A low-modulus flexible gelatin electrospun fiber membrane and its preparation method

Abstract

The present invention belongs to the technical field of biomedical composite materials, and particularly relates to a low-modulus flexible gelatin electrospun fiber membrane and a preparation method thereof. The fiber membrane comprises the following components: modified gelatin, a material loaded with zinc ions, and a solvent; the modified gelatin includes any one or more of methacrylate gelatin, methacrylamide gelatin, acrylate gelatin, and acrylamide-modified gelatin. When used for cell culture, through the synergistic effect of mechanical signals and metal ions, the adhesion and osteogenic differentiation of mesenchymal stem cells in vitro and the regeneration of new bone in vivo are induced, demonstrating its good bone induction and bone regeneration capabilities, providing a new idea for improving the functional reconstruction of defective bone tissue through the bionic design of multiple regulatory factors.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical composite materials, and relates to a low-modulus flexible gelatin electrospun fiber membrane and a preparation method thereof. Background Art

[0002] The incidence of bone and cartilage defects caused by diseases such as trauma, inflammation, and tumors is high, and it continues to increase with the aggravation of the aging degree. Extracellular matrix (ECM) remodeling is an important link in defect repair, which is regulated by cells and growth factors. However, due to the limited supply of endogenous cells and growth factors in the bone and cartilage defect area, it is necessary to transplant or replace materials to deliver exogenous cells and growth factors to assist defect repair. Hydrogel materials are the most commonly used bone repair materials at present. However, they need to reach a relatively high hardness to promote cell force, and when the material reaches a very high hardness, it is likely to induce inflammation due to its too slow degradation. Therefore, it is necessary to develop a class of easily degradable "soft materials" for bone and cartilage repair and regeneration. Summary of the Invention

[0003] The purpose of the present invention is to provide a low-modulus flexible gelatin electrospun fiber membrane and a preparation method thereof.

[0004] Firstly, gelatin, as a common component in natural ECM, has good biocompatibility and biodegradability. The gelatin fiber membrane prepared by electrospinning can deform under the action of cell force on the one hand, generate a greater reaction force, promote cell adhesion, and then induce the osteogenic differentiation of stem cells; on the other hand, the fiber structure can better simulate the morphology of natural ECM. Secondly, there are various metal trace elements in bones, which play an important role in promoting the growth and functional differentiation of osteoblast-related cells, and metal trace elements are also essential trace elements for maintaining bone metabolism. In recent years, the role of metal trace elements in bone tissue repair has been increasingly emphasized, and more and more materials loaded with zinc ions have been used in bone repair. Among them, metal-organic frameworks (MOFs) are a class of crystalline materials constructed by metal ions and organic frameworks, which have the characteristics of diverse structures, large specific surface areas, and uniform cavities. Based on this, the present invention provides a low-modulus flexible gelatin electrospun fiber membrane based on modified gelatin, materials loaded with multiple zinc ions, and the combination of electrospinning technology.

[0005] On the one hand, the present invention provides a flexible fiber membrane, which is prepared from the following components: modified gelatin, a material loaded with zinc ions, and a solvent; the modified gelatin includes any one or more of methacrylated gelatin, methacrylamide gelatin, acrylated gelatin, and acrylamideated gelatin.

[0006] In the above solution provided by the present invention, first, compared with ordinary gelatin, the above-mentioned fiber membrane provided by the present invention has a lower modulus and better cell adhesion ability; second, compared with other electrospun material fiber membranes, because the gelatin fiber membrane provided by the present invention is easily degradable and has a low modulus, it can avoid inducing inflammation; the fiber membrane provided by the present invention has good enrichment ability and can be well enriched by cells, achieving good cell proliferation and adhesion promotion ability.

[0007] In some embodiments, the zinc ion-loaded material includes any one or more of: a salt containing zinc ions, a zinc ion chelating structure, and a zinc-based metal-organic framework material.

[0008] In some embodiments, calculated in mg:mg:ml, the addition ratios of the modified gelatin, the zinc ion-loaded material, and the solvent are: 50 - 250:0.1 - 100:0.5 - 2; preferably, the addition ratios of the modified gelatin, the zinc ion-loaded material, and the solvent are: 80 - 200:1 - 20:0.5 - 2; preferably, the addition ratios of the modified gelatin, the zinc ion-loaded material, and the solvent are: 120 - 150:1 - 5:1 - 2.

[0009] In some embodiments, the salt containing zinc ions includes any one or more of: ZnCl2, ZnSO4, Zn(NO3)2, Zn(ClO4)2.

[0010] In some embodiments, the zinc ion chelating structure includes any one or more of: zinc amino acid chelate, zinc citrate chelate.

[0011] In some embodiments, the zinc-based metal-organic framework material includes any one or more of: Zn-MOF-74, ZIF-67, ZIF-8, ZIF-11.

