Bioelectric effect bone repair material, preparation method and application thereof

Abstract

The application discloses a bioelectric effect bone repair material and a preparation method and application thereof, relates to the field of biomedical materials, and discloses the preparation method, which comprises the following steps: obtaining piezoelectric collagen fibrils with an intact triple helix structure by purifying commercially available collagen, and dispersing the piezoelectric collagen fibrils in a solvent to obtain a dispersion liquid; mixing the dispersion liquid with an auxiliary forming material under low-temperature conditions to obtain a forming precursor; and preparing a predetermined structure through a two-dimensional or three-dimensional forming process, and then cross-linking and fixing to obtain the bone repair material. The prepared bone repair material takes piezoelectric collagen fibrils as a core component, has a two-dimensional film structure or a three-dimensional porous scaffold structure, can generate an open-circuit voltage of 1.3-1.6 V under a physiological load, realizes self-driven electric stimulation to promote bone regeneration, and has excellent biocompatibility and degradability. In a rat skull and alveolar bone defect model, the bone volume fraction reaches 33% and 27% respectively at 8 weeks after operation. The material has a wide application prospect in the field of bone defect repair and can actively induce bone repair without an external power supply.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and particularly relates to a bioelectric effect bone repair material, its preparation method and application. Background Technology

[0002] Bone defects are a common clinical condition caused by trauma, tumor resection, infection, or congenital malformations, severely impacting patients' limb function and quality of life. Large bone defects have extremely limited self-healing ability and usually require intervention with bone repair materials to restore the structural integrity and mechanical support function of the bone tissue. Currently, clinically used bone repair materials mainly include four categories: autologous bone, allogeneic bone, synthetic bone substitutes, and natural polymer-based materials. Among them, autologous bone, due to its natural osteoconductivity, osteoinductive properties, and osteoblast origin, is considered to have the best osteointegration characteristics and is the gold standard for bone defect repair. However, the clinical application of autologous bone is significantly limited by the limited availability of donor bone, the need to create a second surgical site causing secondary trauma, postoperative donor site pain, and a high risk of complications. While allogeneic bone has a relatively abundant source and avoids donor site damage associated with autologous bone transplantation, it still faces the dual risks of immune rejection and potential disease transmission. Synthetic bone substitutes, such as hydroxyapatite, tricalcium phosphate, and bioactive glass, possess good biocompatibility and osteoconductivity, providing a mineral matrix at the defect site to guide new bone ingrowth. However, these traditional synthetic materials generally lack bioactive signals that actively induce bone regeneration; their bone repair primarily relies on passive structural support and surface mineralization, making precise control over the entire bone regeneration process difficult. For patients with large bone defects or poor healing ability, they often fail to achieve ideal repair results. Natural polymer-based materials, such as collagen, gelatin, and chitosan, have received widespread research and attention as biomimetic scaffold materials in bone tissue engineering due to their highly similar chemical composition and bioactive sites to the natural extracellular matrix. However, purely natural polymer materials typically suffer from insufficient mechanical strength, a mismatch between degradation rates and bone regeneration rates, and limited bioactive functions, restricting their clinical translational potential as independent bone repair materials.

[0003] In recent years, multifunctional biomimetic materials that deeply simulate the complex microenvironment of natural bone tissue have become a research hotspot and cutting-edge direction in the field of bone repair. Of particular note is that when natural bone tissue is subjected to physiological and mechanical loads such as tension, compression, and bending, the collagen fibers within it can generate piezoelectric potentials, converting mechanical energy into electrical signals. This endogenous electrical signal is widely considered one of the key biophysical signals regulating osteoblast proliferation, migration, differentiation, and bone tissue remodeling and homeostasis. This force-to-electric conversion function of bone tissue essentially originates from the piezoelectric effect of collagen fibers in its organic matrix; the unique non-centrosymmetric triple helix structure of collagen molecules endows it with inherent shear piezoelectricity. Based on this biomimetic principle, researchers have begun to focus on developing intelligent bone repair materials with piezoelectric effects, aiming to actively promote bone regeneration through the electrical signals autonomously generated by the materials under physiological loads, thereby overcoming the limitations of traditional bone repair materials that only provide passive structural support. Current research on piezoelectric bone repair materials mainly involves synthetic piezoelectric polymers such as polyvinylidene fluoride (PVDF) and its copolymers, as well as composite material systems formed by polymers and inorganic piezoelectric ceramics such as barium titanate (BaTiO3) and zinc oxide (ZnO). However, these materials generally suffer from several unavoidable problems: synthetic piezoelectric polymers have insufficient bioactivity, making it difficult to form a strong chemical bond interface with host bone tissue, and their degradation rate in vivo is extremely slow or even negligible, posing a risk of long-term complications as long-term implants; while inorganic piezoelectric ceramics have high piezoelectric constants, their inherent brittleness, stress shielding effect due to severe mismatch between elastic modulus and natural bone tissue, potential cytotoxicity of nanoparticles, and unclear metabolic pathways for the accumulation of degradation products in vivo all pose substantial challenges to their clinical safety and efficacy as bone repair materials. Furthermore, the preparation of these material systems often involves high-temperature sintering, treatment with highly polar solvents, or complex multi-step modification processes, resulting in high process complexity, difficulty in ensuring batch-to-batch consistency, and hindering large-scale production and clinical translation.

