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

Abstract

The invention discloses application of an electrostatic spinning membrane in preparation of an induction material for promoting cartilage 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 the electrospinning membrane in preparing an inducing material for promoting chondrogenic 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 cartilage 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 chondrogenic 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 spun 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 75-85%, and the pore size distribution is 3um-15um.

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

[0011] In some embodiments of the present invention, 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.

[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 the gelatin solution: a. Preparation of 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, and obtain a gelatin solution after complete dissolution.

[0015] (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 equipment, 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 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, and the total electrospinning time is 2 - 3 h to obtain the gelatin fiber membrane layer.

[0016] (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.

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

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

[0019] (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.

[0020] (3) Drying of the polycaprolactone fiber membrane: Place the obtained 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 chondrogenic 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 fibers 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 chondrogenic differentiation of human mesenchymal stromal cells can be effectively induced. Description of the Drawings

[0022] Figure 1 Shows the intensity - calibrated immunofluorescence intensity of the chondrogenic differentiation marker COL2A1 of hMSC cultured on the electrospun composite membrane of Example 1 of the present invention for 7 days.

[0023] Figure 2A Shows the molecular copy number of the chondrogenic differentiation marker COL2A1 of hMSC cultured on the electrospun composite membrane of Example 1 of the present invention for 7 days analyzed by droplet digital PCR (ddPCR);

[0024] Figure 2B is Figure 2A The molecular copy number normalized by the control group.

[0025] Figures 3A to 3F Shows the comparison of the quantitative morphological characteristics of hMSC cultured on the electrospun composite membrane of Example 1 of the present invention for 7 days with those of hMSC cultured on a control plastic bottom plate for 7 days. Among them, Figure 3A Shows the comparison of the cell area (Area), Figure 3B Shows the comparison of the major axis (Major), Figure 3C Shows the comparison of the minor axis (Minor), Figure 3D Shows the comparison of the ratio of the major axis to the minor axis (AR), Figure 3F Shows the comparison of the roundness, Figure 3F Shows the comparison of the circularity.

[0026] Figure 4 Shows the intensity - calibrated immunofluorescence intensity of the chondrogenic differentiation marker COL2A1 of hMSC cultured on the electrospun membranes of Example 2 and Example 3 of the present invention.

[0027] Figure 5Shows a comparison diagram of the intensity-calibrated immunofluorescence intensity of the chondrogenic differentiation marker COL2A1 of hMSCs cultured on the electrospun membranes of Example 1, Example 2, and Example 3 of the present invention. Detailed implementation manners

[0028] The technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments and the accompanying drawings. The described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. The following description of some exemplary embodiments is only illustrative and does not constitute any limitation to the protection scope of the present invention. All other implementation manners that those of ordinary skill in the art can obtain without creative efforts based on the specific implementation manners recorded in the present invention belong to the protection scope of the present invention.

[0029] Example 1.

[0030] The electrospinning used in this example is a composite membrane, which includes 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 to 300 nm, and the fiber diameter range on the side away from the polycaprolactone fiber membrane is 700 nm to 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.

[0031] The preparation of the electrospun composite membrane in this example includes the following steps:

[0032] (1) Preparation of the gelatin solution

[0033] a, Prepare the dissolving solution:

[0034] In the dissolving 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%;

[0035] b, Mix and dissolve gelatin and the dissolving solution in a mass ratio of 1:8. After complete dissolution, a gelatin solution is obtained; the gelatin is bovine bone gelatin, and the gel strength range is 160 - 180 bloom g;

[0036] (2) Preparation of the gelatin fiber membrane

[0037] Add crosslinking agent glutaraldehyde to the prepared gelatin solution, with the addition ratio of the crosslinking agent being 0.5 wt%, and 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 parameters of electrospinning 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, and then the electrospinning ends to obtain a gelatin fiber membrane;

[0038] The fiber diameter in the gelatin fiber membrane prepared thus gradually increases along the thickness direction, and the diameter range of the fibers is 200 - 800 nm;

[0039] (3) Drying of the gelatin fiber membrane

[0040] Place the gelatin fiber membrane prepared by electrospinning in an oven and dry it at 110 °C for 8 h, and then take it out to obtain a dried electrospun composite membrane.

