CN116271220A - Preparation method and application of a tissue engineering scaffold for rapidly inducing proliferation and differentiation of stem cells - Google Patents

Abstract

The invention discloses a preparation method and application of a tissue engineering scaffold for rapidly inducing proliferation and differentiation of stem cells, wherein the scaffold is prepared by the following steps: respectively dissolving a natural high polymer and a sacrificial agent in an organic solvent to prepare spinning solutions, mixing the two spinning solutions, and carrying out electrostatic spinning to obtain a two-dimensional scale trend fiber scaffold; the two-dimensional fiber scaffold is reacted with a foaming agent solvent to prepare the three-dimensional scaffold which has a trend structure and different elastic moduli. According to the invention, the three-dimensional scaffold with a trend structure and different elastic moduli can be obtained by controlling the preparation conditions, stem cells are planted in the three-dimensional scaffold, the expression of key genes PIP2, ALP and Vav is regulated and controlled by directly regulating the elastic moduli or further regulating the expression of BCL-6 and MiR-126-5p, so that the stem cells are induced to proliferate and differentiate efficiently in different spaces, and the scaffold after cell-carrying treatment is planted on the surface of a damaged wound to accelerate wound healing.

Description

A preparation method of a tissue engineering scaffold for rapidly inducing proliferation and differentiation of stem cells and its application

technical field

The invention belongs to a tissue engineering scaffold, and relates to a preparation method and application of a tissue engineering scaffold for rapidly inducing proliferation and differentiation of stem cells.

Background technique

Effective wound healing is a common concern of the global medical community. Typically, wounds undergo several sequential, overlapping phases of cellular and biochemical activity to heal. Depending on time to heal, wounds can be classified as chronic or acute. Chronic wounds are wounds that cannot heal completely within a short period of time. In general, wounds that do not fully heal within 4-8 weeks can be considered chronic wounds. The normal wound healing process consists of four phases: hemostasis, inflammation, proliferation and remodeling. However, wound healing may stall at any of these stages, most commonly inflammatory or proliferative. At this point, a normal wound develops into a chronic wound, resulting in impaired wound cell function and hindering normal healing progression. For example, diabetic foot ulcers have been clinically common and serious types of chronic wounds. Diabetic foot ischemic, neurological, and neuroischemic diseases can lead to the occurrence of diabetic foot, resulting in varying degrees of foot infection and ulcers, and even amputation.

Small wounds can usually be healed by skin tissue repair, depending on the tissue's ability to proliferate. However, extensive burns and mechanical injuries require treatment with external wound dressings or other medical means. Clinical approaches to treating chronic wounds include debridement, orthopedics, casts, negative pressure therapy, and infection management. For local wound care, feasible methods include cleansing with saline, applying modern wound dressings to promote a moist environment. However, these methods are only effective for treating very small chronic wounds. In the case of negative pressure wound therapy, although it has produced significant clinical benefits, its mechanism of action remains unclear. Furthermore, orthopedics and castings involve higher medical costs and cause greater suffering to patients. Therefore, it is crucial to develop alternative methods for wound treatment. Tissue-engineered skin substitutes are considered a very promising approach for the treatment of wounds not amenable to primary closure. These substitutes offer an ideal alternative to traditional repair mechanisms through skin regeneration. This procedure does not produce any scars or helps to reduce them while allowing the skin's structure and function to be fully restored. According to 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, which open up new possibilities for treating chronic wounds.

As an electrostatic fiber formation technique, electrospinning can be used to tailor equipment to suit desired fiber morphology and structure, including high-voltage power supply units, dosing syringe pumps, and fiber collection devices. Under the action of a high-voltage electric field, the electrospinning device gradually pulls polymer droplets from Taylor cones into filament fibers with diameters of microns or even nanometers. The obtained fibrous scaffold has high porosity, good mechanical properties and excellent biocompatibility, and is beneficial to cell respiration, skin regeneration, wound moisturizing, internal and external metabolism, and hemostasis. Fibrous scaffolds fabricated by electrospinning devices can provide structural and morphological cues for the adhesion of various cell types in wounds and serve as templates for wound tissue regeneration. The extracellular matrix is a complex of fibrin and matrix proteins that provides the mechanical and biological properties required for bone growth, skin regeneration, vascular remodeling, and other tissue growth. With a similar microstructure to extracellular matrix proteins, electrospun fibrous scaffolds play an important role in maintaining cell growth and infiltration, and thus are suitable for tissue engineering.

Oriented nanofibrous scaffolds have received increasing attention in recent years because many natural extracellular matrices have highly oriented structures that contribute to high strength and well-guided cell growth, migration, and differentiation. Highly oriented nanofibers can be obtained by electrospinning. However, this technology is difficult to generate three-dimensional scaffolds, and the traditional cell planting only stays on the surface of two-dimensional materials and three-dimensional scaffolds, and the cell proliferation and differentiation are not complete enough. At present, the biological research on the difference between three-dimensional disordered and ordered scaffolds on the proliferation and differentiation of stem cells is still shallow. How to use these findings to efficiently play the role of living cells in wound healing has become a focus.

Contents of the invention

Purpose of the invention: Aiming at the problems existing in the prior art, the present invention provides a method for preparing a tissue engineering scaffold that rapidly induces the proliferation and differentiation of stem cells. The scaffold with a three-dimensional surface tendency-space anisotropic structure prepared by the present invention can be adjusted by adjusting the elastic modulus Or the way of gene induction can improve the proliferation and differentiation efficiency of stem cells on the surface of traditional random fibers and tropism fibers with higher elastic modulus. It provides strong theoretical and technical support for solving the problem of full extension and differentiation of stem cells in three-dimensional space.

The invention also provides the application of the prepared tissue engineering scaffold.

Technical solution: In order to achieve the above object, a method for preparing a tissue engineering scaffold for rapidly inducing stem cell proliferation and differentiation according to the present invention comprises the following steps:

(1) Select natural high molecular polymer and sacrificial agent as the raw material of scaffold; dissolve natural high molecular polymer and sacrificial agent in organic solvent respectively to prepare spinning liquid, and carry out electrospinning after mixing the two spinning liquids Obtain a two-dimensional scale-tropic fiber scaffold; (2) react the two-dimensional scale-tropic fiber scaffold obtained in step (1) with a foaming agent solvent, so that the sacrificial agent can be quickly dissolved to obtain a tropism structure and an elastic modulus Different 3D brackets.

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

Wherein, in the step (1), the natural polymer is any one or more of collagen, chitosan, gelatin, casein, cellulose acetate, silk fibroin, chitin, and fibrin; the sacrificial agent For polyethylene oxide.

