An implantable self-powered bio-battery for bone regeneration, a preparation method and application thereof

Abstract

The application discloses an implantable self-powered bio-battery for bone regeneration, a preparation method and application thereof, and belongs to the field of biomedical materials and tissue engineering. The bio-battery is composed of a degradable metal electrode and a three-dimensional porous scaffold. The degradable metal electrode comprises a magnesium-based material as an anode and a zinc-based material as a cathode. The three-dimensional porous scaffold is a polylactic acid-glycolic acid copolymer scaffold. By utilizing the electrode potential difference of magnesium and zinc, a continuous and stable electric field is spontaneously generated after being implanted in a bone defect site. The application converts chemical energy into bioavailable electric energy in situ, provides direct energy supply for the energy-deficient microenvironment of bone defects, and the magnesium and zinc ions can be completely degraded and absorbed without secondary surgery. The bio-battery exhibits excellent repair effect in animal bone defect experiments, is also effective in elderly individuals, has excellent biocompatibility and safety, and provides a new strategy for the treatment of refractory bone defects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and tissue engineering, specifically relating to an implantable self-powered bio-battery for bone regeneration, its preparation method, and its application. Background Technology

[0002] Repairing critical-sized bone defects is a major challenge in orthopedics, especially in the context of an aging society, where age-related bone defects often fail to heal due to poor regenerative capacity. Currently, while autologous bone grafting is the gold standard, it is limited by issues such as donor availability, donor site complications, and shape matching. Allogeneic bone, on the other hand, faces constraints including ethical regulations, high screening costs, and insufficient inventory. Therefore, the development of bioactive materials that can actively promote osteogenesis has become a research hotspot.

[0003] Recent studies have shown that insufficient local energy is a key factor hindering the healing of large bone defects. Bone formation is a highly energy-intensive process; osteoblasts require large amounts of ATP to synthesize collagen, secrete mineralization vesicles, and maintain ion pump activity. When injury, hypoxia, or aging impairs mitochondrial oxidative phosphorylation, a self-sustaining cycle of impairment is created, preventing bone marrow mesenchymal stem cells from completing osteogenic differentiation. Therefore, breaking this cycle by supplementing available energy may become a core therapeutic strategy.

[0004] Clinical observations have confirmed that an external electric field can accelerate bone formation through local energy conversion. However, traditional electrical stimulation devices suffer from hardware-related complications (such as wire breakage, unstable output, and poor device portability) and the need for secondary surgery for removal. While piezoelectric and triboelectric nanogenerators developed in recent years can achieve in-situ electrical stimulation, their output fluctuates with physiological activity, resulting in insufficient stability. Therefore, there is an urgent need to develop an in-situ electrical stimulation technology that can provide stable, continuous, external hardware-free, and fully degradable in-situ electrical stimulation. Summary of the Invention

[0005] Purpose of the Invention: The primary objective of this invention is to propose an implantable, self-powered bio-battery for bone regeneration. This bio-battery can directly convert chemical energy into a stable, biocompatible electric field, providing a continuous energy supply for the bone repair process.

[0006] Another object of the present invention is to provide a method for preparing the self-powered bio-battery.

[0007] Another object of the present invention is to provide the application of the self-powered bio-battery in the preparation of treatments for bone defects, osteoporotic bone defects and diseases related to promoting bone regeneration.

[0008] Technical solution: In order to achieve the above objectives, the present invention provides an implantable self-powered bio-battery for bone regeneration, wherein the self-powered bio-battery is composed of a biodegradable metal electrode and a three-dimensional porous scaffold; the biodegradable metal electrode includes a magnesium-based material as the anode and a zinc-based material as the cathode, and the three-dimensional porous scaffold is a polylactic acid-glycolic acid copolymer scaffold.

[0009] Furthermore, the three-dimensional porous scaffold is a polylactic acid-glycolic acid copolymer scaffold modified with calcium sulfate surface.

[0010] Furthermore, the magnesium-based anode is rod-shaped and coaxially disposed at the center of the support; the zinc-based cathode is filament-shaped and spirally wound around the outer periphery of the support; the anode and cathode are connected by a wire to form a closed circuit.

