An electroactive fiber membrane for promoting osteogenesis and methods of making and using the same

Abstract

The application discloses an electroactive fiber membrane for promoting osteogenesis and a preparation method and application thereof. Experimental results show that the electroactive fiber membrane can directly promote macrophages to polarize to an anti-inflammatory M2 phenotype, and can inhibit the inflammatory induction of lipopolysaccharide (LPS) on macrophages, so as to realize the transformation of macrophages from a pro-inflammatory M1 phenotype to an M2 phenotype, and has the effects of inhibiting inflammation and promoting tissue regeneration. Meanwhile, the improvement of the electroactivity of the fiber membrane is beneficial to promoting an electro-physiological microenvironment for osteogenesis, and the fiber membrane can also adsorb cationic substances such as calcium ions in the environment, which is conducive to the occurrence of biological mineralization, so that the fiber membrane can promote osteogenesis.

Description

Technical Field

[0001] This invention belongs to the field of medical materials technology, and relates to an electroactive fiber membrane for promoting osteogenic formation, its preparation method and application. Technical Background

[0002] The most fundamental function of guided tissue regeneration membranes is to repair damaged alveolar bone tissue. This requires materials that can stimulate stem cells to differentiate and mature into osteoblasts, forming new bone tissue. Among the many biomaterials used in bone regeneration, piezoelectric materials have attracted increasing attention from researchers. This is because natural bone tissue possesses piezoelectric properties, and the implantation of piezoelectric materials can mimic the piezoelectric effect of bone, reconstructing the local electrophysiological microenvironment and promoting bone tissue regeneration. Moreover, compared to other electroactive materials, piezoelectric materials do not require an external power source; they can generate electroactivity only under stimulation such as movement or pressure, making them very convenient for patients.

[0003] As research into bone regeneration deepens, it has been discovered that immune cells play a crucial role in this process, and bone immune regulation has become another way to promote bone regeneration. Besides the pro-inflammatory M1 phenotype and the anti-inflammatory M2 phenotype, macrophages can also form multinucleated osteoclasts, which mainly participate in the later stages of bone healing and are responsible for bone resorption. Premature osteoclast formation and high expression of osteoclast factors can interfere with the bone repair process; therefore, inhibiting osteoclast formation is of great significance for bone regeneration. Summary of the Invention

[0004] To overcome the limitations of poly-L-lactic acid (PLA) in terms of hydrophobicity and insufficient cell affinity, this invention prepares an electroactive fiber membrane for promoting osteogenic growth. This membrane not only possesses improved hydrophilicity and biocompatibility but also enhances electroactivity by activating the piezoelectricity of the fiber membrane material through corona polarization treatment, thereby enriching the positive and negative charges on the two surfaces of the material. The negatively charged surface of the electroactive fiber membrane is then directed towards the bone defect area, thereby exerting its role in bone immunomodulation and promoting osteogenic growth. Specifically, this invention provides the following:

[0005] In a first aspect, the present invention provides an electroactive fiber membrane for promoting osteogenic formation, which is formed by electrospinning a mixture of poly-L-lactic acid and natural polymers, wherein the electroactive fiber membrane is subjected to corona polarization treatment to enrich the side surface of the electroactive fiber membrane with charge.

[0006] The electroactive fiber membrane provided by this invention, after being treated with corona polarization, has an accumulation of negative charge on its inner side, which is more conducive to promoting osteogenic differentiation of cells.

[0007] In one specific embodiment of the present invention, the electroactive fiber membrane is formed by electrospinning a mixture of poly-L-lactic acid and a natural polymer. The spinning parameters are not particularly limited; those skilled in the art will understand that the fiber morphology can be controlled by adjusting the spinning parameters to change the fiber diameter, porosity, fiber arrangement, etc., to meet different application requirements.

[0008] The electroactive fiber membrane provided by this invention can have a random or parallel arrangement after electrospinning, without particular limitation. Asymmetrical distribution of positive and negative charges on both sides of the fiber membrane is achieved through corona polarization treatment, with negative charges enriched on the inner side of the fiber layer, thereby promoting osteogenic differentiation and better meeting the clinical needs of electroactive fiber membranes. The distribution of positive and negative charges can be determined using methods known in the art, without particular limitation; for example, it can be easily determined based on the electrode direction during polarization.

[0009] As used in this invention, the term "inner side" refers to the side of the electroactive fiber membrane that is away from the gingival tissue or faces the alveolar bone tissue, and this side is close to the alveolar bone tissue and plays a role in promoting bone growth. The term "outer side" refers to the side of the electroactive fiber membrane that is relative to or faces the gingival tissue.

[0010] In one specific embodiment of the present invention, the weight ratio of poly-L-lactic acid to natural polymer is 1:9-9:1. For example, the mass ratio of poly-L-lactic acid to natural polymer is 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, etc. The ratio of poly-L-lactic acid to natural polymer should not be too large or too small; too small a ratio will affect film formation, while too large a ratio will affect the electrical properties of the resulting fiber membrane.

[0011] In this invention, the organic solvent is not particularly limited; any organic solvent suitable for use in this invention can simultaneously dissolve poly-L-lactic acid and natural polymers. In a preferred embodiment, the organic solvent is trifluoroethanol.

[0012] In one specific embodiment of the present invention, the natural polymer is one of collagen and gelatin.

[0013] In one specific embodiment of the present invention, the natural polymer is gelatin. Preferably, the gelatin has a gel strength of 100-400 g Bloom, more preferably 150-300 g Bloom, and even more preferably 200-280 g Bloom, such as 200, 220, 230, 240, 250, 260, 270, or 280 g Bloom. The gel strength of the gelatin should not be too high or too low; too low a value will affect film formation, while too high a value will affect the electrical properties of the resulting composite film.