Wherein, the voltage of electrospinning in step (1) is 12-15KV; the propulsion speed of the propulsion pump is 0.8-1.0mL/h; the rotational speed of the roller of the collecting device is 1000-1500rpm.

Wherein, the blowing agent described in step (2) is sodium borohydride or fluorodichloroethane, and its concentration is 0.5-1.0M.

Preferably, in step (2), place the two-dimensional scale-oriented fiber scaffold obtained in step (1) and a foaming agent solvent in a mold to react, so that the thickness of the prepared three-dimensional scaffold is 1.0-5.0 mm.

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

Among them, the application is to plant adult stem cells on tissue engineering scaffolds, and then directly adjust the elastic modulus after culture to regulate the expression of key genes PIP2, ALP, and Vav, thereby inducing the efficient proliferation and differentiation of stem cells in heterogeneous space.

Preferably, it is verified by experiments that the differences in the proliferation and differentiation of tropism and anisotropy (randomness) structures are derived from the expression regulation of key genes PIP2, ALP, and Vav. Further, by adjusting the elastic modulus of the scaffold, the genes PIP2, ALP, and Vav of the cells are efficiently expressed, thereby promoting the proliferation and differentiation of the cells.

Further, the gene that has the greatest impact on the proliferation and differentiation of stem cells is the PIP2 gene.

Wherein, the adult stem cells include adult human or mammalian neural stem cells, blood stem cells, bone marrow mesenchymal stem cells or epidermal stem cells.

Preferably, the stem cells are sourced from a single donor or a cell bank, using primary to 10th generation cells, preferably primary to 3rd generation cells.

The application of the tissue engineering scaffold for rapidly inducing proliferation and differentiation of stem cells prepared by the preparation method of the invention in accelerating wound healing on damaged wound surfaces.

The tissue engineering scaffold that rapidly induces stem cell proliferation and differentiation prepared by the preparation method of the present invention regulates the expression of BCL-6 and MiR-126-5p of cells seeded on the scaffold and then regulates the expression of key genes PIP2, ALP, and Vav to thereby Induce efficient proliferation and differentiation of stem cells in heterogeneous space.

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.

The polymer scaffold with three-dimensional structure prepared by the present invention can induce the efficient proliferation and differentiation of stem cells in the heterotropic space by directly regulating the expression of key genes PIP2, ALP, and Vav by directly adjusting the elastic modulus, and further by regulating the expression of the cells seeded on the scaffold. The expression of BCL-6 and MiR-126-5p regulates the expression of key genes PIP2, ALP, and Vav to induce the efficient proliferation and differentiation of stem cells in heterogeneous space, and the scaffolds treated with cells are planted on the damaged wound surface Accelerates wound healing.

Wherein, the three-dimensional scaffold can achieve the best conditions for biological application of wound healing by adjusting its spatial thickness, elastic modulus, expression of related genes, etc., wherein the most important thing is the elastic modulus.

The thickness of the specific mold designed in the present invention varies according to the wound healing application, generally 1.0-5.0mm. The concentration of foaming agent determines the porosity inside the scaffold, and the porosity greatly affects the growth and extension of cells. The concentration of foaming agent is generally 0.5-1M.

In the present invention, for three-dimensional scaffolds with surface tendency-space anisotropic structure and different elastic modulus, the direct intervention measure is the reduction of elastic modulus (reducing the elastic modulus of the scaffold through a specific preparation method) will lead to more obvious effects of proliferation and differentiation, Wherein the mass ratio of the spinning solution of silk fibroin and polyethylene oxide is 7:3, 6:4 and 5:5, and the elastic modulus of the prepared three-dimensional scaffold is respectively 27.2MPa, 14.1MPa and 0.7MPa, in fact Under the conditions of the mechanical properties of the stent allowed by the specific application, the smaller the elastic modulus, the better the effect.

The present invention verified through experiments that the differences in the proliferation and differentiation of tropism and anisotropic structures come from the expression regulation of key genes PIP2, ALP, and Vav, and further found that the expression of genes PIP2, ALP, and Vav can be significantly regulated by changing the elastic modulus of the scaffold , so as to realize the efficient and rapid proliferation and differentiation of cells.

The three-dimensional scaffolds prepared by the present invention have a tendency structure and different elastic modulus, through further intervention measures, directly overexpress BCL-6 and MiR-126-5p of cells planted on the scaffold, and then regulate key genes PIP2, ALP, The expression of Vav can induce the efficient proliferation and differentiation of stem cells in heterogeneous space.

The present invention can prepare three-dimensional scaffolds with a tendency structure and different elastic modulus through a specific method. The raw material of the scaffold is a typical natural polymer including collagen, chitosan, gelatin, casein, cellulose acetate, silk fibroin, chitin, fibrinogen, etc., and the sacrificial material is polyethylene oxide. It is dissolved in trifluoroethanol according to a certain proportion to prepare a spinning solution, and a film-like polymer composite scaffold with a surface tropism structure is prepared by electrospinning; the scaffold is foamed with sodium borohydride, fluorodichloroethane, etc. Place the agent in the pre-3D printed device, and dissolve the sacrificial agent to obtain a three-dimensional scaffold with surface orientation-space anisotropic structure and different elastic modulus; various adult stem cells including neural stem cells, blood stem cells, bone marrow Mesenchymal stem cells, epidermal stem cells, etc. are planted on the three-dimensional polymer scaffold, and then regulate the expression of key genes PIP2, ALP, and Vav by directly regulating the elastic modulus or further regulating the expression of BCL-6 and MiR-126-5p to induce Efficient proliferation and differentiation of stem cells in heterogeneous space, and planting the scaffold treated with cells on the damaged wound surface to accelerate wound healing. The tissue engineering scaffold of the present invention can achieve rapid recellularization after implantation in the body, restructure new tissues in situ in the body, improve medical performance related to wound healing, and meet the clinical requirements of "on-demand and ready-to-use".

The tissue engineering scaffold for rapidly inducing stem cell proliferation and differentiation prepared by the present invention is found to cause the differentiation of PIP2, ALP and Vav genes through the change of elastic modulus, porosity and tropism of the scaffold, which fundamentally changes the proliferation and differentiation of stem cells , by adjusting physical parameters or indirectly interfering with the expression of BCL-6 and MiR-126-5p to regulate the expression of key genes, thereby inducing the efficient proliferation and differentiation of stem cells in heterogeneous spaces, and planting the scaffolds treated with cells on A damaged wound surface accelerates wound healing.

