A bio-bionic osteochondral bio-battery and a preparation method and application thereof

Abstract

The application provides a kind of biological bionic osteochondral bio-battery and its preparation method and application, belong to biological medical material and tissue engineering technical field.The biological battery includes anode layer, cathode layer and diaphragm layer;The anode layer is composed of zinc powder and hydrogel framework, the cathode layer is composed of manganese dioxide powder and hydrogel framework, the diaphragm layer is composed of polyvinylidene fluoride powder and hydrogel framework.The biological battery provided by the application can simulate the natural osteochondral tissue electric microenvironment and chemical signal, has bionic layered structure, and can realize programmed electric stimulation and ion release.The battery can not only provide stable and durable endogenous electric stimulation, but also guide endogenous stem cells to carry out partition directional differentiation, so as to realize the integration of bone and cartilage defect and functional regeneration.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials and tissue engineering technology, and in particular to a biomimetic osteocartilage biobattery, its preparation method, and its application. Background Technology

[0002] Articular cartilage is a highly specialized connective tissue that covers the surface of joint bones, playing a crucial physiological role in cushioning stress and reducing friction. The physiological characteristics of articular cartilage—namely, its avascular, lymphatic, and nerve-free nature—mean that its self-repair capacity is extremely limited once damaged. If cartilage defects are not treated promptly and effectively, they typically worsen progressively, leading to persistent pain, joint dysfunction, and ultimately, disabling osteoarthritis. Currently, clinical treatments for articular cartilage defects mainly include microfracture surgery, autologous / allogeneic osteochondral transplantation, and autologous chondrocyte transplantation (ACI). However, these traditional treatments suffer from problems such as poor biomechanical properties of the repaired tissue, donor site damage, immune rejection, dedifferentiation risks, and poor integration, highlighting the urgent need for novel cartilage repair strategies.

[0003] The development of tissue engineering and regenerative medicine has provided new ideas for cartilage repair, but its traditional strategies rely on exogenous cells and growth factors, which face challenges such as high cost, complex approval processes, immunogenicity, and uncontrollable cell survival and differentiation. Therefore, the research focus has gradually shifted to "in situ regeneration," that is, using functionalized biomaterials to recruit endogenous stem cells and guide their differentiation.

[0004] Among these methods, applying exogenous electrical stimulation is an important means of regulating cell behavior, but its application is limited by the invasiveness, infection risk, and inconvenience of traditional devices. Although self-powered stents based on piezoelectric or triboelectric effects can achieve passive electrical stimulation, their output depends on joint movement, and the current is weak and unstable, which limits their effectiveness for postoperative immobilization patients.

[0005] Therefore, developing biodegradable implants that can actively and continuously provide power is crucial. Summary of the Invention

[0006] In view of this, the present invention aims to provide a biomimetic osteocartilage bio-battery, its preparation method, and its application. The bio-battery provided by the present invention can simulate the electrical microenvironment and chemical signals of natural osteocartilage tissue, possesses a biomimetic layered structure, and can realize programmed electrical stimulation and ion release. This battery can not only provide stable and long-lasting endogenous electrical stimulation, but also guide endogenous stem cells to differentiate in a regional and directed manner, thereby achieving integrated and functional regeneration of osteocartilage defects.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] One of the technical solutions of the present invention is a biomimetic osteocartilage bio-battery, comprising an anode layer, a cathode layer, and a separator layer; the anode layer is composed of zinc powder and a hydrogel framework, the cathode layer is composed of manganese dioxide powder and a hydrogel framework, and the separator layer is composed of PVDF and a hydrogel framework.

[0009] The second technical solution of the present invention is a method for preparing the above-mentioned biomimetic bone cartilage bio-battery, comprising the following steps:

[0010] (1) Dissolve sodium alginate and chitosan in water to prepare a hydrogel precursor solution;

[0011] (2) Divide the hydrogel precursor solution into three parts, and then disperse zinc powder, manganese dioxide powder and PVDF in the hydrogel precursor solution respectively to obtain zinc powder-containing anode printing ink, manganese dioxide-containing cathode printing ink and PVDF-containing diaphragm printing ink.

[0012] (3) Using 3D printing technology, the anode layer, separator layer and cathode layer are printed sequentially to form a three-layer battery precursor;

[0013] (4) The battery precursor is immersed in calcium chloride solution to carry out cross-linking reaction to obtain the biomimetic osteocartilage bio-battery.

[0014] The third technical solution of the present invention is the application of the above-mentioned biomimetic osteocartilage bio-battery in the preparation of osteocartilage defect repair materials and tissue engineering scaffolds.

[0015] The fourth technical solution of the present invention is the application of the above-mentioned biomimetic osteocartilage bio-battery in the preparation of medical dressings or implants for treating articular cartilage damage or osteoarthritis.

[0016] The present invention discloses the following technical effects:

[0017] (1) Continuous and stable endogenous electrical stimulation: Based on the mature electrochemical system of Zn-MnO2, the bio-battery of the present invention can provide an initial DC output of about 0.6V and a continuous output for more than 14 days when activated by body fluids in vivo. This endogenous electric field, which does not depend on external devices and the patient's activity level, provides a powerful and lasting driving force for the directional migration (electrotylation) of mesenchymal stem cells (BMSCs), solving the pain points of unstable current and inconvenience of use in traditional electrical stimulation methods.

[0018] (2) Intelligent programmed signal release: This invention achieves the coordinated programmed release of physical electrical signals and chemical ion signals over time. In the early stage of repair (approximately 0-7 days), the bio-battery outputs high electrical stimulation, its main function being to strongly recruit endogenous stem cells; in the middle and late stages of repair (approximately 7-14 days), Zn 2+ and Mn 2+The release of ions reaches its peak at this time, when the recruited cells are in the differentiation window, and the ion signal can most effectively guide them to differentiate into osteogenic or chondrogenic lineages. This intelligent timing logic of "recruit first, differentiate later" greatly improves repair efficiency.

[0019] (3) Excellent biocompatibility and biodegradability: All components are made of biocompatible materials. The hydrogel matrix provides a moist, suitable three-dimensional microenvironment for cell growth and can gradually degrade and be replaced by new tissue after completing its function. The released Zn... 2+ and Mn 2+ It not only acts as a differentiation signal, but also has functions such as anti-oxidation, scavenging reactive oxygen species, and regulating macrophage polarization towards the M2 type that promotes repair, together creating an immune microenvironment conducive to regeneration.

