Multifunctional guided tissue regeneration membrane and preparation method and application thereof

Abstract

The application belongs to the field of medical materials, and particularly discloses a multifunctional guided tissue regeneration membrane, a preparation method and application thereof. The regeneration membrane comprises an antibacterial fiber layer with positive charge enrichment on the outer side and an osteogenic fiber layer with negative charge enrichment on the inner side. The multifunctional guided tissue regeneration membrane has a double-layer asymmetric structure, and the outer membrane material has certain piezoelectric characteristics through corona polarization treatment, thereby showing intrinsic antibacterial activity. The antibacterial activity does not decrease significantly with the extension of time, and the antibacterial activity not only has a significant effect on common gram-negative escherichia coli, but also can play a good role on periodontitis pathogenic bacteria porphyromonas gingivalis. In addition, the multifunctional guided tissue regeneration membrane has good cell affinity, the negative charge enrichment surface formed through corona polarization treatment can further improve the osteogenic activity of the material, and can promote the regeneration of periodontal tissues such as alveolar bone and jaw bone in an inflammatory environment.

Description

Technical Field

[0001] This invention belongs to the field of medical materials technology, and relates to a multifunctional tissue regeneration guiding membrane, its preparation method and application. Technical Background

[0002] Periodontal tissues, including the gingiva, alveolar bone, periodontal ligament, cementum, and jawbone, are the supporting tissues of teeth, playing a vital role in bearing chewing forces and protecting the integrity of the oral masticatory mucosa. Periodontitis is a chronic, destructive inflammatory disease of the periodontal tissues caused by plaque microorganisms. Its main pathological change is inflammation and destruction of the periodontal supporting tissues. The incidence of periodontal disease in adults in my country is as high as 85%. Periodontal disease often causes irreversible damage to periodontal tissues, seriously affecting people's quality of life and physical and mental health. In addition, trauma or tumor surgery can also cause alveolar bone and jawbone defects, affecting the normal structure and function of maxillofacial tissues. The increasing aging of society further exacerbates the incidence of periodontal tissue defects. The ideal treatment goal for periodontal disease is the complete regeneration of periodontal tissues, including the periodontal ligament, alveolar bone, and cementum.

[0003] Currently, guided tissue regeneration and bone grafts are commonly used in clinical practice to treat alveolar bone and jawbone defects caused by various reasons. Among bone grafts, autologous bone grafting requires creating a donor site on the patient's own body, and the cutting and recovery of the donor site can cause additional pain for the patient. Commercially available allogeneic bone or decellularized xenogeneic bone matrix filling often requires the use of guided tissue regeneration to achieve bone augmentation.

[0004] Guided tissue regeneration (BTR) selectively separates different periodontal tissues by placing a membrane material as a physical barrier. This prevents gingival epithelial cells and gingival connective tissue from growing towards the root surface during wound healing, providing space for periodontal bone regeneration. Simultaneously, it can induce the migration and differentiation of periodontal ligament stem cells with periodontal tissue regeneration potential to the defect site, achieving regeneration of the periodontal ligament, alveolar bone, and cementum. Bioabsorbable BTR membrane materials are widely used in BTR due to their good biocompatibility, barrier effect, and ease of clinical application.

[0005] Currently, guided tissue regeneration membranes used clinically are mainly collagen-based medical membranes. While they offer good isolation and promote new bone regeneration, the unique oral environment makes it easy for bacteria to accumulate and form plaque. Periodontal bone defects caused by severe periodontitis often result in unsatisfactory repair due to bacterial infection. However, there is currently no guided tissue regeneration membrane product on the market with antibacterial and anti-inflammatory functions. Research on the antibacterial properties of guided tissue regeneration membranes often employs loading antibiotics, antibacterial ions, or other drugs or antibacterial agents. These are released locally to reduce the risk of bacterial infection and promote tissue regeneration. However, the antibacterial properties of these release-type antibacterial materials decrease or even disappear as the antibacterial components are released, failing to achieve long-lasting antibacterial effects. Furthermore, the loading and release process of antibacterial components is complex and difficult to control precisely; short-term burst release may cause cytotoxicity and harm to the human body. Summary of the Invention

[0006] To address the adverse side effects of release-type antibacterial materials, this invention provides a tissue regeneration-guided membrane that achieves intrinsic antibacterial properties by endowing the material with piezoelectric characteristics. This membrane material does not require loading with antibacterial drugs or agents; instead, it possesses long-lasting antibacterial properties through pre-corona polarization treatment and the local pressure generated by oral chewing, while also guiding new bone regeneration. Specifically, this invention provides the following:

[0007] In a first aspect, the present invention provides a multifunctional guided tissue regeneration membrane comprising an outer fibrous layer with antibacterial properties and an inner fibrous layer with osteogenic properties, wherein the outer fibrous layer and the inner fibrous layer are pressed together to form a composite fibrous layer, the composite fibrous layer being subjected to corona polarization treatment to enrich the outer fibrous layer with positive charge and the inner fibrous layer with negative charge. The distribution of positive and negative charges can be determined using methods known in the art, and is not particularly limited thereto; for example, it can be easily determined based on the electrode orientation during polarization.

[0008] The tissue regeneration membrane provided by this invention, after being treated with corona polarization, forms an asymmetrical structure with charge distribution on both sides. The inner fibrous layer, which is rich in negative charge, is more conducive to promoting osteogenic differentiation of cells, while the outer fibrous layer, which is rich in positive charge, can achieve antibacterial effect through charge interaction. Thus, the regeneration membrane can further enhance osteogenic activity without loading antibacterial drugs or agents to achieve antibacterial effect. Moreover, the antibacterial property is non-release and will not decrease over time.

