Preparation method and application of a tissue engineering scaffold for rapidly inducing stem cell proliferation and differentiation

Abstract

The application discloses a preparation method of a tissue engineering scaffold for rapidly inducing stem cell proliferation and differentiation and application of the scaffold. The scaffold is prepared as follows: natural high polymer and a sacrificing agent are respectively dissolved in an organic solvent to prepare spinning liquid, two kinds of spinning liquid are mixed, and electrostatic spinning is carried out to obtain a two-dimensional scale tropotaxis fiber scaffold; the two-dimensional fiber scaffold is reacted with a foaming agent solvent to obtain a three-dimensional scaffold with a tropotaxis structure and different elastic moduli. The three-dimensional scaffold with the tropotaxis structure and the different elastic moduli can be obtained by controlling preparation conditions. Stem cells are planted in the three-dimensional scaffold, the expression of key genes PIP2, ALP and Vav is regulated by directly regulating the elastic modulus or further regulating the expression of BCL-6 and MiR-126-5p, stem cell efficient proliferation and differentiation in a heterotropic space are induced, and the scaffold treated by the cells is planted on a damaged wound surface to accelerate wound healing.

Description

Technical Field

[0001] This invention pertains to tissue engineering scaffolds, specifically a method for preparing a tissue engineering scaffold that rapidly induces stem cell proliferation and differentiation, and its application. Background Technology

[0002] Effective wound healing is a global medical concern. Typically, wounds undergo several consecutive, overlapping stages of cellular and biochemical activity to heal. Based on healing time, wounds can be classified as chronic or acute. Chronic wounds are those that do not heal completely within a short period. Generally, a wound that fails to heal completely within 4-8 weeks is considered chronic. The normal wound healing process includes four stages: hemostasis, inflammation, proliferation, and remodeling. However, wound healing may halt at any of these stages, most commonly the inflammation or proliferation stage. At this point, a normal wound develops into a chronic wound, leading to impaired wound cell function and hindering normal healing progress. For example, diabetic foot ulcers have been a common and serious type of chronic wound in clinical practice. Ischemic, neurogenic, and neurogenic ischemic diseases of the diabetic foot can lead to diabetic foot, resulting in varying degrees of foot infection and ulceration, and even requiring amputation.

[0003] Minor wounds can often heal through skin tissue repair, depending on the tissue's proliferative capacity. However, large-area burns and mechanical injuries require treatment with external wound dressings or other medical interventions. Clinical approaches to treating chronic wounds include debridement, orthopedics, plaster casts, negative pressure wound therapy, and infection control. For local wound care, feasible methods include saline cleansing and the application of modern wound dressings to promote a moist environment. However, these methods are only effective for treating very small chronic wounds. For example, while negative pressure wound therapy has shown significant clinical benefits, its mechanism of action remains unclear. Furthermore, orthopedics and castings involve higher medical costs and greater patient discomfort. Therefore, developing alternative methods for wound treatment is crucial. Tissue-engineered skin substitutes are considered a very promising approach for treating wounds unsuitable for initial closure. These substitutes offer an ideal alternative to traditional repair mechanisms through skin regeneration. This process produces no scarring or helps reduce scarring while fully restoring the skin's structure and function. By our definition, a practical wound dressing should promote rapid hemostasis, have good biocompatibility, and promote cell growth. Electrospun nanofibers in tissue engineering have been shown to possess these properties, opening up new possibilities for the treatment of chronic wounds.

[0004] As an electrospinning fiber-forming technology, electrospinning can be used to customize equipment to suit desired fiber morphologies and structures, including high-voltage power supplies, metering pumps, and fiber collection devices. Under the influence of a high-voltage electric field, electrospinning devices gradually draw polymer droplets from a Taylor cone into fine fibers with diameters in the micrometers or even nanometers. The resulting fiber scaffolds possess high porosity, good mechanical properties, and excellent biocompatibility, which are beneficial for cellular respiration, skin regeneration, wound moisturizing, internal and external metabolism, and hemostasis. Fiber scaffolds manufactured by electrospinning devices can provide structural and morphological clues for the adhesion of various cell types in wounds and serve as templates for wound tissue regeneration. The extracellular matrix, a complex of fibrin and matrix proteins, provides the mechanical and biological properties required for bone growth, skin regeneration, vascular reconstruction, and other tissue growth. With a microstructure similar to extracellular matrix proteins, electrospun fiber scaffolds play a crucial role in maintaining cell growth and infiltration, thus making them suitable for tissue engineering.

[0005] In recent years, oriented nanofiber scaffolds have attracted increasing attention because many natural extracellular matrices possess highly oriented structures, which facilitate high strength and effective guidance of cell growth, migration, and differentiation. Highly oriented nanofibers can be obtained using electrospinning. However, this technique struggles to generate three-dimensional scaffolds, and traditional cell seeding methods only reach the surface of two-dimensional materials and the superficial layer of three-dimensional scaffolds, resulting in incomplete cell proliferation and differentiation. Current biological research on the differences in stem cell proliferation and differentiation between disordered and ordered three-dimensional scaffolds is still limited. Utilizing these findings to efficiently leverage the role of living cells in wound healing has become a key focus. Summary of the Invention

[0006] Objective of the Invention: Addressing the problems existing in the prior art, this invention provides a method for preparing a tissue engineering scaffold that rapidly induces stem cell proliferation and differentiation. The scaffold with a three-dimensional surface orientation-spatial anisotropy structure prepared by this invention can improve the efficiency of stem cell proliferation and differentiation on the surface of traditional random fibers and orientation fibers with high elastic modulus by adjusting the elastic modulus or through gene induction. This provides strong theoretical and technical support for solving the problem of sufficient extension and differentiation of stem cells in three-dimensional space.

[0007] The present invention also provides applications of the prepared tissue engineering scaffold.

[0008] Technical Solution: To achieve the above objectives, the present invention provides a method for preparing a tissue engineering scaffold that rapidly induces stem cell proliferation and differentiation, comprising the following steps:

[0009] (1) Select natural polymer and sacrificial agent as raw materials for scaffold; dissolve natural polymer and sacrificial agent in organic solvent to prepare spinning solution, mix the two spinning solutions and perform electrospinning to obtain two-dimensional oriented fiber scaffold; (2) react the two-dimensional oriented fiber scaffold obtained in step (1) with foaming agent solvent to make the sacrificial agent dissolve quickly and obtain a three-dimensional scaffold with oriented structure and different elastic modulus.

[0010] In step (1), the organic solvent is trifluoroethanol; the mass-to-volume ratio of the natural polymer to trifluoroethanol is 7-10% mg / mL; the mass-to-volume ratio of the sacrificial agent to trifluoroethanol is 30-50% mg / mL; and the volume ratio of the natural polymer and the sacrificial agent dissolved in the spinning solution in trifluoroethanol is 7:3-5:5. Experiments have verified that the spinning effect of trifluoroethanol is better than that of traditional solvents such as acetone.