[0012] In some embodiments, the solvent includes any one or more of: chloroform, methanol, tetrahydrofuran, hexafluoroisopropanol, and trifluoroethanol.

[0013] In some embodiments, the porosity of the fiber membrane is: 80% - 90%; the density is: 1.2 - 1.4 g / cm -3 .

[0014] In some embodiments, the modulus of the fiber membrane is: 0.5 - 6 Kpa; preferably, the modulus of the fiber membrane is: 0.8 - 3 Kpa; preferably, the modulus of the fiber membrane is: 0.8 - 1.5 Kpa.

[0015] In some embodiments, the grafting rate of the fiber membrane is 20%-70%; preferably, the grafting rate of the fiber membrane is 20%-60%; preferably, the grafting rate of the fiber membrane is 20%-40%; preferably, the grafting rate of the fiber membrane is 30%.

[0016] In some embodiments, the swelling rate of the fiber membrane is 1-3, and the recruitment rate is 1.05-1.50.

[0017] On the other hand, the present invention also provides a method for preparing the fiber membrane, comprising the following steps:

[0018] (1) Dissolve the modified gelatin in the solvent to obtain an electrospinning pre-prepared solution;

[0019] (2) Mix the electrospinning pre-prepared solution with the material loaded with zinc ions to obtain an electrospinning working solution;

[0020] (3) Pass the electrospinning working solution to prepare the fiber membrane.

[0021] In some embodiments, the electrospinning process includes: injecting the electrospinning working solution into an electrospinning syringe, assembling it on an electrospinning machine, adjusting the electrospinning parameters, and preparing the fiber membrane.

[0022] In some embodiments, the parameters of the electrospinning include: applied voltage, receiving distance, flow rate, and spinning time.

[0023] In some embodiments, the applied voltage is: 10-30 KV; the receiving distance is: 10-40 cm; the flow rate is: 0.006-0.012 ml / min; the spinning time is: 2 h-6 h; preferably, the applied voltage is: 25 KV; the receiving distance is: 20 cm; the flow rate is: 0.008 ml / min; the spinning time is: 4 h.

[0024] In some embodiments, the method further includes: crosslinking the fiber membrane prepared in step (3) with a crosslinking agent solution to obtain a crosslinked fiber membrane.

[0025] In some embodiments, the swelling rate of the crosslinked fiber membrane is 1-3, and the recruitment rate is 1.05-1.50.

[0026] In some embodiments, the crosslinking agent is selected from any one or two of: 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone, lithium phenyl-2,4,6-trimethylbenzoylphosphonate.

[0027] In some embodiments, the solvent of the crosslinking agent solution is selected from any one or more of water, methanol, and ethanol.

[0028] In some embodiments, the concentration of the crosslinking agent in the photo-crosslinking agent solution is 50 - 300 mg / ml; preferably, the concentration of the crosslinking agent in the photo-crosslinking agent solution is 80 - 200 mg / ml; preferably, the concentration of the crosslinking agent in the photo-crosslinking agent solution is 150 - 200 mg / ml; preferably, the concentration of the crosslinking agent in the photo-crosslinking agent solution is 140 - 160 mg / ml.

[0029] On the other hand, the present invention also provides an application of the fiber membrane or the method as described above in the preparation of bone or cartilage repair materials.

[0030] The present invention also provides an application of the fiber membrane as described above in artificial bionic periosteum, induced bone regeneration membrane, and guided tissue regeneration membrane.

[0031] In summary, the present application includes at least one of the following beneficial technical effects:

[0032] (1) First, compared with ordinary gelatin, the fiber membrane provided by the present invention has a lower modulus and better cell adhesion ability; second, compared with other electrospun fiber membranes, because the gelatin fiber membrane provided by the present invention is easily degradable and has a low modulus, it can avoid inducing inflammation; and the fiber membrane provided by the present invention has a good enrichment ability and can be well enriched by cells, achieving a good ability to promote cell proliferation and adhesion.

[0033] (2) The present invention provides a low-modulus flexible gelatin electrospun fiber membrane loaded with zinc ions and capable of promoting cell force. The raw materials are dissolved with a solvent, a material loaded with zinc ions is configured in proportion, an electrospinning solution is prepared, and by adjusting the electrospinning parameters, a low-modulus flexible gelatin electrospun fiber membrane material with excellent cell force promotion is prepared.

[0034] (3) When the fiber membrane material provided by the present invention is used for cell culture, through the synergistic action of mechanical signals and metal ions, the adhesion and osteogenic differentiation of mesenchymal stem cells in vitro and the regeneration of new bone in vivo are induced, demonstrating its good bone induction and bone regeneration ability, providing new ideas for improving the functional reconstruction of defective bone tissue through the bionic design of multiple regulatory factors. Description of the Drawings

[0035] Figure 1 It is a scanning electron microscope image of the gelatin fiber membrane prepared in the embodiment of the present invention; wherein, A: the surface morphology of the gelatin fiber membrane in the initial dry state; B: the surface morphology of the crosslinked gelatin fiber membrane.

