CN116350863A - Composite vascular stent coating for regulating endothelial cell growth activity and preparation method thereof - Google Patents
Composite vascular stent coating for regulating endothelial cell growth activity and preparation method thereof Download PDFInfo
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- CN116350863A CN116350863A CN202310282011.2A CN202310282011A CN116350863A CN 116350863 A CN116350863 A CN 116350863A CN 202310282011 A CN202310282011 A CN 202310282011A CN 116350863 A CN116350863 A CN 116350863A
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Classifications
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- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
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- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
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- A61L31/16—Biologically active materials, e.g. therapeutic substances
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- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06B—TREATING TEXTILE MATERIALS USING LIQUIDS, GASES OR VAPOURS
- D06B3/00—Passing of textile materials through liquids, gases or vapours to effect treatment, e.g. washing, dyeing, bleaching, sizing, impregnating
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Abstract
The invention provides a composite vascular stent coating for regulating and controlling endothelial cell growth activity and a preparation method thereof; the composite vascular stent coating takes a silk fibroin nanofiber membrane as an inner layer and an outer layer of the composite vascular stent coating, and adopts a braiding technology to braid a seamless terylene tubular fabric as a core layer on the basis of the inner layer of the composite vascular stent coating; the composite vascular stent coating has a microstructure and matrix-like components imitating a natural vascular basement membrane, is beneficial to mechanical occlusion between the outer surface of the coating and the inner side of a diseased vessel, stabilizes blood flow, avoids turbulent flow formation, remarkably improves the adhesion, spreading, growth and proliferation capacities of endothelial cells on the inner surface of the composite vascular stent coating, rapidly forms a new endothelial layer, inhibits thrombus and inflammation, keeps lumen clear for a long time, and has good application value.
Description
Technical Field
The invention belongs to the technical field of biomedical materials and vascular stent coating preparation, and particularly relates to a composite vascular stent coating for regulating and controlling endothelial cell growth activity and a preparation method thereof.
Background
The incidence, prevalence and mortality of cardiovascular and cerebrovascular diseases have a tendency to rise year by year, and to be younger. Atherosclerosis is a major cause of cardiovascular and cerebrovascular diseases, and is due to thickening, stiffening and narrowing of arterial walls caused by internal membrane stimulation such as lipid or sugar aggregation, etc., and eventually worsening into occlusive arterial diseases including coronary heart disease, cerebral infarction and peripheral vascular diseases. At present, a covered stent is increasingly used clinically to achieve the purpose of treating vascular obstruction, but complications such as stent displacement, internal leakage, thrombus, restenosis and the like still easily occur in middle and long periods after operation, so that a high proportion of interventions are not ideal, the postoperative re-intervention is needed, and death is seriously caused, which are clinical problems to be solved urgently.
At present, the commercial tectorial membrane bracket tectorial membrane material is mainly terylene and polytetrafluoroethylene, and the two synthetic materials have excellent mechanical properties, but have the defects of poor compliance, biological inertia, no degradation and the like. Furthermore, the two synthetic materials are difficult to endothelialise after being implanted into the body, and may induce thrombosis secondary, restenosis and complications. The middle-aged and elderly people have high thrombus incidence rate and weak self tissue regeneration capability, so that higher requirements are put on the bioactivity of the inner surface of the synthetic polymer stent coating, namely the rapid endothelialization is expected to be realized while long-term mechanical support is maintained after implantation, and the occurrence of thrombus and inflammation is fundamentally inhibited. Therefore, aiming at the existing clinical application bottleneck and the application demands of middle-aged and elderly people, it is necessary to develop an active stent coating for regulating the growth of endothelial cells so as to solve the problems.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a composite vascular stent coating for regulating and controlling the growth activity of endothelial cells and a preparation method thereof; the composite vascular stent coating takes a silk fibroin nanofiber membrane as an inner layer and an outer layer of the composite vascular stent coating, and adopts a braiding technology to braid a seamless terylene tubular fabric as a core layer on the basis of the inner layer of the composite vascular stent coating; the composite vascular stent coating has a microstructure and matrix-like components imitating a natural vascular basement membrane, is beneficial to mechanical occlusion between the outer surface of the coating and the inner side of a diseased vessel, stabilizes blood flow, avoids turbulent flow formation, remarkably improves the adhesion, spreading, growth and proliferation capacities of endothelial cells on the inner surface of the composite vascular stent coating, rapidly forms a new endothelial layer, inhibits thrombus and inflammation, keeps lumen clear for a long time, and has good application value.
