Intelligent response type ursodesoxycholic acid powder-based bioactive ceramic hydrogel wound repair system

Abstract

The application discloses an intelligent response type bear gall powder-based bioactive ceramic hydrogel wound repair system and belongs to the technical field of biomedical materials, and solves the problems of split functions, uncontrollable release and unreasonable interface combination of existing wound dressings. The system innovatively combines intelligent response type hydrogel, bioactive ceramic and active components of bear gall powder through dynamic coordination bonds. The coordination bond formed by the active groups of bear gall powder and metal ions released by the ceramic serves as an intelligent switch, can respond to changes in wound pH / ROS, and accurately controls the release of antibacterial ions and anti-inflammatory components. Meanwhile, the bond realizes the stable and dynamic combination of the ceramic and the gel. The application realizes the organic unity of intelligent response, multiple bioactivity and stable structure, can synergistically resist bacteria, inflammation and promote repair, and improves the quality of wound healing.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a smart responsive bear bile powder-based bioactive ceramic hydrogel wound repair system. Background Technology

[0002] In the field of wound dressings, combining environmental responsiveness, long-lasting bioactivity, and stable material structures is key to improving repair efficacy and also a prominent challenge in current material design. Existing technologies typically combine intelligent responsive carriers, bioactive components, and mechanical support matrices as independent modules, resulting in significant shortcomings in functional synergy and structural integration within the material system. These shortcomings are specifically manifested in the following aspects:

[0003] Smart responsive hydrogels, such as those with pH or ROS responses, can sense changes in the wound microenvironment and respond physically or chemically, making it possible to achieve controlled drug release. However, the innovation of these materials often focuses on the response mechanism itself. Their gel networks are usually composed of synthetic or natural polymers, and their inherent biological activity is limited. The gel network usually acts as an inert drug carrier, and its repair effect is highly dependent on the external drug it carries. When multiple repair functions such as antibacterial, anti-inflammatory, and cell migration promotion are required to be achieved simultaneously, multiple drugs need to be carried, which increases the complexity and uncontrollability of the formulation and makes it difficult to achieve synergistic and long-lasting effects of functional components.

[0004] Components with clearly defined biological activities, such as bioactive ceramics that release antibacterial / osteoproliferative ions, and active ingredients of traditional Chinese medicine with anti-inflammatory and antioxidant pharmacological effects, such as bear bile powder, have been proven to be beneficial for wound repair. However, existing technologies mostly use them as simple functional fillers or additives, introducing them into the dressing matrix through physical blending or adsorption. This approach has significant drawbacks. The active components and the substrate are mainly bound by weak physical interactions, which can be easily lost quickly under the flushing of body fluids. The release behavior is passive and uncontrollable, and cannot match the dynamic process of wound healing. At the same time, this simple mixing makes it difficult to achieve synergy in the release kinetics of different active components.

[0005] Composite materials have been extensively studied in order to combine the advantages of different materials. However, traditional methods, such as using chemical coupling agents to establish strong static covalent bonds between components, while enhancing mechanical stability, lead to interfacial rigidity and irreversibility. In the dynamically changing wound microenvironment, this static bond cannot respond to environmental signals such as pH and ROS and undergo reversible adjustment or reconstruction, limiting the material's self-adaptive ability and the potential for intelligent release of active ingredients.

[0006] It is particularly noteworthy that for active substances such as bear bile powder, traditional Chinese medicine, existing technologies almost entirely treat them as drug loading objects, with their value lying solely in their pharmacological activity. However, the value of active groups contained in their molecular structure that may participate in the construction of material networks, such as hydroxyl and sulfonic acid groups, is overlooked. This results in their combination with other material components, such as inorganic ceramics and polymer chains, remaining at the physical level and failing to form a stable and functionally integrated structure at the molecular scale.

[0007] In summary, the core flaws of existing technologies lie in the fragmentation and mechanical superposition of functional modules. The intelligent responsive carrier, bioactive components, and stable composite interface have not been organically unified through a single design concept. As a result, the materials are responsive but lack sufficient activity, or the materials are rich in activity but release it in a coarse manner and cannot be intelligently regulated. Some materials sacrifice the dynamic responsiveness of the environment in order to achieve stable composite. Therefore, there is an urgent need in this field for a new material construction strategy that can integrate intelligent responsiveness, diverse bioactivity, and a dynamically stable interface into a synergistic system, thereby achieving a more precise, efficient, and comprehensive promotion of the wound healing process. Summary of the Invention

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a smart responsive bear bile powder-based bioactive ceramic hydrogel wound repair system, wherein the bear bile powder is in vitro cultured bear bile powder or artificially synthesized bile acids, and does not involve natural bear bile, comprising:

[0010] pH-responsive and / or reactive oxygen species-responsive hydrogel matrices;

[0011] Bioactive ceramic particles dispersed in the hydrogel matrix;

[0012] Bear bile powder or its active extract loaded onto the bioactive ceramic particles;

[0013] The bioactive ceramic particles and the hydrogel matrix are connected by dynamic coordination bonds formed between the active groups in the bear bile powder or its active extract and the metal ions from the bioactive ceramic particles, such as... Figure 1 As shown.

[0014] Preferably, the smart responsive hydrogel matrix is ​​a dual-network hydrogel, which includes a first network formed by chemical crosslinking and a second network formed by physical crosslinking or photocrosslinking.

[0015] Preferably, the bioactive ceramic particles are mesoporous bioactive glass or bioactive ceramic particles, and the composition of the bioactive glass or bioactive ceramic particles is a system of SiO2, CaO, and CuO or ZnO.

[0016] Preferably, the surface of the bioactive ceramic particles is modified with a metal-organic framework material, and the metal nodes of the metal-organic framework material contain Cu. 2+ or Zn 2+ .

[0017] Preferably, the dynamic coordination bonds are reversible in the wound microenvironment with a pH of 5.5 to 7.4 and a reactive oxygen species concentration of 100 μM.

[0018] Preferably, the active groups in bear bile powder or its active extract include hydroxyl and sulfonic acid groups.

[0019] In a second aspect, the present invention provides a method for preparing the above-mentioned intelligent responsive bear bile powder-based bioactive ceramic hydrogel wound repair system, comprising the following steps:

[0020] S1 provides bioactive ceramic particles loaded with bear bile powder or its active extract;

[0021] S2, mix the bioactive ceramic particles obtained in step S1 with the precursor solution of the smart responsive hydrogel to form a homogeneous mixed solution;

[0022] S3 causes the hydrogel precursor in the mixed solution to undergo a cross-linking reaction, forming a smart responsive hydrogel matrix that encapsulates bioactive ceramic particles.

[0023] S4, place the hydrogel composite material obtained in step S3 in a container containing Ca 2+ and / or Cu 2+ Incubation in an ion buffer solution allows bioactive ceramic particles to form dynamic coordination bonds with a smart responsive hydrogel matrix, resulting in a wound repair system.

[0024] In a third aspect, the present invention provides the use of the above-described system in the preparation of medical devices or dressings for promoting wound healing, inhibiting wound infection, or reducing scar formation.

