An interpenetrating network antibacterial hydrogel with vascularization function and a preparation method and application thereof

Abstract

This invention discloses an interpenetrating network antibacterial hydrogel with angiogenesis function, its preparation method, and its applications. The hydrogel is formed by in-situ photocrosslinking of a precursor solution to create a three-dimensional interpenetrating network structure. The precursor solution comprises the following components by mass fraction: 0.5%–1.5% gallic acid-modified low-viscosity chitosan, 0.5%–1.5% decellularized liver matrix, 8%–12% methacrylic acid gelatin, 0.5%–1.0% photoinitiator, and the balance being phosphate-buffered saline. This invention uses low-viscosity chitosan as the modifying unit, combined with a strategy of precise acid adjustment before blending. This not only effectively avoids the risks of polymer phase separation and flocculation that are easily caused during multi-component blending, but also fundamentally solves the problem of the influence of the acidic microenvironment on the interpenetrating crosslinking of polymers. The precursor solution of this hydrogel has excellent fluidity, can precisely adapt to irregular defects, and can solidify rapidly in situ. It integrates antibacterial properties and induces angiogenesis, showing broad application prospects in the repair of complex skin tissues such as chronic diabetic wounds.

Description

Technical Field

[0001] This invention relates to the field of medical biomaterials technology, and in particular to an interpenetrating network antibacterial hydrogel with angiogenesis function, its preparation method and application. Background Technology

[0002] The skin is the body's first physical barrier against external pathogens. For diabetic patients, the chronically high blood sugar environment leads to an imbalance in the wound microenvironment, making wounds prone to bacterial infection, insufficient angiogenesis, and chronic inflammation, resulting in a pathological state that is extremely difficult to heal. Therefore, researching materials that simultaneously meet the multiple needs of antibacterial, angiogenesis-promoting, and biomimetic microenvironment is of great significance for skin repair in diabetic patients.

[0003] Hydrogels, with their three-dimensional structure similar to the natural extracellular matrix (ECM), show great potential for application in the repair of chronic diabetic wounds. For example, invention patent CN15501376A discloses a gel-type antibacterial medical bandage and its preparation method, which polymerizes gelatin methacrylate (GelMA) and thiolated chitosan under ultraviolet light to obtain an antibacterial gel. However, existing technologies in this category mostly use conventional high-molecular-weight chitosan, which suffers from two major insurmountable drawbacks in practical applications: first, conventional chitosan solutions have extremely high viscosity and poor fluidity, making it difficult to achieve adaptive and seamless filling of irregular, deep diabetic wounds; second, conventional chitosan is extremely dependent on an acidic environment for dissolution, and attempts to adjust it to a near-neutral physiological microenvironment often result in rapid and severe flocculation and precipitation, losing the material's homogeneity. Invention patent CN116063584A discloses a chitosan-gallic acid copolymer and its application in the preparation of hydrogels and wound repair, showing that hydrogels obtained by modifying chitosan with gallic acid (GA) exhibit better antibacterial properties. However, this method often uses chemical cross-linking methods such as sodium periodate, which poses a potential risk of cytotoxicity; and it lacks effective synergy with matrices such as gelatin methacrylate, resulting in a relatively simple hydrogel structure.

[0004] As the only regenerative organ in the human body, the liver's decellularized matrix (DLM) is rich in unique tissue-specific components (such as type VI collagen) and endogenous pro-angiogenic factors, giving it a unique pro-angiogenic capacity. However, under acidic conditions, decellularized liver matrix cannot be effectively cross-linked directly with gelatin methacrylate, failing to form a stable network structure. Furthermore, while the introduction of decellularized matrix components enhances bio-inducible activity, it typically significantly compromises the mechanical strength of the hydrogel. Compensating for the resulting mechanical losses and maintaining gel stability when DLM is introduced remains a major challenge in the field of tissue engineering. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the technical problem this invention aims to solve is: how to provide an interpenetrating network antibacterial hydrogel with angiogenesis function, its preparation method, and its applications. This addresses the problems of traditional modified hydrogels in multi-component composites, such as incomplete cross-linking and inactivation due to acidic environments, poor biocompatibility, and a sharp decline in mechanical properties caused by the introduction of decellularized matrix. It also addresses the limitations of existing dressings, which have limited functionality and are difficult to adapt to irregular diabetic chronic wounds in situ. Ultimately, this invention aims to precisely regulate each stage of wound repair under the complex conditions of an imbalanced microenvironment in diabetic wounds, thereby comprehensively meeting the tissue repair needs of diabetic chronic wounds.

