CN113274561B - Medical intravascular stent material and preparation method thereof - Google Patents
Medical intravascular stent material and preparation method thereof Download PDFInfo
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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
- A61L31/04—Macromolecular materials
- A61L31/041—Mixtures of macromolecular compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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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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- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
- D01D10/00—Physical treatment of artificial filaments or the like during manufacture, i.e. during a continuous production process before the filaments have been collected
- D01D10/06—Washing or drying
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- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
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- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
- D01D5/00—Formation of filaments, threads, or the like
- D01D5/28—Formation of filaments, threads, or the like while mixing different spinning solutions or melts during the spinning operation; Spinnerette packs therefor
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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
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
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Abstract
A medical blood vessel support material is a double-layer composite material prepared by taking fibroin powder, polylactic acid, polycaprolactone, chitosan and recombinant human collagen as raw materials and sequentially soaking ammonia water and warm water through coaxial electrostatic spinning, wherein the fiber diameter of the material is distributed between 130 nm and 160nm, and the average value is about 144 nm. The polymer scaffold composite material prepared by the invention has excellent structural stability, does not generate layering and falling off in the using process, has good biomechanical property and cell compatibility, has the supporting force of 3.125-3.035 MPa, and can effectively form support and protect blood vessels from being damaged when being used for a vascular scaffold, thereby reducing the risk of intimal hyperplasia.
Description
Technical Field
The invention relates to the technical field of medical materials, in particular to a medical intravascular stent material and a preparation method thereof.
Background
Scaffold materials are often used in surgical procedures, such as vascular scaffolds, intestinal scaffolds to provide support, and in addition, artificial bone scaffolds, artificial organ scaffolds, etc. to serve as carriers of nutrients and to provide a dimensional structure.
After the stent is implanted into a vascular lesion, the blood vessel is remodeled, and once the blood vessel is repaired by self, the existence of the stent is meaningless. Early stent materials usually adopt metal or other non-degradable materials, usually need to be taken out by secondary operation, and simultaneously have some negative effects, which cause injury to blood vessels and increase the occurrence probability of long-term thrombosis and restenosis after operation; the bio-absorbable stent is made of a material which can be dissolved in a human body, and can be degraded and absorbed by the human body after being placed in the human body.
The high molecular biodegradable stent is completely absorbed by a human body after the blood vessel is self-repaired, keeps the blood vessel smooth and can inhibit early blood vessel formation and late neointimal hyperplasia, thereby realizing the positive remodeling of the blood vessel. The high molecular degradable stent is soft and easy to deform, and has smaller shearing stress on blood vessels, thereby reducing the risk of intimal hyperplasia. Existing materials for degradable stents include polylactic acid (PLA), polyglycolic acid (PGA), and the like.
An ideal stent would have the following advantages: (1) has high porosity and specific surface area to accommodate cell adhesion, growth and differentiation, intercellular signaling, nutrient transport, and excretion of degradation and metabolism products; (2) has good mechanical property to support cell differentiation and proliferation; (3) has good biocompatibility, and the self and degradation products thereof are nontoxic to cells and matrixes and do not cause inflammation or rejection reaction; (4) has moderate biodegradation rate and can be matched with tissue regeneration rate.
However, the polymer biodegradable stent has certain limitations, such as polylactic acid material with excellent biodegradability, but because the polymer material has no skeleton inside, the radial support force and the pressure resistance of the polymer material are not as good as those of a metal stent, and although the blood vessel is protected from being damaged by the stent, the support performance of the stent is also reduced, and the blood vessel can not be ensured to be unblocked before the benign remodeling of the blood vessel is completed.
Disclosure of Invention
The invention aims to provide a medical blood vessel stent material.
The invention also aims to provide the preparation method of the medical vascular stent material, and the prepared material can protect blood vessels from being damaged when being used for supporting blood vessels, can ensure that the stent has excellent supporting performance and has excellent biocompatibility and biodegradability.
The purpose of the invention is realized by the following technical scheme:
a medical blood vessel stent material is characterized in that: the medical intravascular stent material is a double-layer composite material prepared by taking fibroin, polylactic acid, polycaprolactone, chitosan and recombinant human collagen as raw materials and sequentially soaking ammonia water and warm water through electrostatic spinning, wherein the fiber diameter of the material is distributed between 130 nm and 160nm, and the average value is about 144 nm.
