Body temperature responsive shrinking hydrogel and preparation method and application thereof

Abstract

The present application relates to hydrogel, specifically relates to a body temperature response contraction type hydrogel and a preparation method and application thereof, which comprises dopamine modified gelatin, polyvinyl alcohol, N-isopropyl acrylamide, N-[three (hydroxymethyl) methyl] acrylamide, N,N'-methylene bisacrylamide, initiator and promoter.The composite hydrogel obtains an optimal formula; in-vitro cell experiments prove that it has excellent cell compatibility, and full-thickness skin defect animal model experiments show that it can effectively resist mechanical stress interference and significantly shorten the wound healing period.The present application provides an innovative solution for clinical treatment of wounds in a mechanical stress environment, and has important conversion application prospects in the field of biomedical materials.

Description

Technical Field

[0001] This invention relates to hydrogels, specifically to a thermo-responsive contractile hydrogel, its preparation method, and its applications. Background Technology

[0002] Wound dressings are medical materials used to temporarily cover wounds, physically isolating them from the external environment and creating a suitable microenvironment for wound healing. Traditional dressings, such as cotton, gauze, and linen products, only provide passive physical protection and cannot dynamically adapt to changes in wound condition. They also lack controlled drug release capabilities, resulting in limited healing effects. Gauze, in particular, has poor antibacterial properties and is easily soaked by bodily fluids, leading to pathogen invasion. Fibrous dressings pose a risk of fiber shedding. Furthermore, traditional dressings struggle to maintain a moist wound environment, and newly formed granulation tissue easily adheres to the dressing, causing pain and secondary damage during dressing changes and exacerbating patient discomfort.

[0003] In recent years, hydrogel dressings have developed rapidly, with functional products such as biodegradable, self-adhesive, antibacterial and anti-inflammatory, controlled release of active factors, and intelligent response emerging one after another. However, existing research mainly focuses on the loading and release of chemical drugs, relying on the physiological reaction between drugs and tissue cells to exert their effects. There are still many shortcomings: First, the dressing does not fit the wound well enough, and gaps are easily formed in complex wounds, which weakens the protective effect and increases the probability of infection. Second, the mechanical properties are fixed and singular, which cannot match the dynamic changes in the tissue mechanical environment during wound healing and cannot provide stable and suitable mechanical support. Third, it is still mainly passive protection and lacks the ability to actively regulate the healing process, and cannot efficiently promote wound contraction and tissue regeneration.

[0004] In addition to these limitations, traditional dressings also suffer from four major constraints: their mechanical properties are incompatible with soft tissue, resulting in insufficient elasticity leading to curling and poor adhesion causing easy detachment; their passive protection mode cannot precisely regulate the wound microenvironment, making it difficult to actively accelerate wound closure; they lack active antibacterial efficacy, as physical isolation alone cannot effectively inhibit multidrug-resistant bacteria; and they lack antioxidant properties, failing to improve the oxidative stress state of chronic wounds. These problems significantly prolong the healing period, increase the risk of infection recurrence, and severely limit clinical efficacy. Therefore, the development of novel dressings that combine high conformability and dynamic mechanical properties, capable of actively contracting the wound and accelerating healing, has become an urgent need in the field of wound repair. Summary of the Invention

[0005] Purpose of the invention Addressing the current clinical challenges in wound repair, this invention utilizes N-isopropylacrylamide thermosensitive polymer materials and interdisciplinary theories of materials science and biomedicine to construct a multifunctional composite hydrogel system with differentiated functional properties. Through a systematic solution elucidating the thermosensitive contraction mechanism and verifying the efficacy of wound repair, this invention aims to solve the clinical treatment challenges of different types of wounds, including acute, chronic, and difficult-to-heal wounds.

[0006] Technical solution A thermo-responsive contractile hydrogel, characterized in that it comprises dopamine-modified gelatin, polyvinyl alcohol, N-isopropylacrylamide, N-[tris(hydroxymethyl)methyl]acrylamide, N,N'-methylenebisacrylamide, an initiator, and an accelerator; The thermo-responsive shrinkage hydrogel is characterized by comprising 0.05 g-0.25 g of dopamine-modified gelatin, 0.1-0.3 g of polyvinyl alcohol, 0.25 g-1 g of N-isopropylacrylamide monomer, 0.05 g-0.5 g of N-[tris(hydroxymethyl)methyl]acrylamide, N,N'-methylenebisacrylamide at 0.1 wt%-1 wt% of the mass of N-isopropylacrylamide monomer, an appropriate amount of initiator, and an appropriate amount of accelerator.

[0007] The thermo-responsive contractile hydrogel is characterized by comprising 0.1 g dopamine-modified gelatin, 0.15 g polyvinyl alcohol, 0.75 g N-isopropylacrylamide monomer, 0.1 g N-[tris(hydroxymethyl)methyl]acrylamide, 4 mg N,N'-methylenebisacrylamide, 40 mg initiator, and 15.5 mg accelerator.

[0008] The initiator is ammonium persulfate or potassium persulfate, and the accelerator is tetramethylethylenediamine, N,N-dimethylaniline (DMA), sodium bisulfite, or triethanolamine; Dopamine-modified gelatin was prepared as follows: gelatin was added to MES buffer, heated and stirred for 30 minutes until the gelatin was completely dissolved; the gelatin solution was cooled to 37°C, and 0.5 g EDC and 0.3 g NHS were added, and the pH was adjusted to 5.4; stirring was continued for 30 minutes, and then 0.5 g dopamine hydrochloride was dissolved in 2 mL of deionized water and slowly added dropwise to the above solution; the reaction mixture was kept at 37°C and pH between 5.0 and 6.0 for 24 hours to obtain dopamine-modified gelatin (DAGel).

[0009] A method for preparing a body temperature-responsive contractile hydrogel, comprising the following steps: Dopamine-modified gelatin and polyvinyl alcohol were heated and dissolved in water. N-isopropylacrylamide monomer and N-[tris(hydroxymethyl)methyl]acrylamide were added. N,N'-methylenebisacrylamide was added at room temperature and stirred until completely dissolved. Ammonium persulfate was added to the solution and stirred evenly. After adding the accelerator, the mixture was stirred rapidly. The mixture was poured into a mold and placed at 4°C for 24 hours to complete the polymerization, resulting in a gel.

[0010] Preparation of PTPD hydrogel: 0.1 g of dopamine-modified gelatin (DAGel) and 0.15 g of polyvinyl alcohol (PVA) were heated to 70°C and dissolved in 5 mL of purified water. Then, 0.75 g of N-isopropylacrylamide (NIPAM) monomer and 0.1 g of N-[tris(hydroxymethyl)methyl]acrylamide (THMA) were added. Next, 4 mg of crosslinking agent N,N'-methylenebisacrylamide (MBA) was added under stirring at room temperature until completely dissolved. Then, 40 mg of initiator ammonium persulfate was added to the solution and stirred until homogeneous. After adding 20 µL of accelerator tetramethylethylenediamine (TEMED), the mixture was stirred rapidly to ensure homogeneity. The mixture was poured into a mold and placed in a refrigerator at 4°C for 24 hours to complete polymerization. Finally, the resulting gel was soaked in purified water for 24 hours to remove unreacted monomers, thus obtaining the PTPD hydrogel.

[0011] Application of the thermo-responsive shrinkage hydrogel in the preparation of wound dressings.

