Nanofiber membrane as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biomedical materials, and particularly discloses a nanofiber membrane as well as a preparation method and application thereof. The nanofiber membrane is an ordered polycaprolactone-molybdenum disulfide cross-linked laminin electrostatic spinning nanofiber membrane, the in-vivo nervous tissue microenvironment is simulated through an ordered topological structure of the nanofiber membrane, topographic clues and biological clues are transmitted, adhesion and proliferation of stem cells and differentiation of the stem cells to Schwann-like cells are promoted, and the stem cells can be effectively separated from the Schwann-like cells. Thus, myelination and neurite regeneration are promoted by continuously secreting neurotrophic factors. The molybdenum disulfide nanosheets are added to enhance the binding affinity of the nanofiber membrane to laminin, so that the hydrophilicity of the nanofiber membrane is improved, and a good microenvironment is provided for adhesion and proliferation of adipose-derived mesenchymal stem cells. The nanofiber membrane provided by the invention can effectively solve the problems of low cell survival rate, incomplete differentiation and the like in stem cell transplantation, and is expected to provide a promising solution for treating peripheral nerve injury by stem cell transplantation.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and particularly relates to a nanofiber membrane, a preparation method thereof and an application thereof. Background Art

[0002] Peripheral nerve injury caused by various factors such as trauma, infection and metabolic disorders remains a major clinical challenge worldwide, which seriously reduces people's quality of life. Different from the central nervous system, neurons in the peripheral nervous system have a certain degree of regeneration and growth ability after injury, but the slow axon elongation speed and inaccurate direction hinder the repair of peripheral nerves. At present, autologous nerve transplantation is still the gold standard for repairing peripheral nerve defects. However, factors such as insufficient donor nerves, functional limitations of the donor site, nerve fiber mismatch, postoperative nerve distortion and dislocation, and immunosuppression severely limit its clinical application.

[0003] During the regeneration process of peripheral nerves, Schwann cells (SCs) play a crucial role. After nerve injury, SCs first recruit macrophages to clear myelin debris, and then proliferate and longitudinally arrange to form Büngner bands, providing a longitudinal guiding channel for axon regeneration at the nerve stump. In addition, SCs can also secrete neurotrophic factors to support axon growth and myelin formation, thereby further promoting nerve regeneration. Unfortunately, in most experimental and clinical studies, the source of donor SCs is limited by the low proliferation ability of adult peripheral nerve cells and the high morbidity of the donor site. Interestingly, various types of mesenchymal stem cells can be induced into SCs-like cells, such as adipose-derived mesenchymal stem cells (ADSCs), bone marrow mesenchymal stem cells (BMSCs), umbilical cord mesenchymal stem cells (UCSCs), and dental pulp mesenchymal stem cells (DPSCs). In particular, ADSCs have become promising seed cells for the treatment of peripheral nerve injury repair due to their great differentiation potential and easy access through autologous and minimally invasive methods. However, there are still problems such as low cell survival rate and incomplete differentiation during the transplantation of ADSCs, which limit their effect in nerve repair. Summary of the Invention

[0004] The purpose of the present invention is to provide a nanofiber membrane, a preparation method thereof and an application thereof, aiming at the deficiencies in the prior art. By its ordered topological structure and simulating the microenvironment of nerve tissue in vivo, it transmits topographical cues and biological cues to promote the adhesion, proliferation and neuronal differentiation of adipose-derived mesenchymal stem cells (ADSCs), thereby promoting myelin formation and neurite regeneration by continuously secreting neurotrophic factors. The A PCL-MoS prepared by the present invention 2-LN nanofiber membranes can effectively solve a series of problems in stem cell transplantation, such as low cell survival rate, incomplete differentiation, and limited growth of neurites in vivo, etc., and are expected to provide a promising solution for the treatment of peripheral nerve injury by stem cell transplantation.