A preparation method of a medical blood vessel stent material is characterized by comprising the following steps: sequentially adding fibroin powder, polylactic acid and polycaprolactone into a mixed solution composed of acetone and acetic acid according to a mass ratio of 4-6.5: 3-4.8, stirring at a speed of 400-500 rpm for 30-40 min at 30-45 ℃ to prepare an inner layer spinning solution, dissolving chitosan, polycaprolactone and recombinant human collagen in a trifluoroacetic acid solution with a mass concentration of 0.5-2% at 50-60 ℃ to prepare an outer layer spinning solution, performing coaxial electrostatic spinning to prepare a double-layer composite material, soaking in ammonia water at normal temperature, heating and soaking in purified water, washing, and freeze-drying.
Based on the fact that the fibroin has good biocompatibility and biodegradability, the fibroin is considered to be used for a vascular stent, is beneficial to the proliferation of Human Aortic Endothelial Cells (HAEC) and Human Coronary Artery Smooth Muscle Cells (HCASMC), well keeps the cell phenotype and promotes cell recombination. The material prepared by electrostatic spinning has excellent porosity and specific surface area, and is beneficial to cell adhesion and proliferation, but the fibroin scaffold material prepared by electrostatic spinning has high brittleness, and the electrostatic action of fibroin causes serious fiber aggregation, so that the application of the fibroin scaffold material as a tissue engineering scaffold material is limited. Polycaprolactone has excellent toughness, and is compound with fibroin, can modify fibroin, reduces fibroin fragility, but polycaprolactone adds the back and carries out electrostatic spinning, can lead to in the material of preparation fibroin and polycaprolactone's compatibility to reduce, takes place easily that component separation, structural stability worsen.
In the preparation process, the solvent for preparing the spinning solution has great influence on the structure of the fiber formed by spinning, so that the prepared material has great difference in performance. The crystalline region of the fibroin is mainly a secondary structure with a relatively compact beta-sheet conformation, wherein peptide chains are arranged layer by layer, a stable structure is maintained by virtue of a hydrogen bond formed by amino and carboxyl on adjacent peptide chains, and the fibroin is expanded but not dissolved in water and is the basis of being used as a scaffold material.
The fibroin, polylactic acid and polycaprolactone are mixed and dissolved in a mixed solvent of acetone and acetic acid, and inner-layer spinning solution is formed by stirring, so that the phase change of an atactic curling structure in the fibroin into a beta folding structure is promoted, the content of the beta folding structure in a fibroin crystallization area is increased, and the structural stability of the fibroin crystal area is enhanced. The polylactic acid increases the conductivity of the inner layer spinning solution under the premise that the pH environment of the system is adjusted by the solvent, so that the formed fiber has the advantages of small diameter, narrow diameter distribution and excellent dispersibility in the electrostatic spinning process, and the viscosity of the spinning solution is increased, so that the silk protein and the polycaprolactone fiber are bonded, and the component separation is not easy to occur. When preparing the outer-layer spinning solution, the invention adopts polycaprolactone, chitosan and recombinant human collagen to dissolve in trifluoroacetic acid solution at 50-60 ℃ to form the outer-layer spinning solution, the chitosan can not form fibers, but the conductivity and viscosity of the spinning solution are increased, so that the diameter of the fibers formed by spinning is small, the fibers are uniformly distributed, meanwhile, in the electrostatic spinning process, groups such as hydroxyl, carbonyl, ether bond and the like contained in the chitosan and the polycaprolactone and carboxyl and amino groups contained in the recombinant human collagen form stronger intermolecular attraction, the inner layer and the outer layer of the prepared composite material both contain polycaprolactone, the nano composite material prepared by spinning is firstly soaked in ammonia water at normal temperature, the organic solvent is removed, then the nano composite material is soaked in warm water at 50-60 ℃, the polycaprolactone in the inner layer and the outer layer of fibers is melted to a certain degree, so that the cross contact interface of the inner layer and the outer layer of fiber structures is bonded, the structural stability of the inner layer and the outer layer is enhanced, the condition that the inner layer and the outer layer fall off is avoided, the crystallinity of fibers is improved, the mechanical property of the composite material is enhanced, and the problems that the degradability is reduced due to the increase of the crystallinity are solved because the inner layer and the outer layer are respectively provided with the fibroin and the recombinant human collagen.
