WO2022241852A1

WO2022241852A1 - Preparation method for bioactive substance coated polyester mesh layer scaffold

Info

Publication number: WO2022241852A1
Application number: PCT/CN2021/097993
Authority: WO
Inventors: 李阳; 高毅; 张贵锋; 彭青
Original assignee: 南方医科大学珠江医院
Priority date: 2021-05-19
Filing date: 2021-06-02
Publication date: 2022-11-24
Also published as: CN113318270B; CN113318270A

Abstract

A preparation method for a bioactive substance coated polyester mesh layer scaffold, which comprises the following steps: forming a mesh layer from a polyester material by means of spun bonding and reinforcement; washing and drying the mesh layer; utilizing a strong alkaline solution to perform carboxylation treatment on the washed and dried mesh layer; performing activation and crosslinking on the mesh layer; performing repeated coupling on the mesh layer to form a bioactive substance coating on the mesh layer; performing blocking on the coated mesh layer, and obtaining a bioactive substance coated polyester mesh layer scaffold.

Description

Preparation method of bioactive substance-coated polyester mesh sheet scaffold

technical field

The present application relates to the technical field of tissue engineering, in particular to a method for preparing a biologically active substance-coated polyester mesh sheet scaffold.

Background technique

Physiological activities such as cell proliferation, differentiation, and metabolism are seriously affected by the regulation of the microenvironment. A bioreactor is a bioreactor for large-scale production of cells or for large-scale production of cells or for harvesting cell culture products. Among them, anchorage-dependent cells mainly add scaffold materials to the reactor to provide a cell growth surface, and realize high-density cell culture through the high specific surface area of the material.

At present, commonly used scaffold materials in bioreactors mainly include Cytodex microcarriers, Cytopore microcarriers, and fibrous scaffolds. The Cytodex microcarrier as a cell surface attachment matrix is a spherical two-dimensional cell culture material. The cells naturally settle on the surface of the microcarrier under the action of gravity, and the shape changes from spherical to flat. In this culture mode, the cells will undergo dedifferentiation, so that the cultured cells gradually lose many physiological characteristics of their source tissues. Although large-scale production of cells can be achieved, it is difficult to effectively maintain the activity and function of cells. At the same time, in this mode of cell outsourcing material, the liquid shear force of the reactor will damage the viability and function of the cells. Cytopore microcarrier is a kind of porous microcarrier. Cells grow in the pores of the material, which can realize three-dimensional culture and reduce the damage to cells caused by shear force. The reactor culture mode based on Cytopore microcarriers is stirring type. Although this porous structure can reduce the damage of shear force to cells to a certain extent, it cannot fundamentally eliminate the influence of shear force on cells. Fibrous scaffolds such as Fibra Disk carriers have a certain mechanical strength and good mechanical properties, and can realize the three-dimensional culture structure of cells in vitro. However, the biocompatibility of pure polymer materials is still certain compared with natural extracellular matrix components. Defects.

Contents of the invention

The purpose of this application is to provide a biologically active substance-coated polyester mesh sheet that combines the advantages of synthetic polyester polymer materials with certain mechanical properties and plasticity with the good biocompatibility of natural polymer materials The method of preparation of the scaffold,

In order to achieve the above object, the application provides the following technical solutions:

A method for preparing a biologically active substance-coated polyester mesh sheet stent, comprising the following steps:

The polyester material is spunbonded and reinforced into a mesh sheet;

Cleaning and drying the mesh sheet;

Using a strong alkaline solution to carboxylate the washed and dried mesh sheet;

activating and crosslinking the network sheet;

performing multiple couplings to the mesh sheet to form a biologically active material coating on the mesh sheet;

The coated mesh sheet is sealed to obtain the biologically active substance-coated polyester mesh sheet support.

