Double-network semi-cross chain stacking bionic cartilage magnetoelectric gel, preparation method and application thereof

Abstract

The application relates to the field of biomedical engineering and specifically relates to a double-network semi-crossed chain stacking biomimetic cartilage magnetoelectric gel, a preparation method and application thereof. The biomimetic cartilage magnetoelectric gel is prepared by adding 0.5-2 parts of superparamagnetic ferroferric oxide nanoparticles, 2-8 parts of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and 2-8 parts of sodium alginate into 100 parts of a solvent, wherein the solvent is prepared according to a proportion of water:N,N-dimethylformamide:dichloromethane=4-6:1-2:2-4. The biomimetic cartilage magnetoelectric gel has good biocompatibility and a functional environment of biomimetic cartilage, can release electromagnetic stimulation to promote cell proliferation and migration, promote chondrocyte induced differentiation, provide mechanical bearing and support for the repair of damaged cartilage, promote the generated new cartilage tissue to have transparent cartilage characteristics similar to normal cartilage tissue, is superior to an AFe group induced by simple magnetic stimulation and a VA group induced by simple piezoelectric stimulation, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical engineering, and particularly relates to a double-network semi-crossed chain stacking biomimetic cartilage magnetoelectric gel, a preparation method and application thereof. BACKGROUND

[0002] Articular cartilage plays an important role in functional movement due to its load-bearing lubrication and mechanical cushioning. However, due to the properties of no blood vessels, low cell density, and slow cell matrix update frequency, the cartilage tissue lacks self-repairing ability. The long-term mechanical wear or sudden injury of cartilage defects will lead to the occurrence of osteoarthritis (OA). At present, the clinical treatment of osteoarthritis includes anti-inflammatory or analgesic drug intervention, stem cell therapy, growth factor assisted induction, and surgical replacement implantation. The above treatment methods have the disadvantages of limited effect, few safety basis, and uncontrollable complications, and it is difficult to achieve rapid and stable repair of the defective cartilage.

[0003] Cartilage tissue engineering is a research hotspot in recent years, and the ultimate goal is to select appropriate materials to construct a biomimetic cartilage scaffold or gel, to mimic the mechanical properties, porous structure, and high water content of the cartilage, and to provide an appropriate induction environment and functional compensation for the repair of defective cartilage. However, the selection and application of materials are difficult for those skilled in the art, which increases the difficulty of cartilage tissue engineering research. Moreover, the biomimetic cartilage scaffold or gel prepared in the previous cartilage tissue engineering research needs to be combined with exogenous cells or growth factors to provide cartilage regeneration activity, which has the disadvantages of high cost, disturbance of internal environment homeostasis, and immunological rejection, thereby limiting the treatment and in-situ regeneration and repair of defective cartilage.

[0004] The basis of studying the biomimetic cartilage scaffold is to have a deep understanding of the cartilage tissue environment. The extracellular matrix (ECM) of the cartilage tissue maintains the principle of electrical neutrality. When subjected to external functional stimulation, the uneven ion flow movement in the liquid environment, the uneven charge exposure in the solid environment, and the piezoelectric response of the collagen fibers make the cartilage tissue release varying endogenous electrical stimulation, which, combined with Faraday's law, exhibits endogenous electromagnetic effects. The electromagnetic effects are maintained or weakened with the change of the tissue state, and regulate the expression of cell behavior. On the basis of this theory, it has been proved that the electromagnetic stimulation given to the defective cartilage can simulate and amplify the endogenous electromagnetic effects, regulate the expression of COL2A1 and other expressions related to the repair of hyaline cartilage, and thus achieve rapid and stable repair of the defective cartilage. This is expected to solve the potential drawbacks of the application of exogenous cells or growth factor-based biomimetic cartilage scaffolds or gels. However, the electromagnetic field stimulation given in vitro is easily weakened by the tissue, and has the disadvantages of uncontrollable penetration and limited quantification of the effect strength. 2+ backflow, promote the expression of COL2A1 and other expressions related to the repair of hyaline cartilage, and thus achieve rapid and stable repair of the defective cartilage. This is expected to solve the potential drawbacks of the application of exogenous cells or growth factor-based biomimetic cartilage scaffolds or gels. However, the electromagnetic field stimulation given in vitro is easily weakened by the tissue, and has the disadvantages of uncontrollable penetration and limited quantification of the effect strength.

[0005] Therefore, smart responsive materials have emerged to provide functional modifications for implanted biomimetic cartilage scaffolds that directly respond to and release multiple physical fields at the defect site. Among them, piezoelectric smart responsive materials have attracted widespread attention because they can sense mechanical loads and release electrical stimulation to promote the repair of defective tissue. However, although the various piezoelectric composite materials currently under study have good piezoelectric healing-promoting properties, their healing-promoting effects are limited by the inherent disadvantages of the piezoelectric model. The timeliness of the traditional simple motion-stimulated piezoelectric response model may be affected by the piezoelectric coefficient of the material, the implantation site during surgery, the movement state of the experimental animal after surgery, and the intensity of the exercise load, and there is huge uncertainty. In addition, the ultrasonic stimulation piezoelectric response model proposed in recent reports also has disadvantages such as high cost, complex operation, and interference with motor function. In recent years, relevant reports on the application of piezoelectric materials in the field of cartilage tissue engineering have shown that traditional piezoelectric materials that are non-degradable or highly toxic are gradually transitioning to new piezoelectric materials that are degradable and have good biocompatibility. Among them, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) has attracted widespread attention and application due to its degradation rate that matches the long-term repair cycle of defective tissue, its high mechanical properties, and the safety of its degradation products. However, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) lacks the viscoelasticity required for cartilage tissue engineering materials, and its inherent hydrophobicity and low bioactivity also limit its conventional application in the field of cartilage tissue engineering.

[0006] In addition, magnetoelectric materials composed of piezoelectric materials and magnetostrictive materials can produce a magnetoelectric coupling cyclic conversion effect by sensing an external magnetic field or electric field, thereby realizing the mutual conversion of mechanical energy, electrical energy and magnetic energy, enhancing the piezoelectric response strength and magnetism of the composite material, and obtaining a better tissue repair effect. However, traditional magnetoelectric materials usually have certain biotoxicity and need to be modified to obtain appropriate biosafety. Moreover, traditional magnetoelectric materials require the use of an external electric field or an external magnetic field as a starting stimulus to activate the magnetoelectric coupling cyclic conversion effect, which has the disadvantages of high cost, complex operation, uncontrollable efficacy, and difficulty in adjusting the intensity of action.

[0007] In response to the above technical problems, the present invention provides a double-network semi-cross chain stacked biomimetic cartilage magnetoelectric gel. The biomimetic cartilage magnetoelectric gel has a motion-driven magnetoelectric coupling cyclic conversion effect in a three-phase solvent system, which provides a suitable physical and chemical environment and electromagnetic stimulation for the repair of defective cartilage while compensating for the functional loss of the defective cartilage. The biomimetic cartilage magnetoelectric gel drives the magnetoelectric coupling cyclic conversion effect through motion when sensing light force, including a motion-stimulated piezoelectric response model, a magnetostrictive piezoelectric response model and a magnetoelectric coupling cyclic conversion effect, providing a freely excitable and cyclically enhanced piezoelectric response and piezomagnetic response for inducing in situ regeneration and repair of defective cartilage. Combined with Faraday's law, it releases changing electromagnetic stimulation, simulates and amplifies endogenous electromagnetic effects, cascade-regulates the expression of genes related to defective cartilage repair, and promotes the in situ regeneration of hyaline cartilage tissue. Summary of the Invention

[0008] The present invention achieves the above-mentioned purpose through the following technical scheme. The primary purpose of the present invention is to provide a double-network semi-cross chain stacked biomimetic cartilage magnetoelectric gel. The biomimetic cartilage magnetoelectric gel is prepared by adding 0.5-2 parts of superparamagnetic ferrosoferric oxide nanoparticles, 2-8 parts of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) and 2-8 parts of sodium alginate to 100 parts of solvent. The solvent is prepared by water: N,N-dimethylformamide: dichloromethane in a ratio of 4-6:1-2:2-4.

