3D printed core-shell hydrogel scaffold as well as preparation method and application thereof

Abstract

The invention provides a 3D printed core-shell hydrogel stent as well as a preparation method and application thereof, and belongs to the field of medical dressings. The core-shell hydrogel stent comprises an outer layer and an inner layer; the outer layer and the inner layer are integrally formed through coaxial output 3D printing to prepare the core-shell hydrogel stent; the outer layer is made of methacrylated polysaccharide loaded with antibacterial nano enzyme; the material of the inner layer is methacrylic anhydride modified gelatin loaded with an exosome drug; the polysaccharide is selected from hyaluronic acid, sodium alginate, chitosan and carboxymethyl cellulose; the antibacterial nano-enzyme adopts a metal organic framework compound. The core-shell hydrogel stent is used as a chronic wound (diabetic foot ulcer) repair dressing. Methacrylic anhydride modified polysaccharide and gelatin serve as double-layer ink materials, the antibacterial and anti-inflammatory effects of antibacterial nano-enzyme and the angiogenesis promoting and oxidation resisting effects of exosome loaded medicine are combined, the core-shell hierarchical structure support is constructed through 3D biological printing, and time-space sequential release of the medicine is achieved.

Description

Technical Field

[0001] This invention belongs to the field of medical dressings, and specifically relates to 3D printed core-shell hydrogel scaffolds, their preparation methods, and applications. Background Technology

[0002] Chronic wounds are wounds that last a long time and have a low tendency to heal. They involve complex diseases or complications that are difficult to manage, such as diabetic foot ulcers, pressure sores, venous ulcers, limb arterial ischemic necrosis, burns, and radiation injuries.

[0003] Diabetic foot ulcer (DFU) is a typical chronic wound and a common and serious complication of diabetes. It is primarily caused by neuropathy and vascular damage resulting from underlying diabetes, leading to ischemic necrosis or infection of the skin tissue, ultimately forming an ulcer. Furthermore, the persistently high-sugar environment not only hinders the healing of diabetic foot ulcers but also promotes bacterial growth, suppresses the bactericidal function of the immune system, and keeps the wound in a chronic inflammatory state. This prolonged infection and inflammation also induces high levels of oxidative stress in immune cells, producing a large number of reactive free radicals that damage the tissues and cells of the diabetic foot wound. Therefore, diabetic foot ulcers are characterized by a long course, difficulty in healing, and a high recurrence rate.

[0004] Antibacterial dressings are currently a commonly used clinical method for repairing chronic wounds. Hydrogels are widely used in wound dressings due to their good hydrophilicity, biocompatibility, and three-dimensional porous structure such as extracellular matrix. Among them, natural hydrogels such as hyaluronic acid and gelatin have good biocompatibility, but poor mechanical strength and rapid degradation. To solve this technical problem, existing research uses 3D printing of hydrogel scaffolds, but it still has the following shortcomings: (1) lack of ideal materials with both excellent mechanical properties and biocompatibility; (2) limited scaffold function, making it difficult to meet the complex and multi-stage treatment needs of DFU wounds; (3) single drug release mode, making it impossible to achieve a sequential and controllable release strategy; (4) some natural drugs have pharmacokinetic defects, making it difficult to fully exert their therapeutic effects. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a 3D-printed core-shell hydrogel scaffold, its preparation method, and its application, aiming to solve at least one technical problem in the background art.

[0006] This invention is implemented as follows: The first aspect of the present invention provides a 3D-printed core-shell hydrogel scaffold, the core-shell hydrogel scaffold comprising an outer layer and an inner layer; the outer layer and the inner layer are integrally formed by coaxial output 3D printing to obtain the core-shell hydrogel scaffold. The outer layer material is a methacrylamide polysaccharide loaded with antibacterial nanozymes; The inner layer material is methacrylic anhydride-modified gelatin loaded with exosome drugs; The polysaccharide in the methacrylamide is selected from at least one of hyaluronic acid, sodium alginate, chitosan, and carboxymethyl cellulose, and the antibacterial nanozyme is a metal-organic framework complex.

[0007] Furthermore, the antibacterial nanozyme is selected from at least one of Ag@ZIF-8, Cu@ZIF-8, and Zn@MOF.

[0008] Furthermore, the drug in the exosome is selected from at least one of curcumin, astragalus polysaccharide, resveratrol, and growth factor, and the drug is loaded in the exosome.

[0009] Furthermore, the core-shell hydrogel scaffold also includes a PEG anti-adhesion coating that covers the outer layer.