[0006] To address the aforementioned technical problems, the present invention employs the following technical solution: an interpenetrating network antibacterial hydrogel with angiogenesis function, wherein the hydrogel is formed by in-situ photocrosslinking of a precursor solution to create a three-dimensional interpenetrating network structure; the precursor solution comprises the following components by mass fraction: 0.5%~1.5% gallic acid-modified low-viscosity chitosan, 0.5%~1.5% decellularized liver matrix, 8%~12% methacrylic acid gelatin, 0.5%~1.0% photoinitiator, and the balance being phosphate-buffered saline. It should be noted that those skilled in the art will understand that "the balance being phosphate-buffered saline" does not exclude the possibility that the system may contain trace amounts of conventional additives or impurities that do not affect the basic properties of the hydrogel. This hydrogel network incorporates CS-GA to construct a durable antibacterial functional unit, achieving a synergistic effect of both physical and chemical antibacterial mechanisms: on the one hand, the positive charge carried by CS itself can strongly electrostatically adsorb onto the negative charge of the bacterial cell wall, physically disrupting the bacterial structure; on the other hand, the polyphenolic hydroxyl groups abundant in GA molecules can penetrate and destroy the bacterial cell membrane, chemically blocking bacterial metabolic processes. This hydrogel exhibits extremely significant inhibitory and bactericidal effects against common pathogens; simultaneously, all antibacterial components are derived from natural polymer materials, achieving long-lasting anti-infection while having no significant toxic side effects on normal tissue cells, providing an excellent biosafety microenvironment for tissue repair. Furthermore, decellularized liver matrix with extremely strong tissue regeneration capacity is introduced. Compared with decellularized tissues derived from skin, heart, or cornea, liver-derived DLM retains unique tissue-specific components and the natural extracellular matrix (ECM) conformation, especially its rich content of type VI collagen, laminin, and glycosaminoglycans. These well-preserved natural structural proteins and adhesion sites can provide vascular endothelial cells with a three-dimensional microenvironment that is closest to the physiological state, significantly promoting endothelial cell chemotaxis, adhesion, proliferation and deep migration, thereby actively inducing angiogenesis. At the same time, DLM can also form an interpenetrating structure with the GelMA network, so that a biomimetic microenvironment with a porous structure is formed inside the hydrogel, which greatly improves the regeneration efficiency of ischemic tissue.

[0007] In the preparation of the precursor solution, the pH of the gallic acid-modified low-viscosity chitosan (CS-GA) and the decellularized liver matrix is ​​pre-adjusted to 7.0-7.4 before being mixed with other components. This ensures that DLM can form an interpenetrating structure with the GelMA network and maintain the bioactivity of the decellularized liver matrix under near-neutral conditions.

[0008] Thus, low-viscosity chitosan was chosen as the modifying unit. On the one hand, considering the inherent acid-soluble properties of chitosan, low-viscosity chitosan, due to its shorter molecular chains, can maintain excellent liquid homogeneity even after being precisely adjusted to the physiological pH (7.0~7.4) through a pre-neutralization strategy. This not only effectively avoids agglomeration and phase separation that easily occur during multi-component blending, ensuring excellent fluidity of the gel precursor solution, but also gives it suitable tissue adhesion when in contact with wounds. On the other hand, the short-chain structure endows the molecules with higher spatial flexibility. The low-viscosity CS-GA molecular chains can uniformly penetrate into the GelMA network, achieving effective mechanical compensation through sufficient hydrogen bonding and electrostatic interactions. Furthermore, the innovative pre-neutralization strategy of precisely adjusting the pH (7.0~7.4) before blending not only overcomes the influence of the acidic environment on polymer interpenetrating crosslinking, but also successfully constructs a highly stable three-dimensional physical-chemical interpenetrating polymer network. This allows it to retain the activity of natural angiogenesis factors in the decellularized liver matrix while providing mechanical strength, achieving a highly efficient synergy between antibacterial properties, specific angiogenesis capabilities, and a highly stable three-dimensional mechanical network.