[0009] In one specific embodiment of the present invention, the outer fiber layer is formed by electrospinning poly-L-lactic acid; the inner fiber layer is formed by electrospinning poly-L-lactic acid and natural polymer. The spinning parameters are not particularly limited, but 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.

[0010] The multifunctional guided tissue regeneration membrane provided by this invention comprises an outer fibrous layer formed of poly-L-lactic acid (PLA) and an inner fibrous layer formed of PLA and natural polymers, resulting in asymmetric differences in composition and fiber arrangement. Corona polarization treatment achieves an asymmetric distribution of positive and negative charges on both sides of the composite fibrous layer. This results in the outer fibrous layer, rich in positive charges, possessing intrinsic antibacterial activity, while the inner fibrous layer, rich in negative charges, promotes osteogenic differentiation. This facilitates the multifunctionalization of the guided tissue regeneration membrane, better meeting the clinical needs of guided tissue regeneration.

[0011] As used in this invention, the term "lateral" refers to the side of the guiding tissue regeneration membrane relative to or facing the gingival tissue, which, in application, is close to the gingival tissue and exerts an antibacterial effect. The term "medial" refers to the side of the guiding tissue regeneration membrane away from the gingival tissue or facing the alveolar bone tissue, which, in application, is close to the alveolar bone tissue and exerts an osteogenic function.

[0012] In one specific embodiment of the present invention, the concentration of poly-L-lactic acid in the outer fiber layer in the organic solvent is 0.05-0.4 g / mL.

[0013] In one specific embodiment of the present invention, the concentration of poly-L-lactic acid in the inner fiber layer is 0.05-0.4 g / mL, and the concentration of the natural polymer in the organic solvent is 0.05-0.4 g / mL.

[0014] 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.

[0015] 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.

[0016] In one specific embodiment of the present invention, the natural polymer is one of collagen and gelatin.

[0017] In one specific embodiment of the present invention, the natural polymer is gelatin.

[0018] In one specific embodiment of the present invention, the diameter of the fibers in the inner fiber layer is 500-1500nm, preferably 800-1400nm, for example 800, 900, 1000, 1100, 1200, 1300, 1400nm.

[0019] In one specific embodiment of the present invention, the diameter of the fibers in the outer fiber layer is 500-1500 nm, preferably 800-1400 nm, for example 800, 900, 1000, 1100, 1200, 1300, 1400 nm.

[0020] A second aspect of the present invention provides a method for preparing the above-mentioned multifunctional guided tissue regeneration membrane, comprising the following steps:

[0021] (1) The outer fiber layer is obtained by electrospinning a poly-L-lactic acid solution dissolved in an organic solvent;

[0022] (2) After mixing the natural polymer solution dissolved in an organic solvent with the poly-L-lactic acid solution dissolved in an organic solvent evenly, electrospinning is performed, and cross-linking is carried out under suitable conditions to obtain the inner fiber layer.

[0023] (3) The inner and outer fiber layers are pressed into a composite fiber membrane, and after corona polarization treatment, a multifunctional tissue regeneration membrane is obtained.

[0024] In the preparation method of this invention, the organic solvent is not particularly limited; the 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.

[0025] In one specific embodiment of the present invention, the electrospinning process conditions in (1) and / or (2) 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 outer fiber layer has a smooth fiber surface, an average diameter between 500-1500 nm, and no beading.

[0026] In one specific embodiment of the present invention, the concentration of poly-L-lactic acid 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.

[0027] In one specific embodiment of the present invention, the concentration of the natural polymer 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.

[0028] In one specific embodiment of the present invention, when preparing the inner fiber layer, the mass 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. Preferably, the mass ratio of poly-L-lactic acid to natural polymer is 5:5.

[0029] 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%.

[0030] In one specific embodiment of the present invention, the process of drying the fiber membrane prepared by electrospinning is further included before corona polarization.

[0031] In one specific embodiment of the present invention, the drying is vacuum drying.

[0032] 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.

[0033] In one specific embodiment of the present invention, the drying conditions are: constant temperature 37°C, vacuum degree 30Pa, drying for 24 hours.

[0034] In one specific embodiment of the present invention, the corona polarization voltage is 8-15kV, and the polarization time is 15-30 minutes. Fibers polarized under these conditions exhibit superior antibacterial properties.

[0035] In a specific embodiment of the present invention, (1) after electrospinning, the fiber layer is annealed. The annealing process involves first annealing the electrospinned fiber layer at 100-110°C for 8-12 hours, then naturally cooling it to room temperature, and then annealing it at 155-165°C for 8-12 hours and then naturally cooling it to room temperature.

[0036] A third aspect of the invention provides the application of the aforementioned multifunctional guided tissue regeneration membrane in oral tissues.

[0037] A fourth aspect of the invention provides the use of the multifunctional guided tissue regeneration membrane in the preparation of a repair material that promotes bone regeneration, wherein the promotion of bone regeneration includes at least one of the following:

[0038] a. Increased bone density;

[0039] b. Reduce the distance from the enamel-dentin junction to the alveolar bone ridge;

[0040] c. Promotes the formation of new bone tissue;

[0041] d. Promotes the expression of osteogenic-related genes.

