Absorbable vascular stent covering facilitating endothelialization and preparation method thereof

Abstract

The application provides an absorbable vascular stent covering facilitating endothelialization and a preparation method thereof, and belongs to the technical fields of biomedical materials and vascular stent covering preparation. The absorbable vascular stent covering facilitating endothelialization is prepared based on modified silk fibroin and domestic silkworm silk. The inner surface of the absorbable vascular stent covering facilitating endothelialization has a nano topological structure and a matrix-like component simulating a vascular basement membrane, and the outer surface has a micro topological structure. The absorbable vascular stent covering facilitating endothelialization has good mechanical properties and excellent endothelial cell activity, and can be used for producing a vascular stent capable of rapidly forming a new endothelial layer, maintaining stable blood flow, and inhibiting thrombosis and inflammation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials and vascular stent covering film preparation, and particularly relates to an absorbable vascular stent covering film beneficial to endothelialization and a preparation method thereof. BACKGROUND

[0002] The incidence, prevalence and mortality of cardiovascular and cerebrovascular diseases are increasing year by year, and are showing a trend of younger. Atherosclerosis is the main cause of cardiovascular and cerebrovascular diseases, and atherosclerosis is caused by intimal stimulation such as lipid or sugar accumulation, which leads to thickening and hardening of the arterial wall and stenosis, and eventually worsens to occlusive arterial disease including coronary heart disease, cerebral infarction and peripheral vascular disease. At present, more and more covering stents are used in clinic to achieve the purpose of treating vascular obstruction, but postoperative medium and long term complications such as stent displacement, internal leakage, thrombosis and restenosis are still prone to occur, resulting in a high proportion of unsatisfactory intervention, which needs postoperative re-intervention, and even causes death. These are clinical problems that need to be solved urgently.

[0003] At present, the covering film materials of commercial covering stents are mainly polyester and polytetrafluoroethylene. Although these two synthetic materials have excellent mechanical properties, they have defects such as poor compliance, biological inertia and non-degradation. And the above two synthetic materials are difficult to achieve endothelialization after being implanted into the body, which may induce thrombosis, restenosis and complications. In addition, since the above two synthetic materials are difficult to degrade, long-term medication is needed to inhibit immune rejection, which is a great clinical challenge to the increasing incidence of young and middle-aged population, and the defects of commercial covering stent covering film materials need to be solved by reoperation or multiple operations.

[0004] This is the root cause of inducing postoperative thrombosis, restenosis and complications. And this is a great clinical challenge to the increasing incidence of young and middle-aged population, and needs reoperation or multiple operations. Therefore, in view of the existing clinical application bottleneck and the increasing incidence of young people, it is necessary to develop an absorbable vascular stent covering film to solve the above problems. SUMMARY

[0005] In view of the deficiencies in the prior art, the present application provides an absorbable vascular stent covering film beneficial to endothelialization and a preparation method thereof. The absorbable vascular stent covering film beneficial to endothelialization is prepared based on modified silk fibroin and domestic silkworm silk, the inner surface of the absorbable vascular stent covering film beneficial to endothelialization has a nano topological structure and a matrix-like composition which imitate the vascular basement membrane, and the outer surface has a micro topological structure. The absorbable vascular stent covering film beneficial to endothelialization has good mechanical properties and excellent endothelial cell activity, and can be used to produce a new endothelial layer, maintain stable blood flow, and inhibit thrombosis and inflammation of the vascular stent.

[0006] The present application first provides an absorbable vascular stent covering facilitating endothelialization, which has a tubular fabric woven by treated domestic silkworm raw silk as a core layer, and modified silk fibroin nanofibers as inner and outer layers; the inner surface of the absorbable vascular stent covering has a nano topological structure and matrix-like components simulating a vascular basement membrane, and the outer surface has a micro topological structure.