[0011] Furthermore, both the magnesium and zinc materials are metals with a purity of ≥99.9%, and after completing their electrochemical function, they can be completely degraded into harmless magnesium and zinc ions in vivo.

[0012] Furthermore, the electric field strength generated by the self-powered bio-battery in a physiological environment is 0.1-0.6 V, preferably 0.4 V.

[0013] Furthermore, the discharge duration exceeds 4 weeks, preferably exceeding 6 weeks.

[0014] Furthermore, the percentage of the polylactic acid-glycolic acid copolymer is 75:25.

[0015] Furthermore, the pore size of the three-dimensional porous scaffold is 200-300 µm, and the porosity is 60-70%.

[0016] The present invention discloses a method for preparing an implantable self-powered bio-battery for bone regeneration, the preparation steps of which are as follows: Step (1): Fabricate a PLGA three-dimensional porous scaffold using 3D printing technology; Step (2): The PLGA scaffold is surface activated and then immersed in a saturated calcium sulfate solution. A calcium sulfate coating is formed on the scaffold surface by ion adsorption. After cleaning and drying, a calcium sulfate-modified polylactic acid-glycolic acid copolymer scaffold is obtained. Step (3): Use a high-purity magnesium rod as the anode and insert it coaxially into the center hole of the support obtained in step (2); use a high-purity zinc wire as the cathode and spirally wind it around the outer periphery of the support; connect one end of the magnesium rod and the zinc wire to form a complete galvanic cell circuit, thus obtaining the self-powered bio-battery.

[0017] Furthermore, in step (1), the 3D printing is melt extrusion molding, and the printing temperature is 200-210℃.

[0018] Further, in step (2), the surface activation treatment involves immersing the polylactic acid-glycolic acid copolymer scaffold in a silane coupling agent solution. The silane coupling agent solution is a 2% (v / v) anhydrous toluene solution of trimethoxysilane.

[0019] Furthermore, in step (3), the purity of both the high-purity magnesium rod and the high-purity zinc wire is not less than 99.9%, the diameter of the magnesium rod is 3 mm, and the diameter of the zinc wire is 0.5 mm.

[0020] The self-powered bio-battery described in this invention is used in the preparation of treatments for bone defects, osteoporosis, or diseases that promote bone regeneration.

[0021] Furthermore, the self-powered bio-battery can promote the proliferation, migration, and osteogenic differentiation of bone marrow mesenchymal stem cells.

[0022] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. This invention is the first to propose the use of a biodegradable bio-battery based on a magnesium-zinc galvanic cell for bone repair. By converting chemical energy in situ into bioavailable electrical energy, it provides a direct "energy supply station" for the energy-deficient microenvironment of bone defects, fundamentally solving the energy barrier problem in the bone repair process.

[0023] 2. This invention uses magnesium and zinc as electrode materials. Both are essential trace elements for the human body and have excellent biocompatibility and biodegradability. After completing the electrochemical function, they can be safely degraded in the body without the need for secondary surgery to remove them, thus avoiding the complications associated with traditional electrostimulation devices.

[0024] 3. This invention constructs a PLGA scaffold using 3D printing and combines it with calcium sulfate surface modification. This not only provides mechanical support and osteogenic active ions that match cancellous bone, but also provides precise positioning and fixation for the electrodes, achieving a synergistic effect of structural support and functional electrical stimulation.

[0025] 4. The bio-battery prepared by this invention can generate a stable (approximately 0.4V) and continuous (over 6 weeks) electric field, which can significantly promote the proliferation, migration and osteogenic differentiation of bone marrow mesenchymal stem cells in vitro. In vivo, it has shown excellent repair effects on critical-size bone defect models in rats and goats, and is also effective in elderly individuals, providing a new strategy for the clinical treatment of refractory bone defects. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the bio-battery design concept of the present invention. Figure 2 This is the process of exploring the electric field strength that is most suitable for promoting BMSC proliferation, migration, and osteogenic differentiation in the embodiments of the present invention. Figure 3 This is a schematic diagram of the structure of the bio-battery in the embodiment of the present invention, and a scanning electron microscope image, thermogravimetric analysis diagram, elastic modulus diagram, and physical image of the PLGA scaffold and the scaffold modified with calcium sulfate in the embodiment of the present invention. Figure 4 These are electrochemical performance test diagrams of the bio-battery in the embodiments of the present invention; Figure 5 This is a diagram showing the in vitro experimental results of the effect of the bio-battery on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells in this embodiment of the invention. Figure 6 This is an evaluation diagram of the therapeutic effect of bio-batteries on a rat distal femoral bone defect model in an embodiment of the present invention; Figure 7 This is an evaluation diagram of the therapeutic effect and safety of bio-batteries on a goat ulna proximal bone defect model in an embodiment of the present invention. Detailed Implementation