[0042] The multifunctional guided tissue regeneration membrane provided by this invention has at least one of the following beneficial effects:

[0043] The multifunctional guided tissue regeneration membrane provided by this invention has a double-layer asymmetry in composition, fiber arrangement and charge distribution, which can better meet the requirements of periodontal defect repair.

[0044] The tissue regeneration membrane provided by this invention is formed by the accumulation of fibers prepared by electrospinning process, which can effectively block the entry and growth of gingival tissue into the bone defect area.

[0045] The antibacterial guided tissue regeneration membrane provided by this invention does not require loading with antibiotics, antibacterial ions, or other antibacterial agents. Its antibacterial activity comes from the material itself, which has piezoelectric properties, namely the positive charge enrichment characteristic obtained after corona polarization treatment. This antibacterial activity is non-released and will not significantly decrease with the extension of implantation time. Moreover, this intrinsic antibacterial activity not only has a significant effect on common Gram-negative Escherichia coli, but also plays a good role in the periodontitis pathogen Porphyromonas gingivalis, which can significantly improve the treatment effect of periodontal disease caused by periodontitis.

[0046] The multifunctional tissue regeneration membrane inner layer provided by this invention has good cell affinity and excellent osteogenic effect. The inner membrane has a certain osteogenic effect when used alone without corona polarization treatment. The surface enriched with negative charge after corona polarization can further enhance the osteogenic effect and promote the regeneration of periodontal bone tissue.

[0047] Furthermore, the inner and outer layers of the multifunctional guided tissue regeneration membrane of the present invention are both biodegradable materials, containing no inorganic piezoelectric materials or exogenous growth factors, and can reach a physiological potential of -56.87±0.48mV, thereby achieving a level matching that of the endogenous physiological potential (the physiological potential of natural bone is -62mV), making the multifunctional guided tissue regeneration membrane of the present invention more conducive to osteogenic formation. Attached Figure Description

[0048] Figure 1 This is a scanning electron microscope (SEM) image of the morphology of the electrospun random fiber membrane in Embodiment 1 of the present invention.

[0049] Figure 2 This is a SEM image of the morphology of the electrospun random fiber membrane after annealing treatment in Embodiment 2 of the present invention.

[0050] Figure 3 This is a SEM image of the morphology of the electrospun oriented fiber membrane in Example 3 of the present invention.

[0051] Figure 4 This is a SEM image of the morphology of the electrospun composite fiber membrane in Example 4 of the present invention.

[0052] Figure 5 The figures show the current-voltage characteristic curves of Embodiments 1 and 2 and Comparative Example 1 of the present invention.

[0053] Figure 6 The effect of annealing on fiber properties was demonstrated.

[0054] Figure 7 The figures show the current-voltage characteristic curves of Embodiment 4 and Comparative Example 2 of the present invention.

[0055] Figure 8 This is a SEM image of the cross-sectional morphology of the electrospun fiber membrane after pressing according to Example 6 of the present invention.

[0056] Figure 9 These are live-dead staining laser confocal microscope images of Escherichia coli after 24 hours of co-culturing on the fibrous membranes of Examples 2, 4, 6 and Comparative Example 1.

[0057] Figure 10 The images show the SEM images of Escherichia coli after 24 hours of co-culturing on the fiber membranes in Examples 2, 4, 6 and Comparative Example 1.

[0058] Figure 11 The results are quantitative analysis of live and dead bacteria after co-culturing Escherichia coli with the fiber membranes of Examples 1-7 and Comparative Examples 1-3 for 24 hours.

[0059] Figure 12 SEM images of the bacterial morphology of *Porphyromonas gingivalis* after co-culturing on the fiber membranes of Examples 2, 4, 6 and Comparative Example 1 for 24 hours.

[0060] Figure 13 These are laser confocal microscopy images of rat bone marrow mesenchymal stem cells after being co-cultured on the fibrous membranes of Examples 2, 4, 6 and Comparative Example 1 for 24 hours, stained with live and dead cells.

[0061] Figure 14Quantitative detection results of the activity of alkaline phosphatase (ALP), an early osteogenic marker, after 14 days of osteogenic differentiation culture of rat bone marrow mesenchymal stem cells on the fibrous membranes in Examples 1-7 and Comparative Examples 1-3.

[0062] Figure 15 Shows the expression of inflammation-related genes in mouse gingival crevicular fluid, (A) IL-1β, (B) IL-6, (C) IFN-γ. Each column in the figure represents the control group (Control), untreated group (UT), commercial Oral repair membrane (Heal-All), composite fibrous membrane of Comparative Example 3, composite fibrous membrane of Example 6 from left to right in the figure.

[0063] Figure 16 Shows the Micro-CT models of mouse maxillary alveolar bone: (A) 2D; (B) 3D.

[0064] Figure 17 Shows the Micro-CT quantitative analysis of mouse maxillary alveolar bone: (A) Bone mineral density (BMD), (B) Distance from the alveolar bone crest to the enamel-dentin junction (CEJ-ABC). Each column in the figure represents the control group (Control), untreated group (UT), commercial Oral repair membrane (Heal-All), composite fibrous membrane of Comparative Example 3, composite fibrous membrane of Example 6 from left to right in the figure. Detailed implementation manners

[0065] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which this invention pertains.

[0066] The technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only a part of the embodiments of this invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of this invention without creative efforts belong to the protection scope of this invention.Particular conditions not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments not specified by the manufacturer can all be obtained as conventional products through commercial purchase.