[0007] The present application also provides a preparation method of the absorbable vascular stent covering facilitating endothelialization, which specifically comprises the following steps:

[0008] (1) mixing a silk fibroin aqueous solution with a hydrophilic and flexible crosslinking agent to obtain a modified silk fibroin solution; then using the modified silk fibroin as an electrospinning liquid to spin silk fibroin nanofibers on an auxiliary rod by electrospinning to form an inner layer of the absorbable vascular stent covering;

[0009] (2) weaving a tubular fabric with a weaving angle of 30-150° and an axial weaving density of 1-20 strands / cm on the basis of the inner layer of the absorbable vascular stent covering facilitating endothelialization by weaving technology using a silk thread treated based on domestic silkworm raw silk by a process of doubling, twisting and degumming, and / or a silk thread treated by gamma ray irradiation treatment after degumming; 60 (3) placing the obtained tubular fabric in the modified silk fibroin solution in a rotary-dipping manner for 10-60 seconds, then taking it out and rotating it in an environment at 20-50°C for 10-30 minutes, which is recorded as one time, and repeating the above steps several times to form a core layer of the absorbable vascular stent covering;

[0010] (4) using the modified silk fibroin solution in step (1) as an electrospinning liquid to electrospin silk fibroin nanofibers on the surface of the core layer of the absorbable vascular stent covering by electrospinning to form the absorbable vascular stent covering; and washing the absorbable vascular stent covering in sterile deionized water to remove unreacted crosslinking agents and silk molecules to obtain the absorbable vascular stent covering facilitating endothelialization.

[0011] Preferably, in step (1), the method for obtaining the silk fibroin aqueous solution is as follows: degumming domestic silkworm raw silk by using any one of boiling water, sodium carbonate, sodium bicarbonate or biological enzymes to obtain degummed silk fibroin; completely dissolving the degummed silk fibroin in a lithium bromide solution to obtain a silk fibroin dissolution solution, then perfusing the silk fibroin dissolution solution into a dialysis bag and dialyzing it with deionized water, filtering and evaporating to obtain the silk fibroin aqueous solution.

[0012]

[0013] ​The concentration of the lithium bromide solution is 9.3M; the dialysis bag is a semi-permeable membrane with a molecular weight cut-off of 3-50kDa; the dialysis time is 3 days; and the concentration of the obtained silk fibroin aqueous solution is 10-200mg / mL.

[0014] Preferably, in step (1), the mass ratio of the silk fibroin aqueous solution to the crosslinking agent is 1.0:(0.3-1.0); and the concentration of the modified silk fibroin solution is 20-180mg / mL.

[0015] Preferably, in step (1), the crosslinking agent is polyethylene glycol diglycidyl ether.

[0016] Preferably, in step (2), the auxiliary rod is preferably a stainless steel rod.

[0017] Preferably, in step (1), the rotation speed of the auxiliary rod is 100-3000rpm, the diameter of the auxiliary rod is 1-30mm, the average diameter of the silk nanofiber is 100-5000nm, the average porosity of the silk nanofiber is 35-90%, and the average orientation degree of the silk nanofiber is 30-90%.

[0018] Preferably, in step (2), the method for obtaining the degummed silk thread is as follows:

[0019] The degummed silk thread is obtained by twisting 2-10 strands of 20 / 22D domestic silkworm silk in an S / Z direction to a twist degree of 100-2000 twists per meter, and then degumming the twisted silk thread using any one of boiling water, sodium carbonate, sodium bicarbonate or a biological enzyme.

[0020] The method for obtaining the irradiated silk thread is as follows:

[0021] The degummed silk thread is irradiated using a 60 Co γ-ray to obtain an irradiated silk thread, wherein the 60 The Co γ-ray irradiation dose is 25-300kGy.

[0022] Preferably, in step (2), the silk thread is a degummed silk thread and an irradiated silk thread arranged uniformly at a ratio of 6:0-0:6.

[0023] Preferably, in step (3), the rotation speed of the rotation-dipping is 10-100rpm, the rotation direction is along the circumferential direction of the auxiliary rod, and the dipping area accounts for 10-90% of the side area of the auxiliary rod.

[0024] The number of times is 2-10.

[0025] Preferably, in step (4), the washing is: soaking the vascular stent covering film in 4-37 DEG C sterile deionized water for 1-3 days.

[0026] Compared with the prior art, the application has the beneficial effects that:

[0027] The silk fibroin used in the application is a natural protein derived from animals, which is composed of 20 kinds of amino acids and has the same composition as the extracellular matrix. The artificial blood vessel constructed by the silk fibroin can support the adhesion, growth and proliferation of vascular cells, induce endothelialization after implantation, maintain unobstructed blood flow, and induce in-situ regeneration of vascular tissue, and therefore attracts great attention in the field of vascular tissue engineering materials. Based on good blood compatibility, the silk fibroin can be used for the preparation of absorbable vascular stent covering film which is beneficial to endothelialization.