[0009] Preferably, the molar ratio of chitosan to gallic acid in the gallic acid-modified low-viscosity chitosan is 1:0.4~0.6, and the viscosity of the chitosan at room temperature is 10~200 mPa·s. By chemically grafting gallic acid molecules into the low-viscosity chitosan backbone, a significant synergistic effect is achieved, thereby endowing the modified material with excellent antibacterial properties.

[0010] Preferably, the decellularized liver matrix is ​​derived from mammalian liver, preferably from pigs, cattle, or mice. Liver-derived DLM is rich in natural type VI collagen and specific angiogenesis factors. Compared to conventional skin matrix, it provides a superior chemotactic, adhesion, and proliferative microenvironment for vascular endothelial cells, significantly accelerating vascular network reconstruction in ischemic wounds.

[0011] Preferably, the photoinitiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone (Irgacure 2959) or lithium phenyl-2,4,6-trimethylbenzoyl phosphate (LAP). These initiators exhibit excellent biocompatibility and high initiation efficiency, enabling rapid in-situ solidification of the hydrogel even at extremely low concentrations, effectively ensuring the safety of the encapsulated cells and surrounding tissues.

[0012] Another object of the present invention is to provide a method for preparing the above-mentioned interpenetrating network antibacterial hydrogel with angiogenesis function, comprising the following steps: (1) Using EDC and NHS as activators, gallic acid was grafted onto low-viscosity chitosan. Gallic acid-modified low-viscosity chitosan was obtained by dialysis and freeze drying. It was then completely dissolved in acetic acid solution, and the pH value was adjusted to 7.0~7.4 to obtain gallic acid-modified low-viscosity chitosan solution (CS-GA).

[0013] (2) Grind the decellularized liver tissue into powder, digest it with pepsin in acetic acid solution, and adjust the pH of the solution to 7.0~7.4 after the reaction is completed to obtain decellularized liver matrix solution (DLM).

[0014] (3) Dissolve gelatin methacrylate (GelMA) in phosphate buffered saline, then add a photoinitiator, then add the gallic acid modified low viscosity chitosan solution obtained in step (1) and the decellularized liver matrix solution obtained in step (2), stir evenly, and obtain a precursor solution with good fluidity.

[0015] (4) The precursor solution is transferred to a mold and photocrosslinked and cured to obtain the antibacterial hydrogel.

[0016] In this way, the acid-dependent dissolution components (CS-GA and DLM) are prepared and neutralized independently before being blended with the photocrosslinked substrate GelMA. This preserves the biological signal and angiogenesis efficacy of DLM and solves the problems of component aggregation, phase separation or inactivation of active ingredients that are easily caused by one-pot mixing.

[0017] Preferably, in step (1), the volume concentration of the acetic acid solution is 1-5%, and the mass-to-volume ratio of the gallic acid-modified low-viscosity chitosan to the acetic acid solution is 1-3 g: 100 mL.

[0018] Preferably, in step (2), the molar concentration of the acetic acid solution is 0.4~0.6 mol / L, and the mass-to-volume ratio of the decellularized liver tissue to acetic acid is 1~3 g: 100 mL.

[0019] Preferably, the wavelength in the photocrosslinking process is 300~400 nm, the power is 15~50 W, and the irradiation time is 5~6 min.

[0020] Another objective of this invention is to provide the application of the antibacterial hydrogel obtained by the above method in the preparation of materials for repairing diabetic skin tissue. This hydrogel can seal wounds in situ, provide long-lasting anti-infection, and actively induce vascular network reconstruction, significantly improving the speed and quality of tissue repair. It is particularly suitable for the clinical treatment needs of complex and difficult-to-heal wounds such as chronic diabetic skin wounds and deep soft tissue injuries.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. The antibacterial hydrogel with angiogenesis function provided by this invention uses GelMA as the base material for repair. It innovatively introduces gallic acid-modified low-viscosity chitosan (CS-GA) and decellularized liver matrix (DLM). Through precise acid adjustment (pH 7.0~7.4), CS-GA can undergo partial covalent cross-linking with GelMA. Its abundant polyphenolic hydroxyl and amino groups can also form strong hydrogen bonds and electrostatic interactions with the GelMA network. Furthermore, the physical interpenetrating structure formed by DLM and the GelMA network constructs a stable three-dimensional interpenetrating network, solving the problems of structural instability and difficulty in cross-linking caused by simple mixing of multiple components. Simultaneously, the hydrogen bonds and electrostatic interactions formed by the polyphenolic hydroxyl groups of CS-GA with the GelMA molecular chains compensate for the decrease in mechanical properties caused by the addition of DLM. Therefore, this invention constructs a hydrogel with a high-strength, highly stable three-dimensional physical-chemical interpenetrating network structure.

