A double-sided hydrogel with hemostatic, anti-adhesion and wound repair properties, its preparation and application

Abstract

This invention discloses a double-sided hydrogel with hemostatic, anti-adhesion, and wound repair properties, as well as its preparation and application, belonging to the field of biomedical materials technology. The double-sided hydrogel comprises a repair layer and an anti-adhesion layer. The repair layer is a hydrogel formed by photocuring with OHA, GelMA, AAc, AAc-NHS, and p(APMA-co-THMA) as core components. p(APMA-co-THMA) is obtained by polymerizing THMA and APMA. The anti-adhesion layer is formed by immersing one side of the repair layer in an aqueous solution containing SBMA, GelMA, and MBAA, followed by a thermal cross-linking reaction. The double-sided hydrogel provided by this invention possesses ideal mechanical properties and wet adhesion ability, and exerts anti-inflammatory and wound-healing effects on liver wounds, which can solve the problems of postoperative bleeding, adhesion, and liver regeneration disorders after partial hepatectomy in clinical practice.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a double-sided hydrogel that combines hemostasis, anti-adhesion, and wound repair, as well as its preparation and application. Background Technology

[0002] In the treatment of liver diseases, partial hepatectomy is a crucial procedure widely used to treat various serious liver conditions, including liver tumors and intrahepatic bile duct stones. While this surgery is remarkably effective in saving patients' lives, it also faces numerous challenges, particularly complications such as bleeding, adhesions, and impaired liver regeneration. These issues significantly increase the difficulty of clinical treatment and have a profound impact on patient prognosis. Numerous clinical studies have shown a high incidence of complications after hepatectomy. Reports indicate that the bleeding rate after partial hepatectomy can be as high as 10.2%, and the incidence of postoperative adhesions ranges from 20% to 30%.

[0003] Postoperative bleeding can lead to hemorrhagic shock, increase the need for blood transfusions, significantly prolong hospital stays, and even endanger the patient's life. Wound healing is a complex biological process involving hemostasis, inflammation, cell proliferation, and tissue remodeling. Therefore, the rapid formation of a hemostatic barrier is the first priority in emergency care. For hemostasis, medical hydrogel adhesives such as fibrin glue have shown advantages such as portability, ease of use, hemostatic effect, biocompatibility, and biodegradability, and are widely used in clinical practice. However, because they are derived from human plasma, there is a potential risk of viral transmission, and they are relatively expensive.

[0004] In addition, the biggest challenge in in vivo wound repair is the interaction between wet tissue surfaces and different organs in a continuously dynamic in vivo environment. Most hydrogels mentioned in previous studies have shown uniform adhesion properties, but their indiscriminate adhesion can lead to postoperative adhesion problems, which may cause a series of problems such as intestinal obstruction and chronic abdominal pain, further increasing the risk of secondary surgery and seriously affecting the patient's quality of life.

[0005] This requires that the side of the hydrogel adjacent to the injured tissue should have good bioadhesion and bioactivity, providing mechanical support and a favorable microenvironment for tissue repair; the other side should ideally be non-adhesive to surrounding tissues to physically prevent the formation of fibrotic scars. For example, Zhang Liming's research group developed a Jauns hydrogel patch that simultaneously possesses adhesive and anti-adhesive properties. This patch combines an adhesive hydrogel (HGO) and a non-adhesive hydrogel (CGC) to form a hydrogel with asymmetric adhesion properties on both sides (HGO-C), used for emergency hemostasis and wound healing (Novel Natural Polymer-Based Hydrogel Patches with Janus Asymmetric-Adhesion for Emergency Hemostasis and Wound Healing). Advanced Functional Materials , 2024, 34(36).).

[0006] Furthermore, impaired liver regeneration affects the recovery of liver function and may lead to liver failure, further increasing the mortality and morbidity of patients.

[0007] Clinically, there is a lack of hydrogel materials for preventing complications after partial hepatectomy, and published research on hydrogel materials has limited functionality. For example, while hemostatic hydrogels can control bleeding to some extent, they have little effect on preventing adhesions and promoting liver regeneration; anti-adhesion materials mainly focus on reducing tissue adhesions, and their effects on hemostasis and liver regeneration are not good.

[0008] Therefore, developing a medical material that integrates hemostasis, regeneration, and anti-adhesion functions has become an urgent and pressing need. Summary of the Invention

[0009] The purpose of this invention is to provide a biomedical material for hemostasis, anti-adhesion and wound repair after partial liver resection, so as to overcome the above and / or other potential problems existing in the prior art.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a double-sided hydrogel that combines hemostasis, anti-adhesion, and wound repair functions. The double-sided hydrogel includes a repair layer and an anti-adhesion layer. The repair layer (RL) is a hydrogel formed by photocuring with oxidized hyaluronic acid (OHA), gelatin methacrylate (GelMA), acrylic acid (AAc), N-hydroxysuccinimide acrylate (AAc-NHS) and p(APMA-co-THMA) as the core components. The p(APMA-co-THMA) is obtained by polymerization of N-[tris(hydroxymethyl)methyl]acrylamide (THMA) and N-(3-aminopropyl)methacrylamide hydrochloride (APMA). The anti-adhesion layer (AAL) is formed by immersing one side of the repair layer in an aqueous solution containing [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (SBMA), gelatin methacrylate (GelMA), N,N-methylenebisacrylamide (MBAA) and an initiator, followed by a thermal crosslinking reaction.

[0011] The double-sided hydrogel provided by this invention has a double-layer structure. The repair layer has excellent tissue adhesion and can achieve hemostasis and promote healing by being applied to the wound. The anti-adhesion layer can prevent postoperative tissue adhesion.

