Electroactive bone repair membrane material with imitated bone unit structure as well as preparation method and application of electroactive bone repair membrane material

Abstract

The invention discloses an electroactive bone repair membrane material with a bone-imitating unit structure as well as a preparation method and application of the electroactive bone repair membrane material. Research shows that the bioactivity of the bone repair membrane material can be improved by forming a bone-imitating unit structure on the electroactive bone repair membrane material. The electroactive bone repair membrane material with the imitated bone unit structure can effectively promote mesenchymal stem cell differentiation and up-regulate expression of tissue repair related genes, and can promote vascularized bone regeneration in vivo through an osteogenesis-vascularization synergistic effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and particularly relates to an electroactive bone repair membrane material with a bone unit structure and a preparation method and application thereof. BACKGROUND

[0002] Bone defect repair is a major clinical challenge in the fields of oral medicine, orthopedics, and craniofacial surgery. An ideal bone repair not only requires an implant material to fill the physical space, but also needs to actively guide or induce bone tissue regeneration and establish a rich vascular network in the process, i.e., the synergistic effect of osteogenesis and angiogenesis. The generation of new bone depends on sufficient blood supply to provide nutrition and oxygen for osteoblasts; conversely, the newly formed vascular network also needs stable hard tissue support. Therefore, the development of a dual-functional biomaterial that can simultaneously promote osteogenesis and angiogenesis has become the core direction of current bone tissue engineering research.

[0003] Currently, the widely used guided tissue regeneration membranes (such as polytetrafluoroethylene membranes, collagen membranes, etc.) in clinical practice mainly serve as physical barriers to isolate soft tissue from rapidly growing into the bone defect area, thereby gaining time and space for bone tissue regeneration. However, most of these materials are biologically inert and do not have biological activity, which cannot actively stimulate cell osteogenic differentiation or angiogenesis, and the bone regeneration effect is limited.

[0004] In order to endow biomaterials with biological activity, researchers have tried various strategies, such as: combining membrane materials with growth factors (such as bone morphogenetic protein BMP-2, vascular endothelial growth factor VEGF). However, growth factors have problems such as high cost, easy inactivation in vivo, and the need for high-dose use which may cause side effects (such as ectopic ossification, inflammatory response).

[0005] In recent years, the electrical activity of biomaterials has attracted increasing attention in regulating cell behavior. It is well known that natural bone tissue is a dynamic electrical environment, for example, the skeleton generates a piezoelectric potential (i.e., piezoelectric effect) when subjected to stress, and this endogenous bioelectric signal plays a crucial role in bone remodeling and fracture healing. Studies have shown that moderate exogenous electrical stimulation can promote the proliferation, differentiation, and mineralized nodule formation of osteoblasts, and also enhance the migration and tubular structure formation of vascular endothelial cells, thereby synergistically promoting bone regeneration.

[0006] Piezoelectric polymers have shown great potential in bone tissue engineering due to their good biocompatibility, adjustable piezoelectric constant and easy processing into films. Compared with traditional bio-inert barrier membranes, piezoelectric polymer membranes can generate weak surface charges under external mechanical stress (such as muscle contraction, fluid flow or micro-vibration), simulating the bioelectric environment of natural bone, and thus are expected to achieve "active" regulation of cell behavior without external power supply. However, there are still many challenges in directly applying piezoelectric polymer membranes to bone defect repair and fully exerting their dual promotion of osteogenesis and angiogenesis: first, the piezoelectric response and bioactivity of pure P(VDF-TrFE) membranes are not enough to achieve optimal bone regeneration; second, the surface chemical properties, hydrophilicity and hydrophobicity of the piezoelectric polymer membranes are not the most conducive to cell adhesion and growth; third, how to accurately optimize the biological properties of the piezoelectric polymer membranes through material methods (such as polarization process, microstructure regulation, composite modification, etc.) is a technical problem to be solved. Therefore, it is of great significance to develop a high-performance electroactive bone repair membrane material with significant osteogenic and angiogenic activity to promote the development of electroactive bone regeneration medicine. SUMMARY

[0007] The present application is directed to the problem of poor osteogenic effect and inability to balance osteogenesis and angiogenesis of piezoelectric polymer bone repair membrane materials in the prior art. Through a large number of experimental researches, it is found that preparing piezoelectric polymer into bone repair membrane materials with bone unit structure can significantly improve the osteogenic effect of piezoelectric polymer bone repair membrane materials and has a synergistic effect of osteogenesis and angiogenesis. Specifically, the present application includes the following contents.

[0008] In a first aspect, the present application provides an electroactive bone repair membrane material with bone unit structure, wherein the bone unit structure is a micro-morphology structure on the surface of the bone repair membrane material, which comprises a central part and a plurality of annular patterns around the central part; and the bone repair membrane material comprises a piezoelectric polymer.

[0009] In some embodiments, the electroactive bone repair membrane material with bone unit structure according to the present application, wherein the diameter of the bone unit structure is 10-50 μm, and the surface of the electroactive bone repair membrane material comprises a plurality of the bone unit structures.

[0010] In some embodiments, the electroactive bone repair membrane material with bone unit structure according to the present application, wherein the piezoelectric polymer comprises at least one of polyvinylidene fluoride-based polymer, polylactic acid, polyhydroxybutyrate and natural biopolymer.

[0011] In some embodiments, the electroactive bone repair membrane material with bone unit structure according to the present application, wherein the polyvinylidene fluoride-based polymer comprises polyvinylidene fluoride, polyvinylidene fluoride Hexafluoropropylene, polyvinylidene fluoride At least one of trifluoroethylene, polyvinylidene fluoride-polyacrylic acid, poly(vinylidene fluoride-trifluorochloroethylene), and poly(vinylidene fluoride-trifluoroethylene-trifluorochloroethylene).