Further, the method for extracting the fibroin specifically comprises the steps of heating a sodium bicarbonate ethanol solution to 60-70 ℃, adding the silkworm cocoons, preserving heat, continuously stirring for 30-50 min, filtering, washing with clear water, drying to obtain fibroin fibers, completely dissolving the fibroin fibers in a calcium chloride aqueous solution with the mass concentration of 40-55%, filtering, dialyzing, concentrating the fibroin fibers after dialysis to the concentration of 6-9 g/mL, centrifuging, filtering to obtain a purified fibroin liquid, and freeze-drying to obtain fibroin powder.
In the process of extracting the fibroin, the temperature of the sodium bicarbonate ethanol solution is raised to 60-70 ℃, sericin is removed, the beta-folded structure in the prepared fibroin is tighter, the spinning solution is prepared by using a mixed organic solvent of acetone and acetic acid, the content of the beta-folded structure is promoted, the structural stability is improved, the brittleness of the fibroin is reduced during electrostatic spinning, and performance change does not occur during ammonia water and warm water soaking in the later period.
Further, the mass concentration of the sodium bicarbonate ethanol solution is 0.3-1.5%, and the mass ratio of the silkworm cocoon to the sodium bicarbonate ethanol solution is 1-1.5: 10.
Further, the ammonia water concentration is 0.05mol/L, the soaking time is 20min, and residual acetone and trifluoroethanol in the composite material are removed.
Further, the above-mentioned purified water is heated and soaked at 50-60 deg.C for 2-3 h.
Further, the mass ratio of the fibroin powder to the mixed liquid of polylactic acid, polycaprolactone and acetone acetic acid in the inner layer spinning solution is 1-10: 1-5: 0.1-2: 40-73.
Further, the mass ratio of the chitosan to the polycaprolactone to the recombinant human collagen to the trifluoroacetic acid solution in the outer-layer spinning solution is 1-8: 8-12: 15-22: 80-120.
Further, the extrusion speed of the electrostatic spinning inner layer spinning solution is 2-10 mL/h, the extrusion speed of the outer layer spinning solution is 7-18 mL/h, and the receiving distance is 15-20 cm.
Further, the positive voltage of the electrostatic spinning is 18-25 KV, the negative voltage is 4-6 KV, the rotating speed is 120-150 rpm, the temperature is 37-45 ℃, and the humidity is 20-35%.
Further, the washing is to wash the inner layer of the soaked double-layer composite material by sequentially adopting 75% by mass, 50% by mass and 15% by mass of ethanol and purified water.
Further, the dialysis specifically comprises the steps of adding absolute ethyl alcohol into the filtrate after filtration, transferring the filtrate into a dialysis bag with the molecular weight of 8-14 kDa, dialyzing the filtrate with distilled water for 72 hours, and replacing the distilled water every 2-3 hours to obtain the salinolysis fibroin solution.