[0012] The wound can be an acute or chronic wound. The acute wounds include trauma, burns, scalds, and surgical wounds; the chronic wounds include ulcers, bedsores, leg ulcers, diabetic foot ulcers, and radiation-induced wounds. Beneficial effects Dr. Winter's theory of moist wound healing states that a moist environment can accelerate granulation tissue development and epidermal cell regeneration, reducing wound discomfort and adhesion. Therefore, hydrogel wet dressings can create and maintain a moist microenvironment in the wound. Low critical phase transition temperature (LCST) hydrogels possess unique advantages: their hydrophilicity and hydrophobicity change reversibly with temperature. Below the LCST, they exhibit a hydrophilic swelling state, while above the LCST, they exhibit a hydrophobic contraction state. This temperature-sensitive response characteristic can precisely adapt to the dynamic needs of wound repair, and combined with high water content and good biocompatibility, it can effectively protect wound nerve endings, dissolve necrotic tissue, prevent dressing adhesion, and provide an optimal microenvironment for wound healing.

[0013] To address the technical bottlenecks of existing wound repair materials, such as insufficient mechanical compliance, low mechanical properties matching wound tissue, and poor healing effects, this invention innovatively designs and prepares a composite hydrogel system based on dopamine-modified gelatin (DAGel), polyvinyl alcohol (PVA), N-isopropylacrylamide (NIPAM), and N-[tris(hydroxymethyl)methyl]acrylamide (THMA) using the LCST regulation theory.

[0014] In this invention, the NIPAM monomer endows the system with LCST-type thermosensitive properties, enabling it to undergo specific thermal response contraction at a physiological temperature of 37°C (higher than LCST). This allows it to actively pull on wound edge tissue, provide mechanical stimulation, and directly promote tissue regeneration and repair. The synergistic cross-linking of NIPAM and PVA constructs a three-dimensional network structure with excellent mechanical properties. Its elastic modulus is highly matched with that of biological soft tissue. At the same time, relying on the LCST-type thermosensitive response mechanism, the material is endowed with dynamic mechanical regulation capabilities. It can intelligently adjust its own properties according to changes in the wound microenvironment temperature, further promoting wound closure and shortening the healing period.

[0015] After optimizing the group ratios through orthogonal experiments, the composite hydrogel obtained its optimal formulation. In vitro cell experiments confirmed its excellent cell compatibility, and animal model experiments with full-thickness skin defects showed that it can effectively resist mechanical stress interference and significantly shorten the wound healing period. This invention provides an innovative solution for the clinical treatment of wounds under mechanical stress environments and has important translational application prospects in the field of biomedical materials.

[0016] The specific advantages are as follows: First, the structure is scientifically designed and has excellent mechanical properties. Dopamine-modified gelatin (DAGel) is synthesized through EDC coupling and polyvinyl alcohol (PVA) is introduced to construct a semi-interpenetrating network. Combined with the synergistic effect of NIPAM thermosensitive monomer and crosslinking agent, the hydrogel has good mechanical strength, toughness and flexibility, with a tensile elongation at break of 208.1%. It can be adapted to dynamic wounds such as joints and avoids fracture caused by activity.

[0017] Second, it exhibits outstanding thermo-responsiveness and can actively promote wound closure. Based on the thermo-sensitive properties of PNIPAM, it can undergo directional contraction at a physiological temperature of 37°C, driving the wound edges to converge through mechanical stress, significantly accelerating wound closure, which is superior to commercially available traditional dressings.

[0018] Third, it has excellent adhesion and strong conformability. The dopamine phenolic hydroxyl groups in DAGel can form non-covalent interactions with functional groups on the tissue surface, with an adhesion strength of 1.19 kPa. It can closely conform to complex wounds and is not easy to detach even with repeated joint movements.

[0019] Fourth, it exhibits excellent biocompatibility and high safety. In cytotoxicity experiments, cell survival rate exceeded 85%, and hemolysis rate was less than 5%, meeting medical material standards. It showed no significant cell damage or hemolytic reaction and is suitable for clinical applications.

[0020] Fifth, it has suitable swelling properties, adapting to the wound microenvironment. With a balanced swelling rate as low as 4.07 g / g, it can effectively absorb wound exudate, maintain a moist wound environment, and at the same time avoid mechanical property degradation caused by excessive swelling, thus providing favorable conditions for wound healing. Attached Figure Description

[0021] Figure 1 Design scheme of thermo-responsive contractile hydrogel. (A) Schematic diagram of hydrogel wound dressing inspired by the active contraction of embryonic wounds; (B) Composition structure of hydrogel network; (C) Hydrogel actively closing wound surface under the influence of body temperature; Figure 2 Synthesis of dopamine-modified gelatin. (A) Synthetic scheme of modified gelatin conjugate; (B) NMR spectra of gelatin and modified gelatin; (C) ATR infrared spectra of gelatin and modified gelatin; (D) UV-Vis spectra of gelatin and dopamine-modified gelatin. Figure 3 (A) Strain scan diagrams of hydrogels with different polyvinyl alcohol contents; (B) Strain scan diagrams of hydrogels with different modified gelatin contents; (C) Strain scan diagrams of hydrogels with different N-isopropylacrylamide contents. Figure 4 Strain scanning spectroscopy (SSS) results of hydrogels. (A) Rheological properties of hydrogels with different formulations; (B) Rheological properties of PTPD hydrogels at different temperatures; Figure 5 Mechanical properties of PTPD composite hydrogels. (A) Stress-strain curves; (B) Fracture strength, elastic modulus, toughness, and elongation at break of the hydrogels; Figure 6 Swelling rate of hydrogels. (A) Changes in swelling rate of hydrogels with different formulations after 36 h; (B) Equilibrium swelling rate of hydrogels with different formulations; Figure 7 SEM images of PTPD hydrogel. (A) 30 μm standard ruler; (B) 20 μm standard ruler; Figure 8 Adhesion properties and skin fit of composite hydrogels. (A) Hydrogels adhere to different materials: (A1) Adhesive to an EP tube containing 35 mL of copper sulfate aqueous solution; (A2) Adhesive to a 20 g weight; (A3) (A4) Adhesive to a finger; (A5) (A6) Hydrogels are bent and twisted on pigskin; (B) Different hydrogels are adhered to simulated joint wounds: (B1) (B2) are the initial state of simulated joint wounds; (B3) is after bending the joint 100 times with acrylamide hydrogel; (B4) is after bending the joint 100 times with PTPD hydrogel; (B5) is the wound state after bending the joint 100 times with acrylamide hydrogel; (B6) is the wound state after bending the joint 100 times with PTPD hydrogel. Figure 9 The shrinkage area ratio of hydrogels with different formulations; Figure 10Biocompatibility of hydrogels with different formulations. (A) Cell viability; (B) Hemolysis test; Figure 11 Evaluation of the in vivo wound healing performance of thermosensitive hydrogel. (A) Changes in mouse wounds treated with thermosensitive hydrogel, sterile gauze, and commercially available dressings from 0 to 14 days; (B) Dynamic changes in mouse wounds under different treatment conditions; (C) Wound healing rate of mice under different treatment conditions; Detailed Implementation

[0022] Example 1 1. Experiment Content 1.1 Preparation of Dopamine-Modified Gelatin (DAGel) Dopamine-modified gelatin (DAGel) is a typical adhesive macromolecule, and its synthesis involves a chemical reaction using ethyl-dimethyl-aminopropyl-carbodiimide (EDC) and N-hydroxy-succinimide (NHS) as coupling agents. First, 100 mL of a 50 mM MES buffer solution with a pH of 5.4 was prepared using 2-(N-morpholine)ethanesulfonic acid reagent and transferred to a 250 mL round-bottom flask. Next, 2 g of type A gelatin (derived from pigskin) was added to this solution, and the mixture was heated and stirred in an oil bath at 60 °C for 30 minutes to ensure complete dissolution. After the gelatin solution cooled to 37 °C, 0.5 g of EDC and 0.3 g of NHS were added, and the pH of the mixture was adjusted to 5.4. Stirring continued for 30 minutes, and then 0.5 g of dopamine hydrochloride was dissolved in 2 mL of deionized water and slowly added dropwise to the above solution. The reaction mixture was maintained at 37°C and pH between 5.0 and 6.0 for 24 hours. Afterward, the reaction solution was dialyzed against deionized water for two days and then lyophilized.