[0005] To achieve the above object, the present invention adopts the following technical solutions: The first aspect of the present invention is to provide a preparation method of a nanofiber membrane, comprising the following steps: S1. Disperse MoS 2 nanosheet powder in a hexafluoroisopropanol solution to obtain a MoS 2 nanosheet dispersion liquid, and then mix polycaprolactone particles into the MoS 2 nanosheet dispersion liquid, stir overnight at room temperature to prepare a spinning solution; S2. Inject the spinning solution into an electrospinning machine for electrospinning. The parameters of electrospinning are: flow rate 0.5 - 1.5 mL / h, voltage 8 - 15 kv, distance between the needle tip and the receiving plate 10 - 15 cm, and electrospinning duration 1 - 2 h; S3. Use a high - speed rotating mandrel at 2800 rpm to collect ordered nanofibers to obtain a fiber membrane, denoted as A PCL; S4. Cut the fiber membrane into circular pieces, after irradiating with ultraviolet light, soak it in a NaOH solution for a period of time to generate a surface with reactive functional groups; S5. Dissolve the sample obtained in S4 in a MES solution, remove the MES solution, and then add a MES / EDC / NHS solution for incubation at room temperature for a preset time; S6. Immerse the sample obtained in S5 in a laminin solution and gently oscillate overnight at 4 °C to obtain the nanofiber membrane.

[0006] Further, in step S1, 0.7 - 1.2 g of MoS 2 nanosheets, 10 ml of hexafluoroisopropanol, and 1.2 g of polycaprolactone.

[0007] Further, in step S4, the concentration of the NaOH solution is 0.04 - 0.05 M, and the soaking time is 20 - 25 min.

[0008] Further, in step S4, the ultraviolet light irradiation time is 1 h - 2 h.

[0009] Further, in step S5, the concentration of the MES solution is 0.1 M, the concentration of NHS is 6 mg / mL, and the concentration of EDC is 4 mg / mL.

[0010] Further, in step S6, the concentration of the laminin solution is 0.2 - 2 mg / mL.

[0011] The second aspect of the present invention is to provide a nanofiber membrane obtained by the above preparation method.

[0012] The third aspect of the present invention is to provide the application of the above nanofiber membrane in the treatment of peripheral nerve injury by stem cell transplantation.

[0013] The fourth object of the present invention is to provide the application of the above nanofiber membrane in in vitro cell culture.

[0014] Furthermore, the cell is an adipose-derived mesenchymal stem cell.

[0015] Compared with the prior art, the beneficial effects brought by the technical solution provided by the present invention are as follows: (1) The nanofiber membrane provided by the present invention is an ordered polycaprolactone-molybdenum disulfide cross-linked laminin electrospun nanofiber membrane. Through its ordered topological structure, it mimics the microenvironment of nerve tissue in vivo to transmit topographical cues and biological cues, so as to promote the adhesion, proliferation and differentiation of stem cells into Schwann-like cells, thereby promoting myelin formation and neurite regeneration by continuously secreting neurotrophic factors. Adding MoS 2 nano-sheets enhances the binding affinity of the nanofiber membrane for laminin, thereby improving the hydrophilicity of the nanofiber membrane and providing a good microenvironment for the adhesion and proliferation of adipose-derived mesenchymal stem cells (ADSCs).

[0016] (2) The average diameter of the nanofiber membrane provided by the present invention is below 300 nm, which is close to the diameter of collagen fibers (10 - 300 nm) that dominate the extracellular matrix structure.

[0017] (3) The nanofiber membrane provided by the present invention has good biosafety, can promote the adhesion and proliferation of ADSCs and induce their differentiation into Schwann-like cells, thereby promoting myelin formation and neurite regeneration by continuously secreting neurotrophic factors.

