Asymmetric anti-blocking composite film, and preparation method and application thereof

Abstract

The application provides an asymmetric anti-adhesion composite film and a preparation method and application thereof. The composite film comprises a bioactive layer and an electrospun fiber layer, and the electrospun fiber layer comprises a first fiber layer and a second fiber layer. The first fiber layer comprises fibers with a first diameter. The second fiber layer comprises fibers with the first diameter and fibers with a second diameter, and the first diameter is larger than the second diameter. The composite film has strong mechanical and degradation resistance properties, and is suitable for anti-adhesion applications of various moving organoids or tissues, so that the composite film still has structural integrity and functional integrity for a long time in these environments. One side of the composite film has adhesive fibers, and has stronger adhesion in a wet state, so that the composite film cannot be easily displaced when applied to moving organoids or tissues.

Description

Technical Field

[0001] This application belongs to the field of biomembrane material technology, and in particular relates to an asymmetric anti-adhesion composite membrane, its preparation method and application. Background Technology

[0002] Currently, materials used for anti-adhesion applications in organs / tissues are mainly of two types: gels and films. Gel materials primarily involve directly injecting polymer solutions, such as sodium hyaluronate gel, polylactic acid gel, or chitosan gel, into the adhesion site. These liquid gel materials can also be prepared into films, which are moistened with liquid before application to form a gel that adheres to the easily adhesive area, ultimately achieving an anti-adhesion effect. In addition, some polymer materials, such as animal-derived collagen, oxidized cellulose (e.g., carboxymethyl cellulose), or chitosan, can be freeze-dried or air-dried into films to create anti-adhesion materials. Films prepared using weaving or electrospinning processes are also being explored for anti-adhesion applications.

[0003] Currently, anti-adhesion membranes used for organ or tissue anti-adhesion applications have the following drawbacks / problems:

[0004] (1) It lacks sufficient adhesion and is prone to displacement during use in some musculoskeletal adhesion scenarios;

[0005] (2) Due to the rough surface and porous nature of (woven / electrospun) fibrous materials, cells are easy to adhere to and penetrate, which can easily lead to strong tissue adhesions.

[0006] (3) Some films formed after the polymer solution is air-dried are prone to rapid degradation and decreased mechanical properties in the presence of tissue fluid. Summary of the Invention

[0007] This application provides an asymmetric anti-adhesion composite film, its preparation method, and its application to solve the problems existing in related technologies. The technical solution is as follows:

[0008] In a first aspect, embodiments of this application provide an asymmetric anti-adhesion composite film, comprising a bioactive layer and an electrospun fiber layer, wherein the electrospun fiber layer comprises a first fiber layer and a second fiber layer; the bioactive layer, the first fiber layer, and the second fiber layer are disposed sequentially.

[0009] The first fiber layer includes fibers of a first diameter; the second fiber layer includes fibers of a first diameter and fibers of a second diameter, wherein the first diameter is larger than the second diameter.

[0010] In one embodiment, the diameter of the first diameter fiber ranges from 0.5 to 2 μm; the diameter of the second diameter fiber ranges from 0.2 to 0.5 μm.

[0011] In one embodiment, the ratio of the number of fibers with the first diameter to the number of fibers with the second diameter in the second fiber layer is (1-3):1.

[0012] In one embodiment, the material of the fiber of the first diameter is one or a combination of two or more of polylactic acid, polymethyl methacrylate, polyvinylidene fluoride, polyurethane or polycaprolactone.

[0013] In one embodiment, the material of the fiber with the second diameter is polyvinylpyrrolidone.

[0014] In one embodiment, the thickness of the bioactive layer is 0.5-1 mm.

[0015] In one embodiment, the bioactive layer is made of one or more of collagen, silk fibroin, oxidized cellulose, chitosan, or hyaluronic acid.

[0016] In one embodiment, the bioactive layer is a collagen layer; the collagen is prepared from animal Achilles tendon tissue through steps including enzymatic extraction, salting out, cross-linking, and protein fixation.