[0004] Collagen is a core structural protein and major component of the extracellular organic matrix of bone tissue, accounting for approximately 90% of the organic matrix. It possesses naturally superior biocompatibility, biodegradability, and abundant cell recognition sites. These inherent biological properties make collagen an ideal substrate for constructing bone repair materials. More importantly, collagen is the natural source of the piezoelectric effect in bone tissue; its hierarchical ordered arrangement at the molecular and fibril scales endows bone tissue with unique endogenous mechanoelectric conversion functions. However, conventional collagen extraction and processing often lead to the destruction of its natural triple helix structure and the loss of molecular orientation, causing a significant attenuation or even complete annihilation of collagen's piezoelectric function during material molding. How to preserve or effectively restore collagen's piezoelectric function while endowing it with the mechanical support properties and structural diversity required for clinical use is a key scientific and technological bottleneck that urgently needs to be overcome in this field. Achieving an organic unity between collagen's inherent piezoelectric properties and its excellent bioactivity in material design, and constructing novel intelligent bone repair materials that combine natural bone biomimetic structures with active electrical stimulation functions, has significant clinical translational value and broad application prospects for achieving a technological leap from passive filling repair to active induced regeneration. Summary of the Invention

[0005] The technical problem this invention aims to solve is to address the shortcomings of existing bone repair materials, such as insufficient bioactive signals and lack of active bone regeneration induction function, by providing a bioelectric effect bone repair material, its preparation method, and its applications. This bone repair material uses piezoelectric collagen fibrils with a preserved triple helix structure as its core functional component. It possesses good biocompatibility, biodegradability, and piezoelectric effect, and can autonomously generate electrical signals under physiological loads. Active electrical stimulation promotes bone tissue regeneration, demonstrating broad application prospects in the field of bone repair.

[0006] To solve the above-mentioned technical problems, the present invention is achieved by including the following technical solutions.

[0007] The present invention first provides a method for preparing a bioelectric effect bone repair material, comprising the following steps: (1) repeatedly washing and centrifuging commercially available collagen to obtain piezoelectric collagen fibrils with intact triple helix structure, and uniformly dispersing them in a solvent to obtain a piezoelectric collagen fibril dispersion; (2) mixing the piezoelectric collagen fibril dispersion with an auxiliary molding material under low temperature conditions to obtain a molding precursor; (3) molding the molding precursor into a predetermined structure through a two-dimensional molding process or a three-dimensional molding process, and using a crosslinking agent for crosslinking fixation during or after molding to obtain the final product.

[0008] Further, in step (1), the solvent is water or dilute acetic acid, and the mass fraction of piezoelectric collagen fibrils in the piezoelectric collagen fibrils dispersion is 0.01%~6.5%. In step (2), the auxiliary molding material is selected from one or more of polycaprolactone, polylactic acid-glycolic acid copolymer, polyglycolic acid, poly(3-hydroxybutyrate), poly(4-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), polylactic acid, gelatin, collagen, chitosan, hyaluronic acid, and alginate; the low temperature condition is an ice-water bath condition to ensure that the piezoelectric collagen fibrils maintain their natural triple helix structure and piezoelectric activity during the composite process.

[0009] Further, in step (3), the two-dimensional molding process is selected from one or more of electrospinning, spin coating, scraping, spraying, dip coating, and casting techniques; the two-dimensional molding process may also include corona polarization treatment of the material surface after molding to further enhance the surface charge density and piezoelectric response of the material. The three-dimensional molding process is selected from one or more of ice crystal template method, freeze drying technology, supercritical drying technology, 3D printing technology, and gas foaming technology. When gas foaming technology is used, ammonium bicarbonate can be used as the foaming agent, which decomposes to produce gas at a specific temperature to form a porous structure. The crosslinking agent can be glutaraldehyde or a 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide crosslinking system. After crosslinking fixation, the process also includes a step of cleaning to remove residual crosslinking agent to ensure the biosafety of the material.

[0010] This invention also provides a bioelectric effect bone repair material prepared by any of the above-described methods. The core component of this bone repair material is piezoelectric collagen fibrils, which have a complete triple helix structure. Utilizing their inherent piezoelectricity, they generate electrical signals under physiological loads, mimicking endogenous electrical signals in biological organisms, and constructing an electrophysiological microenvironment to promote bone regeneration and repair.

[0011] Furthermore, the bone repair material has a two-dimensional or three-dimensional structure. When it is a two-dimensional structure, its macroscopic form is a membrane, fiber felt, or coating with a thickness of 0.01–1 mm; when it is a three-dimensional structure, its macroscopic form is a porous scaffold material with an average pore size of 20–500 μm and a porosity of over 85%. Under biomechanical energy simulation cycle testing conditions, this material exhibits an open-circuit voltage of 1.3–1.6 V and an open-circuit voltage of 1.0–1.4 nA / cm². 2The short-circuit current indicates its ability to efficiently convert physiological mechanical energy into electrical signals. The piezoelectric collagen fibrils have a complete triple helix structure, utilizing their inherent piezoelectricity to generate electrical signals under physiological loads to promote bone regeneration and repair. The bone repair material achieved a bone volume fraction of 30%–33% after implantation in a rat skull defect model for 8 weeks, and a bone volume fraction of 24%–27% after implantation in a rat alveolar bone defect model for 8 weeks.

[0012] The present invention also provides the application of the above-mentioned bioelectric effect bone repair material in the preparation of bone defect repair implants.

[0013] Compared with the prior art, the present invention has the following advantages and technical effects:

[0014] The bioelectric effect bone repair material in this invention constructs a multi-modal, osteoblast-inspired extracellular microenvironment, establishing a synergistic matrix microenvironment and electrophysiological microenvironment. Piezoelectric collagen fibrils, as the core functional component, retain the excellent biocompatibility and biodegradability of natural collagen while endowing the material with autonomous force-to-electric conversion capabilities. It can generate electrical signals through daily physiological loads alone, without relying on external power sources or external stimuli such as ultrasound, thus achieving self-driven bone tissue regeneration and repair.