[0023] 1.2 Preparation of hydrogels Preparation of PTPD hydrogel (containing DAGel, PVA, NIPAM, and THMA): 0.1 g of dopamine-modified gelatin (DAGel) and 0.15 g of polyvinyl alcohol (PVA) were heated to 70°C and dissolved in 5 mL of purified water. Then, 0.75 g of N-isopropylacrylamide (NIPAM) monomer and 0.1 g of N-[tris(hydroxymethyl)methyl]acrylamide (THMA) were added. Next, 4 mg of crosslinking agent N,N'-methylenebisacrylamide (MBA) was added under stirring at room temperature until completely dissolved. Then, 40 mg of initiator ammonium persulfate was added to the solution and stirred until homogeneous. After adding 20 µL (15.5 mg) of accelerator tetramethylethylenediamine (TEMED), the mixture was stirred rapidly to ensure homogeneity. The mixture was poured into a mold and placed in a refrigerator at 4°C for 24 hours to complete polymerization. Finally, the resulting gel was soaked in purified water for 24 hours to remove unreacted monomers, thus obtaining the PTPD hydrogel.

[0024] Preparation of PTD hydrogel (containing DAGel, NIPAM, and THMA): 0.1 g of dopamine-modified gelatin (DAGel) was heated to 70°C and dissolved in 5 mL of purified water. Then, 0.75 g of N-isopropylacrylamide (NIPAM) monomer and 0.1 g of N-[tris(hydroxymethyl)methyl]acrylamide (THMA) were added. Next, 4 mg of crosslinking agent N,N'-methylenebisacrylamide (MBA) was added under stirring at room temperature until completely dissolved. Then, 40 mg of initiator ammonium persulfate was added to the solution and stirred until homogeneous. After adding 20 µL of accelerator tetramethylethylenediamine (TEMED), the mixture was stirred rapidly to ensure homogeneity. The mixture was poured into a mold and placed in a refrigerator at 4°C for 24 hours to complete polymerization. Finally, the resulting gel was immersed in purified water for 24 hours to remove unreacted monomers, thus obtaining the PTD hydrogel.

[0025] Preparation of PTP hydrogel (PVA, NIPAM, and THMA): 0.15 g of polyvinyl alcohol (PVA) was heated to 70°C and dissolved in 5 mL of purified water. Then, 0.75 g of N-isopropylacrylamide (NIPAM) monomer and 0.1 g of N-[tris(hydroxymethyl)methyl]acrylamide (THMA) were added. Next, 4 mg of crosslinking agent N,N'-methylenebisacrylamide (MBA) was added under stirring at room temperature until completely dissolved. Then, 40 mg of initiator ammonium persulfate was added to the solution and stirred until homogeneous. After adding 20 µL of accelerator tetramethylethylenediamine (TEMED), the mixture was stirred rapidly to ensure homogeneity. The mixture was poured into a mold and placed in a refrigerator at 4°C for 24 hours to complete polymerization. Finally, the resulting gel was immersed in purified water for 24 hours to remove unreacted monomers, thus obtaining the PTP hydrogel.

[0026] Preparation of PT hydrogel (NIPAM and THMA): 0.75 g of N-isopropylacrylamide (NIPAM) monomer and 0.1 g of N-[tris(hydroxymethyl)methyl]acrylamide (THMA) were dissolved in 5 mL of purified water. Next, 4 mg of crosslinking agent N,N'-methylenebisacrylamide (MBA) was added under stirring at room temperature until completely dissolved. Then, 40 mg of initiator ammonium persulfate was added to the solution and stirred until homogeneous. After adding 20 µL of accelerator tetramethylethylenediamine (TEMED), the mixture was rapidly stirred to ensure homogeneity. The mixture was poured into a mold and placed in a refrigerator at 4°C for 24 hours to complete polymerization. Finally, the resulting gel was immersed in purified water for 24 hours to remove unreacted monomers, thus obtaining the PT hydrogel.

[0027] 1.3 Single-factor optimization of hydrogel formulation The formation of the hydrogel network structure mainly depends on the cross-linking polymerization of monomers and the interpenetration between added polymers. Therefore, in this invention, the mass of NIPAM monomer, PVA, and DAGel were selected as the research objects, and the optimal ratio of these three reagents was preliminarily determined through single-factor experiments. The rheological properties of hydrogels can reflect their viscoelastic characteristics. Storage modulus (G') is one of the key indicators for evaluating hydrogel performance. It represents the ratio of the degree of deformation of the material under external force to the external force, reflecting the material's resistance to external force and describing its elastic properties. Specifically, storage modulus reflects the material's ability to store energy during the elastic deformation stage and is an important parameter for measuring the elasticity of the material. Therefore, we use it as a preliminary standard for evaluating hydrogel performance.

[0028] (1) Optimization of polyvinyl alcohol quality Accurately weigh 0.1 g, 0.15 g, 0.2 g, 0.25 g, and 0.3 g of polyvinyl alcohol and 0.1 g of DAGel, and dissolve them in 5 mL of water by heating to 70 °C. Then, add 0.25 g of NIPAM and 0.1 g of THMA to the mixed solution and stir until homogeneous at room temperature. Next, add the crosslinking agent, initiator, and accelerator sequentially, stirring rapidly to form a prepolymer solution. Pour this solution into a six-well plate and react at 4 °C for 24 hours to obtain a 1.5 mm thick hydrogel sheet. The rheological properties of the hydrogel were then measured.

[0029] (2) Optimization of modified gelatin quality Accurately measure 0.05 g, 0.1 g, 0.15 g, 0.2 g, 0.25 g DAGel, and 0.1 g PVA, and dissolve them in 5 mL of water by heating to 70 °C. Then, add 0.25 g NIPAM and 0.1 g THMA to the above mixed solution and stir until homogeneous at room temperature. Next, add the crosslinking agent MBA, the initiator APS, and the accelerator TEMED sequentially, and stir rapidly to form a prepolymerization solution. Pour the solution into a six-well plate and react at 4 °C for 24 hours to obtain a 1.5 mm thick hydrogel sheet. The rheological properties of the hydrogel were then measured.

[0030] (3) Optimization of the quality of N-isopropylacrylamide monomer Accurately weigh 0.25 g, 0.5 g, 0.75 g, and 1 g of N-isopropylacrylamide monomer (NIPAM) and add them to 5 mL of aqueous solutions containing a certain amount of DAGel, PVA, and THMA, respectively. Stir until homogeneous at room temperature. Then, add the crosslinking agent MBA, the initiator APS, and the accelerator TEMED sequentially, and stir rapidly to form a prepolymerization solution. Pour the solution into a six-well plate and react at 4 °C for 24 hours to obtain a gel sheet with a thickness of 1.5 mm. The rheological properties of the hydrogel were then measured.