[0027] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0028] Example 1: Exploring the appropriate electric field strength to promote BMSC proliferation, migration, and osteogenic differentiation To determine the optimal intensity range of the electric field used in this invention, an external DC electric field device was used for in vitro experiments. Bone marrow mesenchymal stem cells (BMSCs) were exposed to electric fields of 0, 50, 150, 300, and 450 mV / mm in six-well plates supplemented with DMEM medium (this part and subsequent in vitro cell experiments involved electric field stimulation for 6 hours daily). Live / dead cell staining results showed ( Figure 2 A) Cell viability was good in the 150 mV / mm group, and the cell number was significantly increased compared with the control group (0 mV / mm group), with no increase in dead cells; when the electric field strength was ≥300 mV / mm, the number of dead cells increased in a dose-dependent manner. ATP content was measured in cells after stimulation, and it was found that the ATP content of both senescent and young cells increased after stimulation with a 150 mV / mm electric field. Figure 2 C). Quantitative staining further confirmed the promoting effect of the 150 mV / mm group on cell proliferation ( Figure 2 D). Therefore, 150 mV / mm was determined as the safe upper limit. This was determined by cytoskeleton staining and Ki67 proliferation gene staining (…). Figure 2 E) This further confirms that 150 mV / mm is the optimal electric field strength for promoting BMSC proliferation. A uniform, straight scratch was created in each culture dish using a sterile pipette tip. Images of the scratch healing were taken at the initial time point (0 hours) and at regular intervals thereafter (12 and 24 hours). The experiment showed that the scratch healing area in the 150 mV / mm group was significantly higher than that in the control group at 12 hours. Figure 2I). ALP and Alizarin Red staining results both showed that the 150 mV / mm group exhibited the best cell proliferation, early osteogenic differentiation, and matrix mineralization capabilities. Figure 2 F and G), and the expression of osteogenic-related genes (ALP, OCN, OSX, Runx2) was also significantly upregulated. Figure 2 In summary, an appropriate electric field strength (150 mV / mm) can effectively promote the proliferation, migration, and osteogenic differentiation of BMSCs, providing key parameter basis for the design of the electric field strength of the bio-battery described in this invention.

[0029] Example 2: 3D Printing Fabrication of PLGA Scaffold

[0030] Polylactic acid-glycolic acid copolymer (PLGA, 75:25) particles were dried in a vacuum drying oven at 40°C for 12 hours. The dried PLGA particles were then loaded into the heated barrel of a 3D bioprinter and heated to 210°C to melt them. Using a pneumatic extrusion system, the particles were printed layer by layer through a 200 µm inner diameter stainless steel conical nozzle, following a pre-set computer model (a porous mesh structure with 0 / 90° staggered stacking). Printing parameters included: platform temperature 25°C, nozzle movement speed 2 mm / s, extrusion pressure 15 bar, and layer height 200 µm to ensure uniform filament diameter and interlayer adhesion. After printing, the particles were allowed to cool naturally to room temperature, yielding a three-dimensional porous PLGA scaffold with a pore size of 200-300 µm and a porosity of approximately 70%.