[0028] The absorbable vascular stent covering film prepared in the application has an ultra-thin and uniform thickness, excellent endothelialization function, absorbability and stability of the covering film, which can fundamentally solve the problems that the clinical application of the dacron or polytetrafluoroethylene vascular stent cannot be endothelialized, cannot be degraded and is prone to produce thrombosis and slip after surgery. The inner surface of the absorbable vascular stent covering film has a nano topological structure and a matrix-like composition which mimic the vascular basement membrane, can induce the homing and proliferation of endothelial (progenitor) cells, and the silk fibroin has the property of bioabsorbability, so that the new endothelial tissue is quickly formed while the multi-level (silk fibroin electrospun silk, self-assembled membrane layer, irradiated silk thread, non-irradiated silk thread) gradient degradation and absorption are carried out after implantation, the formation of thrombus and inflammation is effectively inhibited, the blood coagulation balance is maintained, and the long-term patency of the blood vessel is maintained. The micro-topological structure on the outer surface of the absorbable vascular stent covering film is beneficial to the close adhesion of the covering film to the inner wall of the blood vessel without slipping, and the micro-topological structure on the inner surface is also beneficial to maintaining the stability of the blood flow and avoiding the formation of turbulent blood flow.

[0029] In the absorbable vascular stent covering film, the silk fibroin macromolecular chain has rich surface charge characteristics and chemical reaction sites, and various bioactive factors such as anti-inflammatory / anticoagulant drugs and growth factors are loaded through coaxial electrospinning technology, which are released slowly with the gradient degradation of the covering film, further guide the cell behavior and promote the regeneration of endothelial tissue.

[0030] The multi-level (silk fibroin electrospun silk, self-assembled membrane layer, irradiated silk thread, non-irradiated silk thread) gradient degradation of the absorbable vascular stent covering film after implantation can meet the needs of personalized groups with different ages, physical conditions and symptoms, regulate endothelialization and blood flow homeostasis, and completely solve the occurrence of thrombosis and restenosis after surgery suitable for different groups of people. DETAILED DESCRIPTION

[0031] The application will be further described in conjunction with specific examples. However, the scope of protection of the application is not limited to the following examples. In the following examples, the auxiliary rod is selected to be a stainless steel rod for illustration, but the auxiliary rod can be of any material. In the following examples, various processes and methods not described in detail are conventional methods known in the art. The source of the reagents used, trade name, and necessary composition components are indicated at the first occurrence, and the same source is used for the same reagents without special indication thereafter; the reagents, materials, etc. involved are commercially available without special indication.

[0032] Example 1

[0033] (1) Bombyx mori raw silk was placed in a 0.1% sodium carbonate aqueous solution at a bath ratio of 1:50 g / mL, treated at a temperature of 98-100°C for three times, each for 30 minutes, then washed thoroughly with deionized water, and dried in an oven at 60°C for 12 hours to obtain degummed silk fibroin fibers.

[0034] Degummed silk fibroin fibers were weighed at a bath ratio of 1:10 g / mL, completely dissolved in a 9.3M lithium bromide solution in a water bath environment at 65±5°C to obtain a silk fibroin dissolution solution. The silk fibroin dissolution solution was poured into a dialysis bag with a molecular weight cut-off of 14 kDa, and deionized water was used for dialysis for 3 days to obtain a purified silk fibroin aqueous solution. The purified silk fibroin aqueous solution was concentrated and adjusted to the desired concentration.

[0035] The silk fibroin aqueous solution was mixed with a hydrophilic and flexible crosslinking agent to obtain a modified silk fibroin solution, and the mass ratio of silk fibroin to crosslinking agent was 1.0:0.5. The crosslinking agent was polyethylene glycol diglycidyl ether.

[0036] The modified silk fibroin solution with a silk fibroin concentration of 40 mg / mL was used as an electrospinning solution, and silk fibroin nanofibers were spun on a stainless steel rod (diameter 10 mm) with a rotation speed of 200 rpm to form an inner layer of an absorbable vascular stent covering membrane.

[0037] (2) Bombyx mori raw silk was treated by plying, twisting, and degumming to obtain degummed twisted silk threads. The Bombyx mori raw silk had a fineness of 20 / 22D, the number of plies was 2, the twist direction was S, and the twist degree was 500 twists per meter. The degumming process was the same as step (1).

[0038] The degummed twisted silk threads were then treated by 60 Co γ-ray irradiation, the 60 Co γ-ray irradiation dose was 100 kGy to obtain irradiated silk threads, which were used as needed.