[0031] 1.4 Orthogonal experiments were used to optimize the hydrogel formulation. Based on the preliminary analysis results of single-factor experiments, this invention selects the peak value of storage modulus and the ratio of shrinkage area as key performance evaluation indicators. For the three core parameters affecting hydrogel performance (monomer feed amount, polyvinyl alcohol addition amount, and modified gelatin content), a three-level variable design is adopted, and the specific parameter combinations are shown in Table 1. By constructing an L9(3)3 orthogonal experimental matrix, the influence of each factor and its interaction on the composite hydrogel performance is systematically investigated, thereby achieving the optimization and screening of preparation process parameters.

[0032] Table 1. Factor-level table for orthogonal experiments

[0033] 1.5 Characterization of Gelatin-Dopamine Macromonomer To verify whether dopamine was successfully grafted into gelatin, proton nuclear magnetic resonance spectroscopy was used. 1 Gelatin-dopamine conjugates were analyzed using 1.5% (g / mL) gelatin-dopamine deuterium oxide (D2O) solution in a 5 mm NMR tube and the data were recorded on an NMR spectrometer.

[0034] Furthermore, this invention evaluated the presence of unoxidized catechol groups in dopamine modification using ultraviolet-visible spectroscopy. 10% (g / mL) gelatin and gelatin-dopamine solutions in ultrapure water were scanned using a spectrophotometer in the wavelength range of 250 nm to 500 nm.

[0035] 1.6 Attenuated Total Reflectance Infrared Spectroscopy (ATR) Test The composition of the composite hydrogel was analyzed using attenuated total reflectance infrared spectroscopy. Before analyzing the test samples, background data was collected, and then gelatin and dopamine-modified gelatin were sequentially placed into the infrared spectrometer for detection.

[0036] 1.7 Scanning Electron Microscopy (SEM) Testing The prepared hydrogel samples were quenched with liquid nitrogen and then dried in a freeze dryer for 24 hours. After removal, the gel was cut into thin slices and fixed on a stage with conductive tape. The conductivity was improved by vacuuming and gold sputtering. Finally, the microstructure of the samples was observed using an EVO18 scanning electron microscope at a test voltage of 3.00 kV.

[0037] 1.8 Rheological property testing The dynamic rheological properties of the composite hydrogel were tested using a rotational rheometer equipped with a 50 mm parallel plate at 20 °C. Hydrogel samples with a diameter of 25 mm and a thickness of 1.5 mm were fixed on the test stage, and the distance between the rotor and the sample was adjusted. The strain amplitude scanning mode was used, with a strain range of 0.1%–1000% and a frequency of 10 rad / s. Each sample was tested three times. Based on the test data, the storage modulus G' and loss modulus G'' curves of the samples at room temperature were plotted and analyzed.

[0038] 1.9 Swelling Performance Test First, hydrogel samples with a diameter of 20 mm and a thickness of 1.5 mm were prepared, ensuring uniform size. After freeze-drying to constant weight, the initial mass of the samples was measured, followed by immersion in deionized water at 25°C. The samples were removed after 0.5, 1, 2, 4, 8, 12, 24, and 36 hours, and weighed after surface drying. Each group of samples was tested three times, and the swelling rate was calculated using the average value according to the formula:

[0039] In the formula W d The original mass in its dry state, in grams; W t The mass after water absorption and swelling is expressed in grams.

[0040] 1.10 Tensile property test The tensile stress-strain characteristics of the hydrogels were determined using a universal testing machine. Different formulations of hydrogel prepolymers were injected into dumbbell-shaped molds to prepare dumbbell-shaped gel samples with dimensions of 50 mm × 8.5 mm × 2.5 mm, followed by tensile tests. The tensile speed was set to 10 mm / min, thus obtaining the tensile stress-strain curves of the hydrogels.

[0041] 1.11 Thermal Response Behavior The hydrogel prepolymer solution was poured into six-well plates and polymerized at 4°C for 24 h. The volume change was measured by placing the sample in a 37°C water bath to evaluate the thermal response. The initial and final dimensions are denoted as L0 and L, respectively, and the shrinkage area ratio was calculated as follows:

[0042] Cytotoxicity assay The cytotoxicity of PT, PTP, PTD, and PTPD was evaluated using the MTT assay. The prepared hydrogel samples were placed in DMEM medium at a specific concentration of 60 mg / mL. -1 The culture medium was then incubated in a constant temperature incubator at 37°C for 24 hours. Afterward, the extract was filtered through a 0.22 µm filter membrane.

[0043] Mouse fibroblasts (L929) were cultured to a concentration of 5 × 10⁻⁶. 4 Cells / mL, 100 μL was seeded into 96-well plates and incubated at 37℃ in a 5% CO2 incubator for 24 hours. After removing the original culture medium, the experimental group was added to DMEM medium containing sample extract, while the control group was incubated with PBS, and cultured for another 12 hours. 50 μL of MTT reagent was added to each well and incubated for 4 hours, and the absorbance at 490 nm was measured using a microplate reader.

[0044]

[0045] In the formula, OD sample The absorbance of the sample, OD blank The absorbance and OD of the blank control group are shown. control The absorbance is for the control group.

[0046] 1.12 Hemolysis test Anticoagulated animal blood (rabbit blood) was centrifuged at 1000 rpm for 10 min to obtain red blood cell pellet. The pellet was then collected, and 10 volumes of 0.9% sodium chloride injection solution were added. The mixture was centrifuged at 1500 rpm for 10 min, and the supernatant was discarded. The washing process was repeated 2-3 times until the supernatant was colorless, resulting in a 2% red blood cell suspension.

[0047] Mix each hydrogel extract (500 μL, 0.05 g / mL) with an equal volume of 2% erythrocyte suspension and incubate at 37°C for 1 hour. Add 10 μL of erythrocytes to 1 mL of distilled water (positive control) and sodium chloride injection (negative control), respectively. After incubation, centrifuge at 1500 rpm for 10 minutes, and transfer 100 μL of supernatant to a 96-well plate. Measure the absorbance at 540 nm. The hemolysis rate can be calculated using the following formula:

[0048] Among them, OD sam The absorbance value of the sample is measured, OD. pos OD neg These represent the absorbance values ​​for the positive and negative controls, respectively.

[0049] 1.13 In vivo wound healing experiment This invention is based on a randomized controlled trial design. Fifteen 8-week-old male BALB / c mice were selected as experimental subjects and randomly divided into a blank control group, a commercial dressing control group, and an experimental group, with a sample size of 5 mice in each group (n=5). The experimental animals were anesthetized by intraperitoneal injection of 1.25% tribromoethanol solution, with the injection dose strictly controlled at 0.2 mL / 10g body weight. In the preoperative preparation stage, the hair on the back of the mice was first initially removed using an electric shaver, followed by a second hair removal using depilatory cream to ensure complete removal of hair from the surgical area. Subsequently, the surgical area was strictly disinfected using povidone-iodine. Following aseptic procedures, a full-thickness skin defect wound was prepared on the back of the mice using a scalpel, reaching the myofascia layer. During postoperative management, the blank control group used sterile gauze to cover the wound, the commercial dressing control group used Kefu hydrocolloid dressing purchased from Taobao, and the experimental group used the PTPD hydrogel dressing independently developed in this invention. Throughout the entire wound healing cycle, intelligent digital devices are used to continuously record dynamic images of the wound healing process, and ImageJ image analysis software is used to quantitatively measure and analyze the wound area, providing accurate quantitative basis for subsequent data statistics and result analysis.