[0020] (4) Biomimetic Structure and Multifunctional Integration: This invention precisely constructs a three-layer structure simulating natural osteochondral structure, consisting of "cartilage-calcified cartilage-subchondral bone," using 3D printing technology. The cathode layer corresponds to the cartilage region, releasing Mn. 2+ Induces chondrogenesis; the anodic layer corresponds to the bone region, releasing Zn. 2+ It promotes osteogenic differentiation; the middle septum not only acts as an electrical insulator to prevent short circuits, but its dense structure also mimics the barrier function of the natural "tidal line", effectively inhibiting abnormal ingrowth of blood vessels from the subchondral bone into the cartilage area, and ensuring the purity of the repaired tissue.

[0021] (5) Advanced manufacturing process and great potential for personalization: The method provided by this invention utilizes multi-material 3D printing technology to achieve integrated, personalized, and rapid prototyping of battery structures, solving the problems of complex traditional battery manufacturing processes and difficulty in matching tissue morphology. This process is simple, easy to control, and scalable. Personalized scaffolds can be designed and manufactured rapidly based on the patient's specific defect imaging data (such as CT / MRI), which has broad prospects for clinical translation. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1The following are characterization diagrams of the precursor hydrogel for the bio-battery of this invention, wherein: (A), (B), and (C) are the mechanical properties of the hydrogel skeleton (SC hydrogel) with different SA / CS ratios, where S3C1 represents an SA:CS ratio of 3:1, and so on; (D), (E), and (F) are schematic diagrams of the bio-battery printing process; (G) and (H) are the rheological properties of the precursor bio-ink, where Mn-H refers to the cathode printing ink containing manganese dioxide powder, and so on; (I) Fourier transform infrared spectrum (FTIR) of the hydrogel; (J) X-ray diffraction (XRD) pattern of the Zn electrode (anode); (K) XRD pattern of the Mn electrode (cathode); (L) XRD pattern of the PVDF hydrogel;

[0024] Figure 2 The images show the physical and microscopic representations of the bio-battery of this invention, wherein: (A) and (B) are macroscopic photographs, scanning electron microscope (SEM) images, and energy-dispersive X-ray spectroscopy (EDS) distribution maps (C, O, Mn) of the Mn electrode (cathode); (C) and (D) are macroscopic photographs, scanning electron microscope (SEM) images, and energy-dispersive X-ray spectroscopy (EDS) distribution maps (C, O, Zn) of the Zn electrode (anode); (E) and (F) are macroscopic photographs, scanning electron microscope (SEM) images, and energy-dispersive X-ray spectroscopy (EDS) distribution maps (C, O, F) of the separator layer; (G) and (H) are images of the assembled complete bio-battery, cross-sectional SEM views, and elemental (Zn, Mn, F) distribution maps of the bio-battery.

[0025] Figure 3 The following are characterization diagrams of the discharge performance of the bio-battery of the present invention, including: (A) the discharge current curve of the bio-battery over 336 hours; (B) the discharge voltage curve of the bio-battery over 336 hours; (C) a physical image of the bio-batteries connected in series lighting an LED bulb; (D), (E), and (F) Nyquist and Bode plots of the electrochemical impedance spectroscopy (EIS) of the bio-battery (with or without carboxyl carbon nanotubes CNTs); (G) the full-range X-ray photoelectron spectroscopy (XPS) spectrum of the bio-battery after one week of discharge; (H) the valence state analysis of zinc (Zn) in the bio-battery after one week of discharge; (I) the valence state analysis of manganese (Mn) in the bio-battery after one week of discharge; and (J) the manganese ion Mn during the discharge process of the bio-battery. 2+ Release curve; (K) Zinc ions (Zn) during the discharge process of the bio-battery. 2+ The release curve;

[0026] Figure 4The diagram shows the free radical scavenging and immune regulation functions of the bio-battery of the present invention, wherein: (A1)-(D2) the scavenging effects of the bio-battery on DPPH free radicals, hydrogen peroxide (H2O2) and hydroxyl radicals (•OH), and its peroxidase-like activity; (E) flow cytometry analysis of the regulation of macrophage response (M1 / M2 phenotype) by the bio-battery and its components.

[0027] Figure 5 To assess the in vitro biocompatibility and migration-promoting effect of the bio-battery of this invention, the following parameters are included: (A) quantitative analysis of the cell compatibility (MTT method) of the bio-battery and its components; (B) staining results of live / dead cells of the bio-battery (scale bar = 200 μm); (C) hemolysis analysis of the bio-battery; (D) in vivo biocompatibility of the bio-battery (H&E staining sections of major organs: heart, liver, spleen, lung, and kidney) (scale bar = 800 μm); (E) and (F) scratch assay (wound healing) results of BMSCs treated with the bio-battery (scale bar = 400 μm).

[0028] Figure 6 The diagram shows the in vitro differentiation-inducing effect of the bio-battery of this invention, where: (A) Alcian Blue (AB) staining results of the bio-battery and its components on BMSCs on days 14 and 21 (scale bar = 500 μm); (B) Immunofluorescence staining of type II collagen (Col-II) on day 7 (scale bar = 50 μm); (C) Alkaline phosphatase (ALP) staining results on days 7 and 14; (D) Alizarin Red S (ARS) staining results on days 7 and 14 (scale bar = 500 μm); (E) Immunofluorescence staining of type I collagen (Col-I) on day 7; (F)-(J) Real-time quantitative polymerase chain reaction (RT-qPCR) results, showing the expression of chondrogenic genes (ACAN, COL2A1, SOX9) and osteogenic genes (COL1A1, RUNX2) in BMSCs induced by the bio-battery;

[0029] Figure 7 Figure 1 shows the in vivo animal experiment results of the bio-battery of the present invention, including: (A) macroscopic photographs and Micro-CT images of the rabbit knee joint healing results at week 6; (B) macroscopic photographs and Micro-CT images of the rabbit knee joint healing results at week 12; (C) and (F) CD31 immunofluorescence staining (vascular markers) and statistical results of the repaired tissue sections at week 12 (scale bar = 200 μm); (D) and (E) measurements of bone surface area to bone volume ratio (BV / TV) and bone mineral density (BMD) of the rabbit knee joint healing at week 12; and (G) the stiffness value of the repaired cartilage tissue.

[0030] Figure 8 Figure (II) shows the in vivo animal experiment results of the bio-battery of this invention, including: (A) and (B) hematoxylin-eosin (H&E) staining, safranin O-Fast Green staining, and Alcian blue staining of each experimental group (Control, SA / CS, Zn, Mn, Zn io, Mn-Zn, Battery) at weeks 6 and 12; (C) and (E) Sirius Red staining and statistical diagram of the arrangement of newly generated cartilage collagen fibers in each experimental group at weeks 6 and 12 (scale bar = 1000 μm); (D) International Cartilage Repair Society (ICRS) histological score;

[0031] Figure 9 Figure 3 shows the in vivo animal experiment results of the bio-battery of this invention, where: (A), (B) immunohistochemical (IHC) staining of type II collagen (Col-II), SOX-9, type I collagen (Col-I), and Runx-2 in each experimental group at weeks 6 and 12 (scale bar = 500 μm); (C), (D), (E), and (F) quantitative analysis of immunohistochemical (SOX-9, RUNX-2, COL-1, COL-2) positive staining; (G) Western blot (WB) results of tissue proteins;

[0032] Figure 10 Figure A is a schematic diagram of the preparation and application of the bio-battery of the present invention, and Figure B is a schematic diagram of the bio-battery of the present invention in a New Zealand white rabbit model. Detailed Implementation

[0033] 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.