[0025] The beneficial effects of this invention are as follows:

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] To achieve precise intelligent response and release, the system uses a dynamic coordination bond composed of active groups from bear bile powder and ceramic metal ions as an intelligent switch. This allows it to specifically respond to characteristic microenvironmental signals during the inflammatory phase of the wound. The response mechanism is as follows: Figure 2As shown, low pH and high reactive oxygen species concentrations can cause the antibacterial metal ion Cu to... 2+ / Zn 2+ The active ingredients TUDCA / UDCA in bear bile powder can be synergistically released at the most needed time and site, avoiding the problems of blind burst release or insufficient release of traditional dressings;

[0028] Leveraging multiple synergistic bioactivities, this invention is not a simple superposition of functions, but rather achieves synergistic functional interaction between inorganic ions and organic components of traditional Chinese medicine in time and space through dynamic release. The intelligently released Cu... 2+ / Zn 2+ Provides immediate antibacterial effects; the simultaneously released bear bile powder exerts anti-inflammatory and antioxidant effects, breaking the infection-inflammation cycle; and the continuously released Ca... 2+ Plasma promotes fibroblast migration and angiogenesis, systematically driving the healing process;

[0029] Constructing a stable and dynamic composite structure, the design of dynamic coordination bonds replaces the traditional physical mixing or static chemical coupling, ensuring a sufficiently strong interfacial bond between bioactive ceramic particles and hydrogel matrix, and endowing the interface with the ability to reversibly reconstruct under specific environments, so that the material has both good structural stability and intelligent environmental response.

[0030] Expanding the application dimensions of active ingredients in traditional Chinese medicine, the active ingredients of bear bile powder are designed from traditional drug loading objects into structural and functional building blocks for constructing intelligent response networks. Their active groups directly participate in the formation of dynamic coordination bonds, enabling the traditional Chinese medicine ingredients to be deeply embedded in the material system, thereby improving utilization efficiency and functional integration. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the structure of a smart responsive bear bile powder-based bioactive ceramic hydrogel wound repair system provided by the present invention;

[0032] Figure 2 This is a schematic diagram illustrating the mechanism by which dynamic coordination bonds respond to changes in the wound microenvironment in this invention.

[0033] Figure 3 A flowchart of a method for a smart responsive bear bile powder-based bioactive ceramic hydrogel wound repair system provided by the present invention;

[0034] Figure 4 This is a comparison graph of the in vitro cumulative release curves of the embodiments and comparative examples of the present invention in different simulated wound microenvironments;

[0035] Figure 5 This diagram serves as an integrated representation of the biological functions of this invention in antibacterial, cell migration-promoting, and anti-inflammatory aspects. Detailed Implementation

[0036] To further illustrate the technical means and effects adopted by the present invention to achieve its intended purpose, exemplary embodiments will be described in detail below, examples of which are illustrated in the accompanying drawings. In the following description relating to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this application. Rather, they are merely examples of methods and systems consistent with some aspects of this application as detailed in the appended claims.

[0037] The terminology used in this application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The singular forms “a” and “the” as used in this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any or all possible combinations of one or more of the associated listed items.

[0038] To better illustrate the purpose, technical solution, and advantages of this application, the following description, in conjunction with specific embodiments and comparative examples, aims to provide a detailed understanding of the content of this application, rather than limiting it. All other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this application. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. In this application, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0039] The following detailed description of the specific implementation methods, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided in detail.

[0040] Example 1

[0041] Smart responsive hydrogel matrix raw materials:

[0042] Sodium alginate oxide, as a precursor of the pH-responsive first network, was purchased from Qingdao Mingyue Algae Group Co., Ltd. The sodium alginate had a viscosity of 200±20 mPa·s. Following the industrial-scale method disclosed in patent CN103554300A, a method for preparing high-viscosity sodium alginate oxide, the sodium alginate oxide was oxidized. This method uses sodium periodate solution for a light-protected, heat-preserved reaction, and removes impurities by solvent washing, thus stably obtaining high-molecular-weight sodium alginate oxide. Finally, 4.0 g of the treated sodium alginate oxide ALG-CHO was weighed for subsequent experiments.

[0043] Adipic acid dihydrazide, as a crosslinking agent for the pH-responsive first network, was purchased from Sigma-Aldrich, and 2.0 g was used in this example;

[0044] Methacrylamide gelatin, used as a raw material for the second photocrosslinking network, was purchased from Xi'an Ruixi Biotechnology Co., Ltd. as GelMA product with a substitution degree of 80%. This product has good photocuring efficiency and biocompatibility. In this example, 10.0 g was used and added in the form of a 10% w / v solution.

[0045] The photoinitiator LAP was purchased from Tianjin Xinsheng Biochemical Technology Co., Ltd., and 0.05 g was used in this example.

[0046] In this embodiment, bioactive ceramic particles are prepared with a mesoporous structure containing SiO2, CaO, and CuO, and the surface is modified with Cu-containing materials. 2+ MOFs of nodes;

[0047] Tetraethyl orthosilicate, calcium nitrate, copper nitrate, and structure-directing agent P123 were all analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. The materials were fed in a SiO2:CaO:CuO ratio of 70:25:5 mol% particles. The final ceramic particles had a measured BET specific surface area of ​​215 m². 2 / g, with an average pore size of 12nm;

[0048] Tristyrene was used to grow Cu-BTC (HKUST-1) MOFs in situ on ceramic surfaces, which are Cu 2+ The nodes are metallic, and the MOF loading, determined by thermogravimetric analysis, is 18 wt% of the ceramic mass.

[0049] The active ingredient in bear bile powder is tauroursodeoxycholic acid (TUDCA), and the standard of tauroursodeoxycholic acid with a purity of ≥98.0% is purchased from Chengdu Mansite Biotechnology Co., Ltd.

[0050] Its active groups, including hydroxyl and sulfonic acid groups, are key to the formation of dynamic coordination bonds. In this embodiment, the loading of TUDCA is 20 mg / g ceramic particles.

[0051] Other reagents containing Ca 2+ and Cu 2+ The ion buffer is used to promote dynamic coordination bonds and is prepared as a Tris-HCl buffer containing 10 mm CaCl2 and 1 mm CuCl2, pH 7.4.

[0052] Please refer to Figure 3 The preparation steps are as follows:

[0053] S1. Prepare bioactive ceramic particles loaded with TUDCA. Mesoporous calcium copper silicate ceramic powder was synthesized by sol-gel method. 1.0 g of ceramic powder was impregnated in 20 mL of ethanol solution containing 20 mg TUDCA, shaken at room temperature for 12 hours, centrifuged, and dried under vacuum at 50 °C to obtain ceramic particles loaded with TUDCA.

[0054] S2, Prepare a mixed solution by dissolving 4.0 g of sodium alginate in 80 mL of deionized water to obtain solution A, dissolving 2.0 g of adipate dihydrazide in 20 mL of deionized water to obtain solution B, and dissolving 10.0 g of GelMA powder and 0.05 g of LAP photoinitiator in 90 mL of PBS at 37 °C to obtain solution C.

[0055] Solutions A, B, C and 3.0 g of ceramic particles loaded with TUDCA (30 wt% of the total mass of the final gel system) were mixed in a beaker and mechanically stirred for 1 hour to form a homogeneous suspension mixture.