[0012] A second aspect of the present invention provides a method for preparing an electroactive bone repair membrane material having a bone-mimicking unit structure according to the first aspect of the present invention, comprising the following steps: (1) Provide a substrate; (2) The electroactive bone repair membrane material is obtained by spin-coating the casting liquid onto the substrate, and then subjected to high temperature treatment for 1-30 min and then cooled, thereby forming at least one bone-like unit structure on the surface of the obtained membrane material.

[0013] In some embodiments, according to the preparation method of the present invention, the substrate comprises at least one selected from polyethylene terephthalate, silicon substrate, polyethylene naphthalate, polydimethylsiloxane, metal substrate, and glass.

[0014] In some embodiments, according to the preparation method of the present invention, the spin coating speed is 4000-6000 rpm.

[0015] In some embodiments, according to the preparation method of the present invention, the cooling rate is 0.05-10°C / min.

[0016] A third aspect of the present invention provides a method for improving the bioactivity of a bone repair membrane material, comprising the step of forming a bone-like unit structure on the surface of the bone repair membrane material, the bone-like unit structure including a central portion and a plurality of annular patterns surrounding the central portion.

[0017] A fourth aspect of the present invention provides the use of an electroactive bone repair membrane material having a bone-mimicking unit structure as described in the first aspect of the present invention in the preparation of bone repair products and / or vascular repair products.

[0018] In some embodiments, according to the application described in this invention, the bone defect repair product includes bone defect repair materials for different parts such as the jawbone, skull, and long bones.

[0019] The present application has found through experimental research that the bioactivity of the electroactive bone repair membrane material can be improved by forming a bone unit structure in the electroactive bone repair membrane material. The electroactive bone repair membrane material with a bone unit structure of the present application can effectively promote the differentiation of mesenchymal stem cells and up-regulate the expression of tissue repair-related genes, and can promote vascularized bone regeneration in vivo through osteogenesis-angiogenesis synergistic effect. Moreover, the electroactive bone repair membrane material of the present application has a ring polarization characteristic, good surface potential stability, higher and more uniform negative charge distribution, and perfect matching with the physiological potential of bone, can reconstruct the electrical microenvironment of bone defect, thereby improving the bone repair effect. The electroactive bone repair membrane material with a bone unit structure of the present application breaks through the limitation of single performance optimization of traditional biological materials, and provides guidance for the development of new bone repair materials. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 AFM morphology of the surface of the electroactive bone repair membrane material of Example 1 and Comparative Example 1.

[0021] Figure 2 Expression of osteogenic differentiation specific genes of bone marrow mesenchymal stem cells promoted by the electroactive bone repair membrane material of Example 1 and Comparative Example 1 (Example 1 is significantly higher than Comparative Example 1, ***p<0.001).

[0022] Figure 3 Micro-CT results of the electroactive bone repair membrane material of Example 1 and Comparative Example 1 after repairing the critical size defect of rat skull for 12 weeks.

[0023] Figure 4 Histological staining results of the electroactive bone repair membrane material of Example 1 and Comparative Example 1 after repairing the critical size defect of rat skull for 12 weeks.

[0024] Figure 5 Expression of vascular endothelial cell marker (CD31, HIF1a, VEGF) specific genes of human umbilical vein endothelial cells (HUVEC) promoted by the electroactive bone repair membrane material of Example 1 and Comparative Example 1 (Example 1 is significantly higher than Comparative Example 1, ***p<0.001).

[0025] Figure 6 Changes in neovascularization during the process of repairing skull defects in mice using the electroactive bone repair membrane material of Example 1 and Comparative Example 1.

[0026] Figure 7 A is the piezoelectric response force microscopy (PFM) characterization results of the electroactive bone repair membrane material of Example 1 and Comparative Example 1, and B is the Kelvin probe force microscopy (KPFM) characterization results of the electroactive bone repair membrane material of Example 1 and Comparative Example 1.

[0027] Figure 8 The surface AFM morphology of the electroactive bone repair membrane material in Example 2 is shown.

[0028] Figure 9 The surface AFM morphology of the electroactive bone repair membrane material without bone-mimicking unit structure in Comparative Example 2 is shown.

[0029] Figure 10 The AFM morphology of the surface of the electroactive bone repair membrane material without bone-mimicking unit structure in Comparative Example 3 is shown.

[0030] Figure 11 The surface AFM morphology of the electroactive bone repair membrane material without bone-mimicking unit structure in Comparative Example 4 is shown.

[0031] Figure 12 This study compares the expression of osteogenic differentiation-specific genes in bone marrow mesenchymal stem cells using electroactive bone repair membrane materials with bone-mimicking unit structures from Examples 1 and 2, and electroactive bone repair membrane materials without bone-mimicking unit structures from Comparative Examples 1 and 4 (*p<0.001).

[0032] Figure 13 The expression of HUVEC markers (CD31, HIF1a, VEGF) specific genes was compared between the electroactive bone repair membrane materials with bone-mimicking unit structures in Examples 1 and 2 and the electroactive bone repair membrane materials without bone-mimicking unit structures in Comparative Examples 1 and 4 (*p<0.001).

[0033] Figure 14 To compare the expression of osteogenic differentiation-specific genes of bone marrow mesenchymal stem cells with the electroactive bone repair membrane materials without bone-mimicking unit structures in Examples 2-4 and Example 1 (*p<0.001).

