Tannic acid-enhanced anisotropic polyvinyl alcohol hydrogel, and preparation method and application thereof

Abstract

This invention discloses a tannic acid-reinforced anisotropic polyvinyl alcohol hydrogel, its preparation method, and its applications. The preparation method of the hydrogel includes: subjecting the polyvinyl alcohol hydrogel to sequential mechanical stretching, salting-out fixation, freeze-drying, and annealing to construct an anisotropic structure, followed by immersion in a tannic acid solution for reinforcement. The hydrogel exhibits high crystallinity, excellent mechanical properties, and an extremely low equilibrium swelling rate, with a swelling rate of only 4.8% in phosphate buffered saline. The introduction of tannic acid endows the hydrogel with broad-spectrum antibacterial, antioxidant, and anti-inflammatory functions. The anisotropic structure, in conjunction with tannic acid, can significantly promote the directional migration of endothelial cells, angiogenesis, and the orderly regeneration of epithelium and smooth muscle. Animal experimental results show that this hydrogel can effectively reduce the recurrence rate of urethral stricture and achieve scarless functional repair.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a tannic acid-reinforced anisotropic polyvinyl alcohol hydrogel, its preparation method, and its application. Background Technology

[0002] Urethral stricture is a disease characterized by fibrosis and scarring of the urethral mucosa and surrounding corpus cavernosum, as well as tissue contracture. It can lead to segmental narrowing of the urethral lumen, severely affecting urinary function. The incidence is higher in men, with a global prevalence of approximately 0.2% to 0.9%. Common causes include postoperative complications of hypospadias, urethral catheterization or instrumentation, trauma, and transurethral surgery. Currently, there are no effective drug treatments; surgery remains the first-line treatment. Autologous buccal mucosal transplantation is widely used due to its advantages such as easy tissue acquisition, good epithelial barrier function, and strong resistance to mechanical friction, which helps reduce postoperative fibrosis and recurrence of stricture to some extent.

[0003] However, autologous tissue transplantation inevitably involves donor site complications such as bleeding, infection, and functional impairment, and the limited availability of tissue makes it difficult to meet the needs of large-area or complex urethral reconstruction. Therefore, tissue engineering methods are highly anticipated, hoping to replace autologous grafts with biodegradable biomaterials to achieve functional urethral repair. It is important to note, however, that the repair environment for urethral strictures differs fundamentally from that of ordinary skin wounds: the urethra is constantly exposed to the chemical stimulation and flushing of urine, while simultaneously enduring the continuous dynamic mechanical load of expansion and contraction during urination. This places stringent requirements on implant materials, requiring both extremely low swelling rates to avoid secondary obstruction due to water absorption and expansion, and excellent mechanical stability and durability in a moist environment.

[0004] To address the aforementioned physical and mechanical challenges, anisotropic hydrogels have attracted widespread attention in tissue engineering in recent years due to their highly ordered microstructure and superior mechanical properties. Studies have shown that anisotropic structures can mimic the orientation of natural tissues, regulate cell behavior, and promote orderly tissue regeneration, demonstrating application potential in the repair of tissues such as the cornea, skin, muscle, and nerves. However, no systematic research has yet explored the applicability of anisotropic hydrogels in the repair of urethral strictures, and their performance advantages in the complex dynamic mechanical environment of the urethra remain to be fully explored.

[0005] Besides optimizing mechanical properties, imbuing hydrogels with bioactive functions is also a key direction for improving the effectiveness of urethral repair. Tannic acid, as a natural polyphenol compound, possesses multiple bioactivities, including broad-spectrum antibacterial, strong antioxidant, anti-inflammatory, and tissue integration-promoting effects. Introducing tannic acid into the hydrogel network not only enhances the material's mechanical properties and structural stability but also provides a local microenvironment that resists infection, inflammation, and promotes angiogenesis, helping to inhibit excessive postoperative fibrosis and scar formation. Therefore, developing a hydrogel material with anisotropic structure, excellent mechanical properties, low swelling rate, and multiple bioactivities is of great significance for meeting the specific physiological needs of urethral stricture repair and achieving scarless functional reconstruction. Summary of the Invention

[0006] This invention provides a tannic acid-reinforced anisotropic polyvinyl alcohol hydrogel, its preparation method, and its applications. The hydrogel is prepared through mechanical training, salting-out fixation, freeze-drying, annealing, and tannic acid impregnation. This hydrogel possesses anisotropic structure, high crystallinity, low swelling ratio (4.8%), and excellent mechanical properties. It also exhibits antibacterial, anti-inflammatory, and antioxidant functions, synergistically promoting directional migration of endothelial cells and angiogenesis, achieving orderly regeneration of epithelium and smooth muscle, and effectively reducing the recurrence rate of urethral stricture.

[0007] On the one hand, the present invention provides a method for preparing tannic acid-reinforced anisotropic polyvinyl alcohol hydrogel, using the following technical solution: A method for preparing tannic acid-reinforced anisotropic polyvinyl alcohol hydrogel includes the following steps: (1) Polyvinyl alcohol is dissolved in hot water to prepare a polyvinyl alcohol solution, which is then subjected to freeze-thaw cycle treatment to form a polyvinyl alcohol hydrogel; (2) Mechanical stretching training is performed on the polyvinyl alcohol hydrogel obtained in step (1) to orient the polymer chains; (3) The hydrogel trained in step (2) is fixed by salting out while maintaining the stretching state to stabilize the orientation structure; (4) The hydrogel fixed in step (3) is subjected to freeze-drying and annealing treatment in sequence to obtain polyvinyl alcohol hydrogel with an anisotropic structure. (5) The anisotropic polyvinyl alcohol hydrogel obtained in step (4) is immersed in tannic acid solution for reinforcement to obtain tannic acid reinforced anisotropic polyvinyl alcohol hydrogel.

[0008] Preferably, in step (1), the mass concentration of the polyvinyl alcohol solution is 10%; the freeze-thaw cycle treatment is a three-cycle freeze-thaw treatment, each cycle including freezing at -20°C for 9 hours and then thawing at room temperature for 3 hours.

[0009] Preferably, in step (2), the mechanical stretching training is to perform cyclic stretching for 30 minutes within the strain range of 0~200%.

[0010] Preferably, in step (3), the salting-out agent used for salting-out fixation is sodium sulfate solution with a concentration of 1.5 M; the tensile state is maintained at a constant strain of 200% for 24 hours.

[0011] Preferably, in step (4), the annealing temperature is 100°C and the time is 90 minutes.

[0012] Preferably, in step (5), the mass concentration of the tannic acid solution is 1.25% to 5%.

[0013] Preferably, the mass concentration of the tannic acid solution is 2.5%.

[0014] On the other hand, the present invention also provides a tannic acid-reinforced anisotropic polyvinyl alcohol hydrogel, which adopts the following technical solution: A tannic acid-reinforced anisotropic polyvinyl alcohol hydrogel prepared by the above-described method has an anisotropic fiber arrangement structure and a structure in which tannic acid is stably bonded to the polyvinyl alcohol network through hydrogen bonds.

[0015] Preferably, the equilibrium swelling ratio of the hydrogel in phosphate buffered saline is no greater than 4.8%.