[0012] Specifically, the repair layer uses oxidized hyaluronic acid, gelatin methacrylate, acrylic acid, N-hydroxysuccinimide acrylate, and p(APMA-co-THMA) as its core components. These components work synergistically to give the hydrogel strong hemostatic and repair-promoting functions. Oxidized hyaluronic acid (OHA) is prepared by modifying hyaluronic acid (HA) with sodium periodate (NaIO4). It not only retains the excellent biological properties and repair-promoting ability of hyaluronic acid (HA), but its aldehyde group can also form Schiff bond with the amino group of the system, thereby improving the mechanical stability of the hydrogel. Gelatin methacrylate (GelMA) is derived from collagen and rapidly cross-links into a gel under ultraviolet light mediated by photoinitiators (such as lithium phenyl-2,4,6-trimethylbenzoyl phosphate LAP). It can not only build an immediate protective barrier for liver wounds, but also significantly promote cell proliferation. p(APMA-co-THMA) enhances tissue adhesion through multiple hydrogen bonds, while acrylic acid (AAc) constructs a stable three-dimensional network through free radical copolymerization. N-hydroxysuccinimide acrylate (AAc-NHS) further strengthens interfacial adhesion and network stability through the covalent reaction between the N-hydroxysuccinimide (NHS) ester group and the amino group of liver tissue. Studies have confirmed that N-hydroxysuccinimide (NHS) can form stable covalent bonds with the amino group on the tissue surface and achieve strong adhesion through the hydrogen bonding of carboxyl and hydroxyl groups, laying a solid foundation for efficient hemostasis and wound repair.

[0013] The anti-adhesion layer (AAL) is mainly composed of [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (SBMA). Its unique hydrophilicity can form a dense hydration layer on the surface of the hydrogel, effectively preventing the adhesion of the tissues around the liver to the hydrogel.

[0014] The aforementioned materials are biocompatible, providing material support for the realization of hydrogel functions.

[0015] In this invention, the repair layer uses oxidized hyaluronic acid, gelatin methacrylate, acrylic acid, N-hydroxysuccinimide acrylate and p(APMA-co-THMA) as core components, which crosslink into a gel under light irradiation mediated by a photoinitiator.

[0016] In this invention, oxidized hyaluronic acid is prepared by modifying hyaluronic acid (HA) with sodium periodate (NaIO4). The preparation method includes: dissolving hyaluronic acid in water, then adding sodium periodate aqueous solution dropwise. After the addition is complete, the mixture is placed at room temperature in the dark and stirred continuously. Then, ethylene glycol is added to the system to remove excess sodium periodate. Subsequently, the reaction solution is transferred to a dialysis bag with a molecular weight cutoff of 3500 Da for dialysis. After the dialysis solution is dried, oxidized hyaluronic acid is obtained.

[0017] In this invention, the preparation method of methacrylic gelatin includes: adding gelatin to phosphate buffer and stirring until completely dissolved. When the gelatin solution is homogeneous and transparent, methacrylic anhydride (MA) is slowly added dropwise to the system while continuously stirring. After the reaction is completed, the solution is transferred to a dialysis bag with a molecular weight cutoff of 3500 Da for dialysis. After the dialysis solution is dried, methacrylic gelatin is obtained.

[0018] In this invention, p(APMA-co-THMA) is prepared by polymerization of N-[tris(hydroxymethyl)methyl]acrylamide (THMA) and N-(3-aminopropyl)methacrylamide hydrochloride (APMA). The preparation method includes dissolving N-[tris(hydroxymethyl)methyl]acrylamide and N-(3-aminopropyl)methacrylamide hydrochloride in water, and then adding an initiator to initiate the monomer polymerization reaction.

[0019] Preferably, the mass ratio of N-[tris(hydroxymethyl)methyl]acrylamide to N-(3-aminopropyl)methacrylamide hydrochloride is 51-62:10-16, and the two undergo polymerization under the action of an initiator. The initiator can be, but is not limited to, ammonium persulfate.

[0020] More preferably, the mass ratio of N-[tris(hydroxymethyl)methyl]acrylamide to N-(3-aminopropyl)methacrylamide hydrochloride is 55:14.

[0021] In this invention, the photoinitiator is an ultraviolet photoinitiator, which may be, but is not limited to, lithium phenyl-2,4,6-trimethylbenzoyl phosphate (LAP).

[0022] Preferably, the mass ratio of gelatin methacrylate, acrylic acid, N-hydroxysuccinimide acrylate, and p(APMA-co-THMA) is 1-3:200-400:10-30:1-3; the mass ratio of oxidized hyaluronic acid to acrylic acid is 1-4:45-70. Studies have shown that the hydrogel prepared under the above formulation conditions has good compressive and tensile strength as well as excellent wet tissue adhesion properties, and the addition of oxidized hyaluronic acid helps to improve the mechanical and adhesive properties of the hydrogel.

[0023] More preferably, the mass ratio of gelatin methacrylate, acrylic acid, N-hydroxysuccinimide acrylate, and p(APMA-co-THMA) is 1:150:10:1. The mass ratio of oxidized hyaluronic acid to acrylic acid is 1:45-67.5.

[0024] In one specific embodiment of the present invention, the mass ratio of oxidized hyaluronic acid, gelatin methacrylate, acrylic acid, N-hydroxysuccinimide acrylate and p(APMA-co-THMA) is 20:9:1350:90:9.

[0025] In this invention, the anti-adhesion layer uses [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (SBMA) as the main component, and gelatin methacrylate (GelMA) as a crosslinkable biomacromolecule. GelMA contains polymerizable double bonds and copolymerizes and crosslinks with SBMA and MBAA under initiation conditions to jointly construct a stable three-dimensional network, improving gel strength, formability, and stability. N,N-methylenebisacrylamide (MBAA) serves as a chemical crosslinking agent, forming a three-dimensional hydrogel network through free radical polymerization and covalent crosslinking under the thermal initiation mediated by the initiator. The initiator can be, but is not limited to, ammonium persulfate.