[0010] Furthermore, the thickness ratio of the inner layer to the outer layer is 2 to 4:1.

[0011] A second aspect of the present invention provides a method for preparing the above-mentioned 3D-printed core-shell hydrogel scaffold, the method comprising the following steps: S1, based on the in-situ photocrosslinking blending method of ultrasonic dispersion, antibacterial nanozymes are loaded into methacrylamide polysaccharides to obtain the outer layer raw material; S2, Exosome drugs were loaded into methacrylic anhydride-modified gelatin using a photocrosslinking embedding method to obtain the inner layer raw material; S3: Load the inner and outer layer materials into the coaxial feeding unit of the 3D printing device, use coaxial output 3D printing, and then solidify to obtain the final product.

[0012] Further, the preparation steps of the methacrylic anhydride modified gelatin are as follows: dissolve gelatin at 50℃~55℃, maintain constant temperature and pH=7.4~7.8, slowly add methacrylic anhydride, react, purify and freeze dry to obtain methacrylic anhydride modified gelatin; The preparation steps of the methacrylamide polysaccharide are as follows: dissolve the polysaccharide under ice bath conditions and activate it with alkali. Under light-protected conditions, add methacrylic anhydride dropwise, raise the temperature to room temperature at an initial temperature of 4℃~8℃ and continue the reaction. After dialysis purification and freeze drying, the methacrylamide polysaccharide is obtained.

[0013] Furthermore, the preparation method includes: S4, applying an anti-adhesion coating to the outer wall of the scaffold; S4 specifically includes: immersing the scaffold obtained in S3 in the coating solution for 5s~10s, removing it and removing the residual liquid, and curing it to form an anti-adhesion coating. The coating solution is a mixture of polyethylene glycol diacrylate (PEGDA), lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP), and PBS buffer.

[0014] Furthermore, the curing method employs photocuring, thermal coagulation, or ionic crosslinking.

[0015] A third aspect of this invention provides the application of the aforementioned 3D-printed core-shell hydrogel scaffold, which is used as a dressing for chronic wound repair. Chronic wounds include, but are not limited to, diabetic foot ulcers, venous ulcers, pressure ulcers, burns, and radiation injuries.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention utilizes methacrylic anhydride-modified polysaccharides (such as hyaluronic acid HAMA) and gelatin (GelMA) as bilayer ink materials, combined with the antibacterial and anti-inflammatory effects of anti-inflammatory nanoenzymes (such as Ag@ZIF-8) and the angiogenesis-promoting and antioxidant effects of exosome-loaded drugs (such as curcumin CUR-EXO), to construct a core-shell hierarchical scaffold using 3D bioprinting. This enables the spatiotemporal sequential release of drugs, breaking through the bottlenecks of existing technologies and providing a new strategy for the comprehensive treatment of chronic wounds such as diabetic foot ulcers.

[0017] 2. This invention uses methacrylic anhydride-modified polysaccharides and gelatin as scaffold substrates. This composite material not only inherits the biocompatibility and cell adhesion of natural polymers, but also significantly improves mechanical properties and degradation controllability through cross-linking reactions, overcoming the problems of easy degradation and poor strength of existing natural materials.

[0018] 3. The core-shell hydrogel scaffold of the present invention is manufactured using 3D printing, which is simple to operate and can be personalized according to the patient's wound morphology. The core-shell hierarchical structure can be mass-produced, which is feasible for industrialization and clinical promotion. It is expected to significantly reduce the amputation rate and treatment cost of diabetic foot ulcers.

[0019] 4. This invention overcomes the problems of insufficient biocompatibility, single function, uncontrollable drug release, and limited efficacy of natural drugs in the prior art through multiple innovations of material modification, drug delivery, and structural design, and provides a new comprehensive treatment solution for diabetic foot ulcers that combines personalization, sequential release, and multifunctional synergy. Attached Figure Description