[0036] Figure 2Modulus statistical graph of two materials, gelatin gel in Comparative Example 1 and the gelatin fiber membrane of the present invention. Among them, fiber: gelatin fiber membrane; hydrogel: gelatin gel;

[0037] Figure 3 Cell adhesion and spreading situation diagram on two materials in Comparative Example 1; among them, A: cell spreading on gelatin gel; B: cell spreading on gelatin fiber membrane;

[0038] Figure 4 Fluorescence graph of adhesion-related protein p-MyosinⅡa of cells in two materials in Comparative Example 1; among them, A: protein expression of p-MyosinⅡa of cells on gelatin gel; B: protein expression of p-MyosinⅡa of cells on gelatin fiber membrane;

[0039] Figure 5 Fluorescence graph of adhesion-related protein LaminA / C of cells in two materials in Comparative Example 1; among them, A: protein expression of LaminA / C of cells on gelatin gel; B: protein expression of LaminA / C of cells on gelatin fiber membrane;

[0040] Figure 6 Statistical graph of adhesion-related protein expression data of cells in two materials in Comparative Example 1; among them, A: statistical graph of relative expression amount data of p-MyosinⅡa protein; B: statistical graph of relative expression amount data of LaminA / C protein;

[0041] Figure 7 Modulus statistical graph of two materials with different grafting rates in Comparative Example 2; among them, Fiber-30%: gelatin fiber membrane with a grafting rate of 30%; Fiber-60%: gelatin fiber membrane with a grafting rate of 60%;

[0042] Figure 8 Comparison diagram before and after swelling of gelatin fiber membrane with a grafting rate of 30% in Comparative Example 2; among them, A: before swelling of gelatin fiber membrane with a grafting rate of 30%; B: after swelling of gelatin fiber membrane with a grafting rate of 30%; C: before swelling of gelatin fiber membrane with a grafting rate of 60%; D: after swelling of gelatin fiber membrane with a grafting rate of 60%;

[0043] Figure 9 Swelling rate statistical graph of two grafting rate fiber membranes in Comparative Example 2; among them, Fiber-30%: gelatin fiber membrane with a grafting rate of 30%; Fiber-60%: gelatin fiber membrane with a grafting rate of 60%;

[0044] Figure 10Comparison diagrams of the gelatin fiber membranes in Comparative Example 2 before and after recruitment; where, A: before recruitment of the gelatin fiber membrane with a grafting rate of 30%; B: after recruitment of the gelatin fiber membrane with a grafting rate of 30%; C: before recruitment of the gelatin fiber membrane with a grafting rate of 60%; D: after recruitment of the gelatin fiber membrane with a grafting rate of 60%.

[0045] Figure 11 Statistical chart of the recruitment rates of the two fiber membranes in Comparative Example 2; where, Fiber-30%: gelatin fiber membrane with a grafting rate of 30%; Fiber-60%: gelatin fiber membrane with a grafting rate of 60%.

[0046] Figure 12 Fluorescence images of the protein p-MyosinⅡa related to cell adhesion on the two materials in Comparative Example 2; where, A: expression of the protein p-MyosinⅡa in cells on the gelatin fiber membrane with a grafting rate of 30%; B: expression of the protein p-MyosinⅡa on the gelatin fiber membrane with a grafting rate of 60%.

[0047] Figure 13 Fluorescence images of the protein LaminA / C related to cell adhesion on the two materials in Comparative Example 2; where, A: expression of the protein LaminA / C in cells on the gelatin fiber membrane with a grafting rate of 30%; B: expression of the protein LaminA / C on the gelatin fiber membrane with a grafting rate of 60%.

[0048] Figure 14 Statistical chart of the moduli of the two fiber membranes in Comparative Example 3; where, Fiber-0mg / ml: gelatin fiber membrane of the material without zinc ion loading; Fiber-2mg / ml: gelatin fiber membrane of the material with zinc ion loading.

[0049] Figure 15 Fluorescence images of the protein p-MyosinⅡa related to cell adhesion on the two materials in Comparative Example 3; where, A: expression of the protein p-MyosinⅡa in cells on the gelatin fiber membrane of the material without zinc ion loading; B: expression of the protein p-MyosinⅡa in cells on the gelatin fiber membrane of the material with zinc ion loading.

[0050] Figure 16 Fluorescence images of the protein p-FAK related to cell adhesion on the two materials in Comparative Example 3; where, A: expression of the protein p-FAK in cells on the gelatin fiber membrane of the material without zinc ion loading; B: expression of the protein p-FAK in cells on the gelatin fiber membrane of the material with zinc ion loading.