[0026] Specifically, the repair layer hydrogel was immersed on one side in an aqueous solution containing SBMA, GelMA, MBAA, and an initiator, and a thermal cross-linking reaction was carried out to form an anti-adhesion layer. Studies have shown that the repair layer and the anti-adhesion layer effectively bond together as a whole through immersion and thermal cross-linking.

[0027] In this invention, GelMA, as a crosslinkable biomacromolecule, enhances the biocompatibility of the material and the bonding strength of the bilayer interface; MBAA, as a chemical crosslinking agent, constructs a stable three-dimensional network through double bond copolymerization, ensuring gel formation and mechanical properties. SBMA's unique hydrophilicity allows it to form a dense hydrated layer on the hydrogel surface, imparting anti-adhesion properties.

[0028] Preferably, the mass ratio of [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, gelatin methacrylate, and N,N-methylenebisacrylamide is 95-103:8-13:1-5. Studies have shown that the hydrogel prepared under the above ratio conditions has good anti-adhesion properties.

[0029] More preferably, the mass ratio of [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, gelatin methacrylate, and N,N-methylenebisacrylamide is 100:10:3.

[0030] Another object of the present invention is to provide a method for preparing the double-sided hydrogel, the method comprising the following steps: (1) N-[tris(hydroxymethyl)methyl]acrylamide and N-(3-aminopropyl)methacrylamide hydrochloride were dissolved in water and polymerized under the action of an initiator to obtain p(APMA-co-THMA); (2) Dissolve p(APMA-co-THMA), gelatin methacrylate and photoinitiator in water, then add acrylic acid and N-hydroxysuccinimide acrylate to dissolve and obtain RL precursor solution. Then mix RL precursor solution with oxidized hyaluronic acid solution and place it under 365 nm ultraviolet light to crosslink and form repair layer gel. (3) The repair layer gel is immersed on one side in an aqueous solution containing [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, gelatin methacrylate, N,N-methylenebisacrylamide and an initiator, and thermal crosslinking is performed to form an anti-adhesion layer, thereby obtaining the double-sided hydrogel.

[0031] In step (1), N-[tris(hydroxymethyl)methyl]acrylamide (THMA) and N-(3-aminopropyl)methacrylamide hydrochloride (APMA) undergo monomer polymerization under the action of an initiator to obtain p(APMA-co-THMA).

[0032] Preferably, in the polymerization reaction system, the concentration of N-[tris(hydroxymethyl)methyl]acrylamide is 110-135 mg / mL; and the concentration of N-(3-aminopropyl)methacrylamide hydrochloride is 25-45 mg / mL.

[0033] Preferably, the initiator is ammonium persulfate, and the polymerization reaction conditions are: heating to 60°C for 1 hour, then heating to 70°C for 7 hours.

[0034] Preferably, after the reaction is complete, dialysis is performed using a dialysis bag with a molecular weight cutoff of 3500 Da.

[0035] In step (2), oxidized hyaluronic acid, gelatin methacrylate, acrylic acid, N-hydroxysuccinimide acrylate and p(APMA-co-THMA) crosslink into a gel under light irradiation mediated by a photoinitiator.

[0036] Preferably, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoyl phosphate; the photocuring conditions are: crosslinking under 365 nm ultraviolet light for 20-30 min.

[0037] Preferably, the concentrations of p(APMA-co-THMA), gelatin methacrylate, lithium phenyl-2,4,6-trimethylbenzoyl phosphate, acrylic acid, and N-hydroxysuccinimide acrylate in the RL precursor solution are 0.001-0.003 g / mL, 0.001-0.004 g / mL, 0.001-0.005 g / mL, 0.20-0.40 g / mL, and 0.01-0.03 g / mL, respectively.

[0038] More preferably, the concentrations of p(APMA-co-THMA), gelatin methacrylate, lithium phenyl-2,4,6-trimethylbenzoyl phosphate, acrylic acid, and N-hydroxysuccinimide acrylate in the RL precursor solution are 0.002 g / mL, 0.002 g / mL, 0.002 g / mL, 0.30 g / mL, and 0.02 g / mL, respectively.

[0039] Preferably, the mass percentage concentration of the oxidized hyaluronic acid solution is 2-4%.

[0040] Preferably, the volume ratio of the RL precursor solution to the oxidized hyaluronic acid solution is 9:1, 8:2, 7:3, or 6:4.

[0041] Preferably, the molecular weights of methacrylic gelatin and oxidized hyaluronic acid are both ≥3500 Da.

[0042] Preferably, after photocuring, the resulting gel is soaked in deionized water and then dried at 35-45°C for 15-25 minutes to obtain the repair layer.

[0043] In step (3), the repair layer gel is immersed on one side in an aqueous solution containing [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, gelatin methacrylate, and a crosslinking agent, and thermally crosslinked under the initiator to form an anti-adhesion layer.

[0044] Preferably, the concentrations of [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, gelatin methacrylate, and N,N-methylenebisacrylamide in the aqueous solution are 0.15-0.25 g / mL, 0.01-0.04 g / mL, and 0.004-0.008 g / mL, respectively.

[0045] More preferably, the concentrations of [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, gelatin methacrylate, and N,N-methylenebisacrylamide in the aqueous solution are 0.2 g / mL, 0.02 g / mL, and 0.006 g / mL, respectively.

[0046] Preferably, the initiator is ammonium persulfate, and the thermal crosslinking conditions are 50-70°C for 50-80 min.

[0047] More preferably, the thermal crosslinking conditions are 60°C for 60 min.

