Micro-nano fiber composite biomembrane based on electrospinning technology, and preparation method and application thereof

Abstract

The application discloses a kind of micro-nano fiber composite biological membranes based on electrospinning technology, by electrospinning process by uniformly distributed micron fiber and uniformly distributed nanometer fiber alternately stacked composite formation, micro-nano fiber composite biological membrane is micron fiber layer formed by micron fiber and nanofiber layer formed by nanometer fiber alternately stacked and formed porous membrane structure, the porous membrane structure thickness is 50-100 μm, average pore size is 2.4-3.6 μm, micron fiber diameter range is 1-3 μm;Nanometer fiber diameter range is 50-1000nm, micro-nano fiber membrane breaking elongation is greater than or equal to 1.2%, tensile strength is 5.1-7MPa, Young's modulus is 1313.2-2442.6MPa.By regulating a concentration and b concentration of A polymer at any time, the distribution of micron fiber and nanometer fiber in micro-nano fiber composite biological membrane can be regulated at any time, so as to regulate the specific surface area of micro-nano fiber membrane, to obtain adjustable specific surface area, enhanced mechanical properties, and simple and flexible technology, spinning process is easy to control.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, and particularly relates to a micro-nano fiber composite biomembrane based on electrospinning technology and a preparation method and application thereof. BACKGROUND

[0002] Electrospinning technology refers to a process in which a polymer solution or melt is jetted and stretched into fibers. It is a new method for preparing polymer nanofibers, and can prepare ultrafine fibers with a nanometer level diameter, and the minimum diameter can be 1 nm. Compared with polymer fibers prepared by traditional methods, polymer nanofibers prepared by electrospinning have the characteristics of simple equipment, easy operation and high efficiency, and the ultrafine fiber membrane prepared by electrospinning has a large specific surface area, high porosity and light weight, and has potential application value in many fields such as tissue engineering, drug and catalyst carriers, wound dressings, filtration, sensors, templates, protective fabrics, nano electronic components and the like.

[0003] In the past few decades, scientific and technological progress has given rise to many innovative single or composite tissue scaffolds for use as drug carriers, filling materials, guiding materials and the like to achieve rapid healing and regenerative repair of damaged tissues. Micro-nano fiber membranes have been widely used in drug carriers, biological scaffolds, tissue engineering and chemical industries due to their high specific surface area. In tissue repair applications, the fiber scaffolds prepared by micro / nano fibers can simulate the structure of human extracellular matrix, provide a micro-growth environment for cell adhesion, proliferation and differentiation, and play an important role in the repair and regeneration of damaged tissues or organs. The micro-nano fibers prepared by electrospinning form a grid-like structure that is stacked on each other during the collection process. This structure is very similar to the biological structure of the natural extracellular matrix, and can well simulate the inherent protein fibers of the natural extracellular matrix in terms of fiber size, morphology and structure, and performance. The specific surface area and porosity can directly affect cell adhesion and proliferation, and a large number of articles have shown that the smaller the average pore size of the micro-nano fiber membrane, the larger the specific surface area, and the micro-nano fiber can significantly promote cell proliferation and differentiation, extracellular matrix deposition and remodeling, and new tissue regeneration. Therefore, the research on micro-nano fibers is widely applied in biological scaffolds.

[0004] The main influencing factors of the function of micro-nano fiber membranes for wound dressings, filling materials and biological scaffolds include specific surface area, pore size, porosity and mechanical properties. At present, there are many micro-nano fiber membranes prepared by electrospinning technology for medical biological products, including the following technologies:

[0005] CN114713038A discloses a micro-nanofiber membrane, which is mainly composed of polymeric nanofibers, the polymer in the polymeric nanofibers includes polyvinylidene fluoride and polystyrene, and the micro-nanofiber membrane includes nanoscale fibers and micrometer scale fibers. The micro-nanofiber membrane has better hydrophobic and oleophilic properties, can effectively remove oil from oil-containing water, and can also filter and remove solid particles in an oil-water mixture. In addition, the application also discloses a preparation method and application of the micro-nanofiber membrane.

[0006] CN116617438A discloses a micro-nanofiber membrane for wound dressing. The invention is a porous membrane structure formed by random stacking of micro-nanofibers, the membrane thickness is adjustable, the micro-nanofibers are composed of chitosan and polyethylene oxide, the distribution of chitosan and polyethylene oxide inside and outside the fiber is different, the content of chitosan in the outer layer is greater than that in the inner layer, and the content of chitosan in the outer layer is ≥70%, and the average diameter of the fibers in the micro-nanofiber membrane is 100-1500nm. The invention has good mechanical properties, thermal stability and antioxidant activity, etc.; the outer layer of the fiber is mainly chitosan, and the antibacterial rate of staphylococcus aureus and escherichia coli is ≥95% without adding other antibacterial agents; no chemical crosslinking agent is used for crosslinking, the structure has good stability, and has no biological toxicity, which does not reduce the biocompatibility of the fiber membrane; the stability in water is ≥10 days, it does not adhere to the wound and does not cause secondary damage to the wound, and it can be used for wound dressing.

