Application of electrostatic spinning membrane in preparation of induction material for promoting adipogenic differentiation of human mesenchymal stem cells

Abstract

The invention discloses application of an electrostatic spinning membrane in preparation of an induction material for promoting adipogenic differentiation of human mesenchymal stem cells (hMSCs), the electrostatic spinning membrane comprises a gelatin fiber membrane layer, the gelatin fiber layer is prepared from a gelatin solution through electrostatic spinning, and the diameter of gelatin spinning fibers is 200-1200 nm.

Description

Technical Field

[0001] The invention relates to the application of an electrospinning membrane, and in particular to the application of an electrospinning composite membrane in preparing an inducing material for promoting adipogenic differentiation of human mesenchymal stromal cells. Background Art

[0002] Electrospinning membranes are widely used in the field of tissue engineering as tissue repair materials. For example, the barrier membrane for guided bone regeneration disclosed in Chinese invention patent CN111330083A is pressed in the form of "gelatin fiber membrane-polycaprolactone fiber membrane-gelatin fiber membrane" to obtain a pressed membrane with a three-layer stacked structure, which is made by an electrospinning process and can effectively guide the growth of keratinized gums, as well as other oral and maxillofacial soft tissue repair. However, it has not yet been found that the electrospinning membrane is used to induce adipogenic differentiation of human bone marrow mesenchymal stem cells (hMCS). Summary of the invention

[0003] In view of the above technical background, the object of the present invention is to provide an application of an electrospun membrane in the preparation of an inducing material for promoting adipogenic differentiation of human bone marrow mesenchymal stem cells hMSC, wherein the electrospun membrane comprises a gelatin fiber membrane layer, wherein the gelatin fiber layer is made of a gelatin solution by electrospinning, and the diameter of the gelatin spinning fiber is 200nm-1200nm.

[0004] In some embodiments of the present invention, the electrospinning membrane comprises a laminated structure of a gelatin fiber membrane layer-a polycaprolactone fiber membrane layer-a gelatin fiber membrane layer, and the polycaprolactone fiber membrane is made from a polycaprolactone solution by electrospinning.

[0005] In some embodiments of the present invention, the fiber diameter of the gelatin fiber membrane layer is 300nm to 1000nm.

[0006] In some embodiments of the present invention, the fiber diameter of the gelatin fiber membrane layer is 200nm to 800nm.

[0007] In some embodiments of the present invention, the fiber diameter of the gelatin fiber membrane layer is 300nm-700nm.

[0008] In some embodiments of the present invention, the fiber diameter of the polycaprolactone fiber membrane layer is in the range of 3 to 5 microns.

[0009] In some embodiments of the present invention, the porosity of the gelatin fiber membrane layer is 82% to 87%, and the pore size distribution is 4um-15um.

[0010] In some embodiments of the present invention, the porosity of the polycaprolactone is 55% to 65%, and the pore size distribution is 15 um to 30 um.

[0011] In some embodiments of the present invention, the fiber diameter of the gelatin fiber membrane layer gradually changes in the thickness direction, gradually increasing from the side facing the polycaprolactone fiber membrane layer to the side away from the polycaprolactone fiber membrane layer.

[0012] In some embodiments of the present invention, the fiber diameter range of the side of the gelatin fiber membrane layer facing the polycaprolactone fiber membrane layer is 200 nm to 300 nm, and the fiber diameter range of the side away from the polycaprolactone fiber membrane layer is 700 nm to 800 nm.

[0013] In some embodiments of the present invention, the preparation of the gelatin fiber membrane layer includes the following steps:

[0014] (1) Preparation of gelatin solution: a. Prepare the dissolution solution: In the dissolution solution, the proportion of purified water is 35 - 40 wt%, the proportion of acetic acid is 25 - 30 wt%, and the proportion of butyl acetate is 30 - 40 wt%; b. Mix and dissolve gelatin and the dissolution solution at a mass ratio of 1:8 - 1:6. After complete dissolution, a gelatin solution is obtained;