[0017] In one embodiment, the enzyme used for enzymatic hydrolysis is a mixture of pepsin and lipase; the mass ratio of pepsin to lipase is (1-3):1.

[0018] In one embodiment, protein immobilization is performed in a glycine solution.

[0019] In one embodiment, the degree of crosslinking is 5-10%.

[0020] Secondly, embodiments of this application provide a method for preparing an asymmetric anti-adhesion composite film, comprising the following steps:

[0021] A second fiber layer is formed by simultaneously electrospinning fibers of a first diameter and fibers of a second diameter on a substrate; electrospinning of the second diameter fibers is stopped; electrospinning of the first diameter fibers is continued on the second fiber layer to form a second fiber layer; a bioactive layer is loaded on one side of the second fiber layer, and vacuum drying is performed to obtain the asymmetric anti-adhesion composite film.

[0022] In one embodiment, the electrospun fiber preparation conditions are as follows: the spinning needle is 21G, the temperature of the spinning environment is 35-40℃, the relative humidity is 35-45%RH, the distance between the spinning needle and the collector is 20cm, and the spinning solution is injected at a rate of 1mL / h; the positive electrode voltage of the first diameter fiber spinning needle is set to +16kV, and the positive electrode voltage of the second diameter fiber spinning needle is set to +14kV.

[0023] Thirdly, embodiments of this application provide the application of asymmetric anti-adhesion composite membranes in the anti-adhesion of organs / tissues.

[0024] The advantages or beneficial effects of the above technical solutions include at least the following:

[0025] The asymmetric anti-adhesion composite membrane of this application has a bioactive layer loaded on a fiber layer composed of fibers of different diameters. The fibers dissolve to generate adhesive force, and the mechanical strength of the fibers makes the adhesiveness of the fibers long-lasting. The bioactive layer can prevent cell / tissue ingrowth and achieve anti-adhesion. The bioactive layer and the fiber layer of the asymmetric anti-adhesion composite membrane are interwoven, which makes the overall performance of the membrane outstanding.

[0026] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of this application will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0027] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the various drawings denote the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings depict only some embodiments disclosed in this application and should not be construed as limiting the scope of this application.

[0028] Figure 1 SEM images of different parts of the asymmetric anti-adhesion composite film of this application;

[0029] Figure 2 The image shows the number of cells in the asymmetric anti-adhesion composite membrane after 4 days of co-culturing fibroblasts in the example and comparative examples. Detailed Implementation

[0030] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of this application. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0031] This application provides an asymmetric anti-adhesion composite membrane, comprising a bioactive layer and an electrospun fiber layer, wherein the electrospun fiber layer comprises a first fiber layer and a second fiber layer; the bioactive layer, the first fiber layer, and the second fiber layer are arranged sequentially.

[0032] The first fiber layer includes fibers of a first diameter; the second fiber layer includes fibers of a first diameter and fibers of a second diameter, wherein the first diameter is larger than the second diameter.

[0033] The composite membrane of this application consists of an anti-adhesion bioactive layer and an adhesive fiber layer. The surface of the bioactive layer is smooth, preventing adhesion caused by washing / tissue ingrowth. The fiber layer is composed of biodegradable polymer fibers. Serving as the adhesion surface, the fibers dissolve to generate adhesive force, and the mechanical properties of the polymer material in this application control degradation to a certain extent, thus maintaining adhesion for a long time. The fiber layer is constructed by layering fibers of different diameters, providing varying degradation capabilities, making the service life of the composite membrane controllable. The electrospun fibers and bioactive layer are composited, with each layer having an interwoven structure; for example, the collagen film layer embeds parts of the coarse fiber layer, and the coarse fiber layer and the coarse / fine fiber composite layer also have an integrated structure. The fibers significantly enhance the membrane's degradation resistance, allowing the composite membrane to maintain a good anti-adhesion effect for a long time.

