Self-polarization degradable gradient electroactive hydrogel and preparation method and application thereof

Abstract

The application belongs to the field of biomedical materials, and particularly relates to a self-polarization degradable gradient electroactive hydrogel as well as a preparation method and application thereof. The application develops a novel degradable electroactive scaffold based on a high-electroactive natural macromolecule hydrogel, and through material component regulation and control and structure design, the piezoelectric performance of the scaffold is significantly improved, and the synergistic precise regulation and control of the scaffold on bone and cartilage regeneration are realized. Through the combination of electron transfer and ion transfer, the pathological electrical requirements of bone and cartilage regions are respectively adapted, and through the differential regulation and control of the electrical microenvironment and the material structure, the efficient bionics and bidirectional repair of the bone-cartilage interface are realized.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a self-polarizing degradable gradient electroactive hydrogel, its preparation method, and its application. Background Technology

[0002] Osteoarthritis (OA) is the most common joint disease worldwide, primarily characterized by degeneration and damage to cartilage tissue, leading to severe pain and significantly limiting patients' daily functions. Currently, commonly used non-surgical treatments (such as analgesics and anti-inflammatory drugs) can only relieve symptoms and cannot achieve a complete cure. Surgical interventions such as subchondral bone microfractures, joint lavage, and debridement are mainly suitable for small-scale cartilage defects. For severe joint lesions, the current gold standard treatment is the surgical implantation of autologous or allogeneic cartilage grafts. However, such transplantation surgeries carry risks such as donor site complications, immune rejection, and infection, and the availability of tissue is limited.

[0003] In recent years, novel materials possessing both electroactivity and biodegradability have emerged in the field of biomedical engineering, providing new directions for the innovation and iteration of orthopedic implant materials. In the clinical application of osteochondral repair, the non-degradability of traditional piezoelectric ceramics (such as PZT and BaTiO3) has led to secondary surgical risks and biotoxicity issues caused by long-term retention, becoming a major bottleneck limiting their widespread application. Therefore, the development of biodegradable electroactive materials has become an important research direction in the field of osteochondral repair.

[0004] For bone repair, the appropriate output voltage of piezoelectric materials is typically between a few millivolts and tens of millivolts. This range effectively stimulates osteoblast activity and promotes bone tissue regeneration and repair. For cartilage repair, however, because cartilage tissue is more sensitive to electrical stimulation, the required output voltage of piezoelectric materials is relatively lower, generally between a few millivolts and twenty millivolts, to avoid excessive electrical stimulation that could damage chondrocytes. Bone and cartilage tissue possess natural electrical charge properties under physiological conditions, especially when subjected to mechanical stress, generating an electric field that plays a crucial role in cell growth, differentiation, and repair. Current research indicates that hydrogels constructed solely using materials such as methacryloyl gelatin (GelMA) and sodium alginate (SA) cannot fully meet the needs of bone and cartilage tissue regeneration, particularly due to insufficient electrical activity. Summary of the Invention

[0005] To address at least some of the technical problems existing in the prior art, this invention provides a self-polarizing degradable gradient electroactive hydrogel material. Based on the biomimetic perspective of bioelectric signals and the core concept of osteochondral tissue engineering repair, it selects GelMA and SA as main raw materials and introduces polar fluorine-containing molecular materials with high voltage response capabilities to form a multi-component degradable electroactive hydrogel. Through, for example, calcium ion crosslinking of SA, and blue light crosslinking technology, the GelMA / SA mixture is further solidified into a gel, resulting in a self-polarizing degradable gradient electroactive hydrogel material with good biocompatibility, a biomimetic osteochondral physiological electroactive environment, and further promoting the repair of osteochondral defects. Specifically, this invention includes the following:

[0006] In a first aspect, the present invention provides a self-polarizing, degradable, gradient electroactive hydrogel for osteochondral repair, comprising a hydrogel substrate and a fluorinated molecular material, wherein the fluorinated molecular material forms a non-covalent network with the hydrogel substrate and causes spontaneous dipole orientation within the hydrogel substrate to generate an electrical response.

[0007] In some embodiments, the self-polarizing degradable gradient electroactive hydrogel for osteochondral repair according to the present invention, wherein when the hydrogel is subjected to external stimulation, the amplitude of the output voltage of the hydrogel in response to the external stimulation is not less than 500 mV. Preferably, the external stimulus is ultrasound stimulation, and the ultrasound conditions include an ultrasound power of 0.1-100 W and / or a frequency of 0.1-100 MHz.

[0008] In some embodiments, the self-polarizing degradable gradient electroactive hydrogel for osteochondral repair according to the present invention comprises a first polymer network and a second polymer network interpenetrating each other. Preferably, the first polymer network is formed by crosslinking a precursor solution containing a photocrosslinkable polymer through light irradiation, and the second polymer network is formed by crosslinking a precursor solution containing an ionicly crosslinkable polysaccharide under the action of polyvalent metal ions.

[0009] In some embodiments, the self-polarizing degradable gradient electroactive hydrogel for osteochondral repair according to the present invention, wherein the photocrosslinking polymer comprises at least one of methacrylamide gelatin, methacrylamide hyaluronic acid, methacrylamide silk fibroin, methacrylamide elastin, methacrylamide chitosan, and methacrylamide alginate.