[0034] Figure 15 To compare the expression of HUVEC marker (CD31, HIF1a, VEGF) specific genes of the electroactive bone repair membrane materials without bone-mimicking unit structure in Comparative Examples 2-4 with that in Example 1 (*p<0.001). Detailed Implementation

[0035] 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.

[0036] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention.

[0038] Electroactive bone repair membrane material with bone-like unit structure In one aspect, the present invention provides an electroactive bone repair membrane material having a bone-mimicking unit structure, wherein the bone-mimicking unit structure is a microstructure located on the surface of the bone repair membrane material, comprising a central portion and a plurality of annular patterns surrounding the central portion; and the bone repair membrane material comprises a piezoelectric polymer. The microstructure of the bone-mimicking unit structure is also referred to as a "concentric circle microstructure".

[0039] In this invention, bone repair refers to the repair of bone defects, which includes bone repair and cartilage repair. The bone and cartilage are not particularly limited. Examples of bone include, but are not limited to, the skull, jawbone, zygomatic bone, alveolar bone, femur, tibia, and ribs. Examples of cartilage include, but are not limited to, costal cartilage and articular cartilage.

[0040] In this invention, the diameter of the bone-like unit structure is 10-50 μm, preferably 10-48 μm, even more preferably 10-46 μm, further preferably 10-44 μm, more preferably 10-42 μm, and even more preferably 10-40 μm, for example 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40 μm, and the surface of the electroactive bone repair membrane material contains multiple bone-like unit structures.

[0041] In this invention, the piezoelectric polymer is not particularly limited, and examples include, but are not limited to, at least one of polyvinylidene fluoride polymer, polylactic acid, polyhydroxybutyrate, natural biopolymers (e.g., but not limited to collagen, chitosan, cellulose, etc.), or piezoelectric composite materials formed by the above materials and other inorganic or organic polymers (e.g., but not limited to poly(L-lactic acid / barium titanate). In a preferred embodiment, the piezoelectric polymer is a polyvinylidene fluoride polymer.

[0042] In this invention, the polyvinylidene fluoride polymer is not particularly limited, and examples include, but are not limited to, polyvinylidene fluoride and polyvinylidene fluoride. Hexafluoropropylene, polyvinylidene fluoride Trifluoroethylene, polyvinylidene fluoride-polyacrylic acid, poly(vinylidene fluoride-chlorotrifluoroethylene), poly(vinylidene fluoride-trifluoroethylene-chlorotrifluoroethylene), etc. In a preferred embodiment, the polyvinylidene fluoride polymer is polyvinylidene fluoride-trifluoroethylene.

[0043] In this invention, the thickness of the repair film is not particularly limited, but it preferably has a nanometer-scale size, preferably 50-150 nm, even more preferably 55-145 nm, further preferably 60-140 nm, more preferably 65-135 nm, and even more preferably 70-130 nm, for example 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130 nm.

[0044] Preparation method One aspect of the present invention provides a method for preparing an electroactive bone repair membrane material with a bone-mimicking unit structure as described in the present invention, comprising the following steps: (1) Provide a substrate; (2) The electroactive bone repair membrane material is obtained by spin-coating the casting liquid onto the substrate, and then subjected to high temperature treatment for 1-30 min and then cooled, thereby forming at least one bone-like unit structure on the surface of the obtained membrane material.

[0045] In this invention, the substrate is not particularly limited, and examples include, but are not limited to, polyethylene terephthalate, silicon substrate, polyethylene naphthalate, polydimethylsiloxane, metal substrate (e.g., but not limited to titanium substrate, titanium alloy substrate, aluminum foil, etc.), glass, etc. In a preferred embodiment, the substrate is polyethylene terephthalate. In another preferred embodiment, the substrate is a platinum-plated silicon substrate.

[0046] It is understandable that the high-temperature processing temperature can be adjusted accordingly when different materials are selected as the substrate. For example, when the substrate is a rigid substrate such as a silicon substrate or a metal substrate, the high-temperature processing temperature can be 250-280℃, while when the substrate is a flexible substrate such as polyimide, the high-temperature processing temperature can be 200-250℃.

[0047] In a preferred embodiment, the casting solution comprises a piezoelectric polymer and an organic solvent. The piezoelectric polymer is as described above. The organic solvent is not particularly limited, and examples include, but are not limited to, acetone, butanone, methyl ethyl ketone, cyclohexanone, and N,N... Dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, dimethyl sulfoxide, triethyl phosphate, butyrolactone, tetrahydrofuran, ethyl acetate, etc. In a preferred embodiment, the organic solvent is butanone.

[0048] In a preferred embodiment, the preparation method includes: (1) Provide a substrate and coat it with an anti-adhesive (e.g., but not limited to silicone oil, vegetable oil, etc.), dissolve an adhesive (e.g., but not limited to polyvinylpyrrolidone, sodium carboxymethyl cellulose, sodium polyacrylate, starch, gelatin, polyvinyl alcohol, etc.) in an organic solvent (e.g., but not limited to alcohols, carboxylic acids, amines, halogenated hydrocarbons, lactams, etc.) to obtain an adhesive solution, and drop it onto the substrate coated with the anti-adhesive, and lay a support film (e.g., but not limited to polyimide, polyetheretherketone, polyphenylene sulfide, polytetrafluoroethylene, polyethylene film, etc.) flat on the substrate; (2) Dissolve the piezoelectric polymer in an organic solvent to obtain a casting solution; (3) Spin-coating the casting solution onto the substrate at a speed of 4000-6000 rpm and drying; (4) After being treated at high temperature for 1-30 min, it is cooled at a rate of 0.05-10℃ / min to form at least one bone-like unit structure on the surface of the resulting membrane material.