[0034] In one embodiment, the diameter of the first diameter fiber ranges from 0.5 to 2 μm, and the diameter of the second diameter fiber ranges from 0.2 to 0.5 μm. The first diameter fibers form a fiber layer, arranged with coarser diameter fibers. The pores between the fibers are filled with active substances from a bioactive layer, enabling the bioactive layer and fiber layer to interweave. The bioactive layer embeds some of the coarser fibers, and the coarser diameter provides stronger mechanical support and a longer degradation time. The second fiber layer is composed of a mixture of coarse and fine fibers. The second fiber layer acts as an adhesion surface, generating adhesion through dissolution. Therefore, the combination of coarse and fine fibers provides the basis for mechanical strength while controlling dissolution to provide adhesion. The first diameter fibers in the second fiber layer can be continuously manufactured with the first diameter fibers in the first fiber layer. Thus, the first and second fiber layers can interweave through the continuity of the first diameter fibers, providing sufficient bonding strength.

[0035] In one embodiment, the ratio of the number of fibers with the first diameter to the number of fibers with the second diameter in the second fiber layer is (1-3):1.

[0036] In one embodiment, the material of the fiber with the first diameter is one or a combination of two or more of polylactic acid, polymethyl methacrylate, polyvinylidene fluoride, polyurethane, or polycaprolactone.

[0037] In one embodiment, the material of the second diameter fiber is polyvinylpyrrolidone.

[0038] In one implementation method, the thickness of the bioactive layer is 0.5-1 mm.

[0039] In one embodiment, the bioactive layer is made of one or more of collagen, silk fibroin, oxidized cellulose, chitosan, or hyaluronic acid.

[0040] In one embodiment, the bioactive layer is a collagen layer; the collagen is prepared from animal Achilles tendon tissue through steps including enzymatic extraction, salting out, cross-linking, and protein fixation.

[0041] In this embodiment, the animal Achilles tendon tissue is obtained from the Achilles tendon of a pig or cow, and its type I collagen is obtained.

[0042] In this embodiment, before enzymatic extraction, the animal Achilles tendon is first cleaned, sliced, disinfected, cleaned and crushed.

[0043] Specifically, the Achilles tendon tissue is trimmed to remove fat and impurities, then sliced. In this embodiment, the slice thickness is 0.5-1 mm. The sliced ​​slices are drained and set aside. The sterilization process involves immersing the slices in an alkaline solution; preferably a 5% sodium hydroxide solution, for 1-3 hours, with intermittent stirring during soaking. After soaking, a preliminary rinse with pure water is performed. Further cleaning after sterilization is carried out in a saline solution. The sterilized slices are immersed in a 10-20% wt NaCl solution at a slice-to-salt-solution weight ratio of 1:(0.5-2) for 10-60 minutes for further cleaning. The cleaned slices can then be pulverized in a grinder.

[0044] In one implementation method, the enzyme used for enzymatic hydrolysis is a mixture of pepsin and lipase; the mass ratio of pepsin to lipase is (1-3):1. The specific enzymatic hydrolysis process is as follows: the pulverized tissue is hydrolyzed with the complex enzyme in an aqueous solution containing glacial acetic acid; the hydrolysis is then terminated. The hydrolysis conditions are 28-32℃ for 30-90 hours.

[0045] To terminate the reaction, simply stir the reaction mixture with an EDTA solution at 30-40 Hz.

[0046] In one implementation method, collagen is extracted by salting out after enzymatic hydrolysis. The hydrolyzed product is stirred in 10% sodium chloride for 10-30 minutes; solid collagen is obtained by solid-liquid separation. Solid-liquid separation can be achieved by centrifugation or filtration.

[0047] In one embodiment, prior to crosslinking, the collagen is washed with ethanol and water and further purified in a dialysis bag. First, the collagen solids are washed with 95% ethanol, then with pure water; subsequently, they are dialyzed in a dialysis bag with a molecular weight of 100 kDa.