The invention provides a composite vascular stent tectorial membrane for regulating endothelial cell growth activity, wherein a composite vascular stent tectorial membrane silk fibroin nanofiber membrane is an inner layer and an outer layer of the composite vascular stent tectorial membrane, and a seamless polyester tubular fabric is woven on the basis of the inner layer of the composite vascular stent tectorial membrane by adopting a weaving technology to serve as a core layer; the composite vascular stent coating has a microstructure and matrix-like composition that mimics the natural vascular basement membrane.
The invention also provides a preparation method of the composite vascular stent coating for regulating and controlling the growth activity of endothelial cells, which comprises the following steps:
(1) Mixing the silk fibroin aqueous solution with a hydrophilic flexible cross-linking agent to obtain a modified silk fibroin solution for later use;
deoiling and desizing the polyester yarns in a sodium carbonate solution to obtain pretreated polyester yarns, and placing the pretreated polyester yarns in a sodium hydroxide solution for surface activation treatment to obtain alkali-treated polyester yarns for later use;
(2) Using the modified silk fibroin solution as an electrospinning solution, and spinning silk fibroin nanofibers on an auxiliary rod through electrostatic spinning to form an inner layer of the composite vascular stent coating;
braiding polyester tubular fabrics with braiding angles of 30-150 degrees and axial braiding densities of 1-20 yarns/cm on the basis of an inner layer of a composite vascular stent tectorial membrane by adopting a braiding technology;
dipping the terylene tubular fabric in the modified silk fibroin solution for 10-60 seconds, placing the terylene tubular fabric in an environment of 20-50 ℃ and rotating the terylene tubular fabric in the circumferential direction for film forming, wherein the rotating speed is 10-100 rpm, and repeating the dipping step for 2-10 times to form a core layer of the vascular stent coating;
(3) And taking the modified silk fibroin solution as an electrospinning solution, electrospinning a silk nanofiber on the surface of a core layer of the vascular stent coating to form a composite vascular stent coating for regulating the growth activity of endothelial cells, immersing the composite vascular stent coating for regulating the growth activity of endothelial cells in sterile deionized water, and removing unreacted cross-linking agent and silk fibroin molecules.
Preferably, in the step (1), the method for obtaining the aqueous silk fibroin solution comprises the following steps: degumming raw silk of silkworm by using any one of boiling water, sodium carbonate, sodium bicarbonate or biological enzyme to obtain degummed silk fibroin fiber; and completely dissolving degummed silk fibroin fibers in a lithium bromide solution to obtain silk fibroin solution, then pouring the silk fibroin solution into a dialysis bag, dialyzing with deionized water, filtering, and evaporating to obtain a silk fibroin aqueous solution.
Wherein the concentration of the lithium bromide solution is 9.3M; the dialysis bag is a semipermeable membrane, and the molecular weight cut-off is 3-50 kDa; the dialysis time was 3 days; the concentration of the obtained silk fibroin aqueous solution is 10-200 mg/mL.
Preferably, in the step (1), the mass ratio of the silk fibroin aqueous solution to the cross-linking agent is 1.0 (0.3-1.0); the concentration of silk fibroin in the modified silk fibroin solution is 20-180 mg/mL.
Preferably, in step (1), the crosslinking agent is polyethylene glycol diglycidyl ether.