Most specifically, the preparation method of the medical blood vessel stent material is characterized by comprising the following steps:
step 1, preparing fibroin powder
Heating a sodium bicarbonate ethanol solution with the mass concentration of 0.3-1.5% to 60-70 ℃, adding silkworm cocoons, preserving heat, continuously stirring for 30-50 min, wherein the mass ratio of the silkworm cocoons to the sodium bicarbonate ethanol solution is 1-1.5: 10, filtering, washing with clear water, drying to obtain silk fibroin fibers, completely dissolving the silk fibroin fibers in a calcium chloride aqueous solution with the mass concentration of 40-55%, filtering, adding absolute ethanol into the filtrate, transferring into a dialysis bag with the mass concentration of 8-14 kDa, dialyzing with distilled water for 72h, replacing distilled water every 2-3 h to obtain a hydrolyzed silk fibroin solution, concentrating the dialyzed solution to the concentration of 6-9 g/mL, centrifuging, filtering to obtain a purified silk fibroin solution, and freeze-drying to obtain silk fibroin powder;
step 2, preparing the double-layer composite material
(1) Preparation of the spinning dope
Sequentially adding fibroin powder and polylactic acid into a mixed solution consisting of acetone and acetic acid according to a mass ratio of 4-6.5: 3-4.8, stirring at a speed of 400-500 rpm for 30-40 min at 30-45 ℃ to prepare an inner layer spinning solution, wherein the mass ratio of the fibroin powder to the mixed solution of polylactic acid, polycaprolactone and acetone-acetic acid is 1-10: 1-5: 0.1-2: 40-73;
dissolving chitosan, polycaprolactone and recombinant human collagen in a trifluoroacetic acid solution with the mass concentration of 0.5-2% at 50-60 ℃ to prepare an outer-layer spinning solution, wherein the mass ratio of the chitosan, the polycaprolactone, the recombinant human collagen and the trifluoroacetic acid solution is 1-8: 8-12: 15-22: 80-120;
(2) electrostatic spinning
Under the parameter environment that the positive voltage is 18-25 KV, the negative voltage is 4-6 KV, the rotating speed is 120-150 rpm, the temperature is 37-45 ℃, and the humidity is 20-35%, the extrusion speed of the spinning solution at the inner layer is 2-10 mL/h, the extrusion speed of the spinning solution at the outer layer is 7-18 mL/h, the receiving distance is 15-20 cm, and the double-layer composite material is formed by spinning;
step 3, post-treatment
Soaking the double-layer composite material prepared by spinning in ammonia water with the concentration of 0.05mol/L for 20min, then transferring to purified water, heating to 50-60 ℃, reacting for 2-3 h, then sequentially washing the inner layer of the double-layer composite material by using ethanol with the mass concentration of 75%, 50% and 15% and the purified water, and freeze-drying.
The invention has the following technical effects:
the invention solves the problems of large brittleness, poor compatibility with polycaprolactone, easy component separation and falling off, poor structural stability, large diameter of prepared fiber and wide distribution range of the electrostatic spinning scaffold material prepared by adopting fibroin as a raw material, so that the polymer medical intravascular scaffold composite material prepared by the invention has the advantages of small fiber diameter with the mean value of 144nm, diameter distribution of 130-160 nm and porosity of 77.9 percent, excellent specific surface area and porosity, excellent mechanical property, good biomechanical property and cell compatibility, and supporting force of 3.125-3.305 MPa, and can effectively form support and protect blood vessels from being damaged when being used for intravascular scaffolds, thereby reducing the risk of intimal hyperplasia. In addition, the composite material has excellent degradability, and after the degradation product is absorbed by a human body, vascular remodeling is further promoted.
Detailed Description
The present invention is described in detail below by way of examples, it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and those skilled in the art can make some insubstantial modifications and adaptations of the present invention based on the above-mentioned disclosure.
Example 1
A preparation method of a medical blood vessel stent material comprises the following steps:
step 1, preparing fibroin powder
Heating a sodium bicarbonate ethanol solution with the mass concentration of 0.3-1.5% to 60 ℃, adding silkworm cocoons, keeping the temperature and continuously stirring for 50min, wherein the mass ratio of the silkworm cocoons to the sodium bicarbonate ethanol solution is 1.5:10, filtering, washing with clear water, drying to obtain silk fibroin fibers, completely dissolving the silk fibroin fibers in a calcium chloride aqueous solution with the mass concentration of 55%, filtering, adding absolute ethyl alcohol into the filtrate, transferring the filtrate into a dialysis bag with the concentration of 14kDa, dialyzing with distilled water for 72h, replacing the distilled water every 3h to obtain a hydrolyzed silk fibroin liquid, concentrating the dialyzed silk fibroin liquid to the concentration of 9g/mL, centrifuging, filtering to obtain a purified silk fibroin liquid, and freeze-drying to obtain silk fibroin powder;
step 2, preparing the double-layer composite material
(1) Preparation of the spinning dope
Sequentially adding fibroin powder, polylactic acid and polycaprolactone into a mixed solution composed of acetone and acetic acid according to a mass ratio of 6.5:4.8, and stirring at a speed of 400rpm at 45 ℃ for 30min to prepare an inner-layer spinning solution, wherein the mass ratio of the fibroin powder, the polylactic acid, the polycaprolactone and the mixed solution of acetone and acetic acid is 10:5:2: 73;
dissolving chitosan, polycaprolactone and recombinant human collagen in 2% trifluoroacetic acid solution at 50 ℃ to prepare outer-layer spinning solution, wherein the mass ratio of the chitosan to the polycaprolactone to the recombinant human collagen to the trifluoroacetic acid solution is 8:12:22: 120;
(2) electrostatic spinning
Under the parameter environment that the positive voltage is 25KV, the negative voltage is 6KV, the rotating speed is 150rpm, the temperature is 37 ℃ and the humidity is 35%, the extrusion speed of the spinning solution of the inner layer is 10mL/h, the extrusion speed of the spinning solution of the outer layer is 18mL/h, the receiving distance is 20cm, and the spinning forms a double-layer composite material;
step 3, post-treatment
Soaking the double-layer composite material prepared by spinning in ammonia water with the concentration of 0.05mol/L for 20min, then transferring to purified water, heating to 60 ℃, reacting for 2h, then washing the inner layer of the double-layer composite material by using ethanol with the mass concentration of 75%, 50% and 15% and the purified water in sequence, and freeze-drying.