[0015] (2) Preparation of gelatin fiber membrane layer: Add a crosslinking agent to the prepared gelatin solution. The proportion of the crosslinking agent in the gelatin solution is 0.5 - 0.6 wt%. After mixing evenly, inject the solution into the syringe pump of the electrospinning device. The distance between the spinning nozzle and the receiving mandrel is 70 - 100 mm; then start electrospinning to obtain a gelatin fiber membrane; the initial parameters of the electrospinning are: the rotational speed of the receiving mandrel is 100 - 200 rpm, the positive voltage is 12 - 15 kV, the negative voltage is 0.1 kV, and the solution flow rate is 3 - 4 mL / h; subsequently, every 25 - 35 min, increase the voltage by 1 - 1.5 kV and increase the flow rate by 0.1 - 0.2 mL / h. The total electrospinning time is 2 - 3 h to obtain the gelatin fiber membrane layer;

[0016] (3) Drying of gelatin fiber membrane layer: Place the obtained gelatin fiber membrane in an oven and dry it at 110 - 140 °C for 6 - 8 h, and then take it out to obtain the dried gelatin fiber membrane.

[0017] In some embodiments of the present invention, the preparation of the polycaprolactone fiber membrane layer includes the following steps:

[0018] (1) Preparation of polycaprolactone solution: Add polycaprolactone to the solvent chloroform. The mass ratio of the added polycaprolactone is 20 wt%. After complete dissolution, a polycaprolactone solution is obtained;

[0019] (2) Preparation of polycaprolactone fiber membrane: Add the prepared polycaprolactone solution to the syringe pump of the electrospinning device, and then start electrospinning to obtain a polycaprolactone fiber membrane;

[0020] (3) Drying of the polycaprolactone fiber membrane: Place the prepared polycaprolactone fiber membrane in an oven and dry it at 50 - 55 °C for 1 - 2 h, then take it out to obtain a dried polycaprolactone fiber membrane layer.

[0021] The present invention has the following beneficial technical effects compared with the prior art: The electrospun membrane of the present invention can promote the adipogenic differentiation of human mesenchymal stromal cells. The electrospun membrane includes a gelatin fiber membrane layer. Among them, the gelatin fiber layer is made by electrospinning a gelatin solution, and the diameter of the gelatin electrospun fiber is 200 nm - 1200 nm. In addition, it is further found that by regulating the fiber diameter of the gelatin fiber membrane layer therein, the adipogenic differentiation of human mesenchymal stromal cells can be effectively induced. Brief Description of the Drawings

[0022] Figure 1 Shows the intensity-calibrated immunofluorescence intensity of the adipogenic differentiation marker PPAR gamma of hMSC cultured on the electrospun membranes of Example 2 and Example 3 of the present invention.

[0023] Figure 2 Shows a comparison diagram of the intensity-calibrated immunofluorescence intensity of the adipogenic differentiation marker PPAR gamma of hMSC cultured on the electrospun membranes of Example 1, Example 2, and Example 3 of the present invention. Detailed Description of the Embodiments

[0024] The technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments and the accompanying drawings. The embodiments described herein are only a part of the embodiments of the present invention, not all of them. The following description of some exemplary embodiments is merely illustrative and does not constitute any limitation to the protection scope of the present invention. All other embodiments that can be obtained by those of ordinary skill in the art based on the specific embodiments recorded in the present invention without creative efforts belong to the protection scope of the present invention.

[0025] Example 1.

[0026] The electrospun composite membrane used in this example has a laminated structure of a gelatin fiber membrane layer - a polycaprolactone fiber membrane layer - a gelatin fiber membrane layer pressed together. Among them, the gelatin fiber layer is made by electrospinning a gelatin solution, and the diameter of the gelatin electrospun fiber is 200 nm - 800 nm. The side with a smaller fiber diameter in the gelatin fiber membrane layer faces the polycaprolactone fiber membrane. The fiber diameter range on the side facing the polycaprolactone fiber membrane is 200 nm - 300 nm, and the fiber diameter range on the side away from the polycaprolactone fiber membrane is 700 nm - 800 nm. The polycaprolactone fiber membrane layer is made by electrospinning a polycaprolactone solution, and the diameter of the polycaprolactone fiber is 3 - 5 microns.