[0020] Figure 1 Macroscopic and microscopic images of the core-shell hydrogel scaffold prepared in Example 1 of this invention; Figure 2 This is a cell staining diagram for biocompatibility evaluation in Example 2 of the present invention; Figure 3This is a colony diagram for the antibacterial evaluation in Example 2 of the present invention; Figure 4 This is the water absorption curve diagram in Embodiment 2 of the present invention; Figure 5 This is a degradation curve diagram from Example 2 of the present invention; Figure 6 This is a sustained-release curve diagram from Embodiment 2 of the present invention; Figure 7 This is a fluorescence detection image from the in vitro antioxidant experiment in Example 2 of the present invention; Figure 8 This is a comparison chart of the wound healing status of each group of animals in Example 3 of the present invention; Figure 9 This is a comparison image of H&E staining and Masson staining of wounds in each group of animals in Example 3 of the present invention. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0022] A 3D-printed core-shell hydrogel scaffold comprises an outer layer and an inner layer, as well as a PEG anti-adhesion coating covering the outer layer. The outer and inner layers are integrally formed using coaxial output 3D printing; the thickness ratio of the inner to outer layer is 2–4:1 to match the healing speed of different wounds. The inner layer is made of methacrylic anhydride-modified gelatin loaded with exosome drugs; the outer layer is made of methacryloyl polysaccharide loaded with antibacterial nanozymes.

[0023] The outer layer has high porosity and a fast degradation rate, enabling early and rapid drug release; the inner layer has low porosity and a slow degradation rate, enabling long-term sustained release and tissue repair. The outer and inner layers are formed in one piece by photopolymerization 3D printing, resulting in a continuous core-shell coaxial structure that can stably adhere to chronic wounds, especially diabetic foot ulcers.

[0024] The outer layer typically degrades in 3 to 7 days. Its function is to rapidly release antibacterial factors in the early stages of wound healing, providing antibacterial, anti-inflammatory, and reactive oxygen species (ROS) scavenging effects, clearing infection and inflammation, improving the adverse microenvironment, and creating a repair microenvironment. The inner layer typically degrades in 10 to 20 days. Its function is to slowly degrade and release drugs in the later stages of wound repair, promoting angiogenesis, reducing inflammation, and promoting tissue regeneration to achieve long-term repair. The outer and inner layers exhibit sequential and complementary effects in time, enabling comprehensive treatment of chronic wounds such as diabetic foot ulcers.

[0025] A method for preparing a 3D-printed core-shell hydrogel scaffold, comprising steps S1 to S4.

[0026] S1, preparation of outer layer raw materials; Specifically, based on the in-situ photocrosslinking blending method of ultrasonic dispersion, antibacterial nanozymes with a particle size of 50nm to 200nm are loaded into methacrylamide polysaccharides to obtain the outer layer raw material.

[0027] The antibacterial nanozyme uses a metal-organic framework complex, such as at least one of Ag@ZIF-8, Cu@ZIF-8, and Zn@MOF. The following examples preferably use Ag@ZIF-8, but are not limited to the listed antibacterial nanozymes. Other unlisted antibacterial nanozymes are also applicable.

[0028] The polysaccharide in the methacrylamide polysaccharide is selected from at least one of hyaluronic acid, sodium alginate, chitosan, and carboxymethyl cellulose. Hyaluronic acid is preferred in the following examples, but it is not limited to the listed polysaccharides. Other unlisted polysaccharides are also applicable.

[0029] In practice, methacrylated polysaccharides can be purchased or prepared by the user. For example, the polysaccharide can be dissolved in an ice bath and activated with alkali. Under light-protected conditions, methacrylic anhydride can be added dropwise, and the mixture can be raised to room temperature from an initial temperature of 4°C to 8°C and allowed to continue reacting. After dialysis purification and freeze-drying, methacrylated polysaccharides can be obtained. The grafting rate can be controlled at 20% to 40% to balance mechanical strength and degradation rate.

[0030] S2, preparation of inner layer raw materials; Specifically, the method involves using photocrosslinking and embedding to load exosome drugs into methacrylic anhydride-modified gelatin to obtain the inner layer material.

[0031] The exosome drug is selected from at least one of curcumin exosomes, astragalus polysaccharide, resveratrol, and growth factors (VEGF, bFGF), and can be loaded onto exosomes using electroporation. The exosomes in the exosome drug can be derived from human bone marrow mesenchymal stem cells (hBMSC), adipose-derived stem cells (ADSC), or umbilical cord mesenchymal stem cells (UC-MSC). The following example preferably uses curcumin exosomes (CUR-EXO). Specifically, an electric field with a strength of 300V / cm to 600V / cm is applied for 5ms to 10ms, and curcumin CUR is loaded onto the exosomes by electroporation, with a drug loading efficiency of 20% to 40%. However, it is not limited to the listed drugs, and other unlisted drugs are also applicable.

[0032] Methacrylic anhydride-modified gelatin can be purchased or prepared in-house. For example, gelatin is dissolved at 50℃~55℃; methacrylic anhydride is slowly added dropwise at a constant temperature and pH of 7.4~7.8; the reaction is carried out; after purification and freeze-drying, methacrylic anhydride-modified gelatin is obtained, with the grafting rate controlled at 30%~50%.

