the surface marker IL-10 of M2 macrophages to varying degrees, indicating that the combined adhesives STU-10wt%HMn@8DSS, STU-10wt%HCu@VEGF, and STU-10wt%HCa@EGF provided in this application all exhibit anti-inflammatory properties.

[0130] 4.3 In vitro angiogenesis activity assessment

[0131] Human umbilical vein endothelial cells (HUVECs) were treated with the STU adhesion precursor, STU-10wt%HCu@VEGF, STU-HCu, and STU-VEGF prepared above, respectively, as follows:

[0132] First, HUVECs cells were starved in serum-free D-MEM medium for 24 h. Simultaneously, Matrigel was thawed overnight at 4°C. 24-well plates, 200 μL pipette tips, and centrifuge tubes were pre-chilled at -20°C. Then, the plates were plated on ice, with 100 μL of Matrigel added to each well, with 5 replicates per group. The Matrigel-coated plates were incubated at 37°C for 30 min to allow the Matrigel to solidify. After starvation, the HUVECs cells were centrifuged. The control group and each experimental group were resuspended in serum-free D-MEM medium and different serum-free extracts, respectively, and then seeded onto Matrigel, approximately 1 × 10⁶ cells per well. 5 Cells were individually cultured, and the wells were gently shaken to ensure uniform cell distribution. The cells were then incubated in a 5% CO2 incubator at 37°C. After 6 hours of culture, cells were stained with Calcein-AM, and tube formation was observed and images were taken using a fluorescence microscope. Finally, ImageJ software was used to calculate parameters such as total tube length and number of nodes. The results were as follows: Figure 10 As shown.

[0133] according to Figure 10 It can be seen that, compared with the control group, the STU-10wt%HCu@VEGF group formed the most complete tubular structure network, and the number and total length of vascular knots were significantly increased (P<0.05), indicating that it has excellent angiogenesis-promoting ability.

[0134] 4.4 In vitro assessment of osteogenic activity

[0135] The STU-10wt%HMn@8DSS, STU-10wt%HMn, STU-10wt%8DSS and STU adhesion precursor prepared above were used to induce osteogenic culture in MC3T3-E1 cells, respectively.

[0136] MC3T3-E1 cells were seeded in 24-well plates, with 5 replicates per group. When cell confluence reached 70%, the culture medium for each group was replaced with osteogenic induction medium (DMEM medium supplemented with 10% fetal bovine serum, 1% penicillin / streptomycin (double antibiotic), 7.5 mM β-glycerophosphate sodium, 100 μg / mL ascorbic acid, and 0.1 μM dexamethasone). The experimental groups were replaced with an extract of the material and osteogenic induction medium. After 14 days of induction culture, alizarin red staining was performed. The culture medium was aspirated from the wells, and the cells were washed three times with PBS for 1 min each time. The cells were then fixed with 4% paraformaldehyde at room temperature for 10 min, washed three times with PBS, and washed once with distilled water. Subsequently, an appropriate amount of alizarin red staining solution was added, and the cells were incubated at 37°C for 30 min. The cells were then washed thoroughly four times with distilled water for 5 min each time. After washing, gross and microscopic images were observed. The results were as follows: Figure 11 As shown.

[0137] according to Figure 11 It can be seen that STU-10wt%HMn@8DSS has the highest strength (P<0.05), indicating that it has the most significant ability to promote bone differentiation.

[0138] 4.5 Verification of in vivo fracture repair effect

[0139] S401: Establishment and Treatment of Animal Models

[0140] An acute TSAFs model of the rabbit knee joint was established and randomly divided into four groups: (A) absorbable suture fixation group (clinical control); (B) CA adhesive group (commercial material control); (C) STU adhesive group (basic formula control); (D) STU-HMn@8DSS group (the group in this example).

[0141] S402: Imaging Assessment

[0142] Eight weeks post-surgery, fracture healing in each group was assessed using Micro-CT, and the results were as follows: Figure 12 As shown.

[0143] S403: Histological Assessment

[0144] Eight weeks post-surgery, joint samples from each group were stained with hematoxylin and eosin (HE), and the results were as follows: Figure 13 As shown.

[0145] according to Figure 12 It can be seen that at 8 weeks postoperatively, the STU-10wt%HMn@8DSS group showed good recovery of cortical bone continuity, abundant trabeculae, and complete fracture healing, with significantly better healing results than other groups.