[0009] Preferably, the ratio of water:N,N-dimethylformamide:dichloromethane is 6:1:3.

[0010] Preferably, the ratio of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) to sodium alginate in the bionic cartilage magnetoelectric gel is 1:1-3.

[0011] Preferably, the ratio of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) to sodium alginate in the bionic cartilage magnetoelectric gel is 1:2-3.

[0012] Preferably, the bionic cartilage magnetoelectric gel is prepared by the following method:

[0013] S1. Prepare a solvent by mixing water, N,N-dimethylformamide, and dichloromethane according to a ratio of 4-6:1-2:2-4;

[0014] S2. Take the solvent prepared in step S1, add superparamagnetic ferroferric oxide nanoparticles in a ratio of 0.5-2:100, ultrasonically disperse uniformly, and magnetically mix the solution;

[0015] S3. Adding poly (3-hydroxybutyrate-co-3-hydroxyvalerate) to the magnetic mixed solution obtained in step S2 to obtain a superparamagnetic ferrosoferric oxide nanoparticle / poly (3-hydroxybutyrate-co-3-hydroxyvalerate) composite solution, wherein the addition ratio of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) to solvent is 2-8:100;

[0016] S4. Adding sodium alginate powder to the superparamagnetic ferroferric oxide nanoparticles / poly(3-hydroxybutyrate-co-3-hydroxyvalerate) composite solution obtained in step S3 to obtain a superparamagnetic ferroferric oxide nanoparticles / poly(3-hydroxybutyrate-co-3-hydroxyvalerate) / sodium alginate composite sol, wherein the addition ratio of sodium alginate to solvent is 2-8:100;

[0017] S5. Take the superparamagnetic ferroferric oxide nanoparticles / poly (3-hydroxybutyrate-co-3-hydroxyvalerate) / sodium alginate composite sol prepared in step S4, draw it out and spread it into a mold, soak it in calcium chloride solution for cross-linking, and then dialyze it with deionized water to obtain a double-network semi-cross chain stacked biomimetic cartilage magnetoelectric gel.

[0018] A second object of the present invention is to provide a method for preparing the double-network semi-cross chain stacked biomimetic cartilage magnetoelectric gel, comprising the following steps:

[0019] S1. Prepare a solvent by mixing water, N,N-dimethylformamide, and dichloromethane according to a ratio of 4-6:1-2:2-4;

[0020] S2. Take the solvent prepared in step S1, add superparamagnetic ferroferric oxide nanoparticles in a ratio of 0.5-2:100, ultrasonically disperse uniformly, and magnetically mix the solution;

[0021] S3. Adding poly (3-hydroxybutyrate-co-3-hydroxyvalerate) to the magnetic mixed solution obtained in step S2 to obtain a superparamagnetic ferrosoferric oxide nanoparticle / poly (3-hydroxybutyrate-co-3-hydroxyvalerate) composite solution, wherein the addition ratio of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) to solvent is 2-8:100;

[0022] S4. Adding sodium alginate powder to the superparamagnetic ferroferric oxide nanoparticles / poly(3-hydroxybutyrate-co-3-hydroxyvalerate) composite solution obtained in step S3 to obtain a superparamagnetic ferroferric oxide nanoparticles / poly(3-hydroxybutyrate-co-3-hydroxyvalerate) / sodium alginate composite sol, wherein the addition ratio of sodium alginate to solvent is 2-8:100;

[0023] S5. Take the superparamagnetic ferroferric oxide nanoparticles / poly (3-hydroxybutyrate-co-3-hydroxyvalerate) / sodium alginate composite sol prepared in step S4, draw it out and spread it into a mold, soak it in calcium chloride solution for cross-linking, and then dialyze it with deionized water to obtain a double-network semi-cross chain stacked biomimetic cartilage magnetoelectric gel.

[0024] Preferably, the mass ratio of calcium chloride to deionized water in the calcium chloride solution of step S5 is 2:100.

[0025] The third object of the present invention is to provide the application of the double-network semi-cross chain stacked biomimetic cartilage magnetoelectric gel in the preparation of artificial cartilage materials.

[0026] The beneficial effects of the present invention are:

[0027] (1) The present invention provides a double-network semi-cross chain stacked biomimetic cartilage magnetoelectric gel, which is prepared by mixing 100 parts of a solvent with a ratio of 4-6:1-2:2-4 of water:N,N-dimethylformamide:dichloromethane, to which 0.5-2 parts of superparamagnetic ferrosoferric oxide nanoparticles, 2-8 parts of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and 2-8 parts of sodium alginate are added. The biomimetic cartilage magnetoelectric gel utilizes N,N-dimethylformamide, which is mutually soluble in deionized water and dichloromethane, to neutralize their repulsive polarities, thereby uniformly mixing the hydrophilic sodium alginate sol and the hydrophobic poly (3-hydroxybutyrate-co-3-hydroxyvalerate) piezoelectric plastic. The mixture is then cross-linked with a calcium chloride solution to form a double-network semi-cross chain stacking structure, thereby obtaining a repair-promoting gel with biomimetic cartilage composition, structure, and performance. This gel can effectively address the limitations of the application of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) in cartilage tissue engineering.

[0028] (2) The biomimetic cartilage magnetoelectric gel prepared in the present invention is creatively endowed with a motion-driven magnetoelectric coupling cyclic conversion effect, including a motion-stimulated piezoelectric response model, a magnetostrictive piezoelectric response model, and a magnetoelectric coupling cyclic conversion effect. Under light force conditions, it provides a freely excitable and cyclically enhanced piezoelectric response and piezomagnetic response for inducing in situ regeneration and repair of defective cartilage. Combined with Faraday's law, it releases variable electromagnetic stimulation, simulates and amplifies endogenous electromagnetic effects, cascade-regulates the expression of genes related to defective cartilage repair, and promotes the in situ regeneration of hyaline cartilage tissue. It can effectively solve the drawbacks of electromagnetic field stimulation given in vitro, such as being easily weakened by tissues, uncontrollable penetration, and limited quantitative intensity of action. It can also effectively solve the problem that the healing-promoting effect of traditional piezoelectric materials is limited by the inherent drawbacks of the piezoelectric model, and can also effectively solve the application limitations of traditional magnetoelectric materials.

[0029] (3) The biomimetic cartilage magnetoelectric gel of the present invention has good biocompatibility and a biomimetic cartilage functional environment. It can release electromagnetic stimulation to promote cell proliferation and migration, promote chondrocyte differentiation, and provide mechanical bearing and support for the repair of cartilage defects. The newly generated cartilage tissue has hyaline cartilage characteristics similar to those of normal cartilage tissue, which is superior to the AFe group induced by pure magnetic stimulation and the VA group induced by pure piezoelectric stimulation.

[0030] (4) The preparation method of the bionic cartilage magnetoelectric gel described in the present invention is simple and clear, the preparation conditions are easy to meet, and batch production is easy to achieve, which has broad market prospects in the field of cartilage tissue engineering; and the bionic cartilage magnetoelectric gel has good biocompatibility, stable piezoelectric properties, excellent piezomagnetic properties and superior magnetoelectric coupling cycle conversion ability, showing the advantages of significant healing effect, multifunctional action mode, economic efficiency, simple operation and low cost; it expands the application scope of piezoelectric materials and magnetoelectric materials in the field of cartilage defect repair, and also provides a new treatment plan for accelerating cartilage defect repair and hyaline cartilage in situ regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The present invention will be described in further detail below with reference to the accompanying drawings.