[0033] The specific steps of the photocrosslinking embedding method are as follows: methacrylic anhydride-modified gelatin is dissolved in PBS, and the photoinitiator LAP is added under light-protected conditions; the exosome drug solution is slowly added and mixed under ice bath and light-protected conditions to obtain the inner layer raw material of exosome drug loaded in methacrylic anhydride-modified gelatin.

[0034] S3, 3D printing; Specifically, the inner layer material obtained by S1 and the outer layer material obtained by S2 are loaded into the coaxial feeding unit of the 3D printing device, and the 3D printing is carried out using coaxial output. After curing, a bracket is obtained.

[0035] The curing method can be light curing, thermal coagulation or ion crosslinking, with ultraviolet light curing being preferred.

[0036] S4, prepare an anti-adhesion coating; Specifically, the scaffold obtained in S3 is immersed in the coating solution for 5 to 10 seconds, then removed to remove residual liquid, and cured to form an anti-adhesion coating.

[0037] The coating solution is a mixture of polyethylene glycol diacrylate (PEGDA), lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP), and PBS buffer.

[0038] The aforementioned 3D-printed core-shell hydrogel scaffold is used as a dressing for the repair of chronic wounds, including but not limited to diabetic foot ulcers, venous ulcers, pressure sores, burns, and radiation injuries.

[0039] Example 1 A method for preparing a 3D-printed core-shell hydrogel scaffold, comprising steps S1 to S4.

[0040] S1, methacrylamide hyaluronic acid (HAMA) powder was dissolved in sterile PBS buffer, and under light-protected conditions, photoinitiator LAP and antibacterial nanozyme Ag@ZIF with a particle size of 50nm-200nm were added. 8. Gently sonicate to defoam for approximately 1.5 minutes, then add sterile PBS buffer to achieve a final HAMA concentration of 2.5% w / v and a final LAP concentration of 0.1% w / v. Ag@ZIF The final concentration of 8 was 0.25 mg / mL, and the outer layer raw material was obtained.

[0041] S2, at 37℃~40℃, dissolve methacrylated gelatin (GelMA) powder in sterile PBS buffer, add photoinitiator LAP under light-protected conditions, and then replenish sterile PBS buffer to make the final concentration of GelMA 10% w / v and the final concentration of LAP 0.05% w / v. Add 100µg / mL curcumin exosomes (CUR-EXO) dropwise under ice bath and light-protected conditions and mix slowly to obtain the inner layer raw material.

[0042] S3: The inner layer material obtained in S2 and the outer layer material obtained in S1 are loaded into the coaxial feeding unit of the 3D printing device. The temperature of the coaxial feeding unit is controlled at 20℃, and the temperature of the platform is controlled at 12℃. The scaffold preform is obtained by coaxial output 3D printing. In-line curing (layer by layer) is performed during 3D printing. The in-line curing conditions are: 405nm ultraviolet light and illuminance of 10mW / cm². 2 Each layer has a curing time of 3 seconds; after 3D printing, a final curing process is performed under the following conditions: 405nm ultraviolet light and an illuminance of 25mW / cm². 2 Curing is performed on both sides for 40 seconds each; after curing, a support is obtained.

[0043] S4, prepare an anti-adhesion coating; Specifically, the scaffold obtained in S3 was immersed in a PEGDA / LAP / PBS mixed coating solution for 10 seconds, then removed, excess solution was scraped off, and photocuring was performed (405nm UV light, 25mW / cm²). 2 After 30 seconds, an anti-adhesion coating is formed, resulting in a core-shell hydrogel scaffold HAMA / Ag@ZIF-8 & GelMA / CUR-EXO, as shown in the figure. Figure 1 As shown, it is denoted as AgZMG-CE.

[0044] Figure 1 Image A is a SEM image of a core-shell hydrogel scaffold, while images B, C, and D are actual images of core-shell hydrogel scaffolds of different sizes and shapes.

[0045] Comparative Example 1 The difference between this comparative example and Example 1 is that only HAMA / Ag@ZIF-8 (i.e., the outer layer material) is used, and a single-structure hydrogel scaffold is 3D printed. The preparation method is as follows: S1, the same as S1 in Example 1, yields the raw material.