[0048] This invention prepares a double-sided hydrogel with synergistic effects of hemostasis, anti-adhesion, and wound repair. This hydrogel possesses excellent mechanical properties and significant asymmetric adhesion properties, with one side exhibiting strong adhesion and the other side exhibiting anti-adhesion. It also demonstrates superior mechanical properties and antibacterial activity, exhibiting good contact inhibition against Gram-negative bacteria (such as E. coli) and Gram-positive bacteria (such as S. aureus). Furthermore, this hydrogel exhibits excellent hemostatic effects and can be used to prepare tissue wound repair materials. While repairing wounded tissue, this hydrogel can prevent adhesion to surrounding tissues.

[0049] Another object of the present invention is to provide the application of the aforementioned double-sided hydrogel in the preparation of tissue trauma repair materials after partial hepatectomy.

[0050] Studies have shown that the double-sided hydrogel provided by this invention possesses ideal mechanical properties and wet adhesion capabilities, and exerts anti-inflammatory and repair-promoting effects on liver wounds. The double-sided hydrogel provided by this invention can solve clinical complications such as bleeding, adhesion, and impaired liver regeneration after partial hepatectomy; moreover, this material is slowly biodegradable while maintaining long-lasting adhesion.

[0051] The beneficial effects of this invention are as follows: This invention innovatively designs a double-sided hydrogel that combines liver wound repair and anti-tissue adhesion functions. It is prepared by thermal cross-linking a repair layer (RL) and an anti-adhesion layer (AAL) at a specific temperature. The RL's core components are OHA, GelMA, AAc, AAc-NHS, and p(APMA-co-THMA), while the AAL's main component is SBMA. These components synergistically endow the product with multiple core advantages, providing a comprehensive solution for recovery after partial hepatectomy. OHA retains the excellent biological properties of HA and enhances mechanical stability by forming Schiff bonds between aldehyde and amino groups. p(APMA-co-THMA) enhances tissue adhesion through multiple hydrogen bonds. AAc constructs a stable three-dimensional network. AAc-NHS strengthens adhesion and network stability by forming covalent bonds between NHS ester groups and amino groups in liver tissue. Combined with the cell proliferation-promoting effect of GelMA, RL achieves strong tissue adhesion, excellent hemostasis and antibacterial activity by relying on dense hydrogen bonds, positive charge effect and Schiff bond cross-linking structure. At the same time, it effectively promotes hepatocyte proliferation and liver wound healing. AAL utilizes the unique hydrophilicity of SBMA to form a hydration layer, effectively inhibiting adhesion of tissues surrounding the liver. Animal experiments have shown that the double-sided hydrogel provided by this invention can significantly reduce the occurrence of acute bleeding after partial hepatectomy in rats, significantly inhibit the formation of intra-abdominal adhesions, and effectively promote hepatocyte proliferation and liver tissue regeneration.

[0052] This invention comprehensively addresses key clinical pain points such as postoperative bleeding, adhesions, and slow wound healing, providing reliable biomedical material support for improving surgical efficacy and patient prognosis. Attached Figure Description

[0053] Figure 1 Scanning electron microscope (SEM) images of the bilayer hydrogel prepared in Example 7 and its component hydrogels RL and AAL after freeze-drying.

[0054] Figure 2 The compressive stress-strain curves of the repair layer hydrogel and double-sided hydrogel prepared in Examples 1-7 are shown.

[0055] Figure 3 The tensile stress-strain curves are for the repair layer hydrogel and double-sided hydrogel prepared in Examples 1-7.

[0056] Figure 4 The stress-displacement curves are obtained from the shear test of the hydrogel overlap of the repair layer prepared in Examples 1-6.

[0057] Figure 5 Images of live / dead L929 cells incubated with the hydrogel extracts prepared in Examples 2 and 7.

[0058] Figure 6 The hemolysis rate diagrams are for the hydrogels prepared in Examples 2 and 7.

[0059] Figure 7 The figures show a comparison of the antibacterial effects of the hydrogels prepared in Examples 2, 7, and 1.

[0060] Figure 8 The diagram shows the skin degradation test results of the hydrogels prepared in Examples 2 and 7.

[0061] Figure 9 Migration map of mouse AML12 cells in the hydrogels prepared for Examples 2 and 7.

[0062] Figure 10 HE staining images of the hydrogels prepared in Examples 2 and 7 demonstrating their anti-adhesion function in vivo.

[0063] Figure 11 The image shows the amount of bleeding 3 minutes after partial hepatectomy for the hydrogel prepared in Example 7, the control group, and the gelatin sponge.

[0064] Figure 12 Immunohistochemical images of Ki67, PCNA, Cyclin D1, and CD68, and immunofluorescence images of CD31 / αSMA, in the liver resection margin tissue of the sham surgery group, control group, gelatin sponge group, and double-sided adhesive treatment group prepared in Example 7 on day 7 after partial hepatectomy in a partial hepatectomy model. Detailed Implementation

[0065] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0066] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0067] The acrylic acid (AAc, >99%), N-hydroxysuccinimide acrylate (AAc-NHS, >98%), lithium phenyl-2,4,6-trimethylbenzoyl phosphite (LAP, >98%), N-(3-aminopropyl)methacrylamide hydrochloride (APMA, >98%), N-[tris(hydroxymethyl)methyl]acrylamide (THMA, 93%), gelatin, methacrylic anhydride (MA, 94%), [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (SBMA, >97%), and sodium periodate (NaIO4, 99.5%) used in the following examples were from Aladdin Biochemical Technology Co., Ltd. (China). Sodium hyaluronate (HA, 97%), ammonium persulfate (APS, 99.99%), and N,N-methylenebisacrylamide (MBAA, 99%) were supplied by Shanghai Maclean Biochemical Co., Ltd. (China). All reagents were of analytical grade as required.

