Preparation method and application of anti-fatigue double-network composite hydrogel

Abstract

The present application relates to a kind of preparation method and application of anti-fatigue double-network composite hydrogel, and it is related to the field of hydrogel preparation.The composite hydrogel includes two networks, the first network is the semi-crystalline PVA polymer assembled by cyclic freezing and thawing process, and the second network is the pectin calcium network constructed by the ionic interaction between calcium and pectin.The preparation method includes: mixing polyvinyl alcohol and pectin with a weight ratio of 8:1, preparing semi-crystalline polyvinyl alcohol polymer by freezing and thawing, then completely immersing the mixture into calcium chloride solution for 24 hours until the gel reaches equilibrium swelling.Application is the application in the preparation of human tissue engineering scaffold.The present application forms a mechanically robust hydrogel by the interpenetrating network structure composed of primary semi-crystalline polyvinyl alcohol network and secondary pectin calcium physical ionic network, has good elasticity, toughness and anti-fatigue performance, tensile elongation reaches more than 300%, has a certain degree of energy dissipation, and can be used as a buffer pad to resist external impact.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of hydrogel preparation, and particularly relates to a preparation method and application of an anti-fatigue double-network composite hydrogel. BACKGROUND

[0002] Hydrogels are three-dimensional polymer networks containing a large amount of water (>80%), and their structural integrity is similar to that of the natural extracellular matrix (ECM). Hydrogels have inherent biocompatibility due to their high water content, and are considered one of the most attractive scaffold candidates in tissue engineering. Unfortunately, they do not exhibit the ideal fracture toughness and strength required for scaffolds used in load-bearing applications. In order to overcome this obstacle, efforts have been made to strengthen or reinforce the mechanical properties of hydrogels, such as adding inorganic nanoparticles, formulating nanostructures, or preparing composite hydrogels.

[0003] Among these efforts, composite hydrogels based on two different interpenetrating polymer networks have become a unique way to enhance the mechanical properties of hydrogels. "Jiehao Chen, Jiahe Huang, Yuhang Hu. 3D Printing of Biocompatible Shape-Memory Double Network Hydrogels. Acsami. 2012. 0c17622" proposes a one-pot method for printing biocompatible shape-memory DN hydrogels by fused deposition method. Two networks of polyacrylamide (PAAm) and gelatin are combined into a hydrogel ink. The PAAm network is covalently cross-linked and is responsible for the permanent shape, while the gelatin network has thermally reversible cross-linking and is responsible for fixing the temporary shape, realizing 3D printable shape-memory DN hydrogels, which exhibit higher toughness, and further research is needed to realize their application in tissue engineering.

[0004] Chinese patent CN118697942A (published on September 27, 2024) discloses a cartilage repair scaffold and a preparation method thereof. The cartilage repair scaffold comprises a polymer mesh scaffold, and a hydrogel filled in the voids of the polymer mesh scaffold, wherein the hydrogel is dispersed with liposome microspheres. The compression modulus of the polymer mesh scaffold is 7.5-9.5 MPa. The cartilage repair scaffold has mechanical properties matching natural cartilage, good mechanical strength, and good biocompatibility. However, the polymer mesh scaffold still has the problem of poor cell affinity.

[0005] How to prepare a hydrogel with favorable mechanical properties, low cytotoxicity, good synthetic properties, and cell adhesion gelation performance makes it easy to design and develop scaffolds, and further research is needed to apply it to tissue engineering. SUMMARY

[0006] To address the aforementioned issues, this invention prepares a polyvinyl alcohol and pectin composite hydrogel using a freeze-thaw process, providing a method for preparing and applying an anti-fatigue dual-network composite hydrogel.

[0007] A fatigue-resistant dual-network composite hydrogel, wherein the composite hydrogel is composed of polyvinyl alcohol and pectin, and the mass concentration of polyvinyl alcohol and pectin in the gel is 5-15%.

[0008] Furthermore, the composite hydrogel comprises two network structures: the first network is a semi-crystalline PVA polymer assembled through a cyclic freeze-thaw process, and the second network is a pectin-calcium network constructed by ionic interactions between calcium and pectin.

[0009] Furthermore, the composite hydrogel has an egg-box structure, which is used to fix bioactive ingredients or cells within the gel structure.

[0010] Furthermore, the pectin gelation process is repeatable, mainly achieved by introducing calcium ions.

[0011] A method for preparing an anti-fatigue dual-network composite hydrogel includes the following steps:

[0012] S1. Mix polyvinyl alcohol and pectin at a weight ratio of 8:1, and take 5-15g of the mixture and distribute it evenly in 100mL of 95℃ deionized water for 2 hours.