[0050] 2. Results and Discussion 2.1 Design Strategy for Hydrogels In the field of wound repair biology, wound contraction, as a core biological event in the wound healing process, plays a crucial role in tissue repair. This process mainly relies on the biological functions of myofibroblasts. These cells, with their unique contractile properties, migrate directionally to the wound area and generate sustained mechanical contractile force, driving the progressive convergence and closure of the wound edge tissue. This invention demonstrates that the scarless repair characteristics exhibited during embryonic wound healing are significantly correlated with an efficient wound contraction mechanism. In stark contrast, the wound contraction capacity of adult organisms is significantly reduced compared to the embryonic period due to differences in tissue structure and physiological environment, becoming one of the key factors affecting the quality and efficiency of wound repair. This difference in biological phenomena provides important theoretical basis and research directions for in-depth exploration of wound repair mechanisms and the development of novel healing-promoting strategies.

[0051] Based on the aforementioned biological mechanisms, this invention constructs a smart hydrogel system with body temperature-responsive contraction properties using a biomimetic design strategy. (See attached image) Figure 1A. The invention first employs interface functionalization modification technology to chemically modify gelatin molecules, enhancing the adhesion properties of the hydrogel to biological tissues by introducing dopamine groups (see Figure 1 B). Subsequently, dopamine-modified gelatin, thermosensitive monomer N-isopropylacrylamide, and mechanically reinforcing component polyvinyl alcohol are molecularly blended. By adding crosslinking agent N,N'-methylenebisacrylamide and initiator ammonium persulfate, PTPD hydrogel is prepared by one-pot in-situ polymerization.

[0052] As shown in Figure 1C, the biomimetic hydrogel exhibits active wound closure capability. This hydrogel network is constructed through cross-linking polymerization based on thermosensitive N-isopropylacrylamide (NIPAM) monomers. When the ambient temperature rises and exceeds its low critical solution temperature (LCST, approximately 33°C), the hydrophobic interactions between hydrophobic groups such as isopropyl groups on the hydrogel molecular chains significantly increase, while the hydrogen bonding between the molecular chains and water molecules gradually weakens. This thermodynamically driven change in intermolecular forces prompts water molecules to rapidly drain from the gel network, leading to the densification and contraction of the hydrogel network structure. Due to the strong adhesion between the hydrogel and the wounded skin, its contractile behavior effectively transfers mechanical stress to the skin tissue, driving synergistic contraction of the skin tissue through mechanical force mediation, ultimately achieving active wound closure and providing an innovative mechanical intervention strategy for wound repair.

[0053] 2.2 Preparation and Characterization of Dopamine-Modified Gelatin Using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as coupling agents, dopamine molecules were chemically modified with the gelatin backbone via a carbodiimide coupling reaction, successfully synthesizing dopamine-modified gelatin (DAGel) (see...). Figure 2 A). For example Figure 2 B, Figure 2 C and Figure 2 As shown in Figure D, the chemical structure of DAGel is obtained through... 1 Determined by HNMR, ATR infrared spectroscopy, and ultraviolet spectroscopy. 1 ¹H NMR spectroscopy analysis revealed a set of characteristic peaks at a chemical shift of 6.67 ppm, which are attributed to hydrogen protons on the benzene ring of the dopamine molecule. Furthermore, a characteristic peak at 2.79 ppm corresponds to the methylene proton adjacent to the phenyl group in the dopamine-gelatin (DAGel) conjugate, whereas in pure gelatin… 1 The peak did not appear in the HNMR spectrum, indicating that the formation of the DAGel conjugate introduced a new chemical environment. In attenuated total reflectance infrared (ATR-IR) spectral characterization, the peak at 3078 cm⁻¹... -1The absorption peak at 1630 cm⁻¹ is attributed to the CH stretching vibration of the dopamine aromatic group, and the appearance of this characteristic peak proves the existence of the dopamine structural unit. Meanwhile, the absorption peak at 1630 cm⁻¹... -1 The absorption peak of amide I at [location] indicates the formation of HN-CO bonds, providing strong evidence for the chemical bonding between dopamine and gelatin. Furthermore, the ATR-IR spectra of pure gelatin and DAGel conjugates both show absorption peaks in the 3550-3200 cm⁻¹ range. -1 A broad and strong absorption peak was observed within the range, attributed to the stretching vibration of hydroxyl groups (-OH) in the samples, indicating the presence of hydroxyl groups in both types of samples. DAGel exhibited a significant UV absorption peak at 275 nm, indicating that the catechol groups had successfully coupled with the gelatin backbone. Furthermore, no absorption peak was detected at 395 nm, suggesting that the catechol groups did not undergo oxidation during the synthesis process.

[0054] 2.3 Optimization of hydrogel formulation 2.3.1 Single-factor optimization of hydrogel formulation (1) Optimization of polyvinyl alcohol quality Variations in polyvinyl alcohol (PVA) content significantly regulate the hydrogel network structure. Based on the experimental data shown in Figure 3A, when the PVA content is in the range of 0.1-0.2 g, the storage modulus (G′) of the hydrogel increases with increasing PVA mass; however, when the PVA content exceeds 0.2 g, the G′ value decreases with increasing PVA mass. This phenomenon can be explained by intermolecular interactions and network structure evolution mechanisms: the hydroxyl groups (-OH) in the PVA molecule form hydrogen bonds with the amide groups (-NH-) of the N-isopropylacrylamide (NIPAM) monomer. This non-covalent bonding effectively fills the internal pores of the hydrogel, increasing the material's hardness. Simultaneously, the introduction of long PVA chains constructs a semi-interpenetrating network (semi-IPN) structure, significantly improving the flexibility of the dual-network hydrogel by enhancing the network's synergistic deformation ability, thereby improving its overall mechanical properties. However, as the amount of PVA increases, excessive PVA molecular chains disrupt the continuity of the gel network, leading to a decrease in network backbone density and weakened support, ultimately resulting in deterioration of mechanical properties. Based on the above experimental results and mechanism analysis, subsequent studies optimized the PVA dosage to 0.2 g to maximize the mechanical properties of the hydrogel.

[0055] (2) Optimization of the quality of modified gelatin Systematic analysis of the experimental data in Figure 3B shows that the dosage of dopamine-modified gelatin (DAGel) has a significant dose-dependent regulatory effect on the storage modulus (G′) of the hydrogel. Within the addition range of 0.05–0.15 g, the G′ value increases linearly with increasing DAGel mass; however, when the dosage exceeds 0.15 g, the G′ value decreases. From a molecular mechanism perspective, the hydroxyl groups (-OH) in the DAGel molecule form intermolecular hydrogen bonds with the amide groups (-NH-) of the N-isopropylacrylamide (NIPAM) monomer. This non-covalent interaction effectively fills the internal pores of the hydrogel, optimizes the network structure density, and significantly improves the mechanical strength and flexibility of the material. However, excessive DAGel will disrupt the ordered structure of the gel network, impair the integrity of the network skeleton, reduce the supporting force, and lead to a decrease in mechanical properties. Based on this, the optimal dosage of DAGel was determined to be 0.15 g to achieve the optimal balance of the hydrogel's mechanical properties.