[0068] Example 1 This embodiment prepares a repair layer (RL) with p(APMA-co-THMA), gelatin methacrylate (GelMA), acrylic acid (AAc), and N-hydroxysuccinimide acrylate (AAc-NHS) as the core components. The specific steps are as follows: 1. Synthesis of p(APMA-co-THMA) 1.21 g of N-[tris(hydroxymethyl)methyl]acrylamide (THMA) and 308 mg of N-(3-aminopropyl)methacrylamide hydrochloride (APMA) were added to 10 mL of deionized water; subsequently, 46 mg of ammonium persulfate (APS) was added, and the temperature was raised to 60°C to initiate the monomer polymerization reaction. After 1 h of reaction, the system temperature was adjusted to 70°C and the reaction was continued. The polymerization was terminated after 7 h. After the reaction was completed, the product was transferred to a dialysis bag (3500 Da) and purified by dialyzing in deionized water for 3 days. After dialysis, p(APMA-co-THMA) was obtained by freeze-drying.

[0069] 2. Synthesis of Gelatin Methacrylate (GelMA) 10 g of gelatin (CAS: 9000-70-8) was added to 100 mL of phosphate-buffered saline (PBS) and stirred at 60 °C until completely dissolved. Once the gelatin solution became homogeneous and transparent, 6 mL of methacrylic anhydride (MA) was slowly added dropwise to the system while continuously stirring to ensure uniform monomer dispersion. After reacting for 1 h, the product was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and purified by dialysis in deionized water for 3 days. After dialysis, the GelMA solution was freeze-dried and stored for later use.

[0070] 3. Synthesis of RL Take 0.02 g of p(APMA-co-THMA), 0.02 g of GelMA and lithium phenyl-2,4,6-trimethylbenzoyl phosphate (LAP), add them to 10 g of deionized water, and stir at 40 °C until completely dissolved. LAP acts as a photoinitiator, causing the solution to gel under ultraviolet light. Then add 3 g of acrylic acid (AAc) and 0.2 g of N-hydroxysuccinimide acrylate (AAc-NHS), and continue stirring until completely dissolved to obtain the RL precursor solution.

[0071] Take 1 mL of RL precursor solution and crosslink it under 365 nm UV light for 30 min to form a gel. The resulting gel is soaked in deionized water to remove toxic monomers and dried at 40 °C for 20 min to obtain RL.

[0072] Example 2 This embodiment prepares a repair layer (RL) with oxidized hyaluronic acid (OHA), p(APMA-co-THMA), gelatin methacrylate (GelMA), acrylic acid (AAc), and N-hydroxysuccinimide acrylate (AAc-NHS) as its core components. The specific steps are as follows: 1. Synthesis of Oxidized Hyaluronic Acid (OHA) First, 2.0 g of HA (molecular weight 1.0 MDa~1.8 MDa) was dispersed in 100 mL of deionized water at a concentration of 10 mg / mL and stirred at room temperature until completely dissolved. Then, 5 mL of a 0.5 mol / L sodium periodate aqueous solution was added dropwise to the HA solution. After the addition was complete, the mixture was stirred continuously at room temperature in the dark for 2 h. After the predetermined reaction time was reached, 1 mL of ethylene glycol was added to the system and stirred at room temperature for 1 h. The reaction solution was then transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed for 3 days. After dialysis, the OHA solution was freeze-dried.

[0073] Take 10 g of deionized water and add 0.4 g of OHA to prepare an OHA solution with a mass fraction of 4%.

[0074] 2. Composition of RL The preparation of the RL precursor solution was the same as in Example 1.

[0075] 0.1 mL of OHA solution was added to 0.9 mL of RL precursor solution. The mixture was placed under 365 nm UV light for 30 min to crosslink and form a gel. The resulting gel was soaked in deionized water to remove toxic monomers and dried at 40 °C for 20 min to obtain R9O1.

[0076] Example 3 This embodiment provides a repair layer (RL) prepared by oxidized hyaluronic acid (OHA) and RL precursor solution under different ratio conditions. The specific steps are as follows: 1. The synthesis of oxidized hyaluronic acid (OHA) is the same as in Example 2.

[0077] 2. Composition of RL The preparation of the RL precursor solution was the same as in Example 1.

[0078] 0.2 mL of OHA solution was added to 0.8 mL of RL precursor solution. The mixture was placed under 365 nm UV light for 30 min to crosslink and form a gel. The resulting gel was soaked in deionized water to remove toxic monomers and dried at 40 °C for 20 min to obtain R8O2.

[0079] Example 4 This embodiment provides a repair layer (RL) prepared by oxidized hyaluronic acid (OHA) and RL precursor solution under different ratio conditions. The specific steps are as follows: 1. The synthesis of oxidized hyaluronic acid (OHA) is the same as in Example 2.

[0080] 2. Composition of RL The preparation of the RL precursor solution was the same as in Example 1.

[0081] 0.3 mL of OHA solution was added to 0.7 mL of RL precursor solution. The mixture was placed under 365 nm UV light for 30 min to crosslink and form a gel. The resulting gel was soaked in deionized water to remove toxic monomers and dried at 40 °C for 20 min to obtain R7O3.

[0082] Example 5 This embodiment provides a repair layer (RL) prepared by oxidized hyaluronic acid (OHA) and RL precursor solution under different ratio conditions. The specific steps are as follows: 1. The synthesis of oxidized hyaluronic acid (OHA) is the same as in Example 2.

[0083] 2. Composition of RL The preparation of the RL precursor solution was the same as in Example 1.

[0084] 0.4 mL of OHA solution was added to 0.6 mL of RL precursor solution. The mixture was placed under 365 nm UV light for 30 min to crosslink and form a gel. The resulting gel was soaked in deionized water to remove toxic monomers and dried at 40 °C for 20 min to obtain R6O4.