The tissue engineering scaffold for rapidly inducing stem cell proliferation and differentiation prepared by the present invention is found to cause the differentiation of PIP2, ALP and Vav genes through the change of the elastic modulus and tropism of the scaffold, which fundamentally changes the proliferation and differentiation of stem cells, and can be achieved by Regulate physical parameters such as elastic modulus or further interfere with the expression of BCL-6 and MiR-126-5p to regulate the expression of key genes to induce efficient proliferation and differentiation of stem cells in heterogeneous space. The reason why stem cells differentiate and proliferate slower on random and high elastic modulus fibers than on low elastic modulus and tropism fibers is fundamentally resolved; and a method to change this difference is proposed. Previous studies only found that tropism fibers can promote the differentiation of stem cells, but did not really find that the changes in tropism and elastic modulus caused the changes of PIP2, Vav and ALP genes (the higher the PIP2, the more promoted, the ALP and Vav genes in the early Elevation will promote differentiation in advance), which eventually leads to differences in the growth of stem cells on different scaffolds. At the same time, the present invention proposes the influence of elastic modulus on cell proliferation and differentiation. The present invention utilizes differential preparation of specific three-dimensional scaffolds to control the expression of target genes through direct control of elastic modulus or indirectly through exogenous genes to finally affect the behavior of cells. The specific preparation method of the invention prepares a tissue engineering scaffold with low elastic modulus, high porosity and good tropism to rapidly induce proliferation and differentiation of stem cells.

Beneficial effect: compared with the prior art, the present invention has the following advantages:

(1) The present invention provides a tissue engineering scaffold for rapidly inducing the proliferation and differentiation of stem cells. In order to allow stem cells to proliferate and differentiate better in the three-dimensional scaffold, natural polymers such as silk fibroin and sacrificial agent polyoxidized Vinyl was used as the raw material for the stent. The present invention focuses on the preparation of scaffolds with different elastic moduli by using specific different ratios of sacrificial agents. These scaffolds with different gradients of elastic modulus have excellent potential to induce stem cell proliferation and differentiation. The electron microscope pictures and elastic modulus pictures in the present invention can be prove.

(2) The present invention provides a tissue engineering scaffold for rapidly inducing stem cell proliferation and differentiation. At present, the biological principle that the tropism structure is superior to the anisotropic row structure proliferation and differentiation has not yet been explained clearly, resulting in the separation of cells in the three-dimensional scaffold space. There is a large lag in growth. The present invention finds that the changes in elastic modulus, porosity and tropism lead to the differentiation of PIP2, ALP and Vav genes, which fundamentally changes the proliferation and differentiation of stem cells. The present invention can adjust the physical parameters when preparing scaffolds, especially The elastic modulus further regulates the expression of key genes to induce the efficient proliferation and differentiation of stem cells in heterogeneous space, and plant the scaffold treated with cells on the damaged wound surface to accelerate wound healing.

(3) The present invention provides a tissue engineering scaffold for rapidly inducing the proliferation and differentiation of stem cells, including but not limited to: adult human or mammalian neural stem cells, blood stem cells, bone marrow mesenchymal stem cells, epidermal stem cells, etc. Tissue-engineered skin with specific thickness, porosity, orientation, and customized stem cells can be designed and mass-produced in a laboratory dish to maximize the simulation of the real wound environment.

(4) In the present invention, by regulating the expression of BCL-6 and MiR-126-5p of the cells seeded on the scaffold and then regulating the expression of key genes PIP2, ALP, and Vav, the efficient proliferation and differentiation of stem cells in the heterogeneous space are induced.

Description of drawings

Fig. 1 is the mold of the specific thickness that the embodiment of the present invention provides;

Fig. 2 is the SEM topography diagram of the two-dimensional support provided by the embodiment of the present invention;

Fig. 3 is the SEM topography diagram of the three-dimensional scaffold provided by the embodiment of the present invention after seeding cells;

Fig. 4 is the influence of three-dimensional scaffold tropism and elastic modulus provided by the embodiment of the present invention on stem cell differentiation; A is the influence of tropism and random fibers on stem cell osteogenic differentiation indicators; B is caused by dissolving sacrificial agents with different contents The influence of elastic modulus changes on the osteogenic differentiation indicators of stem cells;

Figure 5 is the PCR data of the key genes related to the three-dimensional scaffold cell function and differentiation genes provided by the embodiment of the present invention;

Fig. 6 is the Western blot expression of key genes in the three-dimensional scaffold seeded cells provided by the embodiment of the present invention;

Fig. 7 is the SEM topography diagram of the three-dimensional scaffold prepared after 30% and 50% sacrificial agent dissolution provided by the embodiment of the present invention;

Figure 8 is the elastic modulus data of the three-dimensional scaffold after 30% and 50% sacrificial agent dissolution provided by the implementation of the present invention;

Fig. 9 is a topographical view of three-dimensional scaffold seeded on cells after 30% and 50% sacrificial agent dissolution provided by the embodiment of the present invention;

Fig. 10 shows VCAM-1 staining and crystal violet staining after seeding cells in the three-dimensional scaffold provided by the embodiment of the present invention and after culturing for regulating PIP2 gene.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

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

The electrospinning instrument in the examples was purchased from Beijing Xinrui Baina Technology Co., Ltd., model: NanoyarnTEADFS-700.

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

The third-generation bone marrow mesenchymal stem cells: rat bone marrow mesenchymal stem cells were purchased from the Cell Bank of the Chinese Academy of Sciences. Bone marrow from 3-6 week old Sprague Dawley (SD) rats was isolated mechanically in a sterile environment, cultured using SD Rat Mesenchymal Stem Cell Complete Medium (Cat. No. SCSP-615) for adherent culture, and passaged Amplify to the third generation (P3 generation) for future use.

The mold used in the embodiment is to ensure that the thickness of the scaffold film is 1.0-5.0mm after adding two-dimensional scale-oriented fiber scaffold and foaming agent solvent for foaming. The mold shown in Figure 1 can be used, made of polylactic acid The preparation can be prepared by 3D printing, surrounded by a membrane with small holes, and the thickness of the inner diameter is 1.0-5.0mm.

DMEM low-glucose complete medium (containing 10% FBS) was purchased from Shannen Biology, Wuhan City, product number: SNM-003E.

Example 1

S1: Choose silk fibroin and polyethylene oxide as the raw material of the scaffold. Mix silk fibroin and trifluoroethanol at a mass volume ratio of 8% mg/mL to prepare solution A, and polyethylene oxide and trifluoroethanol at a mass volume ratio of 40% mg/mL to prepare solution B. For the spinning instrument, set the electrospinning voltage to 13KV, the propulsion speed of the propulsion pump to 0.9mL/h, and the spinning time to 3 hours. The two spinning solutions A and B were mixed according to the volume ratio of 7:3 for electrospinning (total volume 10mL), and a scaffold film with a two-dimensional orientation structure was prepared on a roller with a rotation speed of 1000rpm.