[0046] S2, the raw material obtained in S1 is loaded into the feeding unit of the 3D printing device. The temperature of the coaxial feeding unit is controlled at 20℃, and the temperature of the platform is controlled at 12℃. The scaffold preform is obtained by 3D printing. During 3D printing, online curing (layer by layer) is performed. The online curing conditions are: 405nm ultraviolet light and illuminance of 10mW / cm². 2Each layer has a curing time of 3 seconds; after 3D printing, a final curing process is performed under the following conditions: 405nm ultraviolet light and an illuminance of 25mW / cm². 2 Curing is performed on both sides for 40 seconds each; after curing, a support is obtained.

[0047] S3, the scaffold obtained in S2 is immersed in a PEGDA / LAP / PBS mixed coating solution for 10 seconds, then removed, excess solution is scraped off, and photocuring is performed (405nm UV light, 25mW / cm²). 2 After 30 seconds, an anti-adhesion coating is formed, resulting in a hydrogel scaffold, denoted as HAMA / Ag@ZIF-8.

[0048] Comparative Example 2 The difference between this comparative example and Example 1 is that only GelMA / CUR-EXO (i.e., the inner layer material) is used, and a single-structure hydrogel scaffold is 3D printed. The preparation method is as follows: S1, the same as S2 in Example 1, yields the raw material.

[0049] S2, the raw material obtained in S1 is loaded into the feeding unit of the 3D printing device. The temperature of the coaxial feeding unit is controlled at 20℃, and the temperature of the platform is controlled at 12℃. The initial preform of the scaffold is obtained by 3D printing. During 3D printing, online curing (layer by layer) is performed. The online curing conditions are: 405nm ultraviolet light and illuminance of 10mW / cm². 2 Each layer has a curing time of 3 seconds; after 3D printing, a final curing process is performed under the following conditions: 405nm ultraviolet light and an illuminance of 25mW / cm². 2 Curing is performed on both sides for 40 seconds each; after curing, a support is obtained.

[0050] S3, Preparation of an anti-adhesion coating; specifically: the scaffold obtained in S2 is immersed in a coating solution of PEGDA / LAP / PBS for 10 seconds, then removed, excess solution is scraped off, and photocuring is performed (405nm UV light, 25mW / cm²). 2 After 30 seconds, an anti-adhesion coating is formed, resulting in a hydrogel scaffold, denoted as GelMA / CUR-EXO.

[0051] Comparative Example 3 The difference between this comparative example and Example 1 is that unloaded blank HAMA and GelMA were used to 3D print a core-shell structured hydrogel scaffold. The preparation method is as follows: S1, methacrylamide hyaluronic acid (HAMA) powder is dissolved in sterile PBS buffer to obtain the outer layer material.

[0052] S2, at 37℃~40℃, dissolve methacrylamide gelatin (GelMA) powder in sterile PBS buffer to obtain the inner layer material.

[0053] S3, same as Example 1.

[0054] S4, same as in Example 1, yields a core-shell hydrogel scaffold, denoted as HAMA&GelMA.

[0055] Example 2 Example 1: The hydrogel scaffolds prepared in Comparative Examples 1 to 3 were subjected to performance characterization tests.

[0056] 1. Biocompatibility The hydrogel scaffolds prepared in Example 1 and Comparative Examples 1 to 3 were immersed in cell culture medium for 48 hours, filtered through a 0.22 μm filter, and the leachate was obtained; an equal volume of cell culture medium served as the control group. Human skin fibroblasts (HDFs) were used as test cells. After culturing for 48 hours, cell viability / deadness staining was used to evaluate cell compatibility. Figure 2 As shown in the figure. After performing cell liveness and death staining experiments, the results showed that the cells cultured in the leachate of each group exhibited a large amount of green fluorescence, indicating a large number of live cells. These results indicate that the core-shell hydrogel scaffold prepared in Example 1 has good biocompatibility.

[0057] 2. Antibacterial properties In bacteria (Staphylococcus aureus) S.aureus Escherichia coli E. coli The hydrogel scaffolds prepared in Example 1, Comparative Examples 1 to 3 were added to the solution and treated for 4 hours. The bacterial colony images and colony count results after treatment are shown below. Figure 3 As shown; Figure 3 (a) shows colony photos of each group; (b) shows the ratio of Staphylococcus aureus colony count after treatment to that before treatment in each group; and (c) shows the ratio of Escherichia coli colony count after treatment to that before treatment in each group.