[0085] Example 6 This embodiment provides a repair layer (RL) prepared by oxidized hyaluronic acid (OHA) and RL precursor solution under different ratio conditions. The specific steps are as follows: 1. The synthesis of oxidized hyaluronic acid (OHA) is the same as in Example 2.

[0086] 2. Composition of RL The preparation of the RL precursor solution was the same as in Example 1.

[0087] Add 0.5 mL of OHA solution to 0.5 mL of RL precursor solution. Place the mixture under 365 nm UV light for 30 min to crosslink and form a gel. Immerse the resulting gel in deionized water to remove toxic monomers, and dry at 40 °C for 20 min to obtain R5O5.

[0088] Example 7 This embodiment provides a method for preparing a double-sided hydrogel (DAR) by thermal crosslinking of a repair layer (RL) and an anti-adhesion layer (AAL), specifically the following steps: 1. The synthesis of RL is the same as in Example 2.

[0089] 2. Synthesis of double-sided tape (DAR) Take 2 g of [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (SBMA), 0.2 g of GelMA, 0.06 g of N,N-methylenebisacrylamide (MBAA) and 0.1 g of APS and add them to 10 g of deionized water. Stir well to obtain AAL solution.

[0090] Dry RL (R9O1) was placed in a mold, immersed in AAL solution on one side for 30 min, and then thermally crosslinked at 60 °C for 60 min to obtain DAR. AAL hydrogel formed by thermal crosslinking with AAL solution alone was used as a control.

[0091] Performance Test Example 1: Microstructure Characterization To evaluate the microstructure of DAR, RL, and AAL, the bilayer hydrogel prepared in Example 7 and its components (RL and AAL) were frozen at -80°C for 12 hours, and then freeze-dried to obtain dried samples. Their microstructure was observed using a scanning electron microscope (SEM, TM-3000, HITACHI).

[0092] SEM results ( Figure 1 The results show that AAL exhibits a loose, porous structure, while RL has a smooth, flat surface. The surface structure of DAR changes from smooth and flat to loose and porous, indicating that AAL and RL are effectively bonded together as a whole through immersion and thermal cross-linking.

[0093] Performance Test Example 2: Mechanical Performance Test To evaluate the mechanical properties of the hydrogels prepared in Examples 1-6 and the DAR prepared in Example 7, tensile and compression tests were performed on them. Each test for each sample was repeated 5 times.

[0094] For tensile testing, a soft matter mechanical testing machine (INSTRON) equipped with a 10 N force sensor was used. In the tensile test, the patches were tested in a dumbbell shape at a constant displacement rate of 20 mm / min. The length, width, and thickness of each hydrogel were 75 mm × 4 mm × 2 mm.

[0095] For the compression test, a soft matter mechanical testing machine (INSTRON) equipped with a 1000 N force sensor was used. In the compression test, the sample was tested in a cylindrical shape and at a constant displacement rate of 20 mm / min.

[0096] Figure 2 The compressive stress-strain curves of the hydrogels prepared in Examples 1-7 are shown. Figure 3 The tensile stress-strain curves of the hydrogels prepared in Examples 1-7 show that Examples 2 and 7 have superior compressive and tensile strengths. Figure 2 It can be seen that the hydrogel prepared in Example 2 has better compressive strength than that in Example 1, indicating that the addition of oxidized hyaluronic acid improves the mechanical properties of the hydrogel.

[0097] Performance Test Example 3: Adhesion Performance Test To determine the adhesion properties of the hydrogels prepared in Examples 1-6, an overlap shear test was conducted. Pigskin was cut to a size of 25 mm × 75 mm, and RL was glued onto two pieces of pigskin, with an overlap size of 25 mm × 30 mm. Using a soft matter mechanical testing machine (INSTRON) equipped with a 1000 N force sensor, the two pieces of pigskin were clamped at opposite ends of the machine's fixtures, and the test was conducted at a constant displacement rate of 20 mm / min.

[0098] The formula for calculating the lap shear strength is as follows: Lap-stress = F max / (w×l); where F max It is the greatest force during the overlapping shearing process, and w and l are the width and length of the hydrogel.

[0099] For the wet tissue adhesion properties of each embodiment, the surfaces of two pieces of pigskin were moistened with a certain amount of liquid, and then the hydrogel was adhered to the two pieces of pigskin. The two pieces of pigskin were clamped at both ends of the fixture of the testing machine, and the process was carried out at a constant displacement rate of 20 mm / min.

[0100] The results are as follows Figure 4As shown, in the dry pigskin overlap shear test, Examples 1-5 all exhibited better adhesion than commercially available fibrin glue (commonly used for intraoperative adhesion and hemostasis), with the repair layer (RL) hydrogel corresponding to Example 2 showing the best adhesion performance. The adhesion performance of the hydrogel prepared in Example 2 was better than that in Example 1, indicating that the addition of oxidized hyaluronic acid improved the wet tissue adhesion performance of the hydrogel.

[0101] Performance Test Example 4: In Vitro Cell Compatibility and Antibacterial Test 1. In vitro cell compatibility To evaluate the in vitro cell compatibility of the single hydrogel prepared in Example 2 and the double-sided hydrogel (DAR) prepared in Example 7, the two materials were first placed in complete culture medium (containing 89% DMEM, 10% FBS and 1% antibiotics) and extracted at 37°C for 24 h to prepare a culture medium containing hydrogel extract. Then, L929 cells were treated with the extract culture medium and pure complete culture medium respectively, and co-cultured in parallel.

[0102] In the Live / Dead staining assay, cells were stained with live / dead fluorescent dyes at fixed time points on days 1 and 3 of co-culture, and the stained images were subsequently acquired and observed using a confocal microscope.