[0016] The present invention also provides the application of tannic acid-reinforced anisotropic polyvinyl alcohol hydrogel in the preparation of medical devices for urethral stricture repair.

[0017] In summary, the beneficial effects of the present invention are as follows: This invention utilizes the synergistic effects of mechanical training, salting-out fixation, freeze-drying, and annealing processes to construct an anisotropic polyvinyl alcohol hydrogel with a highly oriented structure, significantly improving the material's crystallinity and wet mechanical stability. In a simulated body fluid environment, the hydrogel's equilibrium swelling rate is only 4.8%, far lower than conventional hydrogel materials, effectively preventing urethral compression and secondary tissue damage caused by excessive water absorption and swelling after implantation. Simultaneously, the introduction of tannic acid, through multiple hydrogen bonds, stably binds to the polymer network, further enhancing the material's mechanical properties, enabling it to maintain good structural integrity and fatigue resistance under repeated dynamic mechanical conditions.

[0018] Furthermore, this invention endows the hydrogel with multiple biological functions, including broad-spectrum antibacterial, strong antioxidant, and anti-inflammatory properties, significantly inhibiting bacterial colonization and inflammatory responses, and reducing the risk of postoperative infection and fibrosis. The anisotropic structure provides physical topological guidance for the directional alignment of endothelial cells, synergistically upregulating ITGA2 and VEGFA expression with tannins, promoting directional cell migration and vascular network formation, thereby providing nutritional support for the orderly regeneration of epithelial and smooth muscle cells. Animal experiments showed that the urethral obstruction rate after repair with this hydrogel was only 12.6%, achieving simultaneous regeneration of epithelium, blood vessels, and smooth muscle, and achieving significant therapeutic effects of scarless functional repair. Attached Figure Description

[0019] Figure 1 A schematic diagram illustrating the synthesis and urethral reconstruction application of APVA / TA hydrogel; Figure 2 The composition, structure, and thermodynamic characterization of the hydrogel; in, Figure 2 Image A is a macroscopic photograph of the flexibility of APVA / TA2.5 hydrogel; Figure 2 B in the figure shows the Fourier transform infrared (FTIR) spectra of different hydrogel samples; Figure 2 In the middle, C represents the X-ray diffraction (XRD) patterns of different hydrogel samples; Figure 2 D represents the thermogravimetric (TG) curves of the hydrogel and pure TA; Figure 2 E represents the thermogravimetric differential (DTG) curves of the hydrogel and pure TA; Figure 2 F in the middle represents the differential scanning calorimetry (DSC) thermograms of different hydrogel samples; Figure 2 G represents a quantitative comparison of the crystallinity of different hydrogel samples; Figure 2 In this context, H represents the equilibrium water content of different hydrogel samples; Figure 2 Figure I shows the swelling curves of four hydrogels in PBS at 37°C; Figure 3 Characterization of the microstructure and mechanical properties of hydrogels; in, Figure 3 Image A shows scanning electron microscope (SEM) images of PVA, PVA / TA2.5, APVA, and APVA / TA2.5 hydrogels. Figure 3 Image B shows the SEM images of the cross-section and longitudinal section of the APVA / TA2.5 hydrogel. Figure 3In the middle, C represents a three-dimensional confocal microscope image of PVA and APVA / TA2.5 hydrogels; Figure 3 In the middle, D represents atomic force microscopy (AFM) phase images of PVA and APVA / TA2.5 hydrogels; Figure 3 Image E is a macroscopic photograph of the load-bearing capacity of APVA / TA2.5 hydrogel; Figure 3 F represents the tensile stress-strain curves of different hydrogel samples; Figure 3 G represents the Young's modulus of different hydrogel samples; Figure 3 In the figure, H represents the toughness of different hydrogel samples; Figure 3 In Figure I, the continuous cyclic loading-unloading curve of APVA / TA2.5 hydrogel under increasing strain is shown. Figure 3 J represents the cyclic loading-unloading curves of APVA / TA2.5 hydrogel under constant strain; Figure 3 K represents the reactive oxygen species (ROS) scavenging efficiency of different hydrogel samples; Figure 4 The effect of tannic acid (TA) on lipopolysaccharide (LPS)-induced inflammatory response; in, Figure 4 In the figure, A represents the relative mRNA expression level of cyclooxygenase-2 (COX-2) in HUVECs; Figure 4 B represents the relative mRNA expression level of interleukin-6 (IL-6) in HUVECs; Figure 4 C represents the relative mRNA expression level of inducible nitric oxide synthase (iNOS) in HUVECs; Figure 4 D represents the relative mRNA expression level of tumor necrosis factor-α (TNFα) in HUVECs; Figure 4 E in the middle represents the protein expression bands of iNOS, COX-2, NF-κB, and TNFα in HUVECs; Figure 4 Quantitative analysis of the relative protein expression levels of iNOS, COX-2, NF-κB and TNFα in the middle F; Figure 4 Image G in the middle is an immunofluorescence staining image of COX-2 and NF-κB in HUVECs; Figure 5 This demonstrates the synergistic effect of anisotropic structures and TA on endothelial cell angiogenesis and migration. in, Figure 5 A represents a volcano plot of differentially expressed genes between the PVA / TA2.5 group and the APVA / TA2.5 group; Figure 5 B represents the gene ontology (GO) analysis of HUVEC biological processes; Figure 5 The middle section (C) shows the enrichment map of angiogenesis pathways between the PVA / TA2.5 group and the APVA / TA2.5 group; Figure 5 The middle section (D) shows the enrichment map of the epithelial-mesenchymal transition pathway between the PVA / TA2.5 group and the APVA / TA2.5 group; Figure 5 Image E in the middle represents scratch healing experiment images and quantitative analysis of cell migration rate in different treatment groups; Figure 5 Image F in the middle shows experimental images of lumen formation and quantitative analysis of relative tube length for different treatment groups; Figure 5 G represents Transwell migration assay images and quantitative analysis of the number of migrating cells in different treatment groups; Figure 5 In the middle H, the expression bands of ITGA2 and VEGFA proteins in different treatment groups and the quantitative analysis of their relative expression levels are shown. Figure 6 Evaluation of the antibacterial properties and tissue compatibility of APVA / TA hydrogel; in, Figure 6 Images of inhibition zones of AAPVA / TA hydrogel against Escherichia coli and Staphylococcus aureus; Figure 6 In Figure B, the diameter of the inhibition zone of APVA / TA hydrogel against Escherichia coli and Staphylococcus aureus is quantitatively analyzed. Figure 6 In the middle (C), there are live fluorescence images of HUVEC cells on day 1 and day 3 after co-culturing with different concentrations of APVA / TA hydrogels. Figure 6 D represents the cell viability on day 1 and day 3 of co-culture with different concentrations of APVA / TA hydrogels; Figure 6 The middle E represents the H&E staining images of the major organs (heart, liver, spleen, lung, and kidney) in the control group and the APVA / TA2.5 group; Figure 6 In the middle F group, the serum biochemical levels of AST, ALT, and ALP were compared between the control group and the APVA / TA2.5 group. Figure 7 To evaluate the in vivo repair effect of hydrogel in a rabbit urethral defect model; in, Figure 7Image A shows a macroscopic surgical image of four types of hydrogels used for in situ repair of urethral defects; Figure 7 Image B shows urethrography images of different hydrogel treatment groups 4 weeks post-surgery. Figure 7 C represents the quantitative analysis of urethral obstruction rate in different hydrogel treatment groups; Figure 7 Middle D shows H&E staining images of urethral tissue from different hydrogel treatment groups; Figure 7 Image E shows Masson's trichrome staining of urethral tissue from different hydrogel treatment groups. Figure 8 Immunofluorescence analysis to promote scarless urethral repair using hydrogels; in, Figure 8 Image A shows immunofluorescence staining images of pan-CK, CD31, and α-SMA in urethral tissues from different hydrogel treatment groups; Figure 8 In Figure B, the relative fluorescence intensity of pan-CK in different hydrogel treatment groups is quantitatively analyzed. Figure 8 In the middle, C represents the quantitative analysis of the relative fluorescence intensity of CD31 in different hydrogel treatment groups; Figure 8 D represents the quantitative analysis of the relative fluorescence intensity of α-SMA in different hydrogel treatment groups. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to the embodiments.