[0058] Figure 3 The results showed that the number of Staphylococcus aureus and Escherichia coli colonies after treatment with the AgZMG-CE hydrogel scaffold in Example 1 was significantly lower than that after treatment with the hydrogel scaffold HAMA / Ag@ZIF-8 in Comparative Example 1, the hydrogel scaffold GelMA / CUR-EXO in Comparative Example 2, and the hydrogel scaffold HAMA&GelMA in Comparative Example 3. The results indicate that the core-shell hydrogel scaffold prepared in Example 1 has good anti-Staphylococcus aureus and anti-Escherichia coli effects, and the effect is superior to that of Comparative Examples 1 to 3.

[0059] 3. Water absorption The hydrogel scaffold prepared in Example 1 was freeze-dried, its initial weight W0 was measured, and then it was immersed in physiological saline. At a specific time point t, it was removed, the surface moisture was absorbed, and its weight W was measured. tCalculate the water absorption ratio (W) at each time point. t Plot the water absorption curve with time (t) on the x-axis and water absorption ratio on the y-axis; for example, Figure 4 As shown, the hydrogel scaffold prepared in Example 1 has good water absorption.

[0060] 4. Degradation performance The hydrogel scaffold prepared in Example 1 was freeze-dried, and its initial weight was measured. Using 10 U / mL collagenase-PBS solution as the degradation medium, the freeze-dried hydrogel scaffold was immersed in a centrifuge tube containing the degradation medium and slowly shaken in a constant temperature shaker at 37°C. Half of the degradation medium was replaced periodically to maintain enzyme activity and pH stability. At preset time points, the sample was removed, gently rinsed with deionized water, then freeze-dried, and its dry weight was measured again. A plot of time (t) on the x-axis and dry weight (g) on ​​the y-axis was shown below. Figure 5 The degradation curves shown indicate that the faster the curve decreases, the faster the degradation rate. The results show that the mass retention rate of the hydrogel scaffold prepared in Example 1 in PBS gradually decreases over time, indicating good degradability. The degradation behavior matches the wound healing cycle.

[0061] 5. Sustained-release properties The controlled release ability of the active ingredient curcumin was tested using an in vitro drug release experiment, specifically as follows: The hydrogel scaffold prepared in Example 1 was freeze-dried and used as the test sample (the total mass of the active ingredient curcumin was M0). Using PBS (pH=7.4) as the release medium, the freeze-dried hydrogel scaffold was immersed in a container containing the release medium, with the volume of the release medium being 50 times that of the hydrogel scaffold. The container was then placed in a constant-temperature shaker at 37°C and slowly shaken. At preset time points, a certain volume (e.g., 1 mL) of the release medium was removed from the container, and an equal volume of fresh release medium at the same temperature was immediately added to maintain a constant total volume. The concentration C of the drug in the removed sample was determined by HPLC. n Calculate the amount of active ingredient released, M, at this time point. t ; Calculate the cumulative release rate CR (Cur Release, %) at each time point.

[0062]

[0063]

[0064] V represents the concentration of the active ingredient measured at the nth time point; V is the total volume of the release medium; V s This represents the volume of each sample taken. This represents the total amount of active ingredients extracted at all previous time points.

[0065] Plot a graph with time (t) on the x-axis and cumulative release rate (%) on the y-axis as shown below. Figure 6 The release curve shown is as follows: Figure 6 As shown, the cumulative release rate of curcumin (Cur) from the hydrogel scaffold prepared in Example 1 reached approximately 35% on day 8, followed by slow release. The results indicate that the combination of exosomes (Exos) with the polyphenol metal network of antibacterial nanozymes can effectively delay drug release, enabling continuous drug delivery and meeting the temporal requirements of wound healing.

[0066] 6. In vitro antioxidant experiment In vitro antioxidant experiments were conducted on the hydrogel scaffolds prepared in Example 1 and Comparative Examples 1 to 3 using the DCFH-DA method. DCFH-DA working solution and free radical solution were prepared according to the DCFH-DA kit instructions. The hydrogel scaffolds and DCFH-DA working solution were loaded with DCFH-DA probes, and free radical attack and antioxidant reactions were performed. Fluorescence detection and data acquisition were conducted, and the results are as follows: Figure 7 As shown, the hydrogel scaffold prepared in Example 1 exhibits strong antioxidant activity. In the experiment, hydrogen peroxide stimulation of 3T3 cells resulted in the production of large amounts of ROS, oxidizing DCFH to DCF with strong fluorescence intensity. Cells cultured using the leachate from the hydrogel scaffold prepared in Example 1 (AgZMG-CE) showed a significant decrease in intracellular fluorescence intensity. The fluorescence intensity of the hydrogel scaffold group in Comparative Example 1 (HAMA / Ag@ZIF-8) was also significantly lower than that of the hydrogel scaffold group in Comparative Example 2 (GelMA / CUR-EXO) and the hydrogel scaffold group in Comparative Example 3 (HAMA&GelMA). Figure 7 The in vitro antioxidant effects were shown in Example 1 > Comparative Example 1 > Comparative Example 2 > Comparative Example 3. Comparative Example 1, with its phenolic hydroxyl structure, can efficiently capture free radicals and reduce the oxidation of DCFH. Comparative Example 2's Exos, by delivering antioxidant-related miRNAs and proteins, regulates the intracellular antioxidant enzyme system and enhances the cell's ability to clear ROS.

