Bioactive bionic gradient hydrogel as well as preparation method and application thereof

Abstract

The invention relates to the technical field of hydrogel, in particular to bioactive bionic gradient hydrogel as well as a preparation method and application thereof.The preparation method comprises the following steps: dissolving quaternary ammonium salt chitosan, 3-acrylamidophenylboronic acid, platelet-rich plasma and phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate in deionized water, stirring until complete dissolution, centrifuging, and drying to obtain a hydrogel solution containing chitosan, 3-acrylamidophenylboronic acid, platelet-rich plasma and phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate; pre-polymerization liquid is obtained; preparing a tannic acid-iron ion (TA-Fe < 3 + >) mixed solution; adding the pre-polymerized liquid into a mold, and carrying out ultraviolet radiation crosslinking to obtain hydrogel; the hydrogel part is soaked in a tannic acid-iron ion mixed solution, and the gradient hydrogel is obtained. According to the biologically active bionic gradient hydrogel as well as the preparation method and the application thereof, the dual-network hydrogel is soaked in a TA-Fe < 3 + > solution to form a TA-Fe < 3 + > coordinated concentration gradient, so that the hard-to-soft mechanical gradient of tissues such as bone-cartilage, rotator cuff, achilles tendon and the like is accurately simulated.

Description

Technical Field

[0001] This invention relates to the field of hydrogel technology, and in particular to a bioactive biomimetic gradient hydrogel, its preparation method, and its applications. Background Technology

[0002] Osteochondral cartilage, rotator cuff, Achilles tendon, and other tissues possess unique gradient structures. These structures not only enhance the mechanical properties of the tissues but also optimize their physiological functions, enabling organisms to better cope with the challenges of the external environment. For example, the mechanical gradient from soft to hard and the cellular composition gradient exhibited by natural articular cartilage from the surface to the depths are core to achieving functional regeneration, posing a significant challenge to traditional homogeneous biomaterials. Therefore, how to construct tissue repair materials with gradient structures has important research and application value in the fields of tissue engineering and regenerative repair. Hydrogels, due to their high water content and three-dimensional network structure similar to the extracellular matrix, have become ideal scaffolds for tissue regeneration and repair. Therefore, developing hydrogels that can biomimeticly simulate this gradient structure is currently a core frontier in the field.

[0003] To address the challenge of gradient construction, researchers have developed various methods. For example, Song et al. induced phase separation in a polyacrylamide / polyacrylic acid system using phytic acid, creating an ion gradient in a gravitational field; Xu and Yang's teams constructed gradient hydrogels containing metal elements using magnetic field induction and one-step coordination strategies, respectively. Inspired by biomineralization, Zhu's team used hyaluronic acid to regulate calcium phosphate polymer precursors, forming an organic-inorganic nanogradient. Yao's team used an electrochemical method to drive ion migration, thereby training hydrogels to form robust gradient structures. Although these methods have successfully replicated gradients, they generally rely on complex preparation processes, non-physiological external fields such as magnetic fields and electric fields, or artificial materials with poor biocompatibility, severely limiting their clinical translation prospects.

[0004] Therefore, it is urgent to develop a gradient hydrogel preparation strategy that is easy to prepare and whose components have good biocompatibility. Summary of the Invention

[0005] The purpose of this invention is to provide a bioactive biomimetic gradient hydrogel, its preparation method, and its application, wherein the hydrogel is immersed in TA-Fe 3+ In solution, TA-Fe can be spontaneously formed inside the hydrogel using simple diffusion control. 3+ The concentration gradient of the coordination is used to accurately simulate the mechanical gradient from soft to hard in tissues such as bone-cartilage, rotator cuff, and Achilles tendon.

[0006] To achieve the above objectives, the present invention provides a method for preparing a bioactive biomimetic gradient hydrogel, comprising the following steps: S1. Quaternary ammonium salt chitosan, 3-acrylamidophenylboronic acid, platelet-rich plasma and phenyl (2,4,6-trimethylbenzoyl) lithium phosphate were dissolved in deionized water, stirred until completely dissolved and then centrifuged to obtain a prepolymer solution. S2. Dissolve tannic acid granules in deionized water, stir until completely dissolved, then add anhydrous ferric chloride and stir evenly to obtain a tannic acid-ferric ion mixture. S3. Add the prepolymer obtained in S1 into the mold and crosslink it by ultraviolet irradiation to obtain a hydrogel. S4. Immerse the hydrogel portion obtained in S3 in the tannic acid-iron ion mixture in S2 to obtain a biomimetic gradient hydrogel.