[0021] Example Example 1 The specific steps for preparing a PVA hydrogel are as follows: A predetermined amount of polyvinyl alcohol (PVA) powder was added to deionized water heated to 90°C. The mixture was stirred vigorously until a clear, transparent 10 wt% PVA solution was obtained. The solution was poured into a mold and subjected to three freeze-thaw cycles. Each cycle consisted of freezing at -20°C for 9 hours, followed by thawing at room temperature for 3 hours, ultimately forming a PVA hydrogel.

[0022] Example 2 The specific steps for preparing an APVA hydrogel are as follows: The PVA hydrogel prepared in Example 1 was immersed in a water bath and cyclically stretched for 30 minutes at a deformation rate of 40% / s within a strain range of 0 to 200%. Immediately after training, the water bath was replaced with a 1.5M sodium sulfate (Na2SO4) solution, and the hydrogel was held at a constant strain of 200% for 24 hours to induce salting-out fixation and polymer chain orientation. The oriented hydrogel was then freeze-dried. The freeze-dried sample was annealed at 100°C for 90 minutes to improve crystallinity and stabilize the microstructure.

[0023] Example 3 The specific steps for preparing an APVA / TA2.5 hydrogel are as follows: The PVA hydrogel prepared in Example 1 was immersed in a water bath and cyclically stretched for 30 minutes at a deformation rate of 40% / s within a strain range of 0 to 200% using a mechanical stretcher. Immediately after training, the water bath was replaced with a 1.5 M sodium sulfate (Na2SO4) solution, and the hydrogel was held at a constant strain of 200% for 24 hours to induce salting-out fixation and polymer chain orientation. The oriented hydrogel was then rapidly frozen and lyophilized in liquid nitrogen. The lyophilized sample was annealed at 100°C for 90 minutes to improve crystallinity and stabilize the microstructure. Finally, the annealed anisotropic APVA hydrogel was immersed in a 2.5 wt% TA solution to obtain an APVA / TA2.5 hydrogel. Figure 1 The diagram shown illustrates the synthesis of APVA / TA hydrogel and its application in urethral reconstruction.

[0024] Example 4 The preparation of an APVA / TA1.25 hydrogel differs from that in Example 3 in that the 2.5 wt% TA solution is replaced with a 1.25 wt% TA solution, and the resulting hydrogel is designated as APVA / TA1.25. The remaining steps are the same as in Example 3.

[0025] Example 5 The preparation of an APVA / TA5 hydrogel differs from that in Example 3 in that the 2.5 wt% TA solution is replaced with a 5 wt% TA solution, and the resulting hydrogel is designated as APVA / TA5. The remaining steps are the same as in Example 3.

[0026] Comparative Example Comparative Example 1 The specific steps for preparing a PVA / TA1.25 hydrogel are as follows: The PVA hydrogels prepared in Example 1 were immersed in a 1.25 wt% tannic acid (TA) aqueous solution for 2 hours. Subsequently, the hydrogels were transferred to excess deionized water and soaked frequently with water changes to remove unbound TA, yielding the final PVA / TA1.25 hydrogels.

[0027] Comparative Example 2 The preparation of a PVA / TA2.5 hydrogel differs from Comparative Example 1 in that the concentration of the tannic acid (TA) aqueous solution is changed from 1.25 wt% to 2.5 wt%, and the resulting hydrogel is denoted as PVA / TA2.5. The remaining steps are the same as those in Comparative Example 1.

[0028] Comparative Example 3 The preparation of a PVA / TA5 hydrogel differs from Comparative Example 1 in that the concentration of the tannic acid (TA) aqueous solution is changed from 1.25 wt% to 5 wt%, and the resulting hydrogel is denoted as PVA / TA5. The remaining steps are the same as those in Comparative Example 1.

[0029] Test case Test Example 1 The chemical structure, crystallinity, thermodynamic properties, and swelling behavior of the hydrogels prepared in Examples 1-3 and Comparative Example 2 were comprehensively evaluated. The chemical structure and interactions were characterized using Fourier transform infrared spectroscopy (FTIR, ThermoFisher Scientific Nicolet iS20); the crystal structure was characterized using X-ray diffraction (XRD, RigakuSmartLab SE); and the thermal stability and crystallinity were analyzed using thermogravimetric analysis and differential scanning calorimetry (TG-DSC, Netzsch STA 409PC) on a simultaneous thermal analyzer. The crystallinity was calculated based on the DSC data, determining the enthalpy of fusion (ΔH) by integrating the melting endothermic peak of the PVA crystalline phase, using the formula: X dry = ΔH / ΔH 100% (ΔH) 100% (Take 138.6 J / g) Moisture content and swelling rate were determined by gravimetric method.

[0030] like Figure 2 As shown in Figure A, macroscopic observation confirms that this hydrogel possesses excellent flexibility. Figure 2 As shown in B, the characteristic broad peak of the OH stretching vibration starts from 3486 cm⁻¹. -1 Gradually moved to 3204 cm -1 At lower wavenumbers. This pronounced redshift indicates a significant increase in hydrogen bonding, attributed to the formation of the oriented structure and the successful grafting of tannins. Furthermore, at 1710 cm⁻¹... -1 1607 cm -1 and 755 cm -1 New characteristic peaks appeared at the point, corresponding to C=O stretching vibration, aromatic C=C stretching vibration and OH in-plane bending vibration, respectively, providing direct evidence for the introduction of tannic acid (TA) into the polymer network. Figure 2The X-ray diffraction (XRD) pattern in C shows that the diffraction peaks of the original polyvinyl alcohol (PVA) are relatively broad. In contrast, the peaks of the other three groups of hydrogels are significantly sharper and stronger. This change indicates that both anisotropic processing and the introduction of TA contribute to improving the overall crystallinity of the hydrogel matrix. Figure 2 The TG curves in D show that the decomposition temperature of TA itself is lower than that of PVA. Therefore, the hydrogel containing TA begins to show significant mass loss earlier. Figure 2 The thermogravimetric derivative curve in E shows two distinct peaks, corresponding to the individual thermal decomposition stages of TA and PVA, respectively, confirming the composite nature of the material. Figure 2 The DSC thermal analysis plot in F shows that the melting peak of the PVA group appears at the lowest temperature. The melting peaks of the other three groups shift to higher temperatures. This shift indicates that stronger and more numerous hydrogen bonds have formed in these networks, requiring additional energy to dissociate upon melting. Figure 2 The quantitative analysis of crystallinity shown in G is consistent with the XRD observation results. The PVA sample has the lowest crystallinity. The crystallinity of the intermediate group increases, while the APVA / TA2.5 group reaches the maximum crystallinity, indicating a higher content of crystalline domains. The hydration properties of hydrogels are closely related to their crystalline structure. Figure 2 The water content measurements shown in Figure H indicate an inverse relationship between water content and crystallinity. The APVA / TA2.5 hydrogel with the highest crystallinity exhibited the lowest equilibrium water content. Finally, the swelling behavior at different times was investigated. Figure 2 As shown in Figure I, all groups reached swelling equilibrium within 36 hours. The maximum swelling rate of the original PVA hydrogel was 21.0%. In stark contrast, the swelling rate of the APVA / TA2.5 group was significantly lower, at only 4.8%, highlighting their excellent anti-swelling ability in the PBS environment.