[0010] In some embodiments, the self-polarizing degradable gradient electroactive hydrogel for osteochondral repair according to the present invention, wherein the ionic cross-linked polysaccharide includes at least one of sodium alginate, carrageenan, gellan gum, xanthan gum, pectin, gum arabic, chondroitin sulfate, and sodium hyaluronate. Preferably, the ions include at least one of calcium ions, barium ions, and strontium ions; Preferably, the ion is a calcium ion, which is derived from at least one of calcium chloride, calcium gluconate, calcium lactate, calcium sulfate, or calcium hydrogen phosphate.

[0011] A second aspect of the present invention provides a method for preparing a self-polarizing, degradable, gradient electroactive hydrogel for osteochondral repair, comprising the steps of mixing and crosslinking a precursor solution for forming a first polymer network, a precursor solution for forming a second polymer network, and a fluorinated molecular material.

[0012] In some embodiments, according to the preparation method of the present invention, the precursor solution for forming the first polymer network has a mass percentage concentration of 1-10 wt% in the mixed system, and the precursor solution for forming the second polymer network has a mass volume concentration of 1%-10% in the mixed system.

[0013] In some embodiments, according to the preparation method of the present invention, the mixing volume ratio of the precursor solution for forming the first polymer network to the precursor solution for forming the second polymer network is 1:1 to 1:5.

[0014] In some embodiments, according to the preparation method of the present invention, the mass-volume concentration of the fluorinated molecular material in the mixed system is greater than 10%, preferably greater than 15%, even more preferably greater than 20%, and further preferably greater than 30%. Preferably, the mass-volume concentration of the fluorinated molecular material in the mixed system is less than 50%, and even more preferably less than 45%.

[0015] A third aspect of the invention provides the use of the self-polarizing degradable gradient electroactive hydrogel of the invention in the preparation of repair products for osteoarthritis or osteochondritis.

[0016] The beneficial effects of this invention include: (1) The hydrogel material used in this invention has the characteristics of both natural and synthetic biomaterials. It has excellent biocompatibility and biodegradability, and can promote cell adhesion, proliferation and differentiation. By adjusting the proportion of each component and the cross-linking conditions, the mechanical properties can be precisely controlled so that its mechanical properties match those of natural osteochondral tissue.

[0017] (2) Polar fluorine-containing molecular materials endow hydrogels with excellent piezoelectric properties, which can exhibit excellent properties without polarization treatment. They can effectively improve the electroactivity of the material. Furthermore, polar fluorine-containing molecular materials can form a stable hydrogen bond network in the hydrogel. By enhancing the self-polarization performance of the material and optimizing its biomimetic microstructure, the synergistic regulation of electricity and structure can be achieved, thereby improving its bone-cartilage tissue regeneration capacity.

[0018] (3) All components in the material system of the present invention have good biocompatibility and degradability, which meet the requirements of human implant materials, can reduce immune rejection reaction, and gradually degrade with tissue regeneration.

[0019] (4) In an exemplary implementation, the mechanical responsiveness of calcium ion crosslinking is combined with the further stabilization of photocrosslinking to make the material injectable. Then, by adjusting the content of polar fluorine-containing molecular materials, the mechanical and electrical properties can be precisely controlled. After injection, it can quickly adapt to complex osteocartilage defects and provide immediate support. Attached Figure Description

[0020] Figure 1 Gel SEM and EDS images of different parts of a self-polarized, degradable, gradient electroactive hydrogel are shown.

[0021] Figure 2 The piezoelectric output diagram of the biodegradable gradient electroactive hydrogel is shown.

[0022] Figure 3 The biodegradability of the degradable gradient electroactive hydrogel was demonstrated.

[0023] Figure 4 The biocompatibility of the biodegradable gradient electroactive hydrogel was demonstrated.

[0024] Figure 5 The study demonstrated the promoting effect of degradable gradient electroactive hydrogels on bone-related differentiation proteins.

[0025] Figure 6 The effect of biodegradable gradient electroactive hydrogel on osteochondral defect repair was demonstrated. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Self-polarizing degradable gradient electroactive hydrogel In a first aspect, the present invention provides a self-polarizing, degradable, gradient electroactive hydrogel for osteochondral repair, comprising a hydrogel substrate and a fluorinated molecular material, wherein the fluorinated molecular material forms a non-covalent network with the hydrogel substrate and causes spontaneous dipole orientation within the hydrogel substrate to generate an electrical response.

[0030] In a preferred embodiment, the electrical response refers to the fact that when the hydrogel is subjected to an external stimulus, in response to the external stimulus, the amplitude of the output voltage of the hydrogel is not less than 500 mV, for example, not less than 550, 600, 650, 700, 750, 800, 850, 900 mV, or even greater than 900 mV. The external stimulus is not particularly limited, and may be ultrasound stimulation or ultrasound treatment. Ultrasound conditions include ultrasound power of 0.1-100 W (e.g., 0.1-50 W, 0.1-25 W, 0.1-20 W, 0.1-10 W, 0.1-5 W, such as 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 W) and / or frequency of 0.1-100 MHz (e.g., 0.1-50 MHz, 0.1-25 MHz, 0.1-20 MHz, 0.1-10 MHz, 0.1-5 MHz, such as 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 MHz).