[0007] Preferably, in the prepolymer solution of S1, the mass ratio of quaternary ammonium chitosan, 3-acrylamidophenylboronic acid, platelet-rich plasma and phenyl (2,4,6-trimethylbenzoyl) lithium phosphate is 5-10:0.5-1:10-20:0.2-0.4.

[0008] Preferably, in S1, the stirring is carried out at room temperature, with a stirring speed of 800-1200 rpm and a stirring time of 60-90 min.

[0009] Preferably, in S1, the relative centrifugal force is 1000-1200g and the centrifugation time is 5-12min.

[0010] Preferably, in S2, the mass ratio of tannic acid particles to anhydrous ferric chloride is 30-50:3.

[0011] Preferably, in S2, the stirring temperature is room temperature, the stirring time is 0.5-2h, and the stirring speed is 300-500rpm.

[0012] Preferably, in S3, the ultraviolet irradiation time is 45-90s.

[0013] Preferably, in step S4, during immersion, the liquid level of the tannic acid-iron ion mixture is one-third to one-half the height of the hydrogel, and the immersion time is 18h-24h.

[0014] The biomimetic gradient hydrogel was prepared using the above-described method for preparing a bioactive biomimetic gradient hydrogel.

[0015] The aforementioned bioactive biomimetic gradient hydrogel is applied in the preparation of tissue repair materials with biomimetic gradient structures.

[0016] Therefore, the present invention employs the above-mentioned bioactive biomimetic gradient hydrogel, its preparation method, and its application, and its beneficial effects are as follows: 1. In the preparation method of this invention, a quaternary ammonium salt chitosan (QCS)-acrylamidophenylboronic acid (AAPBA) / platelet-rich plasma (PRP) dual network is first obtained, and then processed through tannic acid-iron ions (TA-Fe) 3+ (Diffusion crosslinking constructs biomimetic gradient hydrogels;) 2. In the dual-network hydrogel provided by the present invention, QCS-AAPBA not only provides excellent biocompatibility and initial framework, but its phenylboronic acid groups can also form dynamic covalent bonds to enhance the material's adhesion and self-healing ability. The fibrin network in PRP further regulates the gradient mechanical properties while introducing a variety of growth factors to provide active biological signals for cell proliferation and differentiation. 3. The gradient hydrogel provided by this invention is obtained by immersing the hydrogel in TA-Fe 3+ In solution, TA-Fe can be spontaneously formed inside the hydrogel using simple diffusion control. 3+ The concentration gradient of the coordination is used to accurately simulate the mechanical gradient of natural cartilage from soft to hard.

[0017] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0018] Figure 1 These are photographs of the gradient hydrogel and nanoindentation test images from embodiments of the present invention; Figure 2 This is a SEM image of the portion of the gradient hydrogel top that was not soaked in the tannic acid-iron ion mixture in an embodiment of the present invention; Figure 3 This is a SEM image of the gradient hydrogel soaking tannic acid-iron ion mixture in an embodiment of the present invention; Figure 4 This is a SEM image of the portion of the top of the comparative bilayer hydrogel of the present invention that was not soaked in the tannic acid-iron ion mixture. Figure 5 This is a SEM image of the portion of the comparative bilayer hydrogel of the present invention soaked in a tannic acid-iron ion mixture. Figure 6 This is the Fourier transform infrared spectrum of the gradient hydrogel in this invention. Detailed Implementation

[0019] The present invention will be further described below with reference to the accompanying drawings and embodiments. Unless otherwise defined, the technical or scientific terms used in this invention should be understood in their ordinary sense by those skilled in the art. The features mentioned above or in the specific examples mentioned in this invention can be combined arbitrarily, and these specific embodiments are only used to illustrate the invention and are not intended to limit the scope of the invention.