[0067] Example 3 To examine the therapeutic effect of hydrogel scaffolds on wound healing, an SD rat skin wound model was used.

[0068] 1. Modeling and Grouping Experimental animals: Male SPF-grade SD rats aged 6-8 weeks, weighing 200g ± 20g, were selected. The rats were acclimatized to the environment in a standard laboratory setting (temperature 22℃ ± 2℃, humidity 50 ± 10%, 12-hour light-dark cycle) for at least one week.

[0069] Diabetes model establishment: SD rats were fasted for 12 hours but allowed free access to water. Streptozotocin (STZ) was injected intraperitoneally at a dose of 100 mg / kg. Four hours after injection, drinking water containing 5% glucose was provided to prevent hypoglycemic shock in some rats. Then, 50 mg / kg was injected for three consecutive days. Random blood glucose or fasting blood glucose was measured by blood sample taken from the tail vein using a blood glucose meter. A diabetes model was considered successfully established if random blood glucose ≥16.7 mmol / L and / or fasting blood glucose ≥11.1 mmol / L, accompanied by symptoms such as polydipsia, polyphagia, polyuria, and weight loss.

[0070] Constructing a skin wound model of diabetic foot ulcers: SD rats were anesthetized with isoflurane inhalation. The hair on the back of the rat's hind foot was shaved and cleaned to expose the skin. Then, a circular punch with a diameter of about 8 mm was used to gently press an imprint on the skin and draw a marking line. Along the marking line, sterile ophthalmic scissors and forceps were used to precisely cut away the full-thickness skin tissue within the circular area until the underlying fascia layer was exposed. The wound was gently pressed with sterile cotton swabs or gauze for a moment to achieve mild hemostasis. The skin wound model of diabetic foot ulcers was thus successfully constructed.

[0071] Under aseptic conditions, the wound was first cleaned and disinfected. Then, pre-cut dressings were placed over the wound, ensuring complete coverage and slightly extending beyond the edges. Finally, sterile gauze, breathable medical tape, and elastic bandages were used to gently wrap the wound, ensuring the dressing was secure and would not fall off due to rat movement, while maintaining unobstructed blood flow. The dressing was changed every 24 hours.

[0072] Mice were randomly divided into 5 groups based on different dressings, as follows: (1) AgZMG-CE group: The dressing was the core-shell hydrogel scaffold prepared in Example 1; (2) HAMA / Ag@ZIF-8 group: The dressing was a hydrogel scaffold prepared in Comparative Example 1; (3) GelMA / CUR-EXO group: The dressing was the hydrogel scaffold prepared in Comparative Example 2; (4) HAMA & GelMA group: The dressing was the core-shell hydrogel scaffold prepared in Comparative Example 3. (5) Positive control group: The dressing was an alginate dressing that has been clinically proven to be effective; (6) Negative control group: The dressing was petroleum jelly gauze, which could keep the skin moist but had no active therapeutic effect.

[0073] After treating the wounds according to the above grouping method, the wound size was photographed on days 0, 3, 6, 9, and 12. The wound size was measured and analyzed using a standard ruler and image processing software. The wound healing effect of different groups of mice was observed, and the results are as follows: Figure 8As shown in the figure. The results indicate that the AgZMG-CE hydrogel group from Example 1 exhibited the fastest wound healing speed and the most effective healing effect.

[0074] To further confirm the wound healing capabilities of each hydrogel scaffold group, histological analysis was performed on skin tissue collected on day 12. H&E staining was used to analyze the morphology of regenerated granulation tissue after wound healing, and Masson staining was used to analyze collagen deposition in the wound to assess wound healing. The results are as follows: Figure 9 As shown.