Preferably, in the step (1), the treatment temperature of the deoiling and desizing treatment is 95-100 ℃, the treatment time is 2 hours, the deionized water is used for washing, and the steps are repeated for 2-5 times;
the concentration of the sodium hydroxide solution is 20-50 g/L;
the surface activation temperature is 30-80 ℃, the treatment time is 1-3 hours, and deionized water is used for washing after the activation is finished.
Preferably, the auxiliary rod is preferably a stainless steel rod.
Preferably, in the step (2), the rotation speed of the auxiliary rod is 100-3000 rpm, the diameter of the auxiliary rod is 1-30 mm, the average diameter of the silk fibroin nanofibers is 100-5000 nm, the average porosity of the silk fibroin nanofibers is 35-90%, and the average orientation degree of the silk fibroin nanofibers is 30-90%.
Preferably, in the step (2), the polyester filaments are any one of polyester monofilaments and polyester multifilaments, and the specification is 5-200D.
Preferably, in step (3), the soaking is: the vascular stent is covered and soaked in sterile deionized water at the temperature of 4-37 ℃ for 1-3 days.
Compared with the prior art, the invention has the beneficial effects that:
the silk fibroin adopted by the invention is natural protein derived from animals, consists of 20 amino acids and has the same components as extracellular matrix. The artificial blood vessel constructed by the silk fibroin can support the adhesion, growth and proliferation of vascular cells, can induce endothelialization after being implanted into a body, can keep blood flow smooth, can induce regeneration of vascular tissues in situ, is greatly concerned in the aspect of vascular tissue engineering materials, and can be used for preparing composite vascular stent membranes for regulating and controlling the growth activity of endothelial cells based on good blood compatibility.
The composite vascular stent coating for regulating and controlling the endothelial cell growth activity has excellent endothelialization promoting function and coating stability, and fundamentally solves the problems that clinically applied terylene or polytetrafluoroethylene vascular stents cannot be endothelialized, and thrombus and slippage are easy to generate after operation. The composite vascular stent coating for regulating and controlling the growth activity of endothelial cells can regulate and control the position stability, the blood flow stability and the activity of endothelial cells of the coating by regulating the microscopic topological structures of inner-layer and outer-layer silk fibroin.
The silk fibroin nano topological structure constructed on the inner surface and the outer surface of the composite vascular stent coating for regulating and controlling the growth activity of endothelial cells has a microscopic morphology imitating vascular basement membrane, is beneficial to homing and proliferation of endothelial (progenitor) cells, can quickly form new endothelial tissues while gradually degrading the silk fibroin, inhibits the formation of thrombus and inflammation, effectively maintains blood coagulation balance, and keeps blood vessels clear for a long time. In addition, the micro-topology structure of the nanometer fiber on the outer surface of the composite vascular stent coating for regulating and controlling the growth activity of endothelial cells is also beneficial to mechanical occlusion of the coating and the inner wall of a blood vessel, stabilizes the coating position and avoids the possibility of slippage.
Detailed Description
The present invention will be further described with reference to specific examples, but the scope of the present invention is not limited thereto. In the following embodiments, the auxiliary rod is a stainless steel rod, but the auxiliary rod may be made of any material. In the following examples, various processes and methods, which are not described in detail, are conventional methods well known in the art. The sources of the reagents used, the trade names and the components of the reagents are shown when the reagents appear for the first time, and the reagents which are the same as the sources shown for the first time are not specially indicated; the reagents, materials, etc. involved are commercially available without any particular explanation.
Example 1:
(1) The silk is put into sodium carbonate aqueous solution with the mass percent of 0.1 percent according to the bath ratio of 1:50g/mL, treated for three times at the temperature of 98-100 ℃ for 30 minutes each time, then fully washed by deionized water, and dried for 12 hours in a baking oven with the temperature of 60 ℃ to obtain degummed silk fibroin fiber.