[0031] Considering that hydrogel substrates alone cannot fully meet the needs of osteochondral tissue regeneration, especially due to their insufficient electroactivity, this invention modifies the hydrogel substrate by incorporating biodegradable polar fluorinated molecular materials. These functional polar fluorinated molecular materials, possessing unique electroactivity and biodegradability, can form hydrogen bond networks within the hydrogel, enhancing the material's self-polarization properties. Different concentrations of polar fluorinated molecular materials can form uniformly distributed electroactive sites within the gel, mimicking electrical signals in the physiological environment and promoting cellular electroresponsiveness and tissue regeneration.

[0032] In this invention, the polar fluorine-containing molecular material can be synthesized by methods known in the art or purchased from commercially available products. In a preferred embodiment, the polar fluorine-containing molecular material is a material having an orthorhombic crystal system and a space group of P1. In a more preferred embodiment, it is prepared by the following steps: (1) Dissolve the polyfluorinated alkane-α,ω-diol compound shown in Formula I in an organic solvent to form a solution with a concentration of 40%-90% (w / v); (2) Place the solution in a container and add a non-polar hydrocarbon solvent to it, wherein the volume of the non-polar hydrocarbon solvent is 0.5-5 times that of the solution, to form a liquid-liquid two-phase system; (3) The container is sealed and left to stand for 7-40 days at a temperature of 15℃-35℃ and a relative humidity of 70%-95% to obtain a polar fluorine-containing molecular material.

[0033] In the above preparation method, the compound of formula I is HO-Rf-OH, wherein Rf is a C2-C10 alkylene group substituted with at least one fluorine atom, preferably a perfluoroalkylene group or a partially fluoroalkylene group. The organic solvent is preferably an ester solvent, more preferably a C1-C4 alkyl acetate, and even more preferably ethyl acetate, methyl acetate, or isopropyl acetate. The nonpolar hydrocarbon solvent is preferably a mixture of C5-C8 alkanes, such as petroleum ether or n-hexane. The finally prepared fluorinated molecular material can be identified by methods known in the art, such as, but not limited to, nuclear magnetic resonance spectroscopy (NMR). 1 H NMR, 13 C NMR, 19 Spectroscopy methods include: Fiber spectroscopy (F NMR), Fourier transform infrared spectroscopy (FT-IR), mass spectrometry (MS), X-ray powder diffraction (PXRD), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC).

[0034] In this invention, the hydrogel substrate comprises an interpenetrating first polymer network and a second polymer network. Preferably, the first polymer network is formed by crosslinking a precursor solution containing a photocrosslinkable polymer under light irradiation, and the second polymer network is formed by crosslinking a precursor solution containing an ionicly crosslinkable polysaccharide under the action of polyvalent metal ions.

[0035] In a preferred embodiment, the photocrosslinking polymer comprises at least one of methacrylamide gelatin, methacrylamide hyaluronic acid, methacrylamide silk fibroin, methacrylamide elastin, polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, methacrylamide chitosan, and methacrylamide alginate. In a specific embodiment, the photocrosslinking polymer is methacrylamide gelatin.

[0036] In a preferred embodiment, the ionicly cross-linked polysaccharide comprises at least one selected from sodium alginate, carrageenan, gellan gum, xanthan gum, pectin, gum arabic, chondroitin sulfate, and sodium hyaluronate. In a specific embodiment, the ionicly cross-linked polysaccharide is sodium alginate.

[0037] Preparation method A second aspect of the present invention provides a method for preparing the aforementioned hydrogel, comprising the steps of mixing and crosslinking a precursor solution for forming a first polymer network, a precursor solution for forming a second polymer network, and a fluorinated molecular material.

[0038] In a preferred embodiment, the preparation method of the present invention includes: (1) Preparation of methacrylamide gelatin porous solid Gelatin was mixed with a buffer solution and stirred until dissolved. An acylating agent capable of polymerizing olefin bonds was added, and the mixture was reacted at a suitable temperature to obtain crude methacrylated gelatin. The crude product was purified to remove unreacted small molecules, and the purified product was dried to obtain porous solid methacrylated gelatin.

[0039] (2) Preparation of prepolymer mixture Alginate is dissolved in an aqueous solvent to obtain an alginate solution; the porous solid methacrylamide gelatin obtained in step (1) is dissolved in an aqueous solution containing a water-soluble photoinitiator to obtain a methacrylamide gelatin solution; the alginate solution and the methacrylamide gelatin solution are mixed in a preset ratio and stirred at 40-90°C to obtain a prepolymer mixture.

[0040] (3) Double crosslinking Add a fluorine-containing molecular material solution to the prepolymer mixture in step (2) and dissolve and mix at a temperature not lower than 50°C. Then add a multivalent metal cation crosslinking agent solution for initial crosslinking. Then use photochemical radiation to promote crosslinking. Place the obtained hydrogel precursor in an environment of 20-40°C for complete crosslinking to obtain a self-polarized degradable gradient electroactive hydrogel.