[0049] In another preferred embodiment, the preparation method includes: (1) Provide a substrate (e.g., but not limited to polyethylene terephthalate, silicone substrate, polyethylene naphthalate, polydimethylsiloxane, metal substrate, glass, etc.); (2) Dissolve the piezoelectric polymer in an organic solvent to obtain a casting solution; (3) Spin-coating the casting solution onto the substrate at a speed of 4000-6000 rpm and drying it; (4) After being treated at high temperature for 1-30 min, it is cooled at a rate of 0.05-10℃ / min to form at least one bone-like unit structure on the surface of the resulting membrane material.

[0050] In yet another preferred embodiment, the preparation method includes: (1) Provide a substrate; (2) Dissolve 0.05 parts by weight of polyvinylidene fluoride polymer in 2.01 parts by weight (or 2.5 parts by volume) of butanone to obtain a casting solution; (3) Spin-coating the casting solution onto the substrate at a speed of 4000-6000 rpm and drying it; (4) After being treated at high temperature for 1-30 min, it is cooled at a rate of 0.05-10℃ / min to form at least one bone-like unit structure on the surface of the resulting membrane material.

[0051] To form an electroactive bone repair membrane material with a concentric microstructure resembling bone units and improve its bone repair and / or vascular repair performance, the spin coating speed must be controlled within a suitable range. Excessive speed can lead to excessive loss of the casting solution applied to the substrate, and the substrate may also detach from the spin coater. Insufficient speed can result in the casting solution not being properly spread on the substrate. Therefore, both excessively high and low speeds can lead to the casting solution on the substrate being too thin or too thick, ultimately preventing the formation of a concentric microstructure and resulting in poor bone repair and / or vascular repair performance. The spin coating speed used in this invention is 4000-6000 rpm, preferably 4100-5900 rpm, even more preferably 4200-5800 rpm, further preferably 4300-5700 rpm, more preferably 4400-5600 rpm, and even more preferably 4500-5500 rpm, for example 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500 rpm or any speed within the above range. In a preferred embodiment, the spin coating speed is 5000 rpm.

[0052] To form an electroactive bone repair membrane material with a concentric microstructure resembling bone units and improve its bone repair and / or vascular repair performance, the concentration of the casting solution can be controlled within a suitable range. In this invention, the concentration of the piezoelectric polymer in the casting solution is 0.01-0.1 g / ml, preferably 0.011-0.09 g / ml, even more preferably 0.012-0.08 g / ml, further preferably 0.013-0.07 g / ml, more preferably 0.014-0.06 g / ml, more preferably 0.015-0.05 g / ml, more preferably 0.016-0.04 g / ml, more preferably 0.017-0.03 g / ml, more preferably 0.018-0.0208 g / ml, more preferably 0.0192-0.206 g / ml, and even more preferably 0.0195-0.206 g / ml. g / ml, for example 0.0195, 0.0196, 0.0197, 0.0198, 0.0199, 0.200, 0.201, 0.202, 0.203, 0.204, 0.205, 0.206 g / ml.

[0053] To form an electroactive bone repair membrane material with a concentric microscopic morphology and a bone-like unit structure, and to improve the bone repair and / or vascular repair performance of the electroactive bone repair membrane material, it is necessary to control the high-temperature treatment temperature, time, and cooling rate within a suitable range. In this invention, the high-temperature treatment temperature is 200-300℃, preferably 205-295℃, even more preferably 210-290℃, further preferably 215-285℃, and more preferably 220-280℃, for example 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280℃, or any temperature within the above ranges. In this invention, the high-temperature time is 5-60 min, preferably 5-55 min, even more preferably 5-50 min, further preferably 5-45 min, more preferably 5-40 min, even more preferably 5-35 min, even more preferably 5-30 min, even more preferably 5-25 min, even more preferably 5-20 min, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 min. In this invention, the cooling rate is 0.05-10℃ / min, preferably 0.05-9℃ / min, even more preferably 0.05-8℃ / min, even more preferably 0.05-7℃ / min, even more preferably 0.05-6℃ / min, even more preferably 0.05-5℃ / min, for example 0.05, 0.06, 0.07, 0.08, 0.09, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5℃ / min. In a preferred embodiment, the high-temperature treatment temperature is 250°C, the high-temperature treatment time is 10 min, and the cooling rate is 1°C·min. -1 .

[0054] In this invention, the high-temperature treatment can be performed in one step or in multiple stages, and there is no particular limitation on this. For example, a high-temperature treatment can be performed for a period of time at a higher temperature within the above temperature range, followed by a high-temperature treatment for a period of time at a lower temperature within the above temperature range.

[0055] Method In one aspect, the present invention provides a method for improving the bioactivity of a bone repair membrane material, comprising the step of forming a bone-like unit structure on the surface of the bone repair membrane material, wherein the bone-like unit structure includes a central portion and a plurality of annular patterns surrounding the central portion.

[0056] In this invention, the bioactivity refers to bone repair and / or vascular repair activity. Bone repair includes at least one of the following: increased volume / area of ​​new bone; increased bone thickness; increased bone density; recovery and / or improvement of bone function; increased expression levels of osteogenic specific genes or related proteins, including but not limited to… RUNX2 , BMP-2 , OCN , OPN wait.

