CN116271260A - Absorbable vascular stent coating with bionic interface and preparation method thereof - Google Patents
Absorbable vascular stent coating with bionic interface and preparation method thereof Download PDFInfo
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Classifications
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- 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
- A61L31/148—Materials at least partially resorbable by the body
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- 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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- D04C1/06—Braid or lace serving particular purposes
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Abstract
The invention discloses an absorbable vascular stent coating with a bionic interface and a preparation method thereof, comprising the following steps: firstly, preparing a silk fibroin aqueous solution and a modified silk fibroin solution, adopting an electrostatic spinning technology to spin nano fibers on the surface of a core rod, then spin-dipping the nano fibers in the modified silk fibroin solution, spin-drying the nano fibers, and repeating dipping operation for a plurality of times to form an inner layer; and finally, rotationally dipping the stent core layer in modified silk fibroin solution, rotationally drying and repeating to form a layer-by-layer closely combined vascular stent coating, soaking and eroding nanofibers by sterile deionized water to form a coating inner surface with a tightly connected flat island-shaped microcosmic appearance, thus obtaining an absorbable vascular stent coating with a bionic interface, and solving the fundamental problem that the synthetic polymer coating material applied clinically cannot be endothelialized and degraded so as to induce thrombosis and restenosis.
Description
Technical Field
The invention belongs to the field of biomedical materials, relates to preparation of a vascular stent coating, and in particular relates to an absorbable vascular stent coating with a bionic interface and a preparation method thereof.
Background
Atherosclerosis is the main cause of cardiovascular and cerebrovascular diseases, and starts from the excitation of the intima such as lipid or sugar accumulation, etc., thus leading to thickening, stiffening and stenosis of the arterial wall, and finally, the malignant 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.
Silk fibroin is a natural protein derived from animals, consists of 20 amino acids and has the same components as extracellular matrix, and the existing literature research shows that 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, keep blood flow smooth, can induce regeneration of vascular tissues in situ, is greatly focused on vascular tissue engineering materials, and is a preferred material for an absorbable coating based on good blood compatibility.
At present, the commercial tectorial membrane stent 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, and the endothelialization is difficult to realize after the stent is implanted into a body, which is the main cause of postoperative complications. And because the material is difficult to degrade, thrombus secondary and immune rejection reactions need to be inhibited by taking medicines for a long time, clinical application tracking investigation shows that the occurrence rate of thrombus secondary and restenosis of the material implanted in a covered stent for 5 years is very high, which is not suitable for middle-aged and young people with higher and higher incidence rate. Therefore, for the bottleneck and increasingly younger occurrence of the existing clinical application, there is a need to develop an absorbable stent graft to solve the above problems.
Disclosure of Invention
The invention provides a vascular stent coating with a bionic interface, which can be gradually absorbed, and a preparation method thereof, wherein the microstructure of the vascular stent coating is regulated while the inner surface of the coating is designed, so that the coating is endowed with good mechanical property and excellent biological activity, and the vascular stent coating is used for producing vascular stents which can rapidly form a new endothelial layer, maintain blood flow stability and inhibit thrombosis secondary and inflammation.
In order to achieve the technical purpose, the invention adopts the following specific steps:
(1) Degumming raw silk of silkworm by using any one of boiling water, sodium carbonate, sodium bicarbonate or biological enzyme to obtain degummed silk fibroin (hereinafter referred to as silk fibroin) fiber; completely dissolving the degummed silk fibroin fibers in a lithium bromide solution to obtain silk fibroin dissolving solution, pouring the silk fibroin dissolving solution into a dialysis bag, filtering the silk fibroin dissolving solution by deionized water to obtain purified silk fibroin aqueous solution, and evaporating and concentrating the purified silk fibroin aqueous solution to adjust the concentration of the silk fibroin aqueous solution to 10-200 mg/mL;
mixing a silk fibroin aqueous solution with a hydrophilic flexible cross-linking agent to obtain a modified silk fibroin solution, wherein the concentration of silk fibroin in the modified silk fibroin solution is 20-160 mg/mL;
(2) Preparation of a stent coating inner layer:
(a) Spinning nano fibers on a cylindrical auxiliary rod by using a water-soluble polymer solution as an electrospinning solution through electrostatic spinning to obtain an auxiliary rod with nano fibers; wherein the rotation speed of the cylindrical auxiliary rod is 100-3000 rpm, the average diameter of the nanofiber is 100-5000 nm, and the average orientation degree of the nanofiber is 30-90%;
(b) Soaking an auxiliary rod with nano fibers in the modified silk fibroin solution prepared in the step (1) in a rotary-soaking mode, taking out the soaked auxiliary rod, and rotating the auxiliary rod at 20-50 ℃ to form a film, thereby completing primary soaking treatment; repeating the dipping treatment for several times to obtain an inner layer of the stent coating;
(3) Preparation of a vascular stent coating core layer:
(a) The method comprises the steps of combining, twisting and degumming raw silk of silkworms to obtain degummed twisted silk; the degummed twisted yarn is adopted 60 Co gamma-ray irradiation treatment of 60 Co gamma-ray irradiationThe dose is 25-300 kGy, and the silk thread after irradiation treatment is obtained;
(b) Braiding a tubular fabric with a braiding angle of 30-150 degrees and an axial braiding density of 1-20 threads/cm on the basis of the stent tectorial membrane inner layer in the step (2) through a braiding technology to form a core layer of the vascular stent tectorial membrane, wherein the silk thread is degummed twisted silk or/and silk thread after irradiation treatment;
(4) Placing the core layer of the vascular stent coating in the step (3) in the modified silk fibroin solution obtained in the step (1) in a rotary-dipping mode for treatment, taking out the treated core layer, placing the treated core layer in an environment of 20-50 ℃ and keeping the rotary speed for continuous rotation for 10-30 minutes, namely finishing one dipping treatment, controlling the surface of the membrane to keep wet, and repeating the dipping treatment for a plurality of times to form an outer layer of the vascular stent coating; finally, soaking the stent graft in sterile deionized water to remove unreacted cross-linking agent and silk fibroin molecules and erode nanofibers, thus obtaining the absorbable stent graft with a bionic interface.