[0014] In one specific embodiment of the present invention, the concentration of the poly-L-lactic acid solution is 0.05-0.4 g / mL, preferably 0.08-0.4 g / mL, for example 0.08, 0.1, 0.2, 0.3, 0.4 g / mL.

[0015] In one specific embodiment of the present invention, the concentration of the natural polymer solution is 0.05-0.4 g / mL, preferably 0.08-0.4 g / mL, for example 0.08, 0.1, 0.2, 0.3, 0.4 g / mL.

[0016] In one specific embodiment of the invention, the total mass ratio of the poly-L-lactic acid and the natural polymer to the volume of trifluoroethanol is 5-40%. For example, the total mass-to-volume percentage concentration of the poly-L-lactic acid and gelatin in trifluoroethanol is 5%, 8%, 10%, 13%, 15%, 18%, or 20%, etc. Preferably, the total mass-to-volume percentage concentration of the poly-L-lactic acid and gelatin in trifluoroethanol is 10%.

[0017] In one specific embodiment of the present invention, the diameter of the fiber is 500-1500nm, preferably 800-1400nm, for example 800, 900, 1000, 1100, 1200, 1300, 1400nm.

[0018] A second aspect of the present invention provides a method for preparing an electroactive fiber membrane for promoting osteogenic formation, comprising the following steps:

[0019] (1) After mixing poly-L-lactic acid solution dissolved in organic solvent and natural polymer solution dissolved in organic solvent evenly, electrospinning is performed, and cross-linking is carried out under suitable conditions to obtain cross-linked fiber membrane.

[0020] (2) The fiber membrane is obtained by corona polarization treatment.

[0021] In the preparation method of this invention, the organic solvent is not particularly limited, and any organic solvent suitable for this invention can simultaneously dissolve poly-L-lactic acid and natural polymers. In a preferred embodiment, the organic solvent is trifluoroethanol.

[0022] In one specific embodiment of the present invention, the electrospinning process conditions are as follows: voltage of 10-30 kV, receiving distance of 10-30 cm, feed speed of 0.7-2.0 mL / h, and receiving roller rotation speed of 300-1000 rpm. Under these conditions, complete continuous fibers can be formed, and the prepared fiber layer has a smooth fiber surface, an average diameter between 500-1500 nm, and no beading.

[0023] In one specific embodiment of the present invention, the process of drying the fiber membrane prepared by electrospinning is further included before corona polarization.

[0024] In one specific embodiment of the present invention, the drying is vacuum drying.

[0025] In one specific embodiment of the present invention, the drying conditions are: constant temperature of 25-37°C, vacuum degree below 40Pa, drying for 12-48 hours.

[0026] In one specific embodiment of the present invention, the drying conditions are: constant temperature 37°C, vacuum degree 30Pa, drying for 24 hours.

[0027] In one specific embodiment of the present invention, the corona polarization voltage is 8-15kV, for example 8, 9, 10, 11, 12, 13, 14, 15kV, and the polarization time is 15-30 minutes, for example 15, 18, 20, 22, 24, 26, 28, 30 minutes.

[0028] A third aspect of the present invention provides a guided tissue regeneration composite membrane for the repair of periodontal tissue defects, comprising the electroactive fiber membrane for promoting osteogenic formation described herein. The term "composite membrane" as used herein preferably refers to a restorative material with a bilayer membrane structure, particularly a bilayer membrane structure with an asymmetric structure. Therefore, in a preferred embodiment, the electroactive fiber membrane for promoting osteogenic formation of the present invention serves as the inner monolayer fiber membrane structure of the composite membrane. In another preferred embodiment, the present invention employs an electroactive antibacterial fiber membrane as the outer monolayer fiber membrane structure of the composite membrane. In yet another preferred embodiment, the present invention uses an inner fiber layer with osteogenic properties and an electroactive fiber membrane to obtain a composite membrane material by pressing, followed by corona polarization to enrich the outer fiber membrane with positive charge and the inner fiber layer with negative charge, thereby achieving multifunctional guided tissue regeneration.

[0029] In a specific embodiment, the electroactive antibacterial fiber membrane is prepared by electrospinning a poly-L-lactic acid solution dissolved in an organic solvent. The organic solvent is not particularly limited; any organic solvent suitable for this invention can dissolve poly-L-lactic acid and natural polymers. In a preferred embodiment, the organic solvent is trifluoroethanol. Preferably, the electrospinning process conditions are: voltage of 10-30 kV, receiving distance of 10-30 cm, feed speed of 0.7-2.0 mL / h, and receiving roller rotation speed of 300-1000 rpm. Under these conditions, complete continuous fibers can be formed, and the prepared fiber layer has a smooth fiber surface, an average diameter between 500-1500 nm, and no beading. More preferably, the concentration of the poly-L-lactic acid solution is 0.05-0.4 g / mL, more preferably 0.08-0.4 g / mL, for example 0.08, 0.1, 0.2, 0.3, or 0.4 g / mL. More preferably, the electrospun fiber layer is annealed after electrospinning. The annealing process involves first annealing the electrospun fiber layer at 100-110℃ for 8-12 hours, then naturally cooling it to room temperature, and then annealing it at 155-165℃ for 8-12 hours before naturally cooling it to room temperature.

[0030] A fourth aspect of the present invention provides a method of using an electroactive fiber membrane for promoting osteogenesis, comprising the step of bringing a side of the electroactive fiber membrane having a negative charge enrichment close to and in contact with alveolar bone tissue.

[0031] A fifth aspect of the invention provides the use of an electroactive fiber membrane for promoting osteogenic formation in the preparation of a repair material that promotes tissue regeneration.