S2: Mix all the stent films prepared in step S1 with a 0.8M foaming agent sodium borohydride solution and place them in a mold with a thickness of 2.5mm for foaming. The foaming process is accompanied by the dissolution of the sacrificial agent. A three-dimensional scaffold film with a tendency structure and a certain elastic modulus.

S3: Cut the three-dimensional scaffold film prepared in step S2 into a circular film with the same diameter as a 24-well plate, place it in a ²⁴ - ^well plate, and divide the third-generation bone marrow mesenchymal Stem cells were planted in 24-well plates containing three-dimensional scaffolds, resuspended in DMEM low-sugar complete medium (containing 10% FBS), and placed in a 37°C, 5% CO ₂ incubator for static culture for 3 and 7 days.

Example 2

S1: Choose silk fibroin and polyethylene oxide as the raw material of the scaffold. Mix silk fibroin and trifluoroethanol at a mass volume ratio of 8% mg/mL to prepare a solution A, and mix polyethylene oxide and a solvent at a mass volume ratio of 40% mg/mL to prepare a solution B, and use electrospinning In the instrument, set the electrospinning voltage to 13KV, the propulsion pump speed to 0.9mL/h, and the spinning time to 3 hours. The two spinning solutions A and B were mixed according to the volume ratio of 5:5 for electrospinning (the total volume was 10 mL), and a scaffold film with a two-dimensional orientation structure was prepared on a roller with a rotation speed of 1000 rpm.

S2: Place all the stent films prepared in step S1 and the foaming agent sodium borohydride solution with a concentration of 0.8M in a mold with a thickness of 2.5mm for foaming. The foaming process is accompanied by the dissolution of the sacrificial agent to obtain a A three-dimensional scaffold film with a tendency to structure and a certain elastic modulus.

S3: Cut the three-dimensional scaffold film prepared in step S2 into a circular film with the same diameter as a 24-well plate, place it in a ²⁴ - ^well plate, and divide the third-generation bone marrow mesenchymal Stem cells were planted in 24-well plates containing three-dimensional scaffolds, resuspended in DMEM low-sugar complete medium (containing 10% FBS), and placed in a 37°C, 5% CO ₂ incubator for static culture for 3 and 7 days.

Example 3

S1: Choose silk fibroin and polyethylene oxide as the raw material of the scaffold. Silk fibroin and trifluoroethanol were prepared into solution A according to the mass volume ratio of 8% mg/mL, and polyethylene oxide and solvent were prepared into solution B according to the mass volume ratio of 40% mg/mL. Electrospinning equipment was used. Set the voltage of electrospinning to 13KV, the propulsion speed of the propulsion pump to 0.9mL/h, and the spinning time to 3 hours. The two spinning solutions A and B were mixed according to the volume ratio of 5:5 for electrospinning (the total volume was 10 mL), and a scaffold film with a two-dimensional orientation structure was prepared on a roller with a rotation speed of 1000 rpm.

S2: Place all the stent films prepared in step S1 and the foaming agent sodium borohydride solution with a concentration of 0.5M in a mold with a thickness of 2.5 mm for foaming. The foaming process is accompanied by the dissolution of the sacrificial agent. The surface tends to be a three-dimensional scaffold film with a spatially heterogeneous structure and a certain elastic modulus.

S3: Cut the three-dimensional scaffold film prepared in step S2 into a circular film with the same diameter as a 24-well plate, place it in a ²⁴ - ^well plate, and divide the third-generation bone marrow mesenchymal Stem cells were planted in 24-well plates containing three-dimensional scaffolds, resuspended in DMEM low-sugar complete medium (containing 10% FBS), and placed in a 37°C, 5% CO ₂ incubator for static culture for 3 and 7 days.

Example 4

S1: Choose silk fibroin and polyethylene oxide as the raw material of the scaffold. Silk fibroin and trifluoroethanol were prepared into solution A according to the mass volume ratio of 8% mg/mL, and polyethylene oxide and solvent were prepared into solution B according to the mass volume ratio of 40% mg/mL. Electrospinning equipment was used. Set the voltage of electrospinning to 13KV, the propulsion speed of the propulsion pump to 0.9mL/h, and the spinning time to 3 hours. The two spinning solutions A and B were mixed according to the volume ratio of 5:5 for electrospinning (the total volume was 10 mL), and a scaffold film with a two-dimensional orientation structure was prepared on a roller with a rotation speed of 1000 rpm.

S2: Place all the stent films prepared in step S1 and the foaming agent sodium borohydride solution with a concentration of 1.0M in a mold with a thickness of 2.5mm for foaming. The foaming process is accompanied by the dissolution of the sacrificial agent. The surface tends to be a three-dimensional scaffold film with a spatially heterogeneous structure and a certain elastic modulus.

S3: Cut the three-dimensional scaffold film prepared in step S2 into a circular film with the same diameter as a 24-well plate, place it in a ²⁴ - ^well plate, and divide the third-generation bone marrow mesenchymal Stem cells were planted in 24-well plates containing three-dimensional scaffolds, resuspended in DMEM low-sugar complete medium (containing 10% FBS), and placed in a 37°C, 5% CO ₂ incubator for static culture for 3 and 7 days.

Example 5

S1: Choose silk fibroin and polyethylene oxide as the raw material of the scaffold. Silk fibroin and trifluoroethanol were prepared into solution A according to the mass volume ratio of 8% mg/mL, and polyethylene oxide and solvent were prepared into solution B according to the mass volume ratio of 40% mg/mL. Electrospinning equipment was used. Set the voltage of electrospinning to 13KV, the propulsion speed of the propulsion pump to 0.9mL/h, and the spinning time to 3 hours. The two spinning solutions A and B were mixed according to the volume ratio of 5:5 for electrospinning (the total volume was 10mL), and a two-dimensional orientation structure scaffold film was prepared on a roller with a rotation speed of 1000rpm.

S2: Place all the stent films prepared in step S1 and the foaming agent sodium borohydride solution with a concentration of 0.8M in a mold with a thickness of 1.0mm for foaming. The foaming process is accompanied by the dissolution of the sacrificial agent to obtain a The surface tends to be a three-dimensional scaffold film with a spatially heterogeneous structure and a certain elastic modulus.