[0032] Figure 1 Detailed pictures of VA gels of various systems of the present invention;

[0033] Note: a. Actual picture of VA-2-2 sol; b. Actual picture of VA-4-4 sol; c. Actual picture of VA-6-6 sol; d. Actual picture of VA-8-8 sol; e. Actual picture of VA-3-6 sol; f. Actual picture of VA-2-6 sol;

[0034] Figure 2 The figures are the physical pictures of the VA gel of the present invention and the cross-sectional pictures of the VA gels of various systems;

[0035] Note: a. VA gel; b. Cross-section of VA-6-6 gel; c. Cross-section of VA-3-6 gel; d. Cross-section of VA-2-6 gel;

[0036] Figure 3 Characterization diagram of the mechanical properties of VA gels of various systems of the present invention;

[0037] Note: a. Tensile curves of VA-2-2, VA-4-4, and VA-6-6 gels in 1:1 system; b. Tensile curves of VA-6-6, VA-3-6, and VA-2-6 gels in 1:1, 1:2, and 1:3 systems; c. Compression curves of VA-2-2, VA-4-4, and VA-6-6 gels in 1:1 system; d. Compression curves of VA-6-6, VA-3-6, and VA-2-6 gels in 1:1, 1:2, and 1:3 systems; e. Compression modulus of VA-2-2, VA-4-4, and VA-6-6 gels in 1:1 system; f. Compression modulus of VA-6-6, VA-3-6, and VA-2-6 gels in 1:1, 1:2, and 1:3 systems;

[0038] Figure 4 Detailed illustration of the VAFe sol and gel of the present invention;

[0039] Figure 5 This is the MTT test of VAFe gel containing superparamagnetic ferroferric oxide nanoparticles of the present invention at different concentrations;

[0040] Figure 6 : is a microstructural characterization diagram of the VAFe gel of the present invention;

[0041] Note: ab: Surface microstructure of VAFe gel; cd: Cross-sectional microstructure of VAFe gel; eh: Distribution of characteristic elements (C, O, Ca, Fe) of VAFe gel;

[0042] Figure 7 The chemical composition and basic physical and chemical properties of the VAFe gel of the present invention are characterized;

[0043] Note: a. FTIR; b. XRD; c. Porosity, water content and water loss of VAFe gel; d. Water contact angle of SA, PHBV, VA and VAFe; e. Flexibility of VAFe gel; f. Swelling ratio of SA, VA and VAFe gel; g. Tensile curves of SA, VA and VAFe gel; h. Compression curves of SA, VA and VAFe gel; i. Degradation rate of VAFe gel;

[0044] Figure 8 This is a detection diagram of the motion-driven magnetoelectric coupling cyclic conversion effect of the VAFe gel of the present invention;

[0045] Note: ab. Output voltage and output current of SA, AFe, VA and VAFe gels under the piezoelectric response model of biomimetic motion stimulation; cd. Output voltage and output current of SA, AFe, VA and VAFe gels under the magnetostrictive piezoelectric response model;

[0046] Figure 9The output voltage and output current of the VAFe gel of the present invention are detected under varying forces and varying magnetic fields;

[0047] Note: a. The piezoelectric response output voltage and output current of the VAFe gel when sensing a changing pressure; b. The piezoelectric response output voltage and output current of the VAFe gel when sensing a changing magnetic field;

[0048] Figure 10 hysteresis curves and magnetic saturation intensities of the VAFe gel and AFe gel of the present invention;

[0049] Figure 11 The biocompatibility test diagram and cytological behavior regulation diagram of the VAFe gel of the present invention are shown;

[0050] Note: a. MTT analysis of SA, AFe, VA, and VAFe gels; b. Live-dead cell staining of SA, AFe, VA, and VAFe gels; c. Hemolysis rate of SA, AFe, VA, and VAFe gels; d. Cell scratch test of SA, AFe, VA, and VAFe gels; e. Cell-level chondrogenic differentiation assay of SA, AFe, VA, and VAFe gels;

[0051] Figure 12 This is a diagram showing the cartilage healing induction experiment in a rabbit osteochondral defect model using the VAFe gel of the present invention;

[0052] Note: ab. Gross appearance and Micro-CT scans of the healing tissues in the Control, SA, AFe, VA, and VAFe groups; c. ICRS macrohistological scores; d. Bone volume ratio of the healing tissues in the Control, SA, AFe, VA, and VAFe groups; e. HE staining, Alcian blue staining, safranin staining, and COL2A1 staining.

[0053] Figure 13 Preparation and working principle of double-network semi-cross chain stacking biomimetic cartilage magnetoelectric gel DETAILED DESCRIPTION

[0054] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0055] Example 1. Preparation of 1:1 poly (3-hydroxybutyrate-co-3-hydroxyvalerate) / sodium alginate composite gel

[0056] (1) N,N-dimethylformamide, deionized water and dichloromethane were mixed uniformly at a volume ratio of 2:4:4 (100 ml);

[0057] (2) Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) powder was weighed and dissolved in the mixed solvent obtained in step (1) to obtain a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) solution, and the mass ratio of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) to solvent was 2:100, 4:100, 6:100, 8:100;

[0058] (3) Sodium alginate powder was weighed and dissolved in the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) solution obtained in step (2) to obtain a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) / sodium alginate composite sol, and the mass ratio of sodium alginate to solvent was 2:100, 4:100, 6:100, 8:100;

[0059] (4) The poly(3-hydroxybutyrate-co-3-hydroxyvalerate) / sodium alginate composite sol prepared in step (3) was filled into a mold, crosslinked and shaped with a calcium chloride solution (the mass ratio of calcium chloride to deionized water was 2:100), and finally soaked in deionized water to remove excess impurities to obtain a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) / sodium alginate composite gel, which was named VA-2-2 (the mass fraction of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and sodium alginate was both 2:2), VA-4-4 (the mass fraction of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and sodium alginate was both 4:4), VA-6-6 (the mass fraction of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and sodium alginate was both 6:6), and VA-8-8 (the mass fraction of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and sodium alginate was both 8:8).

[0060] Preparation of a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) / sodium alginate composite gel in a 1:2 system according to Example 2

[0061] (1) N,N-dimethylformamide, deionized water and dichloromethane were mixed uniformly at a volume ratio of 1:6:3 (100 ml);

[0062] (2) Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) powder was weighed and dissolved in the mixed solvent obtained in step (1) to obtain a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) solution, and the mass ratio of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) to solvent was 3:100;

[0063] (3) weighing sodium alginate powder and dissolving it in the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) solution obtained in step (2) to obtain a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) / sodium alginate composite sol, wherein the mass ratio of sodium alginate to solvent is 6:100;

[0064] (4) The poly (3-hydroxybutyrate-co-3-hydroxyvalerate) / sodium alginate composite sol prepared in step (3) is filled into a mold, cross-linked and shaped with a calcium chloride solution (the mass ratio of calcium chloride to deionized water is 2:100), and finally soaked in deionized water to remove excess impurities to obtain a poly (3-hydroxybutyrate-co-3-hydroxyvalerate) / sodium alginate composite gel, which is named VA or VA-3-6.

[0065] Example 3: Preparation of 1:3 poly (3-hydroxybutyrate-co-3-hydroxyvalerate) / sodium alginate composite gel

[0066] (1) Mix N,N-dimethylformamide, deionized water, and dichloromethane in a volume ratio of 2:6:2 (100 ml).