[0067] In the following embodiments, SPSS software is used to perform mean-one-way analysis on the experimental results. Each experiment is set with at least 3 parallel samples, and the final results are expressed as mean ± variance. When 0.01 < P < 0.05, it is considered that there is a significant difference between the two groups of data, denoted as *, when 0.001 < P < 0.01, it is considered that there is a more significant difference between the two groups of data, denoted as **, and when P < 0.001, it is considered that there is an extremely significant difference between the two groups of data, denoted as ***.

[0068] Example 1 Antibacterial Guided Tissue Regeneration Membrane 1

[0069] The method for preparing the antibacterial tissue regeneration membrane 1 in this embodiment is as follows:

[0070] Poly(L-lactic acid) (molecular weight 100,000) was dissolved in trifluoroethanol and magnetically stirred at room temperature for 24 hours to obtain a polymer solution with a concentration of 0.1 g / mL. The polymer solution was then 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 at a speed of 400 rpm, a voltage of 15 kV, a receiving distance of 15 cm, and an injection rate of 0.8 mL / h. Continuous spinning was carried out for 10 hours to obtain a random fiber membrane with randomly arranged fibers.

[0071] The random fiber membrane was placed in a vacuum drying oven and dried at a constant temperature of 37°C and a vacuum degree of 30 Pa for 24 hours. Its morphology was shown in the SEM image below. Figure 1 As shown. From Figure 1 As can be seen, the fiber membrane prepared by electrospinning has a smooth surface and uniform diameter, with an average diameter of 0.82±0.22μm, and has a three-dimensional structure similar to the extracellular matrix.

[0072] The dried random fiber membrane was subjected to corona polarization at 12kV for 30 minutes at room temperature to obtain an antibacterial tissue regeneration membrane 1.

[0073] Example 2 Antibacterial Guided Tissue Regeneration Membrane 2

[0074] The method for preparing the antibacterial tissue regeneration membrane 2 in this embodiment is as follows:

[0075] Poly(L-lactic acid) (molecular weight 100,000) was dissolved in trifluoroethanol and magnetically stirred at room temperature for 24 hours to obtain a polymer solution with a concentration of 0.1 g / mL. The polymer solution was then 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 at a speed of 400 rpm, a voltage of 15 kV, a receiving distance of 15 cm, and an injection rate of 0.8 mL / h. Continuous spinning was carried out for 10 hours to obtain a random fiber membrane with randomly arranged fibers.

[0076] The random fiber membrane described above was annealed first at 105°C for 10 hours, then naturally cooled to room temperature, and finally annealed at 160°C for 10 hours, followed by natural cooling to room temperature. The annealed fiber membrane was obtained, and its morphology was shown in the SEM image below. Figure 2 As shown. From Figure 2As can be seen, the morphology of the annealed fibers did not change significantly, with an average diameter of 500-1500 nm, and still had a three-dimensional structure similar to the extracellular matrix.

[0077] The annealed random fiber membrane was subjected to corona polarization at 12kV for 30 minutes at room temperature to obtain an antibacterial tissue regeneration membrane 2.

[0078] Example 3 Antibacterial Guided Tissue Regeneration Membrane 3

[0079] The method for preparing the antibacterial tissue regeneration membrane 3 in this embodiment is as follows:

[0080] Poly(L-lactic acid) (molecular weight 100,000) was dissolved in trifluoroethanol and magnetically stirred at room temperature for 24 hours to obtain a polymer solution with a concentration of 0.1 g / mL. The polymer solution was then 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 at a speed of 960 rpm, a voltage of 15 kV, a receiving distance of 15 cm, and an injection rate of 0.8 mL / h. Continuous spinning was carried out for 10 hours to obtain an oriented fiber membrane with directional fiber arrangement.

[0081] The above-mentioned oriented fiber membrane was placed in a vacuum drying oven and dried at a constant temperature of 37°C and a vacuum degree of 40 Pa for 24 hours. Its SEM image is shown below. Figure 3 As shown. From Figure 3 As can be seen, the fiber surface of this oriented fiber membrane is smooth, the diameter is uniform, the average fiber diameter is 500-1500nm, and it has a three-dimensional structure similar to the extracellular matrix.

[0082] The dried oriented fiber membrane was subjected to corona polarization at 12kV for 30 minutes at room temperature to obtain an antibacterial tissue regeneration membrane 3.

[0083] Example 4: Osteogenic Guided Tissue Regeneration Membrane

[0084] The preparation method of the osteogenic guided tissue regeneration membrane in this embodiment is as follows:

[0085] Poly(L-lactic acid) (molecular weight 100,000) was dissolved in trifluoroethanol and magnetically stirred at room temperature for 24 hours to obtain polymer solution A with a concentration of 0.1 g / mL. Gelatin (glucopyranosity 250 g Bloom) was dissolved in trifluoroethanol and magnetically stirred at room temperature for 24 hours to obtain polymer solution B with a concentration of 0.1 g / mL. Polymer solution A and polymer solution B were mixed in a 1:1 volume ratio and magnetically stirred at room temperature for 4 hours until homogeneous to obtain polymer solution C.

[0086] The polymer solution C was loaded into a sterile syringe, which was then fixed to a constant flow pump. Electrospinning was performed using a stainless steel roller as the receiving device. The roller speed was 400 rpm, the voltage was 15 kV, the receiving distance was 15 cm, the extrusion speed was 0.8 mL / h, and continuous spinning was carried out for 10 hours to obtain a composite fiber membrane with randomly arranged fibers.