Compared with the prior art, the scheme of the present application has the following advantages:

In the preparation method of the biologically active material-coated polyester mesh sheet stent of the present application, the polyester material is spunbonded and processed into a mesh sheet, and the mesh sheet is carboxylated, Activation of the four steps of cross-linking, coupling and sealing to achieve the coating, by adjusting the parameters to realize the covalent connection between the biomimetic extracellular matrix and the synthetic polymer polyester material, without special equipment such as plasma surface treatment machine, and also does not introduce Toxic chemicals ensure the safety and quality of biological products. And the bioactive substance-coated polyester mesh sheet scaffold made combines the advantages of synthetic polyester polymer materials with certain mechanical properties and plasticity with the good biocompatibility of natural polymer materials, providing a good support for cell culture. The biomimetic culture environment promotes the adhesion, growth and proliferation of cells in the reactor and the maintenance of functional activities. At the same time, the polyester sheet scaffold after coating has higher porosity and specific surface area, which is conducive to mass transfer and can meet the requirements of high-quality cells during production. , high density, and long-term functional maintenance requirements, it can become an ideal loading material for cells in perfusion (packed) bed/stent reactors.

Additional aspects and advantages of the application will be set forth in part in the description which follows, and will become apparent from the description, or may be learned by practice of the application.

Description of drawings

The above and/or additional aspects and advantages of the present application will become apparent and easy to understand from the following description of the embodiments in conjunction with the accompanying drawings, wherein:

Fig. 1 is the process flow chart of the preparation method of the biologically active substance-coated polyester mesh sheet stent of the present application;

Fig. 2 is the XPS analysis result of the ethylene terephthalate mesh sheet scaffold material without biologically active substance coating;

Fig. 3 is the XPS analysis result of the ethylene terephthalate mesh sheet support material after collagen coating;

Fig. 4 is the FTIR spectrogram of the mesh sheet scaffold material before and after the biological active material coating;

Fig. 5 is the measurement structure of the specific surface area of the mesh sheet scaffold material before and after the coating of biologically active substances;

Fig. 6 is the measurement result of the porosity of the reticular sheet scaffold material before and after the biological active material coating;

Fig. 7 is the image observed under the scanning electron microscope of the C3A hepatocytes cultured on the mesh sheet scaffold material after coating on the 7th day;

Fig. 8 is the image observed under the scanning electron microscope of the mesenchymal stem cells cultured on the mesh sheet scaffold material after coating on the 7th day;

Fig. 9 is an ELISA detection image of the difference in the expression of functional activity of C3A hepatocytes after being cultured with the mesh sheet scaffold material before and after coating.

Detailed ways

Embodiments of the present application are described in detail below, examples of which are shown in the drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by referring to the figures are exemplary only for explaining the present application, and are not construed as limiting the present application.

This application relates to a bionic extracellular matrix bioactive substance-coated polyester mesh sheet scaffold, which uses bionic extracellular matrix components to construct a microenvironment that simulates the growth of cells in vivo, has good structural stability and mechanical properties, and is suitable for Large-scale three-dimensional culture of adherent cells in vitro can realize large-scale cell culture and promote cell biological activity and function maintenance.

The biomimetic extracellular matrix biologically active material-coated polyester mesh sheet scaffold (hereinafter referred to as "coated polyester mesh sheet scaffold") includes a mesh sheet and biological tissue covering the surface of the mesh sheet. Active material coating, the mesh sheet is made of polyester material, and the surface of the mesh sheet can be coupled multiple times after carboxylation to form a biologically active substance coating, and at the same time A sealing layer is also provided outside the active material coating.

Specifically, the polyester material forming the mesh sheet includes polycaprolactone, polyester carbonate, ethylene terephthalate, polybutylene terephthalate, propylene terephthalate, polyethylene A combination of one or more of terephthalate-1,4-cyclohexanedimethylester fiber and polyethylene-2,6-naphthalene, and the above-mentioned polyethylene A net-like sheet made of ester material, the thickness of the net-like sheet is 100-500 μm, and the diameter of a single net-like fiber of the net-like sheet is 25-35 μm, and the porosity is between 45% and 85%. , so that the mesh sheet of the present application has good flexibility and plasticity, which is convenient to take and shape, so that the mesh sheet can be designed into a shape of any size, and the material can be enlarged by folding into a certain angle. The stacking area in the reactor is convenient for subsequent coating treatment, and the growth area of cells can be increased in a limited space to achieve high-density culture of cells.