[0067] (2) Weighing poly (3-hydroxybutyrate-co-3-hydroxyvalerate) powder and dissolving it in the mixed solvent obtained in step (1) to obtain a poly (3-hydroxybutyrate-co-3-hydroxyvalerate) solution, wherein the mass ratio of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) to solvent is 2:100;

[0068] (3) weighing sodium alginate powder and dissolving it in the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) solution obtained in step (2) to obtain a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) / sodium alginate composite sol, wherein the mass ratio of sodium alginate to solvent is 6:100;

[0069] (4) The poly (3-hydroxybutyrate-co-3-hydroxyvalerate) / sodium alginate composite sol prepared in step (3) was filled into a mold, cross-linked and shaped with a calcium chloride solution (the mass ratio of calcium chloride to deionized water was 2:100), and finally soaked in deionized water to remove excess impurities to obtain a poly (3-hydroxybutyrate-co-3-hydroxyvalerate) / sodium alginate composite gel, named VA-2-6.

[0070] Example 4. Preparation of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) film

[0071] (1) Mix N,N-dimethylformamide and dichloromethane in a volume ratio of 1:3 (100 ml) until uniform;

[0072] (2) Weighing poly (3-hydroxybutyrate-co-3-hydroxyvalerate) powder and dissolving it in the mixed solvent obtained in step (1) to obtain a poly (3-hydroxybutyrate-co-3-hydroxyvalerate) solution, wherein the mass ratio of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) to solvent is 3:100;

[0073] (3) The poly(3-hydroxybutyrate-co-3-hydroxyvalerate) solution prepared in step (2) was filled into a mold and dried in an oven at 60°C until dry to obtain a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) film, which was named PHBV.

[0074] Example 5. Preparation of sodium alginate gel

[0075] (1) Mix N,N-dimethylformamide and deionized water in a volume ratio of 1:6 (100 ml).

[0076] (2) weighing sodium alginate powder and dissolving it in the mixed solvent obtained in step (1) to obtain a sodium alginate sol, wherein the mass ratio of sodium alginate to solvent is 6:100;

[0077] (3) The sodium alginate sol prepared in step (2) is filled into a mold, cross-linked and shaped with a calcium chloride solution (the mass ratio of calcium chloride to deionized water is 2:100), and finally soaked in deionized water to remove excess impurities to obtain a sodium alginate gel, which is named SA.

[0078] Example 6. Preparation of superparamagnetic ferrosoferric oxide nanoparticles / poly(3-hydroxybutyrate-co-3-hydroxyvalerate) / sodium alginate composite gel

[0079] (1) Mix N,N-dimethylformamide, deionized water, and dichloromethane in a volume ratio of 1:6:3 (100 ml).

[0080] (2) weighing superparamagnetic ferroferric oxide nanoparticles and adding them to the mixed solvent obtained in step (1), and ultrasonically dispersing them until uniform, to obtain a magnetic mixed solution, wherein the mass ratio of superparamagnetic ferroferric oxide nanoparticles to solvent is 0.5:100, 1:100, 1.5:100, and 2:100, respectively;

[0081] (3) Weighing poly (3-hydroxybutyrate-co-3-hydroxyvalerate) powder, dissolving it in the magnetic mixed solution obtained in step (2) to obtain a superparamagnetic ferrosoferric oxide nanoparticle / poly (3-hydroxybutyrate-co-3-hydroxyvalerate) composite solution, wherein the mass ratio of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) to solvent is 3:100;

[0082] (4) Weighing sodium alginate powder, dissolving it in the superparamagnetic ferroferric oxide nanoparticles / poly (3-hydroxybutyrate-co-3-hydroxyvalerate) composite solution obtained in step (3) to obtain a superparamagnetic ferroferric oxide nanoparticles / poly (3-hydroxybutyrate-co-3-hydroxyvalerate) / sodium alginate composite sol, wherein the mass ratio of sodium alginate to solvent is 6:100;

[0083] (5) The superparamagnetic ferroferric oxide nanoparticles / poly (3-hydroxybutyrate-co-3-hydroxyvalerate) / sodium alginate composite sol prepared in step (4) is filled into a mold, cross-linked and shaped with a calcium chloride solution (the mass ratio of calcium chloride to deionized water is 2:100), and finally soaked in deionized water to remove excess impurities to obtain superparamagnetic ferroferric oxide nanoparticles / poly (3-hydroxybutyrate-co-3-hydroxyvalerate) / sodium alginate composite gels, named VAFe-0.5, VAFe-1.0, VAFe-1.5 (or VAFe), and VAFe-2.0, with the mass fraction ratios of superparamagnetic ferroferric oxide nanoparticles being 0.5:100, 1:100, 1.5:100, and 2:100, respectively.

[0084] Example 7. Preparation of superparamagnetic ferroferric oxide nanoparticles / sodium alginate composite gel

[0085] (1) Mix N,N-dimethylformamide and deionized water in a volume ratio of 1:6 (100 ml).

[0086] (2) weighing superparamagnetic ferroferric oxide nanoparticles and adding them to the mixed solvent obtained in step (1), and ultrasonically dispersing them until uniform, to obtain a magnetic mixed solution, wherein the mass ratio of superparamagnetic ferroferric oxide nanoparticles to solvent is 1.5:100;

[0087] (3) Weighing sodium alginate powder and dissolving it in the magnetic mixed solution obtained in step (2) to obtain a superparamagnetic ferroferric oxide nanoparticle / sodium alginate composite sol, wherein the mass ratio of sodium alginate to solvent is 6:100;

[0088] (4) The superparamagnetic ferroferric oxide nanoparticles / sodium alginate composite sol prepared in step (3) was filled into a mold, cross-linked and shaped with a calcium chloride solution (the mass ratio of calcium chloride to deionized water was 2:100), and finally soaked in deionized water to remove excess impurities to obtain a superparamagnetic ferroferric oxide nanoparticles / sodium alginate composite gel, named AFe.

[0089] Figure 1In ac, in a 1:1 system, we dissolved poly (3-hydroxybutyrate-co-3-hydroxyvalerate) and sodium alginate successively. As the concentrations of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) and sodium alginate increased, the liquid-like behavior of VA sol gradually weakened, and the mechanical structure gradually improved, showing stable solid-like properties. 2+ After solution cross-linking, VA gel exhibits a structurally stable viscoelastic behavior, providing a possibility for repairing the mechanical barrier function of cartilage defects ( Figure 2 a). However, when the concentration of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) and sodium alginate increased to 8wt%, the solubility reached saturation. Insoluble flakes with polar repulsion began to appear in the composite sol, and the stable mechanical structure was lost ( Figure 1 d). Therefore, 6 wt% was set as the maximum concentration of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) and sodium alginate in the 1:1 system. However, due to the low boiling point of dichloromethane, poly (3-hydroxybutyrate-co-3-hydroxyvalerate) is easy to volatilize and precipitate, resulting in a rough surface of the VA gel ( Figure 2 b) This does not conform to the surface smoothness that bionic cartilage magnetoelectric gel should have.

[0090] Therefore, on the basis of fixing the sodium alginate concentration at 6 wt% to maintain the viscoelastic structure, the poly (3-hydroxybutyrate-co-3-hydroxyvalerate) concentration was reduced to 3 wt% and 2 wt%, and 1:2 and 1:3 systems were prepared ( Figure 1 ef). Figure 2 As shown in Figures cd, as the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) concentration decreases, the surface of the VA-3-6 gel becomes smoother and more delicate. While the surface roughness of the VA-2-6 gel improves, it exhibits a clumpy structure. Therefore, from a surface structural perspective, the VA-3-6 gel in the 1:2 ratio is superior.

[0091] Example 8

[0092] This experiment uses the different systems of VA gel in Examples 1, 2, and 3 as the implementation objects for the following experiments:

[0093] The mechanical properties of VA gel samples of different systems were tested using a universal material testing machine (DY35-2T, France, strain rate of 5 mm / min at room temperature).