[0032] Preferably, the osteogenesis promotion includes at least one of the following:

[0033] a. Increased bone density;

[0034] b. Reduce the distance from the enamel-dentin junction to the alveolar bone ridge;

[0035] c. Promotes the formation of new bone tissue;

[0036] d. Promotes the expression of osteogenic-related genes.

[0037] A sixth aspect of the invention provides a method for regulating macrophage phenotype, comprising the step of culturing macrophages in vitro using an electroactive fiber membrane. Preferably, the electroactive fiber membrane promotes macrophage polarization toward the M2 phenotype, thereby promoting tissue regeneration. More preferably, the electroactive fiber membrane inhibits lipopolysaccharide (LPS) induction in macrophages, thereby achieving the transformation of macrophages from a pro-inflammatory M1 phenotype to an M2 phenotype.

[0038] This invention, through the determination of voltammetric (CV) curves, confirms that pre-corona polarization treatment can endow the fiber membrane with a certain degree of electroactivity. At the same time, the electroactive fiber membrane, whether positively or negatively charged, can directly promote the polarization of macrophages to the anti-inflammatory M2 phenotype and inhibit the inflammatory induction effect of LPS on macrophages, thereby promoting the transformation of macrophages from the pro-inflammatory M1 phenotype to the M2 phenotype. This indicates that the electroactive fiber membrane of this invention has the effect of inhibiting inflammation and promoting tissue regeneration.

[0039] In this invention, the increased electroactivity of the fibrous membrane is beneficial to promoting the electrophysiological microenvironment of osteogenic formation. In particular, the negatively charged surface can also adsorb cationic substances such as calcium ions in the environment, which helps to cause biomineralization and thus can play a role in improving osteogenic formation. Attached Figure Description

[0040] Figure 1 Scanning electron microscope (SEM) images (A) and fiber diameter distribution (B) of PLLA / Gel fiber membranes.

[0041] Figure 2 The current-voltage (CV) curves of the PLLA / Gel fiber membrane are shown.

[0042] Figure 3 Images showing the live / dead state staining of RAW264.7 macrophages (A) and bone marrow mesenchymal stem cells (BMSCs) on PLLA / Gel fiber membranes (B).

[0043] Figure 4 The proliferation of RAW264.7 (A) and BMSCs (B) on PLLA / Gel fiber membranes.

[0044] Figure 5 Immunofluorescence staining images of arginase-1 (Arg-1) protein (M2 phenotypic marker) expression in RAW264.7 macrophages cultured on the surface of a fiber membrane for 1 day (A) and 5 days (B).

[0045] Figure 6 This study provides a semi-quantitative analysis of the fluorescence intensity of Arg-1 protein expression in macrophages using ImageJ immunofluorescence staining.

[0046] Figure 7 Immunofluorescence staining of interleukin-6 (IL-6) protein (M1 phenotypic marker) expression in macrophages on different fibrous membrane surfaces after 24 hours of treatment with (- / +) LPS (1 μg / mL), (A)-LPS, (B)+LPS.

[0047] Figure 8This study provides a semi-quantitative analysis of the immunofluorescence intensity of IL-6 protein expression in macrophages under LPS stimulation and without using ImageJ.

[0048] Figure 9 After treating with LPS (1 μg / mL) for 24 hours, the cells were replaced with basal culture medium and cultured for another 48 hours for cytoskeleton staining.

[0049] Figure 10 Immunofluorescence staining images of IL-6 protein expression in macrophages on different fibrous membrane surfaces after 24 hours of treatment with LPS (1 μg / mL) followed by 1 day (A) and 3 days (B) of basal culture medium.

[0050] Figure 11 Immunofluorescence staining images of Arg-1 protein expression in macrophages on different fibrous membrane surfaces after treatment with LPS (1 μg / mL) for 24 hours, followed by culturing in basal medium for 1 day (A), 3 days (B), and 5 days (C).

[0051] Figure 12 This study aimed to semi-quantitatively analyze the immunofluorescence intensity of IL-6 and Arg-1 protein expression in macrophages cultured in ImageJ after LPS induction for 24 hours and subsequent replacement with basal medium.

[0052] Figure 13 Alkaline phosphatase (ALP) staining of BMSCs cultured on the surface of electroactive PLLA / Gel fiber membranes.

[0053] Figure 14 Osteogenic differentiation results of BMSCs cultured on the surface of electroactive PLLA / Gel fiber membranes: (A) Quantitative ALP test, (B) Quantitative calcium deposition test.

[0054] Figure 15 To investigate the effects of electroactive PLLA / Gel fiber membranes on the expression of osteogenic differentiation-related genes in BMSCs, (A) ALP, (B) osteopontin (OPN), (C) osteocalcin (OCN), and (D) type I collagen (COL-I).

[0055] Figure 16 Immunofluorescence staining and semi-quantitative analysis of OPN expression in BMSCs cultured on the surface of electroactive PLLA / Gel fiber membranes.

[0056] Figure 17 Immunofluorescence staining and semi-quantitative analysis of OCN expression in BMSCs cultured on the surface of electroactive PLLA / Gel fiber membranes.

[0057] Figure 18 The expression of inflammation-related genes in mouse gingival crevicular fluid is shown in the figures: (A) IL-1β, (B) IL-6, and (C) IFN-γ.

[0058] Figure 19 Micro-CT model of mouse maxillary alveolar bone reconstructed: (A) 2D; (B) 3D.

[0059] Figure 20 Micro-CT quantitative analysis of the maxillary alveolar bone in mice: (A) bone mineral density (BMD), (B) distance from the alveolar ridge to the enamel-dentin junction (CEJ-ABC). Detailed Implementation

[0060] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art described herein.