[0031] Test Example 2 The microstructure, mechanical properties, and antioxidant capacity of the hydrogels prepared in Examples 1-5 and Comparative Example 2 were examined. Microstructure and structure were characterized using scanning electron microscopy (SEM), three-dimensional confocal laser scanning microscopy (3D CLSM), and atomic force microscopy (AFM); mechanical properties were tested using a universal testing machine equipped with a 100 N load sensor; and antioxidant properties were determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical method.

[0032] Figure 3Scanning electron microscopy (SEM) images in Figure A show that the four hydrogels (PVA, APVA, PVA / TA2.5, and APVA / TA2.5) have significantly different surface microstructures. The original polyvinyl alcohol (PVA) exhibits an irregular and isotropic porous network structure. In contrast, the mechanically trained polyvinyl alcohol (APVA) and APVA / TA2.5 hydrogels display clear and well-organized fibrous structures, confirming the successful construction of anisotropic structures through mechanical training and salting-out fixation. The addition of tannic acid (TA) makes the microstructure denser without disrupting the basic orientation. Cross-sectional and longitudinal SEM images of the APVA / TA2.5 hydrogel are also shown. Figure 3 B) The neatly arranged internal structure was displayed from different angles, providing further evidence. This anisotropic structure was confirmed at different scales using complementary techniques. Figure 3 CD displays three-dimensional confocal laser scanning microscopy and atomic force microscopy images of fluorescently labeled PVA networks. The PVA hydrogel exhibits a randomly arranged polymer network, while the APVA / TA2.5 hydrogel shows uniformly arranged fiber bundles along the processing direction, consistent with SEM observations. Figure 3 As shown in Figure E, the APVA / TA2.5 hydrogel can lift an object with a weight approximately 4000 times its own mass, demonstrating its excellent load-bearing capacity. Meanwhile, Figure 3 The tensile stress-strain curves of FH show that the construction of the anisotropic structure primarily improved the tensile strength and Young's modulus of the material. The subsequent introduction of TA further significantly improved the toughness of the material. This enhancement exhibits a concentration-dependent trend; that is, both modulus and strength gradually increase with increasing TA content. Figure 3 I shows a continuous loading-unloading cycle with gradually increasing maximum strain. The increase in hysteresis area indicates continuous energy dissipation, likely due to the reversible breaking of sacrificial hydrogen bonds and the gradual unfolding of semi-crystalline domains. Figure 3 As shown in Figure J, the stress-strain curves closely overlap during multiple cycles, and the mechanical output remains stable, confirming its excellent durability and elastic recovery capability under repeated deformation. Figure 3 The results in K showed that the scavenging efficiency of the PVA hydrogel was similar to that of the control group. In stark contrast, all TA-loaded hydrogels exhibited strong reactive oxygen species scavenging activity, which is attributed to the abundance of phenolic hydroxyl groups in the TA molecule.

[0033] Test Example 3 The anti-inflammatory effects of the hydrogels prepared in Example 1 and Comparative Examples 1-3 were analyzed, and the specific steps are as follows: S1. After resuscitation, human umbilical vein endothelial cells (HUVECs) were cultured in endothelial cell culture medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and routinely passaged in a cell culture incubator at 37°C and 5% CO2. Once the cells reached the logarithmic growth phase, they were digested with 0.25% trypsin-EDTA to collect the cells and seeded at an appropriate density in 6-well plates. The cells were then cultured until confluence reached 70%–80% before further processing.

[0034] S2. Cells were divided into multiple experimental groups: control group (normal culture), PVA group (only PVA hydrogel extract was added), PVA+LPS group (PVA hydrogel extract was added and LPS stimulation was given), and PVA / TA+LPS group (PVA / TA hydrogel extracts of different concentrations were added and LPS stimulation was given). The PVA / TA hydrogel extract was set with three concentration gradients: 1.25%, 2.5% and 5%.

[0035] First, except for the control group, cells in each group were stimulated with lipopolysaccharide (LPS, final concentration 1 μg / mL) for 24 hours to induce an inflammatory response. After 24 hours, the original culture medium was discarded and replaced with fresh culture medium containing the corresponding hydrogel extract, and cultured for another 24 hours.

[0036] S3. After treatment, the culture medium was discarded, and the cells were washed twice with pre-cooled phosphate-buffered saline (PBS). 1 mL of TRIzol reagent was added to each well to lyse the cells and extract total RNA. After determining the RNA concentration and purity, reverse transcription was performed using a one-step RT-qPCR probe kit to synthesize cDNA. Subsequently, mRNA quantification was performed using ChamQ Blue Universal SYBR qPCR Master Mix, and detection was performed on an ABI QuantStudio 6K Flex real-time quantitative PCR system. Inflammation-related genes detected included cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), and tumor necrosis factor-α (TNFα), with β-actin as an internal control gene. 2^ The relative expression levels of each gene were calculated using the ΔΔCt method.

[0037] S4. After treatment, discard the culture medium and wash the cells twice with PBS. Add RIPA lysis buffer containing protease inhibitors to each well, lyse on ice for 30 minutes, collect the lysis buffer, centrifuge at 12,000 rpm for 15 minutes at 4°C, and collect the supernatant. After determining the protein concentration using the BCA method, take an equal volume of protein sample for 10% Tris / SDS polyacrylamide gel electrophoresis (SDS-PAGE) separation, and then transfer the protein to a polyvinylidene fluoride (PVDF) membrane. Block with 5% skim milk powder at room temperature for 1 hour, add primary antibodies against iNOS, COX-2, nuclear factor-κB (NF-κB), TNFα, and the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and incubate overnight at 4°C. The next day, wash the membrane three times with TBST, add horseradish peroxidase-labeled secondary antibody, incubate at room temperature for 1 hour, develop the membrane using enhanced chemiluminescence (ECL) method, acquire images using a gel imaging system, and perform quantitative analysis of the band gray values ​​using ImageJ software.