[0041] In step (1), the buffer solution used includes, but is not limited to, phosphate buffer, borate buffer, etc., with a pH of 7-9. The stirring time is, for example, 1-10 hours, preferably 2-4 hours. The acylating agent that can polymerize the olefin bond can be methacrylic anhydride or methacryloyl halide. Purification can be carried out by any suitable method in the art, such as dialysis bag dialysis. In a preferred embodiment, 1-20% (w / v) of methacrylic anhydride is slowly and uniformly added to a 5-50%, preferably 5-20%, e.g., 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20% gelatin solution, preferably 5-10%, e.g., 5, 6, 7, 8, 9, 10%, while maintaining a water bath heating temperature of 30-60°C, preferably 40-60°C, e.g., 40, 42, 46, 48, 50, 55, 60°C, and the reaction is continued for 1-5 hours, preferably 2-4 hours. After dialysis, the mixture is freeze-dried at a freezing temperature of, for example, -50°C and a vacuum of 4 Pa ​​for three days.

[0042] In step (2), the alginate can be any soluble salt of alginic acid, such as, but not limited to, sodium alginate, ammonium alginate, or potassium alginate. The water-soluble photoinitiator is preferably lithium phenyl-2,4,6-trimethylbenzoylphosphine (LAP) or other acylphosphine oxide photoinitiators. The polyvalent metal cation crosslinking agent is preferably a soluble salt of divalent or trivalent metal ions, such as, but not limited to, calcium, barium, zinc, or iron salts, such as calcium chloride (CaCl2), calcium sulfate (CaSO4), calcium carbonate (CaCO3), calcium lactate, calcium gluconate, etc. In a preferred embodiment, sodium alginate is dissolved in an aqueous solvent, such as ultrapure water, and stirred in a water bath at 30-60°C, preferably 40-60°C, such as 40, 42, 46, 48, 50, 55, 60°C, to form a uniform and transparent 1-10%, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10% (W / V) SA solution. The methacrylamide porous solid obtained in step (1) is fully dissolved in 0.1-1%, preferably 0.1-0.5% (W / V) LAP to obtain a stable 1-10%, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10% (W / V) GelMA solution.

[0043] In step (2), the volume ratio of SA solution and GelMA solution when mixed is not particularly limited, but preferably 1:1 to 1:5, such as 1:1, 1:2, 1:3, 1:4, 1:5, etc.

[0044] This invention discovers that the content of polar fluorine-containing molecular materials has a bidirectional regulatory effect on the pore structure of the scaffold: an appropriate amount of polar fluorine-containing molecular materials can enhance network stability and optimize pore connectivity, while an excessive amount of polar fluorine-containing molecular materials may lead to a decrease in pore size and porosity due to crystal aggregation and local network densification, and too little will result in almost no output voltage, which is not conducive to osteochondral repair. Therefore, in a preferred embodiment, the mass-volume concentration of the fluorinated molecular material added in step (3) in the mixed system is greater than 10% and / or less than 50%, preferably greater than 15% and / or less than 45%, more preferably 15% and / or less than 40%, and even more preferably 15% and / or less than 30%, such as 15-45%, 15-40%, 15-35%, 15-30%, etc., such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45%.

[0045] It is understandable that fluorinated molecular materials can be added in different mass-volume concentrations. For example, a low concentration of fluorinated molecular materials can be added to an SA solution and / or a GelMA solution, while a high concentration of fluorinated molecular materials can be added to another SA solution and / or a GelMA solution. Subsequent ionic crosslinking and photocrosslinking reactions can then be carried out to obtain electroactive hydrogels with different or gradient electrical responses.

[0046] application In one aspect, the present invention provides the use of the aforementioned hydrogel in the preparation of materials for osteoarthritis or osteochondral repair, wherein the repair materials include, but are not limited to, implant materials, bone fillers, or bone tissue engineering scaffolds.

[0047] In some embodiments, the hydrogel is an ultrasonically treated or ultrasonically stimulated hydrogel, wherein the ultrasonic treatment or ultrasonic stimulation includes an ultrasonic power of 0.1-100 W and / or a frequency of 0.1-100 MHz.

[0048] The present invention therefore also provides the use of an ultrasound device in conjunction with a hydrogel in the preparation of medical products for osteoarthritis or osteochondritis, wherein the ultrasound device comprises at least an ultrasound generating unit configured to output ultrasound waves with a power of 0.1-100 W and / or a frequency of 0.1-100 MHz.