[0061] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0062] Example 1

[0063] 1 g of poly-L-lactic acid (molecular weight 100,000) was dissolved in 10 mL of trifluoroethanol and magnetically stirred at 37 °C for 24 hours until the poly-L-lactic acid was completely dissolved, yielding a poly-L-lactic acid solution with a concentration of 0.1 g / mL. 1 g of gelatin (glucopyranosity 250 g Bloom) was dissolved in 10 mL of trifluoroethanol and magnetically stirred at 37 °C for 24 hours, yielding a gelatin solution with a concentration of 0.1 g / mL. The poly-L-lactic acid and gelatin solutions were mixed and magnetically stirred until homogeneous before electrospinning. The polymer solution was loaded into a sterile syringe, which was fixed to an electrospinning injection pump. Electrospinning was performed using a stainless steel roller as the receiving device. The electrospinning voltage was set to 15 kV, the injection rate to 0.8 mL / h, the roller speed to 400 rpm, and the receiving distance to 15 cm. Continuous spinning was used to obtain a fiber membrane, which was then dried in a vacuum oven at 37 °C for 24 hours.

[0064] Since the prepared fiber membrane contains water-soluble gelatin, cross-linking treatment is required to prevent premature dissolution and leaching during later cell culture and use, which could affect the membrane structure. In this embodiment, a chemical cross-linking method is used. The solvent for the cross-linking solution is a mixture of ethanol and water, prepared at a volume ratio of 9:1. The cross-linking agents are 1,3-dimethylaminopropyl-3-ethyldiimide (EDC) and N-hydroxysuccinimide (NHS), which are added to the prepared mixed solution at a molar ratio of 2.5:1 and dissolved completely. The fiber membrane is then placed in the prepared cross-linking solution and cross-linked at 4°C for 12 hours. Subsequently, it is rinsed three times with deionized water and freeze-dried to obtain the PLLA / Gel fiber membrane.

[0065] The cross-linked PLLA / Gel fiber membrane was subjected to corona polarization treatment for 30 minutes and named P-PLLA / Gel. In in vitro cell experiments, positively charged enriched surfaces (PLLA / Gel-P) and negatively charged enriched surfaces (PLLA / Gel-N) were used for culturing.

[0066] Example 2

[0067] 1 g of poly-L-lactic acid (molecular weight 100,000) was dissolved in 10 mL of trifluoroethanol and magnetically stirred at 37 °C for 24 hours until the poly-L-lactic acid was completely dissolved, yielding a poly-L-lactic acid solution with a concentration of 0.1 g / mL. The poly-L-lactic acid solution was loaded into a sterile syringe, which was fixed to a constant flow pump. Electrospinning was performed using a stainless steel roller as the receiving device. The electrospinning voltage was set to 15 kV, the injection rate to 0.8 mL / h, the roller speed to 400 rpm, and the receiving distance to 15 cm. Continuous spinning was used to obtain an R-PLLA fiber membrane with randomly arranged fibers.

[0068] The randomly arranged R-PLLA fiber membrane was dried in a vacuum oven at 37°C for 24 hours. The dried R-PLLA fiber membrane was then annealed in a muffle furnace at 105°C for 10 hours, allowed to cool naturally to room temperature, and then annealed again at 160°C for 10 hours. After cooling naturally to room temperature, the fully annealed F-PLLA fiber membrane was obtained.

[0069] R-PLLA and F-PLLA fiber membranes were composited with unpolarized PLLA / Gel fiber membranes by pressing. The composite membranes were obtained by pressing at 5 MPa for 30 seconds using a tablet press and named R / Gel composite fiber membrane and F / Gel composite fiber membrane, respectively. Subsequently, the bilayer composite membranes were electroactively polarized. By adjusting the orientation of the bilayer membranes in the polarizing electric field, the outer surface of the poly-L-lactic acid cellulose membrane was enriched with positive charge, while the outer surface of the PLLA / Gel fiber membrane was enriched with negative charge, maximizing the effect of the bilayer membranes. The electroactive bilayer fiber membranes obtained after corona polarization were named R / Gel-P composite fiber membrane and F / Gel-P composite fiber membrane, respectively.

[0070] Test Example 1

[0071] In this embodiment, the physicochemical properties of the prepared fiber membrane were characterized, as detailed below.

[0072] I. Experimental Methods

[0073] SEM analysis: A 5mm×5mm fiber membrane was taken, and the microstructure of the fibers was observed and photographed at different magnifications. The fiber diameter and distribution were analyzed and statistically analyzed.

[0074] II. Experimental Results

[0075] Figure 1 The images show the microstructure and diameter distribution of the PLLA / Gel fiber membrane via SEM. The results indicate that the prepared fibers have good morphology, smooth surface, and no disordered structures such as beads. The average diameter of the fibers is 0.88±0.22μm.

[0076] Test Example 2

[0077] In this embodiment, the electroactivity of the prepared fiber membrane was characterized, as detailed below.

[0078] I. Experimental Methods

[0079] Volt-ampere (CV) characteristic curve analysis: The CV curve of the fiber membrane was measured using an electrochemical workstation. The fiber membrane before and after polarization was clamped on a platinum plate, and the electrochemical activity of the material was measured using a three-electrode method.

[0080] II. Experimental Results

[0081] The CV curve measurement results of PLLA / Gel fiber membrane are as follows: Figure 2 As shown in the figure, the P-PLLA / Gel fiber membrane treated with corona polarization exhibits significant current changes and increases. Under the same voltage, the current measured in P-PLLA / Gel is greater, indicating that corona polarization treatment can impart certain electroactivity to the fiber membrane containing poly-L-lactic acid.

[0082] Test Example 3

[0083] In this embodiment, the prepared fiber membrane was characterized by in vitro cell experiments, as detailed below.