[0038] S5. HUVEC cells were seeded in 24-well plates with cell culture slides and subjected to LPS induction and TA treatment as described above. After treatment, the culture medium was discarded, and the cells were washed three times with PBS, fixed with 4% paraformaldehyde at room temperature for 15 minutes, washed with PBS, and permeabilized with 0.1% Triton X-100 for 10 minutes. The cells were blocked with 10% goat serum at room temperature for 30 minutes, and then COX-2 and NF-κB primary antibodies were added, and incubated overnight at 4°C. The next day, the cells were washed three times with PBS, and fluorescently labeled secondary antibodies (such as Alexa Fluor 488 or 594) were added. The cells were incubated at room temperature in the dark for 1 hour, and then the nuclei were stained with DAPI for 5 minutes. After mounting, the cells were observed and images were acquired under a fluorescence microscope.

[0039] S6. All experiments were performed at least three independent biological replicates. ImageJ software was used for image analysis, and GraphPad Prism software was used for statistical analysis. Data are expressed as mean ± standard deviation (SD). Differences between groups were compared using one-way ANOVA and unpaired t-tests for significance. p <0.05 indicates a statistically significant difference.

[0040] Lipopolysaccharide (LPS) stimulation significantly upregulated the mRNA levels of key inflammatory cytokines (including COX-2, IL-6, iNOS, and TNFα) compared to the control group. p <0.001). When cells were co-treated with increasing concentrations of PVA / TA (1.25%, 2.5%, and 5%), a dose-dependent attenuation of the LPS-induced inflammatory response was observed, such as... Figure 4 As shown in AD.

[0041] Further validation at the protein level confirmed the anti-inflammatory activity of TA. Figure 4 As shown in EF, compared with the control group, LPS stimulation significantly enhanced the expression of iNOS, COX-2, NF-κB and TNFα proteins. p <0.001). However, co-treatment with PVA / TA effectively reversed these increases in a concentration-dependent manner. Compared with the PVA+LPS group, 2.5% PVA / TA reduced the protein expression levels of iNOS, COX-2, NF-κB, and TNFα by 33%, 5%, 58%, and 62%, respectively. p <0.001), indicating that TA can interfere with upstream inflammatory signaling pathways. Protein quantification using GAPDH as an internal control further supported these results, confirming the consistency of the observed effects. Figure 4 F). Consistent with these findings, immunofluorescence (IF) staining showed that the expression of COX-2 and NF-κB in HUVECs was significantly reduced after TA treatment. Figure 4 G). Overall, these results indicate that TA effectively reduces the inflammatory response by regulating the transcriptional and translational expression of key inflammatory mediators.

[0042] Test Example 4 The hydrogel extracts prepared in Examples 1-3 and Comparative Example 2 were subjected to experiments on their angiogenesis activity in endothelial cells. The specific steps are as follows: 1. After resuscitation, human umbilical vein endothelial cells (HUVECs) were cultured in endothelial cell culture medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and routinely passaged in a 37°C, 5% CO2 incubator. The experiment was divided into four groups: PVA group (isotropic PVA hydrogel extract), PVA / TA2.5 group (isotropic PVA / TA hydrogel extract, TA concentration 2.5%), APVA group (anisotropic PVA hydrogel extract), and APVA / TA2.5 group (anisotropic PVA / TA hydrogel extract, TA concentration 2.5%). The hydrogel extracts for each group were prepared using the same method: the hydrogels were immersed in serum-free culture medium, incubated at 37°C for 24 hours, and the supernatant was collected, filtered, sterilized, and then used for later use.

[0043] 2. HUVEC cells were co-cultured with PVA / TA2.5 and APVA / TA2.5 hydrogel extracts for 48 hours, respectively. The culture medium was then discarded, and the cells were washed twice with PBS. 1 mL of TRIzol reagent was added to each well to lyse the cells and extract total RNA. RNA integrity was assessed using agarose gel electrophoresis and an Agilent 2100 bioanalyzer. Qualified RNA samples were used for transcriptome sequencing. After constructing cDNA libraries, sequencing was performed on a high-throughput sequencing platform. Sequencing data underwent quality control, alignment, and quantitative analysis to screen for differentially expressed genes (fold change > 2). p <0.05). Gene expression differences between the PVA / TA2.5 group and the APVA / TA2.5 group were analyzed, and gene ontology (GO) enrichment analysis was performed, focusing on biological processes such as "tubule morphogenesis" and "vascular development." Marker pathway analysis was also conducted to examine the enrichment of the "angiogenesis" and "epithelial-mesenchymal transition" pathways.

[0044] 3. Pack HUVEC cells at a density of 5 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 6-well plates and cultured until cell confluence reached ≥90%. A uniform straight line was drawn on the cell monolayer using a 20 μL sterile pipette tip, and detached cells were gently washed with PBS. Serum-free medium treated with hydrogel extracts for each group (PVA group, PVA / TA2.5 group, APVA group, APVA / TA2.5 group) was then added, and the cells were incubated at 37°C in a 5% CO2 incubator. Images of the scratched area were taken using an inverted microscope at 0 and 24 hours, and the scratch healing area was measured using ImageJ software to calculate cell migration rate. The migration rate was calculated using the following formula: (Area of ​​scratches after 0 hours) (Area of ​​scratches in 24 hours) / Area of ​​scratches in 0 hours × 100% 4. Add 50 μL of Matrigel to each well of a 96-well plate beforehand and incubate at 37°C for 30 minutes to allow it to polymerize and form a gel. After starving HUVEC cells in serum-free medium for 24 hours, digest them with 0.05% trypsin-EDTA to collect the cells and resuspend them in serum-free medium treated with the hydrogel extracts of each group. Adjust the cell density to 2 × 10⁶ cells / well. 5 Add 100 μL of cell suspension per well (i.e., 2 × 10⁶ cells / mL) to each well. 4 Cells were incubated at 37°C in a 5% CO2 incubator for 12 hours. After incubation, images of lumen formation in each well were taken using an inverted microscope, and the total tube length and number of branch points were analyzed using ImageJ software for quantitative comparison based on relative tube length ratios.

[0045] 5. HUVEC cells were starved in serum-free medium for 24 hours, then digested with 0.05% trypsin-EDTA to collect the cells, and resuspended in serum-free medium treated with hydrogel extracts from each group. The cell density was adjusted to 2 × 10⁻⁶ cells / year. 5 Cells / mL. Transwell chambers (8 μm pores) were pre-coated with 60 μL of matrix gel and incubated at 37°C for 1–3 hours. 100 μL of cell suspension (2 × 10⁻⁶ cells / mL) was then used. 4 Add 1000 μL of complete culture medium containing 10% FBS to the upper chamber and 1000 μL of complete culture medium to the lower chamber as a chemotactic agent. Incubate at 37°C and 5% CO2 for 12 hours. Remove the chamber, gently wipe away any unmigrated cells from the upper chamber with a cotton swab, fix with 4% paraformaldehyde for 15 minutes, stain with 0.1% crystal violet for 20 minutes, wash with PBS, and photograph under an inverted microscope. Randomly select 5 fields of view to count the number of migrating cells.