[0056] S3 and S4, gradient crosslinking and dynamic coordination bond formation;

[0057] Gradient crosslinking was performed by injecting the mixture into a mold and allowing it to stand at 37°C for 2 hours. A Schiff base reaction occurred between the aldehyde group of sodium alginate and the hydrazide group of adipic acid dihydrazide, forming a pH-responsive first chemical network. Subsequently, a light source with a wavelength of 405 nm and an intensity of 10 mW / cm² was used. 2 Exposure to ultraviolet light for 60 seconds causes GelMA to undergo photocrosslinking, forming a second network;

[0058] Dynamic coordination bond formation was observed, and the initially cross-linked composite hydrogel was removed and immersed in a sufficient amount of water containing 10 mm Ca. 2+ and 1mm Cu 2+ Incubate in Tris-HCl buffer (pH 7.4) at 37°C for 24 hours;

[0059] This process causes the release of Cu from ceramic MOFs. 2+ Ca in the buffer solution 2+ It coordinates with the sulfonic acid groups, hydroxyl groups, and carboxyl groups on the hydrogel network of TUDCA molecules, forming dynamic coordination bonds between ceramic particles and the hydrogel matrix.

[0060] Example 2

[0061] This embodiment provides an active oxygen-responsive hydrogel as a smart responsive hydrogel matrix. The core difference between this embodiment and Embodiment 1 is that the pH / ROS dual-responsive hydrogel matrix is ​​replaced with a single ROS-responsive hydrogel matrix.

[0062] Formula and raw materials: This section only lists the raw materials that are different from those in Example 1, and the same parts are briefly described.

[0063] The intelligent responsive hydrogel matrix material, in this embodiment, adopts a polyvinyl alcohol system containing phenylboronic acid ester bonds that is sensitive to reactive oxygen species (ROS), such as H2O2.

[0064] Polyvinyl alcohol, degree of polymerization 1750±50, degree of alcoholysis 87-89%, purchased from Sinopharm Chemical Reagent Co., Ltd.

[0065] 4-Carboxyphenylboronic acid, used to prepare phenylboronic acid ester bonds, with a purity of ≥98%, was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0066] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, both biochemical reagent grade, were purchased from Sigma-Aldrich and used to catalyze esterification reactions.

[0067] Bioactive ceramic particles, as in Example 1, were made of mesoporous calcium copper silicate SiO2:CaO:CuO=70:25:5mol%, with Cu-BTC(HKUST-1)MOFs grown on the surface and tauroursodeoxycholic acid (TUDCA) loaded. The particles were from Chengdu Mansite Biotechnology Co., Ltd., with a purity ≥98.0% and a loading of 20mg / g ceramic particles.

[0068] To differentiate this example from Example 1, a dynamic coordination bond formation buffer solution was used. This example employed a Tris-HCl buffer solution (pH 7.4) containing only 1 mm CuCl2, without any additional CaCl2 added. The solution primarily relied on the release of Cu from the ceramic itself and the Cu in the MOF nodes. 2+ It forms a coordinate bond with TUDCA;

[0069] The preparation steps are as follows:

[0070] S1, Prepare bioactive ceramic particles loaded with TUDCA. This step is exactly the same as step S1 in Example 1.

[0071] S2, Preparation of ROS-responsive hydrogel precursor solution and mixing. The synthesis of PVA-PBA polymer was carried out using methods known in the art. 10.0 g PVA was dissolved in 90 mL of dimethyl sulfoxide and stirred at 90 °C until completely dissolved. After cooling to 50 °C, 4-carboxyphenylboronic acid was added in a molar ratio of 1:20 to the hydroxyl group of PVA, along with EDC and NHS. The reaction was carried out under nitrogen protection for 24 hours.

[0072] After the reaction was completed, the solution was poured into a large amount of acetone to precipitate, filtered, washed three times with ethanol, and dried under vacuum to obtain phenylboronic acid-modified PVA-PBA. The grafting rate of the phenylboronic acid group was calculated to be approximately 5% by 1H NMR spectroscopy.

[0073] To prepare the precursor solution, the self-made PVA-PBA polymer was dissolved in deionized water at 90°C at a concentration of 8% w / v. The solution was stirred until a uniform and transparent solution was formed, and then cooled to room temperature for later use.

[0074] Mix, weigh 3.0 g of the ceramic particle powder loaded with TUDCA prepared in step S1, which accounts for 30 wt% of the total mass of the final gel system, and add it to 20 mL of the above PVA-PBA solution. Stir mechanically for 1 hour to form a uniform suspension mixture.

[0075] S3 and S4, gelation and dynamic coordination bond formation:

[0076] Physical gelation involves injecting the mixture into a mold and allowing it to stand at 4°C for 12 hours. At this temperature, the PVA chains form a physical cross-linked network through hydrogen bonding, resulting in a primary hydrogel.

[0077] Dynamic coordination bond formation was observed, and the nascent hydrogel was removed and immersed in a sufficient amount of water containing 1 mm Cu. 2+ Incubation was performed at 37°C for 24 hours in Tris-HCl buffer (pH 7.4). During this process, the Cu in the buffer... 2+ And Cu gradually released from ceramics 2+ It diffuses into the hydrogel network and coordinates with the sulfonic acid group, hydroxyl group and the ortho-diol structure that may exist on the PVA-PBA chain of the TUDCA molecule, forming dynamic coordination bonds between the ceramic particles and the hydrogel matrix, giving the gel more stable mechanical properties.

[0078] Example 3

[0079] This embodiment provides the preparation of a zinc-containing bioactive ceramic composite hydrogel wound repair system. By changing the core ceramic component from copper-based CuO in Example 1 to zinc-based ZnO, and accordingly adjusting the metal-organic frameworks (MOFs), the system utilizes zinc ions (ZnO). 2+ Known for its excellent antibacterial properties and good cell compatibility;

[0080] Formula and raw materials: This section only lists the core raw materials that are different from those in Example 1, and the same parts are briefly described.

[0081] Bioactive ceramic particle raw material; in this embodiment, mesoporous bioactive ceramics containing ZnO are prepared.

[0082] The ceramic matrix was synthesized using the sol-gel method for mesoporous calcium zinc silicate ceramics. The raw materials were tetraethyl orthosilicate (TEOS), calcium nitrate tetrahydrate, and zinc nitrate hexahydrate, all of which were analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0083] The ceramic particles were prepared by feeding SiO2:CaO:ZnO with a particle size distribution of 70:25:5 mol%, and the measured BET specific surface area was 198 m². 2 / g, with an average pore size of 11nm;

[0084] Surface MOF modification to introduce Zn onto ceramic surfaces 2+ The nodes were modified using an in-situ growth method for ZIF-8, which is based on Zn. 2+ Typical zinc-based MOFs with a central ion and 2-methylimidazolium as the organic ligand can be gradually degraded and release Zn in aqueous environments. 2+ The ions exert antibacterial and other biological effects; the MOF loading was determined to be 16 wt% of the ceramic mass by thermogravimetric analysis.