[0022] 2. The antibacterial hydrogel with angiogenesis function prepared by this invention possesses excellent biocompatibility, antibacterial properties, and angiogenesis-promoting ability. It can effectively inhibit bacterial infection in the early stage of tissue repair and promote the formation of vascular networks during the repair process. The addition of CS-GA and DLM to this hydrogel system retains the beneficial bioactive properties of GelMA, such as bioactivity, three-dimensional porous morphology, and biodegradability. Meanwhile, the structure provided by DLM is beneficial to cell activities, such as adhesion, migration, and differentiation into various lineages, playing a key role in skin wound healing, morphogenesis, and tissue repair. Furthermore, this hydrogel material is widely available, the preparation process is simple, it can be prepared without complex equipment, the cost is low, it is easy to scale up production, and it has good potential for clinical translation.

[0023] 3. This invention utilizes low-viscosity chitosan and strictly controls the grafting molar ratio, overcoming the shortcomings of conventional chitosan, such as easy flocculation and poor fluidity when adjusted to neutral. This results in a hydrogel precursor solution with excellent fluidity. In clinical applications, it can fill irregular skin defects and achieve rapid in-situ solidification through photocrosslinking after transfer, forming a stable three-dimensional structure. The hydrogel adheres closely to surrounding tissues at the implantation site. Simultaneously, GA-modified CS continuously exerts antibacterial effects, reducing the risk of infection, while DLM creates a pro-angiogenic microenvironment locally, inducing new blood vessel growth towards the defect area, thereby accelerating tissue regeneration and repair. This hydrogel system integrates antibacterial and pro-angiogenic functions, meeting the needs of complex tissue defect repair. It is particularly suitable for the clinical treatment of complex and difficult-to-heal wounds such as diabetic chronic skin lesions and deep soft tissue injuries, possessing extremely broad prospects for industrialization and clinical translation. Attached Figure Description

[0024] Figure 1 GelMA prepared according to the present invention; (A) Schematic diagram of chemical synthesis; (B) Physical image.

[0025] Figure 2 CS-GA prepared according to the present invention; (A) Schematic diagram of chemical synthesis; (B) Physical image.

[0026] Figure 3 Here are schematic diagrams of the antibacterial hydrogel prepared according to the present invention; (A) the precursor solution of the hydrogel; (B) a schematic diagram of the gelation of the precursor solution under ultraviolet irradiation; and (C) a physical image of the antibacterial hydrogel.

[0027] Figure 4 Mechanical properties of the antibacterial hydrogel prepared according to the present invention: (A) stress-strain curve; (B) compressive modulus.

[0028] Figure 5 The microstructure of the antibacterial hydrogel prepared in this invention is shown.

[0029] Figure 6 The degradation and hydrophilic properties of the antibacterial hydrogel prepared in this invention are as follows: (A) degradation properties; (B) water absorption; (C) water retention.

[0030] Figure 7 Live / Dead staining for cells seeded on antibacterial hydrogel.

[0031] Figure 8 To quantify the viability of cells planted on antibacterial hydrogels. Detailed Implementation

[0032] The present invention will be further described in detail below with reference to the embodiments. Unless otherwise specified, the reagents and experimental methods used in the embodiments are all commercially available and conventionally operated.