[0007] CN113737393A discloses an electrospun nanofiber membrane and a preparation method thereof, which is used to solve the problems of poor mechanical strength, short service life, poor uniformity of fibers, consistency of orientation and stability of fiber web of the current electrospun composite nanofiber material. The electrospun nanofiber membrane is prepared by high-voltage electrospinning of a spinning solution, and the spinning solution is a blended solution composed of regenerated silk fibroin, polyvinyl alcohol and polylactic acid with a mass ratio of 75-85:10-20:5 dissolved in a mixed solvent of trifluoroacetic acid and dichloromethane with a volume ratio of 7:3. The invention blends and spins regenerated silk fibroin, polyvinyl alcohol and polylactic acid, and establishes a reasonable mass ratio parameter of regenerated silk fibroin, polyvinyl alcohol and polylactic acid, so as to improve the spinnability of silk fibroin and prepare an electrospun regenerated silk fibroin / polyvinyl alcohol / polylactic acid composite nanofiber membrane with good mechanical properties.

[0008] From the above patent technology, the current technical research focus of nanofiber membrane and micro-nanofiber membrane is mainly the selection of fiber material, dosage and the like, and the corresponding effect is obtained in the filtration and medical material aspects by the function combination of different materials and the dosage. However, in fact, in addition to the component ratio of the spinning solution, the structure of the membrane itself is also an important influencing factor in promoting cell proliferation and differentiation and promoting tissue repair and regeneration function of micro-nanofiber membrane.

[0009] Whether it is a nanofiber membrane or a micro-nanofiber membrane, in order to maximize the promotion of cell proliferation and differentiation, extracellular matrix deposition and remodeling, and new tissue regeneration in the application direction of wound dressings, filling materials and biological scaffolds, and to effectively control the specific surface area, pore size, porosity and mechanical properties of the micro-nanofiber membrane structure, it is relatively difficult in the current micro-nanofiber membrane preparation field. Because the electrospinning process is affected by many parameters, it can be roughly divided into spinning solution parameters, process parameters and environmental parameters. Among them, the spinning solution parameters mainly include the properties of the polymer raw material and the concentration, conductivity, molecular weight and solvent of the spinning solution; the process parameters mainly include the applied voltage (positive and negative voltage), flow rate, receiving distance; the environmental parameters mainly include temperature and humidity. Studies have shown that the above parameters not only significantly affect the appearance and morphology of the fiber, but also interact with each other, and a reasonable set of conditions needs to be established to obtain an ideal membrane structure. In the process of electrospinning using two or more polymer raw material solutions or melts to prepare composite nanofibers, crosslinking points of different polymer raw materials are prone to break, thereby causing the following shortcomings of composite nanofiber materials: first, the mechanical strength is low and the service life is short; second, the uniformity, orientation consistency and stability of the fiber web are poor.

[0010] It can be seen that, on the basis of the existing micro-nanofiber membrane, by controlling the structure of the micro-nanofiber membrane, the mechanical properties can be improved while taking into account the small pore size, high specific surface area and porosity of the micro-nanofiber membrane, and the purpose of assisting in promoting the process of cell proliferation and differentiation and the repair and regeneration of damaged tissues or organs has become a difficult problem for technical personnel in the field of biological scaffold medical treatment. SUMMARY

[0011] In view of the deficiencies of the prior art, the technical problem to be solved by the present application is to provide a micro-nanofiber composite biological membrane based on electrospinning technology, which can be accurately controlled in terms of specific surface area, pore size and porosity, and can significantly improve the mechanical properties, and can effectively improve the cell proliferation and differentiation and the repair and regeneration effect of damaged tissues or organs, and a preparation method and application thereof.

[0012] To solve the above technical problems, the technical scheme adopted by the present application is: a micro-nano fiber composite biological membrane based on electrospinning technology, which is formed by alternating stacking and compounding of uniformly distributed micron fibers and uniformly distributed nanometer fibers through an electrospinning process, the micro-nano fiber composite biological membrane is a porous membrane structure formed by alternating stacking of micron fiber layers formed by micron fibers and nanometer fiber layers formed by nanometer fibers, the porous membrane structure has a thickness of 50-100 μm, an average pore size of 2.4-3.6 μm, and the micron fibers have a diameter range of 1-3 μm; the nanometer fibers have a diameter range of 50-1000 nm, the micro-nano fiber membrane has an elongation at break of ≥1.2%, a tensile strength of 5.1-7 MPa, and a Young's modulus of 1313.2-2442.6 MPa.

[0013] The micro-nano fiber composite biological membrane based on electrospinning technology described above is prepared by synchronously spinning an a-concentration spinning precursor prepared from an A polymer and a b-concentration spinning precursor prepared from the polymer in an electrospinning device, the raw material of the nanometer fibers is an a-concentration spinning solution prepared from the A polymer, the raw material of the micron fibers is a b-concentration spinning solution prepared from the A polymer, the mass percentage of the a-concentration spinning solution is 2%-10%, and the mass percentage of the b-concentration spinning solution is 20%.