[0085] Same as in Example 1, using tauroursodeoxycholic acid (TUDCA) with a purity ≥98.0% (Chengdu Mansite Biotechnology Co., Ltd.), with a loading of 20 mg / g ceramic particles;

[0086] The hydrogel matrix, dynamic coordination bond formation buffer, etc. are exactly the same as in Example 1 to keep the variables singular, only the ceramic composition is changed;

[0087] The preparation steps are as follows:

[0088] S1. Prepare zinc-based bioactive ceramic particles loaded with TUDCA. According to the above molar ratio, use the sol-gel method and combine with the structure directing agent P123 to synthesize mesoporous calcium zinc silicate ceramic powder.

[0089] 1.0 g of the above ceramic powder was immersed in 20 mL of an ethanol solution containing 20 mg TUDCA and shaken at room temperature for 12 hours.

[0090] Centrifugation and vacuum drying at 50°C yielded zinc-based ceramic powder loaded with TUDCA.

[0091] The dried powder was placed in a methanol solution containing 2-methylimidazole and reacted at 65°C for 6 hours to grow ZIF-8 layers in situ on the ceramic surface and in the mesopores. After centrifugation, washing, and drying, the final functional ceramic particles were obtained.

[0092] S2, Prepare the mixed solution. This step is exactly the same as step S2 in Example 1. Weigh 3.0 g, which accounts for 30 wt% of the total mass of the final gel system. The zinc-based ceramic powder loaded with TUDCA prepared in step S1 is dispersed in the dual-network hydrogel precursor mixture with the same formulation as in Example 1.

[0093] Steps S3 and S4 involve gradient crosslinking and dynamic coordination bond formation. These steps are identical to steps S3 and S4 in Example 1, sequentially performing a Schiff base reaction to form the first network, photocrosslinking to form the second network, and finally placing the molded hydrogel in a solution containing 10 mm Ca. 2+ Incubation at 37°C for 24 hours in Tris-HCl buffer (pH 7.4) promotes Zn synthesis. 2+ Metal ions form dynamic coordination bonds with TUDCA and the hydrogel network.

[0094] Example 4

[0095] This embodiment provides the preparation of a bear bile powder crude extract composite hydrogel wound repair system;

[0096] Formula and raw materials: This section only lists the core raw materials that are different from those in Example 1, and the same parts are briefly described.

[0097] The active ingredient of bear bile powder in this embodiment is the crude ethanol-water extract of in vitro cultured bear bile powder. The crude extract is derived from in vitro cultured bear bile powder produced by Jinxiong Pharmaceutical (Zhuhai Hengqin) Co., Ltd. through the conversion of key technologies for mass production of bear bile powder. In order to prepare the crude extract, the bear bile powder raw material produced by the company was taken and extracted with 70% ethanol-water solution according to the HPLC fingerprint chromatographic research method of bear bile powder established in the journal "Chinese Traditional and Herbal Drugs" (2024).

[0098] Characterization of crude extract components: After the above extract was concentrated and dried, the extract was quantitatively analyzed by high performance liquid chromatography (HPLC) using an Agilent 1260 Infinity II.

[0099] The main bile acid components in the extract are ursodeoxycholic acid (40%) and tauroursodeoxycholic acid (25%), and also contain a certain amount of other bile acid components such as tauroursodeoxycholic acid. The crude extract contains various bile acid molecules, such as UDCA and TUDCA, which contain hydroxyl and sulfonic acid groups that can participate in coordination. They are derived from taurine-bound bile acids. In this embodiment, the total loading of the crude extract is 25 mg / g ceramic particles on a solid basis.

[0100] The bioactive ceramic particles, mesoporous calcium copper silicate, surface-grown Cu-BTC MOFs, smart responsive hydrogel matrix materials, oxidized sodium alginate, adipic dihydrazide, GelMA, dynamic coordination bond forming buffer, etc., are all exactly the same as in Example 1.

[0101] The preparation steps are as follows:

[0102] S1, prepare bioactive ceramic particles loaded with crude bear bile powder extract. Take 1.0 g of mesoporous calcium copper silicate ceramic powder, and as in Example 1, dissolve 25 mg of the crude bear bile powder ethanol-water extract prepared above in 20 mL of ethanol-water mixed solvent with a volume ratio of 7:3.

[0103] The ceramic powder was immersed in the solution and shaken at room temperature in the dark for 12 hours to allow various active ingredients in the crude extract to be adsorbed into the interior and surface of the ceramic mesopores. After centrifugation and low-temperature vacuum drying at 40°C, ceramic particles loaded with bear bile powder crude extract were obtained.

[0104] S2, Prepare the mixed solution. This step is exactly the same as step S2 in Example 1. Weigh 3.0 g of ceramic particle powder loaded with bear bile powder crude extract prepared in step S1, which accounts for 30 wt% of the total mass of the final gel system, and disperse it in the double network hydrogel precursor mixture.

[0105] S3 and S4 involve gradient crosslinking and dynamic coordination bond formation, which are identical to steps S3 and S4 in Example 1, sequentially performing chemical crosslinking and photocrosslinking. Finally, the molded hydrogel is placed in a solution containing 10 mm Ca. 2+ and 1mm Cu 2+ Incubate in Tris-HCl buffer (pH 7.4) at 37°C for 24 hours;

[0106] During this process, the metal ions in the buffer solution coordinate with the active groups such as hydroxyl and sulfonic acid groups on various bile acid molecules in the crude extract, thereby forming dynamic coordination bonds between the ceramic particles and the hydrogel matrix.

[0107] Comparative Example 1

[0108] This comparative example provides a control sample that is completely identical in composition to Example 1, but does not form dynamic coordination bonds through an ion buffer incubation step;

[0109] All raw materials used in this comparative example, including their brand, specifications, purity and dosage, are completely consistent with the formulation and raw materials listed in Example 1 to ensure that the variable is singular and only depends on whether dynamic coordination bonds are formed.

[0110] The preparation process of this comparative example is the same as that of Example 1 in the first two steps. The key difference is that the ion buffer incubation step that promotes the formation of dynamic coordination bonds is completely omitted, so that the ceramic particles and the drug are physically dispersed only in the cross-linked and fixed hydrogel network.

[0111] S1, Prepare bioactive ceramic particles loaded with TUDCA. This step is exactly the same as step S1 in Example 1, that is, obtain ceramic particle powder loaded with TUDCA with a loading amount of 20 mg / g.

[0112] S2, prepare the mixed solution and directly crosslink and cure it. The material mixing in this step is exactly the same as step S2 in Example 1. Weigh 3.0 g of ceramic particle powder loaded with TUDCA and disperse it in the double network hydrogel precursor mixture.

[0113] Gradient crosslinking and ion-free incubation were performed. The mixture was injected into a mold and left to stand at 37°C for 2 hours to form the first network through Schiff base reaction. Subsequently, the GelMA was photocrosslinked by irradiation with 405nm ultraviolet light for 60 seconds to form the second network. After crosslinking was completed, the formed hydrogel was directly removed from the mold.

[0114] Comparative Example 2

[0115] This comparative example provides a control sample that is similar in composition to Example 1, but uses traditional static covalent bonds instead of dynamic coordination bonds to connect bioactive ceramic particles to a hydrogel matrix.