The mesh sheet needs to undergo carboxylation treatment and activation crosslinking treatment before coating. Specifically, the cleaned mesh sheet can be soaked in a strong alkaline solution, so that the ester group of the polyester material is broken to expose carboxyl group, and then soak the carboxylated mesh sheet into MES solution (0.1mol MES+0.5mol sodium chloride, pH 4.7~6.0) for activation, and then add EDC and NHS to the MES in proportion solution to further activate and crosslink, and the final concentration of the EDC is 2 to 5mmol/ml, the ratio of EDC and NHS can be 1:2 to 1:9, through the above steps of activation and crosslinking, can adjust the The carboxyl group of the above-mentioned network sheet is activated to an active state, and at the same time, the strength of the subsequent connection of the carboxyl group is enhanced, so as to realize the combination of bionic extracellular matrix bioactive substances and polyester synthetic polymer materials by activating and cross-linking the network sheet. covalently linked, and then a coating of biologically active substances is formed on the mesh sheet.

The biologically active substance coating is formed by sequentially coupling a collagen solution, a mixed solution containing collagen and hyaluronic acid, and an acellular matrix solution, wherein the pH of the collagen solution is 7.2 to 7.5, and the concentration is 3-12 mg/ml, and the collagen in the collagen solution is one or more than one of type I, type II, and type III collagen, for example, type I collagen and type III collagen are mixed and both The ratio is 3:1 to 1:3; the mixed solution contains the above-mentioned collagen and 10 mg/ml hyaluronic acid with a concentration of 3 to 12 mg/ml, and the pH of the mixed solution is also 7.2 to 7.5; The acellular matrix solution is made from animal-derived liver, kidney, heart or umbilical cord, etc., by trimming animal organs and tissues into small pieces, and stirring them with a solution containing 0.01% to 0.04% trypsin and 0.03% to 0.07% EDTA for 1~ 4h, then soaked and stirred in 2%-5% Triton X-100 for 1-4h, and then soaked in 3%-6% sodium deoxycholate solution for 1-4h, so as to obtain the acellular matrix scaffold, and then the The acellular matrix scaffold is freeze-dried and ground into a powder, digested with pepsin hydrochloride for 48-96 hours, and then can be formulated into a 3-12 mg/ml acellular matrix solution. The net-like sheet is coated three times, and the collagen in the collagen solution, the mixed solution, and the acellular matrix solution and the carboxyl group on the surface of the net-like sheet can be formed on the surface of the net-like sheet. The grid structure containing three different active substances enables the polyester mesh sheet of the application to obtain higher porosity and specific surface area, and can also meet the high quality, high density and long-term functional maintenance of cells during production requirements, the coated polyester mesh sheet scaffold of the present application has good biocompatibility, which is conducive to cell adhesion, promotion of cell differentiation and proliferation, and maintenance of cell function.

In addition, the mesh sheet needs to undergo carboxylation treatment before coating. Specifically, the cleaned mesh sheet can be soaked in MES solution (0.1mol MES+0.5mol sodium chloride, pH 4.7-6.0) Activation in MES solution, and then add EDC and NHS in proportion to the MES solution for further activation and crosslinking, and the final concentration of EDC is 2-5mmol/ml, the ratio of EDC and NHS can be 1:2 ~1:9.

Described network sheet layer can form sealing layer on its surface by adding at least one in 20mmol/ml lysine, arginine or glycine after coating, and described sealing layer utilizes lysine, arginine and Amino acid molecules such as glycine seize the position of the group where the coating can undergo chemical reactions, thereby inactivating the activity of the coating, avoiding the consumption of the coating, and prolonging the life of the coating.

The biomimetic extracellular matrix biologically active material-coated polyester mesh sheet scaffold of the present application simulates the physiological environment of the cell body to construct a three-dimensional culture system by coating the bionic extracellular matrix on the mesh sheet, so that the constructed The physiological environment is close to the actual physiological environment of the cell body. The reticular sheet has been coupled three times, that is, three different active substances are covalently connected to the reticular sheet through carboxyl groups, specifically including type I and type II active substances. , type III collagen, fibronectin and other proteins, glycosaminoglycans such as hyaluronic acid and heparan sulfate, and various cell growth factors, so as to form a coating of biomimetic extracellular matrix bioactive substances outside the mesh sheet , can support and connect tissue structures, regulate the occurrence of tissues and the physiological activities of cells, and can affect all life phenomena such as cell morphology, proliferation, function, survival, differentiation, and migration. By cultivating cells in the grid structure of biomimetic extracellular matrix bioactive substances constructed in this application, using the coating as a growth scaffold for cells, cells can differentiate to produce a certain three-dimensional specific structure, simulating the state of the body to the greatest extent . At the same time, the mesh sheet can provide support for the coating of the biomimetic extracellular matrix bioactive substance constructed externally, so that the biomimetic extracellular matrix bioactive substance coating polyester mesh sheet scaffold of the present application has good Structural stability and mechanical properties, with good mechanical properties, can realize three-dimensional culture of cells in vitro.