[0034] 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 each intermediate value between the upper and lower limits of the range is also 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, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] 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. 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.

[0036] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0037] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0038] In this invention, unless otherwise specified, room temperature refers to 15-30°C.

[0039] To provide a biodegradable implant capable of actively, stably, and continuously delivering endogenous electrical energy, a galvanic cell (also known as a "primary cell" or "biocell") based on the electrochemical potential difference of metals offers an ideal solution. Zinc (Zn), as an essential trace element, possesses excellent biocompatibility and biodegradability, enabling it to form a galvanic cell with various cathode materials in a physiological environment, undergoing a mild and stable redox reaction, thus serving as the anode to provide sustained electrical energy. More innovatively, zinc ions (Zn²⁺) released during the degradation process... 2+ Manganese dioxide (MnO2) is itself a potent biosignaling molecule that can significantly promote osteogenic differentiation of bone marrow mesenchymal stem cells, making it an ideal chemical signal for repairing subchondral bone defects. Meanwhile, manganese dioxide (MnO2), as a biocompatible cathode material, produces manganese ions (Mn...) as its degradation product. 2+ ) is a key coenzyme for the synthesis of glycosaminoglycans in the extracellular matrix of chondrocytes, and can effectively promote the differentiation of stem cells into the chondrocyte lineage.

[0040] Therefore, constructing a bio-battery based on a zinc-manganese dioxide (Zn-MnO2) electrochemical system can not only provide active and stable physical electrical stimulation, but also programmatically release chemical ion signals (ZnO2) with specific biological functions during discharge. 2+ Used for osteogenic formation, Mn 2+(For chondrogenesis), providing a highly promising multi-synergistic strategy for integrated, zoned repair of osteochondral tissue. However, translating this electrochemical concept into effective osteochondral repair implants still faces the following major technical challenges:

[0041] Structural biomimicry: Natural osteochondral tissue has a complex layered structure from cartilage to bone, among which the "tidemark" is a key physiological barrier separating the avascular cartilage layer from the vascularized subchondral bone layer. Constructing a functional scaffold that can mimic this layered structure and effectively prevent blood vessels from invading the newly formed cartilage layer is crucial for successful repair.

[0042] Manufacturing process: Traditional material preparation methods make it difficult to accurately construct personalized scaffolds with complex three-dimensional structures that contain multiple functional components (such as anode materials, cathode materials, and insulating barrier materials).

[0043] Material integration: How to seamlessly integrate electrode active materials, conductive materials and structural support materials into a unified, biocompatible and biodegradable hydrogel system is a challenge to ensure battery performance and biological function.

[0044] 3D printing technology offers a revolutionary solution to these problems. It allows for precise control of electrode structures and seamless integration of multiple functional components into a single construct, paving the way for the creation of fully integrated, personalized, implantable power sources. Through multi-material printing, energy storage devices can be customized to match complex tissue morphologies, meeting specific biomechanical and electrochemical requirements.

[0045] In summary, an ideal device should possess the following characteristics: (1) It should be able to provide a stable and persistent directional internal electric field independent of external mechanical stress; (2) It should be able to release metal ions with osteogenic and cartilage-inducing effects in a zoned and programmed manner; (3) It should have a biomimetic three-layer structure, in which the middle layer can effectively simulate the barrier function of the natural "tidal line"; (4) The overall material should be completely biodegradable and have good biocompatibility; (5) The manufacturing process should be flexible and personalized customization should be possible.

[0046] Based on this, the first aspect of the present invention provides a biomimetic osteocartilage bio-battery, comprising an anode layer, a cathode layer, and a separator layer; the anode layer is composed of zinc powder and a hydrogel framework, the cathode layer is composed of manganese dioxide powder and a hydrogel framework, and the separator layer is composed of PVDF and a hydrogel framework.

[0047] In a preferred embodiment of the present invention, the hydrogel framework is a composite hydrogel of sodium alginate and chitosan, wherein the mass ratio of sodium alginate to chitosan is 3:(1~9).

[0048] More preferably, the mass ratio of sodium alginate to chitosan is 3:1, 2:1, 1:1, 1:2, or 1:3.

[0049] In this invention, sodium alginate and chitosan, within the above-mentioned ratio parameters, form a stable polyelectrolyte network through electrostatic interaction, and after being cross-linked by calcium ions, they can obtain optimal mechanical properties and printability.

[0050] In a preferred embodiment of the present invention, zinc powder accounts for 0.3% to 2% of the mass of the hydrogel skeleton in the anode layer; manganese dioxide powder accounts for 0.3% to 2% of the mass of the hydrogel skeleton in the cathode layer; and PVDF accounts for 0.3% to 2% of the mass of the hydrogel skeleton in the membrane layer.

[0051] More preferably, in the anode layer, zinc powder accounts for 0.3%, 0.5%, 0.7%, 0.9%, 1%, 1.2%, 1.5%, 1.7%, or 2% of the mass of the hydrogel skeleton; in the cathode layer, manganese dioxide powder accounts for 0.3%, 0.5%, 0.7%, 0.9%, 1%, 1.2%, 1.5%, 1.7%, or 2% of the mass of the hydrogel skeleton; and in the membrane layer, PVDF accounts for 0.3%, 0.5%, 0.7%, 0.9%, 1%, 1.2%, 1.5%, 1.7%, or 2% of the mass of the hydrogel skeleton.

[0052] In this invention, zinc powder, manganese dioxide powder, and PVDF, within the above parameter range, can provide sufficient electrochemical active materials while ensuring good biocompatibility, thus ensuring that the battery's discharge capacity and ion release kinetics meet the needs of tissue repair.

[0053] In a preferred embodiment of the present invention, the cathode layer further includes CNTs, the CNTs accounting for 0.1% to 0.5% of the mass of the hydrogel skeleton; the membrane layer further includes carboxylated carbon nanotubes, the carboxylated carbon nanotubes accounting for 0.1% to 0.5% of the mass of the hydrogel skeleton.

[0054] More preferably, in the cathode layer, CNTs account for 0.1%, 0.2%, 0.3%, 0.4% or 0.5% of the hydrogel mass, and in the diaphragm layer, CNTs account for 0.1%, 0.2%, 0.3%, 0.4% or 0.5% of the hydrogel mass.