[0011] In step (1), the natural polymer is any one or more of collagen, chitosan, gelatin, casein, cellulose acetate, silk fibroin, chitin, and fibroin; the sacrificial agent is polyethylene oxide.

[0012] In step (1), the voltage of electrospinning is 12-15KV; the propulsion speed of the propulsion pump is 0.8-1.0mL / h; and the rotation speed of the roller of the collection device is 1000-1500rpm.

[0013] In step (2), the foaming agent is sodium borohydride or dichlorofluoroethane, with a concentration of 0.5-1.0 M.

[0014] Preferably, in step (2), the two-dimensional directional fiber scaffold obtained in step (1) is placed in a mold and reacted with a foaming agent solvent, so that the thickness of the prepared three-dimensional scaffold is 1.0-5.0 mm.

[0015] The application of the tissue engineering scaffold prepared by the preparation method described in this invention for rapidly inducing stem cell proliferation and differentiation in the efficient proliferation and differentiation of stem cells in heterogeneous space.

[0016] The application involves planting adult stem cells on a tissue engineering scaffold, and then, through culture, directly regulating the elastic modulus to control the expression of key genes PIP2, ALP, and Vav, thereby inducing efficient proliferation and differentiation of stem cells in heterogeneous space.

[0017] As a preferred approach, experiments were conducted to verify that the differences in proliferation and differentiation between tropism-dependent and anisotropy-dependent (random) structures originate from the expression regulation of key genes PIP2, ALP, and Vav. Furthermore, by adjusting the elastic modulus of the scaffold, the cells achieved efficient expression of genes PIP2, ALP, and Vav, thereby promoting cell proliferation and differentiation.

[0018] Furthermore, the gene that has the greatest impact on stem cell proliferation and differentiation is the PIP2 gene, which describes the tropism-antagonism structure and changes in elastic modulus.

[0019] The adult stem cells include neural stem cells, blood stem cells, bone marrow mesenchymal stem cells, or epidermal stem cells from adult humans or mammals.

[0020] Preferably, the stem cells are sourced from a single donor or cell bank, using primary-10 generation cells, preferably primary-3 generation cells.

[0021] The application of the tissue engineering scaffold prepared by the method described in this invention to rapidly induce stem cell proliferation and differentiation in accelerating wound healing on damaged wound surfaces.

[0022] The tissue engineering scaffold prepared by the method described in this invention rapidly induces stem cell proliferation and differentiation. By regulating the expression of BCL-6 and MiR-126-5p in cells seeded on the scaffold, it further regulates the expression of key genes PIP2, ALP, and Vav, thereby inducing efficient proliferation and differentiation of stem cells in heterogeneous space.

[0023] Among them, the expression of key genes PIP2, ALP, and Vav was regulated by constructing transfection reagents to promote the expression of BCL-6 and inhibit the expression of MiR-126-5p.

[0024] The three-dimensional polymer scaffold prepared by this invention induces the efficient proliferation and differentiation of stem cells in heterogeneous space by directly regulating the expression of key genes PIP2, ALP, and Vav through the regulation of elastic modulus. Furthermore, it regulates the expression of key genes PIP2, ALP, and Vav by regulating the expression of BCL-6 and MiR-126-5p in cells seeded on the scaffold, thereby inducing the efficient proliferation and differentiation of stem cells in heterogeneous space. The scaffold after cell loading is then implanted on the surface of a damaged wound to accelerate wound healing.

[0025] The three-dimensional scaffold achieves optimal conditions for wound healing biology applications by adjusting its spatial thickness, elastic modulus, and expression of related genes, with the elastic modulus being the most important factor.

[0026] The thickness of the specific mold designed in this invention varies depending on the application for wound healing, and is generally 1.0-5.0 mm. The concentration of the foaming agent determines the porosity inside the scaffold, which greatly affects cell growth and extension; the concentration of the foaming agent is generally 0.5-1 M.

[0027] In this invention, the three-dimensional scaffolds with surface-oriented spatial anisotropic structures and different elastic moduli can achieve more significant proliferation and differentiation effects by directly reducing the elastic modulus (through a specific preparation method). Specifically, the elastic moduli of the three-dimensional scaffolds prepared with spinning solutions of silk fibroin and polyethylene oxide at mass ratios of 7:3, 6:4, and 5:5 are 27.2 MPa, 14.1 MPa, and 0.7 MPa, respectively. In practice, under the allowable mechanical performance conditions of the scaffolds in specific applications, the smaller the elastic modulus, the better the effect.

[0028] This invention experimentally verifies that the differences in proliferation and differentiation between tropism and anisotropy structures originate from the expression regulation of key genes PIP2, ALP, and Vav. Furthermore, it discovers that by altering the elastic modulus of the scaffold, the expression of genes PIP2, ALP, and Vav can be significantly regulated, thereby achieving efficient and rapid cell proliferation and differentiation.

[0029] The three-dimensional scaffolds with different orientation structures and elastic moduli prepared by this invention can directly overexpress BCL-6 and MiR-126-5p in cells planted on the scaffolds through further intervention measures, thereby regulating the expression of key genes PIP2, ALP, and Vav and inducing efficient proliferation and differentiation of stem cells in heterogeneous space.

[0030] This invention can produce three-dimensional scaffolds with oriented structures and different elastic moduli through a specific method. The raw materials for the scaffold are typical natural high-molecular polymers including collagen, chitosan, gelatin, casein, cellulose acetate, silk fibroin, chitin, fibrinogen, etc. The sacrificial material is polyethylene oxide. In this invention, polyethylene oxide is dissolved in trifluoroethanol in a certain proportion to prepare a spinning solution. A membrane-like polymer composite scaffold with a surface orientation structure is prepared by electrospinning. The scaffold and foaming agents such as sodium borohydride and dichlorofluoroethane are placed in a pre-3D printed device, and the sacrificial agent is dissolved to obtain a three-dimensional scaffold with a surface orientation-spatial anisotropic structure and different elastic moduli. Various adult stem cells, including neural stem cells, blood stem cells, bone marrow mesenchymal stem cells, and epidermal stem cells, are seeded on the three-dimensional polymer scaffold. By directly adjusting the elastic modulus or further adjusting the expression of BCL-6 and MiR-126-5p, the expression of key genes PIP2, ALP, and Vav is regulated, thereby inducing the efficient proliferation and differentiation of stem cells in anisotropic space. The scaffold after cell loading is seeded on the surface of damaged wounds to accelerate wound healing. The tissue engineering scaffold of the present invention can achieve rapid recellularization after implantation, reconstruct new tissue in situ, improve wound healing-related medical performance, and meet the clinical requirement of "on-demand use".