[0041] (4) Preparation of the polycaprolactone solution: Add polycaprolactone to chloroform, with the mass ratio of the added polycaprolactone being 20 wt%, and after complete dissolution, obtain a polycaprolactone solution; the intrinsic viscosity of the polycaprolactone is 1.0 dL / g (volume percentage 0.1%, chloroform, 25 °C);

[0042] (5) Preparation of the 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 a positive voltage of 16 kV and a negative voltage of 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;

[0043] (6) Drying of the polycaprolactone fiber membrane: Place the polycaprolactone fiber membrane prepared by electrospinning in an oven and dry it at 50 °C for 1.5 h, and then take it out to obtain a dried polycaprolactone fiber membrane;

[0044] (7) Lamination of gelatin fiber membrane and polycaprolactone fiber membrane: Use a lamination device with controllable temperature and pressure to laminate the gelatin fiber membrane and the polycaprolactone fiber membrane; laminate them 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 3-layer laminated structure; the lamination temperature is 65 °C, the pressure is 12 N, and the lamination time is 40 seconds, thereby obtaining the electrospun 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.

[0045] The effect verification of using the electrospun composite membrane of Example 1 to induce chondrogenic differentiation of human mesenchymal stromal cells is as follows:

[0046] 1. hMSC isolation and expansion:

[0047] Collect human bone marrow from the proximal femur during joint replacement surgery, transfer it to a 50 mL centrifuge tube containing 5000 IU heparin, wash it with Dulbecco's phosphate buffered saline (DPBS), and centrifuge at 150 g for 7 min at room temperature. Use lymphocyte separation medium with a density of 1.077 g / mL (GE Healthcare Life Science, Uppsala, Sweden) to perform density gradient separation of hMSC by centrifuging at 150 g for 30 min at room temperature. Add 30 mL of DPBS to wash the interphase, and centrifuge at 350 g for 7 min at room temperature, discard the supernatant. Resuspend the precipitate 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)), transfer it to a culture flask, and continue culturing in an incubator at 37 °C and 5% CO2. After 24 hours and 96 hours, change the medium to remove non-adherent cells, and then change the medium twice a week. After 5-7 days, when the cell density reaches 70-80%, take 5 mL of Accutase cell digestive solution to digest the cells, digest at 37 °C for 5 min, count, and re-inoculate 100,000 cells into a new culture flask. Change the medium twice a week, and after culturing for 5-7 days, inoculate the cells for the experiment.

[0048] 2. Inoculate hMSC onto the electrospun composite membrane for culture:

[0049] The hMSCs were washed three times with 5 mL of DPBS, and then 5 mL of Accutase cell digestive solution was used to digest the cells 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.

[0050] 3. The expression level of single-cell labeled proteins was quantified using an intensity-calibrated immunofluorescence microscope to analyze the chondrogenic differentiation of hMSCs induced by the electrospun composite membrane:

[0051] The 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 the samples used for analysis, after removing DPBS from each sample, the samples were incubated with 1 mL of 0.1% Triton X 100 at room temperature for 30 min; after incubation, the samples were washed three times with DPBS, incubated with 1 mL of 1% bovine serum albumin (BSA) at room temperature for 1 h, 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 the samples were incubated at room temperature for 1 h in the dark. The primary antibody was diluted with 1% BSA according to a specific dilution ratio (collagen type II (COL2A1), Thermo Fischer, MA5-12789, 1:200), and the electrospun composite membrane samples were incubated. The oscillation frequency of the orbital shaker was adjusted to 5 rpm, and the samples were incubated overnight at 4 °C. After incubation, each well was washed three times with DPBS, DPBS was removed, and the samples were co-incubated with the secondary antibody (Alexa Fluor TM 647, Thermo Fisher, A21244) and Orange 555 / 570 nuclear stain (biotium, 41033, 1:1000) in the dark at room temperature for 1 h. Finally, the samples were washed three times with DPBS. A Zeiss Axio Observer Z1 inverted fluorescence microscope (Zeiss Oberkochen, Germany) was used to take microscopic images with manual exposure at a magnification of 10x. The manual exposure value was set using an InSpeck TM Red (580 / 605) microscope image intensity calibration kit. After segmenting single cells using Fiji (a branched application of ImageJ), the fluorescence signal intensity and density of individual cells were determined.