[0084] I. Experimental Methods

[0085] (1) Cells and preparation of culture medium, digestion solution, and cryopreservation solution

[0086] The mouse mononuclear macrophage leukemia cell line (RAW264.7) used in this experiment was purchased from the ATCC cell bank. Mouse bone marrow mesenchymal stem cells (BMSCs) were extracted by ourselves. The proliferation culture medium used for BMSCs consisted of α-MEM medium + 10% fetal bovine serum + 1% penicillin / streptomycin; the osteogenic induction culture medium used for BMSCs consisted of 500 mL proliferation culture medium + 25 mg vitamin C + 1.0802 g β-glycerophosphate sodium + 100 nM dexamethasone; the digestion solution used for BMSCs was 0.25% (w / v) trypsin PBS solution, filtered through a 0.22 μm microporous membrane; the cryopreservation solution used for BMSCs was 90% fetal bovine serum + 10% dimethyl sulfoxide.

[0087] Cell lysis buffer: 1 mL Triton X-100 + 0.315 g Tris-HCl + 0.8775 g NaCl + 100 mL PBS.

[0088] (2) Routine cell culture

[0089] After cell resuscitation, the cells were placed in a constant temperature incubator at 37°C and 5% CO2, with the culture medium changed every two days. The cells were then passaged and cryopreserved.

[0090] (3) Preparation and sterilization of materials

[0091] Cut the fiber membrane to the appropriate size, place the fixed fiber membrane into a well plate, immerse it in 75% alcohol, and irradiate it with ultraviolet light for 6 hours. After soaking in PBS overnight, replace the immersion with an appropriate amount of cell culture medium and place the plate in an incubator to moisten the material.

[0092] (4) Cell inoculation

[0093] Take out the sample, and according to different experimental requirements and cell concentrations, take an appropriate amount of cell suspension and inoculate it on the surface of the material. After the cells have adhered for a period of time, add culture medium.

[0094] (5) Cytotoxicity test of fiber membrane against RAW264.7

[0095] The purpose of this experiment was to determine the cell compatibility of PLLA / Gel fiber membranes with RAW264.7 cells. Quantitative analysis was performed using the CCK-8 assay on days 1, 3, and 5, and qualitative analysis was performed using live / dead cell staining on days 3 and 5.

[0096] (6) Cytotoxicity test of fiber membranes to BMSCs

[0097] The purpose of this experiment was to determine the cytocompatibility of PLLA / Gel fiber membranes with BMSCs. Quantitative analysis was performed using the CCK-8 assay on days 1, 3, and 5, and qualitative analysis was performed using live / dead cell staining on days 3 and 5.

[0098] (7) Effect of fiber membrane on the polarization phenotype of RAW264.7

[0099] The purpose of this experiment was to determine whether electroactive fiber membranes have the ability to directly regulate the polarization of RAW264.7 macrophages toward the tissue regeneration-promoting M2 phenotype. RAW264.7 cells were seeded onto the material, and 1 mL of cell culture medium was added to each well. Tissue culture plates (TCPs) were used as a blank control group and cultured in a constant temperature cell incubator.

[0100] Immunofluorescence staining: Arg-1 primary antibody representing the M2 phenotype was added, followed by FITC-labeled secondary antibody for color development. Protein expression was observed and photographed using a laser confocal microscope (CLSM), and the fluorescence intensity of the captured fluorescence images was quantitatively analyzed using ImageJ.

[0101] (8) Effects of fiber membranes on the phenotype of LPS-induced RAW264.7

[0102] This experiment aims to determine the effect of electroactive fiber membranes on LPS-induced polarization of RAW264.7 macrophages toward the M1 phenotype.

[0103] Normal RAW264.7 cells were seeded onto the material. One group was given 1 mL of macrophage culture medium containing 1 μg / mL LPS, and the other group was given macrophage culture medium without LPS. TCPs were used as a blank control group. The cells were cultured in a constant temperature cell incubator.

[0104] Immunofluorescence staining: After co-culturing for 24 hours, the samples were removed and immunofluorescence staining was performed using the IL-6 primary antibody representing the M1 phenotype marker.

[0105] (9) Effect of fiber membrane on LPS-induced M1 to M2 phenotype conversion of RAW264.7

[0106] This experiment aims to determine whether electroactive fiber membranes can regulate the ability of LPS-induced macrophages to transition from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype.

[0107] RAW264.7 in normal condition was subjected to staining test at 1×10⁻⁶. 4 Cells were seeded onto the material at a density of 1 / well, and 1 mL of macrophage culture medium containing 1 μg / mL LPS was added for induction culture. The medium was then replaced with basal culture medium without LPS for continued culture. TCPs were used as a blank control group. The medium was changed every two days.

[0108] Cytoskeleton staining: After replacing the basal culture medium with LPS-free medium, the samples were cultured for 48 hours and then removed. The samples were fixed, permeabilized and blocked using the same immunofluorescence staining procedure. Then, the staining solution was added and the cytoskeleton was stained at room temperature in the dark for 1 hour. The dye was then aspirated and the macrophage morphology was observed and photographed using CLSM.

[0109] Immunofluorescence staining: After culturing in LPS-free basal medium for 1, 3, and 5 days, the well plates were removed. Immunofluorescence staining was performed on days 1 and 3 using IL-6 (M1 phenotype) primary antibody and on days 1, 3, and 5 using Arg-1 (M2 phenotype) primary antibody.

[0110] (10) Detection of osteogenic properties of fibrous membranes on BMSCs

[0111] The purpose of this experiment is to determine whether electroactive fiber membrane materials have the ability to directly regulate the osteogenic differentiation of BMSCs.