[0033] I. A method for preparing an interpenetrating network antibacterial hydrogel with angiogenesis function Example 1 This example is prepared using the following steps: (1) Synthesis of gallic acid-modified low-viscosity chitosan (CS-GA) Weigh 1 g of low-viscosity chitosan (viscosity 50-200 mPa·s at room temperature) and dissolve it in 50 mL of 1% (v / v) acetic acid aqueous solution. Stir overnight until completely dissolved to obtain a CS solution. Separately, dissolve 0.4224 g of gallic acid (GA) in 50 mL of 50% (v / v) ethanol aqueous solution and stir magnetically until completely dissolved to obtain a GA solution. Subsequently, add 1.19 g of EDC and 0.714 g of NHS to the GA solution at room temperature and activate the reaction for 2 h. Slowly add the activated mixture dropwise to the dissolved CS solution above to carry out the grafting reaction. After the reaction is complete, transfer the mixture to a dialysis bag with a molecular weight cutoff of 8000-14000 Da and dialyze in acidic ultrapure water at pH 5.5 for 72 h (changing the dialysate every 8 h). Finally, dialyze twice more with pure ultrapure water to completely remove residual acid. The dialyzed sample was freeze-dried to obtain CS-GA with a low viscosity chitosan to gallic acid molar ratio of 1:0.4 (its chemical synthesis schematic diagram is shown in Figure 1). Figure 1 As shown in Figure A, the freeze-dried product is as follows: Figure 1 (As shown in B). Before use, the lyophilized CS-GA was redissolved in 50 mL of 1% (v / v) aqueous acetic acid solution, and the pH was neutralized to 7.4 with sodium hydroxide solution under constant stirring to obtain the CS-GA solution.

[0034] (2) Pretreatment of decellularized liver tissue Fresh liver tissue was frozen at -80°C for 12 hours, then thawed in a 37°C water bath for 30 minutes. This freeze-thaw cycle was repeated three times. The treated tissue blocks were then placed in a shaker at 4°C and washed 3-4 times with ultrapure water at 120 rpm for 30 minutes each time. After washing, a mixed decellularization solution containing 1% Triton X-100 and 0.1% ammonia was added to the tissue, and the mixture was shaken at 4°C and 130 rpm for 2-3 days (replacing the solution with fresh solution every 12 hours) until the liver tissue turned completely white. Finally, the tissue was thoroughly washed with deionized water to remove any residual chemicals and stored at 4°C for later use.

[0035] (3) Preparation of decellularized liver matrix (DLM) solution Pretreated decellularized liver tissue was freeze-dried, then minced and ground into powder. 100 mg of tissue powder was weighed, and 100 mg of pepsin (potency 1:10000) and 5 mg of PBS powder were added sequentially. Then, 10 mL of 0.5 mol / L acetic acid solution was added, and the mixture was sealed and stirred at 37°C for 3 days for digestion. The pepsin-digested solution was collected, and its pH was neutralized to 7.4 with sodium hydroxide solution under constant stirring to obtain the DLM solution.

[0036] (4) Synthesis of gelatin methacrylate (GelMA) Dissolve 10g of gelatin in 100mL of PBS and heat to 60℃ until completely dissolved. While stirring vigorously, slowly add 8mL of methacrylic anhydride (MA) and react in the dark for 3 hours (see schematic diagram of chemical synthesis). Figure 2 (As shown in A). After the reaction was complete, 400 mL of preheated PBS was added to terminate the reaction. The reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 8000~14000 Da and dialyzed in deionized water at 40°C for 7 days to completely remove unreacted MA. After dialysis, the solution was placed in a -20°C freezer overnight, then frozen at -80°C for 1 day, and finally freeze-dried in a freeze dryer for 3 days to obtain fibrous white porous foam-like GelMA (as shown in A). Figure 2 As shown in B).

[0037] (5) Preparation of hydrogel precursor solution The lyophilized GelMA was dissolved in PBS (for approximately 10 minutes), followed by the addition of photoinitiator I2959. After the GelMA and photoinitiator were completely dissolved, they were mixed uniformly with the prepared CS-GA and DLM solutions in the specified proportions to obtain the hydrogel precursor solution. The precursor solution was prepared with the following components: gallic acid-modified low-viscosity chitosan 1% (w / v), decellularized liver matrix 1% (w / v), methacrylic acid gelatin 10% (w / v), and photoinitiator 0.5% (w / v).