[0039] The hybrid combination of the degummed silk thread and the irradiation-treated silk thread is used as a thread material for braiding a tubular fabric, wherein the hybrid combination is uniformly arranged in a ratio of 1:1 in number.

[0040] (3) The tubular fabric is placed in a modified silk fibroin solution with a silk fibroin concentration of 60 mg / mL in a rotary-dipping manner for 15 seconds, and after being taken out, the tubular fabric is kept rotating at the above-mentioned rotating speed for 15 minutes in a 35℃ environment, the surface of the film is kept wet, and the step of rotating and compounding the modified silk fibroin is repeated 6 times, so as to form a core layer of the absorbable vascular stent covering film. The rotating speed of the rotary-dipping is 60 rpm, the rotating direction is along the circumferential direction of the stainless steel rod, and the dipping area accounts for 50% of the side area of the stainless steel rod.

[0041] (4) The method in step (1) is used to electrospin the silk fibroin nanofiber on the surface of the core layer of the vascular stent covering film, so as to construct an outer layer of the vascular stent covering film, and the vascular stent covering film is soaked in 37℃ sterile deionized water for 1 day to remove the unreacted crosslinking agent and silk fibroin molecules, so as to obtain the absorbable vascular stent covering film which is beneficial to endothelialization and has the activity of regulating the growth of endothelial cells.

[0042] It is detected that the absorbable vascular stent covering film has excellent mechanical properties and promotes the activity of endothelial cells, the average diameter of the silk fibroin nanofiber is 150±50 nm, the porosity is 80±10%, and the fiber orientation degree is 25±10%. After being implanted in vivo for 1 month, the endothelialization degree of the absorbable vascular stent covering film of the present application is increased by 45% compared with the absorbable vascular stent covering film in the comparative example 1, and the weight loss rate is increased by 37%.

[0043] Example 2:

[0044] (1) The domestic silkworm silk is placed in a 0.1% sodium carbonate aqueous solution in a bath ratio of 1:50 g / mL, treated at a temperature of 98-100℃ for three times, each time for 30 minutes, and then washed with deionized water and dried in a 60℃ oven for 12 hours to obtain the degummed silk fibroin.

[0045] The degummed silk fibroin is weighed and dissolved in a 9.3M lithium bromide solution in a bath ratio of 1:10 g / mL at a water bath temperature of 65±5℃ to obtain a silk dissolution solution. The silk dissolution solution is filled into a dialysis bag with a molecular weight cut-off of 14 kDa, and deionized water is used for dialysis for 3 days to obtain a purified silk fibroin aqueous solution. The purified silk fibroin aqueous solution is concentrated and adjusted to the required concentration.

[0046] Mix the aqueous solution of silk fibroin with a hydrophilic flexible crosslinking agent to obtain a modified silk fibroin solution, the mass ratio of silk fibroin to crosslinking agent being 1.0:0.5, and the crosslinking agent being polyethylene glycol diglycidyl ether.

[0047] The modified silk fibroin solution with a silk fibroin concentration of 160 mg / mL is used as an electrospinning solution to spin the silk fibroin nanofiber on a stainless steel rod (10 mm in diameter) with a rotation speed of 200 rpm to form the inner layer of the absorbable vascular stent covering membrane.

[0048] (2) The domestic silkworm silk is treated by doubling, twisting and degumming to obtain the twisted silk thread after degumming. The domestic silkworm silk has a fineness of 20 / 22D, the doubled strand number is 2, the twisting direction is S, and the twisting degree is 500 twists per meter. The degumming process is the same as step (1).

[0049] Then the twisted silk thread after degumming is treated by 60 Co γ-ray irradiation, the 60 Co γ-ray irradiation dose is 100 kGy, to obtain the irradiated silk thread, which is ready for use.

[0050] The hybrid combination of the above degummed silk thread and the above irradiated silk thread is used as the silk thread raw material for weaving the tubular fabric, wherein the hybrid combination is evenly arranged in a 1:1 ratio of the degummed silk thread and the irradiated silk thread. The silk thread is woven into a tubular fabric with a weaving angle of 120° and an axial weaving density of 4 roots / cm on the outer surface of the inner layer of the absorbable vascular stent covering membrane by using weaving technology.