[0116] The bioactive ceramic particles, the active ingredient TUDCA from bear bile powder, and the sodium alginate and gelatin raw materials used in this comparative example are all identical to those in Example 1 in terms of brand, specifications, purity, and dosage. The difference lies in the addition of chemical reagents used to construct static covalent bonds:

[0117] The silane coupling agent used is (3-aminopropyl)triethoxysilane, commonly abbreviated as APTES, purchased from Merck Germany. The ethoxy group at one end of its molecule can condense with the hydroxyl group on the ceramic surface, and the amino group at the other end can be used for subsequent reaction with glutaraldehyde.

[0118] The covalent crosslinking agent, glutaraldehyde aqueous solution with a concentration of 25%, was purchased from Sinopharm Chemical Reagent Co., Ltd. Its aldehyde group can react with the amino groups on the surface of APTES-modified ceramics and some hydroxyl groups on the sodium alginate chain to form stable covalent bonds.

[0119] The preparation steps are as follows:

[0120] The core of this comparative example preparation is to first modify the surface of ceramic particles with APTES by amination, and then introduce glutaraldehyde when constructing the hydrogel network, so that it can covalently crosslink with the amino groups and sodium alginate on the ceramic surface, thereby locking the ceramic particles in the hydrogel network.

[0121] S1, preparing bioactive ceramic particles with surface amination and TUDCA loading;

[0122] Amination of ceramic surface: 1.0 g of mesoporous calcium copper silicate ceramic powder from Example 1 was dispersed in 50 mL of anhydrous toluene, 1 mL of APTES was added, and the reaction was carried out under nitrogen protection in an oil bath at 80°C for 6 hours under reflux. After the reaction was completed, the ceramic powder was washed three times by alternating centrifugation with toluene and ethanol, and then cured in a vacuum drying oven at 80°C for 2 hours to obtain a ceramic powder with amino-NH2-rich surface.

[0123] By loading TUDCA, the amination-treated ceramic powder was loaded with 20 mg / g of tauroursodeoxycholic acid (TUDCA) in the same manner as step S1 of Example 1, to obtain TUDCA ceramic powder with amino-NH2-rich surface.

[0124] S2, prepare a mixed solution and perform covalent crosslinking;

[0125] Following the method in step S2 of Example 1, a mixture of dual-network hydrogel precursors containing sodium alginate (ALG-CHO), adipic acid dihydrazide (ADH), GelMA, and LAP photoinitiator was prepared.

[0126] Weigh 3.0 g of TUDCA ceramic powder with an amino-NH2-rich surface prepared in step S1, add it to the above mixture, and mechanically stir for 1 hour to form a uniform suspension;

[0127] The key difference step involves introducing covalent crosslinking of glutaraldehyde. While stirring, 0.5 mL of 25% glutaraldehyde aqueous solution is added dropwise to the above mixture, and stirring is continued for 5 minutes to ensure uniform mixing.

[0128] S3, gradient crosslinking and static covalent network fixation;

[0129] The mixture containing glutaraldehyde is quickly injected into the mold;

[0130] Static covalent crosslinking involves placing the mold in a sealed container and allowing it to react at room temperature (approximately 25°C) for 24 hours. During this period, two main reactions occur:

[0131] First, the aldehyde group of glutaraldehyde forms a Schiff base covalent bond with the amino group on the surface of ceramic particles.

[0132] Secondly, glutaraldehyde may also react with hydroxyl groups and other groups on the sodium alginate chain, and these reactions form strong static covalent bonds between the ceramic particles and the hydrogel matrix.

[0133] After the chemical cross-linking is completed, the mold is irradiated with ultraviolet light with a wavelength of 405nm for 60 seconds to cause the GelMA to undergo photocross-linking and form a second network.

[0134] After crosslinking is complete, the hydrogel is removed from the mold and soaked in deionized water for 24 hours instead of the ion buffer solution in Example 1 to remove unreacted impurities such as glutaraldehyde.

[0135] Comparative Example 3

[0136] This comparative example prepared a control sample that was almost identical in preparation process to that of Example 1, but without the addition of any bear bile powder or its active extract.

[0137] The bioactive ceramic particle matrix used in this comparative example, before loading the active ingredient, all raw materials of the smart responsive hydrogel matrix, and the dynamic coordination bond forming buffer solution are completely identical in brand, specification, purity, and amount to those listed in Example 1. The only and crucial difference is:

[0138] No active ingredients of bear bile powder are added. During the preparation process, no tauroursodeoxycholic acid (TUDCA) or any other form of bear bile powder extract is added. Accordingly, no active ingredients are involved in the subsequent loading steps.

[0139] The preparation steps are as follows:

[0140] The preparation process of this comparative example is parallel to that of Example 1 in terms of chemical operation in the first two steps, but it is fundamentally different because it does not contain active ingredients. Although it does not contain TUDCA, the ion buffer incubation step is still performed to observe the effect of this step in the absence of key organic ligands.

[0141] S1, Prepare bioactive ceramic particles without loading active ingredients. Take 1.0 g of the same mesoporous calcium copper silicate ceramic powder as in Example 1, and grow Cu-BTC MOFs on the surface.

[0142] To maintain a consistent solvent environment, the ceramic powder was immersed in 20 mL of anhydrous ethanol and shaken at room temperature for 12 hours.

[0143] Centrifugation and vacuum drying at 50°C yielded blank ceramic particles without any active ingredients. This step is clearly different from the active ingredient loading process in Example 1.

[0144] S2, Prepare the mixed solution. This step is exactly the same as step S2 in Example 1. Weigh 3.0 g of blank ceramic particle powder prepared in step S1 without any active ingredients, which accounts for 30 wt% of the total mass of the final gel system, and disperse it in the double network hydrogel precursor mixture.

[0145] S3 and S4, gradient crosslinking and experimental ion incubation, are exactly the same as step S3 in Example 1. Schiff base reaction is performed sequentially to form the first network, followed by photocrosslinking to form the second network.

[0146] The key difference is the lack of ligand ion incubation; although it does not contain TUDCA, the formed hydrogel is still placed in the same environment as in Example 1 containing 10 mm Ca. 2+ and 1mm Cu 2+ Incubate in Tris-HCl buffer (pH 7.4) at 37°C for 24 hours;

[0147] Theoretically, the metal ions Cu in the buffer solution 2+ Ca 2+ It can weakly coordinate with a few amino and carboxyl groups carried by the hydrogel network itself. Due to the lack of active ingredients in bear bile powder, such as the efficient and specific coordination sites rich in hydroxyl and sulfonic acid groups provided by TUDCA, it is expected that a sufficient number of stable dynamic coordination bonds cannot be formed between the ceramic particles and the hydrogel matrix. As a result, the interfacial bonding between ceramic and gel mainly depends on physical embedding, which is significantly weakened.

[0148] Comparative Example 4

[0149] This comparative example selects a technologically mature, commercially available, and widely used wound repair product as a control. To ensure the objectivity and representativeness of the comparison, this comparative example uses lipid hydrocolloid silver sulfate dressing as a commercially available reference product, based on the following criteria:

[0150] This product is a mature commodity that has obtained medical device registration and is widely used in clinical practice. It represents a mainstream type of functional dressing containing the inorganic antibacterial component silver in the current market.