[0014] The micro-nano fiber composite biological membrane based on electrospinning technology described above, wherein the A polymer is any one of polycaprolactone, polyvinyl alcohol, polypropylene pyrrolidone, polyglycolide-lactide, polylactic acid, and polylactide-glycolide.

[0015] The micro-nano fiber composite biological membrane based on electrospinning technology described above, wherein the electrospinning device comprises a double-power high-voltage electrostatic generator and a receiving drum, two spinning nozzles for preparing micron fiber layers and nanometer fiber layers are symmetrically arranged on the two sides of the receiving drum, and the positive and negative electrodes of the high-voltage electrostatic generator are connected to the two spinning nozzles, respectively.

[0016] The micro-nano fiber composite biological membrane based on electrospinning technology described above, wherein the spinning voltage of the double-power high-voltage electrostatic generator is -12 kV-+12 kV, the receiving distances on the two sides of the receiving drum are 16 cm, respectively, the extrusion speed of the a-concentration and b-concentration spinning solutions is 0.8 mL / h, the spinning temperature is 20°C, and the relative humidity is 40%.

[0017] The micro-nano fiber composite biological membrane based on electrospinning technology described above, wherein the mass percentage of the a-concentration spinning solution prepared from the A polymer is 6%.

[0018] A preparation method of a micro-nano fiber composite biological membrane based on electrospinning technology, characterized by comprising the following steps:

[0019] (1) Prepare a spinning solution with a concentration of a of 2-10% by mass and a concentration of b of 20% by mass, which is prepared from the A polymer, and reserve it;

[0020] (2) Connect the prepared spinning solution to the two spinning nozzles of the electrospinning device respectively, and connect the positive and negative poles of the double-power high-voltage static generator to the two spinning nozzles respectively.

[0021] (3) Turn on the double-power high-voltage static generator to provide opposite high-voltage static electricity to the two spinning nozzles, and perform synchronous spinning under the conditions of a certain spinning voltage, receiving distance, and spinning solution extrusion speed;

[0022] (4) Collect the micron and nanometer fibers formed on both sides of the receiving drum by the two spinning nozzles respectively;

[0023] (5) Form micron fiber mesh and nanometer fiber mesh from the micron fibers and nanometer fibers respectively, and continuously and repeatedly stack them alternately;

[0024] (6) According to the thickness requirement, obtain a micron and nanometer fiber composite biomembrane with continuous, uniform distribution, and no obvious beading.

[0025] The above-mentioned method for preparing a micron and nanometer fiber composite biomembrane based on electrospinning technology, in step (3), the spinning voltage is -12kV-+12kV, the receiving distance is 16cm, the spinning solution extrusion speed is 0.8mL / h, the spinning temperature is 20℃, and the relative humidity is 40%.

[0026] The above-mentioned method for preparing a micron and nanometer fiber composite biomembrane based on electrospinning technology, in step (4), the receiving drum rotation speed is 300rpm.

[0027] Application of the micron and nanometer fiber composite biomembrane based on electrospinning technology in preparing wound dressings for tissue damage wounds, tendon patches, and hernia repair patch materials.

[0028] The application has the advantages that the micro-nano fiber composite biological membrane is prepared by a concentration a of a spinning precursor prepared from an A polymer and a concentration b of a spinning precursor prepared from the polymer in a micro-nano fiber membrane prepared in an improved electrospinning device. The distribution of the micro-nano fiber membrane in the micro-nano fiber membrane can be adjusted at any time by adjusting the concentration a of the A polymer and the concentration b at any time, so that the specific surface area of the micro-nano fiber membrane can be adjusted, the mechanical properties are enhanced, and the micro-nano fiber membrane is simple in technology and easy to control in the spinning process. The micro-nano fiber composite biological membrane with the special structure of the application makes up for the defects of the limited mechanical properties of the traditional micro-nano fiber membrane, and can realize the high directional fiber structure stacking of the micro-nano fiber. The combination of the micro-nano fiber and the nano fiber is equivalent to that the nano fiber fills the gap between the micro-nano fiber and reduces the pore size between the micro-nano fiber, so as to increase the specific surface area of the micro-nano fiber membrane, and the micro-nano fiber and the nano fiber form a micro-nano fiber network and a nano fiber network, respectively, and a continuous and repeated alternating stacking structure, so as to improve the mechanical properties and achieve the dual effects of balancing the specific surface area and the mechanical properties. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 It is a preparation process schematic diagram of the micro-nano fiber composite biological membrane of the application;

[0030] Figure 2 It is a structure schematic diagram of the micro-nano fiber composite biological membrane of the application;

[0031] Figure 3 It is a micro-nano fiber composite biological membrane actual photo of the application;

[0032] Figure 4 It is an electron microscope photo of the surface morphology of the micro-nano fiber composite biological membrane of the application;