[0052] 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 1 shown. Figure 1 It clearly shows the expression level of the single-cell labeled protein of the hMSC chondrogenic differentiation marker COL2A1 after intensity calibration. These data indicate that the electrospun composite membrane can successfully induce the chondrogenic differentiation of hMSC.

[0053] 4. Quantitative analysis of the expression level of the labeled gene using droplet digital PCR (ddPCR) for the chondrogenic differentiation of hMSC induced by the electrospun composite membrane:

[0054] RNA of hMCS cells cultured on the electrospun composite membrane was extracted using the RNeasy Micro kit (Qiagen), and the RNA concentration was determined by measuring the absorbance value at 260 nm. cDNA was synthesized using the oligonucleotide (dT) and random hexamer primers in the Advantage RT-for-PCR kit (Clontech) and the extracted total RNA. Subsequently, absolute quantification of the chondrogenic differentiation marker gene (COL2A1) was performed by ddPCR. An 11 μL ddPCR Supermix (without dUTP, Bio-Rad), 1.1 μL of the single-expression detection probe labeled with HEX or FAM (BioRad), 6.6 μL of cDNA (containing 1.5 ng of RNA), and 2.2 μL of DNase / RNase-free water were used to form a 22 μL system. The PCR reaction was carried out in a QX100 thermal cycler (Bio-Rad), and the set program was: activate the polymerase at 95 °C for 10 min, denature at 94 °C for 30 s, extend at 55 °C for 1 min, and cycle 40 times. Among them, the polymerase was denatured and inactivated at 98 °C for 10 min. The PCR products were stored at 4 °C. The droplet fluorescence was measured using a QX200 droplet reader (Bio-Rad), and normalization was achieved by using a standardized amount of RNA for reverse transcription and a standardized amount of cDNA in each reaction system.

[0055] Data analysis was performed using QuantaSoft software (Bio-Rad) to calculate the absolute concentration of each HEX- and FAM-labeled gene (unit: molecular copy number / μL). The expression of the labeled gene was compared using the molecular copy number / μL or the fold change relative to the control, and the analysis results are as Figures 2A - 2B . Figure 2AIt was shown that hMSCs expressed the chondrogenic differentiation marker gene COL2A1 at molecular copy numbers in the dozens to hundreds, clearly demonstrating the substantial expression of the chondrogenic differentiation marker gene COL2A1. Figure 2B In [reference], the molecular copy numbers were normalized by control. The processed data showed that the electrospun composite membrane induced chondrogenic differentiation. The above results were similar to the protein analysis results, and the copy of the labeled molecule could be detected by substantial expression.

[0056] To analyze the effect of the electrospun composite membrane on cell morphology, quantification was performed at the single-cell level using immunofluorescence microscopy: Cells were stained with 1 μM calcein (Thermo Fisher Scientific) and 1 μg / mL Hoechst (Thermo Fisher Scientific) for 30 min at 37 °C and 5% CO2, fixed with 1% paraformaldehyde solution (PFA) for 15 min, washed 3 times with DPBS, and stored at 4 °C. Microscopic images were taken at 10x magnification using an Axio Observer Z1 microscope (Zeiss Oberkochen, Germany). Single-cell segmentation and morphological measurements were performed using Fiji (a fork application of ImageJ), and the following parameters were analyzed respectively: (a) cell area (Area), (b) major axis (Major), (c) minor axis (Minor), (d) aspect ratio (AR) of the major and minor axes, (e) roundness (Roundness), (f) circularity (Circularity). The corresponding results are as Figures 3A to 3F shown, and it can be seen from Figure 3A that compared with the plastic control, the cell area (Area) of hMSCs on the electrospun composite membrane of Example 1 of the present invention was significantly reduced; it can be seen from Figure 3B that compared with the plastic control, the major axis (Major) of hMSCs on the electrospun composite membrane was significantly reduced; it can be seen from Figure 3C that compared with the plastic control, the minor axis (Minor) of hMSCs on the electrospun composite membrane was significantly reduced; it can be seen from Figure 3D that compared with the plastic control, the aspect ratio (AR) of the major and minor axes of hMSCs on the electrospun composite membrane was significantly reduced; it can be seen from Figure 3E that compared with the plastic control, the roundness (Roundness) of hMSCs on the electrospun composite membrane was significantly higher, and the morphological parameter "roundness (Roundness)" was more sensitive to changes in the main cell body than to changes in cell protrusions; it can be seen from Figure 3FIt can be seen that, compared with the plastic control, the circularity of hMSCs on the electrospun composite membrane is significantly reduced. The morphological parameter "circularity" is sensitive to changes in cell protrusions. In summary, compared with the plastic control, the electrospun composite membrane significantly induces different hMSC morphologies. hMSCs cultured on the electrospun composite membrane are significantly smaller, shorter, narrower, rounder and have fewer protrusions.