[0151] Silver ions have a clear and specific antibacterial effect, which contrasts sharply with the multifunctional integrated design of this invention, making it suitable for analyzing the differences in principle.

[0152] Some of the product's key technical parameters, such as silver content, pH value, and antibacterial rate, are clearly recorded in publicly available information.

[0153] Based on publicly available information, the technical features of this lipid-based hydrocolloid silver sulfate dressing are summarized as follows:

[0154] It is mainly composed of silver-containing polyester mesh, hydrocolloidal particles of sodium carboxymethyl cellulose, and petrolatum, forming a multi-layered physical composite structure. Its key active ingredient is silver ions (Ag). + Silver ions are released upon contact, disrupting bacterial cell membranes and enzyme systems to achieve broad-spectrum antibacterial activity. The product contains approximately 2.98%-4.02% silver and has an antibacterial rate exceeding 99.97% against common pathogenic bacteria such as Staphylococcus aureus.

[0155] The product maintains a neutral pH range of 6.0-7.5, providing a humid environment and having the ability to absorb exudate;

[0156] Table 1 Comparison of commercially available products with Example 1

[0157] Comparison Dimensions Commercially available lipid hydrocolloid silver sulfate dressing Intelligent responsive bear bile powder-based system (Example 1) Design Concept Passive protection and static antibacterial properties rely on the inherent properties of the material to provide a physical barrier and achieve antibacterial effects through the constant release of silver ions. Active response and dynamic synergistic repair: responds to the wound microenvironment through dynamic coordination bonds and intelligently regulates the synergistic release of multiple active ingredients. Intelligent responsiveness No, its silver ion release depends on passive diffusion of concentration gradient and does not have the ability to respond to specific microenvironments of the wound such as pH and ROS. Its function is unrelated to changes in the microenvironment. The system is capable of specifically responding to changes in pH value (5.5-7.4) and reactive oxygen species concentration (10-200 μM), and achieves on-demand release through the reversible dissociation of dynamic coordination bonds. Active ingredients and functions Its composition and function are simple, with silver ions as the only active ingredient. Its core function is antibacterial, but it lacks active ingredients that directly promote cell behavior, such as migration and proliferation, as well as anti-inflammatory, antioxidant, and cell behavior. <![CDATA[The components and functions are synergistic in multiple ways. The active components include: the active substances of bear bile powder, TUDCA / UDCA, which provide pharmacological activities such as anti-inflammatory and antioxidant effects; the ions Ca 2+ , Cu 2+ / Zn 2+ released by bioactive ceramics, which have antibacterial effects of Cu 2+ / Zn 2+ and promote cell proliferation / angiogenesis of Ca 2+ etc.; multiple active groups, which serve as structural parts for forming dynamic bonds.]]> Key structural features Physical composite and static combination: Silver ions exist in the dressing through physical doping or coating, and have a physical mixing or simple adsorption relationship with the substrate. Through chemical coordination and dynamic binding, the hydroxyl and sulfonic acid groups of the active ingredients in bear bile powder are connected to ceramic metal ions through dynamic coordination bonds, making the active ingredients a structural part of the network. Adaptability to complex wounds Limited by its limitations, for chronic, non-healing wounds accompanied by excessive inflammation, oxidative stress, and regenerative disorders, such as diabetic ulcers, antibacterial treatment alone is insufficient to address the root cause of the problem. It is strong, and its multi-component synergistic mechanism can simultaneously address multiple obstacles such as infection, inflammation, oxidative stress, and slow tissue regeneration, and is theoretically more suitable for promoting high-quality healing of complex wounds.

[0158] Experimental Example 1

[0159] Test samples: Example 1, Comparative Example 1, Comparative Example 3;

[0160] The test method involves placing a cylindrical sample with a diameter of 8 mm and a height of 5 mm on the platform of a universal testing machine and performing a compression test at a rate of 1 mm / min. The stress-strain curve is recorded, and the compression modulus, i.e. the slope of the linear elastic zone, is calculated.

[0161] The main instrument is the Instron 5967 benchtop universal testing machine.

[0162] Table 2 Data and Results

[0163] sample Compression modulus (kPa, Mean±SD, n=3) Results Analysis Example 1 15.8±1.5 The high mechanical strength indicates that the strong interfacial bond formed through dynamic coordination bonds can effectively transfer stress. Comparative Example 1 (Physical Mixture) 8.3±1.2 The modulus was lower than that of Example 1, confirming that the lack of chemical bonding led to weak interfacial bonding, which is a significant mechanical weakness. Comparative Example 3 (without bear bile powder) 10.5±1.1 The modulus is between the two but significantly lower than that of Example 1, demonstrating that in the absence of bear bile powder ligands, the interfacial bonding is incomplete and the binding force is weakened.

[0164] Experiment Example 2

[0165] Test sample:

[0166] Example 1: Intelligent responsive TUDCA-calcium copper silicate ceramic composite hydrogel complete system;

[0167] Comparative Example 1: A physically mixed control sample with the same composition as Example 1, but without the ion incubation step;

[0168] Comparative Example 3: Control sample without the active ingredient of bear bile powder;

[0169] After the sample is freeze-dried, a small piece with a flat cross-section is taken, adhered to conductive adhesive, and sent into the sample chamber.

[0170] The test was conducted in an ultra-high vacuum environment using a monochromatic Al Kα X-ray source of 1486.6 eV.

[0171] First, a full-spectrum scan of 0-1350 eV was performed to determine the elemental composition of the sample surface.

[0172] Subsequently, high-resolution narrow-spectrum scanning was performed on key regions: C 1s, O 1s, N 1s and Cu 2p, with a scan step size of 0.05 eV and a pass energy of 20 eV;

[0173] All binding energies were calibrated to the contaminated carbon C 1s peak at 284.8 eV;

[0174] XPSPEAK software was used to perform peak fitting on the high-resolution spectrum to determine the chemical state and precise binding energy of each element.

[0175] The main instrument is the Thermo Fisher Scientific ESCALAB Xi+ X-ray photoelectron spectrometer.

[0176] Analysis of the characteristic peaks of Cu 2p3 / 2 is used to determine the Cu content. 2+ The key to chemical state;

[0177] Table 3 shows the main peak position and full width at half maximum (FWHM) of the fitting results.

[0178] Sample Name Fitting the binding energy of the main peak (eV) Half-width at half maximum (FWHM, eV) The displacement (Δ, eV) relative to Comparative Example 1 Key chemical state analysis Example 1 933.1 2.5 -0.8 <![CDATA[Coordinated Cu 2+ , the binding energy shifts significantly negatively]]> Comparative Example 1 (Physical Mixture) 933.9 2.6 0 <![CDATA[Free / physically adsorbed Cu 2+ , with binding energies in the range of typical uncoordinated or weakly interacting Cu 2+ (such as CuO, Cu(OH)2)]]> Comparative Example 3 (without bear bile powder) 933.8 2.5 -0.1 <![CDATA[Weakly interacting state Cu 2+ , with almost no change in binding energy]]>

[0179] Experimental Example 3

[0180] Test sample:

[0181] Example 1, pH / ROS dual-response TUDCA calcium copper silicate system;

[0182] Example 2, Single ROS-responsive TUDCA calcium copper silicate system;

[0183] Example 3, pH / ROS dual-response TUDCA calcium zinc silicate system;

[0184] Example 4, pH / ROS dual-responsive calcium copper silicate system for crude bear bile powder extract;

[0185] Comparative Example 1: Physically mixed control showed no dynamic bonds;

[0186] Comparative Example 2: Static covalent bond comparison;

[0187] Comparative Example 3, a control group without the active ingredient of bear bile powder;

[0188] For sample preparation, each sample was cut into circular slices with a diameter of about 5 mm and a thickness of about 2 mm, and accurately weighed at 50.0 ± 1.0 mg. Three parallel samples were set up for each condition.