[0015] In a rat model of critically sized skull defects, the two-dimensional bioelectric effect bone repair material of this invention significantly accelerated the formation and maturation of new bone, achieving a bone volume fraction of 33% ± 3% at 8 weeks post-surgery, compared to only 9%–10% in the blank control and control groups. It also effectively bridged the defect during the culture period. In a rat alveolar bone defect model, the three-dimensional bioelectric effect bone repair material of this invention also exhibited excellent bone regeneration capabilities, achieving a bone volume fraction of 24%–27% at 8 weeks post-surgery, significantly superior to the control group. In vitro cell experiments confirmed that this material demonstrated good regulatory effects in both the early differentiation initiation and late mineralization maturation stages of osteogenic induction. Furthermore, by setting up a control group with piezoelectric collagen fibril inactivation, it was confirmed that the bone regeneration effect was specifically mediated by the piezoelectric function of the piezoelectric collagen fibrils, rather than the non-specific bioactive effects of collagen fibrils. Attached Figure Description

[0016] 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.

[0017] Figure 1 The circular dichroism chromatogram of piezoelectric collagen fibrils;

[0018] Figure 2SEM image of piezoelectric collagen fibrils;

[0019] Figure 3 SEM images of the two-dimensional bioelectric effect bone repair materials used in the control and experimental groups;

[0020] Figure 4 Evaluation of the osteogenic differentiation performance of two-dimensional bioelectric effect bone repair materials used in the control and experimental groups;

[0021] Figure 5 The regeneration of skull defects in rats was evaluated in the blank group, control group, and experimental group.

[0022] Figure 6 SEM images of the three-dimensional bioelectric effect bone repair materials used in the control and experimental groups;

[0023] Figure 7 Evaluation of the osteogenic differentiation performance of the three-dimensional bioelectric effect bone repair materials used in the control and experimental groups;

[0024] Figure 8 The study evaluated the regeneration of alveolar bone defects in rats in the blank group, control group, and experimental group. Detailed Implementation

[0025] 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.

[0026] All raw materials used in this invention are not particularly restricted in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0027] There are no particular restrictions on the purity of any of the raw materials used in this invention. However, this invention preferably uses raw materials of analytical grade or purity commonly used in the field of chemical synthesis.

[0028] All piezoelectric collagen fibrils used in the following examples were extracted from commercially available collagen, purchased from Defulle (Nantong) Technology Co., Ltd. (Nantong, China). To remove water-soluble impurities from the collagen and purify it to obtain piezoelectric collagen fibrils with a complete triple helix structure, the collagen was diluted to 0.1 wt% with deionized water, centrifuged at 8000 rpm for 15 minutes at 4°C, and the supernatant was discarded. An equal volume of pre-cooled deionized water was added to the precipitate for resuspending, followed by centrifugation. This washing-centrifugation process was repeated until the collagen fibrils remained as a white flocculent precipitate and no obvious dissolved matter was found in the supernatant. A piezoelectric collagen fibril dispersion with a mass fraction of 6.9 wt% was finally obtained and stored in a sealed container at 4°C for subsequent use. Figure 1As shown, the characteristic positive absorption peak at 222 nm and the characteristic negative absorption peak at 198 nm in the circular dichroism spectrum confirm that the obtained piezoelectric collagen fibrils retain a complete triple helix structure, which is the molecular structural basis for its piezoelectric effect. Figure 2 As shown, the obtained piezoelectric collagen fibrils, observed by scanning electron microscopy, exhibit a typical fibrous micromorphology, with interwoven fibrils and a uniform diameter distribution.

[0029] Example 1

[0030] This embodiment provides a two-dimensional bioelectric effect bone repair material. This material uses polycaprolactone (PCL) as the base framework and piezoelectric collagen fibrils as the functional coating, exhibiting a nanofiber membrane structure. The specific preparation method is as follows:

[0031] Step (1): Prepare the electrospinning solution. At room temperature, dissolve polycaprolactone in hexafluoroisopropanol and stir until completely dissolved to prepare a polycaprolactone spinning solution with a mass fraction of 10 wt%.

[0032] Step (2): Electrospinning to prepare nanofiber membranes. The spinning solution obtained in step (1) was loaded into a 10 mL syringe, with an 18G flat-head stainless steel needle attached to the tip of the syringe, and fixed on the electrospinning machine. The spinning voltage was set to 16 kV, the receiving distance (distance from the needle tip to the surface of the receiving roller) to 15 cm, and the injection speed to 1.0 mL / h. Electrospinning was carried out for 5 hours at an ambient temperature of 25℃ and a relative humidity of 40%±5%, and polycaprolactone nanofiber membranes were collected on the aluminum foil surface of the receiving roller.

[0033] Step (3): Polarization treatment. Carefully peel the nanofiber membrane obtained in step (2) off the aluminum foil and lay it flat on the grounding electrode plate of the polarization device. The polarization medium environment is room temperature air (25℃), and the relative humidity is controlled to be ≤30%. Set the polarization voltage to 18 kV and the polarization distance (distance from the needle tip electrode to the surface of the fiber membrane) to 5 cm, and perform corona polarization treatment for 10 minutes to obtain a nanofiber membrane with a negatively charged surface.

[0034] Step (4): Preparation of piezoelectric collagen fibrillation functional coating. Take the 6.9 wt% piezoelectric collagen fibrillation dispersion prepared above and add it to 2 vol% dilute acetic acid. Stir homogenously for 30 minutes under ice-water bath conditions to ensure that the piezoelectric collagen fibrillation is fully and uniformly dispersed, and obtain a piezoelectric collagen fibrillation dispersion with a mass fraction of 10 wt%. Immerse the polarized nanofiber membrane obtained in step (3) into the piezoelectric collagen fibrillation dispersion for 1 minute, and then pull it out at a uniform speed and drain the excess liquid in an ultra-clean workbench.