[0013] S2. Pour the well-mixed solution into a preheated mold and keep it at -20°C for 72 hours, then keep it at room temperature for 1 hour.

[0014] S3. Repeat the freeze-thaw cycle twice with the solution obtained in step S2 to obtain a semi-crystalline polyvinyl alcohol polymer.

[0015] S4. Then immerse the mixture completely in calcium chloride solution for 24 hours, and then immerse it completely in deionized water until the gel reaches equilibrium swelling.

[0016] Furthermore, there is ionic cross-linking between the pectin and calcium ions. After forming a pectin-calcium network in the semi-crystalline polyvinyl alcohol polymer through step S4, the tensile modulus and fracture toughness increase threefold.

[0017] Furthermore, in step S3, the freezing time for the single freeze-thaw process is 22-23 hours, and the thawing time is 1-2 hours.

[0018] Application of a fatigue-resistant dual-network composite hydrogel in the preparation of human tissue engineering scaffolds.

[0019] Compared with the prior art, the technical solution of this application has the following advantages and effects:

[0020] 1. The fatigue-resistant dual-network composite hydrogel provided in this application is a dual-network composite hydrogel constructed from a semi-crystalline polyvinyl alcohol polymer and a pectin calcium network. It has good elasticity, toughness and fatigue resistance, with a tensile elongation of over 300%. It has a certain degree of energy dissipation and can serve as a buffer against external impacts.

[0021] 2. The interpenetrating network structure provided in this application, consisting of a primary semi-crystalline polyvinyl alcohol network and a secondary pectin calcium physical ion network, forms a mechanically robust hydrogel that can withstand significant deformation without breaking. Moreover, it can recover to its initial state with viscoelasticity after the load is removed. Its crosslinking breakage is weak but reversible under applied load, and the degree of crosslinking dissociation is greater in denser networks.

[0022] 3. The present application provides a method for preparing an anti-fatigue dual-network composite hydrogel, which first prepares a semi-crystalline polyvinyl alcohol polymer and then forms a pectin-calcium network. Compared with a single polyvinyl alcohol hydrogel or uncrosslinked pectin and calcium ions, this composite hydrogel has better Young's modulus and fracture energy mechanical properties, and can be applied to the preparation of human tissue engineering scaffolds.

[0023] The above is merely an overview of the technical solution of this application. In order to better understand the technical means of this application so that it can be implemented in accordance with the contents of the specification, and to make the above and other objects, features and advantages of this application more apparent and understandable, the preferred embodiments of this application are described in detail below with reference to the accompanying drawings.

[0024] The above and other objects, advantages and features of this application will become more apparent to those skilled in the art from the following detailed description of specific embodiments in conjunction with the accompanying drawings. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0026] Figure 1 This is a flowchart of a method for preparing an anti-fatigue dual-network composite hydrogel according to an embodiment of the present invention;

[0027] Figure 2 Figure (a) shows the molecular structure diagrams of polyvinyl alcohol and pectin according to an embodiment of the present invention, wherein Figure (b) shows the molecular structure diagram of polyvinyl alcohol.

[0028] Figure 3 Figure 1 shows the mechanical properties and adhesion ability of a composite hydrogel according to an embodiment of the present invention. Figure 2 shows the relationship between the viscoelastic behavior and oscillatory strain of the composite hydrogel, Figure 3 shows the Young's modulus of the hydrogel under tensile and compression tests, Figure 4 shows the stress-strain curve of the tensile properties, Figure 5 shows the cyclic tensile test results, and Figure 6 shows the cyclic compression test results.

[0029] Figure 4 Figure (a) shows the characterization of the composite hydrogel formation according to an embodiment of the present invention, where Figure (a) is the Young's modulus diagram, Figure (b) is the fracture energy diagram, and Figure (c) is the stress-life behavior diagram.

[0030] Figure 5 Figure 1 shows the recovery capacity of a composite hydrogel according to an embodiment of the present invention. Figure 2 shows the tensile properties of the composite hydrogel scaffold, Figure 3 shows the cyclic tensile test at 100% strain, Figure 4 shows the cyclic compression test at -60% strain, and Figure 5 shows the fatigue life curve.

[0031] Figure 6 This is an adhesion diagram of cells in different scaffolds according to an embodiment of the present invention. Figure 6 a is a diagram showing the adhesion of polyvinyl alcohol hydrogel. Figure 6 b is the adhesion diagram of PVA-Pec-10 hydrogel. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. In the following description, specific details such as specific configurations and components are provided only to help fully understand the embodiments of this application. Therefore, those skilled in the art should understand that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of this application. In addition, for clarity and brevity, descriptions of known functions and structures are omitted in the embodiments. It should be understood that "an embodiment" or "this embodiment" mentioned throughout the specification means that a specific feature, structure, or characteristic related to the embodiment is included in at least one embodiment of this application. Therefore, "an embodiment" or "this embodiment" appearing throughout the specification does not necessarily refer to the same embodiment. Furthermore, these specific features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.