[0033] Figure 5 It is an electron microscope photo of the surface morphology of the micro-nano fiber composite biological membrane of the application;

[0034] Figure 6 It is an electron microscope photo of the surface morphology of the micro-nano fiber composite biological membrane of the application;

[0035] Figure 7 It is an electron microscope photo of the surface morphology of the micro-nano fiber composite biological membrane of the application;

[0036] Figure 8 It is an electron microscope photo of the surface morphology of the micro-nano fiber composite biological membrane of the application;

[0037] Figure 9 Figure 1 is a mechanical property test chart of the micro-nano fiber composite biofilm;

[0038] Figure 10 Figure 2 is a mean pore size and pore size distribution rate test chart of the micro-nano fiber composite biofilm;

[0039] Figure 11 Figure 3 is an immunofluorescence staining photograph of phalloidin and DAPI staining of HDFs cells planted on the micro-nano fiber membrane and cultured for seven days;

[0040] Figure 12 Figure 4 is a real object picture and absorbance value analysis chart of MTT detection of HDFs cells planted on the micro-nano fiber membrane and cultured for 1, 3 and 7 days, respectively. DETAILED DESCRIPTION

[0041] The application will be further described in detail below in combination with the drawings and specific embodiments.

[0042] In the present application, the orientation words such as "upper" and "lower" are generally used to refer to the upper and lower in the actual use or working state of the device, and specifically refer to the drawing direction in the drawings, unless otherwise specified. In addition, in the description of the present application, the term "comprising" means "including but not limited to". The words first, second, third, etc. are only used as labels and do not impose a numerical requirement or establish an order. The word "multiple" means "two or more".

[0043] Various embodiments of the present application can exist in the form of a range; it should be understood that the description in the form of a range is only for the convenience and brevity, and should not be understood as a hard limit on the scope of the present application; therefore, it should be considered that the described range has specifically disclosed all possible sub-ranges and single values within the range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the described range, such as 1, 2, 3, 4, 5 and 6, regardless of the range. In addition, whenever a numerical range is indicated in this text, it refers to any cited number (fraction or integer) within the indicated range.

[0044] As Figure 2 , 3The micro-nano fiber composite biomembrane is formed by alternately stacking and combining the uniformly distributed micron fibers and the uniformly distributed nano fibers through the electrospinning process. The micro-nano fiber composite biomembrane is a porous membrane structure formed by alternately stacking the micron fiber layer formed by the micron fibers and the nano fiber layer formed by the nano fibers. The porous membrane structure has a thickness of 50-100 microns, an average pore diameter of 2.4-3.6 microns, a micron fiber diameter range of 1-3 microns, a nano fiber diameter range of 50-1000 nanometers, a breaking elongation of the micro-nano fiber membrane of greater than or equal to 1.2%, a tensile strength of 5.1-7 MPa, and a Young's modulus of 1313.2-2442.6 MPa.

[0045] Specifically, the micro-nano fiber composite biomembrane is made of a spinning precursor of a concentration a of an A polymer and a spinning precursor of a concentration b of the A polymer, and is made by synchronous spinning in an improved electrospinning device. The spinning solution of the concentration a is used to prepare the nano fibers, and the spinning solution of the concentration b is used to prepare the micron fibers. By adjusting the concentrations a and b of the A polymer at any time, the distribution of the micron fibers and the nano fibers in the micro-nano fiber composite biomembrane can be adjusted at any time, so that the specific surface area of the micro-nano fiber composite biomembrane can be adjusted.

[0046] The mass percentage of the spinning solution of the concentration a for preparing the micro-nano fiber composite biomembrane is 2%-10%, and the mass percentage of the spinning solution of the concentration b is 20%. The A polymer is any one of polycaprolactone, polyvinyl alcohol, polypropylene pyrrolidone, polyglycolide-lactide, polylactic acid, and polylactide-glycolide.