Preferably, the concentration of the lithium bromide solution in step (1) is 9.3M; the dialysis bag is a semipermeable membrane, the molecular weight cut-off is 3-50 kDa, and deionized water is adopted for dialysis for 3 days.
Preferably, in the step (1), the mass ratio of the silk fibroin to the cross-linking agent is 1.0 (0.3-1.0), and the cross-linking agent is polyethylene glycol diglycidyl ether.
Preferably, the cylindrical auxiliary rod in the steps (a) - (b) of the step (2) is a stainless steel rod with the diameter of 1-30 mm.
Preferably, the water-soluble polymer solution in the step (2) is an aqueous silk fibroin solution or an aqueous polyvinyl alcohol solution; the concentration of the aqueous polyvinyl alcohol solution is preferably 50 to 200mg/mL.
Preferably, the time of immersing in the modified silk fibroin solution in the step (b) of the step (2) is 10-60 seconds, the rotation speed is 10-100 rpm, the rotation direction is along the circumferential direction of the auxiliary rod, the immersing treatment is repeated for several times for 0-5 times, and the immersed area accounts for 10-90% of the side area of the stainless steel rod.
Preferably, in the step (3), the titer of the raw silk of the bombyx mori is 20/22D, the number of the combined strands is 2-10 strands, the twisting direction of twisting is S/Z direction, and the twisting twist is 100-2000 twists/m; the degumming process is the same as the operation of the step (1), namely, the raw silk of the silkworm is degummed by any one of boiling water, sodium carbonate, sodium bicarbonate or biological enzyme.
Preferably, the degummed twisted yarn and the irradiated yarn in step (3) are uniformly arranged at a root ratio of 6:0 to 0:6.
Preferably, the time of soaking in the modified silk fibroin solution in the step (4) is 10-60 seconds, the rotating speed is 10-100 rpm, the rotating direction is along the circumferential direction of the auxiliary rod, the repeated soaking treatment is 2-10 times, and the soaking area accounts for 10-90% of the side area of the stainless steel rod; the temperature of the sterile deionized water is 4-37 ℃ and the soaking time is 1-3 days.
The beneficial effects are that:
the absorbable vascular stent coating provided by the invention has ultra-thin and uniform thickness, good mechanical property and biological activity, the inner surface of the absorbable vascular stent coating erodes a tightly connected flat island-shaped nanoscale wire groove, the absorbable vascular stent coating has a high-imitation natural vascular endothelial layer microscopic topological structure, the wire groove size, directivity and order of the absorbable vascular stent coating are adjustable, the homing and proliferation of endothelial (progenitor) cells can be induced, the endothelialization can be induced rapidly, and the high-pressure blood flow can be buffered.
The invention is also innovative in that the degummed silk fibroin fiber or/and the irradiation silk fibroin fiber are hybridized and woven into the vascular stent core layer, after the vascular stent is implanted, the vascular stent coating can cope with different individuals, realize the degradation speed matched with the healing of the vascular lesion part, dynamically keep stable mechanical property, maintain the blood flow steady state, and finally be completely absorbed in situ, reproduce the intimal tissue of the autologous blood vessel, the newly formed complete intimal layer can effectively maintain the balance of the blood coagulation system for a long time, inhibit thrombosis and restenosis, keep long and smooth, has excellent endothelialization promoting function, and solves the fundamental problem that the synthetic polymer coating material applied clinically in the prior art cannot be endothelialized and degraded so as to induce thrombosis and restenosis.