[0087] The composite fiber membrane was placed in a vacuum drying oven and dried at a constant temperature of 37℃ and a vacuum degree of 30Pa for 24 hours. The dried composite fiber membrane was then placed in a 90% ethanol solution of 1,3-dimethylaminopropyl-3-ethyldiimide / N-hydroxysuccinimide (total concentration of 1,3-dimethylaminopropyl-3-ethyldiimide and N-hydroxysuccinimide was 0.02 g / mL, and the mass ratio of 1,3-dimethylaminopropyl-3-ethyldiimide to N-hydroxysuccinimide was 2.5:1) and crosslinked at 4℃ for 24 hours, followed by freeze-drying for 24 hours to obtain the crosslinked composite fiber membrane. Its morphology is shown in the SEM image below. Figure 4 As shown. From Figure 4 It can be seen that the composite fiber membrane has a smooth fiber surface and uniform diameter, with an average diameter of 500-1500 nm, and has a three-dimensional structure similar to the extracellular matrix.

[0088] The dried composite fiber membrane was subjected to corona polarization at 12kV for 30 minutes at room temperature to obtain an osteogenic guided tissue regeneration membrane.

[0089] Examples 5-7: Bilayer Multifunctional Guided Tissue Regeneration Membrane

[0090] The preparation method of the double-layer multifunctional guided tissue regeneration membrane in this embodiment is as follows:

[0091] The unpolarized fiber membranes from Examples 1, 2, and 3 were pressed with the unpolarized fiber membrane from Example 4 at 5 MPa for 15 seconds to form composite fiber membranes.

[0092] The above-mentioned composite fiber membrane was subjected to corona polarization treatment at room temperature with a polarization voltage of 12 kV for 30 minutes to obtain a double-layer multifunctional guided tissue regeneration membrane. The SEM image of the cross-sectional morphology of the guided tissue regeneration membrane in Example 6 is shown below. Figure 8 As shown.

[0093] The difference between Comparative Example 1 and Example 1 is that the random fiber membrane was not polarized after drying.

[0094] The difference between Comparative Example 2 and Example 4 is that the cross-linked composite fiber membrane was not polarized after drying.

[0095] The difference between Comparative Example 3 and Example 5 is that the pressed composite fiber membrane was not polarized.

[0096] Example 8: Detection of Electroactivity of Guided Tissue Regeneration Membrane

[0097] The current-voltage characteristic curves of the fiber membranes in Examples 1, 2, and 4, and Comparative Examples 1 and 2 were detected using an electrochemical workstation. The experimental results are as follows: Figure 5 and Figure 7 As shown.

[0098] from Figure 5 As can be seen, Example 1, after corona polarization treatment, showed a significant change and increase in current compared to Comparative Example 1, which was not corona polarized. At the same voltage, the current measured by the corona-polarized membrane material was greater, indicating that pre-corona polarization treatment can impart certain electroactivity to the piezoelectric poly(L-lactic acid) cellulose membrane. Example 2, after annealing and corona polarization treatment, showed a more significant increase in current measured at the same voltage compared to Example 1 and Comparative Example 1, with a more pronounced change and increase in current, indicating that annealing treatment can further enhance the electroactivity of the poly(L-lactic acid) cellulose membrane after corona polarization.

[0099] from Figure 7 It can be seen that, compared with the untreated Comparative Example 2, Example 4, which underwent corona polarization treatment, showed a significant change and increase in current. Under the same voltage, the membrane material treated with corona polarization had a larger measured current, indicating that corona polarization treatment can impart a certain degree of electroactivity to the poly-L-lactic acid fiber membrane containing piezoelectric properties.

[0100] Furthermore, this invention also explores the effect of annealing treatment on fiber properties. Figure 6 In this study, R-PLLA corresponds to the fiber prepared in Comparative Example 1. F-PLLA corresponds to the fiber prepared in Example 2 before corona polarization treatment. This fiber was obtained by annealing at 105°C for 10 hours, naturally cooling to room temperature, and then annealing at 160°C for 10 hours. The results showed that the nonwoven R-PLLA membrane obtained directly by electrospinning did not exhibit obvious X-ray diffraction (XRD) characteristic peaks and showed an amorphous morphology. The F-PLLA membrane, after complete annealing, showed a β-phase characteristic peak at 17.3°, indicating that complete annealing can induce β-phase crystallization with piezoelectric properties in electrospun poly-L-lactic acid fibers. Therefore, the following examples only show in vitro and animal experiments related to poly-L-lactic acid fiber membranes with β-phase crystallization exhibiting excellent antibacterial properties.

[0101] Example 9: Antibacterial activity of guided tissue regeneration membrane against Escherichia coli

[0102] (1) The fiber membranes from Examples 1-7 and Comparative Examples 1-3 were first sterilized by ultraviolet light irradiation, with both sides irradiated for 6 hours each, and then placed in a culture plate. 100 μL of a 1×10⁻⁶ solution was added. 7 CFU / mL of E. coli was inoculated onto the positively charged surface of the fiber membrane, followed by 900 μL of bacterial culture medium. The membrane was then incubated at 37°C for 24 hours. The fiber membrane was then removed, the culture medium was aspirated, and the membrane was washed twice with PBS.

