Degradable nerve repair membrane and preparation method thereof

Abstract

The invention relates to a degradable nerve repair membrane and a preparation method thereof, and belongs to the technical field of nerve repair membranes, the degradable nerve repair membrane comprises a biological composite structure inner layer, a support outer layer and a bonding layer, and the biological composite structure inner layer and the support outer layer are bonded through the bonding layer. According to the invention, polypyrrole with excellent electrochemical activity, environmental stability, controllable conductivity and poor mechanical properties and collagen nanofibers with high biocompatibility and capable of forming a nano-scale fiber network are adopted to form a conductive-biological composite structure as an inner layer through electrostatic spinning, and the PLGA porous membrane is prepared through 3D printing. A PLGA porous membrane with parallel axial microchannels on the surface is used as an outer layer, so that the membrane body structure can effectively guide directional growth of axons while ensuring conductivity, accurate preparation can be realized according to needs, the degradation period is matched with the nerve regeneration rate, and the degradation time can be regulated and controlled through the proportion of a copolymer; the repairing effect is prevented from being influenced by too fast or too slow degradation.

Description

Technical Field

[0001] This invention relates to the field of nerve repair membrane technology, and in particular to a biodegradable nerve repair membrane and its preparation method. Background Technology

[0002] Peripheral nerve repair membranes are biomedical materials used to promote the repair of peripheral nerves after injury. They primarily support nerve regeneration by providing a protective barrier and a suitable microenvironment.

[0003] In existing technologies, the nerve repair membranes made of natural polymer materials that have been commercially available have good biocompatibility and low immunogenicity, but their drawbacks are insufficient mechanical strength, rapid degradation, poor tear resistance in actual clinical applications, and easy collapse of hollow catheters, which hinders the nerve repair process. Although the nerve repair catheters made of synthetic biodegradable polymer materials have relatively better mechanical strength and slower degradation performance, they still have the disadvantages of the hydrophobicity of polyester itself and relatively poor biocompatibility, which are not conducive to cell attachment and migration and are prone to causing rejection reactions.

[0004] To effectively control the degradation rate and ensure conductivity to guide axonal directional growth, a biodegradable neural repair membrane and its preparation method are proposed. The membrane utilizes polypyrrole (PPG), which exhibits excellent electrochemical activity, environmental stability, and tunable conductivity but poor mechanical properties, and collagen nanofibers with high biocompatibility that can form a nanoscale fiber network. These nanofibers are electrospun to form a conductive-biocomposite structure as the inner layer. A PLGA porous membrane is then prepared using 3D printing, and an outer layer is formed using a PLGA porous membrane with parallel axial microchannels on its surface. This membrane structure ensures conductivity while effectively guiding axonal directional growth. It can be precisely prepared as needed, and the degradation cycle matches the nerve regeneration rate. Furthermore, the degradation time can be controlled by adjusting the copolymer ratio to prevent excessively rapid or slow degradation from affecting the repair effect. Summary of the Invention

[0005] This invention provides a biodegradable nerve repair membrane and its preparation method, which solves the problems mentioned in the background art. It can effectively guide the directional growth of axons while ensuring conductivity. It can be precisely prepared as needed, and the degradation cycle is matched with the nerve regeneration rate to prevent degradation that is too fast or too slow from affecting the repair effect.

[0006] The present invention provides the following solution to the above-mentioned technical problems: a biodegradable nerve repair membrane, comprising a biocomposite inner layer, a supporting outer layer, and an adhesive layer, wherein the biocomposite inner layer and the supporting outer layer are bonded together by the adhesive layer; the biocomposite inner layer is composed of polypyrrole and collagen nanofibers, and NGF@PLGA microspheres are loaded on the surface of the biocomposite inner layer; the supporting outer layer is composed of a PLGA porous membrane, wherein parallel axial microchannels are provided on the surface of the PLGA porous membrane; the LA / GA ratio in the PLGA porous membrane is 75:25; and the adhesive layer is a genipin crosslinked chitosan layer.