And weighing degummed silk fibroin fibers, and completely dissolving the degummed silk fibroin fibers in a 9.3M lithium bromide solution according to a bath ratio of 1:10g/mL in a water bath environment at 65+/-5 ℃ to obtain silk fibroin dissolving solution. The silk fibroin solution is poured into a dialysis bag (the molecular weight cut-off is 14 kDa), and the silk fibroin solution is dialyzed for 3 days by deionized water to obtain purified silk fibroin aqueous solution. Concentrating the purified silk fibroin aqueous solution, and adjusting to the required concentration.
And mixing the silk fibroin aqueous solution with a hydrophilic flexible cross-linking agent to obtain a modified silk fibroin solution for later use. Wherein the mass ratio of the silk fibroin to the cross-linking agent is 1.0:0.5, and the cross-linking agent is polyethylene glycol diglycidyl ether.
And (3) placing the polyester yarns in a sodium carbonate solution with the mass percent of 1% according to the bath ratio of 1:50g/mL, treating for three times at the temperature of 98-100 ℃ for 2 hours each time, and then fully cleaning with deionized water to obtain the pretreated polyester yarns. And (3) placing the pretreated polyester yarn in a 20g/L sodium hydroxide aqueous solution according to a bath ratio of 1:50g/mL for surface activation treatment, wherein the surface activation treatment temperature is 35+/-5 ℃, the treatment time is 1 hour, and washing with deionized water to obtain the alkali-treated polyester yarn for later use.
(2) The modified silk fibroin solution with the silk fibroin concentration of 60mg/mL is used as an electrospinning liquid, silk fibroin nanofibers are spun on a stainless steel rod (with the diameter of 10 mm) with the rotation speed of 300rpm through electrospinning, and an inner layer of the composite vascular stent coating film for regulating and controlling the growth activity of endothelial cells is formed.
Braiding polyester yarn after alkali treatment into polyester tubular fabric with a braiding angle of 120 degrees and an axial braiding density of 4 yarns/cm on the outer surface of an inner layer of a stent coating by adopting a braiding technology, immersing the polyester tubular fabric in a modified silk fibroin solution with a silk fibroin concentration of 60mg/mL for 10 seconds, taking out, placing in a 35 ℃ environment, rotating in the circumferential direction to form a film, repeating the step for 6 times at a rotating speed of 60rpm, and forming a core layer of the composite vascular stent coating for regulating endothelial cell growth activity.
(3) And (3) electrospinning the element nanofiber on the surface of a core layer of the composite vascular stent coating for regulating and controlling the growth activity of endothelial cells by adopting the same method as the step (2), constructing an outer layer of the composite vascular stent coating for regulating and controlling the growth activity of endothelial cells, further soaking the outer layer in sterile deionized water at 37 ℃ for 1 day, and removing unreacted cross-linking agent and silk element molecules to obtain the composite vascular stent coating capable of regulating and controlling the growth activity of endothelial cells, namely the dacron braided fabric/silk element protein composite vascular stent coating.
According to detection, the dacron braided fabric/silk fibroin composite vascular stent coating film of the embodiment has excellent mechanical property and endothelial cell promoting activity, the average diameter of silk fibroin nanofibers is 300+/-50 nm, the porosity is 75+/-10%, the fiber orientation degree is 30+/-8%, the axial tensile strength is 11.5MPa, the breaking elongation is 115%, the circumferential tensile strength is 5.7MPa, the breaking elongation is 51%, the water bursting strength is 3200mmHg, the compliance is 9.6%/100mmHg, and the overall water leakage is less than 10mL/min.cm under 120mmHg water pressure 2 Hemolysis rate<0.1％。
After endothelial cells were cultured on the surface for 3 days, the average spreading area of endothelial cells on the dacron braid/silk fibroin composite vascular stent coating of this example was increased by 38% as compared with comparative example 1, the vascular endothelial growth factor expression amount was increased by 35% as compared with comparative example 1, and the cell proliferation rate was increased by 46% as compared with comparative example 1.