[0103] (2) Add SYTO9 / PI staining solution to the fiber membrane prepared in (1), stain at room temperature in the dark for 15 minutes, and then observe and photograph it under a laser confocal microscope. Then use ImageJ software to perform quantitative analysis on the live and dead staining images.

[0104] (3) Add 500 μL of tissue fixative (2.5% glutaraldehyde) to the fiber membrane prepared in (1) and fix at room temperature for 2 hours. After 2 hours, aspirate the tissue fixative, wash twice with PBS, and dehydrate using a gradient of ethanol (30%, 50%, 75%, 90%, 95%, 100%), treating twice for each concentration for 10 minutes each time. After air-drying the dehydrated material, observe and photograph it using SEM.

[0105] The laser confocal microscopy experimental results of some embodiments, such as Embodiment 2, Embodiment 4, Embodiment 6 and Comparative Example 1, are as follows: Figure 9 As shown, the SEM image is as follows Figure 10 As shown.

[0106] The quantitative analysis results of Examples 1-7 and Comparative Examples 1-3 are as follows: Figure 11 As shown.

[0107] from Figure 10 It can be seen that the E. coli in Comparative Example 1 has a smooth and round surface, normal morphology, and its shape is not affected. However, the E. coli in Examples 2, 4, and 6 have abnormal morphology, with wrinkles and collapses in the bacterial membrane, which leads to bacterial death.

[0108] The morphology of Escherichia coli in Comparative Examples 2 and 3 was similar to that in Comparative Example 1; the morphology of Escherichia coli in Examples 1, 3, 5 and 7 was similar to that in Example 2, with wrinkling, collapse and other defects occurring, resulting in the death of Escherichia coli.

[0109] See Figure 11 In the unpolarized Comparative Examples 1-3, the fiber membranes did not have antibacterial activity, while the polarized fiber membranes in Examples 1-7 all had good antibacterial activity.

[0110] The antibacterial activity of the fiber membranes in Examples 1-3 reached over 77%, with Example 2 exhibiting the highest antibacterial activity, exceeding 85%. This indicates that annealing treatment enhances the electroactivity of the poly-L-lactic acid fiber membrane, thus improving its antibacterial activity. The antibacterial activity of the fiber membrane in Example 4 only reached 52%. In contrast, the antibacterial activity of the fiber membranes in Examples 5-7 reached over 89%, with Example 6 achieving the highest at 94%.

[0111] Comparisons between Examples 1 and 5, Examples 2 and 6, and Examples 3 and 7 showed significant differences (P < 0.001), indicating that the antibacterial activity was further enhanced after the bilayer membrane was composited.

[0112] Example 10: Antibacterial activity of guided tissue regeneration membrane against Porphyromonas gingivalis

[0113] (1) The fiber membranes from Examples 1-7 and Comparative Examples 1-3 were first sterilized by ultraviolet light irradiation, with both sides irradiated for 6 hours each, and then placed in a culture plate. 100 μL of a 1×10⁻⁶ solution was added. 7 A CUF / mL *Porphyromonas gingivalis* bacterial suspension was inoculated onto the positively charged surface of a fiber membrane, followed by 900 μL of bacterial culture medium. The plates were then incubated at 37°C for 24 hours. The culture plates were removed, the culture medium was discarded, and the plates were washed twice with PBS.

[0114] (2) Add 500 μL of tissue fixative (2.5% glutaraldehyde) to the fiber membrane prepared in (1) and fix at room temperature for 2 hours. After 2 hours, aspirate the tissue fixative, wash twice with PBS, and dehydrate using a gradient of ethanol (30%, 50%, 75%, 90%, 95%, 100%), treating twice for each concentration, 10 minutes each time. After air-drying the dehydrated material, observe and photograph it using SEM.

[0115] Some embodiments, such as SEM images of Embodiments 2, 4, 6 and Comparative Example 1, are shown below. Figure 12 As shown. From Figure 12 As can be seen, the surface of *Porphyromonas gingivalis* in Comparative Example 1 is smooth, its morphology is normal, and its form is not affected. However, the *Escherichia coli* in Examples 2, 4, and 6 have abnormal morphology, with wrinkles, collapses, and deformations in the bacterial membrane, which leads to bacterial death.

[0116] The morphology of *Porphyromonas gingivalis* in Comparative Examples 2 and 3 was similar to that in Comparative Example 1; the morphology of *Porphyromonas gingivalis* in Examples 1, 3, 5, and 7 was similar to that in Example 2, all exhibiting wrinkling, collapse, and deformation, leading to the death of *Porphyromonas gingivalis*. This result indicates that the fibrous membrane material containing poly-L-lactic acid, after corona polarization treatment, possesses certain electroactivity. Its positively charged side can interact with *Porphyromonas gingivalis*, a periodontitis pathogen, thereby exerting an antibacterial function.

[0117] Example 11: Effects of Guided Tissue Regeneration Membranes on Cells

[0118] The fibrous membranes from Examples 1-7 and Comparative Examples 1-3 were placed in culture plates and sterilized by immersion in 75% alcohol and irradiation under ultraviolet light for 6 hours. They were then rinsed three times with PBS for 10 minutes each time, soaked overnight in PBS, and then incubated with culture medium for 6 hours. 5000 rat bone marrow mesenchymal stem cells were seeded per well onto each fibrous membrane, and culture medium was added to a final volume of 1 mL. The plates were then incubated at 37°C with 5% CO2 for 24 hours. After removing the culture medium, the plates were rinsed twice with sterile saline, then stained with Calcein-AM / PI dye at room temperature in the dark for 15 minutes. The staining was then observed and photographed under a laser confocal microscope. Results from some examples, such as Examples 2, 4, and 6, and Comparative Example 1, are as follows: Figure 13 As shown.