The mean fiber diameter of the polymer scaffold composite material prepared by the embodiment is 139nm, the diameter is distributed between 120 nm and 160nm, the porosity is 72.6%, and the elongation is 16.92%.
Example 2
A preparation method of a medical blood vessel stent material comprises the following steps:
step 1, preparing fibroin powder
Heating a sodium bicarbonate ethanol solution with the mass concentration of 0.3-1.5% to 60 ℃, adding silkworm cocoons, preserving heat, continuously stirring for 50min, wherein the mass ratio of the silkworm cocoons to the sodium bicarbonate ethanol solution is 1:10, filtering, washing with clear water, drying to obtain silk fibroin fibers, completely dissolving the silk fibroin fibers in a calcium chloride aqueous solution with the mass concentration of 40%, filtering, adding absolute ethanol into filtrate, transferring the filtrate into a dialysis bag with the mass concentration of 8kDa, dialyzing with distilled water for 72h, replacing distilled water every 2h to obtain a hydrolyzed silk fibroin liquid, concentrating the dialyzed silk fibroin liquid until the concentration is 6g/mL, centrifuging, filtering to obtain a purified silk fibroin liquid, and freeze-drying to obtain silk fibroin powder;
step 2, preparing the double-layer composite material
(1) Preparation of the spinning dope
Sequentially adding fibroin powder, polylactic acid and polycaprolactone into a mixed solution consisting of acetone and acetic acid according to a mass ratio of 4:3, stirring at a speed of 500rpm for 30min at 30 ℃ to prepare an inner-layer spinning solution, wherein the mass ratio of the fibroin powder to the mixed solution of polylactic acid, polycaprolactone and acetone-acetic acid is 1:1:0.1: 40;
dissolving chitosan, polycaprolactone and recombinant human collagen in a trifluoroacetic acid solution with the mass concentration of 0.5% at 60 ℃ to prepare an outer-layer spinning solution, wherein the mass ratio of the chitosan to the polycaprolactone to the recombinant human collagen to the trifluoroacetic acid solution is 1:8:15: 80;
(2) electrostatic spinning
Under the parameter environment that the positive voltage is 18KV, the negative voltage is 4KV, the rotating speed is 120rpm, the temperature is 45 ℃, and the humidity is 20%, the extrusion speed of the spinning solution at the inner layer is 2mL/h, the extrusion speed of the spinning solution at the outer layer is 7mL/h, the receiving distance is 15cm, and the spinning forms a double-layer composite material;
step 3, post-treatment
Soaking the double-layer composite material prepared by spinning in ammonia water with the concentration of 0.05mol/L for 20min, then transferring to purified water, heating to 50 ℃, reacting for 3h, then washing the inner layer of the double-layer composite material by using ethanol with the mass concentration of 75%, 50% and 15% and the purified water in sequence, and freeze-drying.
The average diameter of the fiber of the polymer scaffold composite material prepared by the embodiment is 151nm, the diameter is distributed between 125-173 nm, the porosity is 70.9%, and the elongation is 16.57%.