[0048] In one embodiment, the cross-linking of collagen is carried out in the presence of a cross-linking agent. The cross-linking agent is glutaraldehyde; the dialyzed collagen solution is mixed with the cross-linking agent solution, and the mixture is stirred to carry out the cross-linking reaction. Preferably, the mass concentration of the cross-linking agent solution is 5%; the volume ratio of the collagen solution to the cross-linking agent solution is 1:(0.5-2). Preferably, the degree of cross-linking of the collagen reaches 5-10%.

[0049] One implementation method involves fixing the cross-linked collagen. This is done using a glycine solution. Specifically, the collagen is immersed and stirred in a glycine solution to fix the cross-linked collagen.

[0050] As one implementation method, the collagen is washed with purified water before and after fixation.

[0051] This application also provides a method for preparing an asymmetric anti-adhesion composite film, including the following steps:

[0052] A second fiber layer is formed by simultaneously electrospinning fibers of a first diameter and fibers of a second diameter on a substrate; electrospinning of the second diameter fibers is stopped; electrospinning of the first diameter fibers is continued on the second fiber layer to form a second fiber layer; a bioactive layer is loaded on one side of the second fiber layer, and vacuum drying is performed to obtain the asymmetric anti-adhesion composite film.

[0053] In one embodiment, the material of the first diameter fiber is made into an electrospinning solution and the material of the second diameter fiber is made into an electrospinning solution.

[0054] Preferably, the solvent is selected from N,N-methylenebisacrylamide and / or ethanol.

[0055] As one embodiment, the electrospun fiber preparation conditions are as follows: the spinning needle is 21G, the temperature of the spinning environment is 35-40℃, the relative humidity is 35-45%RH, the distance between the spinning needle and the collector is 20cm, and the spinning solution is injected at a rate of 1mL / h; the positive electrode voltage of the first diameter fiber spinning needle is set to +16kV, and the positive electrode voltage of the second diameter fiber spinning needle is set to +14kV.

[0056] This application also provides the application of asymmetric anti-adhesion composite membranes in the anti-adhesion of organs / tissues.

[0057] The following is a further explanation using specific embodiments.

[0058] Example 1: Extraction of Type I Collagen from Porcine Achilles Tendon

[0059] 1) Slicing: Cut the pork Achilles tendon into slices 0.5-1mm thick after trimming off fat and impurities, drain the water, and weigh them;

[0060] 2) Disinfection: Soak the sliced ​​Achilles tendon in a 5% NaOH solution for 2 hours, stirring occasionally, then remove and rinse thoroughly with pure water;

[0061] 3) Cleaning: Soak the slices in a 15% NaCl solution with a weight-to-volume ratio of 1g:1mL for 30 minutes, then rinse with pure water for 10 minutes. Repeat the above salt soaking + water rinsing process 3 times.

[0062] 4) Grinding: Put the slices into a grinder and grind them until you hear a steady grinding sound. Once grinding is complete, you will get a pulverized gel-like substance; freeze for later use.

[0063] 5) Extraction: Take 22.5g of the pulverized gel, add 28.6mL of glacial acetic acid, 0.15g of pepsin and 0.075g of lipase, add water to make the reaction system 1L, and react at 30℃ for 60h; then add 100mL of stop solution, stir at 30-40Hz for 25min to terminate the reaction; the stop solution is prepared by dissolving 7g of EDTA in 100mL of pure water, boiling and filtering through a 300-mesh filter bag;

[0064] 6) Salting out: Filter the reaction solution through a 60-100 mesh filter bag, add an equal volume of 10% NaCl according to the volume of the final product solution, stir for 20 minutes, and filter to collect the solid.

[0065] 7) Washing: Wash the solid extract three times with 95% ethanol, stirring for 1 hour each time, and then wash with pure water;

[0066] 8) Purification: Dialysis was performed using a dialysis bag with a molecular weight of 100 kDa. The water was changed 1-3 times a day. The dialysis process was carried out in a stirred environment. After the dialysis was completed, the contents of the dialysis bag were collected.