[0057] Application This invention further provides the application of an electroactive bone repair membrane material with a bone-mimicking unit structure in the preparation of bone repair products and / or vascular repair products. Vascular repair includes at least one of the following: an increase in at least one of the following: tubule length, number of connection points, vascular lumen volume, and number of lumens; an increase in vascular extension length or budding; formation of new blood vessels; an increase in vascular diameter; accelerated angiogenesis; increased vascular density; promotion of angiogenesis during bone defect repair; and increased expression levels of vascular regulation-related genes or the amount of related proteins. Examples of vascular regulation-related genes include, but are not limited to, those mentioned above. CD31, HIF1a, VEGF wait.

[0058] It is understood that examples of the bone repair products and / or vascular repair products of the present invention include, but are not limited to, orthopedic or dental implant materials, repair scaffolds, drug-loaded controlled-release systems, active coatings, repair devices, and other medical device products with repair functions equivalent to the repair materials of the present invention.

[0059] This invention also provides a method for enhancing the expression of osteogenic specific genes in vitro, comprising: (1) obtaining cells (e.g., but not limited to mesenchymal stem cells, dental pulp stem cells, osteoblasts, etc.); (2) performing in vitro cell expansion culture; (3) contacting the expanded cells with the electroactive bone repair membrane material with a bone-mimicking unit structure described in this invention; and (4) detecting the expression of cell-specific genes. The method is for non-therapeutic purposes, such as drug screening, drug structure optimization, osteogenic mechanism research, etc., and the specific genes include, but are not limited to, those described in this invention. RUNX2 , BMP-2 , OCN , OPN wait.

[0060] This invention further provides a method for improving the expression of specific genes in in vitro vascular endothelial cells, comprising: (1) obtaining vascular endothelial cells; (2) performing in vitro cell expansion culture; (3) contacting the expanded endothelial cells with the electroactive bone repair membrane material with a bone-mimicking unit structure described in this invention; and (4) detecting the expression of vascular endothelial cell-specific genes. The application is a non-therapeutic method, such as drug screening, drug structure optimization, angiogenesis mechanism research, etc., and the specific genes include, but are not limited to, those described above. CD31, HIF1a, VEGF wait.

[0061] In this invention, the method for detecting specific gene expression is not particularly limited, and methods known in the art can be used, including but not limited to polymerase chain reaction.

[0062] Example 1 This embodiment provides a method for preparing an electroactive bone repair membrane material with a bone-mimicking unit structure and its performance testing.

[0063] 1. Preparation method (1) First, prepare the adhesive: Dissolve 1 g of polyvinylpyrrolidone (PVP) in 50 mL of anhydrous ethanol, stir thoroughly, and then add 2-3 drops evenly to the surface of the polyethylene terephthalate (PET) release film substrate coated with silicone oil on one side.

[0064] (2) Then, a 10 mm × 10 mm high transparency polyimide (PI) film is laid flat and bonded to the PET substrate described in step (1) and interlayer air bubbles are removed.

[0065] (3) Preparation of piezoelectric functional layer: Dissolve 0.05 g of P(VDF-TrFE) 70 / 30 mol% in 2.5 mL of butanone solution, stir magnetically for 2 hours to obtain homogeneous casting solution, and then spin coat the solution onto the PI substrate surface described in step (2) using a spin coater at 5000 rpm.

[0066] (4) After the piezoelectric functional layer on the surface of the PI substrate described in step (3) is formed, it is placed in a vacuum oven at 30°C and dried for 1 hour (this step can be omitted if rapid film formation is required). Then, it is subjected to high-temperature treatment in a muffle furnace at 250°C (holding temperature for 10 min, controlling the cooling rate to be 1°C·min). -1 After the high-temperature treatment is complete, turn off the heating switch and remove the sample after it has cooled down.

[0067] 2. Performance Testing The film thickness in this embodiment is approximately 100 nm, and it was characterized using atomic force microscopy (AFM, Asylum MFP-3D). The results are as follows: Figure 1As shown in the left figure, the electroactive bone repair membrane material with a bone-mimicking unit structure in this embodiment exhibits a highly ordered concentric circle micromorphology.

[0068] The gene expression levels of the electroactive bone repair membrane material with a bone-mimicking unit structure in this embodiment were detected by qPCR 3 days after promoting osteogenic differentiation of bone marrow mesenchymal stem cells. The results are as follows. Figure 2 As shown, the gene expression levels of osteogenic transcription factor RUNX2, bone morphogenetic protein BMP2, osteocalcin OCN, and osteopontin OPN in the electroactive bone repair membrane material with a bone-mimicking unit structure in this embodiment are significantly higher than those in the control group.

[0069] MicroCT images were used to characterize the effect of the electroactive bone repair membrane material with a bone-mimicking unit structure in this embodiment on promoting bone regeneration for 2-12 weeks. The results are as follows: Figure 3 As shown, the electroactive bone repair membrane material with a bone-mimicking unit structure in this embodiment has a significantly superior repair effect in promoting bone tissue regeneration compared to the comparative example.

[0070] Use H E-staining and Masson staining images characterize the effect of the electroactive bone repair membrane material with a bone-mimicking unit structure of this embodiment on bone regeneration induced in rat skulls for 12 weeks. The results are as follows: Figure 4 As shown, the electroactive bone repair membrane material with a bone-mimicking unit structure in this embodiment has a significantly superior effect on promoting bone tissue regeneration and repair compared to the comparative example.

[0071] The gene expression level of the electroactive bone repair membrane material with a bone-mimicking unit structure in this embodiment was detected by qPCR 3 days after promoting angiogenesis differentiation of HUVECs. The results are as follows: Figure 5 As shown, the electroactive bone repair membrane material of this embodiment can significantly increase the expression levels of vascular endothelial cell markers (CD31, HIF1a, VEGF) compared to the comparative example.