[0027] The preparation of the electrospun composite membrane in this embodiment includes the following steps:

[0028] (1) Preparation of gelatin solution

[0029] a. Preparation of the dissolution solution:

[0030] In the dissolution solution, the proportion of purified water is 35 wt%, the proportion of acetic acid is 25 wt%, and the proportion of butyl acetate is 40 wt%;

[0031] b. Mix and dissolve gelatin and the dissolution solution at a mass ratio of 1:8. After complete dissolution, a gelatin solution is obtained; the gelatin is bovine bone gelatin, and the gel strength ranges from 160 to 180 bloom g;

[0032] (2) Preparation of gelatin fiber membrane

[0033] Add crosslinking agent glutaraldehyde to the prepared gelatin solution. The proportion of the added crosslinking agent is 0.5 wt%. After mixing evenly, inject the solution into the syringe pump of the electrospinning equipment. The distance between the spinning nozzle and the receiving mandrel is 70 - 100 mm; then start the electrospinning process. The initial electrospinning parameters are that the rotational speed of the receiving mandrel is 100 rpm, the positive voltage is 12 kV, the negative voltage is 0.1 kV, and the solution flow rate is 3 mL / h; then every 25 minutes, increase the voltage by 1 kV and the flow rate by 0.1 mL / h. The total electrospinning time is 3 h. Then the electrospinning ends, and a gelatin fiber membrane is obtained;

[0034] The fiber diameter in the thus-prepared gelatin fiber membrane gradually increases along the thickness direction, and the diameter range of the fibers is 200 - 800 nm;

[0035] (3) Drying of gelatin fiber membrane

[0036] Place the electrospun gelatin fiber membrane in an oven and dry it at 110 °C for 8 h, and then take it out to obtain the dried electrospun composite membrane.

[0037] (4) Preparation of polycaprolactone solution: Add polycaprolactone to chloroform. The mass ratio of the added polycaprolactone is 20 wt%. After complete dissolution, a polycaprolactone solution is obtained; the intrinsic viscosity of the polycaprolactone is 1.0 dL / g (volume percentage 0.1%, chloroform, 25 °C);

[0038] (5) Preparation of polycaprolactone fiber membrane: Add the prepared polycaprolactone solution to the syringe pump of the electrospinning equipment, and set the electrospinning parameters. The voltage parameters are positive voltage 16 kV, negative voltage 1 kV, and the feeding rate range is 1.5 mL / h; the total electrospinning time is 4 h; after the electrospinning ends, a polycaprolactone fiber membrane is prepared;

[0039] (6) Drying of the polycaprolactone fiber membrane: Place the polycaprolactone fiber membrane obtained by electrospinning in an oven and dry it at 50 °C for 1.5 h, and then take it out to obtain the dried polycaprolactone fiber membrane;

[0040] (7) Lamination of the gelatin fiber membrane and the polycaprolactone fiber membrane: Use a lamination device with controllable temperature and pressure to laminate the gelatin fiber membrane and the polycaprolactone fiber membrane; laminate in the form of "gelatin fiber membrane - polycaprolactone fiber membrane - gelatin fiber membrane", and the side of the gelatin fiber membrane with a smaller fiber diameter faces the polycaprolactone fiber membrane to obtain a multi-layer membrane with a three-layer laminated structure; the lamination temperature is 65 °C, the pressure is 12 N, and the lamination time is 40 seconds, thereby obtaining the electrospinning composite membrane of Example 1. Among them, the porosity of the gelatin fiber membrane layer is 82-85%, the pore size distribution is 4-15 μm, and the thickness is 0.20-0.24 mm. The porosity of polycaprolactone is 56-60%, the pore size distribution is 16-21 μm, and the thickness is 0.03-0.04 mm.