[0051] Figure 17 Statistical chart of the protein data related to cell adhesion on the two materials in Comparative Example 3; where, A: statistical chart of the relative expression levels of the protein p-MyosinⅡa; B: statistical chart of the relative expression levels of the protein p-FAK.

[0052] Figure 18 It is a comparison result graph of the osteogenic differentiation of mesenchymal stem cells after 7 days for the alkaline phosphatase (ALP) assay in Comparative Example 3; among them, A: the result of inducing cell differentiation by the gelatin fiber membrane; B: the result of inducing cell differentiation by the gelatin fiber membrane loaded with zinc ions.

[0053] Figure 19 It is a comparison graph of the repair images of bone defects in rats after 30 days of material filling in Comparative Example 4; among them, A: the healing condition of the femur in the blank group; B: the healing condition of the femur in the gelatin fiber membrane group loaded with zinc ions. Specific Embodiments

[0054] The technical solutions of the present invention will be further described below through specific embodiments. The specific embodiments do not represent a limitation on the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0055] In the embodiments provided in this application, the raw material sources are as follows:

[0056] Gelatin: purchased from Aladdin, product number V900863; hexafluoroisopropanol: purchased from Macklin, product number H811026; electrospinning machine: purchased from Beijing Xinrui Baina Technology Co., Ltd., model TADFS-103.

[0057] Example 1 Preparation Method of a Low-Modulus Flexible Gelatin Electrospun Fiber Membrane for Promoting Cell Force

[0058] A preparation method of a gelatin electrospun fiber membrane for promoting cell force, comprising the following steps:

[0059] (1) Dissolve methacrylic anhydride gelatin in a hexafluoroisopropanol solvent at a ratio of 120 mg / ml to obtain an electrospinning pre-solution;

[0060] (2) Mix the zinc ion-loaded material: Zn-MOF-74 with the electrospinning pre-solution obtained in step (1) at a ratio of 2 mg / ml to obtain an electrospinning working solution;

[0061] (3) Inject the electrospinning working solution obtained in step (2) into an electrospinning syringe, assemble it on an electrospinning machine, and adjust the electrospinning parameters: the applied voltage is 25 KV, the receiving distance is 20 cm, the flow rate is 0.008 ml / min, and the spinning time is 4 h, then a low-modulus flexible gelatin electrospun fiber membrane for promoting cell force can be obtained;

[0062] (4) Dissolve the photo-crosslinking agent: 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959) in a methanol solvent to finally prepare a photo-crosslinking solution with a final concentration of 150 mg / mL. Crosslink the fibrous membrane obtained in step (3) in the photo-crosslinking solution to obtain a crosslinked fibrous membrane.

[0063] Example 2

[0064] A method for preparing a gelatin electrospun fibrous membrane that promotes cell force, comprising the following steps:

[0065] (1) Dissolve methacrylated gelatin at a ratio of 150 mg / ml in a hexafluoroisopropanol solvent to obtain an electrospinning pre-polymer solution;

[0066] (2) Mix the zinc ion-loaded material: Zn-MOF-74 at a ratio of 4 mg / ml with the electrospinning pre-polymer solution obtained in step (1) to obtain an electrospinning working solution;

[0067] (3) Inject the electrospinning working solution obtained in step (2) into an electrospinning syringe, assemble it onto an electrospinning machine, and adjust the electrospinning parameters: the applied voltage is 25 KV, the receiving distance is 20 cm, the flow rate is 0.008 ml / min, and the spinning time is 4 h, to obtain a low-modulus flexible gelatin electrospun fibrous membrane that promotes cell force;

[0068] (4) Dissolve the photo-crosslinking agent: 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959) in a methanol solvent to finally prepare a photo-crosslinking solution with a final concentration of 150 mg / mL. Crosslink the fibrous membrane obtained in step (3) in the photo-crosslinking solution to obtain a crosslinked fibrous membrane.

[0069] Example 3

[0070] A method for preparing a gelatin electrospun fibrous membrane that promotes cell force, comprising the following steps:

[0071] (1) Dissolve methacrylated gelatin at a ratio of 120 mg / ml in a hexafluoroisopropanol solvent to obtain an electrospinning pre-polymer solution;

[0072] (2) Mix the zinc ion-loaded material: Zn-MOF-74 at a ratio of 4 mg / ml with the electrospinning pre-polymer solution obtained in step (1) to obtain an electrospinning working solution;

[0073] (3) Inject the electrospinning working solution obtained in step (2) into an electrospinning syringe, assemble it onto an electrospinning machine, and adjust the electrospinning parameters: the applied voltage is 25 KV, the receiving distance is 20 cm, the flow rate is 0.008 ml / min, and the spinning time is 4 h, then a low-modulus flexible gelatin electrospun fiber membrane that promotes cell force can be obtained;

[0074] (4) Dissolve the photo-crosslinking agent: 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959) in a methanol solvent, and finally prepare a photo-crosslinking solution with a final concentration of 150 mg / mL. Crosslink the fiber membrane obtained in step (3) in the photo-crosslinking solution to obtain a crosslinked fiber membrane.