[0051] (3) The tubular fabric is placed in a modified silk fibroin solution with a silk fibroin concentration of 60 mg / mL by rotating-dipping for 15 seconds, and then is taken out and placed in a 35°C environment to continue rotating at the above rotation speed for 15 minutes, so that the surface of the membrane is kept wet. The rotating-dipping step of modifying the silk fibroin is repeated 6 times to form the core layer of the absorbable vascular stent covering membrane. The rotation speed of the rotating-dipping is 60 rpm, the rotation direction is along the circumferential direction of the stainless steel rod, and the dipping area accounts for 50% of the side area of the stainless steel rod.

[0052] (4) The method described in step (1) is used to electrospin the silk fibroin nanofiber on the surface of the core layer of the vascular stent covering membrane to construct the outer layer of the vascular stent covering membrane. The vascular stent covering membrane is soaked in sterile deionized water at 37°C for 1 day to remove the unreacted crosslinking agent and silk fibroin molecules, thereby obtaining the absorbable vascular stent covering membrane which is beneficial to endothelialization and regulates the growth activity of endothelial cells.

[0053] The absorbable vascular stent covering film has excellent mechanical properties and promotes endothelial cell activity. The average diameter of the silk fibroin nanofiber is 700±100 nm, the porosity is 55±10%, and the fiber orientation degree is 25±10%. After 1 month of in vivo implantation, the endothelialization degree of the absorbable vascular stent covering film of the application is increased by 58% compared with that of the absorbable vascular stent covering film in Comparative Example 1.

[0054] Example 3:

[0055] (1) The domestic silkworm silk is placed in a 0.1% by mass sodium carbonate aqueous solution at a bath ratio of 1:50 g / mL, treated at a temperature of 98-100°C for three times, each for 30 minutes, then washed thoroughly with deionized water, and dried in a 60°C oven for 12 hours to obtain degummed silk fibers.

[0056] The degummed silk fibers are weighed and completely dissolved in a 9.3M lithium bromide solution at a bath ratio of 1:10 g / mL in a 65±5°C water bath environment to obtain a silk dissolution solution. The silk dissolution solution is perfused into a dialysis bag with a molecular weight cut-off of 14 kDa, and deionized water dialysis is performed for 3 days to obtain a purified silk protein aqueous solution. The purified silk protein aqueous solution is concentrated and adjusted to the desired concentration.

[0057] The silk protein aqueous solution is mixed with a hydrophilic and flexible crosslinking agent to obtain a modified silk protein solution, and the mass ratio of silk protein to crosslinking agent is 1.0:0.5. The crosslinking agent is polyethylene glycol diglycidyl ether.

[0058] The modified silk protein solution with a silk protein concentration of 160 mg / mL is used as an electrospinning solution, and the electrospinning is performed on a stainless steel rod (diameter of 10 mm) with a rotation speed of 3000 rpm to spin the silk nanofiber and form the inner layer of the absorbable vascular stent covering film.

[0059] (2) The domestic silkworm silk is treated by doubling, twisting, and degumming to obtain degummed twisted silk threads. The domestic silkworm silk has a fineness of 20 / 22D, the number of doubled strands is 2, the twisting direction is S, the twisting degree is 500 twists per meter, and the degumming process is the same as step (1).

[0060] Then the degummed twisted silk threads are subjected to 60 Coγ ray irradiation treatment, and the 60 Coγ ray irradiation dose is 100 kGy to obtain irradiated silk threads, which are used as needed.

[0061] The hybrid combination of the degummed silk thread and the irradiation-treated silk thread is used as a thread material for weaving a tubular fabric, wherein the hybrid combination is uniformly arranged in a ratio of 1:1 in number.

[0062] (3) The tubular fabric is placed in a modified silk fibroin solution with a silk fibroin concentration of 60 mg / mL in a rotary-dipping manner for 15 seconds, and after being taken out, the tubular fabric is kept rotating at the above-mentioned rotating speed for 15 minutes at 35°C, the surface of the membrane is kept wet, and the step of rotating and compounding the modified silk fibroin is repeated 6 times, so as to form a core layer of the absorbable vascular stent covering film. The rotating speed of the rotary-dipping is 60 rpm, the rotating direction is along the circumferential direction of the stainless steel rod, and the dipping area accounts for 50% of the side area of the stainless steel rod.