In summary, the biomimetic extracellular matrix bioactive material-coated polyester mesh sheet scaffold of the present application combines the advantages of synthetic polyester polymer materials with certain mechanical properties and plasticity with the good biocompatibility of natural polymer materials. Combined, the scaffold of this application not only has a certain mechanical strength and good biocompatibility, but also provides a bionic culture environment for cell culture, promotes the adhesion, growth and proliferation of cells in the reactor, and maintains functional activity. The ester sheet scaffold has higher porosity and specific surface area, which is conducive to mass transfer and can meet the requirements of high-quality, high-density, and long-term functional maintenance of cells during production, and can become an ideal for perfusion (packed) bed/stent reactor cells Load material.

This application also relates to a preparation method of a polyester mesh sheet scaffold coated with bioactive substances, specifically the preparation method of the above-mentioned biomimetic extracellular matrix bioactive substance coated polyester mesh sheet scaffold, please refer to Figure 1, Specifically include the following steps:

(1) The polyester material is spunbonded and reinforced into a mesh sheet.

Preferably, the polyester material in this embodiment includes polycaprolactone, polyester carbonate, ethylene terephthalate, polybutylene terephthalate, propylene terephthalate, polyethylene One or more combinations of terephthalate-1,4-cyclohexanedimethylester fiber and polyethylene-2,6-naphthalene diester, the above-mentioned polyester materials are processed by spunbonding and reinforcement Means made of mesh sheet. The thickness of the mesh sheet made in this embodiment is 100-500 μm, the porosity is between 45% and 85%, and the diameter of a single mesh fiber is 25-35 μm. The mesh sheet of this embodiment has A certain degree of softness and plasticity facilitates easy handling, shaping and stacking in the reactor during subsequent processing. The present application can design the shaped net-like sheet into any size shape, and can also be folded into a certain angle to increase the stacking area of the net-like sheet in the reactor.

(2) Washing and drying the mesh sheet.

Wash the above-mentioned mesh sheet with ethanol, acetone and ultrapure water for 8-15 minutes respectively to remove organic impurities and water-soluble impurities on the surface of the mesh sheet, and then dry overnight at 40°C-80°C.

(3) Using a strong alkaline solution to carry out carboxylation treatment on the washed and dried mesh sheet.

Soak the washed and dried mesh sheet in a strong alkaline solution for 20-50 minutes to break the ester group of the polyester mesh sheet and expose the carboxyl group.

Preferably, the strong alkaline solution solute in this embodiment is at least one of sodium hydroxide, potassium hydroxide, and lithium hydroxide, and the strong alkaline solution contains solute % to 20% in mass volume percentage .

(4) Activating and cross-linking the network sheet.

First, soak the above-mentioned carboxylated network sheet in 2-(N-morpholino)ethanesulfonic acid ("MES" for short) solution for one activation, and the activation time is 30 minutes to 4 hours. Preferably, the MES solution of this embodiment includes 0.1 mol/L of 2-(N-morpholino)ethanesulfonic acid and 0.5 mol/L of sodium chloride, and the pH of the MES is adjusted to 4.6-6.0 .

Subsequently, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (abbreviated as EDC, a water-soluble carbodiimide) and N-hydroxysuccinimide ( "NHS" for short) is added in proportion to the above MES solution, among which, EDC is often used as an activation reagent for carboxyl groups and activated phosphate groups, cross-linking of proteins and nucleic acids, and NHS is often used for biological coupling and cross-linking. EDC and NHS can effectively promote coupling efficiency. Preferably, the final concentration of EDC in the MES is 2-5mmol/ml, and the ratio of EDC to NHS is in the range of 1:2-1:9, so as to perform secondary activation on the mesh sheet and cross-linking, the reaction time is 15 minutes to 3 hours, so as to activate the carboxyl group of the mesh sheet to an optimal state, so as to facilitate coupling with the biomimetic extracellular matrix.