[0094] like Figure 3 a. The tensile properties of VA gels in the 1:1 system are generally poor (VA-2-2, VA-4-4, VA-6-6). In contrast, the tensile properties of VA-3-6 and VA-2-6 gels are improved ( Figure 3b). However, due to the slightly lower concentration of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) in VA-2-6 gel, its soft-hard combination synergy is relatively small, and its tensile properties are slightly worse than VA-3-6. In addition, with the increase of sodium alginate and poly (3-hydroxybutyrate-co-3-hydroxyvalerate) concentration, the compressive strength and compression modulus of VA gel gradually increase ( Figure 3 c). The compression moduli of VA-4-4 (about 0.227 MPa), VA-6-6 (about 0.498 MPa), and VA-3-6 (about 0.229 MPa) are all within the range of normal articular cartilage compression modulus (0.2-0.85 MPa) ( Figure 3 ef). However, due to the slightly lower concentration of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) in VA-2-6, its soft-hard combination synergy is relatively small, and its compression performance fails to reach the compression modulus range of normal articular cartilage ( Figure 3 df). Therefore, combining mechanical properties and surface structure, it is determined that the 1:2 system of VA-3-6 is the best. On the basis of fixing the concentration of each component of VA-3-6 gel, composite superparamagnetic ferroferric oxide nanoparticles are prepared to obtain VAFe gel with uniform composition, stable mechanical structure and smooth and delicate surface ( Figure 4 ).

[0095] Example 9

[0096] This experiment uses VAFe-0.5, VAFe-1.0, VAFe-1.5, and VAFe-2.0 in Example 6 as implementation objects for the following experiments:

[0097] Prepare several gel samples of similar size in advance. Thoroughly sterilize the samples by soaking in 75% v / v ethanol and irradiating with ultraviolet light (UV). Collect the extracted and cultured bone marrow mesenchymal stem cells (BMSCs) by trypsin digestion. Centrifuge and resuspend, and count the single cell suspension using a hemocytometer. MTT assays were performed on 24-well plates for 1, 3, 5, and 7 days. Add 1×10 4 Cells were co-cultured with the sample. After a fixed number of days of culture, an appropriate amount of MTT reagent was added and incubated at 37°C for 4 hours. The supernatant was discarded and dimethyl sulfoxide was added to dissolve the purple formazan crystals. The well plate was shaken on a shaker at room temperature for 15 minutes to dissolve the crystals evenly. The liquid in the 24-well plate was then transferred to a 96-well plate. The absorbance was measured at OD = 490 nm using a microplate reader. Three parallel experiments were performed for each group and the average value was calculated.

[0098] It is known that the magnetic induction of the composite gel gradually increases with the increase of the concentration of superparamagnetic ferroferric oxide nanoparticles. The optimal concentration of superparamagnetic ferroferric oxide nanoparticles was determined by MTT test. BMSCs with self-renewal and multidirectional differentiation ability were selected for cell experiments and co-cultured with each group of gel samples. As the concentration of superparamagnetic ferroferric oxide nanoparticles increased, the cell compatibility of VAFe gel first increased and then decreased. Finally, VAFe-1.5 (superparamagnetic ferroferric oxide nanoparticle concentration of 1.5wt%) was determined to be the best biocompatibility ( Figure 5 In subsequent experiments, the concentration of superparamagnetic ferroferric oxide nanoparticles was fixed at 1.5 wt % to prepare VAFe and AFe sol and gel.

[0099] Example 10

[0100] This experiment uses SA, PHBV, VA, and VAFe in Examples 2, 4, 5, and 6 as implementation objects for the following experiments:

[0101] The morphology of the VAFe gel was characterized by scanning electron microscopy (SEM, Hitachi S-4800, Japan). The elemental distribution of the VAFe gel was determined by energy dispersive spectroscopy (EDS, X-MaxN, UK, 20 kV, temperature 25°C, humidity 39%). The crystal structure of the sample was characterized by X-ray diffractometry (XRD, Rigaku D / Max-2400, Germany, using Cu-Ka radiation, voltage 35 kV). Fourier transform infrared spectroscopy was measured using the potassium bromide pellet technique and a Thermo Nicolet Nexus FTIR670 spectrometer (FTIR, IFS66V / S, Germany). The contact angle of water on the VAFe gel surface was measured using a contact angle meter (DSA100, KRUSS, Germany, using 1 drop of 5 μL water and a dwell time of 20 s). The mechanical properties of each gel sample were tested using a universal materials testing machine (DY35-2T, France, with a strain rate of 5 mm / min at room temperature).

[0102] Weigh several similarly sized and regular rectangular SA, VA, and VAFe composite gel blocks. Then, immerse them in PBS buffer for a period of time, remove them, wipe off the surface moisture, and weigh them. Calculate the swelling ratio of the composite gel until swelling equilibrium is reached.

[0103] Several similar-sized and regular rectangular VAFe composite gel blocks were weighed initially and weighed again after freeze-drying to measure the water content of the VAFe composite gel.

[0104] Select several regular rectangular VAFe composite gel blocks of similar size. After freeze-drying, measure their initial weight and volume. Soak them in anhydrous ethanol until swelling equilibrium is reached. Record the weight after swelling equilibrium to calculate the porosity.

[0105] Select several similarly sized, regular rectangular blocks of VAFe composite gel. Wipe dry the surface with filter paper and weigh the initial weight. Place each block in a centrifuge tube and place filter paper of similar size on top. Ultracentrifuge at 5000 rpm for 3 minutes, then weigh the remaining weight to calculate water loss.

[0106] Select several rectangular VAFe composite gel blocks of similar size and regular shape. After freeze-drying, weigh the initial weight. Soak the blocks in PBS buffer and incubate them on a shaker at 37°C. After a fixed period of time, freeze-dry the samples and weigh the remaining weight to calculate the in vitro degradation rate.

[0107] like Figure 6 The surface and cross-section of the ad-VAFe gel are rich in micron-sized pores of varying sizes, providing ample initial aggregation space for cell adhesion, proliferation, and matrix formation. The relatively small pore size on the surface of the gel gives the gel a smooth and even surface. The relatively large pore size in the cross-section of the gel provides suitable initial accumulation space for cells and active substances during cartilage regeneration. The interlaced stacking of the gel network imparts mechanical sensitivity and provides the structural foundation for the construction of a piezoelectric response model.

[0108] The elemental composition of VAFe gel was characterized by EDS. Figure 6 The characteristic elements C, O, Ca, and Fe of sodium alginate, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), and superparamagnetic ferroferric oxide nanoparticles are uniformly distributed throughout the VAFe gel structure, demonstrating the uniform and pure composition of the VAFe gel in the three-phase solvent system. The interwoven stacking of these elements also provides evidence for the construction of a double-network chain stacking structure.

[0109] The molecular interactions and crystal structures of each gel sample were analyzed by FTIR and XRD. Figure 7 ab, the characteristic functional groups and characteristic crystal phase structures of each component in the VAFe gel are present, and no new peaks appear, indicating that the composition of the composite gel is pure and no chemical reaction occurs. In addition, the biomimetic cartilage magnetoelectric gel needs to have physical properties similar to those of the cartilage extracellular matrix to provide a simulated in vivo environment for the expression of cellular behavior for repair of defective cartilage. The porosity of the VAFe gel is approximately 60.75% ( Figure 7 c), providing sufficient initial aggregation space for cell adhesion, proliferation and cell matrix formation. The water content of VAFe gel is about 79.73% ( Figure 7c), which is similar to the water content of cartilage tissue (60-85%), can provide appropriate hydrostatic pressure during exercise and play a mechanical bearing role. After high-speed centrifugation, the water loss of VAFe gel is only about 15.45% ( Figure 7 c). This indicates that VAFe gel has a certain lubricating potential while exhibiting a mechanical buffering effect. However, poly (3-hydroxybutyrate-co-3-hydroxyvalerate) is inherently hydrophobic, which limits its clinical application. The introduction of sodium alginate and superparamagnetic ferroferric oxide nanoparticles solves this problem. Figure 7 d, The water contact angle of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) decreased from 93.80 ± 1.64° to 38.67 ± 2.04°, which provided VAFe gel with suitable cell adhesion polarity. Figure 7 e, VAFe gel can be curled and folded at will, and can bear a weight of 2.41 kg, showing good flexibility and bending resistance. In addition, the inherent hydrophobicity of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) also reduces the swelling performance of SA hydrogel. The addition of superparamagnetic ferroferric oxide nanoparticles makes the structure of VAFe gel more compact by enhancing the intermolecular hydrogen bonding within the chain of the composite gel, and further reduces the swelling performance ( Figure 7 f). Appropriate swelling properties can provide positive surrounding squeezing force to the implant site without interfering with cartilage repair defects, and replenish water content when water is lost under stress, demonstrating cyclic stress bearing and swelling piezoelectric response.