[0049] Example 1 The following describes a method for preparing a self-polarizing, degradable, gradient electroactive hydrogel, comprising the following steps: (1) Polar fluorine-containing molecular materials were synthesized by solvent diffusion method. Hexafluoropentane-1,5-diol was completely dissolved in ethyl acetate to prepare a homogeneous solution with a concentration of 67% (w / v). This solution was then transferred to 15 mL centrifuge tubes. Two volumes of petroleum ether were added to the centrifuge tubes to form a two-layer solution system. After sealing the centrifuge tubes, they were incubated in a constant temperature and humidity chamber at 25°C and RH 85% for 21 days to obtain transparent polar fluorine-containing molecular materials with a uniform orientation and a length of approximately 1.5 cm. (2) Dissolve gelatin in PBS buffer solution and stir magnetically for 4 hours to form a 10% (W / V) gelatin solution. Slowly and evenly add 7% methacrylic anhydride by volume to the gelatin solution, keep the water bath heating temperature at 50°C, and complete the addition within 30 minutes. Continue the reaction for 3 hours, perform dialysis treatment in a dialysis bag for 6 days, and then freeze dry at -50°C and 4 Pa ​​for 3 days to obtain GelMA sponge. (3) Dissolve sodium alginate in ultrapure water and stir in a water bath at 50°C for 4 hours to form a uniform and transparent 4% (W / V) SA solution. Dissolve the GelMA sponge fully in 0.25% (W / V) LAP photocrosslinking agent to obtain a stable 5% (W / V) GelMA solution. (4) Mix 4% (W / V) SA and 5 wt% GelMA in (3) at a ratio of 1:1 to obtain a mixed hydrogel system. Add 15% (W / V) polar fluorine-containing molecular material from (1) as a low-piezoelectric component to the obtained mixture. Dissolve and mix at 70°C for 3 hours. Add 0.0375% (W / V) anhydrous calcium chloride solution and mix thoroughly to carry out preliminary crosslinking. Further promote the crosslinking reaction by irradiating with blue light for 30 seconds. Finally, place the mixed solution in an environment of 37°C to continue crosslinking for 36 hours to obtain a self-polarizing degradable electroactive hydrogel.

[0050] Example 2 The following describes a method for preparing a self-polarizing, degradable, gradient electroactive hydrogel, comprising the following steps: (1) Polar fluorine-containing molecular materials were synthesized by solvent diffusion method. Hexafluoropentane-1,5-diol was completely dissolved in ethyl acetate to prepare a homogeneous solution with a concentration of 67% (w / v). This solution was then transferred to 15 mL centrifuge tubes. Two volumes of petroleum ether were added to the centrifuge tubes to form a two-layer solution system. After sealing the centrifuge tubes, they were incubated in a constant temperature and humidity chamber at 25°C and RH 85% for 21 days to obtain transparent polar fluorine-containing molecular materials with a uniform orientation and a length of approximately 1.5 cm. (2) Dissolve gelatin in PBS buffer solution and stir magnetically for 4 hours to form a 10% (W / V) gelatin solution. Slowly and evenly add 7% methacrylic anhydride by volume to the gelatin solution, keep the water bath heating temperature at 50°C, and complete the addition within 30 minutes. Continue the reaction for 3 hours, perform dialysis treatment in a dialysis bag for 6 days, and then freeze dry at -50°C and 4 Pa ​​for 3 days to obtain GelMA sponge. (3) Dissolve sodium alginate in ultrapure water and stir in a water bath at 50°C for 4 hours to form a uniform and transparent 4% (W / V) SA solution. Dissolve the GelMA sponge fully in 0.25% (W / V) LAP photocrosslinking agent to obtain a stable 5% (W / V) GelMA solution. (4) Mix 4% (W / V) SA and 5 wt% GelMA in (3) at a ratio of 1:1 to obtain a mixed hydrogel system. Add 30% (W / V) polar fluorine-containing molecular material from (1) as a high-voltage electrical component to the mixture. Dissolve and mix at 70°C for 3 hours. Add 0.0375% (W / V) anhydrous calcium chloride solution and mix thoroughly to carry out preliminary crosslinking. Further promote the crosslinking reaction by irradiating with blue light for 30 seconds. Finally, place the mixed solution in a 37°C environment to continue crosslinking for 36 hours to obtain a self-polarizing degradable electroactive hydrogel.

[0051] Example 3 The following describes a method for preparing a self-polarizing, degradable, gradient electroactive hydrogel, comprising the following steps: (1) Polar fluorine-containing molecular materials were synthesized by solvent diffusion method. Hexafluoropentane-1,5-diol was completely dissolved in ethyl acetate to prepare a homogeneous solution with a concentration of 67% (w / v). This solution was then transferred to 15 mL centrifuge tubes. Two volumes of petroleum ether were added to the centrifuge tubes to form a two-layer solution system. After sealing the centrifuge tubes, they were incubated in a constant temperature and humidity chamber at 25°C and RH 85% for 21 days to obtain transparent polar fluorine-containing molecular materials with a uniform orientation and a length of approximately 1.5 cm. (2) Dissolve gelatin in PBS buffer solution and stir magnetically for 4 hours to form a 10% (W / V) gelatin solution. Slowly and evenly add 7% methacrylic anhydride by volume to the gelatin solution, keep the water bath heating temperature at 50°C, and complete the addition within 30 minutes. Continue the reaction for 3 hours, perform dialysis treatment in a dialysis bag for 6 days, and then freeze dry at -50°C and 4 Pa ​​for 3 days to obtain GelMA sponge. (3) Dissolve sodium alginate in ultrapure water and stir in a water bath at 50°C for 4 hours to form a uniform and transparent 4% (W / V) SA solution. Dissolve the GelMA sponge fully in 0.25% (W / V) LAP photocrosslinking agent to obtain a stable 5% (W / V) GelMA solution. (4) Mix 4% (W / V) SA and 5 wt% GelMA in (3) at a ratio of 1:1 to obtain a mixed hydrogel system. Divide the mixture into two parts and add the polar fluorinated molecular material from (1) as the high-voltage component (30% (W / V) polar fluorinated molecular material) and the low-voltage component (15% (W / V) polar fluorinated molecular material) to the two parts respectively. Dissolve and mix at 70°C for 3 hours. Add 0.0375% (W / V) anhydrous calcium chloride solution and mix thoroughly to carry out preliminary crosslinking. Further promote the crosslinking reaction by irradiating with blue light for 30 seconds. Finally, place the mixed solution in an environment of 37°C to continue crosslinking for 36 hours to obtain a self-polarizing degradable gradient electroactive hydrogel.