Example 2:
(1) The silk is put into sodium carbonate aqueous solution with the mass percent of 0.1 percent according to the bath ratio of 1:50g/mL, treated for three times at the temperature of 98-100 ℃ for 30 minutes each time, then fully washed by deionized water, and dried for 12 hours in a baking oven with the temperature of 60 ℃ to obtain degummed silk fibroin fiber.
And weighing degummed silk fibroin fibers, and completely dissolving the degummed silk fibroin fibers in a 9.3M lithium bromide solution according to a bath ratio of 1:10g/mL in a water bath environment at 65+/-5 ℃ to obtain silk fibroin dissolving solution. The silk fibroin solution is poured into a dialysis bag (the molecular weight cut-off is 14 kDa), and the silk fibroin solution is dialyzed for 3 days by deionized water to obtain purified silk fibroin aqueous solution. Concentrating the purified silk fibroin aqueous solution, and adjusting to the required concentration.
And mixing the silk fibroin aqueous solution with a hydrophilic flexible cross-linking agent to obtain a modified silk fibroin solution for later use. Wherein the mass ratio of the silk fibroin to the cross-linking agent is 1.0:0.5, and the cross-linking agent is polyethylene glycol diglycidyl ether.
And (3) placing the polyester yarns in a sodium carbonate solution with the mass percent of 1% according to the bath ratio of 1:50g/mL, treating for three times at the temperature of 98-100 ℃ for 2 hours each time, and then fully cleaning with deionized water to obtain the pretreated polyester yarns. And (3) placing the pretreated polyester yarn in a 20g/L sodium hydroxide aqueous solution according to a bath ratio of 1:50g/mL for surface activation treatment, wherein the surface activation treatment temperature is 35+/-5 ℃, the treatment time is 1 hour, and washing with deionized water to obtain the alkali-treated polyester yarn for later use.
(2) The modified silk fibroin solution with the silk fibroin concentration of 120mg/mL is used as an electrospinning liquid, silk fibroin nanofibers are spun on a stainless steel rod (with the diameter of 10 mm) with the rotation speed of 300rpm through electrostatic spinning, and an inner layer of the composite vascular stent coating film for regulating and controlling the growth activity of endothelial cells is formed.
Braiding polyester yarn after alkali treatment into polyester tubular fabric with a braiding angle of 120 degrees and an axial braiding density of 4 yarns/cm on the outer surface of an inner layer of a stent coating by adopting a braiding technology, immersing the polyester tubular fabric in a modified silk fibroin solution with a silk fibroin concentration of 60mg/mL for 10 seconds, taking out, placing in a 35 ℃ environment, rotating in the circumferential direction to form a film, repeating the step for 6 times at a rotating speed of 60rpm, and forming a core layer of the composite vascular stent coating for regulating endothelial cell growth activity.
(3) And (3) electrospinning the element nanofiber on the surface of a core layer of the composite vascular stent coating for regulating and controlling the growth activity of endothelial cells by adopting the same method as the step (2), constructing an outer layer of the composite vascular stent coating for regulating and controlling the growth activity of endothelial cells, further soaking the outer layer in sterile deionized water at 37 ℃ for 1 day, and removing unreacted cross-linking agent and silk element molecules to obtain the composite vascular stent coating capable of regulating and controlling the growth activity of endothelial cells, namely the dacron braided fabric/silk element protein composite vascular stent coating.
According to detection, the dacron braided fabric/silk fibroin composite vascular stent coating film of the embodiment has excellent endothelial cell promoting activity, the average diameter of silk fibroin nanofibers is 600+/-150 nm, the porosity is 60+/-7%, and the fiber orientation degree is 30+/-10%. After endothelial cells were cultured on the surface for 3 days, the average spreading area of endothelial cells on the dacron braid/silk fibroin composite vascular stent coating of this example was increased by 45% as compared with comparative example 1, the vascular endothelial growth factor expression amount was increased by 43% as compared with comparative example 1, and the cell proliferation rate was increased by 55% as compared with comparative example 1.