[0018] (4) The experimental principle of the preparation method of the nanofiber membrane provided by the present invention is simple, the operation procedure is simple, and the conditions are mild. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 are the scanning electron micrographs of RP CL, RP CL-MoS 2 , A PCL, A PCL-MoS 2 prepared by the present invention; Figure 2 are the graphs of the adsorption of laminin by the PCL and PCL-MoS 2 nanofiber membranes in Example 1; Figure 3 are the PCL and PCL-MoS in Example 12 , PCL-LN, PCL-MoS 2 Water contact angle diagram of -LN nanofiber membrane; Figure 4 SEM image of the nanofiber membrane prepared by electrospinning in Example 2; Figure 5 SEM image of the nanofiber membrane prepared by electrospinning in Example 3; Figure 6 For PCL, PCL-MoS detected at 1 day, 3 days, and 5 days in Example 6 2 , PCL-LN, PCL-MoS 2 CCK-8 results of -LN nanofiber membrane on the viability of ADSCs; Figure 7 For R PCL-LN, R PCL-MoS in Example 7 2 -LN, A PCL-LN, and A PCL-MoS 2 Live / dead cell staining fluorescence images of -LN nanofiber membrane on ADSCs; Figure 8 For ADSCs in R PCL-LN, R PCL-MoS in Example 8 2 -LN, A PCL-LN, and A PCL-MoS 2 After culturing on -LN nanofiber membrane for 14 days in Example 8, its morphology and the expression of S100β and GFAP were identified by immunofluorescence; Figure 9 For R PCL-LN, R PCL-MoS in Example 8 2 -LN, A PCL-LN, and A PCL-MoS 2 Gray value scanning diagrams of S100β, GFAP, NGF, and β-action on -LN; Figure 10 For R PCL-LN, R PCL-MoS in Example 8 2 -LN, A PCL-LN, and A PCL-MoS 2 Expression level diagrams of S100β, GFAP, and NGF on -LN. Detailed implementation manners To make the objectives, technical solutions, and advantages of the present invention clearer, the following further describes in detail the specific implementation manners of the present invention in combination with specific embodiments and the accompanying drawings. For those not specified in the embodiments regarding specific test methods, instrument equipment, or conditions, they shall all be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0020] PCL, MoS 2 , HFIP, MES, EDC, NHS, laminin, β-mercaptoethanol, and all-trans retinoic acid were purchased from Macklin Biochemical Co., Ltd. (China); the CCK-8 kit was purchased from Dongren Chemical Technology Co., Ltd. (Japan); the Calcein-AM / EthD-1 double staining kit was purchased from Invitrogen (USA); NGF, BDNF, and bFGF were purchased from Peprotech (USA); DMEM / F12 medium, fetal bovine serum (FBS), penicillin, streptomycin, and trypsin were purchased from Gibco (CA, USA); NGF, S100β, goat anti-mouse Alexa Fluor® 488, and goat anti-rabbit Alexa Fluor® 594 antibodies were purchased from Abcam (UK); the GFAP antibody was purchased from Sanying Biotechnology Co., Ltd. (China); the β-actin antibody was purchased from Sevier Biotechnology Co., Ltd. (China); the deionized water used in the experiment was from a Milli-Q Gradient A10 pure water system.

[0021] Example 1 This example provides a method for preparing a nanofiber membrane, and the specific steps are as follows: Add 1.2 g of polycaprolactone (PCL) to 10 mL of hexafluoroisopropanol (HFIP), stir overnight to prepare a uniform 12% w / v PCL solution. Additionally, accurately weigh 0.1 g of MoS 2 nanosheet powder, add it to 10 mL of HFIP solution, and ultrasonicate for 30 min to fully disperse it. Mix 1.2 g of PCL particles into the MoS 2 nanosheet dispersion, stir at room temperature overnight to make a spinning solution. Before electrospinning, ultrasonicate the mixture for 30 min, then inject the spinning solution into a 10 mL plastic syringe equipped with a 22-gauge stainless steel needle, and fix it on an electrospinning machine. The parameters of electrospinning are: flow rate: 1 mL / h, voltage 10 kv, the distance between the needle tip and the receiving plate is 15 cm, and the spinning duration is 2 h. Among them, the disordered fibers are collected on the aluminum foil of a 100 rpm slow rotating mandrel, and the ordered nanofibers are collected on the aluminum foil of a 2800 rpm high-speed rotating mandrel. Prepare the PCL nanofiber membrane according to the same method above. After that, the disordered (R) and ordered (A) PCL membranes are respectively named R PCL and A PCL. The disordered and ordered PCL membranes loaded with MoS 2 are respectively named R PCL-MoS 2 and A PCL-MoS 2 .