S3: Cut the three-dimensional scaffold film prepared in step S2 into a circular film with the same diameter as the 24-well plate, place it in a 24-well plate, plant the third-generation bone marrow mesenchymal stem cells in the 24-well plate containing the three-dimensional scaffold, Resuspended in DMEM low-glucose complete medium (containing 10% FBS), placed in a 37°C, 5% CO ₂ incubator for static culture for 3 and 7 days.

Example 6

S1: Choose silk fibroin and polyethylene oxide as the raw material of the scaffold. Silk fibroin and trifluoroethanol were prepared into solution A according to the mass volume ratio of 8% mg/mL, and polyethylene oxide and solvent were prepared into solution B according to the mass volume ratio of 40% mg/mL. Electrospinning equipment was used. Set the voltage of electrospinning to 13KV, the propulsion speed of the propulsion pump to 0.9mL/h, and the spinning time to 3 hours. The two spinning solutions A and B were mixed according to the volume ratio of 5:5 for electrospinning (the total volume was 10mL), and a two-dimensional orientation structure scaffold film was prepared on a roller with a rotation speed of 1000rpm.

S2: Place all the stent films prepared in step S1 and the foaming agent sodium borohydride solution with a concentration of 0.8M in a mold with a thickness of 5.0mm for foaming. The foaming process is accompanied by the dissolution of the sacrificial agent to obtain a The surface tends to be a three-dimensional scaffold film with a spatially heterogeneous structure and a certain elastic modulus.

S3: Cut the three-dimensional scaffold film prepared in step S2 into a circular film with the same diameter as a 24-well plate, place it in a ²⁴ - ^well plate, and divide the third-generation bone marrow mesenchymal Stem cells were planted in 24-well plates containing three-dimensional scaffolds, resuspended in DMEM low-sugar complete medium (containing 10% FBS), and placed in a 37°C, 5% CO ₂ incubator for static culture for 3 and 7 days.

Example 7

S1: Choose silk fibroin and polyethylene oxide as the raw material of the scaffold. Mix silk fibroin and trifluoroethanol according to the mass volume ratio of 8% mg/mL to prepare solution A, use an electrospinning instrument, set the electrospinning voltage to 13KV, and the propulsion speed of the propulsion pump to 0.9mL/h, The spinning time was 3 hours, and the spinning solution A was spun with a total volume of 10 mL, and a two-dimensional random structure scaffold film was prepared on food-grade tin foil.

S2: Place all the stent films prepared in step S1 and the foaming agent sodium borohydride solution with a concentration of 0.8M in a mold with a thickness of 2.5mm for foaming. The foaming process is accompanied by the dissolution of the sacrificial agent to obtain a three-dimensional Stochastic scaffold film.

S3: Cut the three-dimensional scaffold film prepared in step S2 into a circular film with the same diameter as a 24-well plate, place it in a ²⁴ - ^well plate, and divide the third-generation bone marrow mesenchymal Stem cells were seeded in 24-well plates containing three-dimensional scaffolds, resuspended in 10% FBS low-sugar DMEM medium, and placed in a 37°C, 5% CO ₂ incubator for static culture for 3 and 7 days.

Example 8

S1: Choose silk fibroin and polyethylene oxide as the raw material of the scaffold. Mix silk fibroin and trifluoroethanol according to the mass volume ratio of 8% mg/mL to prepare solution A, use an electrospinning instrument, set the electrospinning voltage to 13KV, and the propulsion speed of the propulsion pump to 0.9mL/h, The spinning time was 3 hours, wherein the spinning solution A was spun with a total volume of 10 mL, and a scaffold film with a two-dimensional orientation structure was prepared on a roller with a rotation speed of 1000 rpm.

S2: Place all the stent films prepared in step S1 and the foaming agent sodium borohydride solution with a concentration of 0.8M in a mold with a thickness of 2.5mm for foaming. The foaming process is accompanied by the dissolution of the sacrificial agent to obtain a Three-dimensional scaffold film towards structure.

S3: Cut the three-dimensional scaffold film prepared in step S2 into a circular film with the same diameter as a 24-well plate, place it in a ²⁴ - ^well plate, and divide the third-generation bone marrow mesenchymal Stem cells were planted in 24-well plates containing three-dimensional scaffolds, resuspended in DMEM low-sugar complete medium (containing 10% FBS), and placed in a 37°C, 5% CO ₂ incubator for static culture for 3 and 7 days.

Example 9

S1: Choose silk fibroin and polyethylene oxide as the raw material of the scaffold. Silk fibroin and trifluoroethanol were prepared into solution A according to the mass volume ratio of 8% mg/mL, and polyethylene oxide and solvent were prepared into solution B according to the mass volume ratio of 40% mg/mL. Electrospinning equipment was used. Set the electrospinning voltage to 13KV. The propulsion speed of the propulsion pump was 0.9 mL/h, and the spinning time was 3 hours. The two spinning solutions A and B were mixed according to the volume ratio of 7:3 and 5:5 respectively for electrospinning (the total volume was 10 mL), and the scaffold film with two-dimensional orientation structure was prepared on a roller with a rotation speed of 1000 rpm.

S2: Place all the stent films prepared in step S1 and the foaming agent sodium borohydride solution with a concentration of 0.8M in a mold with a thickness of 2.5mm for foaming. The foaming process is accompanied by the dissolution of the sacrificial agent to obtain a 3D Scaffold Films with Tendency to Structure and Different Elastic Modulus.

S3: Cut the three-dimensional scaffold film prepared in step S2 into a circular film with the same diameter as a 24-well plate, place it in a ²⁴ - ^well plate, and divide the third-generation bone marrow mesenchymal Stem cells were planted in a 24-well plate containing a three-dimensional scaffold, wherein the BCL-6 gene of the stem cells on the fiber scaffold with a volume ratio of 7:3 was overexpressed, and the BCL-6 gene of the stem cells on the fiber scaffold with a volume ratio of 5:5 was treated. For gene suppression treatment, resuspend in DMEM low-sugar complete medium (containing 10% FBS), and place in a 37°C, 5% CO ₂ incubator for static culture for 3 and 7 days.

The overexpression or inhibition of the BCL-6 gene is as follows, refer to the literature (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); for the si-BCL-6 plasmid and BCL-6 overexpression plasmid, please refer to the literature: 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.

1. Plating: the cell density in a 6-well plate is 1×10 ⁶ .

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

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

3. Wash the 6-well plate 2-3 times with serum-free medium.

4. Add the above-mentioned transfection reagent (500 μL) into the 6-well plate containing stem cells, shake gently for 1-2 minutes, and add 2 mL of DMEM low-sugar complete medium. After culturing in an incubator at 37°C for 4 hours, replace it with the original complete medium and continue culturing for 48 hours, and detect the transfection efficiency after 24 or 48 hours to obtain BCL-6 gene overexpression treatment or BCL-6 gene inhibition Treated stem cells.