[0031] Example 3: Calcium sulfate surface modification of PLGA scaffolds

[0032] The PLGA scaffold prepared in Example 2 was immersed in an anhydrous toluene solution of 2% (v / v) silane coupling agent (trimethoxysilane was used in this example) and reacted at room temperature for 2 hours. After the reaction, the scaffold was thoroughly washed with fresh toluene and ethanol to remove unbound silane and dried at room temperature. The surface-silanized scaffold was then immersed in a saturated calcium sulfate aqueous solution and gently shaken for 24 hours. The scaffold was removed, washed with deionized water to remove loosely bound salts on the surface, and dried at room temperature for 24 hours to obtain the calcium sulfate-modified PLGA scaffold (CaPLGA). The preparation process is as follows. Figure 3 As shown in Figure A. The microstructure, surface modification effect, and mechanical properties of the implantable bio-battery scaffold were systematically characterized. Scanning electron microscopy images showed that after surface activation treatment, calcium sulfate microcrystals uniformly coated the surface of the 3D-printed PLGA scaffold struts. The coating was intact and did not block the interconnected macroporous structure inside the scaffold, indicating that this modification strategy introduced bioactive components while preserving the porous network that facilitates cell migration and tissue ingrowth. Figure 3 B). Thermogravimetric analysis results further confirmed the successful loading of calcium sulfate ( Figure 3C). Mechanical compression tests showed that both the unmodified PLGA scaffold and the calcium sulfate-modified composite scaffold maintained an elastic modulus of around 200 MPa, which is highly consistent with the mechanical properties of human cancellous bone. Furthermore, surface mineralization did not weaken the overall stiffness and structural integrity of the scaffold. Figure 3 D). Based on the above results, the scaffold meets the design requirements for bone repair materials in terms of structural biomimicry, surface bioactivity, and mechanical adaptability.

[0033] Example 4: Assembly of a bio-battery

[0034] A high-purity magnesium rod (purity ≥99.9%, diameter 3 mm) was prepared as the anode, and a high-purity zinc wire (purity ≥99.9%, diameter 0.5 mm) was prepared as the cathode. The magnesium rod was coaxially inserted into the pre-drilled hole in the center of the CaPLGA scaffold prepared in Example 3. The zinc wire was evenly and spirally wound around the outer circumferential surface of the scaffold with a height of 1.5 cm. The ends of the magnesium rod and the zinc wire were connected to ensure good electrical contact and form a closed circuit, thus obtaining the implantable self-powered bio-battery MgZn-CaPLGA. All assembly operations were performed under aseptic conditions. The assembled structure is shown below. Figure 3 As shown in E.

[0035] Example 5: Electrochemical Performance Testing of Bio-batteries

[0036] The bio-battery assembled in Example 4 was placed in 1× phosphate-buffered saline (PBS) at 37°C, and its performance was tested using an electrochemical workstation and a Newway battery testing system. Tafel polarization curves were obtained. Figure 4 A) Cyclic voltammetry (CV) curve ( Figure 4 B) LSV curve ( Figure 4 C), and electrochemical impedance spectroscopy (EIS) Figure 4 D) Test results confirmed the electrochemical activity and interfacial stability of the magnesium-zinc galvanic cell. Long-cycle discharge tests showed that the initial open-circuit voltage of this bio-battery in simulated bodily fluids was approximately 0.68 V, the stable operating voltage plateau was approximately 0.4 V (field strength 150 mV / mm), and it could sustain stable discharge for over 1124 hours (approximately 46 days). Figure 4 E). Figure 4Figures F to L systematically demonstrate the electrochemical performance evolution and electrode surface state of the Zn / / Mg bio-battery during long-term discharge in a 1×PBS simulated body fluid environment. Figures F and G present the cyclic voltammetry (CV) and linear sweep voltammetry (LSV) curves of the battery after 15, 30, and 45 days of constant current discharge, respectively. The results show that as the discharge time increases, the integrated area of ​​the CV curve gradually decreases and the reduction peak undergoes a slight negative shift, while the LSV curve shifts upward. These changes indicate a gradual increase in electrode polarization, which is attributed to the formation of a solid electrolyte interface film composed of Mg(OH)₂, ZnO, basic zinc sulfate, and phosphate on the electrode surface during long-term discharge. Figure 4 The electrochemical impedance spectroscopy of H showed that the charge transfer resistance gradually increased in the first 30 days, indicating that the protective surface film was continuously thickening. However, the impedance decreased at 45 days and was accompanied by inductive reactive behavior in the low-frequency region, suggesting that the surface film may have cracked or reconstructed, exposing a fresh metal interface and introducing diffusion restriction. Figure 4 A comparison of scanning electron microscopy results for I and J showed that the original zinc electrode surface was smooth, while the surface of the electrode after discharge was covered with sheet-like deposits and localized pores. Figure 4 XPS full-spectrum analysis of K detected significant O, P, Na, and Cl signals on both electrode surfaces, confirming the formation of oxide, hydroxide, and phosphate corrosion products. Figure 4 The chronopotential curve of L shows that even after different immersion times, the battery can still maintain a stable current output of about 63 μA, verifying the electrochemical reliability and adaptability of the bio-battery in long-term service in complex body fluid environments.