[0047] As shown in FIGS. 1-4, the micro-nano fiber composite biomembrane is formed by alternately stacking and combining the uniformly distributed micron fibers and the uniformly distributed nano fibers through the electrospinning process. The micro-nano fiber composite biomembrane is a porous membrane structure formed by alternately stacking the micron fiber layer formed by the micron fibers and the nano fiber layer formed by the nano fibers. The porous membrane structure has a thickness of 50-100 microns, an average pore diameter of 2.4-3.6 microns, a micron fiber diameter range of 1-3 microns, a nano fiber diameter range of 50-1000 nanometers, a breaking elongation of the micro-nano fiber membrane of greater than or equal to 1.2%, a tensile strength of 5.1-7 MPa, and a Young's modulus of 1313.2-2442.6 MPa. Figure 1As shown, the preparation of the micro-nanofiber composite biomembrane adopts an electrostatic spinning device, and a high-voltage electrostatic generator can generate positive and negative high voltage; the embodiment of the application adopts a double-power high-voltage electrostatic generator, and the positive and negative poles of the high-voltage electrostatic generator are connected with double spinning nozzles respectively. The double spinning nozzles are opposite and stand in a certain distance, the distance is 10-20 cm, and the optimal distance is 16 cm. The structure of the double spinning nozzles is needle type or needle-free type; the needle type nozzle is single needle or multi-needle combination type, and is single shaft, coaxial or multi-channel combination type; the needle-free type nozzle is one or a combination of multiple types of metal wire, slit type, circular ring type, cylindrical type, disc type and spherical type, and the receiving roller is located at the middle position of the double spinning nozzles. The spinning voltage of the double-power high-voltage electrostatic generator is-12 kV-+12 kV, the extrusion speed of the a and b concentration spinning solution is 0.8 mL / h, the spinning temperature is 20 DEG C, and the relative humidity is 40%. Different concentrations of polymers are synchronously prepared by the electrostatic spinning device of the double spinning nozzles, under the above specific spinning conditions, the distribution of the microfiber and nanofiber can be adjusted at any time by adjusting the a concentration and b concentration of the A polymer at any time, and the micro-nanofiber composite biomembrane with uniform distribution of microfiber and nanofiber is obtained, the specific surface area, pore size and porosity can be adjusted, the micro-nanofiber composite biomembrane with large specific surface area and small pore size can be formed, and the process is simple and flexible. The nozzle diameter, distance and height of the application can be used to adjust the spinning of nanofiber materials with different diameters, so that different fiber structure stacks can be obtained, thereby greatly enriching the types of micro-nanofiber materials. In the simulation of the structure of human extracellular matrix, the micro-growth environment for cell adhesion, proliferation and differentiation is provided, and the process of repairing and regenerating damaged tissues or organs is assisted.

[0048] The application will be specifically described below through specific examples, and the following examples are only part of the application and are not a limitation of the application.

[0049] Example 1:

[0050] As Figure 1As shown, the spinning solution configured by polycaprolactone (PCL) is taken as an example to perform spinning. The process includes the following steps: (1) dissolving PCL in hexafluoroisopropanol, and then stirring, dissolving and standing to obtain a concentration of 10% of a concentration spinning solution and a concentration of 20% of b concentration spinning solution; (2) loading the PCL spinning solution with a concentration of 10% into one side of the syringe, and loading the PCL spinning solution with a concentration of 20% into the other side of the syringe, and then performing spinning under the conditions of a spinning voltage of-12kV-+12kV, a receiving distance of 16cm, a spinning solution extrusion speed of 0.8mL / h, a receiving roller rotating speed of 300rpm, a spinning temperature of 20℃ and a relative humidity of 40%; (3) forming micron fiber mesh and nanofiber mesh from the micron fiber and nanofiber respectively, and continuously and repeatedly stacking the micron fiber mesh and nanofiber mesh on the surface of the receiving roller; (4) collecting the PCL nanofiber and micron fiber into a PCL micro-nanofiber membrane without obvious beading, with continuous and uniform fibers and a thickness of 50-100μm, which is the micro-nanofiber composite biomembrane of the present application.

[0051] Example 2

[0052] The same part of the preparation process of the present example and example 1 is not described again, and the difference is that the spinning solution configured by polycaprolactone (PCL) is taken as an example to perform spinning. The process includes the following steps: (1) dissolving PCL in hexafluoroisopropanol, and then stirring, dissolving and standing to obtain a concentration of 6% of a concentration spinning solution and a concentration of 20% of b concentration spinning solution; (2) loading the PCL spinning solution with a concentration of 6% into one side of the syringe, and loading the PCL spinning solution with a concentration of 20% into the other side of the syringe, and then performing spinning under the conditions of a syringe advancing rate of 0.8mL / h, an applied voltage of ±12kV and a receiving roller rotating speed of 300rpm; (3) collecting the PCL nanofiber and micron fiber into a PCL micro-nanofiber membrane without obvious beading, with continuous and uniform fibers and a thickness of 50-100μm.

[0053] Example 3

[0054] The same part of the preparation process of the present example and examples 1 and 2 is not described again, and the difference is that the spinning solution configured by polycaprolactone (PCL) is taken as an example to perform spinning. The process includes the following steps: (1) dissolving PCL in hexafluoroisopropanol, and then stirring, dissolving and standing to obtain a concentration of 2% of a concentration spinning solution and a concentration of 20% of b concentration spinning solution; (2) loading the PCL spinning solution with a concentration of 2% into one side of the syringe, and loading the PCL spinning solution with a concentration of 20% into the other side of the syringe, and then performing spinning under the conditions of a syringe advancing rate of 0.8mL / h, an applied voltage of ±12kV and a receiving roller rotating speed of 300rpm; (3) collecting the PCL nanofiber and micron fiber into a PCL micro-nanofiber membrane without obvious beading, with continuous and uniform fibers and a thickness of 50-100μm.