[0052] Comparative Example 1 The following describes a method for preparing a hydrogel, including the following steps: (1) Dissolve gelatin in PBS buffer solution and stir magnetically for 4 hours to form a 10% (W / V) gelatin solution. Slowly and evenly add 7% methacrylic anhydride by volume to the gelatin solution, keep the water bath heating temperature at 50°C, and complete the addition within 30 minutes. Continue the reaction for 3 hours, perform dialysis treatment in a dialysis bag for 6 days, and then freeze dry at -50°C and 4 Pa ​​for 3 days to obtain GelMA sponge. (2) Sodium alginate was dissolved in ultrapure water and stirred in a water bath at 50°C for 4 hours to form a uniform and transparent 4% (W / V) SA solution. GelMA sponge was fully dissolved in 0.25% (W / V) LAP photocrosslinking agent to obtain a stable 5% (W / V) GelMA solution. (4) Mix 4% (w / v) SA and 5 wt% GelMA in (3) at a ratio of 1:1 to obtain a mixed hydrogel system. Dissolve and mix the obtained mixture at 70°C for 3 hours. Add 0.0375% (w / v) anhydrous calcium chloride solution and mix thoroughly to carry out preliminary crosslinking. Further promote the crosslinking reaction by irradiating with blue light for 30 seconds. Finally, place the mixed solution in an environment of 37°C to continue crosslinking for 36 hours to obtain a hydrogel.

[0053] Comparative Example 2 The following describes a method for preparing a hydrogel, including the following steps: (1) Polar fluorine-containing molecular materials were synthesized by solvent diffusion method. Hexafluoropentane-1,5-diol was completely dissolved in ethyl acetate to prepare a homogeneous solution with a concentration of 67% (w / v). This solution was then transferred to 15 mL centrifuge tubes. Two volumes of petroleum ether were added to the centrifuge tubes to form a two-layer solution system. After sealing the centrifuge tubes, they were incubated in a constant temperature and humidity chamber at 25°C and RH 85% for 21 days to obtain transparent polar fluorine-containing molecular materials with a uniform orientation and a length of approximately 1.5 cm. (2) Dissolve gelatin in PBS buffer solution and stir magnetically for 4 hours to form a 10% (W / V) gelatin solution. Slowly and evenly add 7% methacrylic anhydride by volume to the gelatin solution, keep the water bath heating temperature at 50°C, and complete the addition within 30 minutes. Continue the reaction for 3 hours, perform dialysis treatment in a dialysis bag for 6 days, and then freeze dry at -50°C and 4 Pa ​​for 3 days to obtain GelMA sponge. (3) Dissolve sodium alginate in ultrapure water and stir in a water bath at 50°C for 4 hours to form a uniform and transparent 4% (W / V) SA solution. Dissolve the GelMA sponge fully in 0.25% (W / V) LAP photocrosslinking agent to obtain a stable 5% (W / V) GelMA solution. (4) Mix 4% (W / V) SA and 5 wt% GelMA in (3) at a ratio of 1:1 to obtain a mixed hydrogel system. Add 10% (W / V) polar fluorine-containing molecular material from (1) as a low-piezoelectric component to the obtained mixture. Dissolve and mix at 70°C for 3 hours. Add 0.0375% (W / V) anhydrous calcium chloride solution and mix thoroughly to carry out preliminary crosslinking. Further promote the crosslinking reaction by irradiating with blue light for 30 seconds. Finally, place the mixed solution in an environment of 37°C to continue crosslinking for 36 hours to obtain a hydrogel.