[0007] The preparation method includes the following steps:

[0008] S1: Preparation of the inner layer spinning solution for the biocomposite structure: Type I collagen is magnetically stirred and dissolved in a solvent for four hours. The solvent is a mixture of 0.5M acetic acid solution and ethanol in a volume ratio of 4:1. Polypyrrole nanoparticles and NGF@PLGA microspheres are added. The polypyrrole nanoparticles have a particle size of 50nm. The mixture is ultrasonically dispersed using an ultrasonic disperser for 30 minutes.

[0009] S2: Preparation of the inner layer of the biocomposite structure: The inner layer spinning solution after ultrasonic dispersion is placed in an electrospinning machine. The high-speed rotating drum generates tangential force, which stretches the jet during flight and arranges it along the circumference of the drum. The fibers are collected on the rotating drum to form the inner layer of the biocomposite structure.

[0010] S3: 3D printed support outer layer: PLGA (Mw=80kDa) is dissolved in hexafluoroisopropanol (concentration 15wt%) and loaded into a 3D printer. The support outer layer is constructed by extrusion through a microfluidic printhead with a pore size of 100μm and a construction temperature of 50℃. Axial microchannels are simultaneously laser-engraved.

[0011] S4: Interface cross-linking: Spray 1 wt% genipin cross-linked chitosan layer between the inner layer and the outer supporting layer of the biocomposite structure, and then perform ultraviolet curing to enhance the interlayer bonding force to achieve interface cross-linking;

[0012] S5: Freeze-drying sterilization and packaging: The finished nerve repair membrane is vacuum dried at -80℃ in a vacuum drying oven for 24 hours to retain its porous structure. It is then sterilized using an ethylene oxide sterilizer at a temperature of 50℃, a humidity of 60%, and a time of 6 hours. After sterilization, it is sealed and packaged.

[0013] Based on the above technical solution, the present invention can be further improved as follows.

[0014] Furthermore, the NGF@PLGA microspheres have a particle size of 5-10 μm and a microsphere encapsulation rate of >90%, enabling long-term sustained release of growth factors through microsphere encapsulation technology.

[0015] Furthermore, the porosity of the PLGA porous membrane is 70-80%, which can effectively support the inner layer of the biocomposite structure, and the pores can be used to release growth factors.

[0016] Furthermore, the parallel axial microchannels are 30-50 μm wide, 20-30 μm deep, and 100 μm apart. The microchannels can promote Schwann cell migration and vascularization while blocking the invasion of scar tissue.

[0017] Furthermore, the thickness of the genipin cross-linked chitosan layer is 150-200 μm, the polypyrrole content of the inner layer of the biocomposite structure is 3-5% of the collagen nanofiber mass, and the conductivity is ≥10. -2 The introduction of inner polypyrrole (PPy) increases the conductivity of the repair film to 10 S / cm. -2 With an S / cm ratio far exceeding the insulation properties of pure collagen membranes, it can transmit electrical signals and activate the metabolic activity of nerve cells such as Schwann cells.

[0018] Furthermore, the NGF@PLGA microspheres are prepared by a double emulsion method. The aqueous phase contains 0.1 mg / mL NGF and 5 wt% PLGA, while the oil phase is dichloromethane. The microsphere encapsulation technology enables long-term sustained release of growth factors. NGF promotes the extension and directional growth of neuronal axons by activating TrkA receptors, shortens the repair cycle of nerve defects, promotes axonal regeneration, supports Schwann cell activity, and inhibits neuronal apoptosis.

[0019] Furthermore, in step S2, the electrospinning apparatus operates at a voltage of 18kV, a receiving distance of 15cm, a receiver rotation speed of 2000rpm, an injection rate of 1mL / h, an operating temperature of 25℃±2℃, an operating humidity of 55%±10%RH, and is operated in a fume hood.

[0020] Furthermore, in step S4, the molar ratio of chitosan to genipin in the genipin crosslinked chitosan layer is 1:0.2, and the ultraviolet curing parameters are 365nm wavelength, irradiation intensity of 10mW / cm², and curing time of 60s.