[0055] Comparative example:

[0056] This comparative example serves as a comparison with Examples 1-3 to prepare a microfiber membrane. A spinning solution prepared with polycaprolactone (PCL) was used as an example for spinning. The process included the following steps: (1) PCL was dissolved in hexafluoroisopropanol, and after stirring, dissolving, and settling, a spinning solution with a concentration of 20% was obtained; (2) The 20% PCL spinning solution was loaded into a syringe, and spinning was performed under the following conditions: spinning voltage -12kV to +12kV, receiving distance 16cm, spinning solution extrusion speed 0.8mL / h, receiving roller speed 300rpm, spinning temperature 20℃, and relative humidity 40%; (3) The PCL microfibers were collected into a PCL micro / nanofiber membrane with no obvious beading, continuous and uniform fibers, and a certain thickness.

[0057] 1. Morphological analysis

[0058] The morphology and structure of the micro / nanofiber composite biofilm samples of comparative examples and Examples 1-3 were observed using a scanning electron microscope (TESCAN VEGA3, Czech Republic). To improve the electrical conductivity of the micro / nanofiber composite biofilm samples, gold was deposited on the sample surface for 160 s. The average fiber diameter and fiber diameter distribution were measured using ImageJ software (National Institutes of Health, USA) based on the obtained SEM images. For each sample, 100 different locations were randomly selected to calculate the average fiber diameter and fiber diameter distribution.

[0059] like Figures 5-8 As shown, the average fiber diameters of the coarser microfibers in the comparative examples and Examples 1-3 are 1.71±0.29 μm, 2.13±0.51 μm, 2.20±0.20 μm, and 2.12±0.42 μm, respectively, all exceeding 1 μm and can be defined as microfibers. The average fiber diameters of the nanofibers in Examples 1-3 are 0.42±0.09 μm, 0.20±0.05 μm, and 0.09±0.02 μm, respectively, all less than 1 μm and can be defined as nanofibers. In Examples 1-3, the nanofibers can fill the gaps between the microfibers, thereby increasing the specific surface area of ​​the micro / nanofiber composite biomembrane. Furthermore, pure nanofibers have poor spinnability, resulting in spun fiber membranes with many beads, and the specific surface area of ​​pure microfibers is smaller than that of the examples. Therefore, combining microfibers and nanofibers achieves the optimal specific surface area of ​​the micro / nanofiber composite biomembrane of this invention.

[0060] 2. Mechanical property testing

[0061] The mechanical properties of the comparative example and examples 1-3 were measured using a universal tensile testing machine (Instron 5965, USA). The micro-nano fiber composite biomembrane was clamped at both ends with a fixed distance of 10 mm. Before the formal experiment, a pre-tension of 0.02 N was applied to the sample, and the tensile speed was fixed at 10 mm / min. First, the load-elongation curve was obtained, and then the stress-strain curve, breaking strength, breaking elongation and Young's modulus were calculated, as follows:

[0062] Table 1 is the test results of the mechanical properties of the comparative example and examples 1-3

[0063] Test item Comparative example Example 1 Example 2 Example 3 Young's modulus (MPa) 653.7 1313.2 1399.8 2442.6 Breaking strength (MPa) 2.7 5.1 5.5 7.0 Breaking elongation (%) 8.7 1.4 1.2 1.4

[0064] As Figure 9 shown, in combination with the above table, the stress-strain curve shows that the comparative example exhibits a linear elastic region, followed by a yield platform, and finally leads to fracture. In contrast, examples 1-3 exhibit different tensile curves, with a clear plastic deformation region after the linear elastic region. The Young's modulus and breaking strength of examples 1-3 are significantly improved, but the breaking elongation is significantly reduced. Among them, the Young's modulus of example 3 is improved by 273% compared with the comparative example, and the breaking strength is improved by 159%. It can be seen that under the condition of lower nanofiber spinning concentration, the breaking strength and Young's modulus are larger, and better mechanical properties are exhibited. The smaller the average diameter of the nanofiber in examples 1-3, the tighter the entanglement between the nanofiber and the microfiber, and the better the mechanical properties of the micro-nano fiber membrane can be improved. In the actual electrospinning process, the nanofiber with a small average diameter and the microfiber with a large average diameter produce an entanglement, which promotes the improvement of the mechanical properties. After mixing, the nanofiber and the microfiber have an interlacing, which is equivalent to a decrease in the number of nanofibers, and individual nanofibers may produce beading, but the overall number is greatly reduced, avoiding the phenomenon of beading in nanofiber membranes or microfiber membranes, which affects cell adhesion and proliferation.