Example 10

S1: Choose silk fibroin and polyethylene oxide as the raw material of the scaffold. Silk fibroin and trifluoroethanol were prepared into solution A according to the mass volume ratio of 8% mg/mL, and polyethylene oxide and solvent were prepared into solution B according to the mass volume ratio of 40% mg/mL. Electrospinning equipment was used. Set the electrospinning voltage to 13KV. The propulsion speed of the propulsion pump was 0.9 mL/h, and the spinning time was 3 hours. The two spinning solutions A and B were mixed according to the volume ratio of 5:5 for electrospinning (the total volume is 10mL), and the two-dimensional tendency structure and the random structure were prepared on the roller with a rotation speed of 1000rpm and the food-grade tin foil respectively. film.

S2: Place all the stent films prepared in step S1 and the foaming agent sodium borohydride solution with a concentration of 0.8M in a mold with a thickness of 2.5mm for foaming. The foaming process is accompanied by the dissolution of the sacrificial agent to obtain a A three-dimensional stent film with a certain elastic modulus and a three-dimensional anisotropic structure film.

S3: Cut the three-dimensional scaffold film prepared in step S2 into a circular film with the same diameter as a 24-well plate, place it in a ²⁴ - ^well plate, and divide the third-generation bone marrow mesenchymal Stem cells were planted in a 24-well plate containing a three-dimensional scaffold, in which the BCL-6 gene of the stem cells on the fibers on the tin foil was overexpressed, and the BCL-6 gene of the stem cells on the fibers with a volume ratio of 5:5 was suppressed. After treatment, they were resuspended in DMEM low-sugar complete medium (containing 10% FBS), and placed in a 37°C, 5% CO ₂ incubator for static culture for 3 and 7 days.

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

Example 11

The method of Example 11 is the same as that of Example 9, except that the miR-126a-5p gene is overexpressed or inhibited for stem cells.

Among them, si-miR-126a-5p plasmid, miR-126a-5p overexpression plasmid, see literature: Down-regulation of microRNA-126-5p contributes to overexpression of VEGFA inlipopolysaccharide-induced acute lung injury, (2016), 38: 1277–1284.

Example 12

The method of Example 12 is the same as that of Example 10, except that the miR-126a-5p gene is overexpressed or inhibited for stem cells.

Example 13

The method of Example 13 is the same as that of Example 9, except that the PIP2 gene overexpression treatment or inhibition treatment is performed on the stem cells.

Among them, si-PIP2 plasmid, PIP2 overexpression plasmid, see literature: The Arabidopsis AbioticStress-Induced TSPO-Related Protein Reduces Cell-Surface Expression of the Aquaporin PIP2; 7through Protein-Protein Interactions and AutophagicDegradation, (2014), 26:4974-4990 .

Example 14

The method of Example 14 is the same as that of Example 10, except that the PIP2 gene overexpression treatment or inhibition treatment is performed on the stem cells.

Test example 1

1. The SEM topography of the scaffold prepared in the S1 steps of Examples 1, 2, 7 and 8 is shown in Figure 2, and a' (upper left figure) in Figure 2 is the random two-dimensional fiber scaffold prepared in Example 7 SEM, b' (upper right figure) is the SEM of the tropic two-dimensional fiber scaffold of Example 8, wherein the average fiber diameter of the tropic scaffold prepared in Example 7 is 0.45 μm, the overall fiber arrangement is uniform, the direction is consistent, and the porosity between fibers 41.2%; Example 8 prepared a random (or anisotropic) scaffold with an average diameter of 0.41 μm, an intricate arrangement of the overall fibers, and a porosity of 46.8%. Further, a" (lower left figure) is the two-dimensional scaffold film with oriented structure containing 50% sacrificial agent prepared in Example 2, and b" (lower right figure) is the oriented stent film containing 30% sacrificial agent prepared in Example 1. Structural 2D Scaffold Films. The average fiber diameter of the tropic scaffold containing 50% sacrificial agent prepared in Example 2 is 0.53 μm, the overall fiber arrangement is uniform, the direction is consistent, and the porosity between fibers is 39.7%; the tropic scaffold containing 30% sacrificial agent prepared in Example 1 The average fiber diameter is 0.46μm, the overall fiber arrangement is intricate, and the porosity is 44.3%. Figure 1 mainly reflects the different morphology of scaffolds, indicating that the morphology may affect cell behavior.

Two, the morphology of the cells planted on the scaffold in Example 7 and Example 8 is shown in Figure 3, wherein the left figure is the morphology of the 7th day after the inoculated cells of the three-dimensional random scaffold prepared in Example 7, and the right figure The appearance of the three-dimensional tropism scaffold prepared for Example 8 on the 7th day after seeding cells. From Figure 3, it can be observed that the surface cells of the tropism fiber scaffolds are spindle-shaped and directional, but on the left are random (or anisotropic) surface cells of the fiber scaffolds that are partially circular, and most of the cells are more dispersed high. It shows that the shape of the scaffold has an impact on the growth mode of the cells.

Further, Figure 4 shows the effect of tropism and elastic modulus on stem cell differentiation. Wherein A is the influence of tropism (Example 8) and randomness (Example 7) three-dimensional fiber scaffold on the stem cell osteogenic differentiation index; B is the sacrificial agent (by the method of Example 2 and 1, AB Solution volume ratio (5:5 and 7:3) caused by the change of elastic modulus of the three-dimensional scaffold on the osteogenic differentiation index of stem cells; according to the qPCR data of the differential genes of the cells cultured on the trend/random structure scaffold, it can be seen that, Osteogenic differentiation indicators OCN, RUNX2 and ALP all showed certain differentiation characteristics, but the differentiation indicators of tropism fiber scaffolds were significantly higher than those of random fiber scaffolds. It is obvious that random (or anisotropic) fibers are not conducive to the proliferation and development of stem cells. differentiated. At the same time, different contents of the sacrificial agent also had a significant impact on the osteogenic differentiation index, and the differentiation index of the three-dimensional scaffold prepared by the high-dose sacrificial agent was significantly higher than that of the scaffold prepared by the low-dose sacrificial agent.

However, qPCR was performed on the key genes PIP2, ALP, and Vav related to the cell function and differentiation genes of the seeded cells in the three-dimensional scaffolds prepared in Examples 1, 2, 7, and 8 above. As shown in Figure 5, there are large differences in the qPCR results, among which the difference of PIP2 is the most significant. It can be considered that the gene PIP2 is the key gene that causes differences in tropism and elastic modulus for stem cell differentiation.