[0063] (4) The method in step (1) is used to electrospin the silk fibroin nanofiber on the surface of the core layer of the vascular stent covering film, so as to construct an outer layer of the vascular stent covering film. The vascular stent covering film is soaked in sterile deionized water at 37°C for 1 day, so as to remove the unreacted crosslinking agent and silk fibroin molecules, and an absorbable vascular stent covering film with endothelialization-facilitating endothelial cell growth activity is obtained.

[0064] It is detected that the absorbable vascular stent covering film has excellent mechanical properties and endothelial cell activity, the average diameter of the silk fibroin nanofiber is 650±100 nm, the porosity is 50±10%, and the fiber orientation degree is 75±10%. After being implanted in vivo for 1 month, the endothelialization degree of the absorbable vascular stent covering film of the present application is increased by 43% compared with the absorbable vascular stent covering film in Comparative Example 1.

[0065] Example 4:

[0066] (1) The domestic silkworm silk is placed in a 0.1% sodium carbonate aqueous solution in a bath ratio of 1:50 g / mL, treated at a temperature of 98-100°C for three times, each time for 30 minutes, then washed with deionized water, and dried in a 60°C oven for 12 hours, so as to obtain the degummed silk fibroin.

[0067] The degummed silk fibroin is weighed and completely dissolved in a 9.3M lithium bromide solution in a bath ratio of 1:10 g / mL at a water bath environment of 65±5°C, so as to obtain a silk fibroin dissolution solution. The silk fibroin dissolution solution is perfused into a dialysis bag with a molecular weight cut-off of 14 kDa, and deionized water dialysis is performed for 3 days to obtain a purified silk fibroin aqueous solution. The purified silk fibroin aqueous solution is concentrated and adjusted to a required concentration.

[0068] Mix the aqueous solution of silk fibroin with a hydrophilic flexible crosslinking agent to obtain a modified silk fibroin solution, the mass ratio of silk fibroin to crosslinking agent being 1.0:0.5, and the crosslinking agent being polyethylene glycol diglycidyl ether.

[0069] The modified silk fibroin solution with a silk fibroin concentration of 160 mg / mL is used as an electrospinning solution to electrospin the silk fibroin nanofiber on a stainless steel rod (1-30 mm in diameter) rotating at a speed of 200 rpm to form an inner layer of the absorbable vascular stent covering membrane.

[0070] (2) The domestic silkworm silk is treated by doubling, twisting and degumming to obtain twisted silk thread after degumming. The domestic silkworm silk has a fineness of 20 / 22D, the number of strands of doubling is 2, the twisting direction is S, and the twisting degree is 500 twists per meter. The degumming process is the same as step (1).

[0071] Then the twisted silk thread after degumming is treated by 60 Co γ-ray irradiation, the 60 Co γ-ray irradiation dose is 100 kGy, to obtain the silk thread after irradiation treatment, ready for use.

[0072] The above irradiated silk thread is used as the silk thread raw material for knitting the tubular fabric. The silk thread is knitted into a tubular fabric with a knitting angle of 120° and an axial knitting density of 4 roots / cm on the outer surface of the inner layer of the absorbable vascular stent covering membrane by knitting technology.

[0073] (3) The tubular fabric is placed in a modified silk fibroin solution with a silk fibroin concentration of 60 mg / mL in a rotating-dipping manner for 15 seconds, and then taken out and placed in a 35°C environment to continue rotating at the above-mentioned rotating speed for 15 minutes. The surface of the membrane is kept wet, and the step of rotating and compounding the modified silk fibroin is repeated 6 times to form the core layer of the absorbable vascular stent covering membrane. The rotating speed of the rotating-dipping is 60 rpm, the rotating direction is along the circumferential direction of the stainless steel rod, and the dipping area accounts for 50% of the side area of the stainless steel rod.

[0074] (4) The method described in step (1) is used to electrospin the silk fibroin nanofiber on the surface of the core layer of the vascular stent covering membrane to construct the outer layer of the vascular stent covering membrane. The vascular stent covering membrane is soaked in sterile deionized water at 37°C for 1 day to remove unreacted crosslinking agent and silk fibroin molecules to obtain an absorbable vascular stent covering membrane that is beneficial to endothelialization and regulates the growth activity of endothelial cells.

[0075] The absorbable vascular stent covering film has excellent mechanical properties and promotes endothelial cell activity. The average diameter of the silk fibroin nanofiber is 700±100 nm, the porosity is 55±10%, and the fiber orientation degree is 25±10%. After 1 month of in vivo implantation, the degradation rate of the absorbable vascular stent covering film is significantly accelerated, and the weight loss rate is increased by 60% compared with the comparative example 1.