[0035] Step (5): Crosslinking and fixation. The fiber membrane obtained in step (4) was suspended and laid flat on a polytetrafluoroethylene frame and placed in a sealed desiccator containing a 2.5% (v / v) glutaraldehyde aqueous solution for steam crosslinking at room temperature for 2 hours. After crosslinking, the fiber membrane was removed and transferred to a fume hood for natural evaporation and drying under room temperature airflow for 12 hours to obtain a two-dimensional bioelectric effect bone repair material, the macroscopic morphology of which is a piezoelectric collagen fibrillary nanofiber pad.

[0036] The control group consisted of pure polycaprolactone nanofiber membranes, prepared according to the same steps as described in this example, except that they were not soaked in piezoelectric collagen fibrillary solution or subjected to subsequent treatment.

[0037] The thickness of the two-dimensional bioelectric effect bone repair material prepared in this embodiment was measured to be 0.01 mm. SEM images of the bone repair materials used in the control and experimental groups are shown below. Figure 3 As shown. By Figure 3 As can be seen, the pure polycaprolactone nanofiber membrane (control group) has a smooth surface, uniform fiber thickness, and random orientation. However, after being coated with piezoelectric collagen fibrils (experimental group), a uniformly coated layer of collagen fibrils can be observed on the fiber surface, with localized adhesion and bridging structures between fibers, while maintaining good overall porosity. This microstructure preserves the high specific surface area of ​​the nanofiber membrane and endows the material surface with abundant bioactive sites.

[0038] To verify the piezoelectric function of the material prepared in this embodiment, piezoelectric collagen fibrillated nanofiber pads and pure polycaprolactone nanofiber membranes (control group) were assembled into piezoelectric nanogenerators, and their electrical performance was tested under a biomechanical energy simulation cycle testing system (applying a periodic compressive load with a frequency of 1 Hz and an amplitude of 5 mm). The test results showed that the piezoelectric collagen fibrillated nanofiber pads had an open-circuit voltage of 1.4 V and an open-circuit voltage of 1.2 nA / cm. 2 The short-circuit current was measured, while the pure polycaprolactone nanofiber membrane showed no significant electrical energy output under the same test conditions, with the output signal being comparable to the background noise level. This clearly demonstrates that the two-dimensional bioelectric effect bone repair material prepared in this embodiment possesses the ability to efficiently convert mechanical energy into electrical signals under physiological loads, providing a functional basis for subsequent active electrical stimulation to promote bone repair; while the control group lacked both mechanical energy harvesting and electrical signal conversion capabilities.

[0039] To evaluate the in vitro osteogenic differentiation performance of the materials prepared in this embodiment, rat bone marrow mesenchymal stem cells (BMSCs) were seeded onto the surfaces of piezoelectric collagen fibrillary nanofiber pads (experimental group) and pure polycaprolactone nanofiber membranes (control group), and cultured in osteogenic induction medium. The regulatory effect of the bone repair material on early osteogenic differentiation of cells was evaluated by alkaline phosphatase (ALP) staining, and the terminal mineralization level of cells was evaluated by Alizarin Red S (ARS) staining. Specific experimental results are as follows: Figure 4 As shown in the figure. The staining results show that the area and depth of ALP-positive areas in the experimental group were significantly higher than those in the control group, indicating that the piezoelectric collagen fibrillary nanofiber pads can effectively promote the early differentiation initiation of BMSCs towards osteoblasts. The ARS staining in the experimental group showed large areas of deep red mineralized nodules, while the control group only showed scattered light staining spots, indicating that the experimental group could significantly promote terminal mineralization deposition of cells. In summary, the two-dimensional bioelectric effect bone repair material prepared in this embodiment exhibits good regulatory effects in both the early differentiation initiation and late mineralization maturation stages of osteogenic induction, effectively promoting early osteogenic differentiation and late mineralization deposition of cells, demonstrating excellent osteogenic biological properties.

[0040] To clarify the causal relationship between the osteogenic promoting effect and the piezoelectric function, a supplementary control group was set up. The supplementary control group was a deactivated two-dimensional piezoelectric collagen fibrillary nanofiber pad, which was prepared according to steps (1) to (5) in this embodiment. The difference was that the piezoelectric collagen fibrillary dispersion used in step (4) was preheated in a 60°C water bath for 30 minutes to thermally denature its triple helix structure and lose its piezoelectric activity. The other steps were exactly the same.

[0041] The two-dimensional bioelectric bone repair material (experimental group), pure polycaprolactone nanofiber membrane (control group), and inactivated two-dimensional piezoelectric collagen nanofiber pad (supplementary control group) prepared in this embodiment were implanted into the skull defects of rats to evaluate their osteogenesis effects in vivo. Eight-week-old male SD rats were used in the experiment, and samples were collected at 4 weeks and 8 weeks. Three parallel samples (n=3) were set up for each group at each time point.

[0042] Specific experimental results are as follows: Figure 5 As shown. By Figure 5Micro-CT three-dimensional reconstruction images and quantitative analysis results showed that at 4 weeks post-operation, significant new bone tissue could be observed at the defect edge in the experimental group, with the new bone exhibiting a centripetal growth trend extending from the defect edge towards the center. At 8 weeks post-operation, the defect area in the experimental group had been bridged and filled by a large amount of new bone tissue, with a bone volume fraction (BV / TV) reaching 33%±3% (n=3). In contrast, at 4 weeks post-operation, only a small amount of scattered new bone formation was observed at the outermost edge of the defect in the blank group, control group, and supplementary control group. At 8 weeks post-operation, the bone volume fractions of the three groups were 9%±1%, 10%±1%, and 10%±2% (n=3), respectively, with the central defect area still filled with fibrous soft tissue and no effective bony bridging formed. The osteogenesis effect of the supplementary control group was comparable to that of the control group and significantly worse than that of the experimental group. This result strongly confirms that the bone regeneration effect of the experimental group is specifically mediated by the piezoelectric function of piezoelectric collagen fibrils, rather than the non-specific effect of collagen fibrils as bioactive molecules.