[0046] 6. After co-culturing HUVEC cells with hydrogel extracts from each group for 48 hours, the culture medium was discarded, and the cells were washed twice with PBS. RIPA lysis buffer containing a protease inhibitor was added to each well, and the cells were lysed on ice for 30 minutes. The lysate was collected and centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant was collected. Protein concentration was determined using the BCA method, and equal volumes of protein samples were separated by SDS-PAGE electrophoresis, followed by transfer to PVDF membranes. The membranes were blocked with 5% skim milk powder at room temperature for 1 hour, and primary antibodies against integrin α2 (ITGA2) and vascular endothelial growth factor A (VEGFA) were added, respectively, and incubated overnight at 4°C. The next day, the membranes were washed three times with TBST, and horseradish peroxidase-labeled secondary antibody was added. The membranes were incubated at room temperature for 1 hour, and after washing, enhanced chemiluminescence (ECL) was used for imaging, and images were acquired using a gel imaging system. GAPDH was used as an internal control, and ImageJ software was used to quantitatively analyze the band gray values ​​and calculate the relative protein expression levels of ITGA2 and VEGFA in each group.

[0047] Figure 5 A multidimensional experimental system revealed the molecular mechanism by which anisotropic structures and tannins synergistically enhance endothelial cell angiogenesis and migration. Transcriptome analysis showed that compared with the PVA / TA2.5 group, 2286 genes were significantly upregulated and 1110 genes were downregulated in the APVA / TA2.5 group. Figure 5 A); GO biological process analysis showed significant enrichment in pathways such as "tubule morphogenesis" and "vascular development" (A); Figure 5 B), marker pathway analysis further confirmed that the "angiogenesis" and "epithelial-mesenchymal transition" pathways were significantly activated in the composite hydrogel group. Figure 5CD). Functional experimental results showed that the scratch healing rate of the APVA / TA2.5 group was approximately 48.7% and 35.5% higher than that of the PVA / TA2.5 group and the APVA group, respectively. Figure 5 E), the relative tube length increased by 1.4 times in the lumen formation experiment ( Figure 5 F), the cell penetration ability was enhanced by 3.69 times in the Transwell migration assay (F). Figure 5 G). Western blot analysis further revealed that the expression levels of ITGA2 and VEGFA proteins were significantly upregulated in the APVA / TA2.5 group ( Figure 5 H), providing a molecular basis for integrin-mediated enhanced cell adhesion and angiogenesis signaling. Based on the above results, Figure 5 This study confirmed that anisotropic structures and tannins have a synergistic effect in promoting directional migration of endothelial cells, lumen formation, and vascular network construction.

[0048] Test Example 5 The antibacterial properties and tissue compatibility of the hydrogels prepared in Examples 2-5 were evaluated, and the specific steps are as follows: S1. Collect E. coli ( Escherichia coli ) and Staphylococcus aureus ( Staphylococcus aureus The standard strains were inoculated into lysate (LB) medium and cultured in a shaker at 37°C until the logarithmic growth phase. The bacterial concentration was then adjusted to 1×10⁻⁶ with sterile physiological saline. 8 CFU / mL.

[0049] Pour 20 mL of sterile LB agar medium into a 90 mm diameter sterile petri dish and allow it to solidify at room temperature. Spread 0.1 mL of a bacterial suspension of adjusted concentration evenly onto the agar surface. Use a sterile punch to create 3 mm wells in the agar. Add different concentrations (1.25%, 2.5%, 5%) of APVA / TA hydrogel extract to each well, using sterile physiological saline as a negative control. Incubate the petri dishes at 37°C for 24 hours. After incubation, photograph the petri dishes with a digital camera, measure the diameter of the inhibition zone around each well using ImageJ software, and perform quantitative analysis using GraphPad Prism software.

[0050] S2, human umbilical vein endothelial cells (HUVECs) were introduced into a culture medium of 2 × 10⁶ cells per well. 4 Cells were seeded at a density of 1,000 cells per well in 24-well plates and incubated at 37°C in a 5% CO2 incubator for 24 hours. Subsequently, different concentrations (1.25%, 2.5%, and 5%) of APVA / TA hydrogel extract were added, with untreated cells serving as the control group. Each group had three replicates.

[0051] On days 1 and 3 post-treatment, the original culture medium was discarded, and the cells were gently washed twice with phosphate-buffered saline (PBS). Each well was then incubated with PBS containing Calcein-AM (final concentration 2 μM) for 15–30 minutes at 37°C in the dark. After incubation, the cells were washed once with PBS, and images were immediately observed and acquired under an inverted fluorescence microscope at an excitation wavelength of 488 nm and an emission wavelength of 516 nm.

[0052] ImageJ software was used to perform quantitative analysis of fluorescence images, and the fluorescence intensity of live cells or the number of live cells in each field of view was statistically analyzed. Cell viability was calculated as the ratio of fluorescence intensity (or cell number ratio) of the treatment group to the control group, and the results are expressed as mean ± standard deviation.

[0053] S3. Male C57BL / 6 mice (7 weeks old, weighing 25-30 grams) were randomly divided into a control group (sham surgery) and an APVA / TA2.5 hydrogel implantation group, with 5 mice in each group. Mice were anesthetized with isoflurane inhalation. After skin preparation and disinfection of the back, a longitudinal incision of approximately 1 cm was made, and subcutaneous tissue was bluntly dissected to form a subcutaneous cyst. A pre-sterilized 1 cm × 1 cm × 0.2 cm APVA / TA2.5 hydrogel was implanted into the subcutaneous cyst, and the incision was sutured. The control group underwent only open suturing without implantation. Mice were fed routinely for 28 days post-surgery.

[0054] On day 28 post-implantation, mice were euthanized after deep anesthesia with isoflurane, and major organs (heart, liver, spleen, lung, and kidney) were dissected and collected. Tissues from each organ were fixed in 4% paraformaldehyde for 24 hours, dehydrated with graded ethanol, embedded in paraffin, sectioned (5 μm thick), and stained with hematoxylin and eosin (H&E). After mounting, the tissue morphology and structure were observed under a light microscope to assess for tissue damage, inflammatory cell infiltration, or pathological changes.

[0055] On day 28 post-implantation, whole blood was collected via cardiac puncture before sacrifice. The blood was placed in a blood collection tube without anticoagulant, allowed to stand at room temperature for 30 minutes, and then centrifuged at 3,000×g for 15 minutes at 4°C to separate serum. The supernatant serum was collected, and the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were measured using an enzyme-linked immunosorbent assay (ELISA) kit, strictly following the kit instructions. Each sample was prepared in triplicate, and absorbance values ​​were read using a microplate reader. The activity levels of each enzyme were calculated based on a standard curve.

[0056] Figure 6The antibacterial properties, cell compatibility, and in vivo tissue compatibility of APVA / TA hydrogel were systematically evaluated, revealing its overall safety as a urethral repair material. The inhibition zone assay results showed that APVA / TA hydrogel exhibited concentration-dependent antibacterial activity against both *Escherichia coli* and *Staphylococcus aureus*, with the APVA / TA5 group showing the largest inhibition zone diameters, reaching 15.8 ± 0.5 mm and 14.7 ± 0.3 mm, respectively. Figure 6 (AB), confirming that tannic acid imparts broad-spectrum antibacterial ability to the material. Cell compatibility assessment showed that when the tannic acid concentration was ≤2.5%, the hydrogel had no significant inhibitory effect on HUVEC cell proliferation, and live cell staining on days 1 and 3 showed good cell viability. Figure 6 CD), defining the optimal balance concentration window between antibacterial efficacy and biocompatibility as 2.5%. In vivo tissue compatibility experiments further validated the biocompatibility of the APVA / TA2.5 hydrogel: H&E staining of major organs (heart, liver, spleen, lung, and kidney) showed no significant tissue damage or pathological changes. Figure 6 E), the levels of liver function biochemical indicators (AST, ALT, ALP) were comparable to those in the control group, with no significant differences. Figure 6 F). Comprehensive Figure 6 The results from AF show that APVA / TA2.5 hydrogel, while ensuring excellent antibacterial activity, also exhibits good cell compatibility and in vivo tissue safety, laying a solid safety foundation for its subsequent application in urethral reconstruction.