[0055] In this invention, CNTs can significantly enhance the conductivity of hydrogels and reduce the charge transfer resistance at the electrode interface within the above parameter range, thereby improving the discharge performance and electrical stimulation efficiency of the battery.

[0056] The bio-battery of this invention has an anode corresponding to the bone region of cartilage tissue, a diaphragm layer corresponding to the "tidal line," and a cathode corresponding to the cartilage region of cartilage tissue, simulating the three-layer structure of natural bone and cartilage tissue. After implantation, the bio-battery is activated upon contact with bodily fluids, generating a stable open-circuit voltage with an initial voltage not less than 0.5V (approximately 0.6V), and maintaining effective electrical stimulation output for at least 14 days. This discharge cycle perfectly covers the critical cell recruitment and differentiation stages in the early stages of tissue repair.

[0057] A second aspect of this invention provides a method for preparing the above-mentioned biomimetic bone cartilage bio-battery, comprising the following steps:

[0058] (1) Dissolve sodium alginate and chitosan in water to prepare a hydrogel precursor solution (SC).

[0059] (2) Divide the hydrogel precursor solution into three parts, and then disperse zinc powder, manganese dioxide powder and PVDF in the hydrogel precursor solution respectively to obtain zinc powder-containing anode printing ink, manganese dioxide-containing cathode printing ink and PVDF-containing diaphragm printing ink.

[0060] (3) Using 3D printing technology, the anode layer, separator layer and cathode layer are printed sequentially to form a three-layer battery precursor;

[0061] (4) The battery precursor is immersed in calcium chloride solution to carry out cross-linking reaction to obtain the biomimetic osteocartilage bio-battery.

[0062] In a preferred embodiment of the present invention, the concentration of sodium alginate in the hydrogel precursor solution is (4%-6%) w / v (more preferably, 6% (w / v)), and the mass ratio of sodium alginate to chitosan is 3:(1~9).

[0063] In a preferred embodiment of the present invention, the parameters for 3D printing are: pressure 2.5-4.0 bar, printing speed 8-12 mm / min, and nozzle diameter 0.2-0.3 mm.

[0064] More preferably, the 3D printing parameters are: pressure 3.2 bar, printing speed 10 mm / min, and nozzle diameter 0.26 mm.

[0065] In a preferred embodiment of the present invention, the concentration of the calcium chloride solution is 1%-5% (w / v) (more preferably, 2% (w / v)).

[0066] In a preferred embodiment of the present invention, the crosslinking reaction is carried out at room temperature for 6-24 hours.

[0067] The third aspect of this invention provides the application of the above-mentioned biomimetic osteocartilage bio-battery in the preparation of osteocartilage defect repair materials and tissue engineering scaffolds.

[0068] The fourth aspect of the present invention provides the application of the above-mentioned biomimetic osteocartilage biobattery in the preparation of medical dressings or implants for treating articular cartilage damage or osteoarthritis.

[0069] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0070] To better understand the present invention, the following embodiments further illustrate the content of the present invention, but the content of the present invention is not limited to the following embodiments.

[0071] Example 1

[0072] Preparation and Screening of SA / CS Basic Hydrogels (SC Hydrogels)

[0073] (1) Preparation of basic hydrogel solutions: Sodium alginate (SA) was dissolved in deionized water (DI water) to a final concentration of 6% (w / v). Subsequently, soluble chitosan (CS) was added to prepare a series of hydrogel precursor solutions with different SA:CS weight ratios (SA:CS = 3:1, 2:1, 1:1, 1:2, 1:3). The mixture was continuously stirred at room temperature until a transparent and homogeneous mixed solution (hydrogel precursor solution) was formed.

[0074] (2) Mechanical property screening: The mechanical properties of the material prepared in step (1) and subjected to Ca 2+ Hydrogels with different SA:CS ratios after ion crosslinking (named S3C1, S2C1, S1C1, S1C2, and S1C3 according to SA:CS = 3:1, 2:1, 1:1, 1:2, and 1:3, respectively) were subjected to uniaxial compression tests to evaluate their mechanical integrity. Among them, hydrogels with different SA:CS ratios after Ca2+ crosslinking were subjected to uniaxial compression tests to evaluate their mechanical integrity. 2+ The specific steps for ionic crosslinking are as follows: the hydrogel precursor solution is immersed in calcium chloride solution (2% (w / v)) for crosslinking for 12 hours.

[0075] (3) Screening results: such as Figure 1 As shown in (A)-(C), the analysis of the compressive stress-strain curves and Young's modulus indicates that as the proportion of CS increases from S3C1 to S1C1, both the compressive strength and Young's modulus of the hydrogel gradually increase. This is attributed to the polyanionic (-COO) of sodium alginate. - ) and the polycationic (-NH) of chitosan 3+The enhanced electrostatic interactions between the CS and SA achieve the highest degree of ionic complexation and peak mechanical properties at a 1:1 weight ratio (S1C1). When the concentration of CS exceeds that of SA (S1C2, S1C3), the excessive positive charge generates electrostatic repulsion, leading to network structural defects and a significant decrease in mechanical properties.

[0076] (4) Conclusion: The S1C1 (1:1 weight ratio) formulation was selected as the standard hydrogel matrix for all subsequent bio-ink preparations.

[0077] Example 2

[0078] Preparation of anodic bio-ink (Zn-SC ink)

[0079] (1) Take the SA / CS hydrogel precursor solution with the optimal ratio (S1C1) prepared in Example 1.

[0080] (2) Add zinc (Zn) powder to the above SA / CS hydrogel precursor solution and stir to make it uniformly dispersed; wherein, the concentration of Zn powder added is 0.5% (w / w).

[0081] (3) Store the mixture at 4 °C for later use to obtain anodized bio-ink for 3D printing.

[0082] Example 3

[0083] Preparation of cathode bio-ink (MnO2-CNTs-SC ink)

[0084] (1) Take the SA / CS basic hydrogel precursor solution with the optimal ratio (S1C1) prepared in Example 1.

[0085] (2) Add manganese dioxide (MnO2) powder and carboxyl carbon nanotubes (CNTs) to the above SA / CS hydrogel precursor solution, and stir to uniformly disperse the MnO2 powder and CNTs; wherein the concentration of MnO2 is 0.5% (w / w) and the concentration of CNTs is 0.2% (w / w). MnO2 is used as the cathode active material, and CNTs are used to enhance conductivity. The carboxyl groups of CNTs help to improve their dispersibility and interfacial bonding in the hydrophilic hydrogel matrix.

[0086] (3) Store the mixture at 4 °C to obtain the cathode bio-ink for 3D printing.