Example 3:
(1) The silk is put into sodium carbonate aqueous solution with the mass percent of 0.1 percent according to the bath ratio of 1:50g/mL, treated for three times at the temperature of 98-100 ℃ for 30 minutes each time, then fully washed by deionized water, and dried for 12 hours in a baking oven with the temperature of 60 ℃ to obtain degummed silk fibroin fiber.
And weighing degummed silk fibroin fibers, and completely dissolving the degummed silk fibroin fibers in a 9.3M lithium bromide solution according to a bath ratio of 1:10g/mL in a water bath environment at 65+/-5 ℃ to obtain silk fibroin dissolving solution. The silk fibroin solution is poured into a dialysis bag (the molecular weight cut-off is 14 kDa), and the silk fibroin solution is dialyzed for 3 days by deionized water to obtain purified silk fibroin aqueous solution. Concentrating the purified silk fibroin aqueous solution, and adjusting to the required concentration.
And mixing the silk fibroin aqueous solution with a hydrophilic flexible cross-linking agent to obtain a modified silk fibroin solution for later use. Wherein the mass ratio of the silk fibroin to the cross-linking agent is 1.0:0.5, and the cross-linking agent is polyethylene glycol diglycidyl ether.
And (3) placing the polyester yarns in a sodium carbonate solution with the mass percent of 1% according to the bath ratio of 1:50g/mL, treating for three times at the temperature of 98-100 ℃ for 2 hours each time, and then fully cleaning with deionized water to obtain the pretreated polyester yarns. And (3) placing the pretreated polyester yarn in a 20g/L sodium hydroxide aqueous solution according to a bath ratio of 1:50g/mL for surface activation treatment, wherein the surface activation treatment temperature is 35+/-5 ℃, the treatment time is 1 hour, and washing with deionized water to obtain the alkali-treated polyester yarn for later use.
(2) The modified silk fibroin solution with the silk fibroin concentration of 120mg/mL is used as an electrospinning liquid, silk fibroin nanofibers are spun on a stainless steel rod (with the diameter of 10 mm) with the rotation speed of 2000rpm through electrostatic spinning, and an inner layer of the composite vascular stent coating film for regulating and controlling the growth activity of endothelial cells is formed.
Braiding polyester yarn after alkali treatment into polyester tubular fabric with a braiding angle of 120 degrees and an axial braiding density of 4 yarns/cm on the outer surface of an inner layer of a stent coating by adopting a braiding technology, immersing the polyester tubular fabric in a modified silk fibroin solution with a silk fibroin concentration of 60mg/mL for 10 seconds, taking out, placing in a 35 ℃ environment, rotating in the circumferential direction to form a film, repeating the step for 6 times at a rotating speed of 60rpm, and forming a core layer of the composite vascular stent coating for regulating endothelial cell growth activity.
(3) And (3) electrospinning the element nanofiber on the surface of a core layer of the composite vascular stent coating for regulating and controlling the growth activity of endothelial cells by adopting the same method as the step (2), constructing an outer layer of the composite vascular stent coating for regulating and controlling the growth activity of endothelial cells, further soaking the outer layer in sterile deionized water at 37 ℃ for 1 day, and removing unreacted cross-linking agent and silk element molecules to obtain the composite vascular stent coating capable of regulating and controlling the growth activity of endothelial cells, namely the dacron braided fabric/silk element protein composite vascular stent coating.
The detection shows that the dacron braided fabric/silk fibroin composite vascular stent coating film of the embodiment has excellent endothelial cell promoting activity, the average diameter of silk fibroin nanofibers is 580+/-150 nm, the porosity is 55+/-10%, and the fiber orientation degree is 65+/-10%. After endothelial cells were cultured on the surface thereof for 3 days, the average spreading area of endothelial cells on the dacron braid/silk fibroin composite vascular stent coating of this example was increased by 40% as compared with comparative example 1, the vascular endothelial growth factor expression amount was increased by 38% as compared with comparative example 1, and the cell proliferation rate was increased by 45% as compared with comparative example 1.