In addition, during the above-mentioned secondary activation and cross-linking process, the activation and cross-linking reaction can be terminated by adding 2-mercaptoethanol into the MES solution, so as to proceed to the next step.

(5) Coupling the mesh sheet multiple times to form a biologically active material coating on the mesh sheet.

In this embodiment, the bioactive substance coating is formed on the mesh sheet by coupling the mesh sheet three times, which is specifically divided into three stages:

First, configure a collagen solution, which contains collagen at a concentration of 3-12 mg/ml, and the collagen is one or more than one of collagen types I, II, and III, for example, I The ratio of type collagen: type III collagen is 3:1-1:1, and the pH value of the collagen solution is adjusted to 7.2-7.5. Put the activated mesh sheet into the collagen solution and perform a coupling with the collagen once, the coupling time is 30min-4h, then the collagen can be covalently linked to the carboxyl group through its amino group to coupled to the mesh sheet.

Subsequently, a secondary coupling treatment is performed on the network sheet. Specifically, a mixed solution containing collagen at a concentration of 3-12 mg/ml and glycosaminoglycans at a concentration of 10-80 mg/ml is prepared. The glycosaminoglycans in this embodiment are preferably hyaluronic acid, and the collagen and the above-mentioned The collagen in the primary coupling stage is the same, and the pH value of the mixed solution is adjusted to 7.2-7.5. Put the above-mentioned reticular sheets after the primary coupling into the mixed solution to perform secondary coupling with the collagen and the glycosaminoglycan, and the coupling time is 30 minutes to 4 hours, then the Collagen and glycosaminoglycans are coupled to the network sheet.

Finally, three coupling treatments are performed on the mesh sheet. Specifically, the decellularized matrix solution required for the third coupling is configured. The decellularized tissue solution is a solution that removes cellular components from tissues or organs by physical, chemical, enzymatic and other methods, and retains the structure and components of the extracellular matrix. The extracellular matrix is a complex secreted by cells of tissues and organs, which is a complex of structural and functional proteins, including type I collagen, type III collagen, fibronectin, laminin, and glycosamine Glycans and various cell growth factors, etc., can form an acellular matrix gel coating on the mesh sheet through three couplings of the decellularized tissue solution.

Preferably, the decellularized tissue solution of the present application is prepared using animal-derived organ tissues as tissue materials, such as liver, kidney, heart or umbilical cord, etc., first cut the animal-derived organ tissues into small pieces, and use 0.01% to 0.04 % trypsin and 0.03% to 0.07% ethylenediaminetetraacetic acid (EDTA) solution (EDTA) were stirred for 1 to 4 hours, and then soaked and stirred in 2% to 5% polyethylene glycol octylphenyl ether solution (TritonX-100) for 1 to 4 hours. 4h, then soak in 3%-6% sodium deoxycholate solution for 1-4h, freeze-dry the small decellularized scaffolds after soaking and grind them into powder, and then digest them with pepsin hydrochloride for 48h-96h , can be configured into 3 ~ 12mg/ml acellular matrix solution. The above-mentioned reticular sheets after secondary coupling are put into the decellularized matrix solution to couple with collagen, and the coupling time is 30min-4h.

Through the coupling of different solutions in the above three stages, coatings of three different materials can be formed on the mesh sheet, each coating has different active substances and has different functions.

(6) Sealing the coated mesh sheet to obtain the biologically active substance-coated polyester mesh sheet scaffold.

The net-like sheet after coating is sealed by adding at least one of 20-50 mmol/ml of lysine, arginine or glycine, so as to protect the coating of the net-like sheet, After the sealing is completed, the biomimetic extracellular matrix bioactive substance-coated polyester mesh sheet scaffold of the present application can be obtained.

Finally, the obtained biomimetic extracellular matrix bioactive material-coated polyester mesh sheet scaffold can be sterilized by electron beam radiation of 10-20 kGy, and then stored at 4°C.