[0043] Example 2

[0044] This embodiment provides a two-dimensional bioelectric effect bone repair material, using poly(3-hydroxybutyrate) (P3HB) as the base framework and piezoelectric collagen fibrils as the functional coating, exhibiting a nanofiber membrane structure. The specific preparation method is as follows:

[0045] Step (1): At room temperature, poly(3-hydroxybutyrate) is dissolved in hexafluoroisopropanol and magnetically stirred until completely dissolved to prepare a poly(3-hydroxybutyrate) spinning solution with a mass fraction of 10 wt%.

[0046] Step (2): The spinning solution obtained in step (1) was loaded into a 10 mL syringe, connected with an 18G flat-head stainless steel needle, and fixed on an electrospinning machine. The spinning voltage was set to 16 kV, the receiving distance to 15 cm, and the injection speed to 1.0 mL / h. Electrospinning was carried out for 5 hours at an ambient temperature of 25℃ and a relative humidity of 40%±5% to obtain a poly(3-hydroxybutyrate) nanofiber membrane.

[0047] Step (3): Polarization treatment. The nanofiber membrane obtained in step (2) is laid flat on the grounding electrode plate of the polarization device. The polarization medium environment is room temperature air (25℃), and the relative humidity of the environment is controlled to be ≤30%. The polarization voltage is set to 18 kV and the polarization distance is 5 cm. Corona polarization treatment is performed for 10 minutes to obtain a nanofiber membrane with a negatively charged surface.

[0048] Step (4): Preparation of piezoelectric collagen fibrillation functional coating. Using the same method as step (4) in Example 1, a piezoelectric collagen fibrillation dispersion with a mass fraction of 10 wt‰ was prepared. The polarized nanofiber membrane obtained in step (3) was immersed in the dispersion for 1 minute and then pulled out at a uniform speed to drain off the excess liquid.

[0049] Step (5): Crosslinking fixation. Using the same method as step (5) in Example 1, crosslinking was performed with 2.5% (v / v) glutaraldehyde vapor for 2 hours, followed by natural evaporation drying in a fume hood for 12 hours to obtain the two-dimensional bioelectric effect bone repair material.

[0050] Measurements showed that the thickness of the two-dimensional bioelectric effect bone repair material prepared in this embodiment was 1 mm.

[0051] Piezoelectric functional tests showed that, under biomechanical energy simulation cycle testing conditions, the piezoelectric collagen fibrillary nanofiber pad prepared in this embodiment exhibited an open-circuit voltage of 1.3 V and an open-circuit voltage of 1.0 nA / cm². 2 The short-circuit current was measured, while the pure poly(3-hydroxybutyrate) nanofiber membrane (control group) showed no significant electrical energy output under the same test conditions. This clearly demonstrates that the two-dimensional bioelectric effect bone repair material prepared in this embodiment possesses effective mechanical energy harvesting and electrical signal conversion capabilities.

[0052] In vitro osteogenic differentiation experiments showed that the piezoelectric collagen fibrillary nanofiber pads prepared in this embodiment exhibited strong positive expression in both ALP staining (a marker of early osteogenic differentiation) and ARS staining (terminal mineralization level). The combined staining results indicate that the material in this embodiment demonstrates good regulatory effects in both the early differentiation initiation and late mineralization maturation stages of osteogenic induction, effectively promoting early osteogenic differentiation and late mineralization deposition of cells, and exhibiting excellent osteogenic biological properties.

[0053] The procedures for the in vivo osteogenic experiment were exactly the same as in Example 1. A bilateral skull defect model (5 mm in diameter) was established using 8-week-old male SD rats. The experimental groups were: the two-dimensional bioelectric effect bone repair material prepared in Example 2 (experimental group), pure poly(3-hydroxybutyrate) nanofiber membrane (control group, prepared according to steps (1) to (3) of this example), inactivated two-dimensional piezoelectric collagen fibrillary nanofiber pad (supplementary control group, the piezoelectric collagen fibrillary was pre-treated at 60°C for 30 minutes to inactivate, the remaining steps were the same as in this example), and a blank group without any implanted material. Three parallel samples (n=3) were set up for each group at each time point (4 and 8 weeks post-operation).

[0054] Experimental results showed that 8 weeks post-operation, the bone volume fraction of the group using the two-dimensional bioelectric effect bone repair material designed in Example 2 of this application (experimental group) reached 30%±4% (n=3). Micro-CT three-dimensional reconstruction images showed that the new bone exhibited a centripetal expansion growth pattern, continuously extending from the defect edge to the central region. In contrast, the bone volume fractions of the blank group, control group, and supplementary control group were only 9%±1%, 9%±1%, and 10%±1% (n=3), respectively. Only a sparse amount of new bone was observed at the edge of the defect area, and the bone regeneration effect was not significant.

[0055] Example 3

[0056] This embodiment provides a three-dimensional bioelectric effect bone repair material, prepared using freeze-drying technology. It employs gelatin as the base framework and piezoelectric collagen fibrils as the functional components, exhibiting a three-dimensional porous scaffold structure. The specific preparation method is as follows:

[0057] Step (1): Prepare gelatin solution. Dissolve gelatin powder in 2% (v / v) dilute acetic acid under stirring in a 40℃ water bath to prepare a gelatin solution with a mass fraction of 4 wt%. After complete dissolution, cool to room temperature for later use.