[0057] Test Example 6 The in vivo repair effects of the hydrogels prepared in Examples 1-3 and Comparative Example 2 in a rabbit urethral defect model were evaluated, and the specific steps are as follows: S1. Twenty male New Zealand White rabbits, weighing 3.0-3.5 kg, were selected. The animals were randomly divided into four groups of five each using a random number table: PVA group (isotropic PVA hydrogel), PVA / TA2.5 group (isotropic PVA / TA hydrogel, TA concentration 2.5%), APVA group (anisotropic PVA hydrogel), and APVA / TA2.5 group (anisotropic PVA / TA hydrogel, TA concentration 2.5%). All animals were housed individually in a temperature- and humidity-controlled environment with a 12-hour light-dark cycle and free access to water and food.

[0058] S2. The rabbit was fasted for 12 hours preoperatively, but allowed free access to water. After isoflurane inhalation anesthesia, the rabbit was fixed in a supine position. Routine skin preparation, disinfection, and draping were performed. The skin was incised along the midline of the ventral urethra, and the urethral mucosa was exposed layer by layer. A mucosal defect of approximately 1.0 cm × 0.5 cm was created on the ventral side of the urethra using a scalpel, reaching the submucosa, simulating a urethral stricture defect model. A sterile hydrogel of the appropriate group (approximately 1.0 cm × 0.5 cm × 0.2 cm) was applied to the defect, and the hydrogel was fixed to the wound edge with 6 / 0 absorbable sutures, ensuring a tight fit between the material and the wound surface. Postoperatively, an 8Fr urethral catheter was inserted to maintain urethral patency, and the skin incision was sutured.

[0059] S3. The animal was fed routinely for 4 weeks post-surgery, during which time its urination and mental state were observed. Four weeks post-surgery, retrograde urethrography was performed under general anesthesia. The rabbit was placed in a supine position, and a contrast agent prepared by mixing iodine and physiological saline in a 1:1 ratio was slowly injected into the urethra through an 8Fr catheter. Real-time urethrography images were acquired using an image intensifier to observe the patency of the urethral lumen and the location of any stenosis. The images were saved digitally. Figure 7 B).

[0060] S4. Two independent observers performed blinded analysis of the urethrography images, measuring the minimum internal diameter of the urethral stricture and comparing it with the normal urethral diameter. The obstruction rate for each group was calculated based on the degree of stricture. The obstruction rate calculation formula is: (Normal urethral diameter) Minimum internal diameter of the stenotic site / Normal internal diameter of the urethra × 100%. An obstruction rate ≥50% is defined as significant stenosis. The obstruction rate for each group is statistically analyzed. Figure 7 C).

[0061] S5. After urethrography, the animal was euthanized with an overdose of anesthetic, and the urethral tissue was quickly dissected and separated. A longitudinal incision was made along the midline of the urethra to expose the hydrogel implantation site. The urethral tissue containing the intact defect repair area was harvested, gently rinsed with phosphate-buffered saline (PBS) to remove blood, and immediately fixed in 4% paraformaldehyde for 24 hours.

[0062] S6. After fixation, dehydrate the tissue using graded ethanol, clear with xylene, and embed in paraffin. Section continuously along the longitudinal axis of the urethra, 5 μm thick. After dewaxing and rehydration, stain with hematoxylin for 5-10 minutes, rinse with tap water to regain blue color, stain with 1% eosin for 2-3 minutes, dehydrate with graded ethanol, clear with xylene, and mount with neutral resin. Observe the tissue morphology, epithelial regeneration, inflammatory cell infiltration, and degree of tissue integration under an optical microscope, paying particular attention to the interface between the hydrogel and the host tissue. Figure 7 D).

[0063] S7. After dewaxing and rehydrating the paraffin sections, proceed according to the Masson trichrome staining kit instructions. Stain the sections in Weigert iron hematoxylin staining solution for 10 minutes, rinse with running water, then stain with Ponceau S-acid fuchsin solution for 5-10 minutes, followed by treatment with phosphomolybdic acid solution for 5 minutes, and then counterstain with aniline blue solution for 5 minutes. Dehydrate with graded ethanol, clear with xylene, and mount with neutral resin. Observe collagen fiber deposition under an optical microscope; collagen fibers appear blue, and muscle fibers appear red. Assess the degree of tissue remodeling and fibrosis. Figure 7 E).

[0064] S8. Images of H&E staining and Masson's trichrome staining were taken using an optical microscope at magnifications of 100× and 400×, respectively. The staining results were qualitatively analyzed using ImageJ software to observe tissue integrity, inflammatory response, orderly collagen deposition, and degree of fibrosis.

[0065] Figure 7 The in vivo repair effects of four hydrogel formulations in scarless urethral reconstruction were systematically evaluated using a rabbit urethral defect model. Surgical macroscopic images showed that the patency of the urethral lumen at the repair site was significantly better in the APVA / TA2.5 group than in the other groups, and the narrowing area marked by the yellow circle was the least severe in this group. Figure 7 A). Retrograde urethrography further confirmed that the APVA / TA2.5 group had an intact urethral lumen morphology and the lowest degree of stenosis, while the PVA group showed significant segmental stenosis ( Figure 7 B). Quantitative analysis of obstruction rate showed that the obstruction rate in the APVA / TA2.5 group was only 12.6%, significantly lower than that in the APVA group (47.6%), PVA / TA2.5 group (46.3%), and PVA group (80%). Figure 7 C). In histological evaluation, H&E staining showed that the APVA / TA2.5 group had the best tissue integration, mild inflammatory response, and good epithelial coverage and structural remodeling were visible in the areas of interest marked with red borders. Figure 7 D); Masson's trichrome staining further showed that the APVA / TA2.5 group had the most ordered collagen deposition (blue) and the lowest degree of fibrosis, while the other groups, especially the PVA group, showed obvious collagen disorder and excessive fibrosis. Figure 7 E). Comprehensive Figure 7 According to the results of AE, APVA / TA2.5 hydrogel has the best overall therapeutic effect in promoting urethral defect repair, reducing obstruction rate, and alleviating fibrosis.

[0066] Test Example 7 Immunofluorescence analysis of the simultaneous regeneration of urethral epithelium, blood vessels, and smooth muscle by the hydrogels prepared in Examples 1-3 and Comparative Example 2 was performed as follows: S1. Twenty male New Zealand white rabbits, weighing 3.0-3.5 kg, were selected. The animals were randomly divided into four groups of five each using a random number table: PVA group, PVA / TA2.5 group, APVA group, and APVA / TA2.5 group. After implantation of the corresponding hydrogel in each group, the animals were... Figure 7 The method described above establishes a urethral defect model and performs surgical repair. Postoperatively, the patient is fed routinely for 4 weeks.