[0112] Normal BMSCs were divided into 2×10 4 Cells were seeded onto the material at a density per well and incubated in an incubator for 2 hours until cell attachment was achieved. Then, 1 mL of proliferation culture medium was added to each well. TCPs were used as a blank control group. When the cell density in the TCP group reached 80% or higher under a light microscope, the proliferation culture medium was replaced with osteogenic induction culture medium. The number of days was recorded, and the osteogenic induction culture medium was replaced every two days.

[0113] Alkaline phosphatase (ALP) staining: Samples were taken on days 7 and 14 after the culture medium was changed to osteogenic induction medium for measurement. ALP staining was performed using an ALP staining kit, and photographs were taken for recording.

[0114] BCA Total Protein Assay: Samples were taken on days 7 and 14 after switching to osteogenic induction medium. Standard and working solutions were prepared according to the kit instructions, and absorbance was measured at 562 nm. A BCA standard curve was plotted based on the standard solution values ​​to calculate the total protein content in the samples.

[0115] ALP activity quantification: The absorbance was measured at a wavelength of 520 nm according to the kit instructions, and the ALP activity was calculated in combination with the total protein content of each group of samples.

[0116] Calcium content detection: Prepare the working solution according to the kit instructions, plot the calcium content standard curve based on the standard solution values, and then calculate the calcium content by combining the total protein content of each group of samples.

[0117] Immunofluorescence staining: Samples were collected on days 7, 14, and 21 after the culture medium was changed to osteogenic induction medium. The samples were then subjected to immunofluorescence staining using OPN and OCN as primary antibodies.

[0118] Osteogenesis-related gene expression: A set of samples was taken on days 7 and 14 after the culture medium was changed to osteogenic induction medium for measurement.

[0119] II. Experimental Results

[0120] like Figure 3 The image shows the live / dead staining results of RAW264.7 and BMSCs cells on a PLLA / Gel fiber membrane. It can be seen that neither cell type showed significant red fluorescence on the PLLA / Gel fiber membrane or on its polarized surfaces. The cells exhibited good growth on both positively and negatively charged surfaces. Furthermore, the green fluorescence became increasingly intense over time. Figure 4 The results of CCK-8 cell proliferation also showed good consistency. Both RAW264.7 and BMSCs cells were able to grow continuously on the surface of the fiber membrane, which also demonstrates that the PLLA / Gel fiber membrane of the present invention has good cell compatibility.

[0121] Figure 5 Immunofluorescence staining of Arg-1 protein expression in RAW264.7 cells cultured on fiber membranes for 1 and 5 days. Figure 5 As can be seen, PLLA / Gel treated with corona polarization exhibits higher Arg-1 protein expression on both positively and negatively charged surfaces. The Arg-1 protein expression in the material group is significantly higher than that in the TCPs group, and the number of cells and fluorescence intensity increase with increasing days. Figure 6 Semi-quantitative analysis also showed that the PLLA / Gel-P and PLLA / Gel-N groups, which had high electroactivity after corona polarization treatment, had high Arg-1 protein expression, indicating that the electroactive PLLA / Gel fiber membrane material can promote the transformation of macrophages to the anti-inflammatory M2 phenotype, thereby benefiting tissue regeneration.

[0122] like Figure 7 The image shows immunofluorescence staining images of IL-6 protein expression on different fiber membranes after 24 hours of stimulation with and without LPS (1 μg / mL). Figure 7As can be seen in (A), macrophages in all groups did not show significant IL-6 expression in the absence of LPS stimulation. Figure 8 The results also show that the semi-quantitative analysis of IL-6 protein expression in the material group was similar to that in the TCPs group, meaning that without LPS stimulation, the PLLA / Gel fiber membrane did not induce macrophages to transform into the pro-inflammatory M1 phenotype and did not cause a significant inflammatory response. In the TCPs group, after stimulation with 1 μg / mL LPS for 24 hours, a large amount of IL-6 expression was observed, indicating that LPS stimulation induces the transformation of RAW264.7 macrophages into the pro-inflammatory M1 phenotype. In comparison, the unpolarized PLLA / Gel showed a trend of weakening inflammatory expression, while the corona-polarized PLLA / Gel-P and PLLA / Gel-N significantly weakened LPS-induced inflammatory expression, indicating that the electroactive material can inhibit LPS-induced macrophage inflammatory responses.

[0123] Figure 9 Images of nuclear and cytoplasmic staining of macrophages on the surface of the fibrous membrane are presented after 24 hours of LPS induction followed by 48 hours of culture in basal medium. The images show that RAW264.7 macrophages on the untreated PLLA / Gel surface also exhibit a multipodia morphology, indicating that the macrophages on this fibrous membrane are also in the M1 phenotype. However, macrophages on both groups of electroactive fiber membranes maintained a round shape without significant pseudopodia formation, confirming that the electroactive fiber membrane can inhibit LPS-induced inflammatory expression.

[0124] Figure 10 Immunofluorescence staining images of IL-6 protein expression in macrophages on different fibrous membrane surfaces after 24 hours of treatment with LPS (1 μg / mL) followed by 1 day (A) and 3 days (B) of basal culture medium. Figure 11 Immunofluorescence staining images of Arg-1 protein expression in macrophages on different fibrous membrane surfaces after treatment with LPS (1 μg / mL) for 24 hours, followed by culturing in basal medium for 1 day (A), 3 days (B), and 5 days (C). Figure 12 This study aimed to semi-quantitatively analyze the immunofluorescence intensity of IL-6 and Arg-1 protein expression in macrophages cultured in ImageJ after LPS induction for 24 hours and subsequent replacement with basal medium.