The preparation method of the biologically active substance-coated polyester mesh sheet scaffold of the present application is to spunbond the polyester material, process it into a mesh sheet, and carry out carboxylation and activation crosslinking on the mesh sheet , Coupling and sealing four steps to achieve the coating, by adjusting the parameters to realize the covalent connection between the biomimetic extracellular matrix and the synthetic polymer polyester material, no special equipment such as plasma surface treatment machine is required, and no toxic chemicals are introduced , to ensure the safety and quality of the production of biological products.

In addition, the bionic extracellular matrix biologically active material-coated polyester mesh sheet scaffold for cell culture of the present application was tested and used for animal cell culture experiments. Ethylene glycol diformate network sheet as an example, the results show:

X-ray photoelectron spectroscopy (XPS) analysis and Fourier transform infrared (FTIR) analysis were performed on the ethylene terephthalate network sheet scaffold material coated with biomimetic extracellular matrix bioactive substances, respectively as shown in Figure 1 ~ Figure 5 results. Among them, Fig. 2 is the XPS analysis result of the ethylene terephthalate mesh sheet scaffold material not coated with biologically active substances, and the material is mainly composed of C elements and O elements. Figure 3 is the XPS analysis result of the ethylene terephthalate mesh sheet scaffold material coated with collagen type I collagen: type III collagen at a ratio of 3:1, and N elements appear in the main parts of the material. In the composition, it is suggested that the biologically active substance has been covalently linked to the material. Figure 4 is the FTIR spectrum of the mesh sheet scaffold material before and after coating with biologically active substances. Curve A represents uncoated, and curve B represents after coating. There are obvious differences between the two. Curve B is at 1550cm ^-1 and 1665cm ^- The characteristic absorption peaks of collagen (AmidⅡ, AmidⅠ and AmidA) appeared near ¹ and 3330cm ^-1 , which further suggested that the bioactive substance had been covalently linked to the material. Figure 5 and Figure 6 are the specific surface area and porosity measurement results of the mesh sheet scaffold material before and after coating with biologically active substances, wherein A is uncoated, B represents after coating, and the mesh sheet scaffold can be seen after coating The specific surface area and porosity of the material are significantly improved.

Then fill the materials before and after coating with biologically active substances into the previously constructed circulating perfusion cell culture system and its bioreactor (ZL201510738480.6) for large-scale culture of hepatocytes and mesenchymal stem cells, as shown in Figure 7 and Figure 8 respectively It shows the image observed under the scanning electron microscope on the 7th day of culturing C3A hepatocytes and mesenchymal stem cells on the coated mesh sheet scaffold material. It can be seen that a large number of cells grow and attach tightly to the material, and the connections between cells Compact, some cells showed signs of tissue-like cluster growth. To further evaluate the difference in the expression of functional activity of C3A hepatocytes before and after collagen coating, the culture supernatants were collected on

days

1, 3 and 5, and tested by ELISA. The results are shown in Figure 9 , where A is uncoated, and B represents after coating. The results show that the amount of albumin secreted by the cells in the covered mesh sheet scaffold material is significantly higher than that of the uncoated mesh sheet scaffold material. The mesh sheet scaffold material is beneficial to promote cell differentiation and functional expression.

The above descriptions are only some implementations of the present application. It should be pointed out that for those of ordinary skill in the art, some improvements and modifications can be made without departing from the principle of the application. These improvements and modifications are also It should be regarded as the protection scope of this application.

Claims

A method for preparing a biologically active substance-coated polyester mesh sheet stent, characterized in that it comprises the following steps:

The polyester material is spunbonded and reinforced into a mesh sheet;

Cleaning and drying the mesh sheet;

Using a strong alkaline solution to carboxylate the washed and dried mesh sheet;

activating and crosslinking the network sheet;

performing multiple couplings to the mesh sheet to form a biologically active material coating on the mesh sheet;