[0057] Through the above experimental analysis, it can be known that through the analysis of the chondrogenic differentiation marker COL2A1 of hMSCs at the single-cell protein expression level and the molecular copy number level, the electrospun composite membrane can induce the expression of chondrogenic differentiation markers of hMSCs. In addition, the changes in the expression of these differentiation markers are accompanied by changes in cell morphology.

[0058] Example 2

[0059] The electrospun membrane used in this example is a gelatin fiber electrospun membrane with a fiber diameter of 200nm - 700nm, which can be prepared by the electrospinning process known in the art. In this example, it is prepared by the following steps:

[0060] (i) Provide a spinning solution

[0061] The spinning solution is a composite gelatin solution. Among them, the solvent is a mixed solvent composed of acetic acid and purified water, with acetic acid accounting for 25wt%; the solute is bovine bone gelatin; the gelatin needs to be pretreated before use. The pretreatment method is: first dissolve the gelatin in ultrapure water, use a dialysis bag with a molecular weight of 45000 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 55000 to collect molecules with a molecular weight of 45000 - 50000, and after low-temperature concentration, perform freeze-drying for later use; prepare the gelatin solution with the above mixed solvent after the pretreatment is completed, and spin the gelatin solution after stirring at room temperature for 6h, where the mass ratio of the gelatin mixed solvent is 1:6.

[0062] (ii) The feeding system selects a needle with a diameter of 300um for feeding, and prepares an electrospun fiber membrane 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.

[0063] Specifically, the spinning solution is continuously delivered at a feeding speed of 2mL / h; control the spinning process humidity at 45%RH, spinning voltage at 18KV, spinning distance at 120mm, rotation speed of the receiving device at 150rmp, spinning time at 5h; spinning temperature at 28℃.

[0064] The electrospun membrane thus obtained was placed in an oven and heated at 60 °C for 24 h for crosslinking and removing the excess solvent in the fiber membrane, thereby obtaining an electrospun gelatin fiber membrane. After testing with a scanning electron microscope, the fiber diameter of the obtained fiber membrane was 200 nm - 700 nm.

[0065] Example 3

[0066] In this example, the electrospun membrane used was a gelatin fiber electrospun membrane with a fiber diameter of 800 nm - 1200 nm, which could be prepared by the electrospinning process known in the art. In this example, it was prepared through the following steps:

[0067] (i) Provide a spinning solution

[0068] The spinning solution was a composite gelatin solution. Among them, the solvent was a mixed solvent composed of acetic acid and purified water, where acetic acid accounted for 40 wt%; the solutes were bovine bone gelatin and glyoxal; gelatin, glyoxal, and the mixed solvent were formulated into a composite gelatin solution, and the mass ratio of gelatin, glyoxal, and the mixed solvent was 3:0.01:5.

[0069] The above composite gelatin solution was stirred and dissolved at room temperature for 16 h, pretreated at 40 °C for 2 h, and then spun.

[0070] (ii) Continuously supply the spinning solution to the spinning electrode of the electrospinning equipment 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.