[0038] (6) Photocrosslinking and curing of hydrogels The prepared precursor solution was transferred to a multi-well plate and irradiated under a UV lamp with a wavelength of 365 nm and a power of 15~50 W for 6 min to allow it to fully crosslink and solidify, thus preparing the CS-GA / DLM / GelMA interpenetrating network antibacterial hydrogel.

[0039] Comparative Example 1: No CS-GA and DLM added, other steps are the same as in Example 1.

[0040] Comparative Example 2: Gallic acid and DLM were not added; other steps were the same as in Example 1.

[0041] Comparative Example 3: No DLM added, other steps are the same as in Example 1.

[0042] Comparative Example 4: CS-GA and DLM were not pH-adjusted before mixing with other components, and other steps were the same as in Example 1. The results showed that in the unadjusted system, DLM could not crosslink with GelMA due to its inhibitory effect. Although CS-GA could crosslink with GelMA, the acidic gel microenvironment it constructed significantly impaired cell activity.

[0043] II. Performance Verification and Effect Analysis 1. Observe the morphology of the hydrogels and their scaffolds prepared in Example 1 and Comparative Examples 1-3. The results are as follows: Figure 3 As shown.

[0044] from Figure 3 As can be seen from A, the precursor solutions of Comparative Example 1 (pure GelMA) and Comparative Example 2 (CS / GelMA) are milky white, and CS is uniformly dispersed at the molecular level in the system; while the precursor solutions of Comparative Example 3 (CS-GA / GelMA) and Example 1 (CS-GA / DLM / GelMA), which incorporate gallic acid and decellularized liver matrix, are uniformly light brown, with no obvious aggregation or phase separation, confirming that the pH-controlled preparation process of this invention can effectively ensure the excellent compatibility of multiple components. The above precursor solutions were irradiated under a 365 nm ultraviolet lamp for 6 min (… Figure 3 B), Examples 1 and Comparative Examples 1-3 all underwent rapid free radical polymerization under the action of a photoinitiator, achieving in-situ crosslinking and forming a morphologically stable gel ( Figure 3 C). This indicates that the addition of the modified components did not interfere with the photocrosslinking network forming ability of GelMA.

[0045] 2. The mechanical compressibility properties of the hydrogels prepared in Example 1 and Comparative Examples 1-3 were tested, and the results are as follows: Figure 4 As shown.

[0046] Depend on Figure 4 The stress-strain curves of group A show that, under the same strain conditions, the compressive stress of each gel group exhibits significant differences. Comparative Example 1 has the lowest stress, while Comparative Example 2, with the addition of chitosan (CS), has the highest stress, indicating that the rigid molecular chains of CS can physically interpenetrate with the GelMA network, significantly improving the mechanical strength of the system. Furthermore, the compressive modulus in the 20–30% strain range was extracted. Figure 4B), the compressive moduli of Comparative Examples 1-3 and Example 1 were 14.86±0.62 kPa, 22.15±1.10 kPa, 18.48±1.84 kPa, and 14.60±0.87 kPa, respectively. The data strongly demonstrate the "mechanical compensation mechanism" of this invention: although the introduction of decellularized liver matrix (DLM) objectively weakens the dense network of GelMA (leading to a decrease in mechanical strength), the synergistic introduction of CS and CS-GA in the system effectively offsets the strength loss caused by DLM through hydrogen bonding and electrostatic interactions. Ultimately, the compressive modulus of Example 1 (full component) was perfectly maintained at an excellent level comparable to pure GelMA (approximately 14.60 kPa), fully meeting the mechanical support requirements for skin soft tissue repair.

[0047] 3. The microstructure of the hydrogels prepared in Example 1 and Comparative Examples 1-3 was observed using a scanning electron microscope (SEM), and the results are as follows: Figure 5 As shown.

[0048] from Figure 5 As can be seen, Comparative Examples 1-3 and Example 1 all exhibit typical three-dimensional interconnected porous structures. Comparative Example 1 shows uneven pore size distribution, thinner pore walls, and localized structural collapse. After adding CS (Comparative Example 2), the pore structure becomes more uniform, and the pore walls are significantly thickened, demonstrating microscopic evidence of network mechanical stability. After introducing GA (Comparative Example 3), the increased crosslinking density makes the pore wall structure more dense and continuous. It is worth emphasizing that after further adding DLM (Example 1), the inner surface of the pore walls of the material exhibits a moderate roughness and forms a highly interconnected porous topology. This morphology highly replicates the microscopic characteristics of ECM, which not only helps maintain gel permeability but also provides an ideal biomimetic microenvironment for subsequent adhesion, spreading, deep migration of vascular endothelial cells, and nutrient exchange.