[0020] This invention provides a method for preparing a bioactive biomimetic gradient hydrogel, comprising the following steps: S1. Quaternary ammonium chitosan (QCS), 3-acrylamidophenylboronic acid (AAPBA), platelet-rich plasma (PRP), and lithium phenyl (2,4,6-trimethylbenzoyl) phosphate were dissolved in deionized water, stirred until completely dissolved, and then centrifuged to obtain a prepolymer solution. QCS serves as a cationic polysaccharide backbone, providing biocompatibility and antibacterial properties. AAPBA contains phenylboronic acid groups, which can form dynamic borate ester bonds; it also contains acrylamide groups, participating in photopolymerization. PRP contains fibrinogen and various growth factors; fibrinogen forms a fibrin network after light exposure.

[0021] S2. Dissolve tannic acid (TA) granules in deionized water, stir until completely dissolved, then add anhydrous ferric chloride and stir until homogeneous to obtain tannic acid-ferric ion TA-Fe. 3+ Mixture; S3. Add the prepolymer obtained in S1 into the mold and crosslink it under ultraviolet light to obtain a hydrogel. Use lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (photoinitiator) to quickly cure under ultraviolet light. Ultraviolet light initiates the polymerization of acrylamide groups to form a QCS-AAPBA dynamic covalent network. At the same time, fibrinogen in PRP forms non-covalent bonds such as hydrogen bonds and electrostatic interactions with the hydrogel polymer molecules, and the two form a multi-interaction network hydrogel.

[0022] S4. Immerse the hydrogel obtained in S3 in the tannic acid-iron ion mixture in S2 to obtain a biomimetic gradient hydrogel. Immerse the hydrogel in TA-Fe... 3+ In solution, TA and Fe 3+ It diffuses into the hydrogel via coordination. TA-Fe spontaneously forms within the hydrogel. 3+ The concentration gradient of coordination results in a mechanical gradient from soft to hard.

[0023] In some embodiments of the present invention, the mass ratio of quaternary ammonium chitosan, 3-acrylamidophenylboronic acid, platelet-rich plasma and phenyl (2,4,6-trimethylbenzoyl) lithium phosphate in the prepolymer solution of S1 is 5-10:0.5-1:10-20:0.2-0.4.

[0024] In some embodiments of the present invention, in step S1, stirring is performed at room temperature at a stirring rate of 800-1200 rpm for 60-90 minutes. This ensures that the components are uniformly dispersed and prevents phase separation.

[0025] In some embodiments of the present invention, in S1, the relative centrifugal force is 1000-1200g, and the centrifugation time is 5-12min. This removes air bubbles and improves the uniformity of the pregel.

[0026] In some embodiments of the present invention, in S2, the mass ratio of tannic acid particles to anhydrous ferric chloride is 30-50:3. The multiple phenolic hydroxyl groups of tannic acid can react with Fe... 3+ Metal-phenol coordination bonds are formed.

[0027] In some embodiments of the present invention, in step S2, the stirring temperature is room temperature, the stirring time is 0.5-2 hours, and the stirring speed is 300-500 rpm. Stirring ensures complete dissolution of TA and Fe. 3+ Uniform coordination.

[0028] In some embodiments of the present invention, in S3, the ultraviolet light irradiation time is 45-90s.

[0029] In some embodiments of the present invention, in step S4, during immersion, the liquid level of the tannic acid-iron ion mixture is one-third to one-half the height of the hydrogel, and the immersion time is 18-24 hours. Because the diffusion rate is controlled by the concentration gradient, TA-Fe... 3+ A concentration gradient is formed inside the hydrogel from the surface to the interior, achieving a mechanical gradient structure from highly cross-linked (hard) surface layer to lowly cross-linked (soft) deep layer.

[0030] In some embodiments of the present invention, gradient hydrogels are prepared using the above-described method for preparing a bioactive biomimetic gradient hydrogel.

[0031] In some embodiments of the present invention, the above-mentioned bioactive biomimetic gradient hydrogel is applied in the preparation of tissue repair materials with biomimetic gradient structures.