Comparative example 1:
(1) The silk is put into sodium carbonate aqueous solution with the mass percent of 0.1 percent according to the bath ratio of 1:50g/mL, treated for three times at the temperature of 98-100 ℃ for 30 minutes each time, then fully washed by deionized water, and dried for 12 hours in a baking oven with the temperature of 60 ℃ to obtain degummed silk fibroin fiber.
And weighing degummed silk fibroin fibers, and completely dissolving the degummed silk fibroin fibers in a 9.3M lithium bromide solution according to a bath ratio of 1:10g/mL in a water bath environment at 65+/-5 ℃ to obtain silk fibroin dissolving solution. The silk fibroin solution is poured into a dialysis bag (the molecular weight cut-off is 14 kDa), and the silk fibroin solution is dialyzed for 3 days by deionized water to obtain purified silk fibroin aqueous solution. Concentrating the purified silk fibroin aqueous solution, and adjusting to the required concentration.
And mixing the silk fibroin aqueous solution with a hydrophilic flexible cross-linking agent to obtain a modified silk fibroin solution for later use. Wherein the mass ratio of the silk fibroin to the cross-linking agent is 1.0:0.5, and the cross-linking agent is polyethylene glycol diglycidyl ether.
And (3) placing the polyester yarns in a sodium carbonate solution with the mass percent of 1% according to the bath ratio of 1:50g/mL, treating for three times at the temperature of 98-100 ℃ for 2 hours each time, and then fully cleaning with deionized water to obtain the pretreated polyester yarns. And (3) placing the pretreated polyester yarn in a 20g/L sodium hydroxide aqueous solution according to a bath ratio of 1:50g/mL for surface activation treatment, wherein the surface activation treatment temperature is 35+/-5 ℃, the treatment time is 1 hour, and washing with deionized water to obtain the alkali-treated polyester yarn for later use.
(2) Braiding polyester yarn after alkali treatment into polyester tubular fabric with a braiding angle of 120 degrees and an axial braiding density of 4 yarns/cm on a stainless steel rod by adopting a braiding technology, immersing the polyester tubular fabric in a modified silk fibroin solution with a silk fibroin concentration of 60mg/mL for 10 seconds, taking out, placing in a 35 ℃ environment, rotating in the circumferential direction to form a film, repeating the step for 6 times at a rotating speed of 60rpm, further immersing in sterile deionized water at 37 ℃ for 1 day, and removing unreacted cross-linking agent and silk fibroin molecules to obtain the polyester braided fabric/silk fibroin composite vascular stent coating.
The examples are preferred embodiments of the present invention, but the present invention is not limited to the above-described embodiments, and any obvious modifications, substitutions or variations that can be made by one skilled in the art without departing from the spirit of the present invention are within the scope of the present invention.
Claims (10)
1. The composite vascular stent tectorial membrane for regulating endothelial cell growth activity is characterized in that the composite vascular stent tectorial membrane silk fibroin nanofiber membrane is an inner layer and an outer layer of the composite vascular stent tectorial membrane, and a seamless polyester tubular fabric is woven on the basis of the inner layer of the composite vascular stent tectorial membrane by adopting a weaving technology to serve as a core layer; the composite vascular stent coating has a microstructure and matrix-like composition that mimics the natural vascular basement membrane.