[0049] 4. The degradation, water absorption, and water retention properties of the hydrogels prepared in Example 1 and Comparative Examples 1-3 were tested, and the results are as follows: Figure 6 As shown.

[0050] from Figure 6 In vitro degradation experiments of A showed that the residual degradation rates of Comparative Examples 1-3 and Example 1 were 20.61±8.20%, 40.26±6.21%, 31.56±4.27%, and 37.51±3.12%, respectively. Compared with Comparative Example 1, the residual degradation rates of the other groups increased. This is because the physicochemical dual cross-linking between the multiple components of CS, GA, and DLM enhanced the entanglement between molecular chains, effectively resisting hydrolysis. This moderately delayed degradation characteristic can provide a durable physical barrier and structural support for the long-term healing of diabetic chronic wounds. Figure 6 B and Figure 6 As can be seen from Figure C, the average water absorption rate (approximately 80-84%) and average water retention rate (approximately 71-77%) of each group of hydrogels remained at extremely high levels with little difference. This demonstrates that the introduction of CS / GA / DLM not only did not disrupt the excellent hydrophilic network of GelMA, but also ensured that the hydrogel could continuously absorb excess exudate from the wound and maintain the local moisture balance of the wound, creating an excellent moist microenvironment for scarless tissue healing.

[0051] 5. HUVECs (human umbilical vein endothelial cells) were seeded onto the surface of the hydrogels prepared in Example 1 and Comparative Examples 1-3. After culturing for 1, 3, and 7 days, live / dead cell fluorescence staining was performed. The results are as follows: Figure 7 As shown.

[0052] from Figure 7 As can be seen, at all observation time points, Comparative Examples 1-3 and Example 1 all exhibited large areas of dense green fluorescence (live cells), while red fluorescence (dead cells) was minimal or almost invisible. Furthermore, with increasing planting time, the green fluorescence in Comparative Example 1 increased, indicating an increase in the proportion of live cells, meaning the cells were in good natural growth condition. Simultaneously, the proportion of live cells also increased in Example 1 and Comparative Examples 2-3, indicating that the cells had good proliferative capacity in both Comparative Examples 1-3 and Example 1. Therefore, the hydrogel system in Example 1 did not produce significant inhibitory or toxic effects on the cells.

[0053] 6. The effect of the hydrogels prepared in Example 1 and Comparative Examples 1-3 on the proliferation activity of HUVEC cells was quantitatively evaluated using the MTT assay (with the cell activity of the blank culture medium group as 100%). The results are as follows: Figure 8 As shown.

[0054] from Figure 8As can be seen, after one day of seeding on the hydrogels of Comparative Examples 1-3 and Example 1, the HUVEC cell viability was 69.57±11.49%, 94.60±12.40%, 95.22±9.91%, and 130.75±13.00%, respectively. After two days of seeding on the hydrogels of Comparative Examples 1-3 and Example 1, the HUVEC cell viability was 86.97±10.44%, 97.83±12.39%, 107.14±10.12%, and 130.72±26.07%, respectively. After three days of seeding on the hydrogels of Comparative Examples 1-3 and Example 1, the HUVEC cell viability was 94.01±4.545%, 112.79±11.67%, 101.80±6.20%, and 137.44±15.23%, respectively. It is evident that the cell viability on the hydrogel of Example 1 was significantly higher than that on Comparative Examples 1-3. This demonstrates that CS-GA and DLM form an excellent biological synergy. The natural type VI collagen and pro-angiogenic factors preserved in DLM are successfully released, which greatly activates the proliferation and metabolic capacity of vascular endothelial cells, providing a continuous biological driving force for the early reconstruction of vascular networks in wounds.