[0056] (3) Optimization of the quality of temperature-sensitive monomers According to the experimental data in Figure 3C, the storage modulus of the hydrogel is positively correlated with the mass of N-isopropylacrylamide (NIPAM) monomer. As the mass of NIPAM monomer increases, the storage modulus of the hydrogel gradually increases, indicating a significant enhancement in the structural stability of the hydrogel. Analysis of the reaction mechanism suggests that increasing the monomer concentration increases the density of active sites per unit volume, thereby promoting the full cross-linking effect of the cross-linking agent N,N'-methylenebisacrylamide (MBA). The increased reaction efficiency between MBA and NIPAM monomers leads to an increase in the number of effective cross-linking points, resulting in an increase in molecular chain entanglement density and a more compact gel network structure. This compact network structure effectively improves the mechanical properties and structural stability of the hydrogel. Experimental results show that when the mass of NIPAM is 1.0 g, the storage modulus of the hydrogel reaches its maximum value, at which point the structural stability of the hydrogel is optimal.

[0057] 2.3.2 Orthogonal Experiment to Optimize the Hydrogel Formulation Based on the formulation factor level table, this invention systematically designed an orthogonal experimental scheme. The maximum shear modulus of the hydrogel was accurately measured using a rheometer, and the shrinkage area ratio of the hydrogel under a constant temperature of 37℃ for 24 hours was measured with high precision using ImageJ software. Based on this data acquisition, critical weighted analysis was used to explore the fluctuation characteristics and interrelationships of the two sets of data (detailed analysis results are shown in Table 2). Subsequently, according to the established scoring criteria, a comprehensive evaluation of each experimental group of hydrogels was conducted. Range analysis was used to quantitatively assess the influence of the three reagent masses on the structure and properties of the hydrogel. The results showed that the importance of their influence ranked C > A > B, indicating that the mass of modified gelatin has the most significant impact on the structure and properties of the hydrogel, followed by the monomer mass, while the influence of polyvinyl alcohol mass is relatively weak. After systematic analysis and optimization screening, the optimal synthesis formulation for the hydrogel was finally determined to be A2B1C1, with the corresponding parameters: NIPAM monomer mass 0.75 g, polyvinyl alcohol mass 0.15 g, and modified gelatin mass 0.1 g.

[0058] Table 2. Orthogonal Experiment Table for Hydrogels

[0059] Four types of hydrogels (PT hydrogel, PTP hydrogel, PTD hydrogel and PTPD hydrogel) were prepared according to 1.2 for subsequent research; 2.4 Rheological Performance Analysis Systematic analysis of its rheological properties (Figure 4A) revealed that the storage modulus (G′) and loss modulus (G″) of PTPD hydrogel were significantly higher than those of other hydrogel systems within the strain range of 0.1% to 100%, indicating that PTPD hydrogel possesses superior mechanical strength. This phenomenon is closely related to the cross-linking structure of the hydrogel network: firstly, the introduction of polyvinyl alcohol (PVA) polymer and dopamine gelatin (DAGel) effectively improved the density of the hydrogel network structure, thereby enhancing the overall mechanical load-bearing capacity of the network; secondly, the hydroxyl groups (-OH) in the molecular structures of N-[tris(hydroxymethyl)methyl]acrylamide (THMA), PVA, and DAGel can form hydrogen bonds with the amino groups (-NH2) on the poly(N-isopropylacrylamide) molecule. This non-covalent interaction significantly increases the energy dissipation capacity of the gel system. Through the synergistic effect of multiple enhancement mechanisms, the mechanical properties of the PTPD composite hydrogel were ultimately significantly improved.

[0060] and Figure 4B shows that the hydrogel possesses unique temperature-sensitive properties, exhibiting different mechanical properties at different temperatures. At a physiological temperature of 37°C, the hydrogel exhibits solid-like elastic behavior in the low strain range (0.1% - 2%), with a storage modulus (G′>14000 Pa) significantly higher than the loss modulus (G″), forming a stable physical barrier on the wound surface to resist mechanical impact. When the strain exceeds 20%, G″ exceeds G′, and the material enters a viscosity-dominated "yield" state, dissipating energy through structural micro-damage to adapt to the dynamic mechanical environment of the wound (such as exudate diffusion or joint movement), avoiding rigid fracture. At room temperature of 20°C, the stress slowly decreases from 7000 Pa to 5500 Pa when the strain is 0.1% - 100%, indicating that the material has high structural stiffness at low temperatures, facilitating storage and transportation. At 200% strain, G″>G′ indicates that it still possesses deformation tolerance, ensuring the integrity of operation at room temperature. This combination of low-temperature rigidity and room-temperature deformation capability balances storage stability and clinical operability.

[0061] 2.5 Tensile property analysis Tensile property testing of hydrogels can comprehensively reflect their mechanical behavior and network structure characteristics, and is an important indicator for evaluating the potential of materials for practical applications, such as... Figure 5 Elastic modulus is a material's ability to resist elastic deformation. PTPD composite hydrogel has an elastic modulus of 157.3 kPa, making it suitable for simulating skin tissue (skin stiffness is approximately 1-100 kPa). Tensile strength is the maximum stress a material can withstand before fracture and is a key indicator for evaluating hydrogel wound dressings. PTPD composite hydrogel has a maximum tensile stress of 82.6 kPa and an elongation at break of 208.1%, demonstrating excellent tensile properties. Furthermore, the toughness of hydrogel materials reflects their cyclic loading capacity. PTPD composite hydrogel has a toughness of 106.3 kPa, indicating its potential application in joint wounds without fracture due to joint movement.

[0062] 2.6 Swelling Performance Analysis Hydrogels are hydrophilic gels with a three-dimensional network structure. In an aqueous environment, they can absorb and retain a large amount of water without dissolving. Their water absorption capacity and swelling properties are closely related to their chemical composition and cross-linking density. Generally, increasing the cross-linking density leads to a decrease in water absorption and swelling; conversely, decreasing the cross-linking density increases both water absorption and swelling. Swelling rate is an important indicator of whether a hydrogel can provide a suitable moist environment for the wound and protect it from external physical pressure. Therefore, when applying hydrogels to wound healing, choosing a hydrogel with a lower swelling rate is more beneficial for promoting the healing process of moist wounds.

[0063] like Figure 6A. All hydrogels reached swelling equilibrium after 24 hours. This swelling characteristic indicates that the hydrogel is suitable for use as a wound dressing material, capable of absorbing exudate and maintaining a stable moist environment within a reasonable time, while avoiding problems such as mechanical property deterioration caused by rapid swelling or insufficient absorption caused by slow swelling. Figure 6 As shown in Figure B, PT hydrogel exhibits the highest swelling ratio, with an equilibrium swelling ratio of 7.22 g / g. This is because it contains only thermosensitive PNIPAM and the hydrophilic monomer THMA, resulting in a relatively loose network structure where hydrophilic groups can fully bind water molecules. PTP hydrogel shows a slight decrease in swelling ratio (6.58 g / g) after the introduction of PVA. This is because PVA molecular chains penetrate into the PNIPAM network, forming a semi-interpenetrating structure, increasing physical cross-linking points, and limiting network expansion. The addition of DAGel to PTD hydrogel significantly reduces the swelling ratio (5.56 g / g) because dopamine groups can form covalent cross-links through oxidative self-polymerization, and the hydrophobic aromatic ring structure reduces the proportion of hydrophilic groups. PTPD hydrogel has the lowest swelling ratio (4.07 g / g), which is the result of the synergistic effect of the physical entanglement of PVA and the chemical cross-linking of Gel-DA: PVA hydroxyl groups form hydrogen bonds with PNIPAM, and the catechol groups of Gel-DA construct an additional cross-linking network. The multi-component interaction significantly increases the cross-linking density, and the increased proportion of hydrophobic components reduces the overall hydrophilicity.