[0037] Example 6: Evaluation of in vitro cell compatibility and osteogenic activity of bio-batteries

[0038] The simplified thin-layer model of the bio-battery MgZn-CaPLGA prepared in Example 4, as well as CaPLGA, Zn-CaPLGA, and Mg-CaPLGA, were co-cultured with bone marrow mesenchymal stem cells (BMSCs). Under standard culture conditions (37°C, 5% CO2), BMSCs were seeded on different scaffold surfaces and cultured continuously for 7 days. Cell adhesion, survival, and proliferation were assessed by Calcein-AM staining. Figure 5 A). Live and dead cell staining results showed that cells co-cultured with the MgZn-CaPLGA group exhibited high viability and rapid proliferation (…). Figure 5 B). Alkaline phosphatase (ALP, Figure 5 C) and Alizarin Red (ARS, Figure 5D) Staining results showed that the MgZn-CaPLGA group most significantly promoted early osteogenic differentiation and late matrix mineralization of BMSCs. These results indicate that the bio-battery synergistically enhances the osteogenic performance of BMSCs through the electric field it generates and the active ions it releases.

[0039] Example 7: Evaluation of the therapeutic effect of bio-batteries on critical-size bone defects in the rat femur

[0040] A full-thickness femoral defect model with a diameter of 3.2 mm was established in the distal femur of 6-month-old (young) and 15-month-old (old) rats (both female SD rats, purchased from Suzhou Jingweiyu Biotechnology Co., Ltd.). The procedure involved an incision made medially to the patella to expose the distal femur under general anesthesia. The overlying muscle tissue was bluntly dissected. An incision was made lateral to the periosteum and the defect was opened. A single cortical guide hole was first drilled using a rotary drill. The initial defect was then carefully enlarged and transformed into a full-thickness, continuous bicortical defect. The final defect diameter was standardized using a 3.0 mm drill bit; precise measurements after defect formation confirmed a final diameter of 3.2 mm. In the control group, the defect was left empty after flushing; in the experimental group, the defect was filled with appropriate scaffold material according to a pre-determined grouping scheme. A scaled-down, optimized bio-battery with a diameter of 3 mm and a length of 5 mm was implanted into the defect site. The following groups were selected: an empty defect group, a bio-battery prepared in Example 4 (MgZn-CaPLGA group), a CaPLGA scaffold alone group, a single magnesium electrode group (Mg-CaPLGA), and a single zinc electrode group (Zn-CaPLGA). Finally, the muscle layer and skin were sutured sequentially. Imaging and histological evaluations were performed at 8 weeks (young group) and 12 weeks (old group) postoperatively. Results showed that in young rats, the MgZn-CaPLGA group showed better radiographic performance (X-ray imaging). Figure 6 B) and Micro-CT scan ( Figure 6 C) All experiments showed the best bone healing effect, and Micro-CT quantitative analysis ( Figure 6 D) showed that its bone volume fraction (BV / TV) and trabecular bone number (Tb.N) were significantly higher than those of other control groups, while trabecular bone separation (Tb.Sp) was significantly reduced. Histological staining (H&E and Masson, Figure 6 E and F also confirmed that this group had more mature lamellar bone formation and medullary canal recanalization. OCN fluorescence staining results of the osteogenic components also confirmed their osteogenic role. Figure 6 G). In post-implantation evaluation of aged rat models, X-ray ( Figure 6 H), Micro-CT scan ( Figure 6 I) In the experiment, Micro-CT quantitative analysis ( Figure 6 J), and histological staining (H&E and Masson, Figure 6K and L also exhibited the same experimental phenomena, indicating that this bio-battery can effectively overcome age-related energy metabolism disorders and promote bone repair.