[0075] Example 4

[0076] A preparation method of a gelatin electrospun fiber membrane that promotes cell force, comprising the following steps:

[0077] (1) Dissolve acrylamide-modified gelatin in a hexafluoroisopropanol solvent at a ratio of 120 mg / ml to obtain an electrospinning pre-polymer solution;

[0078] (2) Mix the zinc ion-loaded material: Zn-MOF-74 with the electrospinning pre-polymer solution obtained in step (1) at a ratio of 2 mg / ml to obtain an electrospinning working solution;

[0079] (3) Inject the electrospinning working solution obtained in step (2) into an electrospinning syringe, assemble it onto an electrospinning machine, and adjust the electrospinning parameters: the applied voltage is 25 KV, the receiving distance is 20 cm, the flow rate is 0.008 ml / min, and the spinning time is 4 h, then a low-modulus flexible gelatin electrospun fiber membrane that promotes cell force can be obtained;

[0080] (4) Dissolve the photo-crosslinking agent: 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959) in a methanol solvent, and finally prepare a photo-crosslinking solution with a final concentration of 150 mg / mL. Crosslink the fiber membrane obtained in step (3) in the photo-crosslinking solution to obtain a crosslinked fiber membrane.

[0081] Example 5

[0082] A preparation method of a gelatin electrospun fiber membrane that promotes cell force, comprising the following steps:

[0083] (1) Dissolve methacrylic anhydride-modified gelatin in a hexafluoroisopropanol solvent at a ratio of 120 mg / ml to obtain an electrospinning pre-polymer solution;

[0084] (2) Mix the zinc ion-loaded material: zinc citrate chelate with the electrospinning pre-solution obtained in step (1) at a ratio of 2 mg / ml to obtain an electrospinning working solution;

[0085] (3) Inject the electrospinning working solution obtained in step (2) into an electrospinning syringe, assemble it onto an electrospinning machine, and adjust the electrospinning parameters: the applied voltage is 25 KV, the receiving distance is 20 cm, the flow rate is 0.008 ml / min, and the spinning time is 4 h, then a low-modulus flexible gelatin electrospun fiber membrane that promotes cell force can be obtained;

[0086] (4) Dissolve the photo-crosslinking agent: 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959) in a methanol solvent to finally prepare a photo-crosslinking solution with a final concentration of 150 mg / mL. Crosslink the fiber membrane obtained in step (3) in the photo-crosslinking solution to obtain a crosslinked fiber membrane.

[0087] Effect Example 1

[0088] The porosity of the fiber membranes obtained in Examples 1-5 is: 80%-90%; the density is: 1.2-1.4 g / cm -3 . Further, the uncrosslinked fiber membranes and the crosslinked fiber membranes prepared in the above examples were observed by scanning electron microscopy respectively. The results show that: both the uncrosslinked fiber membranes and the crosslinked fiber membranes have nanofiber morphology and pore structure, which are beneficial for better cell adhesion and spreading. The scanning electron micrographs of the uncrosslinked fiber membrane and the crosslinked fiber membrane in Example 1 observed by scanning electron microscopy are specifically as Figure 1 shown. The results show that: both the uncrosslinked fiber membranes and the crosslinked fiber membranes have nanofiber morphology and pore structure, which are beneficial for better cell adhesion and spreading.

[0089] Comparative Example 1

[0090] This comparative example explored and analyzed the cell adhesion-promoting ability and the expression of related proteins of ordinary gelatin gel disclosed in the prior art and a gelatin fiber membrane (without zinc ion-loaded material) with a grafting rate of 30% after crosslinking, as follows:

[0091] In this comparative example, the preparation method of the gelatin fiber membrane with a grafting rate of 30% was carried out with reference to the method of Example 1. The difference from Example 1 is that: in step (2), the zinc ion material was mixed with the electrospinning pre-solution obtained in step (1) at a ratio of 0 mg / ml to obtain an electrospinning working solution, and the other steps and conditions were the same, and a gelatin fiber membrane (without zinc ion-loaded material) with a grafting rate of 30% was prepared.