[0071] Specifically, the spinning solution was continuously supplied at a supply rate of 3 mL / h; the humidity during the spinning process was controlled at 20% RH, the spinning voltage was 28 KV, the spinning distance was 70 mm, the rotation speed of the receiving device was 100 rmp, and the spinning time was 4 h; the temperature during the spinning process was controlled in 2 stages, the temperature was 38 °C from 0 - 2.5 h, and the temperature was 42 °C from 2.5 - 4 h.

[0072] The electrospun membrane thus obtained was placed in an oven and heated at 60 °C for 24 h for crosslinking and removing the excess solvent in the fiber membrane, thereby obtaining an electrospun gelatin fiber membrane. After testing with a scanning electron microscope, the fiber diameter of the obtained fiber membrane was 800 - 1200 nm.

[0073] Using the induction effect evaluation method described in Example 1, the effects of the electrospun membranes of Example 2 and Example 3 on inducing chondrogenic 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.

[0074] The fluorescence signal intensity and density data of single cells were analyzed using SigmaPlot v.14.0 (Systat, Chicago) and Microsoft Excel (v.2013). 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. The analysis results are as Figure 4 shown. It can be seen from Figure 4 that hMSCs expressed the chondrogenic differentiation marker COL2A1 protein when cultured on the electrospun membranes of Example 2 and Example 3. At the same time, there were statistically significant differences between the two groups (indicated by the symbol "*"), and hMSCs cultured on the electrospun membrane of Example 2 had a higher expression level of the chondrogenic 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 induced a higher expression level of chondrogenic differentiation markers than the electrospun membrane with a relatively larger fiber diameter of the gelatin fiber membrane in Example 3.

[0075] The following comparative analysis was performed on the induction effects of the electrospun membranes of Example 2 and Example 3 and the electrospun composite membrane of Example 1 on hMSC chondrogenic differentiation. The expression levels of single-cell marker proteins were also quantified using an immunofluorescence microscope with the same intensity calibration as in Application Example 1.

[0076] The fluorescence signal intensity and density data of single cells were analyzed using SigmaPlot v.14.0 (Systat, Chicago) and Microsoft Excel (v.2013). 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. For comparisons of more than two groups, analysis of variance using the rank test was used. If the analysis of variance showed significant differences between two groups, a post hoc test (Dunn’s Method) was used to compare each group. Dunn’s test allows comparison of groups with different sample sizes. The analysis results are as Figure 5 shown. From Figure 5As can be seen, hMSCs cultured on the electrospun membranes of Example 1, Example 2, and Example 3 all expressed chondrogenic differentiation markers. At the same time, there were statistically significant differences among the three groups (indicated by the symbol "*"), and hMSCs cultured on the electrospun membrane of Example 2 had higher expression of chondrogenic differentiation marker proteins than hMSCs cultured on the electrospun membrane of Example 3 and the electrospun composite membrane of Example 1. The expression of the chondrogenic differentiation marker protein COL2A1 in hMSCs cultured on the electrospun composite membrane of Example 1 was significantly higher than that on the electrospun membrane of Example 3 but lower than that on the electrospun membrane of Example 2.

[0077] From the analysis of the expression level of the chondrogenic differentiation marker COL2A1 of hMSCs in the above exemplary embodiments, it can be seen that the electrospun membrane of the present invention can successfully induce the chondrogenic differentiation of hMSCs, and when the fiber diameter range in the gelatin fiber membrane layer is controlled within 200 - 700 nm, a more ideal induction effect is obtained.

[0078] 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 chondrogenic differentiation of human mesenchymal stem cells hMSCs, wherein the electrospun membrane comprises a gelatin fiber membrane layer, The gelatin fiber layer is made from a gelatin solution by electrospinning, 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 from a polycaprolactone solution by electrospinning.

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 micrometers.

7. The application according to claim 2, wherein the porosity of the gelatin fiber membrane layer is 75% - 85%, and the pore size distribution is 3 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, and gradually increases 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 crosslinking agent to the prepared gelatin solution, and 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 equipment, 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 increase the flow rate by 0.1 - 0.2 ml / h, and the total electrospinning time is 2 - 3 h to obtain a 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 a completely 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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