[0065] 3. Pore size test

[0066] As Figure 10 shown, the pore size of the comparative example and examples 1-3 was measured using a pore size analyzer. The micro-nano fiber composite biomembrane was cut into a circle with a diameter of 2 cm, and a drop of infiltration solution was added to the material until the liquid completely filled the pores of the sample. The specific test results are as follows:

[0067] Test item Comparative example 1 Example 1 Example 2 Example 3 Average pore size (pm) 5.2 3.6 2.4 3.0

[0068] According to the research of the prior art, the smaller the pore size of the micro-nano fiber membrane, the greater the specific surface area, and the more adhesion sites provided for cells. The average pore size of Examples 1-3 is significantly smaller than that of the comparative examples, with an average reduction of about 30%. It is shown that by combining micro-fibers and nano-fibers, the average pore size of the micro-nano fiber membrane can be reduced, thereby the specific surface area of the micro-nano fiber can be accurately regulated. The average pore size obtained by using a spinning solution concentration of 6% for preparing nano-fibers is the smallest, i.e. Example 2 of the present application.

[0069] 4. Cell experiment

[0070] As shown in Figure 11 , human dermal fibroblasts (HDFs, Chinese Academy of Sciences cell line) were cultured in DMEM medium. The micro-nano fiber membrane was cut into a circle with a diameter of 10 mm. The samples were sterilized, and HDFs were inoculated on the samples at a cell density of 4x10 4 cells / mm2, and the culture medium was replaced every two days until the predetermined time point was reached. The MTT method (Sigma-Aldrich) was used to determine the cell viability and proliferation of the samples after 1, 3 and 7 days of culture. Subsequently, the area of blue-violet Formazan crystals in different samples was observed by taking photos. The absorbance was measured at a wavelength of 490 nm using a microplate reader (Infinite M Nano, Tecan). After 7 days of culture, the cell morphology and nucleus structure were observed by staining with IFluorTM 488 phalloidin and DAPI, and finally, the stained samples were observed using a confocal laser scanning microscope (CLSM, Zeiss 900, Carl Zeiss).

[0071] As shown in Figure 12 , F-actin and DAPI represent the cytoskeleton and nucleus of HDFs, respectively. The results show that the number of cells on the samples of Examples 1-3 is significantly higher than that of the comparative examples, which indicates that HDFs have good cell adhesion, proliferation and migration on the samples of Examples 1-3. Further, the MTT method was used to determine the proliferation of HDFs on the samples within 7 days. The crystalline area of the examples is significantly larger than that of the comparative examples at the three time points. And statistical analysis shows that the absorbance value of Examples 1-3 is significantly greater than that of the comparative examples. In summary, the specific surface area of the micro-nano fiber composite biomembrane with uniform distribution of micro-fibers and nano-fibers is larger, which promotes the adhesion, proliferation and migration of cells.

[0072] It is known that in the field of electrospinning applications, the regulation of spinning parameters, especially the regulation of spinning diameter and directionality, is also a key technology. At present, the regulation of spinning diameter and directionality can be achieved by changing voltage, spinning distance, solution concentration, injection flow and working distance. However, in industrial production, it is generally necessary to fix all other parameters and then adjust a certain parameter. In actual production, it is difficult to achieve stable regulation of all parameters, and it is difficult to mass-produce electrospun fibers with uniform diameter and directionality, which further limits the development of many applications. The concept of the present application ingeniously utilizes the irregular pores generated by the difficulty in controlling the fineness and direction of electrospun fibers. The obtained micro-nano fiber mesh is composed of fibers with different fineness and direction disorder, and the multi-layer structure formed by stacking plays a supporting role as a "skeleton". Not only a larger specific surface area and porosity are obtained, but also good external barrier ability is given.

[0073] Specific application of the micro-nano fiber composite biological membrane of the present application:

[0074] 1. Hydrogel microneedle biological scaffold:

[0075] Although the current hydrogel scaffold has good biocompatibility, can promote cell proliferation and differentiation, extracellular matrix deposition and remodeling, and new tissue regeneration, and has the functions of simulating natural tissue in structure, composition and function, there are still some defects in the function of the fiber membrane or fiber combined with the hydrogel microneedle that affect the biological performance of the scaffold. Although the drug loading capacity of hydrogel is strong and the drug loading efficiency is high, the mechanical properties of the hydrogel scaffold are limited and the external barrier effect is poor. Under external force, the hydrogel will crack and lead to bacterial invasion. The present application is combined with hydrogel microneedles, and the micro-nano fiber composite biological membrane adheres to the outer layer of the hydrogel to support the shape and barrier effect, thereby improving the mechanical properties and external barrier ability of the hydrogel microneedle.

[0076] 2. Tendon patch:

[0077] Tendon injury is one of the common diseases of the motor system, and at present, arthroscopic surgery is one of the most effective means, but tendon healing is interfered by many factors, which seriously affects the healing effect after operation. The use of tendon repair patch for enhanced repair or bridging repair in arthroscopic surgery has become a new treatment option. The current tendon patch requires good biomechanical properties and biocompatibility, induces tissue regeneration and healing, and provides stable mechanical support for the tendon-bone interface. The current main research direction is the degradable function, mainly including the use of degradable warp and weft to weave a fiber structure, or the use of degradable synthetic polymer polymerization to weave a sheet network structure. Compared with the traditional tendon patch, the present application has obvious advantages in promoting tissue regeneration, healing and assisting mechanical support. The spinning solution adopts degradable material, and the structure of the electrospun micro-nanofiber membrane can be more closely combined with the tissue. The combination of microfiber and nanofiber can reduce the average pore size of the micro-nanofiber membrane, so that the specific surface area of the micro-nanofiber can be accurately controlled, which can obviously promote cell proliferation and differentiation, extracellular matrix deposition and remodeling, and new tissue regeneration. The close entanglement structure between nanofiber and microfiber can better improve the mechanical properties of the micro-nanofiber membrane, which can be used for tendon tissue repair and healing. It provides a research direction for the further development of tendon patch technology.