[0092] Focal adhesions connect the intracellular actomyosin cytoskeleton and transmit the traction force of the intracellular molecular motor, myosin, to the ECM, establishing a bidirectional transmission of mechanical force through focal adhesions. Myosin plays an important role in cell movement and intracellular material transport, and to a certain extent, it reflects the cell adhesion situation. Lamin A / C is an intracellular cytoskeletal protein and the main component of the nuclear lamina, belonging to a type of intermediate filament. In recent years, it has been found that lamin A / C can promote cell differentiation and muscle growth, etc. Focal adhesion kinase (FAK) is a cytoplasmic protein tyrosine kinase that plays an important role in the pathogenesis of embryonic development and human diseases (including cancer and cardiovascular diseases) and has long been considered a regulator of cell migration. In most experimental systems, enhanced FAK signaling promotes cell movement, while inhibiting FAK signaling by various methods impairs cell migration. FAK plays a role in controlling the movement of randomly migrating cells and responding to a wide range of stimuli (including chemotaxis, haptotaxis, and directional signals).

[0093] After sterilizing the gelatin fiber membrane with a grafting rate of 30% and ordinary gelatin gel above, and washing them 5 times with sterile PBS, mesenchymal stem cells (hASCs) were inoculated, and then placed in a CO2 incubator for in vitro culture. After 24 hours of culture, the expression of cell adhesion-related proteins was statistically analyzed, and the results are as Figures 2 - 6 shown:

[0094] As Figure 2 shown, through the measurement results of a nanoindenter, it is known that the modulus of the above ordinary gelatin gel is 3 Kpa, and the modulus of the gelatin fiber membrane with a grafting rate of 30% is 0.8 Kpa.

[0095] As Figure 3 shown, compared with the gelatin gel with a modulus of 3 Kpa, the cell spreading situation is better on the gelatin fiber membrane with a modulus of 0.8 Kpa, that is, the cell spreading situation is better on the gelatin fiber membrane with a smaller modulus, which is more conducive to cell adhesion, proliferation, and growth; at the same time, because the gelatin fiber membrane is easily degraded and has a low modulus, compared with other electrospun fiber membranes, it can avoid inducing inflammation.

[0096] As Figure 4 , Figure 5 shown, from the expression of p-Myosin IIa and Lamin A / C in cells in two different materials, gelatin fiber membrane and gelatin gel, compared with the gelatin gel with a modulus of 3 Kpa, the expression levels of cell adhesion-related proteins are higher on the gelatin fiber membrane with a smaller modulus (0.8 Kpa), and the induced cell adhesion situation is better.

[0097] AsFigure 6 As shown, the expression of p-MyosinⅡa and LaminA / C in two different materials, namely gelatin fiber membranes and gelatin gels, was statistically analyzed. The results were the same as those of Figure 4 and Figure 5 . Compared with the gelatin gel (3Kpa), on the gelatin fiber membrane with a smaller modulus (0.8Kpa), the expression level of cell adhesion-related proteins was higher, and the induction of cell adhesion was better.

[0098] Comparative Example 2

[0099] In this comparative example, the fiber swelling and recruitment of gelatin fiber membranes with different grafting rates obtained after cross-linking the gelatin fiber membranes (materials without zinc ion loading) were comparatively analyzed as follows:

[0100] (1) The gelatin fiber membranes with different grafting rates were sterilized respectively, and after being soaked in sterile PBS for 24 h, the fiber swelling was statistically analyzed;

[0101] (2) In in vitro cell experiments, the gelatin fiber membranes with different grafting rates were sterilized respectively, and after being washed 5 times with sterile PBS, mesenchymal stem cells (hASCs) were inoculated, and then placed in a CO2 incubator for in vitro culture. After 24 h, the recruitment of each fiber membrane was statistically analyzed. The results were as Figures 7 - 13 shown:

[0102] As Figure 7 shown, it was measured by a nanoindentation instrument that the modulus of the gelatin fiber membrane with a grafting rate of 30% was 0.8Kpa (the same as the gelatin fiber membrane in Comparative Example 1), while the modulus of the gelatin fiber membrane with a grafting rate of 60% was 3Kpa.

[0103] As Figure 8 and 9 shown, the gelatin fiber membranes with different grafting rates after cross-linking swelled in water. The swelling rate of the fiber membrane with a grafting rate of 30% was 1.2, and the swelling rate of the gelatin fiber membrane with a grafting rate of 60% was 1.07. The swelling rate was calculated based on the change in fiber length before and after swelling.

[0104] As Figure 10 and 11As shown, the recruitment rate of the fiber membrane with a grafting rate of 30% is 2, and the recruitment rate of the gelatin fiber membrane with a grafting rate of 60% is 1.5. The recruitment rate is calculated by the ratio of the fiber density around the cells to the total fiber density. After fiber swelling, it can be recruited under the action of cell forces at the pN-nN level. After fiber recruitment, a greater reaction force is provided, and with the increase of the recruitment rate, the provided reaction force also increases, which can further promote cell adhesion and then induce osteogenic differentiation of stem cells. That is, an appropriate grafting rate can make the fiber membrane have an excellent recruitment rate to further achieve a better ability to promote cell adhesion and induce osteogenic differentiation of stem cells.