The coated mesh sheet is sealed to obtain the biologically active substance-coated polyester mesh sheet support.
The preparation method of bioactive material coating polyester mesh sheet support according to claim 1, is characterized in that, described polyester material comprises polycaprolactone, carbonate polyester, ethylene terephthalate , polybutylene terephthalate, trimethylene terephthalate, poly-1,4-cyclohexanedimethylene terephthalate fiber and polyethylene-2,6-naphthalate One or more compositions, and the diameter of a single mesh fiber of the formed mesh sheet is 25-35 μm.
The preparation method of biologically active substance-coated polyester mesh sheet stent according to claim 1, wherein cleaning and drying the mesh sheet comprises the following steps: using ethanol, acetone and ultrapure Ultrasonic cleaning is performed on the net-shaped sheet layer with water, each cleaning time is 8-15 minutes, and then dried at 40° C.-80° C. overnight.
The preparation method of the biologically active substance-coated polyester mesh sheet stent according to claim 3, characterized in that, using a strong alkaline solution to carry out carboxylation treatment on the cleaned and dried mesh sheet includes the following steps : soak the cleaned and dried net-like sheet in a strong alkaline solution for 20 to 50 minutes to expose the carboxyl group by breaking the ester group of the described net-like sheet, and the solute of the strong alkaline solution is sodium hydroxide, At least one of potassium hydroxide and lithium hydroxide, and the strong alkaline solution contains 5% to 20% of solute in terms of mass volume percentage.
The preparation method of the biologically active substance-coated polyester mesh sheet stent according to claim 1, wherein activating and crosslinking the mesh sheet comprises the following steps:

Immerse the carboxylated network sheet in 2-(N-morpholino)ethanesulfonic acid solution for one activation, and the reaction time is 30min～4h;

Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in proportion to 2-(N-morpholino)ethanesulfonic acid solution Carry out secondary activation and crosslinking, the reaction time is 15min~3h;

Finally, 2-mercaptoethanol was added to terminate the activation and cross-linking reaction described above.
The preparation method of biologically active substance coating polyester mesh sheet support according to claim 5, is characterized in that, described 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt The concentration of acid salt is 2～5mmol/ml, and the mass ratio range of described 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 1:2～1:9.
The preparation method of the biologically active substance-coated polyester mesh sheet stent according to claim 1, wherein the mesh sheet is coupled to form a bioactive substance coating on the mesh sheet , including the following steps:

Collagen solution is configured, and the mesh sheet is put into the collagen solution for one coupling, and the reaction time is 30 minutes to 4 hours;

Configure a mixed solution containing collagen and glycosaminoglycans, put the above-mentioned mesh sheet after the primary coupling into the mixed solution for secondary coupling, and the reaction time is 30 minutes to 4 hours;

The acellular matrix solution is configured, and the above-mentioned reticular sheets after the secondary coupling are dropped into the acellular matrix solution to carry out three couplings, and the reaction time is 30min to 4h.
The preparation method of biologically active substance-coated polyester mesh sheet scaffold according to claim 7, characterized in that the pH value of the collagen solution is 7.2 to 7.5, and it contains collagen at a concentration of 3 to 12 mg/ml Protein, the collagen is one or a mixture of type I, II, and III collagen.
The preparation method of biologically active material-coated polyester mesh sheet scaffold according to claim 7, characterized in that the pH value of the mixed solution is 7.2-7.5, and it contains collagen with a concentration of 3-12 mg/ml Protein and 10mg/ml-80mg/ml hyaluronic acid, the collagen is one or more than one type of collagen I, II, and III.
The preparation method of biologically active material-coated polyester mesh sheet scaffold according to claim 7, characterized in that, the acellular matrix solution is prepared by using animal-derived organ tissue, and first cutting the animal-derived organ tissue into small pieces, stirred with a solution containing 0.01% to 0.04% trypsin and 0.03% to 0.07% ethylenediaminetetraacetic acid for 1 to 4 hours, and then soaked and stirred in a solution of 2% to 5% polyethylene glycol octylphenyl ether 1 to 4 hours, then soaked in 3% to 6% sodium deoxycholate solution for 1 to 4 hours, freeze-dried the small decellularized scaffolds after soaking and ground them into powder, and then digested with pepsin hydrochloride for 48 hours to After 96 hours, it can be prepared into a decellularized matrix solution of 3-12 mg/ml.
The preparation method of the biologically active substance-coated polyester mesh sheet stent according to claim 1, characterized in that, adding 20 to 50 mmol/ml lysine and arginine to the mesh sheet after coating Or at least one of glycine for blocking.
The preparation method of the biologically active substance-coated polyester mesh sheet stent according to claim 1, characterized in that, after obtaining the bioactive substance-coated polyester mesh sheet stent, radiation sterilization is carried out Then store at 4°C.