[0072] The changes in angiogenesis during the repair of skull defects in mice were dynamically observed using an in vivo angiography (OCTA) system. Results are as follows: Figure 6 As shown, in the defect area where the P(VDF-TrFE) membrane with concentric micromorphology of this embodiment is implanted, a richer and more mature vascular network gradually forms over time (3 days to 4 weeks after the operation).

[0073] The electroactive bone repair membrane material with a bone-mimicking unit structure in this embodiment was examined using piezoelectric force microscopy and Kelvin probe force microscopy. The results are as follows: Figure 7As shown, PFM results indicate that the electroactive bone repair membrane material of this embodiment has ring polarization characteristics, and KPFM results indicate that the electroactive bone repair membrane material of this embodiment has good surface potential stability, higher and more uniform negative charge distribution, and perfectly matches the physiological potential of bone, and can reconstruct the electrical microenvironment of bone defects.

[0074] Example 2 This embodiment provides a method for preparing an electroactive bone repair membrane material with a bone-mimicking unit structure and its performance testing.

[0075] 1. Preparation method (1) Use a 10 mm × 10 mm platinum-plated silicon substrate, wipe the substrate with lens paper soaked in anhydrous ethanol, and then clean it with first deionized water, anhydrous ethanol and second deionized water in sequence, and dry it for later use.

[0076] (2) Preparation of piezoelectric functional layer: Dissolve 0.05 g of P(VDF-TrFE) 70 / 30 mol% in 2.5 mL of butanone solution, stir magnetically for 2 hours to obtain homogeneous casting solution, and spin coat the solution onto the surface of the platinum-plated silicon substrate described in step (1) at 5000 rpm using a spin coater.

[0077] (3) After the piezoelectric functional layer on the surface of the platinum-plated silicon substrate described in step (2) is formed, it is placed in a vacuum oven at 30°C and dried for 1 hour. Then, it is subjected to high-temperature treatment in a muffle furnace at 280°C (holding temperature for 10 min, controlling the cooling rate to be 1°C·min). -1 After the high-temperature treatment is complete, turn off the heating switch and remove the sample after it has cooled down.

[0078] 2. Performance Testing Characterization was performed using atomic force microscopy (AFM, Asylum MFP-3D), and the results are as follows: Figure 8 As shown, the electroactive bone repair membrane material with a bone-like unit structure in this embodiment has a thickness of approximately 100 nm and exhibits a highly ordered concentric circle microstructure.

[0079] The gene expression levels of the electroactive bone repair membrane material with a bone-mimicking unit structure in this embodiment were detected by qPCR 3 days after promoting osteogenic differentiation of bone marrow mesenchymal stem cells. The results are as follows. Figure 12 As shown, the gene expression levels of osteogenic transcription factor RUNX2, bone morphogenetic protein BMP2, osteocalcin OCN, and osteopontin OPN in the electroactive bone repair membrane material of this embodiment are significantly higher than those in the control group.

[0080] The gene expression level of HUVEC angiogenesis 3 days after the electroactive bone repair membrane material with a bone-mimicking unit structure in this embodiment was promoted by qPCR was detected. The results are as follows. Figure 13As shown, the expression levels of vascular endothelial cell markers (CD31, HIF1a, VEGF) were significantly increased compared to the control group.

[0081] Comparative Example 1 This comparative example provides a method for preparing and testing the performance of an electroactive bone repair membrane material without a bone-mimicking unit structure.

[0082] 1. Preparation method Weigh 3 g of P(VDF-TrFE) powder and add it to 21 ml of organic solvent DMF. Stir for 3 minutes. The polymer solution was obtained by vacuum degassing for 6 hours until completely dissolved. After degassing, the solution was poured onto a quartz plate and dried. The obtained P(VDF-TrFE) film was then heated to 55°C at a rate of 3.3°C / min and held for 60 minutes, followed by natural cooling to room temperature. Polarization was performed using this annealing-assisted corona polarization method with the following parameters: polarization field strength 21 kV and polarization time 30 min.

[0083] 2. Performance Testing Characterization was performed using atomic force microscopy (AFM, Asylum MFP-3D), and the results are as follows: Figure 1 As shown in the right figure, it does not exhibit a highly ordered concentric circle micromorphology and does not have a bone-like unit structure.

[0084] The gene expression levels of the electroactive bone repair membrane material without a bone-mimicking unit structure in this comparative study were detected by qPCR after promoting osteogenic differentiation of bone marrow mesenchymal stem cells for 3 days. Results are as follows: Figure 2 As shown, the gene expression levels of osteogenic transcription factor RUNX2, bone morphogenetic protein BMP2, osteocalcin, and osteopontin OPN in this comparative electroactive bone repair membrane material are significantly lower than those in the example.

[0085] MicroCT images were used to characterize the effect of the electroactive bone repair membrane material without bone-mimicking unit structure in promoting bone regeneration for 2-12 weeks in this comparative study. The results are as follows: Figure 3 As shown, the electroactive bone repair membrane material in this comparative example exhibits significantly inferior bone tissue regeneration repair effects compared to the examples.

[0086] Use H E-staining and Masson staining images characterize the effect of the electroactive bone repair membrane material without bone-mimicking unit structure in this comparative study on bone regeneration induced in rat skulls for 12 weeks. The results are as follows: Figure 4 As shown, the electroactive bone repair membrane material in this comparative example exhibits significantly inferior bone tissue regeneration repair effects compared to the examples.

[0087] The gene expression levels of HUVECs after 3 days of angiogenesis promotion using electroactive bone repair membrane material without bone-mimicking unit structure in this comparative study were detected by qPCR. Results are as follows: Figure 5 As shown, the expression levels of vascular endothelial cell markers (CD31, HIF1a, VEGF) in this comparative example were significantly lower than those in the example.