[0189] Medium A (simulating normal physiological environment), phosphate buffered saline solution, PBS, 0.01 mol / L, pH 7.4;

[0190] Medium B (simulating an acidic environment of inflammation), acetate-sodium acetate buffer, 0.1 mol / L, pH 5.5;

[0191] Medium C (simulating oxidative stress environment): PBS containing 100 μM hydrogen peroxide (H2O2), pH 7.4;

[0192] Each sample was placed in 10.0 mL of the corresponding release medium and incubated in a constant temperature shaking incubator at 37.0 ± 0.5°C and 100 rpm.

[0193] All released media were collected at predetermined time points of 1, 3, 6, 12, 24, and 48 hours, and immediately replenished with an equal amount of fresh media at the same temperature to maintain the leakage conditions. The collected released liquid was filtered through a 0.22 μM filter membrane and analyzed in two parts:

[0194] HPLC analysis was performed using an Agilent 1260 Infinity II system with a C18 column and a detection wavelength of 210 nm to quantitatively analyze the concentrations of TUDCA and UDCA.

[0195] ICP-OES analysis was performed using a PerkinElmer Avio 500 spectrometer, and the elemental concentration of Cu or Zn was determined using the standard curve method.

[0196] Data processing: Cumulative release rate (%) = (Cumulative release amount / Total load in sample) × 100%, and the results are expressed as mean ± standard deviation;

[0197] The main instrument is a high-performance liquid chromatograph, an Agilent Technologies 1260 Infinity II system;

[0198] Inductively coupled plasma atomic emission spectrometer, PerkinElmer Avio 500;

[0199] Constant temperature shaking incubator, Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd., model ZWYR-2401;

[0200] The table below shows the cumulative release rate of TUDCA or total bile acids from crude extract after 24 hours of incubation in the three media for each sample. For a comparison of the cumulative release curves, please refer to [link to table]. Figure 4 ;

[0201] Table 4 Key data on 24-hour cumulative release rate

[0202] sample PBS (pH 7.4) Acetate buffer (pH 5.5) <![CDATA[PBS+100μMH2O2]]> Release behavior qualitative analysis Example 1 22.3±2.1% 64.7±3.8% 58.2±3.2% Typical pH / ROS dual response: In acidic and oxidative environments, the release rate is increased to ~3 times that in neutral environments. Example 2 18.1±2.9% 20.5±2.0% 69.5±4.5% <![CDATA[Single ROS response: Dramatic increase in release only in the presence of H2O2, confirming the specific response of phenylborate ester bonds.]]> Example 3 20.8±1.9% 60.1±3.5% 55.3±3.8% The zinc-based system exhibits a dual response: it shows a similar response trend to Example 1, demonstrating the universality of metal ions. Example 4 24.9±2.5% 61.5±4.1% 57.8±3.9% Crude extract system response: The release behavior was not statistically different from that in Example 1, demonstrating the effectiveness of the crude extract. Comparative Example 1 84.6±5.7% 87.2±4.9% 85.8±6.1% Uncontrolled burst release: Due to the lack of dynamic bond binding, most of the drug is rapidly released within 12 hours in any medium, with no responsiveness. Comparative Example 2 7.8±1.0% 8.5±1.2% 8.1±1.5% Release severely blocked: Static covalent bonds completely lock in the active ingredient, resulting in extremely low release and no response. Comparative Example 3 15.2±2.0% 17.8±2.5% 16.9±2.1% Weak and unresponsive release: In the absence of releasable TUDCA, only background-level ion release was detected.

[0203] Experiment Example 4

[0204] Test sample:

[0205] Experimental groups: Example 1 and Example 3;

[0206] Control group: Comparative example 1, comparative example 2, comparative example 3;

[0207] Positive control: A fragment of commercially available lipid hydrocolloid silver sulfate dressing, used as a product reference for Comparative Example 4;

[0208] This experiment was conducted strictly in accordance with the oscillation contact method specified in the People's Republic of China National Standard GB / T 21510-2008 Test Method for Antibacterial Properties of Nano-Inorganic Materials.

[0209] Bacterial strain and culture: The test bacterium was Staphylococcus aureus, standard strain ATCC 6538. The strain was inoculated into nutrient broth medium and cultured in a 37℃ constant temperature shaking incubator until the logarithmic growth phase. The bacterial suspension concentration was adjusted to 1.0 × 10⁻⁶ using PBS buffer (0.01 mol / L, pH 7.4). 5 ~5.0×10 5 CFU / mL;

[0210] All hydrogel samples and commercially available dressing fragments were cut into 10.0±0.5mg sizes, sterilized with ethylene oxide, and used for later use. Four replicates were set up for each group of samples.

[0211] For the shaking contact, the sample was placed in a sterile Erlenmeyer flask, and 20.0 mL of the above bacterial suspension was added. The Erlenmeyer flask was fixed on a constant temperature shaker and shaken for 24 h at 37±1℃ and 200 rpm. At the same time, a bacterial suspension without the sample was set up as a blank control group.

[0212] For viable cell count, immediately after 24 hours of contact, take samples from each Erlenmeyer flask and perform 10 μL of PBS buffer. 1 10 2 10 3 10 4 Perform serial dilutions, select 2-3 suitable dilutions, take 100 μL of each bacterial culture, spread evenly on nutrient agar plates, and incubate upside down at 37℃ for 24-48 h;

[0213] The colony-forming units (CFU) on the counting plate are used to calculate the number of viable bacteria per milliliter of the original bacterial solution based on the dilution factor. The antimicrobial rate is calculated using the following formula:

[0214]

[0215] Main instruments and materials: constant temperature shaking incubator, biosafety cabinet, autoclave, electronic balance with an accuracy of 0.1 mg, nutrient broth, nutrient agar, Staphylococcus aureus ATCC 6538, selected from China Industrial Microbial Culture Collection Center;

[0216] Results of antibacterial rate determination of each sample against Staphylococcus aureus after 24 hours of shaking contact culture;

[0217] Table 5 Results of antibacterial rate determination (data are expressed as mean ± standard deviation, n=4)

[0218] Sample Name Antibacterial rate (%) Statistical significance analysis compared with Example 1 Example 1 99.98±0.02 --(Reference Group) Example 3 99.95±0.03 p>0.05, no significant difference Comparative Example 1 (Physical Mixture) 85.4±3.7 p<0.001, indicating a highly significant decrease Comparative Example 2 (Static Covalent) 15.2±4.1 p<0.001, indicating a highly significant decrease Comparative Example 3 (without bear bile powder) 68.9±4.5 p<0.001, indicating a highly significant decrease Comparative Example 4 (Commercially available silver-containing dressing) 98.5±0.8 p<0.01, significantly reduced Blank control group 0 --

[0219] Experimental Example 5

[0220] Test sample:

[0221] Experimental group: Extract from Example 1;

[0222] Control group: extracts from comparative examples 1, 2, and 3;

[0223] Blank control: DMEM complete medium containing 10% fetal bovine serum (FBS);

[0224] All cell experiments were performed under sterile conditions, in accordance with the international standard for in vitro cytotoxicity testing, ISO 10993-5.