[0022] The prepared PCL membranes were cut into 10 mm×10 mm small round pieces, soaked in 75% alcohol overnight, washed three times with PBS, irradiated with ultraviolet light from an ultraviolet lamp in a cell ultra-clean bench for 1 - 2 h, and placed in 1.5 mL EP tubes for later use. All samples were first immersed in 0.04 M NaOH solution and incubated at room temperature for 20 min to generate a surface with reactive functional groups. Subsequently, the samples were immersed in 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES) solution for 30 min. 240 mg of N-hydroxysuccinimide (NHS) and 160 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were weighed using an electronic balance, dissolved in 40 mL of MES solution, mixed evenly, and reserved for later use. The MES solution in the EP tube was removed, and 1 mL of MES / EDC / NHS solution was added to the EP tube and incubated at room temperature for 1 h. Finally, each sample was rinsed 3 times with MES solution. The samples were soaked in 100 μL of LN solution (0.5 mg / mL) and gently shaken overnight at 4 °C. The BCA protein assay kit was used to quantify the percentage of protein absorbed on the fiber membrane. The method was to add the prepared BCA working solution to a 96-well plate, then add 20 μL of the crosslinked LN solution, incubate at 37 °C for 30 min, and use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance at 562 nm to quantify the protein content in each group. The LN standard curve was set, and the laminin absorption percentage was calculated according to the formula.

[0023] As Figure 1 shown, SEM observation showed that the RP CL, RP CL-MoS 2 and AP CL, AP CL-MoS 2 nanofiber membranes prepared by electrospinning, and these fibers were all continuous, smooth, and bead-free, with diameters all below 300 nm. Compared with PCL fibers, the addition of MoS 2 nanosheets did not cause significant changes in the morphology and diameter of the composite fiber membranes, indicating that the loading of MoS 2 nanosheets had little effect on the apparent morphology of the fibers.

[0024] To study the change in the water contact angle values of the nanofiber membranes, 5 μL of deionized water was vertically dropped onto the surfaces of PCL, PCL-MoS 2 and PCL-LN, PCL-MoS 2 -LN fiber membranes crosslinked with LN, and the contact angle values after the water droplets dropped were measured. Each group of samples had at least 5 parallel samples, and the average contact angle values of each group of fiber membranes were photographed, recorded, and calculated.

[0025] As Figure 2As shown, the BCA protein quantification kit was used to quantitatively detect the adsorption of PCL and PCL-MoS 2 nano-fiber membrane LN. Compared with the PCL nano-fiber membrane, the cross-linked LN on the PCL-MoS 2 nano-fiber membrane increased significantly.

[0026] As Figure 3 shown, the water contact angle values of PCL and PCL-MoS 2 were 111.2±2.8° and 101.4±1.8°, respectively, both greater than 90°, indicating that both are hydrophobic materials. However, after cross-linking with LN, the water contact angle of PCL-LN decreased to 41.1±0.8°, and that of PCL-MoS 2 -LN decreased to 15.6±0.3°, both less than 90°, indicating that the material changed from hydrophobic to hydrophilic. The BCA experiment and the water contact angle experiment proved that the addition of MoS 2 nanosheets enhanced the binding affinity of the scaffold for LN, thereby improving the hydrophilicity of the scaffold and providing a good microenvironment for the adhesion and proliferation of ADSCs.

[0027] Example 2 It was basically the same as Example 1, except that the parameters of electrospinning were: flow rate: 0.5 mL / h, voltage 8 kv, the distance between the needle tip and the receiving plate was 10 cm, and the spinning duration was 0.5 h.

[0028] As Figure 4 shown, due to the relatively low flow rate and voltage values used, the electrospun fibers were relatively large (1~2μm), which did not meet the diameter range of collagen fibers in the extracellular matrix structure required by the experiment (10~300nm).

[0029] Example 3 It was basically the same as Example 1, except that the parameters of electrospinning were: flow rate: 1.5 mL / h, voltage 15 kv, the distance between the needle tip and the receiving plate was 15 cm, and the spinning duration was 1.5 h.

[0030] As Figure 5 shown, due to the relatively high flow rate and voltage values used, the electrospun fibers showed a discontinuous state during the experiment, specifically manifested as beading on the fibers and uneven thickness changes in diameter.

[0031] Example 4 It was basically the same as Example 1, except that the concentration of the laminin solution was 0.2 mg / mL.

[0032] Example 5 It was basically the same as Example 1, except that the concentration of the laminin solution was 2 mg / mL.