[0076] (1) The domestic silkworm silk is placed in a 0.1% by mass sodium carbonate aqueous solution at a bath ratio of 1:50 g / mL, treated at a temperature of 98-100°C for three times, each for 30 minutes, then washed with deionized water, and dried in a 60°C oven for 12 hours to obtain degummed silk fibers.

[0077] The degummed silk fibers are weighed and completely dissolved in a 9.3M lithium bromide solution at a bath ratio of 1:10 g / mL in a 65±5°C water bath environment to obtain a silk dissolution solution. The silk dissolution solution is perfused into a dialysis bag with a molecular weight cut-off of 14 kDa, and deionized water dialysis is performed for 3 days to obtain a purified silk protein aqueous solution. The purified silk protein aqueous solution is concentrated and adjusted to the required concentration.

[0078] The silk protein aqueous solution is mixed with a hydrophilic and flexible crosslinking agent to obtain a modified silk protein solution, and the mass ratio of silk protein to crosslinking agent is 1.0:0.5. The crosslinking agent is polyethylene glycol diglycidyl ether.

[0079] (2) The domestic silkworm silk is treated by plying, twisting, and degumming to obtain degummed twisted silk threads. The domestic silkworm silk has a fineness of 20 / 22D, the number of plies is 2, the twisting direction is S, and the twisting degree is 500 twists per meter. The degumming process is the same as step (1). The degummed silk threads are knitted into a tubular fabric with a knitting angle of 120° and an axial knitting density of 4 roots / cm on the outer surface as the inner layer of the absorbable vascular stent covering film.

[0080] (3) The tubular fabric is placed in a modified silk protein solution with a concentration of 60 mg / mL in a rotary-dipping manner for 15 seconds, and then taken out and kept rotating at the above-mentioned rotation speed for 15 minutes in a 35°C environment, so that the surface of the film is kept wet. The rotary-dipping and modification of the silk protein are repeated for 6 times to form the core layer of the absorbable vascular stent covering film. The rotation speed of the rotary-dipping is 60 rpm, the rotation direction is along the circumferential direction of the stainless steel rod, and the dipping area accounts for 50% of the side area of the stainless steel rod. The vascular stent covering film is further soaked in 37°C sterile deionized water for 1 day to remove unreacted crosslinking agents and silk molecules, and an absorbable vascular stent covering film is obtained. The thickness deviation of the absorbable vascular stent covering film is less than 10 μm.

[0081] (4) The tubular fabric is placed in a modified silk fibroin solution with a silk fibroin concentration of 60 mg / mL in a static-dipping manner for 15 seconds, the dipping area accounts for 100% of the area of the stainless steel rod side, and after being taken out, it is placed in a 35°C environment for static-drying or rotary-drying for 15 minutes, the surface of the film is controlled to remain wet, the step of dipping and compounding modified silk fibroin is repeated 6 times, and a vascular stent coating film is formed. The vascular stent coating film is further soaked in 37°C sterile deionized water for 1 day to remove unreacted cross-linking agents and silk molecules, and an absorbable vascular stent coating film is obtained. The thickness deviation of the absorbable vascular stent coating film is > 50 μm (static-drying) and > 20 μm (rotary-drying), respectively.

[0082] In summary, the absorbable vascular stent coating film with endothelialization facilitating function has good mechanical properties and excellent endothelial cell activity, and can be used to produce a vascular stent capable of rapidly forming a new endothelial layer, maintaining stable blood flow, and inhibiting thrombosis and inflammation.

[0083] The embodiments are preferred embodiments of the present application, but the present application is not limited to the above embodiments, and any obvious improvements, replacements or modifications made by those skilled in the art without departing from the essential content of the present application shall fall within the protection scope of the present application.