[0110] The tensile and compressive properties of the samples were tested using a universal material testing machine. The addition of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) made the tensile and compressive properties of VA gel better than those of SA gel. The addition of superparamagnetic ferroferric oxide nanoparticles also further improved the tensile and compressive properties of VAFe gel ( Figure 7 gh), which is consistent with the change in swelling properties ( Figure 7 f). Therefore, from a mechanical perspective, VAFe has better mechanical properties than SA and VA, and is the best, meeting the application requirements of biomimetic cartilage gel. In addition, as time goes by, the mass of VAFe gel slowly decreases, and it has long-term degradation ability, providing a structural basis for the long-term repair of cartilage defects ( Figure 7 i).

[0111] Example 11

[0112] This experiment uses SA, AFe, VA, and VAFe in Examples 2, 5, 6, and 7 as implementation objects for the following experiments:

[0113] The gel was encapsulated with high-purity copper foil and copper wire. It was then connected to a highly sensitive multimeter to measure and record the effective output current and voltage data. Using a multi-purpose materials testing machine, a programmable mode was set to apply varying instantaneous impact forces to the gel to construct a motion-stimulated piezoelectric response model. Furthermore, ten small 280mT magnets were stacked and placed on one side of the encapsulated gel, with the distance adjusted. This magnetized superparamagnetic Fe3O4 nanoparticles generated a magnetostrictive effect, thereby constructing a magnetostrictive piezoelectric response model.

[0114] AFe and VAFe gel samples were freeze-dried and then ground into powder. 1 g of each gel powder was weighed and placed into a sample holder. The hysteresis curves and magnetic saturation intensities of the gel samples were characterized using a vibrating sample magnetometer (VAM, VersaLab, USA). The temperature was maintained at room temperature. The step size was set to 400 Oe. A continuously variable magnetic field was applied between -3T and 3T. Data were recorded and analyzed.

[0115] Figure 8 abFirst, the feasibility of the piezoelectric response model was characterized by a high-precision multimeter. SA gel and AFe gel without piezoelectric effect were used as control groups. A universal material testing machine was used to set the single impact force to 30N to simulate the average force on the joints of New Zealand white rabbits during normal movement. Both VA gel and VAFe gel exhibited good piezoelectric properties when bearing impact force, and the output voltage (about ±175mv) and output current (about ±2.1μA) of VAFe gel were greater than those of VA gel (about ±150mv and about ±1.8μA). In addition, a gradient impact force was set, and the test results showed that the piezoelectric response intensity of VAFe gel changed with the impact force ( Figure 9 a), suggesting that VAFe gel can generate a changing electric field in the complex motion force environment of New Zealand white rabbits. Combined with Faraday's law, it manifests as a changing electromagnetic field, which provides the possibility of magnetoelectric conversion of electromagnetically magnetized superparamagnetic Fe3O4 nanoparticles for magnetoelectric conversion. In order to verify the feasibility of the magnetostrictive piezoelectric response model, as shown in Figure 9 b, Changing the distance between the magnet and the gel sample, the results show that the magnetic field distance is positively correlated with the piezoelectric response intensity of the gel sample. Figure 8 cd. When the distance between the fixed magnetic field and the gel sample is 1 cm, the output voltage and output current of the VAFe gel fluctuate stably within a small range, with average values ​​of about 20.34 mV and about 0.25 μA, respectively.

[0116] In order to verify the feasibility of electromagnetic conversion, the hysteresis curve and magnetic saturation intensity of the gel sample were characterized by a vibrating sample magnetometer. Figure 10, the magnetic saturation intensity of VAFe gel is greater than that of AFe gel containing superparamagnetic ferroferric oxide nanoparticles at the same concentration. This shows that the magnetostrictive piezoelectric electromagnetic stimulation generated by superparamagnetic ferroferric oxide nanoparticles in a magnetic field enhances the magnetic induction intensity of VAFe gel through the magnetoelectric coupling cyclic conversion effect, verifying the feasibility of the magnetoelectric coupling cyclic conversion effect and the possibility of electromagnetic conversion from the magnetic response level. In summary, VAFe gel releases enhanced piezoelectric electromagnetic stimulation and piezomagnetic stimulation through the magnetoelectric coupling cyclic conversion effect under motion drive, showing great potential to induce rapid and stable repair of cartilage defects.

[0117] Example 12

[0118] This experiment uses SA, AFe, VA, and VAFe in Examples 2, 5, 6, and 7 as implementation objects for the following experiments:

[0119] Prepare several gel samples of similar size in advance. Thoroughly sterilize the samples by soaking in 75% v / v ethanol and irradiating with ultraviolet light (UV). Collect the extracted and cultured bone marrow mesenchymal stem cells (BMSCs) by trypsin digestion. Centrifuge and resuspend, and count the single cell suspension using a hemocytometer. MTT assays were performed on 24-well plates for 1, 3, 5, and 7 days. Add 1×10 4 Cells were co-cultured with the sample. After a fixed number of days of culture, an appropriate amount of MTT reagent was added and incubated at 37°C for 4 hours. The supernatant was discarded and dimethyl sulfoxide was added to dissolve the purple formazan crystals. The well plate was shaken on a shaker at room temperature for 15 minutes to dissolve the crystals evenly. The liquid in the 24-well plate was then transferred to a 96-well plate. The absorbance was measured at OD = 490 nm using a microplate reader. Three parallel experiments were performed for each group and the average value was calculated.

[0120] The steps of cell culture and material sterilization are the same as above. 1×10 4 Cells were co-cultured with the materials for 1, 3, and 7 days. The supernatant was then discarded and the cells were washed with PBS buffer. Calcein-AM / PI live-dead cell double staining detection kit was used and the cells were stained in a 37°C incubator for 0.5 h. The staining solution was aspirated to terminate the incubation. An appropriate amount of PBS buffer was added to each well to cover the cells. Live cells (yellow-green fluorescence) and dead cells (red fluorescence) were then observed using an inverted fluorescence microscope and photographed.

[0121] Take an appropriate amount of healthy rabbit whole blood, add 3.8wt% sodium citrate to it for anticoagulation, and dilute it with normal saline at a volume ratio of 4:5 for later use. Place the sample in 10ml of normal saline and incubate at 37°C for 30 minutes. Then add 0.2ml of diluted blood and incubate for 1 hour. Normal saline and deionized water serve as negative and positive control groups, respectively. Centrifuge all test tubes at 1000rpm for 5 minutes. Transfer the supernatant to a 96-well plate. Read the absorbance (optical density, OD) at a wavelength of 545nm using an enzyme reader to calculate the hemolysis rate.