[0041] Example 8: Evaluation of the therapeutic effect and safety of bio-batteries on critical-size bone defects in the ulna of goats.

[0042] A 1.5 cm segmental bone defect model was established in the proximal ulna of 2-year-old (young) and 10-year-old (old) goats (both purchased from Haimen Goat Breeding Base, Jiangsu Province). The method was the same as in Example 7. Corresponding 1.5 cm long and 5 mm diameter bio-cells were implanted, and imaging, histological, and blood biochemical analyses were performed 12 weeks later. CT three-dimensional reconstruction and cross-sectional scanning were also performed. Figure 7 (B and C) show that the MgZn-CaPLGA group successfully achieved bone bridging of defects in both young and old goats, and the morphology of the regenerated bone highly matched the morphology of the implanted bio-battery. Figure 7 As shown in Figure D, there were no significant differences in routine blood tests and biochemical indicators (ALT, creatinine, calcium, phosphorus, etc.) before and after implantation. Figure 7 As shown in Figure E, no significant pathological changes were observed in the tissue sections of major organs such as the heart, liver, spleen, lungs, and kidneys. These results validate the remarkable efficacy and good biosafety of the bio-battery in a large animal model, demonstrating its potential for clinical translation.

Claims

1. An implantable, self-powered bio-battery for bone regeneration, characterized in that, The self-powered bio-battery is composed of a biodegradable metal electrode and a three-dimensional porous scaffold; the biodegradable metal electrode includes a magnesium-based material as the anode and a zinc-based material as the cathode, and the three-dimensional porous scaffold is a polylactic acid-glycolic acid copolymer scaffold.

2. The self-powered bio-battery according to claim 1, characterized in that, The three-dimensional porous scaffold is a polylactic acid-glycolic acid copolymer scaffold modified with calcium sulfate surface.

3. The self-powered bio-battery according to claim 1, characterized in that, The magnesium-based anode is rod-shaped and coaxially arranged at the center of the support; the zinc-based cathode is filament-shaped and spirally wound around the outer periphery of the support; the anode and cathode are connected by a wire to form a closed circuit.

4. The self-powered bio-battery according to claim 1, characterized in that, The percentage of the polylactic acid-glycolic acid copolymer is 75:

25.

5. The self-powered bio-battery according to claim 1, characterized in that, The three-dimensional porous scaffold has a pore size of 200-300 µm and a porosity of 60-70%.

6. A method for preparing an implantable self-powered bio-battery for bone regeneration as described in any one of claims 1-5, characterized in that, Includes the following steps: Step (1): Prepare a three-dimensional porous scaffold of polylactic acid-glycolic acid copolymer using 3D printing technology; Step (2): The polylactic acid-glycolic acid copolymer scaffold is surface activated and then immersed in a saturated calcium sulfate solution. A calcium sulfate coating is formed on the surface of the scaffold by ion adsorption. After cleaning and drying, a calcium sulfate-modified polylactic acid-glycolic acid copolymer scaffold is obtained. Step (3): Use a high-purity magnesium rod as the anode and insert it coaxially into the center pre-drilled hole of the support obtained in step (2); use a high-purity zinc wire as the cathode and spirally wind it around the outer periphery of the support; connect one end of the magnesium rod and one end of the zinc wire through a conductive connector to form a complete galvanic cell circuit, thus obtaining the self-powered bio-battery.

7. The preparation method according to claim 6, characterized in that, In step (1), the 3D printing is melt extrusion molding, and the printing temperature is 200-210℃.

8. The preparation method according to claim 6, characterized in that, In step (2), the surface activation treatment involves immersing the polylactic acid-glycolic acid copolymer scaffold in a silane coupling agent solution.

9. The use of the self-powered bio-battery as described in any one of claims 1-5 in the preparation of treatments for bone defects, osteoporosis, or diseases related to promoting bone regeneration.

10. The application according to claim 9, characterized in that, The self-powered bio-battery can promote the proliferation, migration, and osteogenic differentiation of bone marrow mesenchymal stem cells.

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