[0031] The tissue engineering scaffold prepared in this invention rapidly induces stem cell proliferation and differentiation. It was found that changes in the elastic modulus, porosity, and tropism of the scaffold lead to differential expression of PIP2, ALP, and Vav genes, fundamentally altering stem cell proliferation and differentiation. By regulating physical parameters or indirectly intervening in the expression of BCL-6 and MiR-126-5p, the expression of key genes can be regulated, thereby inducing efficient proliferation and differentiation of stem cells in heterotropic space. Furthermore, the cell-loaded scaffold can be implanted on the surface of damaged wounds to accelerate wound healing.

[0032] This invention provides a tissue-engineered scaffold for rapidly inducing stem cell proliferation and differentiation. It reveals that changes in the scaffold's elastic modulus and tropism lead to differential expression of PIP2, ALP, and Vav genes, fundamentally altering stem cell proliferation and differentiation. This can be achieved by regulating physical parameters such as elastic modulus or further intervening in the expression of BCL-6 and MiR-126-5p, thereby controlling the expression of key genes and inducing efficient stem cell proliferation and differentiation in heterotropic spaces. This fundamentally solves the reason why stochastic and high-elastic-modulus fibers exhibit slower stem cell differentiation and proliferation compared to low-elastic-modulus and tropistic fibers; and proposes methods to modify this difference. Previous studies only found that tropistic fibers promote stem cell differentiation, but did not truly discover that changes in tropism and elastic modulus cause changes in PIP2, Vav, and ALP genes (higher PIP2 promotes differentiation more, and early elevation of ALP and Vav genes accelerates differentiation), ultimately leading to differences in stem cell growth on different scaffolds. This invention also proposes the influence of elastic modulus on cell proliferation and differentiation. This invention utilizes differential fabrication of specific three-dimensional scaffolds to influence cell behavior by directly controlling the elastic modulus or indirectly regulating the expression of target genes through exogenous gene modulation. The specific fabrication method of this invention produces tissue-engineered scaffolds with low elastic modulus, high porosity, and good tropism, which rapidly induce stem cell proliferation and differentiation.

[0033] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0034] (1) This invention provides a tissue engineering scaffold for rapidly inducing stem cell proliferation and differentiation. To enable stem cells to proliferate and differentiate better in a three-dimensional scaffold, natural high-molecular polymers such as silk fibroin and sacrificial agent polyethylene oxide are selected as raw materials for the scaffold. This invention focuses on using specific proportions of sacrificial agents to prepare scaffolds with different elastic moduli. These scaffolds with varying elastic moduli possess excellent potential for inducing stem cell proliferation and differentiation, as demonstrated by the electron micrographs and elastic modulus images presented in this invention.

[0035] (2) This invention provides a tissue engineering scaffold for rapidly inducing stem cell proliferation and differentiation. Currently, the biological principle behind the superiority of tropism-dependent structures over anisotropy-dependent structures in terms of proliferation and differentiation is not yet fully understood, resulting in significant lag in cell growth within the three-dimensional scaffold space. This invention discovers that changes in elastic modulus, porosity, and tropism lead to differentiation of PIP2, ALP, and Vav genes, fundamentally altering stem cell proliferation and differentiation. This invention can regulate the expression of key genes by adjusting the physical parameters during scaffold preparation, especially the elastic modulus, thereby inducing efficient proliferation and differentiation of stem cells in anisotropy space. Furthermore, the cell-loaded scaffold can be implanted onto the surface of damaged wounds to accelerate wound healing.

[0036] (3) This invention provides a tissue-engineered scaffold for rapidly inducing stem cell proliferation and differentiation. The stem cells include, but are not limited to, neural stem cells, blood stem cells, bone marrow mesenchymal stem cells, and epidermal stem cells from adult humans or mammals. Tissue-engineered skin with specific thickness, porosity, orientation, and customized stem cells can be designed and mass-produced in laboratory culture dishes to maximize the simulation of a real wound environment.

[0037] (4) In this invention, the expression of BCL-6 and MiR-126-5p of cells seeded on the scaffold is regulated, thereby regulating the expression of key genes PIP2, ALP and Vav, thereby inducing the efficient proliferation and differentiation of stem cells in heterogeneous space. Attached Figure Description

[0038] Figure 1 It is a mold of a specific thickness provided in the embodiments of the present invention;

[0039] Figure 2 This is a two-dimensional SEM image of the scaffold provided in an embodiment of the present invention;

[0040] Figure 3 This is a SEM image of the three-dimensional scaffold after cell seeding provided in an embodiment of the present invention;

[0041] Figure 4 The three-dimensional scaffold provided in this invention has the effect of tropism and elastic modulus on stem cell differentiation; A is the effect of tropism and random fibers on stem cell osteogenic differentiation indicators; B is the effect of elastic modulus changes caused by dissolving different amounts of sacrificial agent on stem cell osteogenic differentiation indicators.

[0042] Figure 5 These are PCR data of key genes related to the function and differentiation genes of three-dimensional scaffold cells provided in the embodiments of the present invention;

[0043] Figure 6 This invention provides a three-dimensional scaffold seeded cell protein imprint expression method.

[0044] Figure 7 These are SEM images of three-dimensional scaffolds prepared after dissolution of 30% and 50% sacrificial agents, provided in the embodiments of the present invention.

[0045] Figure 8 These are the elastic modulus data of the three-dimensional scaffolds after dissolution of 30% and 50% sacrificial agents provided in this invention.

[0046] Figure 9 These are morphology images of three-dimensional scaffolds seeded in cells after dissolution of 30% and 50% sacrificial agents, as provided in the embodiments of the present invention.

[0047] Figure 10The VCAM-1 staining and crystal violet staining are performed after cell culture in the three-dimensional scaffold provided in this embodiment of the invention and after culture regulating the PIP2 gene. Detailed Implementation

[0048] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0049] The raw materials and reagents used in the examples are all commercially available.

[0050] The electrospinning instrument used in this embodiment was purchased from Beijing Xinrui Baina Technology Co., Ltd., model: NanoyarnTEADFS-700.

[0051] Silk fibroin was purchased from Hubei Hongxin Ruiyu Fine Chemical Co., Ltd., model number 96690-41-4.

[0052] Third-generation bone marrow mesenchymal stem cells: These are rat bone marrow mesenchymal stem cells, purchased from the Cell Bank of the Chinese Academy of Sciences. They were obtained from the bone marrow of 3-6 week old Sprague Dawley (SD) rats, isolated mechanically under sterile conditions, and cultured in SD rat mesenchymal stem cell complete culture medium (catalog number SCSP-615) to reach the third generation (P3) for later use.

[0053] The mold used in this embodiment is designed to ensure that the thickness of the scaffold film is 1.0-5.0 mm after foaming with the two-dimensional directional fiber scaffold and foaming agent solvent. This can be achieved using a mold such as... Figure 1 The mold shown is made of polylactic acid and can be manufactured by 3D printing. It is a membrane with small holes distributed around it, with an inner diameter and thickness of 1.0-5.0 mm.