[0067] S2. Four weeks post-surgery, animals were euthanized with an overdose of anesthetic, and the urethral tissue was rapidly dissected and separated. A 2 cm long urethral tissue sample containing the intact repaired defect area was harvested, gently rinsed with phosphate-buffered saline (PBS) to remove blood, and immediately fixed in 4% paraformaldehyde for 24 hours. The fixed tissue was dehydrated with graded ethanol, cleared with xylene, and embedded in paraffin. Serial sections were prepared along the longitudinal axis of the urethra, 5 μm thick, and mounted on poly-L-lysine-treated slides for storage at room temperature.

[0068] S3. Place the paraffin sections in a 60°C oven for 30 minutes, then dewax them by immersing them sequentially in xylene I and II for 10 minutes each. Next, perform a gradient hydration with anhydrous ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 5 minutes each. Finally, wash three times with PBS for 5 minutes each time. Place the sections in 0.01 M sodium citrate buffer (pH 6.0) and microwave for antigen heat retrieval. Heat on high until boiling, then reduce to medium-low heat and maintain for 10 minutes. Remove and allow to cool to room temperature. Wash three times with PBS for 5 minutes each time. Draw circles around the tissue with an immunohistochemical pen, add 10% goat serum blocking solution, and incubate in a humidified chamber at room temperature for 30 minutes to block non-specific binding sites. Gently shake off the blocking solution and add the next antibody: Pan-CK: Mouse anti-rabbit monoclonal antibody, diluted 1:200 CD31: Rabbit anti-rabbit polyclonal antibody, diluted 1:100 α-Smooth muscle actin (α-SMA): Mouse anti-rabbit monoclonal antibody, diluted 1:200 Place the slides in a humidified chamber and incubate overnight at 4°C (approximately 16-18 hours). The next day, remove and allow to warm to room temperature for 30 minutes. Wash three times with PBS, 5 minutes each time. Add the appropriate fluorescently labeled secondary antibody. pan-CK and α-SMA groups: Alexa Fluor 488-labeled goat anti-mouse IgG (green fluorescence) was added at a dilution of 1:200. CD31 group: Add Alexa Fluor 488-labeled goat anti-rabbit IgG (green fluorescence) at a dilution of 1:200. Place the slides in a humidified chamber and incubate at room temperature in the dark for 1 hour. After incubation, wash three times with PBS, 5 minutes each time. Add DAPI staining solution (final concentration 1 μg / mL), incubate at room temperature in the dark for 5 minutes, and wash three times with PBS, 5 minutes each time. Mount the slides with anti-fluorescence quenching mounting medium, avoiding air bubbles, and store at 4°C in the dark.

[0069] S4. Images were acquired using a laser scanning confocal microscope or a fluorescence microscope. The green fluorescence excitation wavelength for pan-CK, CD31, and α-SMA was 488 nm, and the emission wavelength was approximately 516 nm; the nuclear staining excitation wavelength for DAPI was 358 nm, and the emission wavelength was approximately 461 nm. Quantitative analysis of the immunofluorescence images was performed using ImageJ software.

[0070] Figure 8 Immunofluorescence staining and quantitative analysis were used to systematically evaluate the ability of four hydrogel formulations to promote simultaneous regeneration of epithelium, blood vessels, and smooth muscle in urethral defect repair. Immunofluorescence staining results showed that the APVA / TA2.5 group exhibited significantly higher green fluorescence intensities for pan-keratin (pan-CK, an epithelial cell marker), CD31 (an endothelial cell marker), and α-smooth muscle actin (α-SMA, a smooth muscle cell marker) than the other three groups, indicating that this group performed best in promoting epithelial formation, angiogenesis, and smooth muscle tissue regeneration. The PVA group showed the weakest fluorescence intensity for each marker. Figure 8 A). Quantitative analysis further confirmed that the relative fluorescence intensity of pan-CK in the APVA / TA2.5 group was approximately 3.1-fold, 1.7-fold, and 1.4-fold higher than that in the PVA group, PVA / TA2.5 group, and APVA group, respectively. Figure 8 B); the relative fluorescence intensity of CD31 increased by approximately 3.0 times, 1.6 times, and 1.4 times, respectively. Figure 8 C); the relative fluorescence intensity of α-SMA increased by approximately 2.8-fold, 1.5-fold, and 1.3-fold, respectively. Figure 8 D). Comprehensive Figure 8 The results of AD show that the synergistic effect of anisotropic structures and tannins can effectively promote the orderly and synchronous regeneration of epithelium, blood vessels and smooth muscle, laying a histological foundation for achieving scarless functional repair of the urethra.

[0071] The above are all preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing tannic acid-reinforced anisotropic polyvinyl alcohol hydrogel, characterized in that, Includes the following steps: (1) Polyvinyl alcohol is dissolved in hot water to prepare a polyvinyl alcohol solution, which is then subjected to freeze-thaw cycle treatment to form a polyvinyl alcohol hydrogel; (2) Mechanical stretching training is performed on the polyvinyl alcohol hydrogel obtained in step (1) to orient the polymer chains; (3) The hydrogel trained in step (2) is fixed by salting out while maintaining the stretching state to stabilize the orientation structure; (4) The hydrogel fixed in step (3) is subjected to freeze-drying and annealing treatment in sequence to obtain polyvinyl alcohol hydrogel with an anisotropic structure. (5) The anisotropic polyvinyl alcohol hydrogel obtained in step (4) is immersed in tannic acid solution for reinforcement to obtain tannic acid reinforced anisotropic polyvinyl alcohol hydrogel.

2. The preparation method according to claim 1, characterized in that, In step (1), the mass concentration of the polyvinyl alcohol solution is 10%; the freeze-thaw cycle treatment is a three-cycle freeze-thaw treatment, each cycle including freezing at -20°C for 9 hours and then thawing at room temperature for 3 hours.

3. The preparation method according to claim 1, characterized in that, In step (2), the mechanical stretching training is to cyclically stretch for 30 minutes within the strain range of 0~200%.

4. The preparation method according to claim 1, characterized in that, In step (3), the salting-out agent used for salting-out fixation is sodium sulfate solution with a concentration of 1.5 M; the tensile state is maintained at a constant strain of 200% for 24 hours.

5. The preparation method according to claim 1, characterized in that, In step (4), the annealing process is performed at a temperature of 100°C for 90 minutes.

6. The preparation method according to claim 1, characterized in that, In step (5), the mass concentration of the tannic acid solution is 1.25%~5%.

7. The preparation method according to claim 6, characterized in that, The mass concentration of the tannic acid solution is 2.5%.

8. A tannic acid-reinforced anisotropic polyvinyl alcohol hydrogel prepared by the preparation method according to any one of claims 1-7, characterized in that, The hydrogel has an anisotropic fiber arrangement structure and a structure in which tannic acid is stably bonded to a polyvinyl alcohol network via hydrogen bonds.

9. The tannic acid-reinforced anisotropic polyvinyl alcohol hydrogel according to claim 8, characterized in that, The equilibrium swelling ratio of the hydrogel in phosphate-buffered saline is no greater than 4.8%.

10. The use of the tannic acid-reinforced anisotropic polyvinyl alcohol hydrogel according to any one of claims 8-9 in the preparation of a medical device for urethral stricture repair.

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