[0054] Comparative Example 3 The following describes a method for preparing a self-polarizing, degradable, gradient electroactive hydrogel, comprising the following steps: (1) Polar fluorine-containing molecular materials were synthesized by solvent diffusion method. Hexafluoropentane-1,5-diol was completely dissolved in ethyl acetate to prepare a homogeneous solution with a concentration of 67% (w / v). This solution was then transferred to 15 mL centrifuge tubes. Two volumes of petroleum ether were added to the centrifuge tubes to form a two-layer solution system. After sealing the centrifuge tubes, they were incubated in a constant temperature and humidity chamber at 25°C and RH 85% for 21 days to obtain transparent polar fluorine-containing molecular materials with a uniform orientation and a length of approximately 1.5 cm. (2) Dissolve gelatin in PBS buffer solution and stir magnetically for 4 hours to form a 10% (W / V) gelatin solution. Slowly and evenly add 7% methacrylic anhydride by volume to the gelatin solution, keep the water bath heating temperature at 50°C, and complete the addition within 30 minutes. Continue the reaction for 3 hours, perform dialysis treatment in a dialysis bag for 6 days, and then freeze dry at -50°C and 4 Pa ​​for 3 days to obtain GelMA sponge. (3) Dissolve sodium alginate in ultrapure water and stir in a water bath at 50°C for 4 hours to form a uniform and transparent 4% (W / V) SA solution. Dissolve the GelMA sponge fully in 0.25% (W / V) LAP photocrosslinking agent to obtain a stable 5% (W / V) GelMA solution. (4) Mix 4% (W / V) SA and 5 wt% GelMA in (3) at a ratio of 1:1 to obtain a mixed hydrogel system. Add 50% (W / V) polar fluorine-containing molecular material from (1) as a high-voltage electrical component to the obtained mixture. Dissolve and mix at 70°C for 3 hours. Add 0.0375% (W / V) anhydrous calcium chloride solution and mix thoroughly to carry out preliminary crosslinking. Further promote the crosslinking reaction by irradiating with blue light for 30 seconds. Finally, place the mixed solution in an environment of 37°C to continue crosslinking for 36 hours to obtain a hydrogel.

[0055] Example of effect SEM and EDS of different parts of the self-polarized degradable gradient electroactive hydrogel prepared in the examples are shown in the figures. Figure 1 The red dots represent carbon (C) and the green dots represent phosphorus (F). By controlling the content of polar fluorine-containing molecular materials (F content), the intrinsic pore size and porosity of the scaffold can be effectively regulated, thereby giving the material a better microstructure to simulate the natural tissue characteristics of bone and cartilage. The deposition of polar fluorine-containing molecular materials in the material exhibits a "crystalline" state.

[0056] This invention reveals that the content of polar fluorine-containing molecular materials has a bidirectional regulatory effect on the pore structure of the scaffold. An appropriate amount of polar fluorine-containing molecular materials can enhance network stability and optimize pore connectivity, while excessive amounts lead to a decrease in pore size and porosity due to crystal aggregation and localized network densification (Comparative Example 3). Measurements showed that the hydrogel prepared in Comparative Example 1 had an average pore size of 57.01 μm and a porosity of 14.68%, the hydrogel prepared in Example 1 had an average pore size of 38.28 μm and a porosity of 20.41%, and the hydrogel prepared in Example 2 had an average pore size of 26.08 μm and a porosity of 23.75%. Furthermore, excessively low fluorine content resulted in virtually no output voltage, which is detrimental to osteochondral repair (Comparative Example 2).

[0057] The piezoelectric output of the biodegradable gradient electroactive hydrogel prepared in the examples is shown below. Figure 2 .from Figure 2 As can be seen, the introduction of polar fluorine-containing molecular materials effectively improves the electrical response of the materials.

[0058] The biodegradability of the biodegradable gradient electroactive hydrogel prepared in the examples is shown in the figure. Figure 3 The concentration of polar fluorine-containing molecular materials has a significant impact on their degradation behavior. Hydrogels with high polar fluorine-containing molecular material content showed obvious degradation after 14 days, but about 40% remained on day 48, which well matched the dynamic growth requirements in the bone-cartilage repair process.

[0059] The biocompatibility of the biodegradable gradient electroactive hydrogel prepared in the examples is shown in the figure. Figure 4 .from Figure 4 As can be seen, compared with the control group, the hydrogel has good biocompatibility and can promote the proliferation of bone mesenchymal stem cells.

[0060] To further evaluate the osteogenic differentiation-promoting ability of the described degradable gradient electroactive hydrogel, rat bone marrow mesenchymal stem cells (BMSCs) were seeded at appropriate densities onto the surface of sterilized degradable gradient electroactive hydrogels and cultured at 37°C in a 5% CO2 incubator. After 24 h of cell culture, the cells were stimulated with ultrasound (US) at a frequency of 1 MHz for 10 min daily, according to a pre-defined protocol. On days 7 and 14 of culture, cell viability, cytoskeleton morphology, cell spreading and adhesion, and cell proliferation were evaluated using AO / PI live-cell-dead cell double staining, phalloidin / DAPI fluorescence staining, and the CCK-8 assay, respectively. On day 14 of culture, total RNA and total protein were extracted from each group of cells, and the expression levels of osteogenic markers such as Pro-collagen I, ALP, and OCN were detected by qRT-PCR and Western blot, respectively. The promoting effect of the prepared degradable gradient electroactive hydrogel on bone-related differentiation proteins is described in the example below. Figure 5 .from Figure 5 As can be seen from the results, compared with the control group, under ultrasound stimulation (US) conditions, the expression levels of Pro collagen-I, ALP and OCN in each example group were significantly increased, with Example 1 and Example 2 showing more significant improvement, indicating that the hydrogel can effectively promote osteogenic differentiation of bone mesenchymal stem cells and bone matrix formation.