[0054] DMEM low-glucose complete culture medium (containing 10% FBS) was purchased from Wuhan Shangen Biotechnology Co., Ltd., catalog number: SNM-003E.

[0055] Example 1

[0056] S1: Silk fibroin and polyethylene oxide were selected as the raw materials for the scaffold. Solution A was prepared by mixing silk fibroin with trifluoroethanol at a mass-to-volume ratio of 8% mg / mL, and solution B was prepared by mixing polyethylene oxide with trifluoroethanol at a mass-to-volume ratio of 40% mg / mL. An electrospinning apparatus was used, with the electrospinning voltage set to 13 kV, the feed pump speed set to 0.9 mL / h, and the spinning time set to 3 hours. The two spinning solutions A and B were mixed at a volume ratio of 7:3 and electrospun (total volume 10 mL) on a roller at a rotation speed of 1000 rpm to prepare a two-dimensional oriented scaffold film.

[0057] S2: Mix all the scaffold films prepared in step S1 with a 0.8M foaming agent sodium borohydride solution and place them in a 2.5mm thick mold for foaming. The foaming process is accompanied by the dissolution of the sacrificial agent, thus obtaining a three-dimensional scaffold film with a oriented structure and a certain elastic modulus.

[0058] S3: Cut the three-dimensional scaffold film prepared in step S2 into circular films with the same diameter as the 24-well plate, place them in the 24-well plate, and arrange them at a density of 1.0 x 10⁻⁶. 6 pcs / cm 2 Third-generation bone marrow mesenchymal stem cells were seeded in 24-well plates containing a three-dimensional scaffold, resuspended in DMEM low-glucose complete medium (containing 10% FBS), and statically cultured for 3 and 7 days in a 37°C, 5% CO2 incubator.

[0059] Example 2

[0060] S1: Silk fibroin and polyethylene oxide were selected as the raw materials for the scaffold. Solution A was prepared by mixing silk fibroin with trifluoroethanol at a mass-to-volume ratio of 8% mg / mL, and solution B was prepared by mixing polyethylene oxide with a solvent at a mass-to-volume ratio of 40% mg / mL. An electrospinning apparatus was used, with the electrospinning voltage set to 13 kV, the feed pump speed set to 0.9 mL / h, and the spinning time set to 3 hours. The two spinning solutions A and B were mixed at a volume ratio of 5:5 and electrospun (total volume 10 mL) on a roller at a rotation speed of 1000 rpm to prepare a two-dimensional oriented scaffold film.

[0061] S2: Place all the scaffold films prepared in step S1 and a 0.8M sodium borohydride foaming agent solution in a 2.5mm thick mold for foaming. The foaming process is accompanied by the dissolution of the sacrificial agent, thus obtaining a three-dimensional scaffold film with a oriented structure and a certain elastic modulus.

[0062] S3: Cut the three-dimensional scaffold film prepared in step S2 into circular films with the same diameter as the 24-well plate, place them in the 24-well plate, and arrange them at a density of 1.0 x 10⁻⁶. 6 pcs / cm 2 Third-generation bone marrow mesenchymal stem cells were seeded in 24-well plates containing a three-dimensional scaffold, resuspended in DMEM low-glucose complete medium (containing 10% FBS), and statically cultured for 3 and 7 days in a 37°C, 5% CO2 incubator.

[0063] Example 3

[0064] S1: Silk fibroin and polyethylene oxide were selected as the raw materials for the scaffold. Solution A was prepared by mixing silk fibroin and trifluoroethanol at a mass-to-volume ratio of 8% mg / mL, and solution B was prepared by mixing polyethylene oxide and a solvent at a mass-to-volume ratio of 40% mg / mL. An electrospinning apparatus was used, with the electrospinning voltage set to 13 kV, the feed pump speed set to 0.9 mL / h, and the spinning time set to 3 hours. The two spinning solutions A and B were mixed at a volume ratio of 5:5 and electrospun (total volume 10 mL) on a roller at a speed of 1000 rpm to prepare a two-dimensional oriented scaffold film.

[0065] S2: Place all the scaffold films prepared in step S1 and a 0.5M sodium borohydride foaming agent solution in a 2.5mm thick mold for foaming. The foaming process is accompanied by the dissolution of the sacrificial agent, thus obtaining a three-dimensional scaffold film with a surface orientation-spatial anisotropic structure and a certain elastic modulus.

[0066] S3: Cut the three-dimensional scaffold film prepared in step S2 into circular films with the same diameter as the 24-well plate, place them in the 24-well plate, and arrange them at a density of 1.0 x 10⁻⁶. 6 pcs / cm 2 Third-generation bone marrow mesenchymal stem cells were seeded in 24-well plates containing a three-dimensional scaffold, resuspended in DMEM low-glucose complete medium (containing 10% FBS), and statically cultured for 3 and 7 days in a 37°C, 5% CO2 incubator.

[0067] Example 4

[0068] S1: Silk fibroin and polyethylene oxide were selected as the raw materials for the scaffold. Solution A was prepared by mixing silk fibroin and trifluoroethanol at a mass-to-volume ratio of 8% mg / mL, and solution B was prepared by mixing polyethylene oxide and a solvent at a mass-to-volume ratio of 40% mg / mL. An electrospinning apparatus was used, with the electrospinning voltage set to 13 kV, the feed pump speed set to 0.9 mL / h, and the spinning time set to 3 hours. The two spinning solutions A and B were mixed at a volume ratio of 5:5 and electrospun (total volume 10 mL) on a roller at a speed of 1000 rpm to prepare a two-dimensional oriented scaffold film.

[0069] S2: Place all the scaffold films prepared in step S1 and a 1.0M sodium borohydride foaming agent solution in a 2.5mm thick mold for foaming. The foaming process is accompanied by the dissolution of the sacrificial agent, thus obtaining a three-dimensional scaffold film with a surface orientation-spatial anisotropic structure and a certain elastic modulus.

[0070] S3: Cut the three-dimensional scaffold film prepared in step S2 into circular films with the same diameter as the 24-well plate, place them in the 24-well plate, and arrange them at a density of 1.0 x 10⁻⁶. 6 pcs / cm2 Third-generation bone marrow mesenchymal stem cells were seeded in 24-well plates containing a three-dimensional scaffold, resuspended in DMEM low-glucose complete medium (containing 10% FBS), and statically cultured for 3 and 7 days in a 37°C, 5% CO2 incubator.

[0071] Example 5

[0072] S1: Silk fibroin and polyethylene oxide were selected as the raw materials for the scaffold. Solution A was prepared by mixing silk fibroin and trifluoroethanol at a mass-to-volume ratio of 8% mg / mL, and solution B was prepared by mixing polyethylene oxide and a solvent at a mass-to-volume ratio of 40% mg / mL. An electrospinning apparatus was used, with the electrospinning voltage set to 13 kV, the feed pump speed set to 0.9 mL / h, and the spinning time set to 3 hours. The two spinning solutions A and B were mixed at a volume ratio of 5:5 and electrospun (total volume 10 mL) on a roller at a speed of 1000 rpm to prepare a two-dimensional oriented scaffold film.