[0146] according to Figure 13It can be seen that at 8 weeks postoperatively, the continuity of the bone cortex in the STU-10wt%HMn@8DSS group was completely restored, and the trabeculae in the fracture area were arranged in an orderly manner and had high maturity, indicating that the fracture had been completely healed and its histological repair effect was better than that of other groups.

[0147] In summary, the STU-10wt%HMn@8DSS adhesion-treatment synergistic integrated tissue adhesive provided in this application not only provides effective mechanical fixation, but also actively regulates the injury microenvironment through its multiple bioactivities, thereby significantly accelerating the fracture healing process. Its comprehensive efficacy is superior to existing clinical fixation methods and traditional adhesives.

[0148] 4.6 Verification of in vivo skin repair effect

[0149] S404: Establishment and Treatment of Animal Models

[0150] A diabetic SD rat model of infectious skin defects was established and randomly divided into five groups: (A) blank group; (B) STU group; (C) STU-HCa group; (D) STU-EGF group; (E) STU-10wt%HCa@EGF group. After treatment with different groups, photos were taken at regular intervals to record skin growth. Figure 14 As shown.

[0151] according to Figure 14 The results showed that one week after the operation, the diabetic SD rats in the STU-10wt%HCa@EGF group had the smallest infectious skin defects and showed a trend of imminent healing. This is the advantage of the STU-10wt%HCa@EGF material, which integrates antibacterial, anti-inflammatory and repair-promoting functions.

[0152] The various embodiments in this application are described in a progressive manner. The same or similar parts between the various embodiments can be referred to each other. Each embodiment focuses on describing the differences from other embodiments.

[0153] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of this application.

Claims

1. An adhesive-therapeutic synergistic tissue adhesive, characterized in that, The components include: (1) 100 parts by weight of the adhesion precursor, wherein the adhesion precursor is a mixture of silk fibroin-polyphenol composite hydrogel and urea; (2) 0.1 to 100 parts by weight of medically acceptable metal oxide particles, wherein the metal oxide particles have a hollow mesoporous shell structure and are loaded with tissue-repairing components.

2. The adhesion-therapeutic integrated tissue adhesive according to claim 1, characterized in that, The metal oxide is selected from zinc oxide, magnesium oxide, copper oxide, cuprous oxide, cobalt oxide, manganese oxide, manganese dioxide, and combinations thereof.

3. The adhesion-therapeutic integrated tissue adhesive according to claim 1, characterized in that, The silk fibroin-polyphenol composite hydrogel includes silk fibroin-tannic acid composite hydrogel, silk fibroin-dopamine composite hydrogel, silk fibroin-tea polyphenol composite hydrogel, silk fibroin-caffeic acid composite hydrogel and silk fibroin-chlorogenic acid composite hydrogel.

4. The adhesion-therapeutic integrated tissue adhesive according to claim 1, characterized in that, The tissue repair promoting ingredients are selected from 8DSS peptide, isoflavones, collagen peptide, abapapeptide, teriparatide, amorphous calcium phosphate, demineralized bone matrix, hydroxyapatite, bone morphogenetic protein, NGF nerve growth factor, BDNF brain-derived neurotrophic factor, EGF epidermal growth factor, extracellular matrix, and VEGF vascular endothelial growth factor.

5. The adhesion-therapeutic integrated tissue adhesive according to claim 4, characterized in that, The loading of the tissue repair promoting component is 0.1-15%.

6. A method for preparing an integrated adhesion-therapeutic tissue adhesive according to any one of claims 1 to 5, characterized in that, Include: Adhesion precursors and metal oxide particles with hollow mesoporous shell structures were prepared separately. After loading the metal oxide particles with tissue-repairing components, the adhesion precursor is mixed with the metal oxide particles loaded with tissue-repairing components to form an integrated adhesion-treatment tissue adhesive.

7. The preparation method according to claim 6, characterized in that, The method for loading the metal oxide particles with tissue repair-promoting components includes: The metal oxide particles with hollow mesoporous shell structure are vacuum treated to remove air, then injected into a tissue repair-promoting component solution and ultrasonically vibrated in an ice bath to obtain the final product.

8. The use of the adhesive-therapeutic synergistic integrated tissue adhesive according to any one of claims 1 to 6 in the preparation of therapeutic drugs or implantable devices for tissue defects, infections, and inflammation.

9. A method of using an adhesive-therapeutic synergistic tissue adhesive according to any one of claims 1 to 6, characterized in that it comprises: After the adhesive-therapeutic synergistic tissue adhesive is applied to the tissue defect site, it is cured by water immersion for at least 24 hours to form a stable adhesive support structure.

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