[0058] Step (2): Composite of piezoelectric collagen fibers and gelatin. The 6.9 wt% piezoelectric collagen fiber dispersion prepared above was added to the gelatin solution from step (1) at a mass ratio of 1:4 (piezoelectric collagen fibers to gelatin). The mixture was placed in an ice-water bath and stirred continuously at 300 rpm for 5 hours to ensure that the piezoelectric collagen fibers were fully and uniformly dispersed in the gelatin solution and formed a stable pre-crosslinked physical network.

[0059] Step (3): Chemical crosslinking and mold casting. Slowly add a 2% glutaraldehyde aqueous solution to the mixture obtained in step (2) while gently stirring, so that the final concentration of glutaraldehyde is 0.25% (v / v). Quickly dispense the mixture into polytetrafluoroethylene molds and place them in a refrigerator at 4°C for 24 hours to allow them to naturally defoam and complete the chemical crosslinking reaction.

[0060] Step (4): Freeze-drying molding. The cross-linked sample from step (3), along with the mold, was placed in a Dewar flask containing liquid nitrogen for rapid pre-freezing. After the sample was completely frozen, it was quickly transferred to an ultra-low temperature freezer at -87°C and left to stand for 3 hours to fix the ice crystal morphology. Subsequently, the frozen sample was quickly transferred to a freeze dryer and dried continuously for 48 hours under conditions of cold trap temperature -55°C and vacuum degree <10 Pa to allow the ice crystals to fully sublimate and remove all solvents from the system, thus obtaining a three-dimensional porous scaffold material morphology for bioelectric effect bone repair.

[0061] Measurements showed that the pore size of the three-dimensional bioelectric effect bone repair material prepared in this embodiment was 20 μm, and the porosity was 85%.

[0062] The control group consisted of a three-dimensional gelatin scaffold that did not contain collagen fibrils. The scaffold was prepared according to the same steps as described in this embodiment, except that piezoelectric collagen fibrils were not added to the gelatin solution.

[0063] SEM images of the bone repair materials used in the control and experimental groups are as follows: Figure 6 As shown. By Figure 6 It can be seen that the control group pure gelatin three-dimensional scaffold exhibits a relatively regular lamellar porous structure with smooth pore wall surfaces; while a large number of uniformly distributed piezoelectric collagen fibrils can be observed on the pore wall surface of the experimental group scaffold, which are attached to the gelatin pore wall surface in a fluffy manner, significantly increasing the micro-roughness and bioactive site density of the scaffold inner surface.

[0064] Piezoelectric function tests showed that, under biomechanical energy simulation cycle test conditions, the three-dimensional porous scaffold (experimental group) prepared in this embodiment had an open-circuit voltage of 1.5 V and a short-circuit current of 1.3 nA / cm², while the three-dimensional gelatin scaffold (control group) showed no significant electrical energy output under the same test conditions. This clearly demonstrates that the three-dimensional bioelectric effect bone repair material prepared in this embodiment possesses effective mechanical energy harvesting and electrical signal conversion functions.

[0065] In vitro osteogenic differentiation experiment Figure 7 The results shown are the basis. Rat bone marrow mesenchymal stem cells (BMSCs) were seeded onto three-dimensional porous scaffolds in the experimental group and three-dimensional gelatin scaffolds in the control group, respectively. After culturing in osteogenic induction medium, ALP and ARS staining were performed. The staining results show that the ALP-positive expression and ARS mineralization nodule deposition in the experimental group were significantly stronger than those in the control group. This indicates that the three-dimensional bioelectric effect bone repair material exhibits a good regulatory effect in both the early differentiation initiation and late mineralization maturation stages of osteogenic induction, effectively promoting early osteogenic differentiation and late mineralization deposition, demonstrating excellent osteogenic biological properties.

[0066] The supplementary control group was set as a deactivated three-dimensional piezoelectric collagen fibril porous scaffold, which was prepared according to steps (1) to (4) of this embodiment. The difference is that the piezoelectric collagen fibril dispersion added in step (2) was preheated in a 60°C water bath for 30 minutes to make it lose its piezoelectric activity.

[0067] In vivo osteogenic experiments were conducted using a rat alveolar bone defect model. The pure gelatin three-dimensional porous scaffold described above served as the control group, while the inactivated three-dimensional bioelectric effect bone repair material served as a supplementary control group. Samples were collected at 4 and 8 weeks, respectively. Three parallel samples (n=3) were set up for each group at each time point.

[0068] Specific experimental results are as follows: Figure 8 As shown. By Figure 8 Micro-CT three-dimensional reconstruction images and quantitative analysis results showed that, 8 weeks post-operation, the three-dimensional bioelectric effect bone repair material designed in Example 3 of this application (experimental group) had a significant bone regeneration promoting capacity. A large amount of new bone tissue ingrowth was observed in the defect area, forming a good bony bridge with the host bone margin, with a bone volume fraction reaching 27% ± 7% (n=3). In contrast, the blank group, control group, and supplementary control group had very limited effects on new bone formation; most of the defect area remained empty or filled with fibrous tissue. The bone volume fractions of the three groups were only 10% ± 2%, 9% ± 1%, and 10% ± 3% (n=3), respectively, indicating poor bone repair capacity.

[0069] Example 4

[0070] This embodiment provides a three-dimensional bioelectric effect bone repair material, prepared using 3D printing technology. It uses gelatin as the base material and piezoelectric collagen fibers as the functional components, forming a pre-designed three-dimensional porous scaffold structure. The specific preparation method is as follows:

[0071] Step (1): The 6.9 wt% piezoelectric collagen fibrillation dispersion prepared above was homogenized and stirred for 30 minutes in an ice-water bath using a high-speed homogenizer to uniformly disperse it in 2 vol% dilute acetic acid to obtain the piezoelectric collagen fibrillation dispersion.