[0021] The beneficial effects of this invention are as follows: This invention provides a biodegradable nerve repair membrane and its preparation method, which has the following advantages:

[0022] 1. The synergistic effect of conductivity and controllable degradation, with the introduction of inner polypyrrole enabling the repair membrane to achieve a conductivity of 10. -2With an S / cm ratio far exceeding the insulation of pure collagen membranes, it can transmit electrical signals and activate the metabolic activity of nerve cells. The degradation cycle of the outer PLGA layer with LA / GA=75:25 is 6-8 months, which is highly compatible with the regeneration rate of peripheral nerves (usually 6-12 months). This avoids the structural collapse or foreign body residue problems caused by the excessively rapid degradation of traditional single materials such as pure collagen (3-6 months) or the excessively slow degradation of PLA.

[0023] 2. Guiding axonal directional growth: The inner layer of the biocomposite structure prepared by electrospinning technology is equipped with parallel microchannels with a width of 50μm and a depth of 30μm on the outer layer, which promotes Schwann cell migration and vascularization, while blocking the invasion of scar tissue. The loaded NGF@PLGA microspheres can achieve 14-day sustained release of nerve growth factor NGF, which can effectively maintain the local drug concentration and avoid cytotoxicity caused by sudden changes in concentration.

[0024] 3. The inner layer electrospinning preparation can ensure high fiber orientation, and the outer layer 3D printing combined with laser engraving technology can accurately construct microchannels and ensure preparation accuracy. This process has strong compatibility and is suitable for large-scale production. The use of genipin cross-linked chitosan layer to replace traditional glutaraldehyde cross-linking can effectively reduce cytotoxicity while enhancing the interlayer bonding strength.

[0025] 4. The material's complete degradation time is well-matched to the nerve regeneration cycle error, eliminating the need for surgical removal and effectively reducing treatment costs. The overall preparation process can be customized to different diameters and bifurcation structures, making it suitable for complex injury scenarios such as brachial plexus nerve and diabetic peripheral neuropathy.

[0026] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Specific embodiments of the present invention are given in detail below with reference to the accompanying drawings. Attached Figure Description

[0027] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings:

[0028] Figure 1 This is a flowchart illustrating the preparation method of a biodegradable nerve repair membrane and its preparation method, as provided in an embodiment of the present invention. Detailed Implementation

[0029] The following is in conjunction with the appendix Figure 1The principles and features of the present invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. The invention is described more specifically in the following paragraphs by way of example with reference to the accompanying drawings. The advantages and features of the invention will become clearer from the following description and claims. It should be noted that the drawings are in a very simplified form and use non-precise proportions, and are only used to facilitate and clarify the illustration of the embodiments of the invention.

[0030] It should be noted that when a component is described as "fixed to" another component, it can be directly on the other component or may have a component in between. When a component is considered "connected to" another component, it can be directly connected to the other component or may have a component in between. When a component is considered "set on" another component, it can be directly set on the other component or may have a component in between. The terms "vertical," "horizontal," "left," "right," and similar expressions used in this document are for illustrative purposes only.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0032] like Figure 1 As shown, this invention provides a biodegradable nerve repair membrane, comprising a biocomposite inner layer, a supporting outer layer, and an adhesive layer. The biocomposite inner layer and the supporting outer layer are bonded together by the adhesive layer. The biocomposite inner layer is composed of polypyrrole and collagen nanofibers, and NGF@PLGA microspheres are loaded on the surface of the biocomposite inner layer. The supporting outer layer is composed of a PLGA porous membrane with parallel axial microchannels on the surface of the PLGA porous membrane. The LA / GA ratio in the PLGA porous membrane is 75:25. The adhesive layer is a genipin crosslinked chitosan layer.

[0033] Preferably, the NGF@PLGA microspheres have a particle size of 5-10 μm and a microsphere encapsulation rate of >90%, and the growth factor can be released slowly and continuously through microsphere encapsulation technology.

[0034] Preferably, the porosity of the PLGA porous membrane is 70-80%. The PLGA porous membrane can effectively support the inner layer of the biocomposite structure, and the pores can be used to release growth factors.