[0073] S2: Place all the scaffold films prepared in step S1 and a 0.8M sodium borohydride foaming agent solution in a 1.0mm thick mold for foaming. The foaming process is accompanied by the dissolution of the sacrificial agent, thus obtaining a three-dimensional scaffold film with a surface orientation-spatial anisotropic structure and a certain elastic modulus.

[0074] S3: Cut the three-dimensional scaffold membrane prepared in step S2 into a circular membrane with the same diameter as the 24-well plate, place it in the 24-well plate, seed the third generation bone marrow mesenchymal stem cells in the 24-well plate containing the three-dimensional scaffold, resuspend it in DMEM low-glucose complete medium (containing 10% FBS), and statically culture it in a 37°C, 5% CO2 incubator for 3 and 7 days.

[0075] Example 6

[0076] S1: Silk fibroin and polyethylene oxide were selected as the raw materials for the scaffold. Solution A was prepared by mixing silk fibroin and trifluoroethanol at a mass-to-volume ratio of 8% mg / mL, and solution B was prepared by mixing polyethylene oxide and a solvent at a mass-to-volume ratio of 40% mg / mL. An electrospinning apparatus was used, with the electrospinning voltage set to 13 kV, the feed pump speed set to 0.9 mL / h, and the spinning time set to 3 hours. The two spinning solutions A and B were mixed at a volume ratio of 5:5 and electrospun (total volume 10 mL) on a roller at a speed of 1000 rpm to prepare a two-dimensional oriented scaffold film.

[0077] S2: Place all the scaffold films prepared in step S1 and a 0.8M sodium borohydride foaming agent solution in a 5.0mm thick mold for foaming. The foaming process is accompanied by the dissolution of the sacrificial agent, thus obtaining a three-dimensional scaffold film with a surface orientation-spatial anisotropic structure and a certain elastic modulus.

[0078] S3: Cut the three-dimensional scaffold film prepared in step S2 into circular films with the same diameter as the 24-well plate, place them in the 24-well plate, and arrange them at a density of 1.0 x 10⁻⁶. 6 pcs / cm 2 Third-generation bone marrow mesenchymal stem cells were seeded in 24-well plates containing a three-dimensional scaffold, resuspended in DMEM low-glucose complete medium (containing 10% FBS), and statically cultured for 3 and 7 days in a 37°C, 5% CO2 incubator.

[0079] Example 7

[0080] S1: Silk fibroin and polyethylene oxide were selected as the raw materials for the scaffold. Silk fibroin and trifluoroethanol were mixed at a mass-to-volume ratio of 8% mg / mL to prepare solution A. An electrospinning apparatus was used, with the electrospinning voltage set to 13 kV, the feed pump speed set to 0.9 mL / h, and the spinning time set to 3 hours. Spinning solution A was prepared with a total volume of 10 mL. A two-dimensional random structure scaffold film was then fabricated on food-grade tin foil.

[0081] S2: Place all the scaffold films prepared in step S1 and a 0.8M sodium borohydride foaming agent solution into a 2.5mm thick mold for foaming. The foaming process is accompanied by the dissolution of the sacrificial agent, thus obtaining a three-dimensional random scaffold film.

[0082] S3: Cut the three-dimensional scaffold film prepared in step S2 into circular films with the same diameter as the 24-well plate, place them in the 24-well plate, and arrange them at a density of 1.0 x 10⁻⁶. 6 pcs / cm 2 Third-generation bone marrow mesenchymal stem cells were seeded in 24-well plates containing a three-dimensional scaffold, resuspended in low-glucose DMEM culture medium containing 10% FBS, and statically cultured for 3 and 7 days in a 37°C, 5% CO2 incubator.

[0083] Example 8

[0084] S1: Silk fibroin and polyethylene oxide were selected as the raw materials for the scaffold. Silk fibroin and trifluoroethanol were mixed at a mass-to-volume ratio of 8% mg / mL to prepare solution A. An electrospinning apparatus was used, with the electrospinning voltage set to 13 kV, the feed pump speed set to 0.9 mL / h, and the spinning time set to 3 hours. Spinning solution A was prepared with a total volume of 10 mL on a roller at a rotation speed of 1000 rpm, resulting in a two-dimensional oriented scaffold film.

[0085] S2: Place all the scaffold films prepared in step S1 and a 0.8M sodium borohydride foaming agent solution into a 2.5mm thick mold for foaming. The foaming process is accompanied by the dissolution of the sacrificial agent, thus obtaining a three-dimensional scaffold film with a oriented structure.

[0086] S3: Cut the three-dimensional scaffold film prepared in step S2 into circular films with the same diameter as the 24-well plate, place them in the 24-well plate, and arrange them at a density of 1.0 x 10⁻⁶. 6 pcs / cm 2 Third-generation bone marrow mesenchymal stem cells were seeded in 24-well plates containing a three-dimensional scaffold, resuspended in DMEM low-glucose complete medium (containing 10% FBS), and statically cultured for 3 and 7 days in a 37°C, 5% CO2 incubator.

[0087] Example 9

[0088] S1: Silk fibroin and polyethylene oxide were selected as the raw materials for the scaffold. Solution A was prepared by mixing silk fibroin with trifluoroethanol at a mass-to-volume ratio of 8% mg / mL, and solution B was prepared by mixing polyethylene oxide with a solvent at a mass-to-volume ratio of 40% mg / mL. An electrospinning apparatus was used, with the voltage set to 13 kV. The feed pump speed was 0.9 mL / h, and the spinning time was 3 hours. The two spinning solutions A and B were mixed at volume ratios of 7:3 and 5:5, respectively, and electrospun (total volume 10 mL) on a roller at a speed of 1000 rpm to prepare a two-dimensional orientation scaffold film.

[0089] S2: Place all the scaffold films prepared in step S1 and a 0.8M sodium borohydride foaming agent solution in a 2.5mm thick mold for foaming. The foaming process is accompanied by the dissolution of the sacrificial agent, thus obtaining a three-dimensional scaffold film with a directional structure and different elastic moduli.

[0090] S3: Cut the three-dimensional scaffold film prepared in step S2 into circular films with the same diameter as the 24-well plate, place them in the 24-well plate, and arrange them at a density of 1.0 x 10⁻⁶. 6 pcs / cm 2Third-generation bone marrow mesenchymal stem cells were seeded in 24-well plates containing a three-dimensional scaffold. The stem cells on the 7:3 volume ratio fibrous scaffold were treated with BCL-6 gene overexpression, while the stem cells on the 5:5 volume ratio fibrous scaffold were treated with BCL-6 gene suppression. The cells were then resuspended in DMEM low-glucose complete medium (containing 10% FBS) and statically cultured at 37°C in a 5% CO2 incubator for 3 and 7 days, respectively.