Further, integrin-mediated PIP5K-PIP2-BCL-6 and osteogenic differentiation pathways were verified by Western blot. A in Fig. 6 is PIP5K and PIP2 gene and three differentiation genes after BCL-6 is knocked out and overexpressed, in the stem cell respectively on the expression on arrangement and random three-dimensional fibrous support (using the support of embodiment 7 and 8, gene regulation For the method refer to Example 9). B is after BCL-6 gene knockout and overexpression, the expression of PIP5K and PIP2 genes and three differentiation genes of stem cells on the tropism three-dimensional fiber support after sacrificial agent dissolves respectively (using the support of embodiment 1 and 2, For the method of gene regulation, refer to Example 9). C is the expression of PIP5K, PIP2 and BCL-6 genes of stem cells and three differentiation genes on the arrangement and random three-dimensional fiber scaffolds after miR-126a-5p knockdown and overexpression (using the scaffolds of Examples 7 and 8, For the method of gene regulation, refer to Example 11). D is after miR-126a-5p knockdown and overexpression, the expression of PIP5K, PIP2 and BCL-6 genes and three differentiation genes respectively on the three-dimensional fibrous support of the tendency after the sacrificial agent dissolves (using examples 1 and 2 scaffold, gene regulation method refer to Example 11).

In Figure 6, the PIP5K and PIP2 genes and the three differentiation genes of stem cells showed a trend of decline and rise respectively after BCL-6 knockout and overexpression. However, the PIP5K, PIP2 and BCL-6 genes and the three differentiation genes of stem cells showed an upward and downward trend after miR-126a-5p knockdown and overexpression, respectively. This step further explained the relationship between BCL-6 and miR-126a-5p and PIP2, that is, BCL-6 promotes the expression of PIP2, while miR-126a-5p inhibits the expression of PIP2.

For example, the cells in Example 7 can regulate the expression of BCL-6 and MiR-126-5p, and the cell differentiation index and the expression of PIP2 on the overall scaffold can be restored to a level comparable to that of Example 2. It shows that the gene affects cell growth. At the same time, it can be shown that the proliferation and differentiation effect of the cells inoculated in Example 2 is better, and the expression of the key gene PIP2 can be promoted by promoting the expression of BCL-6 and inhibiting the expression of MiR-126-5p to further promote the proliferation and differentiation of the cells.

3. The SEM morphology of the three-dimensional scaffold prepared in the S2 step of Example 1 and Example 2 is shown in Figure 7, wherein the left figure is the SEM of the tropic three-dimensional fiber scaffold after the dissolution of 30% sacrificial agent (Example 1) The figure on the right is the SEM of the tropic three-dimensional fiber scaffold after 50% sacrificial agent (embodiment 2) dissolves. The average fiber diameter of the scaffold prepared in Example 1 was 0.46 μm, the overall fibers were evenly arranged and in the same direction, and the porosity between fibers was 45.6%; the average diameter of the scaffold prepared in Example 2 was 0.52 μm, the overall fibers were arranged neatly, and the porosity was 43.1%. It can be seen from Figure 8 that the single fiber of the fiber scaffolds prepared by the above two methods has partial loss, and Figure 8 shows the elastic modulus when the volume ratio is 7:3 (Example 1) and 5:5 (Example 2) They are 27.2MPa and 0.7MPa respectively, indicating that the more the sacrificial agent is, the greater the loss of mechanical properties of the fiber will be during dissolution.

4. The morphology of the cells planted on the three-dimensional scaffold in Example 1 and Example 2 is shown in Figure 9, wherein the left figure is the morphology of the scaffold (30% sacrificial agent) prepared in Example 1 on the 7th day after inoculated cells, The right figure is the appearance of the scaffold (50% sacrificial agent) prepared in Example 2 on the 7th day after seeding cells. An extension of the spin cone topography was observed for both, with Example 2 showing significantly better extension based on the day 7 topography. Furthermore, the mechanical properties of the sacrificial agent decrease seriously, which cannot meet the requirements of the cell scaffold.

At the same time, according to the differentiation index in Figure 5, the qPCR results of the key genes PIP2, ALP, and Vav root of the cells on the fiber scaffold are quite different, and the difference of PIP2 before and after culture is the most obvious; further regulation of PIP2 can promote cell proliferation and differentiation.

Further, as shown in Figure 10, the VCAM-1 staining and crystal violet staining of stem cells after inoculation of cells in Example 1, 2, 7 and 8; and overexpression or knockout of PIP2 in Example 1, 2, 7 and 8 VCAM-1 staining and crystal violet staining of stem cells after gene cells were cultured on fibrous scaffolds. Wherein A is VCAM-1 staining and crystal violet staining of stem cells after normal seeding of stem cells (NC) or overexpression of PIP2 gene (OE) on random scaffolds (Example 7). B is VCAM-1 staining and crystal violet staining of stem cells after normal seeding of stem cells (NC) or knockout of PIP2 gene (SI) on the tropism scaffold (Example 8). C is VCAM-1 staining and crystal violet staining of stem cells after normal seeding of stem cells (NC) or knockout of PIP2 gene (SI) on aligned scaffolds with low elastic modulus (Example 2). D is VCAM-1 staining and crystal violet staining of stem cells after normal seeding of stem cells (NC) or overexpression of PIP2 gene (OE) on aligned scaffolds with higher elastic modulus (Example 1).

From the 7d (NC) cell growth in Figure A and the 7d (NC) cell growth in Figure B, it can be seen that the tropism scaffold prepared in Example 2 of the present invention can obviously promote the proliferation and differentiation of cells compared with the random scaffold; The 7d (OE) cell growth in Figure A and the 7d (SI) cell growth in Figure B can be seen: the proliferation and differentiation of cells can be promoted by overexpressing the PIP2 gene, and the proliferation of cells can be inhibited by knocking out the PIP2 gene (SI) and differentiation.

From the 7d (NC) cell growth in Figure C and the 7d (NC) cell growth in Figure D, it can be seen that the low elastic modulus scaffold prepared in Example 2 of the present invention can obviously promote cell proliferation and Differentiation; and through the 7d (SI) cell growth of Figure C and the 7d (OE) cell growth of Figure D, it can be seen that the proliferation and differentiation of cells can be promoted by overexpressing the PIP2 gene, and the proliferation and differentiation of cells can be promoted by knocking out the PIP2 gene (SI). Inhibits cell proliferation and differentiation.