[0033] Furthermore, reference numerals and / or letters may be repeated in different examples within this application. Such repetition is for the purpose of simplification and clarity and does not in itself indicate a relationship between the various embodiments and / or settings discussed.

[0034] In this article, the term "and / or" describes a relationship between related objects, indicating that three relationships can exist. For example, "A and / or B" can mean: A exists alone, B exists alone, or A and B exist simultaneously. The term " / and" describes another type of relationship, indicating that two relationships can exist. For example, "A / and B" can mean: A exists alone, or A and B exist alone. Additionally, the character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0035] It should also be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion.

[0036] Example 1

[0037] like Figure 1 As shown, this embodiment provides a method for preparing an anti-fatigue dual-network composite hydrogel, which is carried out according to the following steps:

[0038] S1. Mix polyvinyl alcohol and pectin at a weight ratio of 8:1. Take 5g, 10g, or 15g of the mixture respectively, and distribute it evenly in 100mL of deionized water at 95℃ for 2 hours. Figure 2 (a) and (b) show the molecular structure diagrams of polyvinyl alcohol and pectin, respectively;

[0039] S2. Pour the well-mixed solution into a preheated mold and keep it at -20°C for 72 hours, then keep it at room temperature for 1 hour.

[0040] S3. Repeat the freeze-thaw cycle twice with the solution obtained in step S2. The freezing time is 23 hours and the thawing time is 1 hour to obtain a semi-crystalline polyvinyl alcohol polymer.

[0041] S4. The mixture is then completely immersed in calcium chloride solution for 24 hours, and then completely immersed in deionized water until the gel reaches equilibrium swelling. Composite hydrogels with a total polymer content of 5, 10, and 15 w / v in solution volume (hereinafter referred to as: PVA-Pec-5, PVA-Pec-10, PVA-Pec-15) are prepared.

[0042] Technical effects of this embodiment: This embodiment provides a method for preparing anti-fatigue dual-network composite hydrogels, which can prepare composite hydrogels with different total amounts of polymer in the solution volume.

[0043] Example 2

[0044] Based on the composite hydrogels with different total polymer contents in the solution volume in Example 1, rheological experiments were conducted in oscillating mode to detect the relationship between the viscoelastic behavior (storage modulus and loss modulus) and oscillating strain of the composite hydrogels with total polymer contents of 5, 10, and 15 w / v in the solution volume. Figure 3 As shown in Figure a, a linear viscoelastic (LVE) region was observed at low oscillatory strain (<0.1%), where the storage modulus (G′) and loss modulus (G″) remained relatively constant. Hydrogels with higher polymer concentrations exhibited higher G′. With increasing oscillatory strain, G′ began to decrease, while G″ began to increase, indicating deformation of the polymer network. After the transition point (G′ = G″ or Tanδ = 1), the hydrogel transitioned from a solid state (G′ > G″) to a liquid state (G′ > G″). <G〃)。

[0045] In addition to viscoelastic behavior, the tensile and compressive properties of the expanded hydrogels were investigated at body temperature (37°C). All three hydrogels exhibited both elasticity and toughness, with tensile elongation exceeding 300%. Figure 3 c). The Young's modulus of the hydrogel increases with increasing polymer concentration, which is in good agreement with the results of viscoelastic behavior. The Young's modulus of PVA-Pec-15 reaches 117.9 ± 9.0 kPa in tensile testing and 79.3 ± 20.6 kPa in compression testing. Figure 3 b).

[0046] The stress-strain curves of the hydrogel under cyclic loading are as follows: Figure 3 As shown in d and e, the curves indicate that all three hydrogels recover their original shape even after 100% stretching or 50% compression. The hysteresis loops observed after unloading reveal the viscoelasticity of the material, likely due to weak but reversible crosslinking breakage under applied load. The degree of hysteresis increases with increasing polymer concentration, indicating greater crosslinking dissociation in denser networks.

[0047] The technical effects of this embodiment are as follows: This embodiment verifies that the composite hydrogel is a mechanically responsive system, and its rheological properties fluctuate with changes in stress or strain. Furthermore, it exhibits viscoelastic behavior, good tensile and compressive properties, and weak but reversible crosslinking properties related to viscoelasticity.

[0048] Example 3

[0049] This embodiment further verifies the crosslinking performance and fatigue resistance of the PVA-Pec-5, PVA-Pec-10, and PVA-Pec-15 composite hydrogels described in the above embodiments.