[0091] Overexpression or inhibition of the BCL-6 gene is as follows, and can be found in the reference (MircoRNA□126□5pinhibits apoptosis of endothelial cell in vascular arterial walls via NFκB / PI3K / AKT / mTOR signaling pathway in atherosclerosis, Journal of Molecular Histology (2022) 53:51–62); the si-BCL-6 plasmid and BCL-6 overexpression plasmid can be found in the reference: Highly efficient genome editing via CRISPR–Cas9 in human pluripotent stem cells is achieved by transient BCL-XL overexpression, Nucleic Acids Research, (2018), 46:10195-10215.

[0092] 1. Plate formation: The cell density in a 6-well plate is 1×10⁻⁶. 6 .

[0093] 2.1) Preparation of transfection inhibition reagent: A. Dissolve a total of 100 pmol si-BCL-6 plasmid in 250 μL of serum-free medium; B. Dissolve 5 μL of Lipo2000 in 250 μL of serum-free medium. Mix thoroughly and let stand at room temperature for 5 min; C. Mix A and B and let them stand at room temperature for 20 min.

[0094] 2) Preparation of overexpression transfection reagents: A. Dissolve a total of 4 μg of BCL-6 overexpression plasmid in 250 μL of serum-free medium; B. Dissolve 10 μL of Lipo2000 in 250 μL of serum-free medium. Mix thoroughly and let stand at room temperature for 5 min; C. Mix A and B and let them stand at room temperature for 20 min.

[0095] 3. Wash the 6-well plate 2-3 times with serum-free culture medium.

[0096] 4. Add the above transfection reagent (500 μL) to a 6-well plate containing stem cells, gently shake for 1-2 minutes, and add 2 mL of DMEM low-glucose complete medium. After culturing at 37°C for 4 hours, replace the medium with the original complete medium and continue culturing for 48 hours. Detect transfection efficiency after 24 or 48 hours to obtain stem cells treated with BCL-6 gene overexpression or BCL-6 gene suppression.

[0097] Example 10

[0098] S1: Silk fibroin and polyethylene oxide were selected as the raw materials for the scaffold. Solution A was prepared by mixing silk fibroin and trifluoroethanol at a mass-to-volume ratio of 8% mg / mL, and solution B was prepared by mixing polyethylene oxide and a solvent at a mass-to-volume ratio of 40% mg / mL. An electrospinning apparatus was used, with the voltage set to 13 kV. The feed pump speed was 0.9 mL / h, and the spinning time was 3 hours. The two spinning solutions A and B were mixed at a volume ratio of 5:5 and electrospun (total volume 10 mL). Two-dimensional scaffold films with oriented and random structures were prepared on a roller rotating at 1000 rpm and on food-grade aluminum foil, respectively.

[0099] S2: Place all the scaffold films prepared in step S1 and a 0.8M sodium borohydride foaming agent solution in a 2.5mm thick mold for foaming. The foaming process is accompanied by the dissolution of the sacrificial agent, which can produce a three-dimensional scaffold film with a oriented structure and a certain elastic modulus, as well as a three-dimensional anisotropic structure scaffold film.

[0100] S3: Cut the three-dimensional scaffold film prepared in step S2 into circular films with the same diameter as the 24-well plate, place them in the 24-well plate, and arrange them at a density of 1.0 x 10⁻⁶. 6 pcs / cm 2 Third-generation bone marrow mesenchymal stem cells were seeded in 24-well plates containing a three-dimensional scaffold. The BCL-6 gene of stem cells on the filaments of the foil was overexpressed, while the BCL-6 gene of stem cells on the tropism filaments (volume ratio 5:5) was suppressed. The cells were then resuspended in DMEM low-glucose complete medium (containing 10% FBS) and statically cultured at 37°C in a 5% CO2 incubator for 3 and 7 days.

[0101] The treatment of BCL-6 gene overexpression or inhibition is the same as in Example 9.

[0102] Example 11

[0103] The method in Example 11 is the same as in Example 9, except that the miR-126a-5p gene of stem cells is either overexpressed or suppressed.

[0104] Among them, the si-miR-126a-5p plasmid and the miR-126a-5p overexpression plasmid are described in the literature: Down-regulation of microRNA-126-5p contributes to overexpression of VEGFA inlipopolysaccharide-induced acute lung injury, (2016), 38:1277–1284.

[0105] Example 12

[0106] The method in Example 12 is the same as in Example 10, except that the miR-126a-5p gene of stem cells is either overexpressed or suppressed.

[0107] Example 13

[0108] The method in Example 13 is the same as in Example 9, except that the PIP2 gene of stem cells is either overexpressed or suppressed.

[0109] Among them, the si-PIP2 plasmid and the PIP2 overexpression plasmid are described in the literature: The Arabidopsis AbioticStress-Induced TSPO-Related Protein Reduces Cell-Surface Expression of the Aquaporin PIP2; 7through Protein-Protein Interactions and Autophagic Degradation, (2014), 26:4974-4990.

[0110] Example 14

[0111] The method in Example 14 is the same as in Example 10, except that the PIP2 gene of stem cells is either overexpressed or suppressed.

[0112] Experimental Example 1

[0113] I. SEM images of the scaffolds prepared in step S1 of Examples 1, 2, 7 and 8 are shown below. Figure 2 As shown, Figure 2In Figure a' (top left), the image shows a SEM image of the random two-dimensional fiber scaffold prepared in Example 7, and in Figure b' (top right), the image shows a SEM image of the directional two-dimensional fiber scaffold prepared in Example 8. The directional scaffold prepared in Example 7 has an average fiber diameter of 0.45 μm, uniform fiber arrangement, consistent direction, and a porosity of 41.2%. The random (or anisotropic) scaffold prepared in Example 8 has an average diameter of 0.41 μm, complex fiber arrangement, and a porosity of 46.8%. Further, Figure a' (bottom left) shows a directional two-dimensional scaffold film containing 50% sacrificial agent prepared in Example 2, and Figure b' (bottom right) shows a directional two-dimensional scaffold film containing 30% sacrificial agent prepared in Example 1. The directional scaffold containing 50% sacrificial agent prepared in Example 2 had an average fiber diameter of 0.53 μm, with uniform fiber arrangement and consistent direction, and a porosity of 39.7% between fibers; the directional scaffold containing 30% sacrificial agent prepared in Example 1 had an average fiber diameter of 0.46 μm, with complex fiber arrangement and a porosity of 44.3%. Figure 1 The main difference was reflected in the morphology of the scaffold, indicating that morphology may affect cell behavior.

[0114] II. Cell morphology planted on the scaffold in Examples 7 and 8 as follows Figure 3 As shown, the left image shows the morphology of the three-dimensional random scaffold prepared in Example 7 on day 7 after cell seeding, and the right image shows the morphology of the three-dimensional directional scaffold prepared in Example 8 on day 7 after cell seeding. Figure 3 The cells on the surface of the directional fibrous scaffold were observed to be spindle-shaped and directional, but on the left side they exhibited randomness (or anisotropy). The cells on the surface of the fibrous scaffold also showed a locally rounded characteristic, with most cells exhibiting high dispersion. This indicates that the morphology of the scaffold influences the cell growth pattern.