[0225] The extract was prepared according to ISO 10993-12 standard. Each sample was extracted at a ratio of 0.1 g / mL in DMEM medium containing 10% FBS at 37°C and 5% CO2 for 24 ± 2 h. The extract was then filtered through a 0.22 μM filter membrane for sterilization before use.

[0226] Cell culture was performed using mouse fibroblasts L929, which were routinely cultured at 37°C in a 5% CO2 incubator in DMEM medium containing 10% FBS at the China Center for Type Culture Collection.

[0227] Cytotoxicity / proliferation assay using the CCK-8 assay: L929 cells were cultured at 5 × 10⁻⁶ cells / year. 3 Cells were seeded at a density per well in 96-well plates and pre-cultured for 24 hours to allow the cells to adhere.

[0228] Discard the original culture medium and add 100 μL of each sample extract or blank control culture medium to each sample.

[0229] After culturing for 1, 3, and 5 days respectively, 10 μL of CCK-8 solution was added to each well, and incubation was continued for 2 hours.

[0230] The absorbance (OD) value of each well was measured using an ELISA reader at a wavelength of 450 nm.

[0231] The formula for calculating the relative cell proliferation rate (RGR) is:

[0232]

[0233] Cell migration test (scratch assay):

[0234] L929 cells were fed at a rate of 2 × 10⁻⁶ 5 Cells were seeded at a density per well in 24-well plates and cultured until a dense monolayer was formed.

[0235] Using a 200μL sterile pipette tip, make three uniform scratches vertically along a ruler on the cell layer in each well;

[0236] Floating cells were gently washed with PBS to remove them. 1 mL of each sample extract or blank control culture medium was added. All of these were low serum culture media containing 2% FBS to reduce the impact of cell proliferation.

[0237] Take photos of the same location at 0h and 24h under an inverted microscope;

[0238] Use ImageJ software to measure the scratch width and calculate the 24-hour scratch closure rate:

[0239]

[0240] The relative proliferation rate (RGR) of L929 cells after culturing the extracts of each sample for 3 days is shown in Table 6, which was used to evaluate cell compatibility and metabolic activity.

[0241] Table 6. Relative cell proliferation rate (RGR) (Day 3)

[0242] Sample group Relative cell proliferation rate (RGR, %) Mean±SD (n=6) Example 1 118.5±6.2 Comparative Example 1 (Physical Mixture) 102.1±5.5 Comparative Example 2 (Static Covalent) 95.3±4.1 Comparative Example 3 (without bear bile powder) 101.2±5.0 Blank control group 100.0

[0243] Scratch assays directly simulate key processes of cell migration during wound healing;

[0244] Table 7 24-hour scratch closure rate

[0245] Sample group 24-hour scratch closure rate (%) Mean±SD (n=6) Example 1 65.2±4.8 Comparative Example 1 (Physical Mixture) 42.3±3.9 Comparative Example 3 (without bear bile powder) 43.8±4.2 Blank control group 40.1±3.5

[0246] In summary, the system of this invention exhibits synergistic advantages in antibacterial, cell migration-promoting, and anti-inflammatory effects. Specific biofunctional verification results are integrated as follows: Figure 5 As shown, Example 1 is non-cytotoxic and can significantly promote the proliferation and migration of fibroblasts. Cell migration is a core link in granulation tissue formation and wound contraction.

[0247] Comparative Example 2 shows that if the active ingredient cannot be released, the biological activity is lost;

[0248] Comparative Example 3 shows that if the active ingredients of bear bile powder are lacking, the effects of promoting proliferation and migration are significantly weakened.

[0249] Comparative Example 1 shows that even with the same composition, the lack of intelligent release regulation results in a less effective biological effect than that of the present invention.

[0250] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A smart responsive bear bile powder-based bioactive ceramic hydrogel wound repair system, characterized in that, include: ppH-responsive and / or reactive oxygen species-responsive hydrogel matrices; Bioactive ceramic particles dispersed in the hydrogel matrix, wherein the bioactive ceramic particles are mesoporous bioactive glass or bioactive ceramic particles, the composition of the bioactive glass or bioactive ceramic particles is a system of SiO2, CaO, and CuO or ZnO, and the surface of the bioactive ceramic particles is modified with a metal-organic framework material, wherein the metal nodes of the metal-organic framework material contain Cu 2+ or Zn 2+ ; Bear bile powder or its active extract loaded onto the bioactive ceramic particles, wherein the active groups in the bear bile powder or its active extract include hydroxyl and sulfonic acid groups; The bioactive ceramic particles and the hydrogel matrix are connected by the hydroxyl and sulfonic acid groups and Cu from the metal-organic framework material. 2+ or Zn 2+ The dynamic coordination bonds formed between them, wherein the dynamic coordination bonds are formed by placing the composite hydrogel in a Ca-containing environment. 2+ and / or Cu 2+ It is formed by incubation in an ion buffer solution.

2. The wound repair system according to claim 1, characterized in that, The smart responsive hydrogel matrix is ​​a dual-network hydrogel, which includes a first network formed by chemical crosslinking and a second network formed by physical crosslinking or photocrosslinking.

3. The wound repair system according to claim 1, characterized in that, The dynamic coordination bonds are reversible in the wound microenvironment with a pH of 5.5 to 7.4 and a reactive oxygen species concentration of 100 μM.

4. A method for preparing the intelligent responsive bear bile powder-based bioactive ceramic hydrogel wound repair system according to claim 1, characterized in that, Includes the following steps: S1, providing bioactive ceramic particles loaded with the bear bile powder or its active extract; The bioactive ceramic particles are mesoporous bioactive glass or bioactive ceramic particles, the composition of which is a system of SiO2, CaO, and CuO or ZnO, and the surface is modified with a metal-organic framework material, wherein the metal nodes of the metal-organic framework material contain Cu. 2+ or Zn 2+ ; S2, mix the bioactive ceramic particles obtained in step S1 with the precursor solution of the smart responsive hydrogel to form a homogeneous mixed solution; S3, causing the hydrogel precursor in the mixed solution to undergo a cross-linking reaction, forming a smart responsive hydrogel matrix that encapsulates the bioactive ceramic particles; S4, place the hydrogel composite material obtained in step S3 in a container containing Ca 2+ and / or Cu 2+ The system is incubated in an ion buffer solution to allow the bioactive ceramic particles to form dynamic coordination bonds with the smart responsive hydrogel matrix, thereby obtaining the wound repair system.

5. The application of the intelligent responsive bear bile powder-based bioactive ceramic hydrogel wound repair system according to claim 1 in the preparation of medical devices or dressings for promoting wound healing, inhibiting wound infection, or reducing scar formation.

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