[0087] Example 4

[0088] Preparation of isolation layer bio-ink (PVDF-CNTs-SC ink)

[0089] (1) Take the SA / CS basic hydrogel precursor solution with the optimal ratio (S1C1) prepared in Example 1.

[0090] (2) Polyvinylidene fluoride (PVDF) powder and carboxylated carbon nanotubes (CNTs) were added to the above SA / CS hydrogel precursor solution, and the PVDF powder and CNTs were uniformly dispersed by stirring; wherein the concentration of PVDF added was 0.5% (w / w), and the concentration of CNTs added was 0.2% (w / w). PVDF is used to construct a dense physical barrier to simulate the function of "tidal line" and maintain electrical insulation, and CNTs are used to assist ion transport.

[0091] (3) Store the mixture at 4 °C to obtain the isolation layer bio-ink for 3D printing.

[0092] Example 5

[0093] Fabrication of 3D-printed biomimetic osteocartilage biobattery scaffold

[0094] (1) The three bio-inks prepared in Example 2 (anodic ink), Example 3 (cathode ink) and Example 4 (isolation layer ink) were respectively loaded into the individual cartridges of the 3D bio-printer (Envision Tec 3D-Bioplotter).

[0095] (2) Design the support structure using 3ds Max software and set the printing parameters as follows: printing pressure 3.2 bar, printing speed 10 mm / min, and nozzle diameter 0.26 mm.

[0096] (3) An alternating multi-nozzle printing process is used to fabricate the scaffold layer by layer from bottom to top (anode-isolation layer-cathode). The bottom layer (corresponding to the bone side) is printed with anode (Zn-SC) ink, the middle layer is printed with isolation layer (PVDF-CNTs-SC) ink, and the top layer (corresponding to the cartilage side) is printed with cathode (MnO2-CNTs-SC) ink. This structure aims to simulate the three-layer structure of natural osteochondral tissue. Figure 1 (D)-(F)).

[0097] (4) After printing, immerse the scaffold in a calcium chloride (CaCl2) solution (2% w / v) to utilize the Ca... 2+ Ions promote complete ionic cross-linking of sodium alginate (SA) components, forming a stable three-layer bio-battery scaffold.

[0098] Example 6

[0099] Rheological and printability characterization of bio-inks

[0100] The three types of bio-inks prepared in Examples 2, 3, and 4 were used as experimental subjects in the following experiments:

[0101] The rheological properties of three bio-inks were evaluated using a strain-controlled rheometer (Anton-Paar) to determine their suitability for 3D printing.

[0102] (1) Shear thinning properties: such as Figure 1 As shown in Figure G (where Mn-H, PVDF-H, and Zn-H represent the cathode ink, separator ink, and anode ink, respectively), all three inks (anode, cathode, and separator) exhibit significant shear-thinning behavior, meaning their viscosity decreases significantly with increasing shear rate. This characteristic is essential for achieving smooth and continuous extrusion and can reduce extrusion pressure.

[0103] (2) Yield stress: such as Figure 1 As shown in Figure (H), the storage modulus (G') and loss modulus (G'') were measured using oscillatory rheology. The yield stress values ​​for the three bio-inks were determined to be in the range of 400–680 Pa. This yield stress value is much higher than the minimum threshold (approximately 100 Pa) required for 3D printing hydrogel structures, ensuring that the deposited fibers can rapidly recover their structural integrity after printing and support subsequent layers without collapsing or spreading.

[0104] (3) Conclusion: This embodiment demonstrates that the bio-inks prepared in Examples 2, 3 and 4 have ideal "printable windows" and possess the rheological properties required for successful additive manufacturing of complex, multi-material structures.

[0105] Example 7

[0106] Physicochemical and microstructure characterization of bio-battery scaffolds

[0107] The bio-battery scaffolds and their components prepared in Examples 1, 2, 3, 4, and 5 were used as experimental subjects and freeze-dried for the following experiments:

[0108] (1) FTIR characterization: such as Figure 1 As shown in (I), the FTIR spectrum reveals sodium alginate (approximately 1594 cm⁻¹). -1 And chitosan (approximately 1427 cm) -1 The characteristic absorption peaks of the base polymers were observed. After incorporation of Zn, MnO2, PVDF, and CNTs, the positions of these base polymer peaks did not shift significantly, indicating that the additives were physically blended and did not form new covalent bonds with the polymer matrix.

[0109] (2) XRD characterization: such as Figure 1 As shown in (J)-(L), the XRD patterns confirm the successful incorporation of the functional additives and the maintenance of their inherent crystal structure. Anodic hydrogel ( Figure 1 The cathode hydrogel (J) exhibits diffraction peaks at 2θ values ​​of 36.2°, 38.9°, and 43.1°, corresponding to the (002), (100), and (101) crystal planes of crystalline zinc (Zn). Figure 1 The medium (K) layer exhibits characteristic peaks of MnO2 at 28.6° and 37.5°. The isolation layer hydrogel ( Figure 1 The middle (L) shows characteristic peaks of PVDF (18.2° and 20.0°) and CNTs (24.6° and 42.7°).

[0110] (3) SEM microstructure: such as Figure 2 As shown in (A), (C), and (E), the SEM images reveal that the scaffold has a well-defined structure and exhibits a highly porous and interconnected network structure, which facilitates cell invasion and nutrient delivery. Figure 2 Cross-sectional SEM analysis shown in (G) confirms the layer-by-layer assembly of the anode, separator, and cathode. Crucially, the anode and cathode layers exhibit a microporous internal structure, while the intermediate separator is observed to be "significantly denser." This dense microstructure serves a dual function: electrochemically, it acts as a physical barrier to prevent short circuits between electrodes; biologically, it can serve as a partition separating bone and cartilage regions.

[0111] (4) Distribution of EDS elements: such as Figure 2 As shown in Figure (H), the EDS elemental analysis of the stent clearly demonstrates the precise location of key functional elements. Zinc (Zn) is uniformly dispersed in the anode layer (bottom layer), manganese (Mn) is confined to the cathode layer (top layer), and fluorine (F) (as a marker for PVDF) is confined to the isolation layer (intermediate layer). The interfaces between layers are clear and distinct, with minimal cross-contamination.

[0112] (5) Conclusion: This embodiment demonstrates that a bio-battery scaffold with porous electrodes and a dense insulating layer, exhibiting layered biomimetic characteristics in both chemistry and structure, was successfully manufactured using the 3D printing method of Example 5.

[0113] Example 8

[0114] Electrochemical performance and ion release characterization of bio-battery scaffolds

[0115] The bio-battery scaffold prepared in Example 5 was used as the experimental subject in the following experiments:

[0116] (1) Electrical performance: The stent was immersed in phosphate-buffered saline (PBS, simulated body fluid), and its output was monitored using a high-precision digital multimeter. For example... Figure 3As shown in (A) and (B), the device provides robust initial power output with a voltage of approximately 0.6 V and a current of 10 mA. The bio-battery exhibits stable and gradual performance degradation, with an effective operating life exceeding 14 days (336 hours). Figure 3 As shown in (C), three bio-cells connected in series are sufficient to power an LED light bulb.