[0067] 9) Crosslinking: Adjust the pH of the dialyzed collagen liquid to ≈7.0 with 20% NaOH; adjust the pH of the crosslinking agent solution to ≈7 with 10% HCl solution; then soak the collected precipitate in 5% glutaraldehyde solution at a mass-volume ratio of 1g:2mL and stir for 14h.

[0068] 10) Washing: Centrifuge the cross-linking product for 3 min and take the precipitate. Then add purified water at a mass-volume ratio of 1 g: 4 mL and stir to wash twice, 10 min each time. After each wash, centrifuge for 3 min and take the precipitate.

[0069] 11) Protein fixation: The cross-linked collagen was soaked in a 1% glycine solution at a mass-volume ratio of 1g:4mL for 2 hours with stirring. Fresh glycine solution was replaced every 30 minutes. Before each replacement of glycine solution, the precipitate was centrifuged for 3 minutes and collected.

[0070] 12) Washing: Add purified water to the precipitate at a mass-volume ratio of 1g:4mL, stir and wash three times, 30min each time, centrifuge for 3min and collect the precipitate. Finally, weigh and record the precipitate to obtain purified porcine Achilles tendon type I collagen.

[0071] Example 2

[0072] Preparation of electrospinning solution: Weigh 300g of polyurethane (PU) and place it in 700g of N,N-methylenebisacrylamide solution. Stir at room temperature to dissolve it and prepare a 30%wt PU electrospinning solution. Weigh 200g of polyvinylpyrrolidone (PVP) into a glass bottle, add 300g of a mixed solution of N,N-methylenebisacrylamide and ethanol in a mass ratio of 9:1, and stir at room temperature to dissolve it and prepare a 20%wt PVP electrospinning solution.

[0073] Electrospun fiber preparation conditions: the spinning needle is 21G, the temperature of the spinning environment is 35-40℃, the relative humidity is 35-45%RH, the distance between the spinning needle and the collector is 20cm, the spinning solution is injected at a rate of 1mL / h, and the positive electrode voltage of the needle is set to +16kV (PU fiber) and +14kV (PVP fiber).

[0074] Preparation of asymmetric anti-adhesion composite film: On the substrate, an electrospinning method was used to prepare a PU and PVP mixed electrospinned film by spinning PU and PVP electrospinning needles for 5 minutes each. The voltage of the PVP electrospinning needle was turned off to stop the PVP spinning process. The electrospinning time of the PU electrospinning fiber layer alone was 2 minutes. Finally, the porcine Achilles tendon type I collagen prepared in Example 1 was brushed / scraped onto one side of the above PU electrospinned film to form a collagen layer with a thickness of 0.7 mm. After completion, the above composite film was placed in a vacuum oven to remove excess organic solvents from the spun film and to air dry the collagen layer into a film, finally obtaining the asymmetric anti-adhesion composite film A.

[0075] Example 3

[0076] Compared to Example 1, the crosslinking agent solution concentration used in this example is 1%, the remaining steps are the same as in Example 1, and the membrane preparation method is the same as in Example 2, resulting in an asymmetric anti-adhesion composite membrane B.

[0077] Example 4

[0078] Compared to Example 1, the crosslinking agent solution concentration used in this example is 3%, the remaining steps are the same as in Example 1, and the membrane preparation method is the same as in Example 2, resulting in an asymmetric anti-adhesion composite membrane C.

[0079] Example 5

[0080] Compared to Example 1, the crosslinking agent solution concentration used in this example is 10%, the remaining steps are the same as in Example 1, and the membrane preparation method is the same as in Example 2, resulting in an asymmetric anti-adhesion composite membrane D.

[0081] Example 6

[0082] Compared to Example 2, in this example, the electrospinning time for the individual PU electrospun fiber layer is 1 minute, and the remaining steps are the same as in Example 2, resulting in an asymmetric anti-adhesion composite film E.