[0075] Granulation tissue is a crucial temporary tissue in the wound repair process. It fills tissue defects, provides a scaffold for epithelial regeneration, and ultimately achieves wound closure through scar formation. Under a microscope, actively regenerating granulation tissue contains three basic components: newly formed capillaries, fibroblasts / myofibroblasts, and inflammatory cells. H&E staining analysis showed that the AgZMG-CE group in Example 1 had abundant capillaries, active fibroblasts, and moderate inflammatory cell infiltration, indicating the formation of healthy granulation tissue.

[0076] The proportion of blue collagen region in the entire tissue area in Masson staining images was calculated using image analysis software, and the amount of collagen deposition was semi-quantitatively analyzed. The results showed that the AgZMG-CE group in Example 1 had the highest level of collagen deposition.

[0077] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A 3D printed core-shell hydrogel scaffold, characterized in that, The core-shell hydrogel scaffold comprises an outer layer and an inner layer; the outer layer and the inner layer are integrally formed by coaxial output 3D printing to prepare the core-shell hydrogel scaffold; The material of the outer layer is antibacterial nanometer enzyme loaded methacrylated polysaccharide; The material of the inner layer is exosome drug loaded methacrylic anhydride modified gelatin; The polysaccharide in the methacrylated polysaccharide is selected from at least one of hyaluronic acid, sodium alginate, chitosan and carboxymethyl cellulose; the antibacterial nanometer enzyme adopts a metal organic framework complex.

2. The 3D printed core-shell hydrogel scaffold of claim 1, wherein, The antibacterial nanometer enzyme is selected from at least one of Ag@ZIF-8, Cu@ZIF-8 and Zn@MOF.

3. The 3D printed core-shell hydrogel scaffold of claim 1, wherein, The drug in the exosome drug is selected from at least one of curcumin, astragalus polysaccharide, resveratrol and growth factor; the drug is loaded in the exosome.

4. The 3D printed core-shell hydrogel scaffold of claim 1, wherein, The core-shell hydrogel scaffold further comprises a PEG anti-adhesion coating covering the outside of the outer layer.

5. The 3D printed core-shell hydrogel scaffold of claim 1, wherein, The thickness ratio of the inner layer to the outer layer is 2-4:

1.

6. The method of producing a 3D printed core-shell hydrogel scaffold according to any one of claims 1 to 5, characterized in that, The preparation method comprises the following steps: S1, in-situ photo-crosslinking blending method based on ultrasonic dispersion is used to load the antibacterial nanometer enzyme in the methacrylated polysaccharide to obtain the outer layer raw material; S2, the exosome drug is loaded in the methacrylic anhydride modified gelatin by photo-crosslinking embedding method to obtain the inner layer raw material; S3, the inner layer raw material and the outer layer raw material are loaded into the coaxial feeding unit of the 3D printing device, and coaxial output 3D printing is adopted, and then solidification is performed to obtain the core-shell hydrogel scaffold.

7. The method of claim 6, wherein the 3D printed core-shell hydrogel scaffold is prepared by, The preparation steps of the methacrylic anhydride modified gelatin are as follows: dissolving gelatin at 50-55°C; slowly adding methacrylic anhydride under constant temperature and maintaining pH=7.4-7.8, and then reacting, purifying, and freeze-drying to obtain the methacrylic anhydride modified gelatin; The preparation steps of the methacrylic anhydride modified gelatin are as follows: dissolving gelatin at 50-55°C; slowly adding methacrylic anhydride under constant temperature and maintaining pH=7.4-7.8, and then reacting, purifying, and freeze-drying to obtain the methacrylic anhydride modified gelatin; 8. The method of claim 6, wherein the 3D printed core-shell hydrogel scaffold is prepared by, The preparation method comprises: S4, modifying an anti-adhesion coating on the outer wall of the scaffold; S4 specifically comprises: immersing the scaffold obtained in S3 in a coating solution for 5-10s, removing the residual liquid after taking out, and forming an anti-adhesion coating after solidification; The coating solution is a mixture of polyethylene glycol diacrylate PEGDA, phenyl(2,4,6-trimethylbenzoyl) lithium phosphate salt LAP and PBS buffer.

9. The method of claim 6, wherein the 3D printed core-shell hydrogel scaffold is prepared by, The solidification method adopts light solidification, heat solidification or ion crosslinking.

10. Use of the 3D-printed core-shell hydrogel scaffold according to any one of claims 1 to 5, characterized in that The core-shell hydrogel scaffold is used as a chronic wound repair dressing.

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