[0117] (2) EIS characterization: Electrochemical impedance spectroscopy (EIS) tests were performed on bio-batteries containing CNTs (as in Examples 3 and 4) and those without CNTs. Figure 3 As shown in (D)-(F), Nyquist plot ( Figure 3 The middle (D) diagram shows that the bio-battery containing CNTs exhibits a “significantly smaller high-frequency semicircle,” which corresponds to a significant reduction in charge transfer resistance. Bode plot ( Figure 3 The results (E) and (F) also show that it has a lower impedance modulus (|Z|) in the low-frequency region. This confirms that the addition of CNTs effectively improves the electron transfer efficiency at the electrode-electrolyte interface.

[0118] (3) Electrochemical Mechanism (XPS): To verify the generator mechanism, XPS analysis was performed on the electrode after discharge in PBS for 7 days. For example... Figure 3 As shown in Figure (I), the Mn 2p spectrum of the cathode shows that the tetravalent manganese in MnO2 is significantly transformed into Mn. 2+ and Mn 3+ The state confirms that it gained electrons as a positive electrode and underwent a reduction reaction. For example... Figure 3 As shown in (G) and (H), the Zn spectrum at the anode shows a transition from the elemental state to the oxidation state, confirming that it lost electrons as the negative electrode and underwent an oxidation reaction.

[0119] (4) Ion release (ICP-OES): ICP-OES was used for quantitative detection of Zn. 2+ and Mn 2+ The release. For example... Figure 3 As shown in (J) and (K), Zn 2+ (from the anode) and Mn 2+ The release (from the cathode) is gradual and continuous, reaching a peak concentration around day 12.

[0120] (5) Conclusion: This embodiment demonstrates that the scaffold of the present invention is a functional bio-battery that can generate a biocompatible electrical signal (approximately 0.6 V) for more than 14 days and, through an electrochemical reaction, programmatically releases therapeutically active Zn in a controllable manner that matches the biological healing cycle (differentiation period from day 7 to 14) (peaking on day 12). 2+ and Mn 2+ ion.

[0121] Example 9

[0122] Free radical scavenging and immune regulation functions of bio-batteries

[0123] This experiment uses the bio-battery scaffold and its components prepared in Example 5 as the experimental subjects for the following experiments:

[0124] (1) Free radical scavenging ability: The antioxidant capacity of the scaffold was assessed by methods such as DPPH free radical scavenging, hydrogen peroxide (H2O2) scavenging, peroxidase-like activity (TMB substrate), and hydroxyl radical (•OH) scavenging (via Fenton reaction). Figure 4 As shown in (A1)-(D2), the bio-battery (primarily due to the release of Mn and Zn ions) exhibits highly efficient scavenging ability against all tested free radicals. In contrast, the control group receiving only electrical stimulation induced the generation of free radicals. This indicates that the bio-battery of the present invention (electrical stimulation + ion release) is better able to maintain the homeostasis of the microenvironment than electrical stimulation alone.

[0125] (2) Immunomodulatory function: The scaffold's regulation of the inflammatory microenvironment was studied using the RAW264.7 macrophage cell line. Macrophages were induced to the M1 phenotype and then co-cultured with the scaffold.

[0126] (3) Flow cytometry analysis: such as Figure 4 As shown in (E), flow cytometry results showed that, compared with the control group, the biocell group significantly promoted the transformation of macrophages from the pro-inflammatory M1 phenotype to the pro-repair M2 phenotype.

[0127] (4) Conclusion: This embodiment demonstrates that, in addition to providing electrical and ion stimulation, the scaffold of the present invention also has a third biological function: by clearing damage-related reactive oxygen species (ROS) and regulating the immune microenvironment (M1 to M2 polarization), it actively creates a microenvironment conducive to tissue repair.

[0128] Example 10

[0129] In vitro biocompatibility and migration-promoting ability of bio-batteries

[0130] In this experiment, the bio-battery scaffold and its components prepared in Example 5 were sterilized by immersion in 75% ethanol and used as experimental subjects for the following experiments:

[0131] (1) Cell compatibility (MTT): Assessed using BMSCs. For example... Figure 5As shown in (A), quantitative analysis using the MTT assay revealed that the cell viability of the biocell, anode, cathode, and isolation layer remained above 80% throughout the culture period (1, 4, 7, 12 days), demonstrating excellent cell compatibility.

[0132] (2) Live / dead cell staining: such as Figure 5 As shown in (B), the staining results clearly show that BMSCs cultured on the scaffold surface exhibit dense and vigorous green fluorescence (live cells), while almost no red fluorescence (dead cells) is observed, confirming that the material is non-cytotoxic.

[0133] (3) Blood compatibility: such as Figure 5 As shown in (C), the hemolysis test results showed that the hemolysis rate of all material groups was lower than the international standard of 5%, demonstrating good blood compatibility.

[0134] (4) In vivo biocompatibility: The stent was implanted subcutaneously into a New Zealand white rabbit. For example... Figure 5 As shown in (D), after 6 weeks, no obvious organ damage, inflammatory response or structural abnormalities were observed in the H&E stained sections of the major organs (heart, liver, spleen, lung and kidney), indicating that it has excellent in vivo biocompatibility.

[0135] (5) Promotes migration (electromigration): To verify the function of the endogenous electric field (physical cue), in vitro scratch experiments were performed on BMSCs. For example... Figure 5 As shown in (E) and (F), the migration rate of BMSCs exposed to the biocell electric field was "significantly accelerated". At 12 hours (12 h), the scratches (cell-deficient areas) in the biocell group were "almost completely healed", while the control group still had obvious cell-deficient gaps.

[0136] (6) Conclusion: This embodiment demonstrates that the scaffold of the present invention has excellent cell, blood and systemic biocompatibility, and the endogenous electric field it generates (as shown in Example 8) can effectively promote the directed migration (electromigration) of BMSCs, which is crucial for recruiting host stem cells to initiate repair.

[0137] Example 11

[0138] Bio-batteries induce specific differentiation of BMSCs in vitro across specific regions.

[0139] This experiment uses the bio-battery scaffold and its electrode components prepared in Example 5 as experimental objects to verify the region-specific differentiation-inducing ability of the "chemical cues" (Zn ions and Mn ions, as shown in Example 8) released by the bio-battery.

[0140] (1) Chondrogenic induction (simulated cathode side): BMSCs were co-cultured with the cathode (Mn) group and the biobattery group, respectively.