The absorbable vascular stent coating has ultrathin and uniform thickness, and the average thickness of the vascular stent coating is 60+/-5 mu m; the absorbable vascular stent coating with the bionic interface has excellent performance, the endothelialization speed of the inner surface of the absorbable vascular stent coating is obviously accelerated after being implanted in a body for 2 weeks, and the coverage rate is increased by 80 percent compared with that of a vascular stent coating of a control example 7; after 6 months of in vivo implantation, the absorbable vascular stent graft of the present invention had a 121% increase in weight loss compared to control example 7.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
Example 1:
(1) Placing raw silkworms into a 0.1% sodium carbonate aqueous solution according to a bath ratio of 1:50g/mL, treating for three times at 98-100 ℃ for 30 minutes each time, then fully cleaning with deionized water, and drying in a 60 ℃ oven for 12 hours to obtain degummed silk fibroin fibers; 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 a required concentration;
mixing the aqueous silk fibroin solution with a hydrophilic flexible cross-linking agent to obtain a modified silk fibroin solution with the concentration of 60mg/mL; 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.
(2) The 50mg/mL aqueous solution of silk fibroin is used as an electrospinning liquid, and the silk fibroin nanofiber is electrospun on a stainless steel rod (with the diameter of 10 mm) with the rotating speed of 500rpm by electrospinning, wherein the average diameter of the silk fibroin nanofiber is 200nm, and the average orientation degree is 35%.
Then placing the stainless steel rod wound with the silk fibroin nano fibers in a modified silk fibroin solution with the concentration of 60mg/mL for 10 seconds in a rotary-dipping mode, wherein the dipping area accounts for 50% of the side area of the stainless steel rod, the rotary speed is 60rpm, taking out, and then placing in an environment at 35 ℃ to keep the rotary speed for continuous rotation for 15 minutes to form an inner layer of the stent coating; wherein the rotating direction is along the circumferential direction of the stainless steel rod;
(3) The method comprises the steps of combining, twisting and degumming raw silk of silkworms to obtain degummed twisted silk; the titer of raw silk of the silkworms is 20/22D, the number of the combined strands is 2, the twisting direction of twisting is S direction, and the twisting twist is 500 twists/m; the degumming process is operated in the same way as the step 1;
the degummed twisted yarn is adopted 60 Co gamma-ray irradiation treatment of 60 The Co gamma-ray irradiation dose is 100kGy, and the wire after irradiation treatment is obtained; and weaving the tubular fabric with the weaving angle of 120 degrees and the axial weaving density of 4 threads/cm on the basis of serving as the inner layer of the stent coating by a weaving technology to form a core layer of the vascular stent coating, wherein the threads are degummed twisted threads and the threads subjected to irradiation treatment are uniformly arranged in a 1:1 ratio.
(4) Placing the core layer of the vascular stent coating in the step (3) in the modified silk fibroin solution in a rotating-dipping mode for 60 seconds, wherein the dipping area accounts for 50% of the side area of the stainless steel rod, the rotating speed is 60rpm, taking out the vascular stent coating, placing the vascular stent coating in a 35 ℃ environment, keeping the rotating speed for 15 minutes, controlling the surface of the film to keep wet, repeating the step of rotating the composite modified silk fibroin for 6 times, wherein the rotating direction is along the circumferential direction of the stainless steel rod, forming the outer layer of the vascular stent coating, and further obtaining the vascular stent coating; soaking the vascular stent coating in sterile deionized water at 37 ℃ for 1 day, removing unreacted cross-linking agent and silk fibroin molecules and eroding the silk fibroin nanofiber to obtain the absorbable vascular stent coating with a bionic interface.
Through detection, the absorbable vascular stent coating has ultrathin and uniform thickness of 60+/-5 mu m;
the absorbable vascular stent coating with the bionic interface has excellent endothelialization promoting capability, and after being implanted in a body for 2 weeks, the endothelialization speed of the inner surface of the absorbable vascular stent coating is obviously increased, and the coverage rate is increased by 55% compared with that of a vascular stent coating of a comparison example 7; after 6 months of in vivo implantation, the absorbable vascular stent graft of the present invention had a 42% increase in weight loss compared to control example 7.
Example 2:
(1) Placing raw silkworms into a 0.1% sodium carbonate aqueous solution according to a bath ratio of 1:50g/mL, treating for three times at 98-100 ℃ for 30 minutes each time, then fully cleaning with deionized water, and drying in a 60 ℃ oven for 12 hours to obtain degummed silk fibroin fibers; 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 a required concentration;
mixing the aqueous silk fibroin solution with a hydrophilic flexible cross-linking agent to obtain a modified silk fibroin solution with the concentration of 60mg/mL; 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.
(2) The 50mg/mL silk fibroin aqueous solution is used as an electrospinning liquid, and the silk fibroin nanofiber is electrospun on a stainless steel rod (with the diameter of 10 mm) with the rotating speed of 2500rpm by electrospinning, wherein the average diameter of the silk fibroin nanofiber is 200nm, and the average orientation degree is 75%.