[0103] Depend on Figure 5 It can be seen that the hydrogel prepared in Example 2 and the L929 cells incubated in DAR medium prepared in Example 7 exhibited normal morphology, and the cell viability and proliferation behavior trends were similar to those of the control group.

[0104] 2. Hemolysis test The hemolysis experiment was performed as follows: The single hydrogel prepared in Example 2 and the DAR prepared in Example 7 were respectively placed in 1 mL of 10×PBS solution and extracted at 37℃ for 24 h to prepare material extracts. 1 mL of rat blood was collected using an EDTA anticoagulant tube, centrifuged at 4℃ and 2000 rpm for 10 min, the supernatant was discarded, and the precipitate was washed four times with 0.9% physiological saline and then prepared as a 2% erythrocyte suspension with PBS. The erythrocyte suspension was mixed with the two material extracts at a 9:1 volume ratio. PBS was used as a negative control, and 1% Triton X-100 solution was used as a positive control. Both were incubated at 37℃ for 2 h. After incubation, the mixture was centrifuged at 4℃ and 2000 rpm for 10 min. 100 μL of the supernatant was added to a 96-well plate, and the absorbance (OD value) at 541 nm was measured using a microplate reader. The hemolysis rate was calculated using the formula: Hemolysis (%) = (ODg) / (ODg)g Sa -OD Ne ) / (OD Po -ODNe ), where OD Sa OD value of material group Ne The OD value is for the negative control group. Po The OD value is for the positive control group.

[0105] Depend on Figure 6 It can be seen that the hemolysis rate of the hydrogel prepared in Example 2 and the DAR prepared in Example 7 is less than 5%, which indicates that no obvious hemolysis was caused.

[0106] 3. Antibacterial test In the antibacterial experiment, the concentrations of Staphylococcus aureus and Escherichia coli bacterial suspensions were adjusted using a turbidimeter (C-WGZ-XT) and diluted to 10⁻⁶. 5 After CFU / mL, the solution was evenly spread on the surface of a culture dish. The single hydrogels prepared in Examples 1 and 2 and the DAR prepared in Example 7 were cut into 6 mm × 6 mm discs, placed in culture dishes inoculated with bacteria, and incubated at 37°C for 24 h to observe the antibacterial effect.

[0107] Depend on Figure 7 It can be seen that, except for the control group and the Example 1 group, no colonies grew around the other hydrogels, and they all had good antibacterial properties. The reason for this phenomenon is that the effective antibacterial activity may be due to the specific reaction between the aldehyde group in DAR (OHA contains aldehyde group) and the bacterial outer membrane, which interferes with its structure or function.

[0108] Performance Test Example 5: Observation of the In vivo Effects of Postoperative DAR Application All animal care and experiments were conducted in accordance with the ethics review of Hangzhou Medical College (ZJCLA-IACUC-20011198). In vivo biocompatibility testing was performed using a skin degradation assay.

[0109] To assess the in vivo biocompatibility of the bilayer hydrogels, the hydrogels prepared in Examples 2 and 7 were implanted into the subcutaneous tissue of the back of male Sprague-Dawley (SD) rats (6-8 weeks old, 180-220 g). The SD rats were anesthetized with isoflurane (Macklin). The skin on the back was removed and the surface was disinfected. Penicillin was administered postoperatively to prevent postoperative infection. Rats were sacrificed and back tissues were harvested at weeks 1, 2, 3, and 4 post-implantation. The tissues were photographed and stained with hematoxylin and eosin (HE) to observe the degradation of the hydrogels prepared in Examples 2 and 7.

[0110] Depend on Figure 8 As can be seen, with the metabolism of organisms, both groups of hydrogels showed good degradation in the fourth week, which indicates that the hydrogels prepared in Example 2 and Example 7 can slowly biodegrade while maintaining long-term adhesion.

[0111] Performance Test Example 6: Cell Migration Effect of Double-Sided Hydrogels Mouse AML12 cells were seeded into 12-well plates, two wells per group. After 24 hours of incubation, once the cells reached 90% confluence, vertical and horizontal scratches were made in each well using a pipette tip. The separated cells were washed away with 1 mL of complete culture medium, and then the complete culture medium was replaced with serum-free medium (120 μg / mL) containing extracts from Examples 2 and 7. Images were captured at 0 h, 12 h, and 24 h post-scratching using an inverted microscope (Leica, Germany).

[0112] Depend on Figure 9 It can be seen that the cell migration rate of the Example 7 (DAR) group at 12 h and 24 h was higher than that of the Control group and the Example 2 group, indicating that DAR can effectively enhance cell migration ability, thereby promoting liver wound repair.

[0113] Performance Test Example 7: Evaluation of the Anti-Tissue Adhesion Function of Double-Sided Hydrogels To systematically evaluate the actual efficacy of the hydrogel prepared in Example 7 in preventing intraperitoneal adhesions, a cecal injury model was constructed. After anesthetizing rats, the abdominal cavity was surgically opened to fully expose the cecal tissue. Subsequently, scattered, visible bleeding points were formed on the cecal serosal surface through repeated rubbing. The experimental animals were divided into a control group (no anti-adhesion intervention was applied after intraperitoneal injury) and a treatment group (treated with the hydrogel patch prepared in Example 7 and the hydrogel patch prepared in Example 2, respectively).

[0114] HE staining results showed that ( Figure 10 When the abdominal cavity was reopened on day 14, obvious adhesions had appeared in the control group and the hydrogel group prepared in Example 2, while no adhesions were observed in the treatment group. Observation of inflammatory cells at 50x magnification revealed that the number of inflammatory cells in the control group and the hydrogel group prepared in Example 2 was significantly higher than that in the hydrogel group prepared in Example 7, which fully demonstrates that DAR has a good anti-abdominal adhesion effect.