[0115] Furthermore, Figure 4 This study demonstrates the influence of tropism and elastic modulus on stem cell differentiation. A represents the effect of tropism (Example 8) and randomness (Example 7) three-dimensional fibrous scaffolds on osteogenic differentiation indices of stem cells; B represents the effect of changes in elastic modulus caused by dissolving different amounts of sacrificial agent (according to the methods of Examples 2 and 1, with A and B solution volume ratios of 5:5 and 7:3) on osteogenic differentiation indices of stem cells. Based on qPCR data of differentially expressed genes in cells cultured on tropism / random scaffolds, it can be seen that osteogenic differentiation indices OCN, RUNX2, and ALP all exhibit certain differentiation characteristics. However, the differentiation indices of tropism-dependent fibrous scaffolds are significantly higher than those of random fibrous scaffolds, clearly indicating that random (or heterotropic) fibers are detrimental to stem cell proliferation and differentiation. Simultaneously, different amounts of sacrificial agent also have a significant impact on osteogenic differentiation indices; the differentiation indices of three-dimensional scaffolds prepared with high doses of sacrificial agent are significantly higher than those prepared with low doses.

[0116] qPCR was performed on key genes related to cell function and differentiation genes, namely PIP2, ALP, and Vav, in the three-dimensional scaffolds prepared in Examples 1, 2, 7, and 8 above. Figure 5 The qPCR results showed significant differences, with the most significant difference observed in PIP2. It can be considered that PIP2 is a key gene leading to the differences in tropism and elastic modulus in stem cell differentiation.

[0117] Furthermore, Western blotting was used to verify the integrin-mediated PIP5K-PIP2-BCL-6 and osteogenic differentiation pathways. Figure 6 A shows the expression of PIP5K and PIP2 genes, and three differentiation genes, in stem cells after BCL-6 knockout and overexpression on arranged and random three-dimensional fiber scaffolds, respectively (using scaffolds from Examples 7 and 8, and gene regulation methods from Example 9). B shows the expression of PIP5K and PIP2 genes, and three differentiation genes, in stem cells after BCL-6 gene knockout and overexpression on directional three-dimensional fiber scaffolds after sacrificial agent lysis (using scaffolds from Examples 1 and 2, and gene regulation methods from Example 9). C shows the expression of PIP5K, PIP2, and BCL-6 genes, and three differentiation genes, in stem cells after miR-126a-5p knockdown and overexpression on arranged and random three-dimensional fiber scaffolds, respectively (using scaffolds from Examples 7 and 8, and gene regulation methods from Example 11). D represents the expression of PIP5K, PIP2, and BCL-6 genes, as well as three differentiation genes, on a tactic three-dimensional fiber scaffold after sacrificial agent dissolution following miR-126a-5p knockdown and overexpression (using scaffolds from Examples 1 and 2, and gene regulation methods as described in Example 11).

[0118] Figure 6 The PIP5K and PIP2 genes, as well as three differentiation genes, in stem cells showed decreasing and increasing trends after BCL-6 knockout and overexpression, respectively. Conversely, the PIP5K, PIP2, and BCL-6 genes, as well as the three differentiation genes, showed increasing and decreasing trends after miR-126a-5p knockdown and overexpression, respectively. This step further explains the relationship between BCL-6 and miR-126a-5p and PIP2: BCL-6 promotes PIP2 expression, while miR-126a-5p inhibits PIP2 expression.

[0119] For example, in Example 7, regulating the expression of BCL-6 and MiR-126-5p in the cells restored cell differentiation indices and PIP2 expression on the overall scaffold to levels comparable to those in Example 2. This demonstrates that the gene influences cell growth. It also shows that Example 2 showed better cell proliferation and differentiation after seeding, and that promoting the expression of the key gene PIP2 by promoting BCL-6 and inhibiting MiR-126-5p can further promote cell proliferation and differentiation.

[0120] III. The SEM morphology of the three-dimensional scaffolds prepared in step S2 of Examples 1 and 2 is shown in the figure. Figure 7 As shown, the left image is a SEM image of the directional three-dimensional fiber scaffold after dissolving 30% sacrificial agent (Example 1); the right image is a SEM image of the directional three-dimensional fiber scaffold after dissolving 50% sacrificial agent (Example 2). The scaffold prepared in Example 1 had an average fiber diameter of 0.46 μm, with uniform fiber arrangement and consistent orientation, and a porosity of 45.6% between fibers; the scaffold prepared in Example 2 had an average diameter of 0.52 μm, with neatly arranged fibers and a porosity of 43.1%. Figure 8 It can be seen that the individual fibers of the fiber scaffolds prepared by both methods exhibit partial loss, and Figure 8 The elastic moduli for volume ratios of 7:3 (Example 1) and 5:5 (Example 2) were 27.2 MPa and 0.7 MPa, respectively, indicating that the more sacrificial agent there is, the greater the loss of fiber mechanical properties during dissolution.

[0121] IV. Cell morphology on three-dimensional scaffolds in Examples 1 and 2 Figure 9 As shown, the left image shows the morphology of the scaffold (30% sacrificial agent) prepared in Example 1 on day 7 after cell seeding and culture, and the right image shows the morphology of the scaffold (50% sacrificial agent) prepared in Example 2 on day 7 after cell seeding and culture. Based on the morphology on day 7, both showed an extension of the spindle-shaped morphology, with Example 2 showing significantly better extension. Furthermore, increasing the sacrificial agent resulted in a severe decrease in mechanical properties, failing to meet the requirements of the cell scaffold.

[0122] At the same time, according to Figure 5 Differentiation indicators, key genes PIP2, ALP, and Vav roots of cells on fiber scaffolds showed significant differences in qPCR results, with PIP2 showing the most significant difference before and after culture; cell proliferation and differentiation can be promoted by further regulating PIP2.

[0123] Furthermore, such as Figure 10The images show VCAM-1 staining and crystal violet staining of stem cells after seeding and culturing in Examples 1, 2, 7, and 8; and VCAM-1 staining and crystal violet staining of stem cells after culturing cells overexpressing or knocking out the PIP2 gene on fibrous scaffolds in Examples 1, 2, 7, and 8. In Example A, VCAM-1 staining and crystal violet staining of stem cells after normal seeding (NC) or PIP2 gene overexpression (OE) on a randomized scaffold (Example 7). In Example B, VCAM-1 staining and crystal violet staining of stem cells after normal seeding (NC) or PIP2 gene knockout (SI) on a directional scaffold (Example 8). In Example C, VCAM-1 staining and crystal violet staining of stem cells after normal seeding (NC) or PIP2 gene knockout (SI) on an aligned scaffold with low elastic modulus (Example 2). In Example D, VCAM-1 staining and crystal violet staining of stem cells after normal seeding (NC) or PIP2 gene overexpression (OE) on an aligned scaffold with high elastic modulus (Example 1).