And then placing the stainless steel rod wound with the silk fibroin nanofibers into a modified silk fibroin solution with the concentration of 60mg/mL for 10 seconds in a rotary-dipping mode, wherein the dipping area accounts for 50% of the side area of the stainless steel rod, the rotary speed is 60rpm, and the stainless steel rod is taken out and placed in an environment of 35 ℃ to keep the rotary speed for continuous rotation for 15 minutes, so that an inner layer of the stent coating is formed. The rotating direction is along the circumferential direction of the stainless steel rod;
(3) The method comprises the steps of combining, twisting and degumming raw silk of silkworms to obtain degummed twisted silk; the titer of raw silk of the silkworms is 20/22D, the number of the combined strands is 2, the twisting direction of twisting is S direction, the twisting twist is 500 twists/m, and the degumming process is the same as that of the step 1;
the degummed twisted yarn is adopted 60 Co gamma-ray irradiation treatment of 60 The Co gamma-ray irradiation dose is 100kGy, and the wire after irradiation treatment is obtained; and weaving the tubular fabric with the weaving angle of 120 degrees and the axial weaving density of 4 threads/cm on the basis of serving as the inner layer of the stent coating by a weaving technology to form a core layer of the vascular stent coating, wherein the threads are degummed twisted threads and the threads subjected to irradiation treatment are uniformly arranged in a 1:1 ratio.
(4) Placing the core layer of the vascular stent coating in the step (3) in the modified silk fibroin solution in a rotating-dipping mode for 60 seconds, wherein the dipping area accounts for 50% of the side area of the stainless steel rod, the rotating speed is 60rpm, taking out the vascular stent coating, placing the vascular stent coating in a 35 ℃ environment, keeping the rotating speed for 15 minutes, controlling the surface of the film to keep wet, repeating the step of rotating the composite modified silk fibroin for 6 times, wherein the rotating direction is along the circumferential direction of the stainless steel rod, forming the outer layer of the vascular stent coating, and further obtaining the vascular stent coating; soaking the vascular stent coating in sterile deionized water at 37 ℃ for 1 day, removing unreacted cross-linking agent and silk fibroin molecules and eroding the silk fibroin nanofiber to obtain the absorbable vascular stent coating with a bionic interface.
Through detection, the absorbable vascular stent coating has ultrathin and uniform thickness of 60+/-5 mu m;
the absorbable vascular stent coating with the bionic interface has excellent endothelialization promoting capability, and after being implanted in a body for 2 weeks, the endothelialization speed of the inner surface of the absorbable vascular stent coating is obviously increased, and the coverage rate is increased by 63% compared with that of a control example 7.
Example 3:
(1) The silkworm raw silk is placed in 0.1 percent sodium carbonate aqueous solution 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 60 ℃ oven, thus obtaining degummed silk 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 a required concentration;
mixing the aqueous silk fibroin solution with a hydrophilic flexible cross-linking agent to obtain a modified silk fibroin solution with the concentration of 60mg/mL; 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.
(2) 150mg/mL of silk fibroin aqueous solution is used as an electrospinning liquid, and the silk fibroin nanofiber is electrospun on a stainless steel rod (with the diameter of 10 mm) with the rotating speed of 2500rpm by electrospinning, wherein the average diameter of the silk fibroin nanofiber is 750nm, and the average orientation degree is 75%.
Then placing the stainless steel rod wound with the silk fibroin nano fibers in a modified silk fibroin solution with the concentration of 60mg/mL for 10 seconds in a rotary-dipping mode, wherein the dipping area accounts for 50% of the side area of the stainless steel rod, the rotary speed is 60rpm, taking out, and then placing in an environment at 35 ℃ to keep the rotary speed for continuous rotation for 15 minutes to form an inner layer of the stent coating; the rotating direction is along the circumferential direction of the stainless steel rod;
(3) The method comprises the steps of combining, twisting and degumming raw silk of silkworms to obtain degummed twisted silk; the titer of raw silk of the silkworms is 20/22D, the number of the combined strands is 2, the twisting direction of twisting is S direction, the twisting twist is 500 twists/m, and the degumming process is the same as that of the step 1;
the degummed twisted yarn is adopted 60 Co gamma-ray irradiation treatment of 60 The Co gamma-ray irradiation dose is 100kGy, and the wire after irradiation treatment is obtained; passing the yarn through a braiding techniqueAnd weaving tubular fabrics with a weaving angle of 120 degrees and an axial weaving density of 4 threads/cm on the basis of the tubular fabrics serving as the inner layer of the stent coating to form a core layer of the vascular stent coating, wherein the threads are degummed twisted threads and the threads subjected to irradiation treatment are uniformly arranged in a 1:1 ratio.
(4) Placing the core layer of the vascular stent coating in the step (3) in the modified silk fibroin solution in a rotating-dipping mode for 60 seconds, wherein the dipping area accounts for 50% of the side area of the stainless steel rod, the rotating speed is 60rpm, taking out the vascular stent coating, placing the vascular stent coating in a 35 ℃ environment, keeping the rotating speed for continuously rotating for 15 minutes, controlling the surface of the film to keep wet, repeating the step of rotating the composite modified silk fibroin for 6 times, and forming the outer layer of the vascular stent coating along the circumferential direction of the stainless steel rod in the rotating direction, thereby obtaining the vascular stent coating; soaking the vascular stent coating in sterile deionized water at 37 ℃ for 1 day, removing unreacted cross-linking agent and silk fibroin molecules and eroding the silk fibroin nanofiber to obtain the absorbable vascular stent coating with a bionic interface.