[0061] To further evaluate the effects of the described biodegradable gradient electroactive hydrogel on new bone formation, bone volume, and defect filling, a femoral condyle cartilage defect model (defect diameter 2 mm, depth 2 mm) was established in male SD rats. Biodegradable gradient electroactive hydrogels from each group were implanted into the defect site. Postoperatively, ultrasound (US) stimulation was administered according to a pre-defined protocol (ultrasound frequency 1 MHz, stimulation for 10 min daily; n=5 per group). Joint specimens were harvested at 1 and 2 months postoperatively for gross morphology imaging to observe tissue repair in the defect area. Subsequently, Micro-CT was used for three-dimensional reconstruction and quantitative analysis of the cartilage repair effect. The biodegradable gradient electroactive hydrogel prepared in the example is shown below for its application in cartilage defects. Figure 6 ,from Figure 6 It can be seen that the degradable gradient electroactive hydrogel group under ultrasound stimulation has the best repair effect, with the richest new tissue and the most complete coverage in macroscopic view. Micro-CT shows the most new bone formation and the most thorough defect filling.

[0062] This invention provides a novel biodegradable electroactive scaffold based on highly electroactive natural macromolecular hydrogels. Through material composition regulation and structural design, its piezoelectric properties are significantly enhanced, enabling synergistic and precise regulation of bone and cartilage regeneration in different parts of the scaffold. This effectively promotes osteochondral tissue regeneration and interfacial integration, enhances osteoblast / chondrocyte activity, and promotes matrix deposition. By combining electron and ion transport, it adapts to the pathological electrical needs of bone and cartilage regions, achieving highly efficient biomimetic and bidirectional repair of the bone-cartilage interface through differentiated regulation of the electrical microenvironment and material structure.

[0063] 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 the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A self-polarizing, biodegradable, gradient electroactive hydrogel for osteochondral repair, characterized in that, The invention comprises a hydrogel substrate and a fluorinated molecular material, wherein the fluorinated molecular material forms a non-covalent network with the hydrogel substrate, causing spontaneous dipole orientation and electrical response within the hydrogel substrate. The fluorinated molecular material is an orthorhombic crystal system with a P1 space group. The hydrogel substrate comprises an interpenetrating first polymer network and a second polymer network. The first polymer network is formed by crosslinking a precursor solution containing methacrylamide gelatin under photoirradiation, and the second polymer network is formed by crosslinking a precursor solution containing alginate under the action of multivalent metal ions. The fluorinated molecular material is prepared through the following steps: (1) Dissolve polyfluoroalkane-α,ω-diol compounds in an organic solvent to form a solution with a concentration of 40%-90% (w / v); (2) Place the solution in a container and add a non-polar hydrocarbon solvent to it, wherein the volume of the non-polar hydrocarbon solvent is 0.5-5 times that of the solution, to form a liquid-liquid two-phase system; (3) The container is sealed and left to stand for 7-40 days at a temperature of 15℃-35℃ and a relative humidity of 70%-95% to obtain the fluorine-containing molecular material.

2. The self-polarizing, degradable, gradient electroactive hydrogel for osteochondral repair according to claim 1, characterized in that, When the hydrogel is subjected to an external stimulus, in response to the external stimulus, the amplitude of the output voltage of the hydrogel is not less than 500 mV.

3. The self-polarizing, degradable, gradient electroactive hydrogel for osteochondral repair according to claim 2, characterized in that, The external stimulus is ultrasound stimulation, and the ultrasound conditions include an ultrasound power of 0.1-100 W and / or a frequency of 0.1-100 MHz.

4. The self-polarizing, biodegradable, gradient electroactive hydrogel for osteochondral repair according to claim 1, characterized in that, The ions include at least one of calcium ions, barium ions, and strontium ions.

5. The self-polarizing, biodegradable, gradient electroactive hydrogel for osteochondral repair according to claim 1, characterized in that, The ion is a calcium ion, which is derived from at least one of calcium chloride, calcium gluconate, calcium lactate, calcium sulfate, or calcium hydrogen phosphate.

6. A method for preparing a self-polarizing, degradable, gradient electroactive hydrogel for osteochondral repair as described in any one of claims 1-5, characterized in that, The method includes the steps of mixing and crosslinking a precursor solution for forming a first polymer network, a precursor solution for forming a second polymer network, and a fluorinated molecular material, wherein the mass-volume concentration of the fluorinated molecular material in the mixed system is greater than 10% and less than 50%.

7. The preparation method according to claim 6, characterized in that, The precursor solution used to form the first polymer network has a mass percentage concentration of 1-10 wt% in the mixing system.

8. The preparation method according to claim 6, characterized in that, The precursor solution used to form the second polymer network has a mass-volume concentration of 1%-10% in the mixed system.

9. The preparation method according to claim 6, characterized in that, The mixing volume ratio of the precursor solution for forming the first polymer network to the precursor solution for forming the second polymer network is 1:1 to 1:

5.

10. The use of the self-polarizing degradable gradient electroactive hydrogel according to any one of claims 1-5 in the preparation of repair products for osteoarthritis or osteochondritis.

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