[0119] from Figure 13 As can be seen, rat bone marrow mesenchymal stem cells can grow well on the fibrous membranes in all embodiments and comparative examples, indicating that the material has good biocompatibility.

[0120] Example 12: The effect of guided tissue regeneration membrane on osteogenicity

[0121] In this embodiment, the effect of the guiding tissue regeneration membrane on osteoogenesis was evaluated by detecting the activity of alkaline phosphatase (ALP), an early osteogenic marker, in rat bone marrow mesenchymal stem cells after 14 days of osteogenic induction culture on a fibrous membrane.

[0122] The fibrous membranes from Examples 1-7 and Comparative Examples 1-3 were placed in culture plates and sterilized by immersion in 75% ethanol and irradiation under UV light for 6 hours. They were then rinsed three times with PBS for 10 minutes each time, soaked overnight in PBS, and then incubated for 6 hours in complete culture medium. 5000 rat bone marrow mesenchymal stem cells were seeded per well onto each fibrous membrane, and complete culture medium was added to a final volume of 1 mL. The plates were incubated at 37°C with 5% CO2 until the cell count reached 80%. The complete culture medium was then replaced with osteogenic induction medium, and the culture was continued for 14 days, changing the medium every two days. After 14 days, the fibrous membranes were removed, and ALP activity was measured according to the instructions of the ALP activity assay kit. The experimental results are as follows: Figure 14 As shown.

[0123] See Figure 14 In Example 1 compared to Comparative Example 1 (P = 0.148, no significant difference), corona polarization of the guided tissue regeneration membrane formed solely by electrospinning of poly-L-lactic acid failed to significantly upregulate the osteogenic differentiation level of cells. Example 4 compared to Comparative Example 2, and Example 5 compared to Comparative Example 3, all showed significant differences (**P < 0.01). This indicates that corona polarization treatment enhances the electroactivity of the guided tissue regeneration membrane formed by electrospinning of poly-L-lactic acid and natural polymers, and its negatively charged surface can promote osteogenic differentiation indicators. Comparative Example 2 compared to Comparative Example 1, and Example 4 compared to Example 1, showed extremely high significant differences (***P < 0.001), indicating that natural polymer gelatin is beneficial for osteogenic regeneration.

[0124] As can be seen from Examples 5-7 compared with Comparative Examples 1-3 and Examples 1-4, the bilayer membrane composite followed by polarization can better promote the regeneration of osteogenic tissue (**P values ​​are all less than 0.01, showing significant differences).

[0125] Example 13: Study on in vivo periodontal defect repair guided by tissue regeneration membrane

[0126] To construct a mouse periodontitis model, two composite fiber membranes, Comparative Example 3 and Example 6, were used as experimental groups, as follows: (1) Control group, (2) Untreated defect group, (3) Commercial product. Oral repair membrane (Heal-All), (4) Comparative Example 3 composite fiber membrane, (5) Example 6 composite fiber membrane. Subsequently, materials of appropriate size were implanted, and the expression levels of inflammation-related genes and Micro-CT analysis were performed on the material samples from mice.

[0127] Upregulation of INF-γ and TNF-α expression in immune cells can stimulate macrophages to polarize 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, we selected the expression of three inflammation-related cytokines—IL-1β, IL-6, and INF-γ—in mouse gingival crevicular fluid using real-time quantitative polymerase chain reaction (RT-qPCR) as an important parameter for assessing the level of periodontal inflammation in mice. The results are as follows: Figure 15 As shown in the figure, the expression levels of the three inflammation-related genes in the UT group were significantly increased compared to the Control group. Heal-All ( 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 filling membrane has a certain therapeutic effect on 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 mice in the Heal-All treatment group was still high, indicating that... Oral repair membranes do not have an anti-inflammatory effect. Neither layer of the R / Gel bilayer fiber membrane (composite fiber membrane of Comparative Example 3) has an anti-inflammatory effect. The expression levels of inflammatory genes in the gingival crevicular fluid of this group of mice were similar to those in the Heal-All treatment group, showing high levels of expression, indicating that after treatment with the R / Gel composite fiber membrane, the mice remained in a state of high inflammatory expression without significant regression. In contrast, the electroactive F / Gel-P bilayer composite fiber membrane (composite fiber membrane of Example 6) prepared in this invention has the ability to regulate immune cells and suppress inflammation on both sides. In the gingival crevicular fluid of periodontitis mice after treatment, the expression levels of three inflammatory genes were significantly lower than those in the UT group. Only IL-1β expression showed a significant difference from normal mice at week four, but after 8 weeks of treatment, its expression level was the same as that of normal mice. The expression levels of IL-6 and IFN-γ did not show significant differences from the Control group at either week four or week eight. Considering the combined expression levels of the three inflammation-related genes, this indicates that the electroactive composite fiber membrane of this invention has the ability to eliminate inflammation in periodontitis mice.