[0115] Performance Test Example 8: Evaluation of the Hemostatic Ability of Double-Sided Hydrogels To evaluate the hemostatic ability of the hydrogel prepared in Example 7, we established a partial hepatectomy model. First, the liver of naked rats was exposed by making an incision along the midline of the abdomen. After removing 60% of the middle lobe of the liver, the wound was covered with the hydrogel prepared in Example 7 and commercially available gelatin sponge, respectively. Prepared filter paper was placed on the substrate for characterization.

[0116] Three minutes later, the gross bleeding conditions of the untreated group, the gelatin sponge group, and the hydrogel group prepared in Example 7 were as follows: Figure 11 As shown, the hydrogel group prepared in Example 7 had the least bleeding, which indicates that DAR has a good hemostatic effect.

[0117] Performance Test Example 9: Wound Healing Promotion Effect of Double-Sided Hydrogel Rapid healing of liver tissue and recovery of liver function after partial hepatectomy are crucial for patient prognosis, especially for patients with chronic liver disease, as the efficiency of this process directly affects treatment outcomes. To investigate the effect of the hydrogel prepared in Example 7 on promoting liver repair after partial hepatectomy, this study constructed an animal model of partial hepatectomy, specifically removing 60% of the middle lobe of the liver. The effects of the hydrogel and gelatin sponge prepared in Example 7 were then analyzed using a series of assays. Rats were sacrificed on day 7 post-surgery, and liver tissue from the sites of adhesion to each group was collected, fixed in formaldehyde, and subjected to immunohistochemical and immunofluorescence analyses. The expression levels of proliferation-related markers Ki67, PCNA, Cyclin D1, CD31 / α-SMA, and the macrophage marker CD68 were detected to assess the repair and regeneration activity of liver tissue.

[0118] Figure 12 The results showed that in the DAR hydrogel group prepared in Example 7, the expression levels of Ki67, PCNA, Cyclin D1 (a marker of hepatocyte proliferation) and CD31 / α-SMA (a marker of angiogenesis) in liver tissue were significantly higher than those in other control groups, while the expression level of CD68 (a macrophage marker associated with inflammatory response) was significantly lower than that in other control groups. The above results fully demonstrate that the DAR of the present invention can effectively promote the liver repair process after partial hepatectomy in rats.

[0119] The above description is merely a specific embodiment of the present invention, intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. All equivalent modifications or substitutions made based on the essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A double-sided hydrogel that combines hemostasis, anti-adhesion, and wound repair functions, characterized in that, The double-sided hydrogel includes a repair layer and an anti-adhesion layer. The repair layer is a hydrogel formed by photocuring with oxidized hyaluronic acid, gelatin methacrylate, acrylic acid, N-hydroxysuccinimide acrylate and p(APMA-co-THMA) as the core components. The p(APMA-co-THMA) is obtained by polymerization of N-[tris(hydroxymethyl)methyl]acrylamide and N-(3-aminopropyl)methacrylamide hydrochloride. The anti-adhesion layer is formed by immersing one side of the repair layer in an aqueous solution containing [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, gelatin methacrylate, N,N-methylenebisacrylamide and an initiator, followed by a thermal crosslinking reaction.

2. The double-sided hydrogel as described in claim 1, characterized in that, The mass ratio of methacrylic gelatin, acrylic acid, N-hydroxysuccinimide acrylate and p(APMA-co-THMA) is 1-3:200-400:10-30:1-3; the mass ratio of oxidized hyaluronic acid to acrylic acid is 1-4:45-70.

3. The double-sided hydrogel as described in claim 1, characterized in that, The mass ratio of N-[tris(hydroxymethyl)methyl]acrylamide and N-(3-aminopropyl)methacrylamide hydrochloride is 51-62:10-16, and the two undergo polymerization under the action of an initiator.

4. The double-sided hydrogel as described in claim 1, characterized in that, The mass ratio of [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, gelatin methacrylate, and N,N-methylenebisacrylamide is 95-103:8-13:1-5.

5. The method for preparing the double-sided hydrogel according to any one of claims 1-4, characterized in that, Includes the following steps: (1) N-[tris(hydroxymethyl)methyl]acrylamide and N-(3-aminopropyl)methacrylamide hydrochloride were dissolved in water and polymerized under the action of an initiator to obtain p(APMA-co-THMA); (2) Dissolve p(APMA-co-THMA), gelatin methacrylate and photoinitiator in water, then add acrylic acid and N-hydroxysuccinimide acrylate to dissolve and obtain RL precursor solution. Then mix RL precursor solution with oxidized hyaluronic acid solution and photocur to form repair layer gel. (3) The repair layer gel is immersed on one side in an aqueous solution containing [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide, gelatin methacrylate, N,N-methylenebisacrylamide and an initiator, and thermal crosslinking is performed to form an anti-adhesion layer, thereby obtaining the double-sided hydrogel.

6. The preparation method according to claim 5, characterized in that, In step (1), the initiator is ammonium persulfate, and the polymerization reaction conditions are: heating to 60℃ for 1 h, then heating to 70℃ for 7 h; after the reaction is completed, dialysis is performed using a dialysis bag with a molecular weight cutoff of 3500 Da.

7. The preparation method according to claim 5, characterized in that, In step (2), the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoyl phosphate; the photocuring conditions are: crosslinking under 365 nm ultraviolet light for 20-30 min.

8. The preparation method according to claim 5, characterized in that, In step (2), after photocuring, the obtained gel is soaked in deionized water and then dried at 35-45°C for 15-25 min to obtain the repair layer.

9. The preparation method according to claim 5, characterized in that, In step (3), the initiator is ammonium persulfate, and the thermal crosslinking conditions are 50-70°C for 50-80 min.

10. The use of the double-sided hydrogel according to any one of claims 1-4 in the preparation of tissue trauma repair materials after partial hepatectomy.

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