[0088] The changes in angiogenesis during the repair of skull defects in mice were dynamically observed using an in vivo angiography system. Results are as follows: Figure 6 As shown, in the defect area where the P(VDF-TrFE) membrane of this comparative example was implanted, the effect of vascular network formation was significantly worse than that of the example over time (3 days to 4 weeks post-operation).

[0089] The electroactive bone repair membrane material without bone-mimicking unit structure in this comparative example was examined using piezoelectric force microscopy and Kelvin probe force microscopy. The results are as follows: Figure 7 As shown, PFM results indicate that the electroactive bone repair membrane material of this comparative example does not have ring polarization characteristics, and KPFM results indicate that the surface potential stability of the electroactive bone repair membrane material of this comparative example is poor, with few negative charges and uneven distribution, and it cannot match the physiological potential of bone well.

[0090] Comparative Example 2 This comparative example provides a method for preparing and testing the performance of an electroactive bone repair membrane material without a bone-mimicking unit structure.

[0091] 1. Preparation method (1) First, prepare the adhesive: Dissolve 1 g of polyvinylpyrrolidone (PVP) in 50 mL of anhydrous ethanol, stir thoroughly, and then add 2-3 drops evenly to the surface of the polyethylene terephthalate (PET) release film substrate coated with silicone oil on one side.

[0092] (2) Then, a 10 mm × 10 mm high transparency polyimide (PI) film is laid flat and bonded to the PET substrate described in step (1) and interlayer air bubbles are removed.

[0093] (3) Preparation of piezoelectric functional layer: Dissolve 0.05 g of P(VDF-TrFE) 70 / 30 mol% in 2.4 mL of butanone solution, stir magnetically for 2 hours to obtain homogeneous casting solution, and then spin coat the solution onto the PI substrate surface described in step (2) using a spin coater at 5000 rpm.

[0094] (4) After the piezoelectric functional layer on the surface of the PI substrate described in step (3) is formed, it is placed in a vacuum oven at 30°C and dried for 1 hour (this step can be omitted if rapid film formation is required). Then, it is subjected to high-temperature treatment in a muffle furnace at 250°C (holding temperature for 10 min, controlling the cooling rate to be 1°C·min). -1After the high-temperature treatment is complete, turn off the heating switch and remove the sample after it has cooled down.

[0095] 2. Performance Testing Characterization was performed using atomic force microscopy (AFM, Asylum MFP-3D), and the results are as follows: Figure 9 As shown, it does not exhibit a highly ordered concentric circle micromorphology and does not have a bone-like unit structure.

[0096] The gene expression levels of the electroactive bone repair membrane material without bone-mimicking unit structure in this comparative study were detected by qPCR after promoting osteogenic differentiation of bone marrow mesenchymal stem cells for 3 days. Results are as follows: Figure 14 As shown, the gene expression levels of osteogenic transcription factor RUNX2, bone morphogenetic protein BMP2, osteocalcin OCN, and osteopontin OPN in the electroactive bone repair membrane material of this comparative example were significantly lower than those in the example.

[0097] The gene expression levels of HUVECs after 3 days of angiogenesis promotion using electroactive bone repair membrane material without bone-mimicking unit structure in this comparative study were detected by qPCR. Results are as follows: Figure 15 As shown, the expression levels of vascular endothelial cell markers (CD31, HIF1a, VEGF) were significantly lower than in the example.

[0098] Comparative Example 3 This comparative example provides a method for preparing an electroactive bone repair membrane material without a bone-mimicking unit structure and its performance testing.

[0099] 1. Preparation method (1) First, prepare the adhesive: Dissolve 1 g of polyvinylpyrrolidone (PVP) in 50 mL of anhydrous ethanol, stir thoroughly, and then add 2-3 drops evenly to the surface of the polyethylene terephthalate (PET) release film substrate coated with silicone oil on one side.

[0100] (2) Then, a 10 mm × 10 mm high transparency polyimide (PI) film is laid flat and bonded to the PET substrate described in step (1) and interlayer air bubbles are removed.

[0101] (3) Then, the piezoelectric functional layer was prepared: 0.05 g of P(VDF-TrFE) 70 / 30 mol% was dissolved in 2.6 mL of butanone solution. After obtaining a homogeneous casting solution by magnetic stirring for 2 hours, the solution was spin-coated onto the PI substrate surface described in step (2) using a spin coater at a speed of 5000 rpm.

[0102] (4) After the piezoelectric functional layer on the surface of the PI substrate described in step (3) is formed, it is placed in a vacuum oven at 30°C and dried for 1 hour (this step can be omitted if rapid film formation is required). Then, it is subjected to high-temperature treatment in a muffle furnace at 250°C (holding temperature for 10 min, controlling the cooling rate to be 1°C·min). -1 After the high-temperature treatment is complete, turn off the heating switch and remove the sample after it has cooled down.

[0103] 2. Performance Testing Characterization was performed using atomic force microscopy (AFM, Asylum MFP-3D), and the results are as follows: Figure 10 As shown, it does not exhibit a highly ordered concentric circle micromorphology and does not have a bone-like unit structure.

[0104] The gene expression levels of the electroactive bone repair membrane material without bone-mimicking unit structure in this comparative study were detected by qPCR after promoting osteogenic differentiation of bone marrow mesenchymal stem cells for 3 days. Results are as follows: Figure 14 As shown, the gene expression levels of osteogenic transcription factor RUNX2, bone morphogenetic protein BMP2, osteocalcin OCN, and osteopontin OPN in the electroactive bone repair membrane material of this comparative example were significantly lower than those in the example.