[0050] The addition of a calcium pectin network to polyvinyl alcohol hydrogels resulted in an approximately threefold increase in tensile modulus (~30±20 kPa to ~100±10 kPa) and fracture toughness (~300±100 Jm⁻² to ~900±100 Jm⁻¹). The tensile modulus and fracture toughness are shown in the figures below. Figure 4 As shown in (a) and (b), the addition of uncrosslinked pectin (PVA-Pec-no Ca2+) or Ca2+ ions (PVA-Ca) alone has a positive effect on the mechanical properties of the polyvinyl alcohol hydrogel system, including Young's modulus and fracture energy. However, the greatest improvement was observed in the PVA-Pec hydrogel with an ionicly crosslinked calcium-pectin network. The stress-life behavior of the polyvinyl alcohol hydrogel and the composite hydrogel is shown in (a) and (b). Figure 4 As shown in (c).

[0051] The composite hydrogel exhibited recovery ability after more than 5 cycles, and energy dissipation and the appearance of hysteresis loops were observed. Figure 5 As shown in Figure (a), the tensile properties of different PVA-Pec stents are shown; (b) shows the cyclic tensile test at 100% strain; (c) shows the cyclic compression test at -60% strain; and (d) shows the fatigue life curve.

[0052] Technical effects of this embodiment: This embodiment verifies that the cross-linked calcium-pectin network has a significant improvement on the Young's modulus and fracture energy of the composite hydrogel, as well as good recovery ability.

[0053] Example 4

[0054] This embodiment relates to the application of the fatigue-resistant dual-network composite hydrogel described in the above embodiments in the preparation of human tissue engineering scaffolds. The preparation process includes placing the gel at the bottom of a confocal dish and adding 1×10⁻⁶ ppm of hydrogel onto the gel surface. 4 After incubating stem cells for 12 hours, adherent cells were fixed and stained for cell microfilaments and nuclei. Images were collected using a confocal microscope. Figure 6 Adhesion diagrams of cells in different scaffolds Figure 6 a. Polyvinyl alcohol hydrogel adhesion diagram Figure 6 The adhesion diagram of bPVA-Pec-10 hydrogel shows that more material adheres to the PVA-Pec-10 hydrogel compared to the polyvinyl alcohol hydrogel.

[0055] Technical effects of this embodiment: This embodiment verifies that the composite hydrogel has good cell adhesion properties, enabling its application in human tissue engineering scaffolds.

[0056] The above description is merely a preferred embodiment of the present invention and does not limit the scope of protection of the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any changes, modifications, substitutions, integrations, and parameter alterations to these embodiments within the spirit and principles of the present invention, achieved through conventional substitutions or by achieving the same function without departing from the principles and spirit of the present invention, fall within the scope of protection of the present invention.

Claims

1. A method for preparing an anti-fatigue dual-network composite hydrogel, characterized in that, Includes the following steps: S1. Mix polyvinyl alcohol and pectin at a weight ratio of 8:1, and take 5-15g of the mixture and distribute it evenly in 100 mL of 95°C deionized water for 2 hours. S2. Pour the well-mixed solution into a preheated mold and keep it at -20°C for 72 hours, then keep it at room temperature for 1 hour. S3. Repeat the freeze-thaw cycle twice with the solution obtained in step S2 to obtain a semi-crystalline polyvinyl alcohol polymer. S4. Then immerse the mixture completely in calcium chloride solution for 24 hours, and then immerse it completely in deionized water until the gel reaches equilibrium swelling. The prepared composite hydrogel is composed of polyvinyl alcohol and pectin, and the mass concentration of polyvinyl alcohol and pectin in the gel is 5-15%. The composite hydrogel comprises two network structures: the first network is a semi-crystalline PVA polymer assembled through a cyclic freeze-thaw process, and the second network is a pectin-calcium network constructed by ionic interactions between calcium and pectin. The composite hydrogel has an egg-box structure, which is used to fix bioactive ingredients or cells within the gel structure. The pectin gelation process is made repeatable by introducing calcium ions.

2. The method for preparing an anti-fatigue dual-network composite hydrogel according to claim 1, characterized in that, There is ionic cross-linking between the pectin and calcium ions. After forming a pectin-calcium network in the semi-crystalline polyvinyl alcohol polymer through step S4, the tensile modulus and fracture toughness increase threefold.

3. The method for preparing an anti-fatigue dual-network composite hydrogel according to claim 1, characterized in that, The freezing time for the single freeze-thaw process in step S3 is 22-23 hours, and the thawing time is 1-2 hours.

4. The application of the fatigue-resistant dual-network composite hydrogel as described in any one of claims 1-3 in the preparation of human tissue engineering scaffolds.

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