[0124] As can be seen from the 7d (NC) cell growth in Figure A and Figure B, the directional scaffold prepared in Example 2 of this invention can significantly promote cell proliferation and differentiation compared to the random scaffold. As can be seen from the 7d (OE) cell growth in Figure A and Figure B, overexpression of the PIP2 gene can promote cell proliferation and differentiation, while knocking out the PIP2 gene (SI) can inhibit cell proliferation and differentiation.

[0125] As can be seen from the 7d (NC) cell growth in Figure C and Figure D, the low elastic modulus scaffold prepared in Example 2 of this invention can significantly promote cell proliferation and differentiation compared to the high elastic modulus scaffold. As can be seen from the 7d (SI) cell growth in Figure C and Figure D, overexpression of the PIP2 gene can promote cell proliferation and differentiation, while knocking out the PIP2 gene (SI) can inhibit cell proliferation and differentiation.

[0126] When the expression of BCL-6 and MiR-126-5p was regulated in the seeded cells of the examples, the PIP2 gene corresponding to cells on anisotropic scaffolds and scaffolds with high elastic modulus was suppressed, and the growth of cells on anisotropic scaffolds and scaffolds with high elastic modulus was accelerated. This indicates that the gene has a compensatory effect on the growth of cells on anisotropic fibers and fibers with lost elastic modulus. Similarly, this shows that better tropism and greater loss of elastic modulus are beneficial to the growth and differentiation of stem cells.

[0127] V. The parameters and performance of the scaffolds prepared in Examples 2, 3, and 4 were determined. The scaffold in Example 4 had an average fiber diameter of 0.41 μm, with uniform fiber arrangement and consistent orientation, and a porosity of 46.8%. The scaffold in Example 3 had an average fiber diameter of 0.49 μm, with uniform fiber arrangement and consistent orientation, and a porosity of 33.9%. The scaffold in Example 2 had an average fiber diameter of 0.45 μm, with uniform fiber arrangement and consistent orientation, and a porosity of 41.2%. This indicates that the foaming agent makes the elastic fibers thinner overall and increases their porosity. A 0.8 M foaming agent is suitable without affecting the overall mechanical properties of the fiber scaffold.

[0128] VI. The scaffolds prepared in Examples 2, 5, and 6 were tested. The scaffold in Example 6 had an average fiber diameter of 0.41 μm, with uniform fiber arrangement and consistent orientation, and a porosity of 46.8%. The scaffold in Example 2 had an average fiber diameter of 0.45 μm, with uniform fiber arrangement and consistent orientation, and a porosity of 41.2%. The scaffold in Example 5 had an average fiber diameter of 0.51 μm, with uniform fiber arrangement and consistent orientation, and a porosity of 39.8%. As the scaffold thickness increased, the foaming agent caused the elastic fibers to become thinner overall and the porosity to increase. A thickness of 2.5 mm is suitable without affecting the overall mechanical properties of the fiber scaffold.

[0129] In summary, the tissue engineering scaffold for rapidly inducing stem cell proliferation and differentiation, its preparation method, and its principle provided by this invention have the following advantages:

[0130] (1) The reason for the large differences in stem cell differentiation and proliferation in tropism-antagonism scaffolds was solved from a genetic perspective.

[0131] (2) The reasons for the large differences in stem cell differentiation and proliferation in the scaffold structure with changing elastic modulus were discovered and resolved from the perspective of genes.

[0132] (3) From a biological perspective, indirectly regulate the level of differentially expressed genes to restore the heterogeneous cells to a level comparable to the differentiation and proliferation of the oriented cells.

[0133] (4) From a biological perspective, indirectly regulate the level of differentially expressed genes to restore cells in structures with differential elastic modulus to a level comparable to that of normal cell differentiation and proliferation.

[0134] (5) It provides suitable methods for fabricating scaffolds for wound repair, foaming agent concentration and scaffold thickness range.

[0135] (6) The three-dimensional scaffold structure of this invention solves the problem of spatial anisotropy and surface orientation of cell differentiation and proliferation, allowing cells to extend and differentiate better within the scaffold. This invention maximizes the achievement of the level of natural skin structure required for wounds in the laboratory stage.

Claims

1. The application of a tissue engineering scaffold with low elastic modulus that rapidly induces stem cell proliferation and differentiation in the preparation of materials that induce efficient proliferation and differentiation of stem cells in heterogeneous space; The method for preparing the tissue engineering scaffold for rapidly inducing stem cell proliferation and differentiation with low elastic modulus includes the following steps: S1: Silk fibroin and polyethylene oxide were selected as raw materials for the scaffold. Silk fibroin and trifluoroethanol were mixed at a mass-volume ratio of 8% mg / mL to prepare solution A. Polyethylene oxide and solvent were mixed at a mass-volume ratio of 40% mg / mL to prepare solution B. An electrospinning instrument was used, with the electrospinning voltage set to 13 KV, the feed pump speed set to 0.9 mL / h, and the spinning time set to 3 hours. The two spinning solutions A and B were mixed at a volume ratio of 5:5 and electrospinned to a total volume of 10 mL. A two-dimensional orientation structure scaffold film was prepared on a roller at a rotation speed of 1000 rpm. S2: Place all the scaffold films prepared in step S1 and a 0.8 M sodium borohydride foaming agent solution in a 2.5 mm thick mold for foaming. The foaming process is accompanied by the dissolution of the sacrificial agent, thus obtaining a three-dimensional scaffold film with a oriented structure and low elastic modulus.

2. The application according to claim 1, characterized in that, The application involves seeding adult stem cells onto a tissue engineering scaffold, and then, through culture, directly regulating the elastic modulus to control the expression of key genes PIP2, ALP, and Vav, thereby inducing efficient proliferation and differentiation of stem cells in heterogeneous space.

3. The application according to claim 2, characterized in that, The adult stem cells include neural stem cells, blood stem cells, bone marrow mesenchymal stem cells, or epidermal stem cells of adult mammals.

4. The application according to claim 1, characterized in that, This application utilizes tissue-engineered scaffolds with low elastic modulus to rapidly induce stem cell proliferation and differentiation, thereby regulating the expression of BCL-6 and MiR-126-5p in cells seeded on the scaffold and promoting the expression of the PIP2 gene, thus inducing efficient in vitro proliferation and differentiation of stem cells.

5. The application of a tissue engineering scaffold with low elastic modulus that rapidly induces stem cell proliferation and differentiation in the preparation of wound healing materials for damaged wound surfaces; the preparation method of the tissue engineering scaffold with low elastic modulus that rapidly induces stem cell proliferation and differentiation is the same as that in claim 1.

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