[0033] Example 6 ADSCs were seeded on PCL, PCL-MoS 4 at a density of 2×10 2 , PCL-LN, PCL-MoS 2 -LN. After culturing for 1, 3, and 5 days, the culture plates were taken out, the culture medium was aspirated, washed 3 times with PBS, 50 μL of CCK-8 and 450 μL of culture medium were added in the dark, and incubated for 2 h. 100 μL of the solution was aspirated into a 96-well plate, with 4 replicate wells for each sample. The absorbance (OD value) of each plate at 450 nm was measured using a microplate reader to indirectly reflect the cell viability.

[0034] As Figure 6 shown, ADSCs were seeded on the surfaces of PCL, PCL-LN, PCL-MoS 2 , PCL-MoS 2 -LN. After culturing for 1, 3, and 5 days, the effects of these materials on the proliferation of ADSCs were evaluated by CCK-8 assay. Compared with the Control group, PCL and PCL-MoS 2 showed no obvious cytotoxicity, while the OD values of PCL-LN and PCL-MoS 2 -LN crosslinked with LN were higher than those of the Control group, indicating good biocompatibility with ADSCs and promoting the proliferation of ADSCs.

[0035] Example 7 After culturing ADSCs on R PCL-LN, R PCL-MoS 2 -LN, A PCL-LN, and A PCL-MoS 2 -LN for 72 h, 300 μL of Calcein-AM / EthD-1 staining working solution (5 mL PBS + 2.5 μL Calcein-AM + 10 μL EthD-1) was added to each well and incubated for 30 min in the dark. 1 mL of PBS was added to each well and washed three times. The imaging effect was observed under a confocal microscope (live cells would be stained green and dead cells would be stained red).

[0036] As Figure 7 shown, the Live / Dead experiment verified the biocompatibility of ADSCs on the scaffolds and its effect on cell morphology by fluorescence excitation, making live cells emit green fluorescence and dead cells emit red fluorescence. There were very few EthD-1 positive (dead cells) on all scaffolds, indicating that ADSCs on R PCL-LN, R PCL-MoS 2-LN, APCL-LN, and APCL-MoS 2 They can all adhere and grow healthily on -LN, showing good biocompatibility. In addition, the experimental results also showed that ADSCs presented a random diffusion state on the disordered fiber membrane without a specific directionality, while on the ordered fiber membrane, ADSCs were arranged along the longitudinal axis of the fiber in an orderly direction.

[0037] Example 8 Take the membrane in Example 1 and place it in a 24 - well plate. Add 2×10 4 ADSCs into each well and culture them in an incubator at 37 °C and 5% CO 2 . Induce differentiation into SCs through the following steps: Add DMEM / F12 complete medium (containing 10% FBS + 1% P / S) with 1 mM β - mercaptoethanol and culture for 24 h; add DMEM / F12 medium containing 35 ng / mL all - trans retinoic acid and treat for 72 h; add nerve induction medium: DMEM / F12 medium containing 10 ng / mL brain - derived neurotrophic factor (BDNF), 10 ng / mL nerve growth factor (NGF), and 20 ng / mL basic fibroblast growth factor (bFGF), and change the medium every 3 d.

[0038] (1) After inducing ADSCs for 14 d, detect the expression of S100β and GFAP by immunofluorescence staining. Fix with 4% paraformaldehyde for 30 min, discard it, wash with PBS 3 times, 10 min each time. Treat the cells with 0.1% TritonX - 100 for 20 min for permeabilization; discard TritonX - 100, add 10% goat serum blocking solution, block at room temperature for 2 h, add the primary antibody rabbit anti - S100β (1:200), and incubate overnight in a 4 °C refrigerator. Wash 3 times with TBST to remove the excess S100β antibody, then add mouse anti - GFAP (1:200) and incubate overnight in a 4 °C refrigerator. Wash 3 times again with TBST, add goat anti - mouse Alexa Fluor® 488 (1:100) and goat anti - rabbit Alexa Fluor® 594 (1:100), incubate at room temperature in the dark for 2 h, then wash 3 times with TBST, 10 min each time, add DAPI, and take pictures under a confocal microscope to observe the cell morphology and the expression of the marker proteins (S100β) and glial fibrillary acidic protein (GFAP) of SCs.