[0055] In summary, the interpenetrating network antibacterial hydrogel with angiogenesis function provided by this invention (represented by Example 1) achieves excellent mechanical strength, high water absorption and retention rate, and perfect biocompatibility while significantly reversing the cell proliferation disadvantage under high glucose environment through a "structural interpenetration and mechanical compensation" mechanism, thus endowing the material with outstanding ability to promote vascular endothelial cell proliferation. Furthermore, the introduction of CS-GA into the hydrogel network of this invention to construct a durable antibacterial active functional unit endows the modified material with excellent antibacterial properties. Therefore, this invention achieves a multi-dimensional synergy of sustained antibacterial activity, mechanical support, biomimetic microenvironment, and active angiogenesis promotion, possessing extremely high clinical translational value and broad application prospects in the fields of diabetic skin chronic wound repair and soft tissue engineering.

[0056] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An interpenetrating network antibacterial hydrogel with angiogenesis function, characterized in that, The hydrogel is formed by in-situ photocrosslinking of the precursor solution to form a three-dimensional interpenetrating network structure; the precursor solution comprises the following components by mass fraction: gallic acid modified low viscosity chitosan 0.5%~1.5%, decellularized liver matrix 0.5%~1.5%, methacrylic acid gelatin 8%~12%, photoinitiator 0.5%~1.0%, and the balance being phosphate buffered saline; In the preparation of the precursor solution, the pH value of the gallic acid-modified low-viscosity chitosan and decellularized liver matrix is ​​pre-adjusted to 7.0-7.4 before being mixed with other components.

2. The interpenetrating network antibacterial hydrogel with angiogenesis function according to claim 1, characterized in that, The gallic acid-modified low-viscosity chitosan has a molar ratio of chitosan to gallic acid of 1:0.4~0.6, and the chitosan has a viscosity of 10~200 mPa·s at room temperature.

3. The interpenetrating network antibacterial hydrogel with angiogenesis function according to claim 1, characterized in that, The decellularized liver matrix is ​​derived from mammalian liver.

4. The interpenetrating network antibacterial hydrogel with angiogenesis function according to claim 1, characterized in that, The photoinitiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone or lithium phenyl-2,4,6-trimethylbenzoyl phosphate.

5. A method for preparing an interpenetrating network antibacterial hydrogel with angiogenesis function as described in claim 1, characterized in that, Includes the following steps: (1) Using EDC and NHS as activators, gallic acid was grafted onto low-viscosity chitosan, and gallic acid-modified low-viscosity chitosan was obtained by dialysis and freeze drying. Then, it was completely dissolved in acetic acid solution, and the pH value was adjusted to 7.0~7.4 to obtain gallic acid-modified low-viscosity chitosan solution. (2) Grind the decellularized liver tissue into powder, digest it with pepsin in acetic acid solution, and adjust the pH of the solution to 7.0~7.4 after the reaction is completed to obtain a decellularized liver matrix solution; (3) Dissolve gelatin methacrylic acid in phosphate buffered saline, then add photoinitiator, then add gallic acid modified low viscosity chitosan solution obtained in step (1) and decellularized liver matrix solution obtained in step (2), stir evenly to obtain precursor solution; (4) The precursor solution is transferred to a mold and photocrosslinked and cured to obtain the antibacterial hydrogel.

6. The method for preparing the interpenetrating network antibacterial hydrogel with angiogenesis function according to claim 5, characterized in that, In step (1), the volume concentration of the acetic acid solution is 1-5%, and the mass-volume ratio of the gallic acid-modified low-viscosity chitosan to the acetic acid solution is 1-3 g:100 mL.

7. The method for preparing the interpenetrating network antibacterial hydrogel with angiogenesis function according to claim 5, characterized in that, In step (2), the molar concentration of the acetic acid solution is 0.4~0.6 mol / L, and the mass-to-volume ratio of the decellularized liver tissue to the acetic acid solution is 1~3 g:100 mL.

8. The method for preparing the interpenetrating network antibacterial hydrogel with angiogenesis function according to claim 5, characterized in that, The wavelength in the photocrosslinking process is 300~400 nm, the power is 15~50 W, and the irradiation time is 5~6 min.

9. The use of the antibacterial hydrogel as described in claim 1 or the antibacterial hydrogel prepared by any one of claims 5 to 8 in the preparation of materials for repairing diabetic skin tissue.

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