[0032] Example S1. Dissolve 5.0 wt% quaternary ammonium chitosan, 1.0 wt% 3-acrylamidophenylboronic acid, 10 wt% platelet-rich plasma, and 0.3 wt% lithium phenyl (2,4,6-trimethylbenzoyl)phosphate in deionized water. Stir at 1000 rpm for 60 min at room temperature until completely dissolved. Centrifuge at 1100 g relative centrifugal force for 8 min to obtain the prepolymer solution.

[0033] S2. Dissolve 40wt% tannic acid granules in deionized water, stir at 300rpm for 1 hour at room temperature until completely dissolved, then add 3wt% anhydrous ferric chloride and stir evenly to obtain a tannic acid-ferric ion mixture.

[0034] S3. Slowly add the prepolymer obtained in S1 into the mold to avoid air bubbles, and crosslink it by UV irradiation for 60 seconds to obtain a shaped hydrogel.

[0035] S4. Immerse the hydrogel obtained in S3 in the tannic acid-iron ion mixture in S2. The liquid level of the tannic acid-iron ion mixture is one-third of the height of the hydrogel. After immersion for 18 hours, a biomimetic gradient hydrogel from soft to hard is obtained.

[0036] Comparative Example S1. Dissolve 5.0 wt% quaternary ammonium chitosan, 1.0 wt% 3-acrylamidophenylboronic acid, and 0.3 wt% phenyl (2,4,6-trimethylbenzoyl) lithium phosphate in deionized water. Stir at 1000 rpm for 60 min at room temperature until completely dissolved. Centrifuge at 1100 g relative centrifugal force for 8 min to obtain the prepolymer solution.

[0037] S2. Dissolve 40wt% tannic acid granules in deionized water, stir at 300rpm for 1 hour at room temperature until completely dissolved, then add 3wt% anhydrous ferric chloride and stir evenly to obtain a tannic acid-ferric ion mixture.

[0038] S3. Slowly add the prepolymer obtained in S1 into the mold to avoid air bubbles, and crosslink it by UV irradiation for 60 seconds to obtain a shaped hydrogel.

[0039] S4. Immerse the hydrogel obtained in S3 in the tannic acid-iron ion mixture in S2. The liquid level of the tannic acid-iron ion mixture is one-third of the height of the hydrogel. Immerse for 18 hours to obtain a double-layer hydrogel.

[0040] Test case a. The gradient hydrogel from the example, after soaking for approximately 18 hours, was subjected to nanoindentation testing. Seven points on the gradient hydrogel were selected from bottom to top for indentation testing. For example... Figure 1 As shown, the elastic modulus decreases with increasing height, indicating that the TA-Fe immersion... 3+ The hydrogel crosslinking density of the solution increased, resulting in a significant improvement in the elastic modulus.

[0041] b. The hydrogels prepared in the comparative example and the gradient hydrogels prepared in the examples were frozen at -80°C for 24 hours, then dried in a freeze dryer and subjected to scanning electron microscopy (SEM) analysis. Figure 2 and Figure 3 As shown. By Figure 2 and Figure 3 The comparison shows that the bottom of the gradient hydrogel in the embodiment is immersed in TA-Fe 3+ The pores in this portion of the solution are smaller, while the pores in the hydrogel portion not soaked in the tannic acid-iron ion mixture are larger. This demonstrates that during the soaking process, TA and Fe... 3+ It penetrates into the hydrogel network to form TA-Fe 3+The coordination complex provides more cross-linking sites, which are added to the original QCS / AAPBA chemical cross-linking network, making the overall polymer network of the gradient hydrogel more compact and exhibiting a smaller pore structure.

[0042] Depend on Figure 4 and Figure 5 The comparison shows that the hydrogel pore size in the comparative example is significantly larger than that in the example, and the bottom of the comparative example is immersed in TA-Fe. 3+ Although the pores in this portion of the solution are smaller, they are not significantly different in size from those in the unsoaked tannic acid-iron ion mixture. This demonstrates that the platelet-rich plasma component plays a key molecular regulatory role in the gradient structure formation of this embodiment.