[0141] (2) Chondrogenic staining: such as Figure 6 As shown in (A), Alcian blue (AB) staining (for detecting glycosaminoglycans) revealed "significantly stronger" staining in both the Mn and Biobattery groups on days 14 and 21. Figure 6 As shown in (B), immunofluorescence of type II collagen (Col-II, the main matrix of hyaline cartilage) also showed that the expression of both groups was "significantly increased".

[0142] (3) Chondrogenic gene detection (RT-qPCR): such as Figure 6 As shown in (F), (H), and (J), RT-qPCR results confirmed that the presence of Mn ions significantly upregulated the expression of chondrogenic marker genes (Col2A1, Sox-9, ACAN).

[0143] (4) Osteogenesis induction (simulated anode side): BMSCs were co-cultured with the anode (Zn) group and the biobattery group, respectively.

[0144] (5) Osteogenic staining: such as Figure 6 As shown in (C), ALP staining (early osteogenic marker) showed "significantly stronger" results in the Zn group on days 7 and 14. Figure 6 As shown in (D), Alizarin Red S (ARS) staining (late-stage calcium deposition) revealed strong red staining in the Zn group on days 14 and 21. Figure 6 As shown in (E), type I collagen (Col-I, bone matrix protein) was significantly highly expressed in both the Zn group and the Biobattery group.

[0145] (6) Osteogenesis gene detection (RT-qPCR): such as Figure 6 As shown in (G) and (I), RT-qPCR results confirmed that the presence of Zn ions significantly upregulated the expression of osteogenic marker genes (Runx2, COL1A1).

[0146] (7) Conclusion: This embodiment demonstrates that the anode (releasing Zn ions) of the bio-battery of the present invention has a significant ability to promote bone differentiation, while the cathode (releasing Mn ions) has a significant ability to promote cartilage differentiation, thus confirming that the “regional chemical instruction set” design of the present invention can effectively induce BMSCs to undergo lineage-specific differentiation.

[0147] Example 12

[0148] Evaluation of in vivo osteochondral defect repair using bio-battery scaffolds (rabbit model)

[0149] This experiment used the bio-battery scaffold prepared in Example 5 and various control scaffolds as experimental subjects for the following experiments:

[0150] (1) Animal model: A full-thickness osteochondral defect (5.0 mm in diameter and 4.0 mm in depth) was established in the trochlear groove of the femur of New Zealand white rabbits.

[0151] (2) Experimental grouping: Seven groups were set up: (1) Control (blank control group); (2) SC (hydrogel matrix only group); (3) Zn (anodide only group); (4) Mn (cathode only group); (5) Zn io (zinc ion group, Zn for hydrogel) 2+ (6) Mn-Zn (without a membrane, direct contact between anode and cathode, without structural partitioning); (7) Battery (complete bio-battery pack of the present invention).

[0152] (3) Animals from the corresponding batches were sacrificed at 6 and 12 weeks for macroscopic and micro-CT evaluation:

[0153] (4) Results at 6 weeks and 12 weeks: such as Figure 7 As shown in (A) and (B), the Control and SA / CS groups showed minimal repair at different time points, with the defect remaining a large cavity. The Zn and Mn groups (single clue) showed some degree of repair, but failed to effectively regenerate the overlying cartilage layer. In contrast, Battery (group 7) showed "undisputed superior regeneration," with the defect almost completely filled by new tissue, and the joint surface "smooth, glossy, and seamlessly integrated with the surrounding natural cartilage."

[0154] (5) Micro-CT quantification: such as Figure 7 As shown in (D) and (E), the Battery group had significantly higher BV / TV and BMD values ​​at 12 weeks than all other groups, confirming its excellent regenerative capacity for the underlying subchondral bone.

[0155] (6) Key control group (Zn io group, to verify the effect of electric field): such as Figure 7 As shown in (A)-(B), the repair process in the Zn io group (group 5, chemical signal only) was "significantly delayed," and the height of newly formed tissue was significantly lower than that in the Battery group. This result provides direct evidence that endogenous electrical stimulation (physical signal) is "a key and indispensable component that can actively accelerate and enhance the overall regeneration process."

[0156] (7) Key control group (Mn-Zn group, to verify the role of the isolation layer): such as Figure 7As shown in (A) (6 weeks), the Mn-Zn group (Group 6, without an isolation layer) exhibited "abnormal upward protrusion of tissue" and disordered tissue morphology on Micro-CT. This indicates that the lack of structural integrity due to the absence of an isolation layer led to abnormal and undesirable (cartilaginous) ossification from the subchondral bone.

[0157] (8) Function of the isolation layer (CD31 staining): To verify the above findings, CD31 (vascular endothelial cell marker) immunofluorescence staining was performed on the repaired tissue at 12 weeks. For example... Figure 7 As shown in (C) and (F), strong expression of CD31 was observed in the newly formed cartilage in all other control groups, including the Mn-Zn group (indicating vascular invasion). In contrast, the cartilage layer in the Battery (Group 7) showed "virtually no CD31 expression." This result strongly demonstrates that the isolation layer of the present invention (derived from Example 4) successfully serves as an "artificial tide line," effectively reducing vascular invasion of the cartilage layer and preventing repair failure and cartilage ossification.

[0158] (9) Mechanical properties: such as Figure 7 As shown in (G), the stiffness test of the repaired tissue at 12 weeks showed that the stiffness of the regenerated cartilage in the Battery group was closest to that of natural cartilage, while the stiffness of all other groups was significantly lower.

[0159] (10) Histological assessment (H&E, Safranbolu O): such as Figure 8 As shown in (A) and (B), at 12 weeks, the defect in the Control group was still a cavity filled with granulation tissue. In contrast, the defect in the Battery group was "almost completely filled with newly formed, hyaline cartilage tissue," and showed strong positive staining with Safranin O (red, representing GAGs), indicating abundant cartilage matrix. The underlying trabecular bone structure "developed from a cavitary defect into an organized trabecular bone structure."

[0160] (11) Collagen arrangement (Sirius red): such as Figure 8 As shown in (C) and (E), Sirius red polarized light staining revealed that the newly formed collagen fibers in the Battery group were "organized in a biomimetic manner," with horizontal arrangement in the superficial layer (simulating lubrication function) and vertical orientation in the deep layer (simulating weight-bearing function), which is consistent with the structure of natural articular cartilage.

[0161] (12) Histological scoring and mechanism (IHC, WB): such as Figure 8 As shown in (D), the Battery group achieved the highest ICRS histological score. Figure 9 As shown, immunohistochemical (IHC) and Western blot (WB) results confirmed that the Battery group activates signaling pathways such as Wnt / β-Catenin and MAPK. Figure 9In vivo, it significantly upregulated the chondrogenic markers SOX-9 and Col-II. Figure 9 C, 9D) and osteogenic markers RUNX-2, Col-I (C, 9D) and osteogenic markers RUNX-2, Col-I ( Figure 9 The expression of (E), (F)).