[0064] 2.7 SEM Analysis If the hydrogel is dried by heating, on the one hand, the rapid evaporation of water molecules can easily lead to significant volume shrinkage; on the other hand, when the temperature exceeds the low critical solution temperature (LCST) of the PNIPAM network, the aggregation of molecular chains can cause the internal structure to collapse; in addition, prolonged high temperatures may cause material degradation. Therefore, freeze-drying is used to preserve the complete microstructure. As shown in Figure 7, the hydrogel exhibits a porous three-dimensional structure, in which water molecules can flow. This structure increases the specific surface area, promoting contact between hydrophilic segments and free water, thereby enhancing water absorption capacity and rate. When used as a dressing, it can absorb wound exudate and maintain a moist environment on the wound surface, creating favorable conditions for healing.

[0065] 2.8 Adhesion Performance Analysis The introduction of dopamine (DA) significantly enhances the adhesive properties of the composite hydrogel. Based on the interaction between the phenolic hydroxyl groups and functional groups such as amino groups in the tissue, the composite hydrogel possesses excellent tissue adhesion properties. As shown in Figures 8A1 and A2, the composite hydrogel exhibits good adhesion to various material surfaces, including plastics and iron. This is mainly attributed to the non-covalent interactions between the phenolic hydroxyl groups in dopamine and the material surface. Figures 8A3 and A4 further demonstrate that the hydrogel has good adhesion to human finger skin tissue, maintaining stable adhesion even under repeated bending, showcasing its excellent adhesion and flexibility on human skin. The adhesion strength, tested using a universal testing machine, reaches 1.19 kPa. This adhesion performance stems from the high binding affinity of the pyrogallol groups within the hydrogel for various peptide and protein nucleophiles (such as amino and thiol groups) on the tissue surface. Figures 8A5 and A6 illustrate the close adhesion effect of the hydrogel under skin bending and torsion conditions, indicating its suitability for complex joint wounds. Figures 8 (B1-B6) illustrate simulated joint wound tearing during movement by applying different hydrogels. Figures 8 (B1, B3, and B5) show that the wound with acrylamide hydrogel applied to it tore after 100 joint flexions, and the hydrogel detached from the wound. Figure 8 Figures B2, B4, and B6 show that the PTPD hydrogel adhered to the wound surface even after 100 bending cycles, and the wound did not tear. This indicates that the shrinkage stress of the PTPD hydrogel can effectively counteract the tearing force and inhibit wound expansion, demonstrating good application potential.

[0066] 2.9 Thermal Response Analysis When the temperature exceeds the phase transition temperature (VPTT) of the polymer network, the poly(N-isopropylacrylamide) (PNIPAM) network undergoes a transition from hydrophilic to hydrophobic. At this point, the polymer molecular chains aggregate and exhibit more coiled conformations, leading to an overall hydrophobic trend in the polymer molecules. This change reduces the intermolecular distance in the cross-linked network, and due to the enhanced hydrophobic interaction, some water molecules are repelled from the network structure. Therefore, on a macroscopic level, the composite hydrogel exhibits a whitening effect and a shrinkage in volume. This invention characterizes the temperature-sensitive shrinkage properties of the hydrogel by simulating a human body temperature (37°C) environment, placing the hydrogel in a constant temperature and humidity incubator, and measuring its area change after a period of heat treatment.

[0067] Figure 9This study presents the comparative results of the area shrinkage rates of PT hydrogel, PTP hydrogel, PTD hydrogel, and PTPD hydrogel at 37℃. The results show that PT hydrogel exhibits the most significant area change, with a shrinkage rate of 69.7% within 24 hours. In contrast, PTP, PTD, and PTPD hydrogels all show significant area reduction, with shrinkage rates of 54.8%, 40.5%, and 36.0%, respectively.

[0068] This phenomenon can be attributed to the excellent hydrophilicity and linear structure of polyvinyl alcohol (PVA) in PTP hydrogels. This allows the gel to effectively adsorb more water molecules during solid hydrogel formation, with the water molecules occupying more voids. Consequently, the hydrogel expels more water below the low critical phase transition temperature (LCST), resulting in a higher area shrinkage rate. In contrast, dopamine gelatin (DAGel) modified hydrogels, due to their three-dimensional network structure, occupy a larger space and therefore exhibit a relatively lower area shrinkage rate.

[0069] 2.10 Biocompatibility Biocompatibility evaluation of hydrogel dressings is a necessary condition for their application as medical materials. The biocompatibility of hydrogel dressings was evaluated through cytotoxicity and blood compatibility tests.

[0070] In assessing cytotoxicity, this invention used the MTT assay and employed L929 mouse fibroblasts as the experimental cell line, using an extraction method for toxicity evaluation. Figure 2-10 As shown in Figure A, after co-culturing cells with extracts from PT hydrogel, PTP hydrogel, PTD hydrogel, and PTPD hydrogel samples, the cell viability exceeded 85%. This result reveals that the composite hydrogel possesses excellent cell compatibility and is suitable for applications in the medical field. These findings provide experimental evidence for further research on composite hydrogels as wound healing materials.

[0071] According to the biological evaluation standards for medical devices, the hemolysis rate of biomedical materials should be less than 5%. Figure 2-10 B shows that the hemolysis rates of the PT hydrogel, PTP hydrogel, PTD hydrogel, and PTPD hydrogel materials of the present invention are 3.08%, 3.50%, 3.39%, and 3.47%, respectively, all below 5%, indicating that the PT hydrogel, PTP hydrogel, PTD hydrogel, and PTPD hydrogel all have good blood compatibility.

[0072] 2.11 Analysis of in vivo wound healing In a standardized experimental model system based on full-thickness skin defects in mice, a rigorous controlled study was conducted to systematically evaluate the differences in wound healing efficacy between PTPD composite hydrogel and commercially available traditional dressings. Figure 11 The results showed that on day 3 post-traumatic injury, the wound healing rate of the PTPD composite hydrogel treatment group reached 29.29%, significantly higher than the 13.19% of the commercially available traditional dressing group, confirming its significant healing-promoting advantage in the early stages of trauma. As the repair process progressed, on day 11 post-traumatic injury, the wound healing rate of the PTPD composite hydrogel group continued to increase to 88.06%, while the healing rate of the commercially available traditional dressing group was only 80.52% at the same time. By day 14 post-traumatic injury, the wound in the PTPD composite hydrogel treatment group had nearly completely healed, with a healing rate as high as 99.78%, while the commercially available traditional dressing group still had significant scab tissue remaining, with a healing rate of 96.76%. These results indicate that the PTPD composite hydrogel is significantly superior to commercially available traditional dressings in accelerating wound healing and promoting wound closure, providing solid experimental evidence for its application in the field of wound repair.

[0073] A deeper exploration of its mechanism of action reveals that the highly efficient wound-healing properties of this composite hydrogel benefit from the synergistic effect of its multi-dimensional properties. On one hand, the NIPAM thermosensitive network undergoes a volume phase transition under physiological temperature conditions, exhibiting contractile behavior and actively providing directional traction force to the wound edges, thereby effectively promoting the early closure process of the wound. On the other hand, the physical entanglement structure formed between PVA molecular chains endows the material with excellent mechanical toughness, providing stable and continuous mechanical support throughout the wound healing process. In addition, the thermosensitive response characteristics of this composite hydrogel can dynamically regulate the humidity of the wound microenvironment, combined with the excellent water retention capacity of PVA, to create a moist environment conducive to cell proliferation, thereby accelerating fibroblast migration and orderly collagen deposition, ultimately promoting wound healing.