Through detection, the absorbable vascular stent coating has ultrathin and uniform thickness of 60+/-5 mu m;
the absorbable vascular stent coating with the bionic interface has excellent endothelialization promoting capability, and after being implanted in a body for 2 weeks, the endothelialization speed of the inner surface of the absorbable vascular stent coating is obviously increased, and the coverage rate is increased by 80 percent compared with that of a control example 7.
Example 4:
(1) The silkworm raw silk is placed in 0.1 percent sodium carbonate aqueous solution 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 60 ℃ oven, thus obtaining degummed silk 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. Pouring the silk fibroin solution into a dialysis bag (with the molecular weight cut-off of 14 kDa), and dialyzing with deionized water for 3 days to obtain purified silk fibroin aqueous solution; concentrating the purified silk fibroin aqueous solution, and adjusting to a required concentration;
mixing the aqueous silk fibroin solution with a hydrophilic flexible cross-linking agent to obtain a modified silk fibroin solution with the concentration of 60mg/mL; 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.
(2) 150mg/mL polyvinyl alcohol aqueous solution is used as an electrospinning liquid, and the polyvinyl alcohol nanofiber is electrospun on a stainless steel rod (with the diameter of 10 mm) with the rotating speed of 2500rpm by electrospinning, wherein the average diameter of the polyvinyl alcohol nanofiber is 550nm, and the average orientation degree is 75%.
Then placing the stainless steel bar wound with the polyvinyl alcohol nanofiber in a modified silk fibroin solution with the concentration of 60mg/mL for 10 seconds in a rotary-dipping mode, wherein the dipping area accounts for 50% of the side area of the stainless steel bar, the rotary speed is 60rpm, taking out, and then placing in an environment at 35 ℃ to keep the rotary speed for continuous rotation for 15 minutes to form an inner layer of the stent coating; the rotating direction is along the circumferential direction of the stainless steel rod;
(3) The method comprises the steps of combining, twisting and degumming raw silk of silkworms to obtain degummed twisted silk; the titer of raw silk of the silkworms is 20/22D, the number of the combined strands is 2, the twisting direction of twisting is S direction, the twisting twist is 500 twists/m, and the degumming process is the same as that of the step 1;
the degummed twisted yarn is adopted 60 Co gamma-ray irradiation treatment of 60 The Co gamma-ray irradiation dose is 100kGy, and the wire after irradiation treatment is obtained. And weaving the tubular fabric with the weaving angle of 120 degrees and the axial weaving density of 4 threads/cm on the basis of serving as the inner layer of the stent coating by a weaving technology to form a core layer of the vascular stent coating, wherein the threads are degummed twisted threads and the threads subjected to irradiation treatment are uniformly arranged in a 1:1 ratio.
(4) Placing the core layer of the vascular stent coating in the step (3) in the modified silk fibroin solution in a rotating-dipping mode for 60 seconds, wherein the dipping area accounts for 50% of the side area of the stainless steel rod, the rotating speed is 60rpm, taking out the vascular stent coating, placing the vascular stent coating in a 35 ℃ environment, keeping the rotating speed for 15 minutes, controlling the surface of the film to keep wet, repeating the step of rotating the composite modified silk fibroin for 6 times, wherein the rotating direction is along the circumferential direction of the stainless steel rod, forming the outer layer of the vascular stent coating, and further obtaining the vascular stent coating; soaking the vascular stent coating in sterile deionized water at 37 ℃ for 1 day, removing unreacted cross-linking agent and silk fibroin molecules, and eroding the polyvinyl alcohol nanofiber to obtain the absorbable vascular stent coating with a bionic interface.
Through detection, the absorbable vascular stent coating has ultrathin and uniform thickness of 60+/-5 mu m;
the absorbable vascular stent coating with the bionic interface has excellent endothelialization promoting capability, and after being implanted in a body for 2 weeks, the endothelialization speed of the inner surface of the absorbable vascular stent coating is obviously increased, and the coverage rate is increased by 68% compared with that of a control example 7.
Example 5:
(1) The silkworm raw silk is placed in 0.1 percent sodium carbonate aqueous solution 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 60 ℃ oven, thus obtaining degummed silk 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 a required concentration;
mixing the aqueous silk fibroin solution with a hydrophilic flexible cross-linking agent to obtain a modified silk fibroin solution with the concentration of 60mg/mL; 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.
(2) The 50mg/mL aqueous solution of silk fibroin is used as an electrospinning liquid, and the silk fibroin nanofiber is electrospun on a stainless steel rod (with the diameter of 10 mm) with the rotating speed of 500rpm by electrospinning, wherein the average diameter of the silk fibroin nanofiber is 200nm, and the average orientation degree is 35%.