[0105] Comparative Example 3

[0106] This comparative example is based on Example 1, and a comparative analysis was carried out on the ability of the gelatin fiber membrane loaded or not with zinc ion material to promote cell adhesion and osteogenic differentiation, as follows:

[0107] Alkaline phosphatase (ALP) is a marker enzyme that reflects the catabolic level in bone tissue and plays a key role in calcification. Calcium ions are deposited on collagen under the action of ALP to complete the matrix mineralization process. Bone tissue is formed by the calcification of the bone matrix, and the bone matrix is synthesized and secreted by osteoblasts; when the matrix begins to calcify, the ALP activity of osteoblasts is the highest, and the activity is the lowest when the calcification is nearly complete. Its activity reflects the differentiation degree and functional state of osteoblasts to a certain extent.

[0108] In this comparative example, the preparation method of the low-modulus flexible gelatin electrospun fiber membrane (grafting rate of 30%) not loaded with zinc ion material refers to the method of Example 1. The difference from Example 1 is that in step (2), the zinc ion material is mixed with the electrospinning pre-prepared solution obtained in step (1) at a ratio of 0 mg / ml to obtain an electrospinning working solution, and the remaining steps and conditions are the same. A gelatin fiber membrane not loaded with zinc ion material is prepared and the following experiments are carried out:

[0109] In vitro osteogenic differentiation experiment: The gelatin fiber membrane loaded with zinc ion material prepared in Example 1 of the present invention and the gelatin fiber membrane not loaded with zinc ion material prepared above were sterilized respectively, and after being washed 5 times with sterile PBS, mesenchymal stem cells (hASCs) were inoculated, and then placed in a CO2 incubator for in vitro culture. After 7 days, they were taken out and the formation of alkaline phosphatase in the cells was measured using a kit. The specific results are as follows:

[0110] As Figure 14 shown, it was measured by a nanoindenter that the modulus of the gelatin fiber membrane not loaded with zinc ion material is 0.8 Kpa, while after being loaded with zinc ion material, the modulus of the gelatin fiber membrane is 1.5 Kpa.

[0111] AsFigure 15 , Figure 16 As shown in Figure 16 , from the expression of p-Myosin IIa and p-FAK in cells on two different fiber membrane materials with and without zinc ion materials, compared with the fiber membrane without zinc ion materials, after loading with zinc ion materials, the gelatin fiber membrane induced better cell adhesion;

[0112] As Figure 17 shown in Figure 17 , statistical analysis of the expression of p-Myosin IIa and p-FAK in two different fiber membrane materials with and without zinc ion materials was carried out, and the results were the same as Figure 14 and Figure 15 the results. Compared with the fiber membrane without zinc ion materials, after loading with zinc ion materials, the gelatin fiber membrane induced better cell adhesion.

[0113] As Figure 18 shown in Figure 18 , from the results of osteogenic differentiation of mesenchymal stem cells measured by alkaline phosphatase (ALP) at 7 days, compared with the gelatin fiber membrane without zinc ion materials ( Figure 18 , A), the gelatin fiber membrane after loading with zinc ion materials ( Figure 18 , B) induced better cell differentiation.

[0114] That is, the above results further showed that compared with the gelatin fiber membrane without zinc ion materials, the gelatin fiber membrane loaded with zinc ion materials had better modulus, induced better cell adhesion ability and cell differentiation effect.

[0115] Comparative Example 4

[0116] To study the effect of the material on osteogenesis in vivo, a rat cranial defect model was constructed. According to different materials, the experiment was divided into 2 groups, namely the control group (blank group, without any treatment) and the gelatin fiber membrane group loaded with zinc ion materials (the same as Example 1). The specific operations are as follows:

[0117] Dormicum was used to sedate the rats, and isoflurane was used as the induction anesthesia and maintenance anesthesia drug for the experimental rats. After the experimental rats were anesthetized and the skin was disinfected, an incision with a size of 1 cm x 1 cm was made on the lateral skin of the femur. The muscle tissue was separated, and after the femur was exposed, a bone drill with a 1 mm diameter bit was used to prepare a bone defect with a diameter of 1 mm and a depth of 15 mm at the distal end of the femur. After washing the bone debris and stopping the bleeding, the scaffold material was completely filled into the bone defect according to the group. After the patella was completely restored, the incision was sutured with 5-0 absorbable suture, and the wound was routinely disinfected. According to the experimental plan, at 30 days, the experimental rats were euthanized with an overdose of isoflurane, and the femurs were taken out for observation. The results are as Figure 19 shown in Figure 19 .