[0072] Step (2): Add gelatin powder to the piezoelectric collagen fiber dispersion from step (1), and continuously stir mechanically in an ice-water bath until completely dissolved and mixed evenly to obtain a uniform mixed solution with a total mass fraction of 5 wt% of piezoelectric collagen fiber and gelatin, which can be used as 3D printing ink.

[0073] Step (3): 3D printing. The printing ink prepared in step (2) is loaded into the printing cartridge and 3D printed on a low-temperature printing platform at 4℃. The printing parameters are set as follows: needle inner diameter is 300 μm, extrusion pressure is 40 psi, printing speed is 10 mm / s, layer height is 0.3 mm, and the printing path adopts an alternating 0° / 90° grid filling pattern to build the preset three-dimensional porous scaffold structure layer by layer to obtain the scaffold preform.

[0074] Step (4): Crosslinking and post-treatment. The preform of the scaffold printed in step (3) was placed in a sealed container containing an aqueous solution of glutaraldehyde and crosslinked with glutaraldehyde vapor at room temperature for 12 hours. After crosslinking, the scaffold was removed, frozen overnight in a -20°C freezer, and then transferred to a freeze dryer. It was dried for 24 hours under conditions of -55°C in a cold trap and a vacuum degree <10 Pa to completely remove residual moisture from the scaffold, thus obtaining a three-dimensional porous scaffold material with bioelectric effect bone repair.

[0075] Measurements showed that the pore size of the three-dimensional bioelectric effect bone repair material prepared in this embodiment was 200 μm, the porosity was 90%, and the scaffold had a regularly interconnected macroporous structure and good overall permeability.

[0076] Piezoelectric function tests showed that, under biomechanical energy simulation cycle testing conditions, the three-dimensional porous scaffold (experimental group) prepared in this embodiment had an open-circuit voltage of 1.6 V and an open-circuit voltage of 1.3 nA / cm². 2 The short-circuit current of the three-dimensional gelatin scaffold (control group, prepared by referring to steps (1) to (4) of this embodiment, the difference being that piezoelectric collagen fibrils were not added in step (1) but replaced with an equal volume of 2 vol% dilute acetic acid) under the same test conditions showed no significant power output.

[0077] In vitro osteogenic differentiation experiments showed that the three-dimensional porous scaffold prepared in this embodiment exhibited strong positive expression in both ALP and ARS staining. The combined staining results indicate that the three-dimensional bioelectric effect bone repair material showed good regulatory effects in both the early differentiation initiation and late mineralization maturation stages of osteogenic induction, effectively promoting early osteogenic differentiation and late mineralization deposition of cells, demonstrating excellent osteogenic biological properties.

[0078] The supplementary control group was set as a deactivated three-dimensional piezoelectric collagen fibril porous scaffold, which was prepared according to steps (1) to (4) of this embodiment. The difference is that the piezoelectric collagen fibril dispersion used in step (1) was preheated in a 60°C water bath for 30 minutes to make it lose its piezoelectric activity.

[0079] In vivo osteogenic experiments were conducted using a rat alveolar bone defect model, with the experimental procedures and group settings identical to those in Example 3. Micro-CT quantitative analysis at 8 weeks post-surgery showed that the three-dimensional bioelectric effect bone repair material designed in Example 4 of this application (experimental group) significantly outperformed the blank group, control group, and supplementary control group in promoting bone regeneration. At 8 weeks post-surgery, the bone volume fraction in the experimental group reached 24% ± 5% (n=3), with good new bone filling in the defect area and significant bone repair effect; while the bone volume fraction in the blank group, control group, and supplementary control group remained at a low level, with very limited impact on new bone formation and poor bone repair capacity.

[0080] Example 5

[0081] This embodiment provides a three-dimensional bioelectric effect bone repair material, prepared using gas foaming technology. It uses gelatin as the base material and piezoelectric collagen fibers as the functional components, exhibiting a three-dimensional porous scaffold structure. The specific preparation method is as follows:

[0082] Step (1): The 6.9 wt% piezoelectric collagen fibrillation dispersion and gelatin solution prepared above were mixed at a mass ratio of piezoelectric collagen fibrillation to gelatin of 3:1. The mixture was mechanically stirred in an ice-water bath to obtain a homogeneous premix. A 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide (EDC / NHS) crosslinking system was added to the premix, wherein the final concentration of EDC was 10 mM and the final concentration of NHS was 5 mM. The pre-crosslinking reaction was carried out under ice-water bath conditions with continuous stirring for 15 minutes.

[0083] Step (2): Add ammonium bicarbonate powder as a foaming agent to the pre-crosslinked mixture from step (1). The amount of ammonium bicarbonate added is 0.3 g per 10 mL of mixture. After rapid and uniform stirring, immediately pour the mixture into a polytetrafluoroethylene mold, and then transfer the mold to a 70℃ oven and let it stand for 15 minutes. At 70℃, ammonium bicarbonate rapidly decomposes upon heating to produce carbon dioxide and ammonia gas. The gas expands in situ in the mixture to form a large number of bubbles. At the same time, the EDC / NHS crosslinking system accelerates the in-situ crosslinking and curing reaction under the heating condition, locking in the foam structure.