[0041] The effect of using the electrospinning composite membrane of Example 1 to induce adipogenic differentiation of human mesenchymal stromal cells was verified as follows:

[0042] 1. hMSC isolation and expansion:

[0043] Human bone marrow was collected from the proximal femur during joint replacement surgery, transferred to a 50 mL centrifuge tube containing 5000 IU heparin, washed with Dulbecco's phosphate buffered saline (DPBS), and centrifuged at 150 g for 7 min at room temperature. Lymphocyte separation medium with a density of 1.077 g / mL (GE Healthcare Life Science, Uppsala, Sweden) was used for density gradient separation of hMSCs by centrifuging at 150 g for 30 min at room temperature. 30 mL of DPBS was added to wash the interphase, and centrifuged at 350 g for 7 min at room temperature, and the supernatant was discarded. The precipitate was resuspended in 1 mL of FBS medium (prepared from low-glucose DMEM medium (1 g / L D-glucose, 1 mM sodium pyruvate, Sigma Aldrich), 10% fetal bovine serum FBS (Biochrom AG), 1000 IU / mL heparin (Carl Roth), 1% L-glutamine (Lonza Walkersville Inc.), 1% penicillin-streptomycin (Gibco)), transferred to a culture flask, and further cultured in an incubator at 37 °C and 5% CO2. After 24 hours and 96 hours, the medium was changed to remove non-adherent cells, and then the medium was changed twice a week. After 5 - 7 days, when the cell density reached 70 - 80%, 5 mL of Accutase cell digestion solution was used to digest the cells, digested at 37 °C for 5 min, counted, and 100,000 cells were re-seeded into a new culture flask. The medium was changed twice a week, and after culturing for 5 - 7 days, the cells were seeded for experiments.

[0044] 2. hMSCs were seeded onto the electrospun composite membrane for culture:

[0045] hMSCs were washed 3 times with 5 mL of DPBS, and 5 mL of Accutase cell digestion solution was used to digest the cells, digested at 37 °C for 5 min. The digested cells were transferred to a 50 mL Falcon tube, counted, and 10,000 cells were seeded onto a new six-well plate containing the electrospun composite membrane, and incubated in an incubator at 37 °C and 5% CO2 for 7 days for subsequent analysis.

[0046] 3. The expression level of single-cell labeled proteins was quantified using an intensity-calibrated immunofluorescence microscope to analyze the adipogenic differentiation of hMSCs induced by the electrospun composite membrane:

[0047] Samples cultured on the electrospun composite membrane were washed three times with DPBS, fixed with 1% paraformaldehyde solution (PFA) for 15 min, washed three times with DPBS again, and stored at 4 °C. For samples used for analysis, after removing DPBS from each sample, it was incubated with 1 mL of 0.1% Triton X 100 at room temperature for 30 min; after incubation, it was washed three times with DPBS, incubated with 1 mL of 1% bovine serum albumin (BSA) at room temperature for 1 hour, and washed three times with DPBS. The cell nuclei were stained with DAPI stain diluted 1:1000, and the cytoskeleton was stained with Phalloidin-iFluor 488 stain diluted 1:500. After removing DPBS, 1 mL of the mixture of the two stains was added to each well, and incubated at room temperature for 1 hour in the dark. The primary antibody was diluted with 1% BSA according to a specific dilution ratio (PPARgamma: Cell Signaling, 24355, 1:200), and the electrospun composite membrane samples were incubated. The oscillation frequency of the orbital shaker was adjusted to 5 rpm, and incubated overnight at 4 °C. After incubation, each well was washed three times with DPBS, DPBS was removed, and the sample was incubated with the secondary antibody (Alexa Fluor TM 647, Thermo Fisher, A21244) and orange 555 / 570 nuclear stain (biotium, 41033, 1:1000) together at room temperature for 1 hour in the dark. Finally, the samples were washed three times with DPBS. Using an Axio Observer Z1 inverted fluorescence microscope (Zeiss Oberkochen, Germany), microscopic images were taken with manual exposure at a magnification of 10x. Using the InSpeck TM Red (580 / 605) microscope image intensity calibration kit to set the manual exposure value. After using Fiji (a branched application of ImageJ) to segment single cells, the fluorescence signal intensity and density of single cells were determined.

[0048] The fluorescence signal intensity and density data of single cells were analyzed using SigmaPlot v.14.0 (Systat, Chicago) and Microsoft Excel (v.2013), and the analysis results are as Figure 2 shown. Figure 2 It clearly shows the intensity-calibrated single-cell labeled protein expression level of the hMSC adipogenic differentiation marker PPAR gamma. These data indicate that the electrospun composite membrane can successfully induce the adipogenic differentiation of hMSC.