[0035] Preferably, the parallel axial microchannels are 30-50 μm wide, 20-30 μm deep, and 100 μm apart. The microchannels can promote Schwann cell migration and vascularization while blocking the invasion of scar tissue.

[0036] Preferably, the thickness of the genipin cross-linked chitosan layer is 150-200 μm, the polypyrrole content of the inner layer of the biocomposite structure is 3-5% of the mass of the collagen nanofibers, and the conductivity is ≥10. -2 The introduction of inner polypyrrole (PPy) increases the conductivity of the repair film to 10 S / cm. -2 With an S / cm ratio far exceeding the insulation properties of pure collagen membranes, it can transmit electrical signals and activate the metabolic activity of nerve cells such as Schwann cells.

[0037] Preferably, NGF@PLGA microspheres are prepared by double emulsion method, with an inner aqueous phase containing 0.1 mg / mL NGF and 5 wt% PLGA, and an oil phase of dichloromethane. The microsphere encapsulation technology enables long-term sustained release of growth factors. NGF promotes the extension and directional growth of neuronal axons by activating TrkA receptors, shortens the repair cycle of nerve defects, promotes axonal regeneration, supports Schwann cell activity, and inhibits neuronal apoptosis.

[0038] The specific working principle and usage method of this invention are as follows:

[0039] S1: Preparation of the inner layer spinning solution for the biocomposite structure: Type I collagen was magnetically stirred and dissolved in a solvent for four hours. The solvent used was a mixture of 0.5M acetic acid solution and ethanol in a volume ratio of 4:1. Polypyrrole nanoparticles and NGF@PLGA microspheres were added. The polypyrrole nanoparticles had a particle size of 50nm. The mixture was ultrasonically dispersed using an ultrasonic disperser for 30 minutes.

[0040] S2: Preparation of the inner layer of the biocomposite structure: The inner layer spinning solution after ultrasonic dispersion was placed in an electrospinning apparatus (MSK-NFES-4 electrospinning system). The high-speed rotating drum generated tangential force, which stretched the jet during flight and arranged it along the circumference of the drum. The fibers were collected on the rotating drum to form the inner layer of the biocomposite structure. The electrospinning apparatus operated at a voltage of 18kV, a receiving distance of 15cm, a receiver rotation speed of 2000rpm, an injection rate of 1mL / h, an operating temperature of 25℃±2℃, and an operating humidity of 55%±10%RH. The operation was carried out in a fume hood.

[0041] S3: 3D printed support outer layer: PLGA (Mw=80kDa) is dissolved in hexafluoroisopropanol (concentration 15wt%) and loaded into a 3D printer. The support outer layer is constructed by extrusion through a microfluidic printhead with a pore size of 100μm and a construction temperature of 50℃. Axial microchannels are simultaneously laser-engraved.

[0042] S4: Interface cross-linking: Spray 1 wt% genipin cross-linked chitosan layer between the inner layer and the outer supporting layer of the biocomposite structure, and then perform UV curing to enhance the interlayer bonding force to achieve interface cross-linking. The molar ratio of chitosan to genipin in the genipin cross-linked chitosan layer is 1:0.2. The UV curing parameters are 365 nm wavelength, irradiation intensity 10 mW / cm², and curing time 60 s.

[0043] S5: Freeze-drying sterilization and packaging: The finished nerve repair membrane is vacuum dried at -80℃ in a vacuum drying oven for 24 hours to retain its porous structure. It is then sterilized using an ethylene oxide sterilizer at a temperature of 50℃, a humidity of 60%, and a time of 6 hours. After sterilization, it is sealed and packaged.

[0044] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Those skilled in the art can readily implement the present invention based on the accompanying drawings and the above description. However, any modifications, alterations, or variations made by those skilled in the art without departing from the scope of the present invention, utilizing the disclosed technical content, are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, or variations made to the above embodiments based on the essential technology of the present invention are still within the protection scope of the present invention.