[0118] Figure 19 The results show that, compared with the bone defect in the blank group of Group A, the cure rate of Group B (the gelatin fiber membrane group loaded with zinc ions) is significantly higher. The above results indicate that the zinc-based metal / gelatin group can effectively promote the formation of new bone in the bone defect area and the osteogenic differentiation of stem cells in the in vivo environment.

[0119] It can be understood that the present invention is described through some embodiments. Those skilled in the art know that, without departing from the spirit and scope of the present invention, various changes or equivalent substitutions can be made to these features and embodiments. Additionally, under the teaching of the present invention, these features and embodiments can be modified to adapt to specific situations and materials without departing from the spirit and scope of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of this application belong to the scope protected by the present invention.

Claims

1. A flexible fiber membrane, characterized in that, It includes the following components: modified gelatin, zinc ion-loaded material, and solvent; The modified gelatin includes any one or more of methacrylate gelatin, methacrylamide gelatin, acrylate gelatin, and acrylamide-modified gelatin; The fiber membrane is prepared by the following preparation method: (1) Dissolve the modified gelatin in the solvent to obtain an electrospinning precursor solution; (2) Mix the electrospinning precursor solution with the zinc ion-loaded material to obtain an electrospinning working solution; (3) Prepare the fiber membrane by electrospinning the electrospinning working solution; (4) Crosslink the fiber membrane prepared in step (3) with a crosslinking agent solution to obtain a crosslinked fiber membrane; Among them, the grafting rate of the crosslinked fiber membrane is 20%-40%, and the modulus of the crosslinked fiber membrane is: 0.8-1.5 Kpa; the concentration of the crosslinking agent in the crosslinking agent solution is 80-200 mg / ml.

2. The fiber membrane according to claim 1, wherein The zinc ion-loaded material includes any one or more of zinc ion-containing salts, zinc ion chelation structures, and zinc-based metal-organic framework materials; 3. The fiber membrane according to claim 1, wherein, Calculated in mg:mg:ml, the addition ratio of the modified gelatin, zinc ion-loaded material, and solvent is: 50-250:0.1-100:0.5-2.

4. The fiber membrane according to claim 1, wherein Calculated in mg:mg:ml, the addition ratio of the modified gelatin, zinc ion-loaded material, and solvent is: 80-200:1-20:0.5-2.

5. The fiber membrane according to claim 2, wherein, The zinc ion-containing salts include any one or more of ZnCl2, ZnSO4, Zn(NO3)2, and Zn(ClO4)2; 6. The fiber membrane according to claim 2, wherein The zinc ion chelation structures include any one or more of amino acid chelated zinc and zinc citrate chelate; 7. The fiber membrane according to claim 2, wherein, The zinc-based metal-organic framework materials include any one or more of Zn-MOF-74, ZIF-67, ZIF-8, and ZIF-11; 8. The fiber membrane according to claim 1, characterized in that, The solvent includes any one or more of chloroform, methanol, tetrahydrofuran, hexafluoroisopropanol, and trifluoroethanol; 9. The fiber membrane according to any one of claims 1-8, characterized in that, The porosity of the fiber membrane is: 80%-90%; 10. The fiber membrane according to any one of claims 1-8, characterized in that, The density of the fiber membrane is: 1.2 - 1.4 g / cm -3 .

11. The fiber membrane according to any one of claims 1-8, characterized in that, The swelling ratio of the fiber membrane is 1-3; 12. The fiber membrane according to any one of claims 1-8, characterized in that, The recruitment rate of the fiber membrane is 1.05-1.50; 13. The fiber membrane according to claim 1, characterized in that, The electrospinning process includes: injecting the electrospinning working solution into an electrospinning syringe, assembling it on an electrospinning machine, adjusting the electrospinning parameters, and preparing the fiber membrane; 14. The fiber membrane according to claim 13, characterized in that, The electrospinning parameters include: applied voltage, receiving distance, flow rate, and spinning time; 15. The fiber membrane according to claim 14, wherein The applied voltage is: 10-30 KV; the receiving distance is: 10-40 cm; the flow rate is: 0.006-0.012 ml / min; the spinning time is: 2 h-6 h; 16. The fiber membrane according to claim 1, characterized in that, The crosslinking agent is selected from any one or two of 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone and lithium phenyl-2,4,6-trimethylbenzoylphosphonate; 17. The fiber membrane according to claim 1, wherein, The solvent of the crosslinking agent solution is selected from any one or more of water, methanol, and ethanol.

18. Use of the fiber membrane according to any one of claims 1-17 in the preparation of bone or cartilage repair materials.

19. Use of the fiber membrane according to any one of claims 1-17 in artificial bionic periosteum, induced bone regeneration membrane and guided tissue regeneration membrane.

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