[0049] Example 2

[0050] In this embodiment, the electrospun membrane used is a gelatin fiber electrospun membrane with a fiber diameter of 200 nm - 700 nm, which can be prepared by the electrospinning process known in the art. In this embodiment, it is prepared through the following steps:

[0051] (i) Provide a spinning solution

[0052] The spinning solution is a composite gelatin solution. Among them, the solvent is a mixed solvent composed of acetic acid and purified water, where acetic acid accounts for 25 wt%; the solute is bovine bone gelatin; the gelatin needs to be pretreated before use. The pretreatment method is as follows: First, dissolve the gelatin in ultrapure water, use a dialysis bag with a molecular weight of 45,000 to dialyze the solution to remove small molecules in the gelatin solution, and then for the retained macromolecules, use a dialysis bag with a molecular weight of 55,000 to collect molecules with a molecular weight of 45,000 - 50,000. After low-temperature concentration, it is freeze-dried and reserved; the pretreated gelatin is formulated into a gelatin solution using the above mixed solvent, and the gelatin solution is stirred at room temperature for 6 h and then electrospun. The mass ratio of the gelatin mixed solvent is 1:6.

[0053] (ii) The feeding system selects a needle with a diameter of 300 um for feeding, and an electrospun fiber membrane is prepared by controlling the feeding speed of the spinning solution, spinning temperature, spinning humidity, spinning voltage, spinning distance, rotation speed of the receiving device, and spinning time.

[0054] Specifically, the spinning solution is continuously fed at a feeding speed of 2 mL / h; the humidity during the spinning process is controlled at 45% RH, the spinning voltage is 18 KV, the spinning distance is 120 mm, the rotation speed of the receiving device is 150 rmp, and the spinning time is 5 h; the spinning temperature is 28 °C.

[0055] The electrospun membrane thus obtained is placed in an oven and heated at 60 °C for 24 h to crosslink and remove excess solvent in the fiber membrane, thereby obtaining an electrospun gelatin fiber membrane. After being tested by scanning electron microscopy, the fiber diameter of the obtained fiber membrane is 200 nm - 700 nm.

[0056] Example 3.

[0057] In this embodiment, the electrospun membrane used is a gelatin fiber electrospun membrane with a fiber diameter of 800 nm - 1200 nm, which can be prepared by the electrospinning process known in the art. In this embodiment, it is prepared through the following steps:

[0058] (i) Provide a spinning solution

[0059] The spinning solution is a composite gelatin solution. Among them, the solvent is a mixed solvent composed of acetic acid and purified water, where acetic acid accounts for 40 wt%; the solutes are bovine bone gelatin and glyoxal; gelatin, glyoxal and the mixed solvent are formulated into a composite gelatin solution, and the mass ratio of gelatin, glyoxal and the mixed solvent is 3:0.01:5.

[0060] The above-mentioned composite gelatin solution is stirred and dissolved at room temperature for 16 h, pretreated at 40 °C for 2 h, and then spun.

[0061] (ii) Continuously supply the spinning solution to the spinning electrode of the electrospinning device for spinning, and prepare an electrospun fiber membrane by controlling the spinning solution supply rate, spinning temperature, spinning humidity, spinning voltage, spinning distance, receiving device rotation speed and spinning time.

[0062] Specifically, the spinning solution is continuously supplied at a supply rate of 3 mL / h; the humidity during the spinning process is controlled at 20% RH, the spinning voltage is 28 KV, the spinning distance is 70 mm, the rotation speed of the receiving device is 100 rmp, and the spinning time is 4 h; the temperature control during the spinning process is carried out in 2 stages, the temperature is 38 °C for 0 - 2.5 h, and the temperature is 42 °C for 2.5 - 4 h.

[0063] The electrospun membrane thus obtained is placed in an oven and heated at 60 °C for 24 h to crosslink and remove the excess solvent in the fiber membrane, thereby obtaining an electrospun gelatin fiber membrane. After being tested by scanning electron microscopy, the fiber diameter of the obtained fiber membrane is 800 - 1200 nm.