Claims

1. A biodegradable neural repair membrane, comprising a biocomposite inner layer, a supporting outer layer, and an adhesive layer, characterized in that: The inner layer and the supporting outer layer of the biocomposite structure are bonded together by an adhesive layer. The inner layer of the biocomposite structure is composed of polypyrrole and collagen nanofibers, and NGF@PLGA microspheres are loaded on the surface of the inner layer. The supporting outer layer is composed of a PLGA porous membrane with parallel axial microchannels on the surface of the PLGA porous membrane. The LA / GA ratio in the PLGA porous membrane is 75:

25. The adhesive layer is a genipin crosslinked chitosan layer. The preparation method includes the following steps: S1: Preparation of the inner layer spinning solution for the biocomposite structure: Type I collagen is magnetically stirred and dissolved in a solvent for four hours. The solvent is a mixture of 0.5M acetic acid solution and ethanol in a volume ratio of 4:

1. Polypyrrole nanoparticles and NGF@PLGA microspheres are added. The polypyrrole nanoparticles have a particle size of 50nm. The mixture is ultrasonically dispersed using an ultrasonic disperser for 30 minutes. S2: Preparation of the inner layer of the biocomposite structure: The inner layer spinning solution after ultrasonic dispersion is placed in an electrospinning machine. The high-speed rotating drum generates tangential force, which stretches the jet during flight and arranges it along the circumference of the drum. The fibers are collected on the rotating drum to form the inner layer of the biocomposite structure. S3: 3D printed support outer layer: PLGA (Mw=80kDa) is dissolved in hexafluoroisopropanol (concentration 15wt%) and loaded into a 3D printer. The support outer layer is constructed by extrusion through a microfluidic printhead with a pore size of 100μm and a construction temperature of 50℃. Axial microchannels are simultaneously laser-engraved. S4: Interface cross-linking: Spray 1 wt% genipin cross-linked chitosan layer between the inner layer and the outer supporting layer of the biocomposite structure, and then perform ultraviolet curing to enhance the interlayer bonding force to achieve interface cross-linking; S5: Freeze-drying sterilization and packaging: The finished nerve repair membrane is vacuum dried at -80℃ in a vacuum drying oven for 24 hours to retain its porous structure. It is then sterilized using an ethylene oxide sterilizer at a temperature of 50℃, a humidity of 60%, and a time of 6 hours. After sterilization, it is sealed and packaged.

2. The biodegradable neural repair membrane according to claim 1, characterized in that, The NGF@PLGA microspheres have a particle size of 5-10 μm and an encapsulation efficiency of >90%.

3. The biodegradable neural repair membrane according to claim 1, characterized in that, The porosity of the PLGA porous membrane is 70-80%.

4. The biodegradable neural repair membrane according to claim 1, characterized in that, The parallel axial microchannels are 30-50 μm wide, 20-30 μm deep, and 100 μm apart.

5. The biodegradable neural repair membrane according to claim 1, characterized in that, The thickness of the genipin crosslinked chitosan layer is 150-200 μm, and the polypyrrole content of the inner layer of the biocomposite structure is 3-5% of the collagen nanofiber mass, with a conductivity ≥10. -2 S / cm.

6. The biodegradable neural repair membrane according to claim 1, characterized in that, The NGF@PLGA microspheres were prepared by double emulsion method, with the inner aqueous phase containing 0.1 mg / mL NGF and 5 wt% PLGA, and the oil phase being dichloromethane.

7. The biodegradable neural repair membrane according to claim 1, characterized in that, In step S2, the electrospinning apparatus operates at a voltage of 18kV, a receiving distance of 15cm, a receiver rotation speed of 2000rpm, an injection rate of 1mL / h, an operating temperature of 25℃±2℃, an operating humidity of 55%±10%RH, and is operated in a fume hood.

8. The biodegradable neural repair membrane according to claim 1, characterized in that, In step S4, the molar ratio of chitosan to genipin in the genipin crosslinked chitosan layer is 1:0.2, and the UV curing parameters are 365nm wavelength, irradiation intensity 10mW / cm², and curing time 60s.

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