[0043] c. The gradient hydrogel samples from the examples, after being lyophilized and soaked for 18 hours, were subjected to FTIR-ATR scanning. The measurement positions of the gradient hydrogel samples were divided into four sampling groups along the sample height direction, labeled 1, 3, 5, and 7. Group 1 was located at the bottom of the gradient hydrogel sample, defined as the zero point of height reference; Group 3 was located at one-third of the height of the gradient hydrogel sample; Group 5 was located at two-thirds of the height of the gradient hydrogel sample; and Group 7 was located at the top of the gradient hydrogel sample, approximately 1.5 cm from the bottom. Figure 6 As shown, it was found at 1031cm -1 The peak heights in the vicinity differ significantly, with the highest peak in group 1 and the lowest in group 7. This peak is a characteristic peak of TA, indicating that the hydrogel successfully introduced phenolic CO stretching, and the results directly prove TA-Fe 3+ The penetration within the gel is gradient-distributed, forming a gradient cross-linked structure.

[0044] Therefore, this invention employs the aforementioned bioactive biomimetic gradient hydrogel, its preparation method, and its application, by immersing the hydrogel in TA-Fe... 3+ In solution, TA-Fe can be spontaneously formed inside the hydrogel using simple diffusion control. 3+ The concentration gradient of the coordination is used to accurately simulate the mechanical gradient of natural cartilage from soft to hard.

[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a bioactive biomimetic gradient hydrogel, characterized in that: Includes the following steps: S1. Quaternary ammonium salt chitosan, 3-acrylamidophenylboronic acid, platelet-rich plasma and phenyl (2,4,6-trimethylbenzoyl) lithium phosphate were dissolved in deionized water, stirred until completely dissolved and then centrifuged to obtain a prepolymer solution. S2. Dissolve tannic acid granules in deionized water, stir until completely dissolved, then add anhydrous ferric chloride and stir evenly to obtain a tannic acid-ferric ion mixture. S3. Add the prepolymer obtained in S1 into the mold and crosslink it by ultraviolet irradiation to obtain a hydrogel. S4. Immerse the hydrogel portion obtained in S3 in the tannic acid-iron ion mixture in S2 to obtain a biomimetic gradient hydrogel.

2. The method for preparing a bioactive biomimetic gradient hydrogel according to claim 1, characterized in that: In the prepolymer solution of S1, the mass ratio of quaternary ammonium chitosan, 3-acrylamidophenylboronic acid, platelet-rich plasma and phenyl (2,4,6-trimethylbenzoyl) lithium phosphate is 5-10:0.5-1:10-20:0.2-0.

4.

3. The method for preparing a bioactive biomimetic gradient hydrogel according to claim 1, characterized in that: In S1, stir at room temperature at a stirring speed of 800-1200 rpm for 60-90 min.

4. The method for preparing a bioactive biomimetic gradient hydrogel according to claim 1, characterized in that: In S1, the relative centrifugal force is 1000-1200g, and the centrifugation time is 5-12min.

5. The method for preparing a bioactive biomimetic gradient hydrogel according to claim 1, characterized in that: In S2, the mass ratio of tannic acid particles to anhydrous ferric chloride is 30-50:

3.

6. The method for preparing a bioactive biomimetic gradient hydrogel according to claim 1, characterized in that: In S2, the stirring temperature is room temperature, the stirring time is 0.5-2 hours, and the stirring speed is 300-500 rpm.

7. The method for preparing a bioactive biomimetic gradient hydrogel according to claim 1, characterized in that: In S3, the ultraviolet irradiation time is 45-90s.

8. The method for preparing a bioactive biomimetic gradient hydrogel according to claim 1, characterized in that: In S4, during immersion, the liquid level of the tannic acid-iron ion mixture is one-third to one-half the height of the hydrogel, and the immersion time is 18-24 hours.

9. A bioactive biomimetic gradient hydrogel, characterized in that: It was prepared using the preparation method of a bioactive biomimetic gradient hydrogel as described in any one of claims 1-8.

10. An application of a bioactive biomimetic gradient hydrogel, characterized in that: The bioactive biomimetic gradient hydrogel according to claim 9 is used in the preparation of tissue repair materials with biomimetic gradient structures.

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