[0064] Using the induction effect evaluation method described in Example 1, the effects of the electrospun membranes of Example 2 and Example 3 on inducing the adipogenic differentiation of hMSC were tested. Specifically, the separation and amplification of hMSC and the inoculation of hMSC onto the electrospun membrane for culture were the same as in Example 1, and the quantitative analysis of the expression level of single-cell marker proteins using an intensity-calibrated immunofluorescence microscope was also the same as in Example 1.

[0065] SigmaPlot v.14.0 (Systat, Chicago) and Microsoft Excel (v.2013) were used to analyze the fluorescence signal intensity and density data of single cells. The Kolmogorov - Smirnov test was used to test the normality of the data. For comparisons between two groups, Student's t-test was used for data that followed a normal distribution, and the Mann - Whitney rank sum test was used for data that did not follow a normal distribution to analyze and test the data. The analysis results are as Figure 1 shown. From Figure 1It can be seen that hMSCs express the adipogenic differentiation marker PPAR gamma protein when cultured on the electrospun membranes of Example 2 and Example 3. At the same time, there are statistically significant differences between the two groups (indicated by the symbol "*"), and hMSCs cultured on the electrospun membrane of Example 2 have a higher expression level of the adipogenic differentiation marker protein than hMSCs cultured on the electrospun membrane of Example 3. These data indicate that the electrospun membrane with a relatively smaller fiber diameter of the gelatin fiber membrane in Example 2 induces a higher expression level of adipogenic differentiation markers than the electrospun membrane with a relatively larger fiber diameter of the gelatin fiber membrane in Example 3.

[0066] The following compares and analyzes the induction effects of the electrospun membranes of Example 2 and Example 3 and the electrospun composite membrane of Example 1 on the adipogenic differentiation of hMSCs. The expression levels of single-cell marker proteins are also quantified using an immunofluorescence microscope calibrated with the same intensity as in Application Example 1.

[0067] The fluorescence signal intensity and density data of individual cells are analyzed using SigmaPlot v.14.0 (Systat, Chicago) and Microsoft Excel (v.2013). The Kolmogorov-Smirnov test is used to test the normality of the data. For comparisons between two groups, Student's t-test is used for data that follow a normal distribution, and the Mann-Whitney rank sum test is used for data that do not follow a normal distribution. For comparisons of more than two groups, analysis of variance with rank tests is used. If the analysis of variance shows significant differences between two groups, a post hoc test (Dunn's Method) is used to compare each group. Dunn's test allows comparisons of groups with different sample sizes, and the analysis results are as Figure 2 shown. As can be seen from Figure 2 , hMSCs cultured on the electrospun membranes of Example 1, Example 2, and Example 3 all express the adipogenic differentiation marker. At the same time, there are statistically significant differences between the three groups (indicated by the symbol "*"), and hMSCs cultured on the electrospun membrane of Example 2 have a higher expression of the adipogenic differentiation marker protein than hMSCs cultured on the electrospun membrane of Example 3 and the electrospun composite membrane of Example 1. The expression of the adipogenic differentiation marker protein PPARgamma in hMSCs cultured on the electrospun composite membrane of Example 1 is significantly higher than that on the electrospun membrane of Example 3 but lower than that on the electrospun membrane of Example 2.

[0068] From the analysis of the expression level of the adipogenic differentiation marker PPAR gamma in hMSCs in the above exemplary embodiments, it can be seen that the electrospun membrane of the present invention can successfully induce the adipogenic differentiation of hMSCs, and when the fiber diameter range in the gelatin fiber membrane layer is controlled within 200 nm - 700 nm, a more ideal induction effect is achieved.

[0069] The terms and phrases used in the description of the present invention are for illustrative purposes only and do not constitute a limitation. Those skilled in the art should understand that various changes can be made to the details of the above embodiments without departing from the basic principles of the disclosed embodiments. Therefore, the protection scope of the present invention is determined only by the claims, and in the claims, unless otherwise specified, all terms should be understood in the broadest and most reasonable sense.

Claims

1. Use of an electrospun membrane in the preparation of an induction material for promoting adipogenic differentiation of human mesenchymal stem cells hMSCs, wherein the electrospun membrane comprises a gelatin fiber membrane layer, where The gelatin fiber layer is made by electrospinning a gelatin solution, and the diameter of the gelatin spun fibers is 200 nm - 1200 nm.