Then placing the stainless steel rod wound with the silk fibroin nano fibers in a modified silk fibroin solution with the concentration of 60mg/mL for 10 seconds in a rotary-dipping mode, wherein the dipping area accounts for 50% of the side area of the stainless steel rod, the rotary speed is 60rpm, taking out, and then placing in an environment at 35 ℃ to keep the rotary speed for continuous rotation for 15 minutes to form an inner layer of the stent coating; the rotating direction is along the circumferential direction of the stainless steel rod;
(3) The method comprises the steps of combining, twisting and degumming raw silk of silkworms to obtain degummed twisted silk; the titer of raw silk of the silkworms is 20/22D, the number of the combined strands is 2, the twisting direction of twisting is S direction, the twisting twist is 600 twists/m, and the degumming process is the same as that of the step 1;
the degummed twisted yarn is adopted 60 Co gamma-ray irradiation treatment of 60 The Co gamma-ray irradiation dose is 100kGy, and the wire after irradiation treatment is obtained; and weaving the tubular fabric with the weaving angle of 120 degrees and the axial weaving density of 4 threads/cm on the basis of serving as the inner layer of the stent coating by a weaving technology to form a core layer of the vascular stent coating, wherein the threads are degummed twisted threads and the threads subjected to irradiation treatment are uniformly arranged in a 1:2 number ratio.
(4) Placing the core layer of the vascular stent coating in the step (3) in the modified silk fibroin solution in a rotating-dipping mode for 60 seconds, wherein the dipping area accounts for 50% of the side area of the stainless steel rod, the rotating speed is 60rpm, taking out the vascular stent coating, placing the vascular stent coating in a 35 ℃ environment, keeping the rotating speed for 15 minutes, controlling the surface of the film to keep wet, repeating the step of rotating the composite modified silk fibroin for 6 times, wherein the rotating direction is along the circumferential direction of the stainless steel rod, forming the outer layer of the vascular stent coating, and further obtaining the vascular stent coating; soaking the vascular stent coating in sterile deionized water at 37 ℃ for 1 day, removing unreacted cross-linking agent and silk fibroin molecules and eroding the silk fibroin nanofiber to obtain the absorbable vascular stent coating with a bionic interface.
Through detection, the absorbable vascular stent coating has ultrathin and uniform thickness of 60+/-5 mu m;
the absorbable vascular stent coating with the bionic interface has excellent performance, and the degradation speed of the absorbable vascular stent coating is obviously accelerated after being implanted in a human body for 6 months, and the weight loss rate is increased by 79 percent compared with that of comparative example 7.
Example 6:
(1) Placing raw silkworms into a 0.1% sodium carbonate aqueous solution according to a bath ratio of 1:50g/mL, treating for three times at 98-100 ℃ for 30 minutes each time, then fully cleaning with deionized water, and drying in a 60 ℃ oven for 12 hours to obtain degummed silk fibroin fibers; 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 a required concentration;
mixing the aqueous silk fibroin solution with a hydrophilic flexible cross-linking agent to obtain a modified silk fibroin solution with the concentration of 60mg/mL; 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.
(2) The 50mg/mL aqueous solution of silk fibroin is used as an electrospinning liquid, and the silk fibroin nanofiber is electrospun on a stainless steel rod (with the diameter of 10 mm) with the rotating speed of 500rpm by electrospinning, wherein the average diameter of the silk fibroin nanofiber is 200nm, and the average orientation degree is 35%.
Then placing the stainless steel rod wound with the silk fibroin nano fibers in a modified silk fibroin solution with the concentration of 60mg/mL for 10 seconds in a rotary-dipping mode, wherein the dipping area accounts for 50% of the side area of the stainless steel rod, the rotary speed is 60rpm, taking out, and then placing in an environment at 35 ℃ to keep the rotary speed for continuous rotation for 15 minutes to form an inner layer of the stent coating; the rotating direction is along the circumferential direction of the stainless steel rod.
(3) And (3) treating the raw silk of the silkworm by adopting the procedures of combining, twisting and degumming to obtain the degummed twisted silk. The titer of raw silk of the silkworms is 20/22D, the number of the combined strands is 2, the twisting direction of twisting is S direction, the twisting twist is 600 twists/m, and the degumming process is the same as that of the step 1; the degummed twisted yarn is adopted 60 Co gamma-ray irradiation treatment of 60 The Co gamma-ray irradiation dose is 200kGy, and the obtained productAnd (3) irradiating the treated silk thread. And weaving the tubular fabric with the weaving angle of 120 degrees and the axial weaving density of 4 threads/cm on the basis of serving as the inner layer of the stent coating by a weaving technology to form a core layer of the vascular stent coating, wherein the threads are degummed twisted threads and the threads subjected to irradiation treatment are uniformly arranged in a 1:2 number ratio.