[0084] Step (3): Cleaning and post-treatment. The solidified foam scaffold from step (2) was removed from the mold and first immersed in a 1 M glycine solution for 30 minutes to seal unreacted EDC / NHS active groups and neutralize residual alkaline substances. Then, it was repeatedly soaked and cleaned three times with a large amount of deionized water, changing the water each time for 2 hours to ensure thorough removal of residual cross-linking byproducts and salts. After cleaning, the scaffold was frozen overnight at -20°C and finally transferred to a freeze dryer. It was continuously dried for 48 hours under conditions of -55°C cold trap temperature and <10 Pa vacuum to fully dehydrate, thus obtaining a three-dimensional porous bioelectric effect bone repair material.

[0085] Measurements showed that the pore size of the three-dimensional bioelectric effect bone repair material prepared in this embodiment was 500 μm, the porosity was 90%, the scaffold exhibited a highly interconnected open-cell foam structure, and abundant through windows were formed between the walls of the large pores.

[0086] Piezoelectric function tests showed that, under biomechanical energy simulation cycle testing conditions, the three-dimensional porous scaffold (experimental group) prepared in this embodiment had an open-circuit voltage of 1.5 V and an open-circuit voltage of 1.4 nA / cm². 2 The short-circuit current of pure gelatin three-dimensional scaffold (control group, prepared by referring to steps (1) to (3) of this embodiment, the difference being that piezoelectric collagen fibrils were not added to the premixed liquid in step (1)) showed no significant power output under the same test conditions.

[0087] In vitro osteogenic differentiation experiments showed that the three-dimensional porous scaffold prepared in this embodiment exhibited strong positive expression in both ALP and ARS staining. The combined staining results indicate that the three-dimensional bioelectric effect bone repair material showed good regulatory effects in both the early differentiation initiation and late mineralization maturation stages of osteogenic induction, effectively promoting early osteogenic differentiation and late mineralization deposition of cells, demonstrating excellent osteogenic biological properties.

[0088] The supplementary control group was set as a deactivated three-dimensional piezoelectric collagen fibril porous scaffold, which was prepared according to steps (1) to (3) of this embodiment. The difference is that the piezoelectric collagen fibril dispersion used in step (1) was preheated in a 60°C water bath for 30 minutes to make it lose its piezoelectric activity.

[0089] In vivo osteogenic experiments were conducted using a rat alveolar bone defect model, with the experimental procedures and group settings identical to those in Example 3. Micro-CT quantitative analysis at 8 weeks post-surgery showed that the three-dimensional bioelectric effect bone repair material designed in Example 5 of this application (experimental group) exhibited excellent bone regeneration-promoting properties. At 8 weeks post-surgery, the bone volume fraction was 25% ± 5% (n=3), demonstrating significant bone defect repair. In contrast, the bone volume fraction in the blank group, control group, and supplementary control group remained at a low level, indicating weak promotion of new bone formation and significantly insufficient bone repair capacity.

[0090] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a bioelectrically effective bone repair material, characterized in that, Includes the following steps: (1) Commercially available collagen was repeatedly washed and centrifuged to obtain piezoelectric collagen fibrils with intact triple helix structure, and then uniformly dispersed in a solvent to obtain a piezoelectric collagen fibril dispersion. (2) The piezoelectric collagen fibrillation dispersion and the auxiliary molding material are mixed evenly under low temperature conditions to obtain the molding precursor; (3) The molding precursor is made into a predetermined structure by a two-dimensional molding process or a three-dimensional molding process, and cross-linking agent is used to fix it during or after molding to obtain the desired structure.

2. The preparation method according to claim 1, characterized in that, In step (1), the solvent is water or dilute acetic acid, and the mass fraction of piezoelectric collagen fibrils in the piezoelectric collagen fibrils dispersion is 0.01%~6.5%.

3. The preparation method according to claim 1, characterized in that, In step (2), the auxiliary molding material is selected from one or more of polycaprolactone, polylactic acid-glycolic acid copolymer, polyglycolic acid, poly(3-hydroxybutyrate), poly(4-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), polylactic acid, gelatin, collagen, chitosan, hyaluronic acid, and alginate; the low temperature condition is an ice-water bath condition.

4. The preparation method according to claim 1, characterized in that, In step (3), the two-dimensional molding process is selected from one or more of electrospinning technology, spin coating technology, scraping coating technology, spraying technology, dip coating technology and casting technology. The two-dimensional molding process also includes corona polarization treatment on the material surface after molding.

5. The preparation method according to claim 1, characterized in that, In step (3), the three-dimensional molding process is selected from one or more of the following: ice crystal template method, freeze drying technology, supercritical drying technology, 3D printing technology and gas foaming technology.

6. A bioelectric effect bone repair material, characterized in that, It is prepared by the preparation method described in any one of claims 1-5.

7. The bioelectric effect bone repair material according to claim 6, characterized in that, When the bone repair material is a two-dimensional structure, its macroscopic form is a membrane, fiber felt, or coating with a thickness of 0.01~1 mm; when the bone repair material is a three-dimensional structure, it is a porous scaffold with an average pore size of 20~500 μm and a porosity of over 85%.

8. The bioelectric effect bone repair material according to claim 6, characterized in that, The bone repair material exhibits an open-circuit voltage of 1.3–1.6 V and an open-circuit voltage of 1.0–1.4 nA / cm² under biomechanical energy simulation cycle testing conditions. 2 The short-circuit current.

9. The bioelectric effect bone repair material according to claim 6, characterized in that, The piezoelectric collagen fibrils have a complete triple helix structure and utilize their inherent piezoelectricity to generate electrical signals under physiological loads to promote bone regeneration and repair. The bone repair material achieved a bone volume fraction of 30% to 33% after 8 weeks of implantation in a rat skull defect model and a bone volume fraction of 24% to 27% after 8 weeks of implantation in a rat alveolar bone defect model.

10. The use of a bioelectric effect bone repair material as described in any one of claims 6-9 in the preparation of bone defect repair implants.

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