2. The application according to claim 1, wherein the electrospun membrane comprises a laminated structure of a gelatin fiber membrane layer - a polycaprolactone fiber membrane layer - a gelatin fiber membrane layer formed by pressing, and the polycaprolactone fiber membrane is made by electrospinning a polycaprolactone solution.

3. The application according to claim 1, wherein the fiber diameter of the gelatin fiber membrane layer is 300 nm to 1000 nm.

4. The application according to claim 1, wherein the fiber diameter of the gelatin fiber membrane layer is 200 nm to 800 nm.

5. The application according to claim 1, wherein the fiber diameter of the gelatin fiber membrane layer is 300 nm - 700 nm.

6. The application according to claim 2, wherein the fiber diameter range of the polycaprolactone fiber membrane layer is 3 - 5 microns.

7. The application according to claim 2, wherein the porosity of the gelatin fiber membrane layer is 82% - 87%, and the pore size distribution is 4 um - 15 um.

8. The application according to claim 7, wherein the porosity of the polycaprolactone is 55% - 65%, and the pore size distribution is 15 um - 30 um.

9. The application according to claim 2, wherein the fiber diameter of the gelatin fiber membrane layer gradually changes along the thickness direction, gradually increasing from the side facing the polycaprolactone fiber membrane layer to the side away from the polycaprolactone fiber membrane layer.

10. The application according to claim 9, wherein the fiber diameter range of the side of the gelatin fiber membrane layer facing the polycaprolactone fiber membrane layer is 200 nm - 300 nm, and the fiber diameter range of the side away from the polycaprolactone fiber membrane layer is 700 nm - 800 nm.

11. The application according to claim 2, wherein the preparation of the gelatin fiber membrane layer comprises the following steps: (1) Preparation of the gelatin solution: a. Preparation of the dissolution solution: The proportion of purified water in the dissolution solution is 35 - 40 wt%, the proportion of acetic acid is 25 - 30 wt%, and the proportion of butyl acetate is 30 - 40 wt%; b. Mix and dissolve gelatin and the dissolution solution at a mass ratio of 1:8 - 1:6, and obtain a gelatin solution after complete dissolution. (2) Preparation of the gelatin fiber membrane layer: Add a cross-linking agent to the prepared gelatin solution, and the proportion of the cross-linking agent in the gelatin solution is 0.5 - 0.6 wt%. After mixing evenly, inject the solution into the syringe pump of the electrospinning device, and the distance between the spinning nozzle and the receiving mandrel is 70 - 100 mm; then start electrospinning to obtain a gelatin fiber membrane; the initial electrospinning parameters are: the rotational speed of the receiving mandrel is 100 - 200 rpm, the positive voltage is 12 - 15 kV, the negative voltage is 0.1 kV, and the solution flow rate is 3 - 4 mL / h; subsequently, every 25 - 35 min, increase the voltage by 1 - 1.5 kV and the flow rate by 0.1 - 0.2 ml / h, and the total electrospinning time is 2 - 3 h to obtain the gelatin fiber membrane layer. (3) Drying of the gelatin fiber membrane layer: Place the obtained gelatin fiber membrane in an oven, dry it at 110 - 140 °C for 6 - 8 h, and then take it out to obtain the dried gelatin fiber membrane. The preparation of the polycaprolactone fiber membrane layer includes the following steps: (1) Preparation of the polycaprolactone solution: Add polycaprolactone to the solvent chloroform, and the mass ratio of the added polycaprolactone is 20 wt%, and a polycaprolactone solution is obtained after complete dissolution; (2) Preparation of the polycaprolactone fiber membrane: Add the prepared polycaprolactone solution to the syringe pump of the electrospinning equipment, and then start electrospinning to obtain a polycaprolactone fiber membrane; (3) Drying of the polycaprolactone fiber membrane: Place the prepared polycaprolactone fiber membrane in an oven and dry it at 50-55 °C for 1-2 h, and then take it out to obtain a completely dried polycaprolactone fiber membrane layer.

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