[0105] The gene expression levels of HUVECs after 3 days of angiogenesis promotion using electroactive bone repair membrane material without bone-mimicking unit structure in this comparative study were detected by qPCR. Results are as follows: Figure 15 As shown, the expression levels of vascular endothelial cell markers (CD31, HIF1a, VEGF) were significantly lower than in the example.

[0106] Comparative Example 4 This comparative example provides a method for preparing an electroactive bone repair membrane material without a bone-mimicking unit structure and its performance testing.

[0107] 1. Preparation method (1) First, prepare the adhesive: Dissolve 1 g of polyvinylpyrrolidone (PVP) in 50 mL of anhydrous ethanol, stir thoroughly, and then add 2-3 drops evenly to the surface of the polyethylene terephthalate (PET) release film substrate coated with silicone oil on one side.

[0108] (2) Then, a 10 mm × 10 mm high transparency polyimide (PI) film is laid flat and bonded to the PET substrate described in step (1) and interlayer air bubbles are removed.

[0109] (3) Then, the piezoelectric functional layer was prepared: 0.05 g of P(VDF-TrFE) 70 / 30 mol% was dissolved in 2.5 mL of butanone solution. After obtaining a homogeneous casting solution by magnetic stirring for 2 hours, the solution was spin-coated onto the PI substrate surface described in step (2) using a spin coater at a speed of 5000 rpm.

[0110] (4) After the piezoelectric functional layer on the surface of the PI substrate described in step (3) is formed, it is placed in a vacuum oven at 30°C and dried for 1 hour (this step can be omitted if rapid film formation is required). Then, it is subjected to high-temperature treatment in a muffle furnace at 180°C (holding temperature for 10 min, controlling the cooling rate to be 1°C·min). -1 After the high-temperature treatment is complete, turn off the heating switch and remove the sample after it has cooled down.

[0111] 2. Performance Testing Characterization was performed using atomic force microscopy (AFM, Asylum MFP-3D), and the results are as follows: Figure 11 As shown, it does not exhibit a highly ordered concentric circle micromorphology and does not have a bone-like unit structure.

[0112] The gene expression levels of the electroactive bone repair membrane material without bone-mimicking unit structure in this comparative study were detected by qPCR after promoting osteogenic differentiation of bone marrow mesenchymal stem cells for 3 days. Results are as follows: Figure 12 and 14 As shown, the gene expression levels of osteogenic transcription factor RUNX2, bone morphogenetic protein BMP2, osteocalcin OCN, and osteopontin OPN in the electroactive bone repair membrane material of this comparative example were significantly lower than those in the example.

[0113] The gene expression levels of HUVECs after 3 days of angiogenesis promotion using electroactive bone repair membrane material without bone-mimicking unit structure in this comparative study were detected by qPCR. Results are as follows: Figure 13 and 15 As shown, the expression levels of vascular endothelial cell markers (CD31, HIF1a, VEGF) were significantly lower than in the example.

[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An electroactive bone repair membrane material with a bone-mimicking unit structure, characterized in that, The bone-like unit structure is a microstructure located on the surface of the bone repair membrane material, which includes a central part and multiple annular patterns surrounding the central part; and the bone repair membrane material contains a piezoelectric polymer.

2. The electroactive bone repair membrane material with a bone-mimicking unit structure according to claim 1, characterized in that, The diameter of the bone-like unit structure is 10-50 μm, and the surface of the electroactive bone repair membrane material contains multiple bone-like unit structures.

3. The electroactive bone repair membrane material with a bone-mimicking unit structure according to claim 1, characterized in that, The piezoelectric polymer includes at least one of polyvinylidene fluoride polymer, polylactic acid, polyhydroxybutyrate, and natural biopolymers.

4. The electroactive bone repair membrane material with a bone-mimicking unit structure according to claim 3, characterized in that, The polyvinylidene fluoride polymer includes polyvinylidene fluoride and polyvinylidene fluoride. Hexafluoropropylene, polyvinylidene fluoride At least one of trifluoroethylene, polyvinylidene fluoride-polyacrylic acid, poly(vinylidene fluoride-trifluorochloroethylene), and poly(vinylidene fluoride-trifluoroethylene-trifluorochloroethylene).

5. The method for preparing an electroactive bone repair membrane material with a bone-mimicking unit structure according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Provide a substrate; (2) The electroactive bone repair membrane material is obtained by spin-coating the casting liquid onto the substrate, and then subjected to high temperature treatment for 1-30 min and then cooled, thereby forming at least one bone-like unit structure on the surface of the obtained membrane material.

6. The preparation method according to claim 5, characterized in that, The substrate includes at least one of polyethylene terephthalate, silicon substrate, polyethylene naphthalate, polydimethylsiloxane, metal substrate, and glass.

7. The preparation method according to claim 5, characterized in that, The spin coating speed is 4000-6000 rpm.

8. The preparation method according to claim 5, characterized in that, The cooling rate is 0.05-10℃ / min.

9. A method for improving the bioactivity of bone repair membrane materials, characterized in that, It includes the step of forming a bone-like unit structure on the surface of the bone repair membrane material, wherein the bone-like unit structure includes a central portion and a plurality of annular patterns surrounding the central portion.

10. The use of the electroactive bone repair membrane material with a bone-mimicking unit structure according to any one of claims 1-4 in the preparation of bone defect repair products and / or vascular repair products; Preferably, the bone defect repair product includes bone defect repair materials for different parts such as the jawbone, skull, and long bones.

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