Claims

1. A method for preparing an absorbable vascular stent graft that facilitates endothelialization, characterized in that, include: (1) Mix the aqueous solution of silk fibroin with a hydrophilic and flexible crosslinking agent to obtain a modified silk fibroin solution; Then, using modified silk fibroin as the electrospinning solution, silk fibroin nanofibers were spun on the auxiliary rod by electrospinning to form the inner layer of the absorbable vascular stent coating. The average porosity of the silk fibroin nanofibers is 35-90%, and the average degree of orientation of the silk fibroin nanofibers is 30-90%. (2) The silk threads processed from silkworm raw silk are woven into a tubular fabric with a braiding angle of 30-150° and an axial braiding density of 1-20 threads / cm on the inner layer of an absorbable vascular stent covering that facilitates endothelialization, using a braiding technique; the silk threads are degummed silk threads obtained by processing silkworm raw silk through a process of combining, twisting, and degumming, and / or irradiated silk threads obtained by irradiating degummed silk threads with 60Co γ-rays. The method for obtaining the irradiated filaments is as follows: The degummed yarn was treated with 60Co γ-ray irradiation to obtain irradiated yarn, wherein the 60Co γ-ray irradiation dose was 25~300kGy; The filaments are degummed filaments and irradiated filaments arranged in a uniform ratio of 6:0 to 0:

6. (3) The obtained tubular fabric is placed in the modified silk fibroin solution for 10-60 seconds by rotation-immersion, then taken out and placed in an environment of 20-50℃ for 10-30 minutes. This is recorded as one operation step. The above operation steps are repeated several times to form the core layer of absorbable vascular stent coating. (4) The modified silk fibroin solution in step (1) is used as an electrospinning solution. Silk fibroin nanofibers are electrospinned on the core layer surface of the absorbable vascular stent membrane by electrospinning to form an absorbable vascular stent membrane. The absorbable vascular stent membrane is then immersed in sterile deionized water for washing to remove unreacted crosslinking agents and silk fibroin molecules, thereby obtaining the absorbable vascular stent membrane that facilitates endothelialization.

2. The method for preparing an absorbable vascular stent coating that facilitates endothelialization according to claim 1, characterized in that, In step (1), the method for obtaining the silk fibroin aqueous solution is as follows: the raw silk of the silkworm is degummed using boiling water, sodium carbonate, sodium bicarbonate or biological enzyme to obtain degummed silk fibroin fibers; the degummed silk fibroin fibers are completely dissolved in lithium bromide solution to obtain silk fibroin solution; then the silk fibroin solution is poured into a dialysis bag and dialyzed with deionized water, filtered, and evaporated to obtain silk fibroin aqueous solution.

3. The method for preparing an absorbable vascular stent coating that facilitates endothelialization according to claim 1, characterized in that, In step (1), the mass ratio of the silk fibroin aqueous solution to the crosslinking agent is 1.0:(0.3~1.0); the concentration of silk fibroin in the modified silk fibroin solution is 20~180mg / mL; The crosslinking agent is polyethylene glycol diglycidyl ether.

4. The method for preparing an absorbable vascular stent coating that facilitates endothelialization according to claim 1, characterized in that, In step (1), the rotation speed of the auxiliary rod is 100~3000rpm, the diameter of the auxiliary rod is 1~30mm, and the average diameter of the silk nanofiber is 100~5000nm.

5. The method for preparing an absorbable vascular stent coating that facilitates endothelialization according to claim 1, characterized in that, In step (2), the method for obtaining the degummed yarn is as follows: 2-10 strands of raw silkworm silk with a fineness of 20 / 22D are combined and then twisted in the S / Z direction with a twist of 100-2000 twists / meter. Then, degumming is performed using any one of boiling water, sodium carbonate, sodium bicarbonate or biological enzymes to obtain degummed silk thread.

6. The method for preparing an absorbable vascular stent coating that facilitates endothelialization according to claim 1, characterized in that, In step (3), the rotation speed of the rotation-impregnation is 10~100rpm, the rotation direction is along the circumference of the auxiliary rod, and the impregnation area accounts for 10~90% of the side area of ​​the auxiliary rod.

7. The method for preparing an absorbable vascular stent coating that facilitates endothelialization according to claim 1, characterized in that, The operation steps are repeated 2 to 10 times.

8. The method for preparing an absorbable vascular stent coating that facilitates endothelialization according to claim 1, characterized in that, In step (4), the washing process involves immersing the vascular stent membrane in sterile deionized water at 4-37°C for 1-3 days.

9. The absorbable vascular stent coating for endothelialization prepared by the method according to any one of claims 1 to 8, characterized in that, The absorbable vascular stent membrane has a core layer of tubular fabric woven from silk threads processed from raw silkworm silk, and oriented modified silk fibroin nanofibers as the inner and outer layers. The inner surface of the absorbable vascular stent membrane has a nano-topological structure and matrix-like components that mimic the vascular basement membrane, and the outer surface has a micro-topological structure.