[0122] like Figure 11 a shows that BMSCs with self-renewal and multidirectional differentiation ability were selected for cell experiments and co-cultured with each group of gel samples. The MTT test data of SA, AFe, VA and VAFe gels were all greater than 90%, showing good cell compatibility. In addition, live-dead cell staining further demonstrated that each gel sample group had good biocompatibility ( Figure 11 b) The results showed that no red fluorescent spots representing dead cells were observed in the fluorescence staining image after the red and green channels were merged, and the number of green fluorescent spots representing living cells was positively correlated with the co-culture time, which was consistent with the results of the cell proliferation experiment. In order to further verify the biocompatibility of each gel sample group, Figure 11 c. The hemolysis rate of each gel sample group was less than 5%, meeting the requirements of international standards and demonstrating good biosafety.

[0123] Example 13

[0124] This experiment uses SA, AFe, VA, and VAFe in Examples 2, 5, 6, and 7 as implementation objects for the following experiments:

[0125] A built-in chamber matching the cell culture plate was fabricated and different groups of gel samples were placed within it. The bottom of the chamber was uniformly porous to facilitate the entry of culture medium and the conduction of electrical stimulation. A horizontal shaker (NMYC-100, China) was set to a speed of 20 and shaken for 15 minutes daily to establish a piezoelectric response model. By sensing the frictional impact force between the gel sample and the water flow, the piezoelectric stimulus was released to conduct subsequent cell experiments.

[0126] The steps of cell culture and material disinfection are the same as above. Use a six-well plate to inoculate cells. Place each group of samples in the built-in chamber. After the cell fusion rate reaches 90%, use a 20μl pipette tip to mark the cell-free area. Add PBS buffer for washing. Then replace it with serum-free basal medium to eliminate the influence of serum. At 0h and 48h, after crystal violet staining, an upright fluorescence microscope (Nikon-ECLIPSE 80i / DS-Ri2 / NIS-ElementsD, Nikon, Japan) was used to observe the healing of the scratches and take pictures.

[0127] The chondrogenic induction medium was prepared with ITS (C0343, Beyotime, China, 1%), L-proline (P120032, Aladdin, China, 0.4 mmol / L), 2-phosphate ascorbic acid (8855609, Aladdin, China, 91.5 μg / ml), dexamethasone (D137736, Aladdin, China, 100 nmol / ml), non-essential amino acids (N1250, Solebao, China, 0.1 mmol / L), TGF-β3 (P02149, Solebao, China, 10 ng / ml), and DMEM. The cells were wrapped in tin foil and stored in the dark. The cell culture and material sterilization procedures were the same as above. To prevent cell floating after fusion during long-term culture, we coated the bottom of the 12-well plate with gelatin (C0136, Bryotime, China). After the culture dish was dried, it was washed with PBS. 5×10 cells were seeded in each well. 4 cells. The culture medium was changed every 24 hours. On day 14, the cells were fixed with 4% paraformaldehyde solution. Alcian blue stain (C0155S, Beyotime, China) was used for staining, the cells were allowed to stand at room temperature for 1 hour, and then washed with PBS. The cells were then observed and photographed using an inverted laser confocal microscope.

[0128] like Figure 11 d. The cell proliferation and migration rates of the AFe, VA and VAFe groups were faster than those of the control group, and the VAFe group had the fastest rate. The cell scratch wounds were almost completely healed after 48 hours. This shows that both simple electricity and magnetism can promote cell proliferation and migration, and also implies that the electromagnetic stimulation released by the magnetoelectric coupling cycle conversion effect has a stronger promoting effect. In addition, in order to verify that the electromagnetic stimulation generated by VAFe gel has the potential to regulate the differentiation of BMSCs into chondrocytes, we co-cultured BMSCs with each group of gel samples in cartilage induction medium for 14 days, and then stained the cells of each group with Alcian blue. The results showed that the staining depth of the AFe, VA and VAFe groups was greater than that of the control group, and the staining depth of the VAFe group was the deepest, indicating that VAFe gel has the strongest ability to promote cartilage induction differentiation ( Figure 11 e).

[0129] Example 14

[0130] This experiment uses SA, AFe, VA, and VAFe in Examples 2, 5, 6, and 7 as implementation objects for the following experiments:

[0131] With the approval of the Animal Ethics Committee, several healthy New Zealand white rabbits (male, approximately 2.5 kg) were selected and randomly divided into five groups for in vivo experiments. After strict analgesia, anesthesia, and disinfection, the knee joint was exposed through the lateral parapatellar incision and the patella was displaced. An osteochondral defect with a diameter of 5 mm and a depth of 4 mm was drilled in the femoral trochlear groove. Several gel samples of similar size from each group were strictly disinfected and sterilized and then filled into the defect. The patella was repositioned and the joint was sutured. After strict disinfection, analgesia, and injection of antibiotics, the rabbits were returned to the marked cages. They were named Control group, SA group, AFe group, VA group, and VAFe group. Close observation was carried out. After 6 or 12 weeks of feeding, the rabbit knee joint tissues were collected and fixed with 4% paraformaldehyde buffer. The healing of the knee joint tissues in each group was scored using the ICRS macroscopic evaluation table. Subsequently, the knee joint tissues in each group were scanned by Micro-CT (80 kV, 100 uA). The single exposure time was 50 ms, the scanning resolution was 25 mm, and the scanning angle interval was 0.5°. HiscanReconstruct software was used for three-dimensional geometric reconstruction and calculation and analysis of new bone formation volume. Additionally, rabbit knee joint tissues collected after 6 or 12 weeks of feeding were fixed in 4% paraformaldehyde buffer. After decalcification, gradient dehydration, paraffin embedding, and sectioning, they were stained with hematoxylin and eosin, Alcian blue, safranin, and immunohistochemistry (COL2A1). The tissues were observed under an optical microscope and the healing status of the defect tissue was evaluated.

[0132] Figure 12 a shows that in order to further verify the ability of VAFe gel to induce in situ healing of defective cartilage in vivo, we constructed an osteochondral defect model (5mm in diameter, 4mm in depth) that exceeded the critical autologous repair size. We chose the trochlear groove of the rabbit knee joint to construct the osteochondral defect model, rather than the femoral head. The purpose is to reduce the postoperative discomfort of experimental animals, provide appropriate exercise stimulation for the implanted gel sample, and amplify its effect as much as possible on the basis of stimulating the exercise-driven magnetoelectric coupling cyclic conversion effect. After 6-12 weeks of treatment, in comparison, the new tissue in the VAFe group had good fusion with the surrounding tissue, and no granulation tissue similar to the Control group and SA group appeared, and the defective cartilage healing was the best. After 12 weeks, the surface of the new tissue was smooth, and the color and morphology were close to those of the surrounding normal tissue. The healing of the VA group and AFe group was second best. The ICRS macroscopic tissue score also further verified this conclusion ( Figure 12c). Micro-CT reconstruction images and bone volume ratio analysis data showed that the amount of subchondral bone healing in the VAFe group was higher than that in the other groups, which could provide mechanical bearing and support for the repair of cartilage defects ( Figure 12 b and Figure 12 d). For histological and immunohistochemical evaluations, we performed hematoxylin and eosin (H&E), safranin, Alcian blue, and COL2A1 staining on the newly formed articular cartilage tissues ( Figure 12 e). The results showed that at week 6, a large amount of newly formed cartilage tissue was accumulated in the VAFe group, and its cell distribution, density and morphology were good. At week 12, the newly formed cartilage tissue in the VAFe group was well integrated with the surrounding normal tissue, showing histological characteristics similar to those of hyaline cartilage tissue. The cartilage healing of the VA group and the AFe group was second best, but there was still a certain degree of cartilage tissue defect. Relatively speaking, the cartilage tissue healing of the Control group and the SA group was poor, and there were still large cartilage tissue defects and granulation tissue at week 12. In addition, strong Safranin-O staining, Alcian blue staining and COL2A1 staining were observed in the newly formed cartilage tissue of the VAFe group, indicating that under the induction of electromagnetic stimulation released by the motion-driven magnetoelectric coupling cyclic conversion effect, the newly formed cartilage tissue in the VAFe group had hyaline cartilage characteristics similar to those of normal cartilage tissue, which was better than the AFe group induced by simple magnetic stimulation and the VA group induced by simple piezoelectric stimulation. This may be attributed to the enhanced piezoelectric electromagnetic stimulation and piezoelectric stimulation released by the motion-driven magnetoelectric coupling cyclic conversion effect, such as Figure 13 shown.