[0083] Example 7

[0084] Compared to Example 2, the electrospinning time for the PU electrospun fiber layer in this example is 3 minutes, and the remaining steps are the same as in Example 2, resulting in an asymmetric anti-adhesion composite film F.

[0085] Example 8

[0086] Compared to Example 2, the electrospinning time for the individual PU electrospun fiber layer in this example is 7 minutes, and the remaining steps are the same as in Example 2, resulting in an asymmetric anti-adhesion composite film G.

[0087] Example 9

[0088] Compared to Example 2, the electrospinning time for mixing PVP fibers and PU fibers in this example is 1 minute, and the remaining steps are the same as in Example 2, resulting in an asymmetric anti-adhesion composite film H.

[0089] Example 10

[0090] Compared to Example 2, the electrospinning time for mixing PVP fibers and PU fibers in this example is 10 minutes, and the remaining steps are the same as in Example 2, resulting in an asymmetric anti-adhesion composite film I.

[0091] Example 11

[0092] Compared to Example 2, in this example, the injection speed of PVP solution during the electrospinning process of PVP fibers is increased to 3 mL / min, and the remaining steps are the same as in Example 2, resulting in an asymmetric anti-adhesion composite film J.

[0093] Example 12

[0094] Compared to Example 2, in this example, the thickness of the collagen solution layer brushed / scraped onto one side of the PU fiber is 3 mm, and the remaining steps are the same as in Example 2, resulting in an asymmetric anti-adhesion composite film K.

[0095] Comparative Example 1

[0096] Compared to Example 2, the electrospinning steps of PU and PVP electrospun fibers were omitted in this comparative example, and a single collagen solution was air-dried into a film (thickness of 0.7 mm) as a control sample.

[0097] Comparative Example 2

[0098] Compared to Example 2, in this comparative example, after electrospun PU fiber and PU and PVP electrospun fiber layers, the step of brushing / scraping a collagen layer on one side of the PU electrospun fiber layer was omitted. The remaining steps were the same as in Example 2, resulting in a comparative asymmetric anti-adhesion composite film 1.

[0099] Comparative Example 3

[0100] Compared to Example 2, in this comparative example, a PU and PVP mixed electrospun fiber membrane was obtained by electrospinning. Then, a collagen layer was brushed / scraped onto both sides of the mixed electrospun fiber membrane to completely encapsulate the mixed electrospun fiber membrane, resulting in a composite film in which the collagen layer completely encapsulates the electrospun fiber layer. The remaining steps were the same as in Example 2, resulting in a comparative asymmetric anti-adhesion composite film 2.

[0101] Comparative Example 4

[0102] Compared to Example 2, this comparative example uses an electrospinning process to obtain a PU electrospun fiber membrane with the same total thickness as the fiber membrane in Example 2. Then, collagen layers are brushed / coated onto both sides of the mixed electrospun fiber membrane to completely encapsulate it, resulting in a composite film where the collagen layer completely embeds the electrospun fiber layer. The remaining steps are the same as in Example 2, yielding a comparative asymmetric anti-adhesion composite film 3.

[0103] Characterization and performance testing

[0104] (1) The asymmetric anti-adhesion composite film prepared in Example 2 was observed in various parts using a scanning electron microscope. The electron microscope image of the PU and PVP mixed electrospun fiber layer (second fiber layer) is shown below. Figure 1 As shown in Figure a; the electron microscope image of the PU electrospun fiber layer (first fiber layer) is shown in Figure a. Figure 1 As shown in Figure b; the electron micrograph of the interface between the PU electrospun fiber layer and the collagen layer is shown in Figure b. Figure 1 As shown in C; electron micrograph of collagen thin film as shown in Figure C. Figure 1 As shown in d.