[0162] (13) Summary of this embodiment: In vivo animal models, especially compared with key control groups (Zn io group and Mn-Zn group), ultimately proved that the bio-battery scaffold of the present invention, through "physical cues" (electric field) and "chemical cues" (Zn 2+ / Mn 2+ The synergistic effect of the combination of the "structural cues" (isolation layer) and the "structural cues" enables complete regeneration of functional osteochondral tissue, with effects superior to any single or partial combination of treatments.

[0163] In summary:

[0164] (1) The present invention uses multi-material 3D printing technology and combined with optimized SA / CS bio-ink to prepare a bio-battery scaffold with a biomimetic three-layer structure of anode, cathode and isolation layer. The scaffold structure (porous electrodes and dense isolation layer) and chemical components (Zn in anode and MnO2 in cathode) are arranged in a partitioned biomimetic manner.

[0165] (2) The bio-battery scaffold prepared in this invention is based on the Zn-MnO2 electrochemical system, which can generate a stable and durable (>14 days) endogenous electric field of about 0.6 V in simulated body fluid. This electric field can serve as a physical cue to effectively recruit and attract host stem cells (BMSCs) to the defect site through electromigration (as shown in Example 10).

[0166] (3) During the electrochemical discharge process, the stent of the present invention can programmatically release partition-specific chemical cues: Zn ions released from the anode (bone side) have been shown to effectively induce BMSCs to differentiate into osteogenic lineage; Mn ions released from the cathode (cartilage side) have been shown to effectively induce BMSCs to differentiate into chondrogenic lineage (as shown in Example 11).

[0167] (4) The PVDF-doped dense isolation layer constructed by 3D printing in this invention successfully simulates the “tidal line” function of the natural osteochondral interface. As a physical barrier, it has been proven to effectively prevent blood vessels (CD31) in the medullary cavity below from invading the cartilage regeneration area above, thus preventing abnormal ossification and ensuring the orderly reconstruction of layered tissues (as shown in Example 12).

[0168] (5) The Zn and Mn metal ions released by the scaffold of the present invention have multiple biological functions, including efficient removal of damage-related reactive oxygen species (ROS) and regulation of macrophage polarization toward the M2 phenotype that promotes repair, thereby actively optimizing the biological microenvironment of the damaged site (as shown in Example 9).

[0169] (6) Through the intelligent synergistic effect of the above-mentioned physical, chemical, biological and structural cues, the present invention has been shown to achieve functional and seamless integration of hyaline cartilage and underlying trabecular bone regeneration in a rabbit osteochondral defect model. The repair effect is significantly better than the control group (as shown in Example 12) which is untreated, uses hydrogel alone, uses electrodes alone or lacks key components (electric field or isolation layer).

[0170] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A biomimetic bone cartilage bio-battery, characterized in that, It includes an anode layer, a cathode layer, and a diaphragm layer; the anode layer is composed of zinc powder and a hydrogel framework, the cathode layer is composed of manganese dioxide powder and a hydrogel framework, and the diaphragm layer is composed of polyvinylidene fluoride powder and a hydrogel framework. The hydrogel framework is a composite hydrogel of sodium alginate and chitosan, wherein the mass ratio of sodium alginate to chitosan is 3:(1~9). In the anode layer, zinc powder accounts for 0.3% to 2% of the mass of the hydrogel skeleton; in the cathode layer, manganese dioxide powder accounts for 0.3% to 2% of the mass of the hydrogel skeleton; in the diaphragm layer, polyvinylidene fluoride powder accounts for 0.3% to 2% of the mass of the hydrogel skeleton. The preparation method of the biomimetic osteocartilage bio-battery includes the following steps: (1) Dissolve sodium alginate and chitosan in water to prepare a hydrogel precursor solution; (2) Divide the hydrogel precursor solution into three parts, and then disperse zinc powder, manganese dioxide powder and polyvinylidene fluoride powder in the hydrogel precursor solution respectively to obtain zinc powder-containing anode printing ink, manganese dioxide-containing cathode printing ink and polyvinylidene fluoride-containing diaphragm printing ink. (3) Using 3D printing technology, the anode layer, separator layer and cathode layer are printed sequentially to form a three-layer battery precursor; (4) The battery precursor is immersed in calcium chloride solution to carry out cross-linking reaction to obtain the biomimetic osteocartilage bio-battery.

2. The biomimetic osteocartilage bio-battery according to claim 1, characterized in that, The cathode layer is replaced with a structure composed of manganese dioxide powder, a hydrogel framework, and carboxylated carbon nanotubes, wherein the manganese dioxide powder accounts for 0.3% to 2% of the mass of the hydrogel framework, and the carboxylated carbon nanotubes account for 0.1% to 0.5% of the mass of the hydrogel framework; the membrane layer is replaced with a structure composed of polyvinylidene fluoride powder, a hydrogel framework, and carboxylated carbon nanotubes, wherein the polyvinylidene fluoride powder accounts for 0.3% to 2% of the mass of the hydrogel framework, and the carboxylated carbon nanotubes account for 0.1% to 0.5% of the mass of the hydrogel framework.

3. A method for preparing the biomimetic osteocartilage bio-battery according to claim 1, characterized in that, Includes the following steps: (1) Dissolve sodium alginate and chitosan in water to prepare a hydrogel precursor solution; (2) Divide the hydrogel precursor solution into three parts, and then disperse zinc powder, manganese dioxide powder and polyvinylidene fluoride powder in the hydrogel precursor solution respectively to obtain zinc powder-containing anode printing ink, manganese dioxide-containing cathode printing ink and polyvinylidene fluoride-containing diaphragm printing ink. (3) Using 3D printing technology, the anode layer, separator layer and cathode layer are printed sequentially to form a three-layer battery precursor; (4) The battery precursor is immersed in calcium chloride solution to carry out cross-linking reaction to obtain the biomimetic osteocartilage bio-battery.

4. The preparation method according to claim 3, characterized in that, The concentration of sodium alginate in the hydrogel precursor solution is (4%-6%) w / v.

5. The preparation method according to claim 3, characterized in that, The parameters for 3D printing are: pressure 2.5-4.0 bar, printing speed 8-12 mm / min, and nozzle diameter 0.2-0.3 mm.

6. The application of the biomimetic osteocartilage bio-battery according to claim 1 in the preparation of osteocartilage defect repair materials and tissue engineering scaffolds.

7. The application of the biomimetic osteocartilage biobattery according to claim 1 in the preparation of medical dressings or implants for treating articular cartilage damage or osteoarthritis.

Images