[0133] In summary, the present invention provides a double-network semi-cross chain stacked biomimetic cartilage magnetoelectric gel. The biomimetic cartilage magnetoelectric gel is prepared by mixing 100 parts of a solvent with a ratio of 4-6:1-2:2-4 of water:N,N-dimethylformamide:dichloromethane, and adding 0.5-2 parts of superparamagnetic ferrosoferric oxide nanoparticles, 2-8 parts of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and 2-8 parts of sodium alginate. The biomimetic cartilage magnetoelectric gel utilizes N,N-dimethylformamide, which is mutually soluble in deionized water and dichloromethane, to neutralize their repulsive polarities, thereby uniformly mixing the hydrophilic sodium alginate sol and the hydrophobic poly (3-hydroxybutyrate-co-3-hydroxyvalerate) piezoelectric plastic. The hydrophilic sodium alginate sol and the hydrophobic poly (3-hydroxybutyrate-co-3-hydroxyvalerate) piezoelectric plastic are cross-linked with a calcium chloride solution to form a double-network semi-cross chain stacking structure, thereby obtaining a repair-promoting gel with composition, structure and performance that mimics cartilage. This can effectively solve the application limitations of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) in the field of cartilage tissue engineering. The biomimetic cartilage magnetoelectric gel prepared by Mingzhong is creatively endowed with a motion-driven magnetoelectric coupling cyclic conversion effect, including a motion-stimulated piezoelectric response model, a magnetostrictive piezoelectric response model, and a magnetoelectric coupling cyclic conversion effect. Under light force, it provides freely excitable and cyclically enhanced piezoelectric and piezomagnetic responses for inducing in situ regeneration and repair of defective cartilage. Combined with Faraday's law, it releases variable electromagnetic stimulation, simulates and amplifies endogenous electromagnetic effects, cascades and regulates the expression of genes related to defective cartilage repair, and promotes the in situ regeneration of hyaline cartilage tissue. It can effectively solve the shortcomings of electromagnetic field stimulation given in vitro, such as easy weakening by tissues, uncontrollable penetration, and limited quantitative intensity of action. It can also effectively solve the problem that the healing-promoting effect of traditional piezoelectric materials is limited by the inherent shortcomings of the piezoelectric model. In addition, it can also effectively solve the application limitations of traditional magnetoelectric materials. The bionic cartilage magnetoelectric gel described in the present invention has a functional environment similar to that of bionic cartilage, can release electromagnetic stimulation to promote cell proliferation and migration, promote the induction of chondrocyte differentiation, and provide mechanical bearing and support for the repair of defective cartilage; the generated new cartilage tissue has the characteristics of hyaline cartilage similar to those of normal cartilage tissue, which is better than the AFe group induced by simple magnetic stimulation and the VA group induced by simple piezoelectric stimulation; the preparation method of the bionic cartilage magnetoelectric gel described in the present invention is simple and clear, the preparation conditions are easy to meet, and mass production is easy to achieve, and it has broad market prospects in the field of cartilage tissue engineering; and the bionic cartilage magnetoelectric gel has good biocompatibility, stable piezoelectric properties, excellent piezomagnetic properties and superior magnetoelectric coupling cycle conversion ability, showing the advantages of significant healing promotion effect, multifunctional action mode, economic efficiency, simple operation and low cost; it expands the application scope of piezoelectric materials and magnetoelectric materials in the field of defective cartilage repair, and also provides a new treatment plan for accelerating defective cartilage repair and hyaline cartilage in situ regeneration.

[0134] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A double-network semi-cross chain stacked biomimetic cartilage magnetoelectric gel, characterized in that: The bionic cartilage magnetoelectric gel is prepared by the following method: S1. Prepare a solvent by mixing water, N,N-dimethylformamide, and dichloromethane according to a ratio of 4-6:1-2:2-4; S2. Take the solvent prepared in step S1, add superparamagnetic ferroferric oxide nanoparticles in a ratio of 0.5-2:100, and ultrasonically disperse them uniformly to obtain a magnetic mixed solution; S3. Adding poly (3-hydroxybutyrate-co-3-hydroxyvalerate) to the magnetic mixed solution obtained in step S2 to obtain a superparamagnetic ferrosoferric oxide nanoparticle / poly (3-hydroxybutyrate-co-3-hydroxyvalerate) composite solution, wherein the addition ratio of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) to solvent is 2-8:100; S4. Adding sodium alginate powder to the superparamagnetic ferroferric oxide nanoparticles / poly(3-hydroxybutyrate-co-3-hydroxyvalerate) composite solution obtained in step S3 to obtain a superparamagnetic ferroferric oxide nanoparticles / poly(3-hydroxybutyrate-co-3-hydroxyvalerate) / sodium alginate composite sol, wherein the addition ratio of sodium alginate to solvent is 2-8:100; S5. Take the superparamagnetic ferroferric oxide nanoparticles / poly (3-hydroxybutyrate-co-3-hydroxyvalerate) / sodium alginate composite sol prepared in step S4, draw it out and spread it into a mold, soak it in calcium chloride solution for cross-linking, and then dialyze it with deionized water to obtain a double-network semi-cross chain stacked biomimetic cartilage magnetoelectric gel.

2. The method for preparing the double-network semi-cross chain stacked biomimetic cartilage magnetoelectric gel according to claim 1, characterized in that: The steps include: S1. Prepare a solvent by mixing water, N,N-dimethylformamide, and dichloromethane according to a ratio of 4-6:1-2:2-4; S2. Take the solvent prepared in step S1, add superparamagnetic ferroferric oxide nanoparticles in a ratio of 0.5-2:100, and ultrasonically disperse them uniformly to obtain a magnetic mixed solution; S3. Adding poly (3-hydroxybutyrate-co-3-hydroxyvalerate) to the magnetic mixed solution obtained in step S2 to obtain a superparamagnetic ferrosoferric oxide nanoparticle / poly (3-hydroxybutyrate-co-3-hydroxyvalerate) composite solution, wherein the addition ratio of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) to solvent is 2-8:100; S4. Adding sodium alginate powder to the superparamagnetic ferroferric oxide nanoparticles / poly(3-hydroxybutyrate-co-3-hydroxyvalerate) composite solution obtained in step S3 to obtain a superparamagnetic ferroferric oxide nanoparticles / poly(3-hydroxybutyrate-co-3-hydroxyvalerate) / sodium alginate composite sol, wherein the addition ratio of sodium alginate to solvent is 2-8:100; S5. Take the superparamagnetic ferroferric oxide nanoparticles / poly (3-hydroxybutyrate-co-3-hydroxyvalerate) / sodium alginate composite sol prepared in step S4, draw it out and spread it into a mold, soak it in calcium chloride solution for cross-linking, and then dialyze it with deionized water to obtain a double-network semi-cross chain stacked biomimetic cartilage magnetoelectric gel.

3. The method for preparing the double-network semi-cross chain stacked biomimetic cartilage magnetoelectric gel according to claim 2, characterized in that: The mass ratio of calcium chloride to deionized water in the calcium chloride solution of step S5 is 2:

100.

4. Use of the double-network semi-cross chain stacked biomimetic cartilage magnetoelectric gel as claimed in claim 1 in the preparation of artificial cartilage materials.

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