[0125] Arg-1 is a marker of anti-inflammatory M2 phenotype macrophages. Macrophages were co-cultured on different fibrous membranes, and Arg-1 protein expression was analyzed by immunofluorescence staining. The results are as follows: Figure 11As shown in the figure, the corona-polarized fiber membranes exhibited higher Arg-1 protein expression, especially the PLLA / Gel-N fiber membrane, whose surface fluorescence intensity was significantly higher than that of other groups. Furthermore, both cell number and fluorescence intensity increased over time. Figure 12 As shown, the fluorescence intensity analysis by ImageJ also revealed that the fibrous membrane had high Arg-1 protein expression, with the M2 phenotype marker being most highly expressed on the PLLA / Gel-N fibrous membrane. This indicates that the electroactive PLLA / Gel-N fibrous membrane material can stimulate macrophages to transform into the anti-inflammatory M2 phenotype, which is beneficial to the tissue regeneration process.

[0126] LPS can induce macrophage polarization towards the pro-inflammatory M1 phenotype. LPS stimulation successfully induced the conversion of RAW264.7 cells to the pro-inflammatory M1 phenotype, while macrophages cultured on highly electroactive fibrous membranes showed significantly lower IL-6 fluorescence expression (e.g., Figure 10 As shown in the figure, this indicates that the highly electroactive fibrous membrane can, to some extent, inhibit the induction of the M1 phenotype of macrophages by LPS, thereby reducing the occurrence of inflammatory responses.

[0127] ALP protein is an early marker of osteogenic development, with expression levels typically peaking at day 14. ALP activity reflects the osteogenic differentiation level of cells; higher activity indicates more pronounced differentiation into mature osteoblasts. After seeding bone marrow mesenchymal stem cells (BMSCs) onto a fibrous membrane, ALP protein expression on the fibrous membrane was qualitatively detected using staining on days 7 and 14 of osteogenic induction culture. Figure 13 As shown in the figure, from day 7 to day 14, the staining of ALP on the fiber membrane gradually deepened, with BMSCs cultured on negatively charged surfaces exhibiting the deepest ALP staining at day 14. Figure 14 (A) The results of quantitative detection of ALP activity showed the same pattern as ALP staining results: electrically active PLLA / Gel fiber membranes exhibited higher ALP expression, with negatively charged surfaces showing a significant osteogenic effect. ALP protein catalyzes the breakdown of intracellular polyphosphates, thereby inducing the formation of calcium nodules. Calcium deposition is a late marker of osteogenic differentiation, gradually accumulating during culture. Its quantitative detection results are as follows... Figure 14As shown in (B), the trend of calcium content changes is consistent with the pattern of ALP activity, increasing with the extension of induction differentiation time, and the calcium content level continues to increase. Among them, the calcium content level of the negatively charged surface PLLA / Gel-N group was the highest on day 21, followed by PLLA / Gel-P, and then PLLA / Gel fiber membrane. This result indicates that the electroactive fiber membrane after corona polarization treatment is more conducive to the differentiation of BMSCs into osteoblasts than the untreated fiber membrane, especially its negatively charged surface has a more significant ability to induce osteogenic differentiation. The negatively charged surface can adsorb proteins, calcium ions, etc., which is more conducive to osteogenic differentiation.

[0128] Subsequently, the ability of different charged surfaces of the electroactive PLLA / Gel fiber membrane to induce osteogenic differentiation was examined at the gene level, selecting four osteogenic-related genes: ALP, OPN, OCN, and COL-Ⅰ. Real-time quantitative polymerase chain reaction (RT-qPCR) results showed that the expression of all four osteogenic-related genes was upregulated from 7 to 14 days. The expression of each gene on the negatively charged surface was significantly higher than that on the unpolarized group and the positively charged surface group, consistent with the aforementioned osteogenic differentiation detection results. This indicates that the negative charge on the PLLA / Gel-N surface can facilitate biomineralization and provide a biomimetic electrophysiological microenvironment, thereby promoting osteogenic differentiation. Figure 15 As shown.

[0129] To further investigate the osteogenic differentiation capacity of PLLA / Gel fibrous membranes in promoting BMSCs osteogenic differentiation, immunofluorescence staining and semi-quantitative analysis of two osteogenic differentiation indices, OPN and OCN, were performed. The results are as follows: Figure 16 and Figure 17 As shown, even unpolarized PLLA / Gel fiber membranes possess a certain ability to promote osteogenic differentiation of BMSCs, enhancing the expression of OPN and OCN during culture. Corona-polarized PLLA / Gel exhibits increased electroactivity, resulting in a more significant increase in the expression of both OPN and OCN with electroactive PLLA / Gel. In particular, PLLA / Gel-N with a negatively charged surface can facilitate biomineralization, thereby further promoting osteogenic differentiation.

[0130] In summary, this invention, through CV curve determination, confirms that pre-corona polarization treatment can endow PLLA / Gel fiber membranes containing poly-L-lactic acid with certain electroactivity. Electroactive PLLA / Gel fiber membranes, regardless of whether their surfaces are positively or negatively charged, can directly promote macrophage polarization towards the anti-inflammatory M2 phenotype and inhibit the inflammatory induction effect of LPS on macrophages, thus promoting the transformation of macrophages from the pro-inflammatory M1 phenotype to the M2 phenotype. This indicates that electroactive PLLA / Gel fiber membranes can inhibit inflammation and promote tissue regeneration. Increased fiber membrane electroactivity is beneficial for promoting the electrophysiological microenvironment of osteogenic tissue, especially since negatively charged surfaces can adsorb cations such as calcium ions from the environment, contributing to biomineralization and thus enhancing osteogenic activity. Therefore, PLLA / Gel-N with negatively charged enriched surfaces exhibits significant bone immunomodulatory and osteogenic effects.