2. The method for preparing a composite vascular stent-graft for regulating endothelial cell growth activity according to claim 1, comprising:
(1) Mixing the silk fibroin aqueous solution with a hydrophilic flexible cross-linking agent to obtain a modified silk fibroin solution for later use;
deoiling and desizing the polyester yarns in a sodium carbonate solution to obtain pretreated polyester yarns, and placing the pretreated polyester yarns in a sodium hydroxide solution for surface activation treatment to obtain alkali-treated polyester yarns for later use;
(2) Using the modified silk fibroin solution as an electrospinning solution, and spinning silk fibroin nanofibers on an auxiliary rod through electrostatic spinning to form an inner layer of the composite vascular stent coating;
braiding polyester tubular fabrics with braiding angles of 30-150 degrees and axial braiding densities of 1-20 yarns/cm on the basis of an inner layer of a composite vascular stent tectorial membrane by adopting a braiding technology;
immersing the terylene tubular fabric in the modified silk fibroin solution in the step (1) for 10-60 seconds, placing the terylene tubular fabric in an environment of 20-50 ℃ and rotating the terylene tubular fabric in the circumferential direction to form a film, wherein the rotating speed is 10-100 rpm, recording the terylene tubular fabric as one operation step, and repeating the operation step for 2-10 times to form a core layer of the vascular stent coating;
(3) And taking the modified silk fibroin solution as an electrospinning solution, electrospinning a silk nanofiber on the surface of a core layer of the vascular stent coating to form a composite vascular stent coating for regulating the growth activity of endothelial cells, immersing the composite vascular stent coating for regulating the growth activity of endothelial cells in sterile deionized water, and removing unreacted cross-linking agent and silk fibroin molecules.
3. The method for preparing a composite vascular stent-graft for regulating endothelial cell growth activity according to claim 2, wherein in the step (1), the method for obtaining the aqueous silk fibroin solution comprises the steps of: degumming raw silk of silkworm by using any one of boiling water, sodium carbonate, sodium bicarbonate or biological enzyme to obtain degummed silk fibroin fiber; and completely dissolving degummed silk fibroin fibers in a lithium bromide solution to obtain silk fibroin solution, then pouring the silk fibroin solution into a dialysis bag, dialyzing with deionized water, filtering, and evaporating to obtain a silk fibroin aqueous solution.
4. The method for preparing a composite vascular stent-graft for regulating endothelial cell growth activity according to claim 2, wherein in the step (1), the mass ratio of the aqueous silk fibroin solution to the crosslinking agent is 1.0 (0.3-1.0); the concentration of silk fibroin in the modified silk fibroin solution is 20-180 mg/mL.
5. The method for preparing a composite vascular stent graft for regulating endothelial cell growth activity according to claim 2, wherein in the step (1), the crosslinking agent is polyethylene glycol diglycidyl ether.
6. The method for preparing a composite vascular stent-graft for regulating endothelial cell growth activity according to claim 2, wherein in the step (1), the treatment temperature of the deoiling and desizing treatment is 95-100 ℃, the treatment time is 2 hours, the washing is performed by deionized water, and the steps are repeated for 2-5 times;
the concentration of the sodium hydroxide solution is 20-50 g/L;
the surface activation temperature is 30-80 ℃, the treatment time is 1-3 hours, and deionized water is used for washing after the activation is finished.
7. The method for preparing a composite vascular stent graft for modulating endothelial cell growth activity according to claim 2, wherein in step (2), the auxiliary rod is a stainless steel rod.
8. The method for preparing a composite vascular stent graft for controlling endothelial cell growth activity according to claim 2, wherein in the step (2), the rotation speed of the auxiliary rod is 100-3000 rpm, the diameter of the auxiliary rod is 1-30 mm, the average diameter of the silk fibroin nanofibers is 100-5000 nm, the average porosity of the silk fibroin nanofibers is 35-90%, and the average orientation degree of the silk fibroin nanofibers is 30-90%.
9. The method for preparing a composite vascular stent graft for regulating endothelial cell growth activity according to claim 2, wherein in the step (2), the polyester filaments are any one of polyester monofilaments and polyester multifilaments, and the specification is 5-200D.
10. The method for preparing a composite vascular stent-graft for modulating endothelial cell growth activity according to claim 2, wherein in step (3), the immersing is: the vascular stent is covered and soaked in sterile deionized water at the temperature of 4-37 ℃ for 1-3 days.
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