(4) Placing the core layer of the vascular stent coating in the step (3) in the modified silk fibroin solution in a rotating-dipping mode for 60 seconds, wherein the dipping area accounts for 50% of the side area of the stainless steel rod, the rotating speed is 60rpm, taking out the vascular stent coating, placing the vascular stent coating in a 35 ℃ environment, keeping the rotating speed for 15 minutes, controlling the surface of the film to keep wet, repeating the step of rotating the composite modified silk fibroin for 6 times, wherein the rotating direction is along the circumferential direction of the stainless steel rod, forming the outer layer of the vascular stent coating, and further obtaining the vascular stent coating; soaking the vascular stent coating in sterile deionized water at 37 ℃ for 1 day, removing unreacted cross-linking agent and silk fibroin molecules and eroding the silk fibroin nanofiber to obtain the absorbable vascular stent coating with a bionic interface.
Through detection, the absorbable vascular stent coating has ultrathin and uniform thickness of 60+/-5 mu m,
the absorbable vascular stent coating with the bionic interface has excellent performance, and the degradation speed of the coating is obviously accelerated after being implanted in a human body for 6 months, and the weight loss rate is increased by 121 percent compared with that of comparative example 7.
Example 7 (comparative example):
(1) The silkworm raw silk is placed in 0.1 percent sodium carbonate aqueous solution 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 60 ℃ oven, thus obtaining degummed silk 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 a required concentration;
mixing the aqueous silk fibroin solution with a hydrophilic flexible cross-linking agent to obtain a modified silk fibroin solution with the concentration of 60mg/mL; 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.
(2) Placing a stainless steel rod (with the diameter of 10 mm) in a modified silk fibroin solution with the concentration of 60mg/mL for 10 seconds in a static-dipping mode, wherein the dipping area accounts for 100% of the side area of the stainless steel rod, taking out, placing in a 35 ℃ environment, rotating and drying for 15 minutes, and forming an inner layer of a stent coating at the rotating speed of 60 rpm; the rotating direction is along the circumferential direction of the stainless steel rod.
(3) The method comprises the steps of combining, twisting and degumming raw silk of silkworms to obtain degummed twisted silk; and weaving the twisted yarn into a tubular fabric with a weaving angle of 120 degrees and an axial weaving density of 4 pieces/cm on the basis of being used as the inner layer of the stent coating by a weaving technology to form the core layer of the vascular stent coating.
(4) Placing the core layer of the vascular stent coating in the step (3) in the modified silk fibroin solution in a static-dipping mode for 60 seconds, wherein the dipping area accounts for 100% of the side area of a stainless steel rod, taking out, placing in a 35 ℃ environment, rotating and drying for 15 minutes, controlling the rotating speed to be 60rpm, controlling the surface of the film to keep wet, and repeating the step of rotating the composite modified silk fibroin for 6 times to form the outer layer of the vascular stent coating so as to obtain the vascular stent coating; soaking the vascular stent coating in sterile deionized water at 37 ℃ for 1 day, and removing unreacted cross-linking agent and silk fibroin molecules to obtain an absorbable vascular stent coating; the thickness of the absorbable vascular stent coating is 60+/-20 mu m.
(5) Placing the core layer of the vascular stent coating in the step (3) in the modified silk fibroin solution in the step (1) in a static-dipping mode for 60 seconds, wherein the dipping area is 100% of the side area of the stainless steel rod, taking out, placing in a 35 ℃ environment, static-drying for 15 minutes, controlling the surface of the film to keep wet, and repeating the step of dipping the composite modified silk fibroin for 6 times to form the outer layer of the vascular stent coating. The vascular stent coating is soaked in sterile deionized water at 37 ℃ for 1 day, unreacted cross-linking agent and silk fibroin molecules are removed, and the absorbable vascular stent coating is obtained, and the thickness of the absorbable vascular stent coating is measured to be 70+/-40 mu m.
Description: the above embodiments are only for illustrating the present invention and not for limiting the technical solution described in the present invention; thus, while the invention has been described in detail with reference to the various embodiments described above, it will be understood by those skilled in the art that the invention may be modified or equivalents; all technical solutions and modifications thereof that do not depart from the spirit and scope of the present invention are intended to be included in the scope of the appended claims.