When the seeded cells in the example were used to regulate the expression of BCL-6 and MiR-126-5p, the PIP2 gene corresponding to the cells on the scaffold with higher elastic modulus was suppressed, and the anisotropic scaffold and elastic Cell growth on scaffolds with higher modulus was accelerated, indicating that the gene has a compensatory effect on cell growth on anisotropic fibers and fibers with loss of elastic modulus. In the same way, it shows that the better the tropism and the more loss of elastic modulus, it is beneficial to the growth and differentiation of stem cells.

5. Measure the parameters and performance of the scaffolds prepared in Examples 2, 3 and 4. The average fiber diameter of the scaffold in Example 4 is 0.41 μm, the overall fibers are evenly arranged and in the same direction, and the porosity between the fibers is 46.8%; the average fiber diameter of the scaffold in Example 3 is The diameter is 0.49 μm, the overall fiber arrangement is uniform, the direction is consistent, and the porosity between fibers is 33.9%; the average fiber diameter of the scaffold in Example 2 is 0.45 μm, the overall fiber arrangement is uniform, the direction is consistent, and the porosity between fibers is 41.2%; it shows that the foaming agent The overall diameter of the elastic fiber becomes thinner and the porosity becomes larger. It is more appropriate to choose 0.8M foaming agent without affecting the overall mechanical properties of the fiber scaffold.

Six, test the scaffolds prepared in Examples 2, 5 and 6, the average fiber diameter of the scaffold in Example 6 is 0.41 μm, the overall fiber arrangement is uniform, the direction is consistent, and the porosity between the fibers is 46.8%; the average fiber diameter of the scaffold in Example 2 is 0.45 μm, The overall fiber arrangement is uniform, the direction is consistent, and the porosity between fibers is 41.2%; the average fiber diameter of the scaffold in Example 5 is 0.51 μm, the overall fiber arrangement is uniform, the direction is consistent, and the porosity between fibers is 39.8%; The foaming agent causes the diameter of the elastic fiber to become thinner overall and the porosity to become larger. It is more appropriate to choose a thickness of 2.5 mm without affecting the overall mechanical properties of the fiber scaffold.

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

(1) From the genetic point of view, the reasons for the large differences in the differentiation and proliferation of stem cells in the tropism-heterotropic scaffold were solved.

(2) From the perspective of genes, the reasons for the large differences in stem cell differentiation and proliferation in the structural scaffolds of elastic modulus changes were discovered and solved.

(3) Indirectly regulate the level of differential genes from a biological point of view to restore heterotropic structural cells to a level comparable to that of structural differentiation and proliferation.

(4) Indirectly regulate the level of differential genes from a biological point of view to restore the cells in the elastic modulus differential structure to a level comparable to normal cell differentiation and proliferation.

(5) Provides the preparation method, foaming agent concentration and thickness range of the scaffold suitable for wound repair.

(6) The three-dimensional structure scaffold of the invention solves the problem that the spatial anisotropy is equivalent to the differentiation and proliferation of surface-oriented cells, so that cells can extend and differentiate better inside the scaffold. The invention maximizes the level of natural skin structure required for wounds at the laboratory stage.

Claims (10)

1. a preparation method of a tissue engineering scaffold for rapidly inducing stem cell proliferation and differentiation, is characterized in that, comprises the following steps: (1) Select natural high molecular polymer and sacrificial agent as the raw material of scaffold; dissolve natural high molecular polymer and sacrificial agent in organic solvent respectively to prepare spinning solution, mix the two spinning solutions and carry out electrospinning Obtain a two-dimensional scale-oriented fiber scaffold; (2) reacting the two-dimensional scale-oriented fiber scaffold obtained in step (1) with a foaming agent solvent, so that the sacrificial agent can be quickly dissolved to prepare a three-dimensional scaffold with a oriented structure and different elastic modulus. 2. the preparation method of the tissue engineering scaffold of rapidly inducing stem cell proliferation and differentiation according to claim 1, is characterized in that, in step (1), organic solvent is trifluoroethanol; The quality of natural macromolecular polymer and trifluoroethanol The volume ratio is 7-10% mg/mL; the mass volume ratio of the sacrificial agent to trifluoroethanol is 30-50% mg/mL; the natural polymer and the sacrificial agent are respectively dissolved in the spinning solution in trifluoroethanol The volume ratio is 7:3-5:5. 3. the preparation method of the tissue engineering scaffold of rapid induction stem cell proliferation and differentiation according to claim 1, is characterized in that, in step (1), natural macromolecular polymer is collagen, chitosan, gelatin, casein, acetic acid Any one or more of cellulose, silk fibroin, chitin, and fibrin; the sacrificial agent is polyethylene oxide; the voltage of electrospinning is 12-15KV; the propulsion speed of the propulsion pump is 0.8-1.0mL /h; the rotating speed of the collecting device roller is 1000-1500rpm. 4. the preparation method of the tissue engineering scaffold of rapidly inducing stem cell proliferation and differentiation according to claim 1, is characterized in that, foaming agent described in step (2) is sodium borohydride or fluorodichloroethane, its The concentration is preferably 0.5-1.0M; in step (2), place the two-dimensional scale-oriented fiber scaffold obtained in step (1) and the foaming agent solvent in a mold to react so that the thickness of the prepared three-dimensional scaffold is 1.0-5.0mm. 5. The application of the tissue engineering scaffold for rapidly inducing stem cell proliferation and differentiation prepared by the preparation method according to claim 1 in inducing the high-efficiency proliferation and differentiation of stem cells in heterogeneous space. 6. The application according to claim 5, characterized in that, the application is planting adult stem cells on a tissue engineering scaffold, and then inducing stem cells by directly regulating the elastic modulus and then regulating the expression of key genes PIP2, ALP, and Vav. Efficient proliferation and differentiation in a heterotropic space. 7. The application according to claim 6, wherein the adult stem cells include adult human or mammalian neural stem cells, blood stem cells, bone marrow mesenchymal stem cells or epidermal stem cells. 8. The application of the tissue engineering scaffold for rapidly inducing stem cell proliferation and differentiation prepared by the preparation method of claim 1 in accelerated wound healing on damaged wound surfaces. 9. A tissue engineering scaffold for rapidly inducing stem cell proliferation and differentiation prepared by the preparation method according to claim 1 regulates key genes PIP2, PIP2, The expression of ALP and Vav induces the high-efficiency proliferation and differentiation of stem cells. 10. The application according to claim 9, characterized in that the expression of key genes PIP2, ALP, and Vav is regulated by constructing a transfection reagent to promote the expression of BCL-6 and inhibit the expression of MiR-126-5p.

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