[0105] from Figure 1 As can be seen in Figure a, the diameter of PVP electrospun fibers is smaller than that of PCL electrospun fibers, with coarse PU fibers intertwined with fine PVP fibers. Figure 1 As can be seen from Figure c, the surface of the collagen film layer is relatively smooth, and there is an interlaced structure between the collagen film layer and the electrospun fiber layer, with some electrospun fibers being embedded in the collagen layer.

[0106] (2) Mechanical properties and in vitro degradation properties of the membranes prepared in the examples and comparative examples were tested:

[0107] Measurement of the friction coefficient of asymmetric anti-adhesion composite film

[0108] The test was conducted in accordance with the GB / T 10006 standard. The prepared asymmetric anti-adhesion composite membrane (5cm×5cm) was taken, and the friction coefficient of five random areas of the fiber membrane was measured using an MXD-02 friction coefficient meter. The measured data were recorded, and the average value and error were calculated.

[0109] Tensile breaking strength determination of asymmetric anti-adhesion composite film

[0110] The asymmetric anti-adhesion composite film was cut into strips 5cm long and 1cm wide. The two ends of the composite film were clamped by the upper and lower clamps of a tensile testing machine. The composite film was then stretched at a rate of 5cm / min. The maximum force detected by the tensile testing machine during the stretching process was recorded, which is the tensile breaking strength of the asymmetric anti-adhesion composite film. Three samples were taken for each embodiment and comparative example for testing, and the average value and error were taken.

[0111] Determination of wet adhesion of asymmetric anti-adhesion composite film

[0112] The test was conducted in accordance with GB 4851 standard. The asymmetric anti-adhesion composite film was cut into square strips 5cm long and 1cm wide. One side of the film fiber was pressed onto the wet substrate, and the adhesion force was tested using an adhesion tester at a stretching speed of 3cm / min. The reading F (in N) of the tensile tester was recorded during the process of the composite film detaching from the substrate. The maximum force value tested during the process is the wet adhesion force of the composite film. Three samples were taken for each example and comparative example for testing, and the average value and error were taken.

[0113] In vitro enzymatic hydrolysis experiment of asymmetric anti-adhesion composite membrane

[0114] Weigh the sample and record the mass as m1. Place it in a 50mL centrifuge tube and immerse it in 5mL of 1mg / mL collagenase solution. Place the tube in a 37℃ constant temperature oven. Take samples every 6 hours, wash and dry them, and record the mass as m2. Calculate the sample mass loss as a mass ratio (%): mass ratio (%) = m2 / m1 × 100%. Record the time period for complete sample degradation (100%).

[0115] The results are shown in Table 1.

[0116] Table 1

[0117]

[0118] As shown in Table 1, the increased cross-linking degree of collagen may increase the friction coefficient of the film surface after film formation to a certain extent. This may be because the increased cross-linking degree leads to an increased degree of collagen fibrillation, resulting in a decrease in the surface smoothness after film formation. However, the friction coefficient of the collagen film is much smaller than that of the electrospun fiber layer, as can be seen from Example 2 and Comparative Examples 2 and 3.

[0119] As can be seen from Example 2 and Comparative Example 1, the presence of the electrospun fiber layer can significantly improve the tensile breaking strength of the asymmetric anti-adhesion composite film, and this strength increases with the increase of the fiber electrospinning time.

[0120] The presence of the PVP electrospun fiber layer can significantly improve the wet adhesion of the asymmetric anti-adhesion composite film, and this adhesion increases with the increase of PVP fiber electrospun time.

[0121] The presence of an electrospun fiber layer can improve the in vitro degradation time of the asymmetric anti-adhesion composite membrane;

[0122] Comparative Examples 3 and 4 show that using a single-sided brush / scraping method to partially embed the electrospun fiber layer in the collagen film can result in a composite film with both strong tensile strength and good wet adhesion.