[0078] 3. Hernia repair patch:

[0079] The hernia repair patch is a short name of hernia repair material. In recent years, with the rapid development of materials science, various hernia repair materials have been widely used in clinical practice, which has fundamentally changed the treatment of hernia. At present, the synthetic materials widely used for hernia repair in the world can be divided into two categories: the first category is non-absorbable polyester patch, polypropylene patch and expanded polytetrafluoroethylene patch; the second category is composite patch. The basic functions of the hernia repair patch are: soft, more resistant to bending and folding; can be cut to the required size; the stimulation of fibrous tissue proliferation is more obvious, the mesh aperture is large, which is more conducive to the growth of fibrous tissue, easy to be infiltrated by connective tissue, can be early embedded with tissue, resistant to infection, has higher tensile strength and low price. The micro-nanofiber composite biological membrane of the present application is more soft compared with the fabric structure by using electrospinning technology, which avoids the distortion of the mesh caused by scar contraction, and the irregular surface may stimulate and damage the surrounding tissue. Moreover, the controllable thickness, specific surface area, pore size and porosity have more advantages in preventing adhesion and promoting healing, and the stacking and entanglement structure of the micro-nanofiber greatly improves the mechanical properties of the membrane itself, so that the special and controllable structure of the micro-nanofiber composite biological membrane is more resistant to bending and folding, and the growth in the abdominal wall after implantation is more firm.

[0080] Of course, the above description is not a limitation of the present application, and the present application is not limited to the above examples. Changes, modifications, additions or substitutions made by those skilled in the art within the spirit of the present application should be within the scope of the present application.

Claims

1. A preparation method of a micro-nanofiber composite biomembrane based on electrospinning technology, the micro-nanofiber composite biomembrane is formed by alternately stacking and combining microfibers and nanofibers with uniform distribution through an electrospinning process, the micro-nanofiber composite biomembrane is a porous membrane structure formed by alternately stacking a micrometer fiber layer formed by microfibers and a nanometer fiber layer formed by nanofibers, the porous membrane structure has a thickness of 50-100 μm, an average pore size of 2.4-3.6 μm, a microfiber diameter range of 1-3 μm, and a nanofiber diameter range of 50-1000 nm, the micro-nanofiber membrane has an elongation at break of ≥1.2%, a tensile strength of 5.1-7 MPa, and a Young's modulus of 1313.2-2442.6 MPa, and the method is characterized in that, It comprises the following steps: (1) preparing a 2% a concentration spinning solution of A polymer and a 20% b concentration spinning solution, for standby; (2) connecting the prepared spinning solutions to two spinning nozzles of an electrostatic spinning device respectively, and connecting the positive and negative poles of a double-supply high-voltage electrostatic generator to the two spinning nozzles respectively; (3) starting the double-supply high-voltage electrostatic generator to provide opposite high-voltage electrostatic to the two spinning nozzles, and performing synchronous spinning under the conditions of a spinning voltage of-12 kV-+12 kV, a receiving distance of 16 cm, a spinning solution extrusion speed of 0.8 mL / h, a spinning temperature of 20℃, and a relative humidity of 40%; (4) collecting the micron and nanometer fibers formed by the two spinning nozzles on both sides of the receiving drum respectively; (5) forming micron fiber mesh and nanometer fiber mesh from the micron and nanometer fibers respectively, and continuously and repeatedly stacking them alternately; (6) obtaining a micron and nanometer fiber composite biomembrane with continuous, uniform distribution and no obvious beading; The A polymer is any one of polycaprolactone, polyvinyl alcohol, polypropylene pyrrolidone, polyglycolide-lactide, polylactic acid, and poly(lactide-co-glycolide).

2. The method of claim 1, wherein the method is characterized by: The electrostatic spinning device comprises a double-supply high-voltage electrostatic generator and a receiving drum, and two spinning nozzles for preparing micron fiber layers and nanometer fiber layers are symmetrically arranged on both sides of the receiving drum, and the positive and negative poles of the high-voltage electrostatic generator are connected to the two spinning nozzles respectively.

3. The method of claim 1, wherein the method is characterized by: In step (4), the rotating speed of the receiving drum is 300 rpm.

4. Application of the micron and nanometer fiber composite biomembrane prepared by the preparation method of any one of claims 1-3 in preparing wound dressings for tissue injury, tendon patches, and hernia repair patch materials.

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