[0128] Next, Micro-CT analysis of the maxillary alveolar bone of mice was used to verify the osteogenic effect of the electroactive bilayer fibrous membrane in periodontitis. Figure 16 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. Over time, a small amount of new bone tissue was formed, but it was far from achieving self-healing. The oral repair membrane, as a commercially available membrane, possesses a certain ability to promote bone regeneration and exhibits some new bone formation after Heal-All treatment. However, some tooth roots are still clearly visible around the second molars in mice, indicating that complete repair is not achieved, and the bone density is far below normal levels. The composite fiber membrane in Comparative Example 3, due to the addition of an inner gelatin layer, also possesses certain osteogenic properties, and its repair level for bone resorption in periodontitis mice is comparable to... Similar to oral repair membranes, these membranes can, to some extent, increase alveolar bone density and reduce the CEJ-ABC distance in mice with periodontitis. The electroactive composite fiber membrane in Example 6, on the one hand, inhibits inflammation, and on the other hand, promotes osteogenic formation, thus having a more significant effect on in-situ bone repair. More new bone tissue was observed around the second molar. The reconstructed 3D images also show that the new bone tissue in this group has a similar structure and morphology to the normal group, and its bone density and CEJ-ABC distance are almost identical to those of normal mice (e.g., ...). Figure 17 (As shown).

[0129] In summary, the multifunctional guided tissue regeneration membrane provided by this invention has an electroactive bilayer asymmetric structure. It not only possesses intrinsic antibacterial properties, exhibiting antibacterial activity against common Gram-negative bacteria such as *Escherichia coli*, but also maintains antibacterial activity against periodontitis pathogens such as *Porphyromonas gingivalis*. This avoids the side effects caused by the release of antibacterial drugs or agents into the human body, solving the problem of limited antibacterial activity in existing guided tissue regeneration membranes. It also eliminates the need for loading antibacterial drugs or agents, saving on their cost. Furthermore, when placed at the periodontal bone defect site, the local pressure generated during oral chewing allows the regeneration membrane to further exert its highly efficient and long-lasting antibacterial properties. The polarization of the composite bilayer membrane further promotes the adhesion, proliferation, and differentiation of bone or periodontal ligament cells, better meeting the requirements for regeneration and repair of periodontal bone defects in inflammatory environments caused by periodontitis.

[0130] 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. A multifunctional guided tissue regeneration membrane, characterized in that, It consists of an outer fibrous layer with antibacterial properties and an inner fibrous layer with osteogenic properties. The outer fibrous layer is formed by electrospinning poly-L-lactic acid, and the inner fibrous layer is formed by electrospinning poly-L-lactic acid and a natural polymer, which is either collagen or gelatin. The outer fibrous layer and the inner fibrous layer are pressed together to form a composite fibrous layer. The composite fibrous layer is corona polarized so that the outer fibrous layer is enriched with positive charge and the inner fibrous layer is enriched with negative charge. The preparation of the regenerated membrane includes the following steps: (1) The outer fiber layer is obtained by electrospinning a poly-L-lactic acid solution dissolved in an organic solvent. After electrospinning, the fiber layer is annealed. The annealing process is to first anneal the electrospinned fiber layer at 100-110℃ for 8-12 hours, then naturally cool it to room temperature, and then anneal it at 155-165℃ for 8-12 hours and then naturally cool it to room temperature. (2) After mixing the natural polymer solution dissolved in the organic solvent with the poly-L-lactic acid solution dissolved in the organic solvent evenly, electrospinning is performed, and cross-linking is carried out under suitable conditions to obtain the inner fiber layer. (3) Press the inner fiber layer and the outer fiber layer into a composite fiber membrane, and obtain a multifunctional guided tissue regeneration membrane after corona polarization treatment. The conditions for corona polarization treatment are: voltage of 8-15 kV and polarization time of 15-30 minutes.

2. The multifunctional guided tissue regeneration membrane according to claim 1, characterized in that, The outer fiber layer has a poly-L-lactic acid concentration of 0.05-0.4 g / mL in an organic solvent; and / or, the inner fiber layer has a poly-L-lactic acid concentration of 0.05-0.4 g / mL in an organic solvent, a natural polymer concentration of 0.05-0.4 g / mL in an organic solvent, and a poly-L-lactic acid to natural polymer weight ratio of 1:9-9:

1.

3. The multifunctional guided tissue regeneration membrane according to claim 1, characterized in that, The diameter of the fibers in the inner fiber layer and / or the outer fiber layer is 500-1500 nm.

4. The multifunctional guided tissue regeneration membrane according to any one of claims 1-3, characterized in that, The electrospinning process conditions in (1) and / or (2) are as follows: voltage is 10-30 kV, receiving distance is 10-30 cm, feed speed is 0.7-2.0 mL / h, and receiving roller rotation speed is 300-1000 rpm.

5. The multifunctional guided tissue regeneration membrane according to claim 4, characterized in that, The conditions suitable for crosslinking refer to crosslinking in a 90% ethanol solution of 1,3-dimethylaminopropyl-3-ethyldiimide / N-hydroxysuccinimide.

6. The use of the multifunctional guided tissue regeneration membrane according to any one of claims 1-3 in the preparation of an antibacterial guided tissue regeneration membrane for the repair of periodontal tissue defects.

7. The use of the multifunctional guided tissue regeneration membrane according to any one of claims 1-3 in the preparation of repair materials that promote bone regeneration, characterized in that, The promotion of bone regeneration includes at least one of the following: a. Increased bone density; b. Reduce the distance from the enamel-dentin junction to the alveolar bone ridge; c. Promotes the formation of new bone tissue; d. Promotes the expression of osteogenic-related genes.

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