Claims (10)
1. The preparation method of the absorbable vascular stent coating with the bionic interface is characterized by comprising the following steps of:
(1) 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; completely dissolving the degummed silk fibroin fibers in a lithium bromide solution to obtain silk fibroin dissolving solution, pouring the silk fibroin dissolving solution into a dialysis bag, filtering the silk fibroin dissolving solution by deionized water to obtain purified silk fibroin aqueous solution, and evaporating and concentrating the purified silk fibroin aqueous solution to adjust the concentration of the silk fibroin aqueous solution to 10-200 mg/mL;
mixing a silk fibroin aqueous solution with a hydrophilic flexible cross-linking agent to obtain a modified silk fibroin solution, wherein the concentration of silk fibroin in the modified silk fibroin solution is 20-160 mg/mL;
(2) Preparation of a stent coating inner layer:
(a) Spinning nano fibers on a cylindrical auxiliary rod by using a water-soluble polymer solution as an electrospinning solution through electrostatic spinning to obtain an auxiliary rod with nano fibers; wherein the rotation speed of the cylindrical auxiliary rod is 100-3000 rpm, the average diameter of the nanofiber is 100-5000 nm, and the average orientation degree of the nanofiber is 30-90%;
(b) Soaking an auxiliary rod with nano fibers in the modified silk fibroin solution prepared in the step (1) in a rotary-soaking mode, taking out the soaked auxiliary rod, and rotating the auxiliary rod at 20-50 ℃ to form a film, thereby completing primary soaking treatment; repeating the dipping treatment for several times to obtain an inner layer of the stent coating;
(3) Preparation of a vascular stent coating core layer:
(a) The method comprises the steps of combining, twisting and degumming raw silk of silkworms to obtain degummed twisted silk; the degummed twisted yarn is adopted 60 Co gamma-ray irradiation treatment of 60 The Co gamma-ray irradiation dose is 25-300 kGy, and the wire after irradiation treatment is obtained;
(b) Braiding a tubular fabric with a braiding angle of 30-150 degrees and an axial braiding density of 1-20 threads/cm on the basis of the stent tectorial membrane inner layer in the step (2) through a braiding technology to form a core layer of the vascular stent tectorial membrane; the silk thread is degummed twisted silk or/and silk thread after irradiation treatment;
(4) Placing the core layer of the vascular stent coating in the step (3) in the modified silk fibroin solution obtained in the step (1) in a rotary-dipping mode for treatment, taking out the treated core layer, placing the treated core layer in an environment of 20-50 ℃ and keeping the rotary speed for continuous rotation for 10-30 minutes, namely finishing one dipping treatment, controlling the surface of the membrane to keep wet, and repeating the dipping treatment for a plurality of times to form an outer layer of the vascular stent coating; finally, soaking the stent graft in sterile deionized water to remove unreacted cross-linking agent and silk fibroin molecules and erode nanofibers, thus obtaining the absorbable stent graft with a bionic interface.
2. The method of claim 1, wherein the concentration of the lithium bromide solution in step (1) is 9.3M; the dialysis bag is a semipermeable membrane, the molecular weight cut-off is 3-50 kDa, and deionized water is adopted for dialysis for 3 days.
3. The preparation method of the absorbable vascular stent coating with the bionic interface according to claim 1, wherein the mass ratio of the silk fibroin to the cross-linking agent in the step (1) is 1.0 (0.3-1.0), and the cross-linking agent is polyethylene glycol diglycidyl ether.
4. The method for preparing an absorbable vascular stent coating with a bionic interface according to claim 1, wherein the cylindrical auxiliary rod in the steps (a) - (b) of the step (2) is a stainless steel rod with the diameter of 1-30 mm.
5. The method of claim 1, wherein the water-soluble polymer solution in step (2) is an aqueous silk fibroin solution or an aqueous polyvinyl alcohol solution; the concentration of the aqueous solution of silk fibroin is 10-200 mg/mL; the concentration of the polyvinyl alcohol aqueous solution is 50-200 mg/mL.
6. The method for preparing an absorbable vascular stent coating with a bionic interface according to claim 1, wherein the time of immersing in the modified silk fibroin solution in the step (b) of the step (2) is 10-60 seconds, the rotation speed is 10-100 rpm, the rotation direction is along the circumferential direction of the auxiliary rod, the repeated immersing treatment is carried out for 0-5 times, and the immersed area accounts for 10-90% of the side area of the stainless steel rod.
7. The method for preparing an absorbable vascular stent coating with a bionic interface according to claim 1, wherein the titer of raw silk of the bombyx mori in the step (a) of the step (3) is 20/22D, the number of the combined strands is 2-10 strands, the twisting direction of twisting is the S/Z direction, and the twisting twist is 100-2000 twists/m; the degumming process is the same as the operation of the step (1), namely, the raw silk of the silkworm is degummed by any one of boiling water, sodium carbonate, sodium bicarbonate or biological enzyme.
8. The method of claim 1, wherein the degummed twisted filaments and the irradiated filaments in step (3) are uniformly arranged at a ratio of 6:0-0:6.
9. The method for preparing an absorbable vascular stent coating with a bionic interface according to claim 1, wherein the time of immersing in the modified silk fibroin solution in the step (4) is 10-60 seconds, the rotation speed is 10-100 rpm, the rotation direction is along the circumferential direction of the auxiliary rod, the number of times of repeated immersing treatment is 2-10 times, and the immersed area accounts for 10-90% of the side area of the stainless steel rod; the temperature of the sterile deionized water is 4-37 ℃ and the soaking time is 1-3 days.
10. The method of any one of claims 1-9 for preparing an absorbable stent graft with a biomimetic interface, wherein the absorbable stent graft is woven into a stent core layer with a thickness of 60 ± 5 μm from a hybrid combination of degummed silk fibers or/and irradiated silk fibers.
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