[0131] Test Example 4

[0132] This embodiment evaluates the alveolar bone regeneration capacity of the prepared composite bilayer membrane, as detailed below.

[0133] A mouse model of periodontitis was designed and treated by filling the area with membrane material using guided tissue regeneration. In this experiment, R / Gel fiber membranes and F / Gel-P fiber membranes were selected for testing, and commercially available membranes were also chosen. The oral repair membrane was used as a control. Samples were taken at 1 month and 2 months to detect periodontal inflammation and alveolar bone regeneration in mice. The results are shown below.

[0134] Upregulation of INF-γ and TNF-α expression in immune cells can stimulate macrophage polarization towards the pro-inflammatory M1 phenotype, leading to bone regeneration failure. M1 macrophages, in turn, secrete pro-inflammatory factors such as IL-1β, IL-6, and TNF-α to enhance inflammation. In this experiment, the expression of three inflammation-related cytokines—IL-1β, IL-6, and INF-γ—in mouse gingival crevicular fluid was detected by RT-qPCR as an important parameter for assessing the level of periodontal inflammation in mice. The results are as follows: Figure 18 As shown in the figure, the expression levels of three inflammation-related genes in the UT group (untreated defect group) were significantly increased compared to the control group. The Heal-All group ( Compared with the UT group, the expression of inflammatory factors in the oral repair membrane treatment group was reduced to a certain extent. This may be because the membrane filling can inhibit alveolar bone resorption, thereby reducing the expression level of inflammatory genes compared with UT. However, compared with normal mice without induced periodontitis, the expression level of inflammation-related genes in the gingival crevicular fluid of the Heal-All group mice was still high, indicating that... Oral repair membranes do not have an anti-inflammatory effect. Preliminary cellular level tests showed that the R / Gel bilayer fiber membrane did not have an anti-inflammatory effect; therefore, this group was similar to the Heal-All group, with high expression levels of inflammatory genes in the gingival crevicular fluid of mice. However, the prepared electroactive F / Gel-P bilayer composite fiber membrane demonstrated the ability to regulate immune cells and suppress inflammation. After 4-8 weeks of treatment, the expression levels of three inflammatory genes in the gingival crevicular fluid of periodontitis mice were significantly lower than those in the UT group, and similar to those in normal mice. In conclusion, only the electroactive F / Gel-P composite fiber membrane has the ability to significantly eliminate inflammation in periodontitis mice.

[0135] Next, we will use Micro-CT analysis of the maxillary alveolar bone of mice to verify the osteogenic effect of the electroactive bilayer fibrous membrane in periodontitis. Figure 19 As can be seen, the mice in the Control group did not undergo ligation-induced bone resorption, while the UT group showed significant alveolar bone resorption. Although a small amount of new bone tissue could form over time, it was far from achieving self-healing. As a commercially available membrane, the oral repair membrane has a certain ability to promote bone regeneration. The Heal-All group showed some new bone formation after treatment, but some tooth root exposure was still clearly visible around the second molars in mice, failing to achieve complete repair, and the bone density was far lower than normal. The R / Gel composite fiber membrane, due to the addition of bone-promoting gelatin to its inner layer, showed a higher level of inhibition of bone resorption in periodontitis mice compared to... Similar to oral repair membranes, these methods can improve alveolar bone density and reduce the CEJ-ABC distance in periodontitis mice to some extent. However, only the electrically active F / Gel-P fiber membrane can both inhibit inflammation and promote osteogenic formation, thus exhibiting the most significant promoting effect on in-situ bone repair. After 4-8 weeks of treatment, the most new bone tissue was observed around the second molar. Reconstructed 3D images showed that the new bone tissue in this group had a similar structure and morphology to the normal group, and its bone density and CEJ-ABC distance were close to the level of normal mice. Figure 20 As shown.

[0136] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. An electroactive fiber membrane for promoting osteogenesis, characterized in that, It is formed by electrospinning of a mixture of poly-L-lactic acid and gelatin, the electroactive fiber membrane is treated by corona polarization to make the side of the electroactive fiber membrane have charge enrichment, the diameter of the fiber is 500-1500 nm, and the preparation of the electroactive fiber membrane comprises the following steps: (1) mixing a solution of poly-L-lactic acid dissolved in an organic solvent and a solution of gelatin dissolved in an organic solvent uniformly, and then performing electrospinning, and cross-linking under conditions suitable for cross-linking, to obtain a cross-linked fiber membrane, the gelatin having a gel strength of 100 400 g Bloom; (2) the electroactive fiber membrane is obtained after the fiber membrane is treated by corona polarization.

2. The electroactive fiber film of claim 1, wherein, One side of the electroactive fiber membrane is enriched with positive charges, and the other side is enriched with negative charges.

3. The electroactive fiber film of claim 2, wherein, The concentration of the poly-L-lactic acid in the organic solvent is 0.05-0.4 g / mL, and the concentration of the gelatin in the organic solvent is 0.05-0.4 g / mL.

4. The electroactive fiber film of claim 1, wherein, The weight ratio of the poly-L-lactic acid to the gelatin is 1:9-9:

1.

5. The electroactive fiber film of claim 1, wherein, The conditions of the corona polarization treatment are as follows: the voltage is 8-15 kV, and the polarization time is 15-30 minutes.

6. A guided tissue regeneration composite membrane for periodontal tissue defect repair, characterized by, The electroactive fiber membrane for promoting osteogenesis according to any one of claims 1-5.

7. Use of the electroactive fiber membrane for promoting osteogenesis according to any one of claims 1-5 in the preparation of a repair material for promoting bone regeneration.

8. A method of modulating a macrophage phenotype, characterized by, The step of culturing macrophages in vitro using the electroactive fiber membrane according to any one of claims 1-5.

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