[0039] (2)After 14 days of induction, the cells were digested from the nanofiber membrane with 0.25% trypsin, an appropriate amount of high-efficiency RIPA cell lysate (containing 1% PMSF) was added, and the cells were lysed on ice for 30 min. The lysate was collected in a 1.5 mL EP tube, grinding beads were added, and the sample was ground in a low-temperature grinder for 30 s. Then, it was centrifuged at 12,000 rpm at 4 °C for 30 min, and the protein supernatant was collected. After protein quantification, 25 μg of total protein from each group of samples was loaded onto a 10% SDS-PAGE lane for electrophoresis separation. Then, the target protein was transferred to a PVDF membrane. After washing the membrane, antibodies S100β (1:800), GFAP (1:1000), NGF (1:1000), and β-action (1:1500) were added respectively, and the membrane was incubated overnight at 4 °C. After rinsing with PBS three times for 10 minutes each time, the corresponding secondary antibody was incubated at room temperature for 2 hours. Chemiluminescent reagent was added, and the membrane was exposed, developed, washed, and scanned for gray value using Image J software.

[0040] As Figure 8 shown, the expressions of S100β and GFAP in non-induced ADSCs were negative, while the induced ADSCs showed a spindle shape, and the expressions of S100β and GFAP were positive. Among them, on the A PCL-MoS 2 -LN nanofiber membrane, ADSCs were arranged in a bipolar slender spindle shape, which was similar to the common morphology of SCs.

[0041] As Figure 9 and Figure 10 shown, the results of Western blotting experiments further confirmed that the expression levels of S100β, GFAP, and NGF (nerve growth factor) in the A PCL-MoS 2 -LN group were higher, indicating that it promoted the differentiation of ADSCs into SCs and induced the secretion of neurotrophic factors.

[0042] Without conflict, the above embodiments and the features in the embodiments in this article may be combined with each other.

[0043] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a nanofiber membrane, characterized in that: The following steps are involved: S1, dispersing MoS2 nanosheet powder in hexafluoroisopropanol solution to obtain MoS2 nanosheet dispersion, then mixing polycaprolactone particles into the MoS2 nanosheet dispersion, stirring at room temperature overnight to prepare a spinning solution; S2, injecting the spinning solution into the electrospinning machine for spinning, the electrospinning parameters are: flow rate 0.5~1.5 mL / h, voltage 8~15 kV, needle tip distance 10~15 cm from the receiving plate, spinning duration 1~2 h; S3, using a high-speed rotating mandrel at 2800 rpm to collect the ordered nanofibers to obtain a fiber membrane, which is recorded as A PCL; S4, cutting the fiber membrane into discs, irradiating them with ultraviolet light, and then soaking them in a NaOH solution for a period of time to generate a surface with active reaction groups; S5, dissolving the sample obtained in S4 in MES solution, removing the MES solution, and then adding MES / EDC / NHS solution to incubate at room temperature for a preset time; S6. Soak the sample obtained in S5 in a laminin solution and gently shake it at 4° C. overnight to obtain the nanofiber membrane.

2. The preparation method according to claim 1, characterized in that In step S1, 0.7-1.2 g MoS2 nanosheets, 10 ml hexafluoroisopropanol, and 1.2 g polycaprolactone.

3. The preparation method according to claim 1, characterized in that In step S4, the concentration of the NaOH solution is 0.04-0.05 M, and the infiltration time is 20-25 min.

4. The preparation method according to claim 3, characterized in that: In step S4, the ultraviolet light irradiation time is 1 h to 1.5 h.

5. The preparation method according to claim 1, characterized in that In step S5, the concentration of the MES solution is 0.1 M, the concentration of NHS is 6 mg / mL, and the concentration of EDC is 4 mg / mL.

6. The preparation method according to claim 1, characterized in that In step S6, the concentration of the laminin solution is 0.2-2 mg / mL.

7. A nanofiber membrane prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the nanofiber membrane as claimed in claim 7 in stem cell transplantation for the treatment of peripheral nerve damage.

9. Use of the nanofiber membrane as claimed in claim 7 in in vitro cell culture.

10. The use according to claim 9, characterized in that The cells are adipose-derived mesenchymal stem cells.

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