[0074] Example 2 Preparation of PTPD hydrogel: 0.05 g of dopamine-modified gelatin (DAGel) and 0.1 g of polyvinyl alcohol (PVA) were heated to 70 °C and dissolved in 5 mL of purified water. Then, 0.05 g of N-isopropylacrylamide (NIPAM) monomer and 0.1 g of N-[tris(hydroxymethyl)methyl]acrylamide (THMA) were added. Next, 1 mg of crosslinking agent N,N'-methylenebisacrylamide (MBA) was added under stirring at room temperature until completely dissolved. Then, 40 mg of initiator ammonium persulfate was added to the solution and stirred until homogeneous. After adding 20 µL of accelerator tetramethylethylenediamine (TEMED), the mixture was stirred rapidly to ensure homogeneity. The mixture was poured into a mold and placed in a refrigerator at 4 °C for 24 hours to complete polymerization. Finally, the obtained gel was soaked in purified water for 24 hours to remove unreacted monomers, thus obtaining the PTPD hydrogel.

[0075] Example 3 Preparation of PTPD hydrogel: 0.25 g of dopamine-modified gelatin (DAGel) and 0.3 g of polyvinyl alcohol (PVA) were heated to 70 °C and dissolved in 5 mL of purified water. Then, 1 g of N-isopropylacrylamide (NIPAM) monomer and 0.5 g of N-[tris(hydroxymethyl)methyl]acrylamide (THMA) were added. Next, 20 mg of crosslinking agent N,N'-methylenebisacrylamide (MBA) was added under stirring at room temperature until completely dissolved. Then, 40 mg of initiator ammonium persulfate was added to the solution and stirred until homogeneous. After adding 20 µL of accelerator tetramethylethylenediamine (TEMED), the mixture was stirred rapidly to ensure homogeneity. The mixture was poured into a mold and placed in a refrigerator at 4 °C for 24 hours to complete polymerization. Finally, the resulting gel was soaked in purified water for 24 hours to remove unreacted monomers, thus obtaining the PTPD hydrogel.

[0076] summary This invention utilizes the EDC coupling method to prepare DAGel, and introduces PVA and DAGel into the crosslinked network using NIPAM and THMA as monomers to enhance the mechanical and adhesive properties of the hydrogel, thus preparing a wound dressing that can accelerate wound healing. The chemical structure of the DAGel was characterized using UV-Vis, and the hydrogel formulation was optimized through single-factor and orthogonal experiments. Finally, the rheological properties, tensile properties, swelling properties, in vitro wound contraction properties, in vivo wound healing properties, and cytotoxicity were characterized.

[0077] The material system has the following advantages: (1) The thermal response characteristics of the material are expected to enable active wound closure and accelerate wound healing; (2) Through the incorporation of PVA chains and the multiple interactions between the phenolic hydroxyl groups of DAGel and the amino groups of PNIPAM chains, a network structure with good toughness and flexibility is constructed, which can closely fit the complex contour of the wound.

Claims

1. A thermo-responsive contractile hydrogel, characterized in that, Including dopamine-modified gelatin, polyvinyl alcohol, N-isopropylacrylamide, N-[tris(hydroxymethyl)methyl]acrylamide, N,N'-methylenebisacrylamide, initiators, and accelerators.

2. The thermo-responsive contractile hydrogel according to claim 1, characterized in that: 0.05 g to 0.25 g dopamine-modified gelatin, 0.1 g to 0.3 g polyvinyl alcohol, 0.25 g to 1 g N-isopropylacrylamide monomer, 0.05 g to 0.5 g N-[tris(hydroxymethyl)methyl]acrylamide, N,N'-methylenebisacrylamide at 0.1 wt% to 1 wt% of the mass of N-isopropylacrylamide monomer, appropriate amount of initiator and appropriate amount of accelerator.

3. The thermo-responsive contractile hydrogel according to claim 1, characterized in that: 0.1 g dopamine-modified gelatin, 0.15 g polyvinyl alcohol, 0.75 g N-isopropylacrylamide monomer, 0.1 g N-[tris(hydroxymethyl)methyl]acrylamide, 4 mg N,N'-methylenebisacrylamide, 40 mg initiator and 15.5 mg accelerator.

4. The thermo-responsive contractile hydrogel according to claim 3, characterized in that: The initiator is ammonium persulfate or potassium persulfate, and the accelerator is tetramethylethylenediamine, N,N-dimethylaniline, sodium bisulfite, or triethanolamine.

5. A thermo-responsive contractile hydrogel according to any one of claims 1 to 3, wherein the dopamine-modified gelatin is prepared by the following steps: adding gelatin to MES buffer, heating and stirring for 30 minutes until the gelatin is completely dissolved; cooling the gelatin solution to 37°C, adding 0.5 g EDC and 0.3 g NHS, and adjusting the pH to 5.4; continuing to stir for 30 minutes, then dissolving 0.5 g dopamine hydrochloride in 2 mL of deionized water and slowly adding it dropwise to the above solution; maintaining the reaction mixture at 37°C and a pH between 5.0 and 6.0 for 24 hours to obtain dopamine-modified gelatin.

6. A method for preparing a body temperature-responsive contractile hydrogel, characterized in that, To achieve this, follow these steps: Dopamine-modified gelatin and polyvinyl alcohol were dissolved in water by heating. N-isopropylacrylamide monomer and N-[tris(hydroxymethyl)methyl]acrylamide were added. N,N'-methylenebisacrylamide was added under stirring at room temperature and stirred until completely dissolved. Ammonium persulfate was added to the solution and stirred evenly. After adding the accelerator, the mixture was stirred rapidly. The mixture was poured into a mold and placed at 4°C for 24 hours to complete the polymerization and obtain a gel.

7. The method according to claim 6, characterized in that, The following steps were performed: 0.1 g of dopamine-modified gelatin and 0.15 g of polyvinyl alcohol were heated to 70°C and dissolved in 5 mL of purified water. Then, 0.75 g of N-isopropylacrylamide monomer and 0.1 g of N-[tris(hydroxymethyl)methyl]acrylamide were added. Next, 4 mg of crosslinking agent N,N'-methylenebisacrylamide was added under stirring at room temperature and stirred until completely dissolved. Then, 40 mg of initiator ammonium persulfate was added to the solution and stirred until homogeneous. After adding 15.5 mg of accelerator tetramethylethylenediamine, the mixture was stirred rapidly to ensure homogeneity. The mixture was poured into a mold and placed in a refrigerator at 4°C for 24 hours to complete the polymerization. Finally, the obtained gel was soaked in purified water for 24 hours to remove unreacted monomers, thereby obtaining a hydrogel.

8. The application of the thermoresponsive contractile hydrogel according to any one of claims 1 to 5 in the preparation of wound dressings.

9. The application according to claim 8, characterized in that, The wound can be an acute or chronic wound.

10. The application according to claim 9, characterized in that: The acute wounds are those caused by trauma, burns, scalds, or surgery; the chronic wounds are those caused by ulcers, bedsores, leg ulcers, diabetic foot ulcers, or radiation-induced wounds.

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