[0123] (3) Evaluation experiment on cell migration and proliferation activity of asymmetric anti-adhesion composite membrane

[0124] Fibroblasts were seeded in 6-well plates (fibroblast density was 1×10⁶). 5 / mL), cultured for 24 hours until cells were fully adhered, then the culture medium was changed. An asymmetric anti-adhesion composite membrane was then cut to fit perfectly to the wells of a six-well plate. One side of the collagen film was attached to the seeded cells. The culture dish was placed in a constant-temperature cell culture incubator and cultured for 4 days. After 4 days, the composite membrane was removed, washed three times with PBS, and then fibroblasts on the sponge were digested with trypsin and counted. Three samples were taken for each example and comparative example, and the average value and error were calculated. Results are as follows: Figure 2 As shown.

[0125] Asymmetric anti-adhesion composite membranes with collagen film layers have a good ability to prevent cell migration and proliferation through the film layer into the composite membrane fiber layer, which means they have ideal potential to prevent tissue adhesion.

[0126] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of those different embodiments or examples.

[0127] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "a plurality of" means two or more, unless otherwise explicitly specified.

[0128] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in this application, and these should all be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. An asymmetric anti-adhesion composite film, characterized in that, It includes a bioactive layer and an electrospun fiber layer, wherein the electrospun fiber layer includes a first fiber layer and a second fiber layer; the bioactive layer, the first fiber layer, and the second fiber layer are arranged sequentially. The first fiber layer includes fibers of a first diameter; the second fiber layer includes fibers of a first diameter and fibers of a second diameter, wherein the first diameter is larger than the second diameter; the diameter of the first diameter fiber ranges from 0.5 to 2 μm; the diameter of the second diameter fiber ranges from 0.2 to 0.5 μm. The bioactive layer is made of one or more of the following materials: collagen, silk fibroin, oxidized cellulose, chitosan, or hyaluronic acid. The material of the first diameter fiber is one or a combination of two or more of polylactic acid, polymethyl methacrylate, polyvinylidene fluoride, polyurethane or polycaprolactone. The material of the second diameter fiber is polyvinylpyrrolidone; The preparation method of the asymmetric anti-adhesion composite film includes the following steps: A second fiber layer is formed by simultaneously electrospinning fibers of a first diameter and fibers of a second diameter on a substrate; electrospinning of the second diameter fibers is stopped; electrospinning of the first diameter fibers is continued on the second fiber layer to form a first fiber layer; a bioactive layer is loaded on one side of the first fiber layer, and vacuum drying is performed to obtain the asymmetric anti-adhesion composite film.

2. The asymmetric anti-adhesion composite film according to claim 1, characterized in that, In the second fiber layer, the ratio of the number of fibers with the first diameter to the number of fibers with the second diameter is (1-3):

1.

3. The asymmetric anti-adhesion composite film according to claim 1, characterized in that, The thickness of the bioactive layer is 0.5-1 mm.

4. The asymmetric anti-adhesion composite film according to claim 1, characterized in that, The bioactive layer is a collagen layer; the collagen is prepared from animal Achilles tendon tissue through steps including enzymatic extraction, salting out, cross-linking and protein fixation.

5. The asymmetric anti-adhesion composite film according to claim 4, characterized in that, The enzyme used for enzymatic hydrolysis is a mixture of pepsin and lipase; the mass ratio of pepsin to lipase is (1-3):1; Protein fixation was performed in a glycine solution.

6. The asymmetric anti-adhesion composite film according to claim 1, characterized in that, Electrospun fiber preparation conditions: the spinning needle is 21G, the temperature of the spinning environment is 35-40℃, the relative humidity is 35-45%RH, the distance between the spinning needle and the collector is 20cm, and the spinning solution is injected at a rate of 1mL / h; the positive electrode voltage of the first diameter fiber spinning needle is set to +16kV, and the positive electrode voltage of the second diameter fiber spinning needle is set to +14kV.

7. The use of the asymmetric anti-adhesion composite membrane according to any one of claims 1-6 in the preparation of organ / tissue anti-adhesion products.

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