Example 3
A preparation method of a medical intravascular stent material comprises the following steps:
step 1, preparing fibroin powder
Heating a sodium bicarbonate ethanol solution with the mass concentration of 1% to 65 ℃, adding silkworm cocoons, preserving heat, continuously stirring for 40min, wherein the mass ratio of the silkworm cocoons to the sodium bicarbonate ethanol solution is 1.2:10, filtering, washing with clear water, drying to obtain silk fibroin fibers, completely dissolving the silk fibroin fibers in a calcium chloride aqueous solution with the mass concentration of 50%, filtering, adding absolute ethanol into filtrate, transferring the filtrate into a dialysis bag with the concentration of 8kDa, dialyzing with distilled water for 72h, replacing distilled water every 2.5h to obtain a hydrolyzed silk fibroin liquid, concentrating the solution after dialysis until the concentration is 8g/mL, centrifuging, filtering to obtain a purified silk fibroin liquid, and freeze-drying to obtain silk fibroin powder;
step 2, preparing the double-layer composite material
(1) Preparation of the spinning dope
Sequentially adding fibroin powder, polylactic acid and polycaprolactone into a mixed solution consisting of acetone and acetic acid according to a mass ratio of 5:4, and stirring at 40 ℃ and a speed of 450rpm for 35min to prepare an inner-layer spinning solution, wherein the mass ratio of the fibroin powder, the polylactic acid, the polycaprolactone and the mixed solution of acetone and acetic acid is 5:2:1: 60;
dissolving chitosan, polycaprolactone and recombinant human collagen in a trifluoroacetic acid solution with the mass concentration of 1% at 55 ℃ to prepare an outer-layer spinning solution, wherein the mass ratio of the chitosan to the polycaprolactone to the recombinant human collagen to the trifluoroacetic acid solution is 5:10:18: 100;
(2) electrostatic spinning
Under the parameter environment that the positive voltage is 20KV, the negative voltage is 5KV, the rotating speed is 130rpm, the temperature is 40 ℃, and the humidity is 25%, the extrusion speed of the spinning solution at the inner layer is 8mL/h, the extrusion speed of the spinning solution at the outer layer is 15mL/h, the receiving distance is 18cm, and the spinning forms a double-layer composite material;
step 3, post-treatment
Soaking the double-layer composite material prepared by spinning in ammonia water with the concentration of 0.05mol/L for 20min, then transferring to purified water, heating to 55 ℃, reacting for 2.5h, then sequentially washing the inner layer of the double-layer composite material by using ethanol with the mass concentration of 75%, 50% and 15% and the purified water, and freeze-drying.
The average fiber diameter of the scaffold composite material prepared by the embodiment is 144nm, the diameter is distributed between 130 nm and 160nm, the porosity is 77.3%, the fiber surface is smooth, and the elongation is 17.39%.
Comparative example 1:
(1) preparing fibroin: placing the silkworm cocoon in Na with the mass fraction of 0.05% 2 CO 3 Boiling the aqueous solution for 30min, repeating the steps for 3 times, removing sericin, completely dissolving the aqueous solution of calcium chloride with the mass concentration of 50%, filtering, adding absolute ethyl alcohol into filtrate, transferring the filtrate into a dialysis bag with 8kDa, dialyzing the filtrate with distilled water for 72 hours, replacing the distilled water every 2.5 hours to obtain salinolysis fibroin liquid, concentrating the dialyzed filtrate until the concentration is 8g/mL, centrifuging the dialyzed solution, filtering the dialyzed solution to obtain purified fibroin liquid, and freeze-drying the purified fibroin liquid to obtain fibroin powder;
the preparation of the spinning solution and the electrostatic spinning are the same as those in example 3, and a double-layer composite material is prepared, and then the same soaking and washing treatment is carried out, and the freeze drying is carried out.
The diameter of the fiber in the scaffold composite material prepared in the comparative example 1 is 150-220 nm, the average diameter is 170nm, the porosity is 63.7%, and the surface is rough. The elongation of the prepared composite material is 7.66% and is more brittle than that of the embodiment 3.
Comparative example 2:
(1) fibroin was prepared as in example 3;
(2) electrostatic spinning: dissolving fibroin powder, polylactic acid and polycaprolactone in hexafluoroisopropanol solution to prepare inner-layer spinning solution, wherein the mass ratio of the fibroin powder to the polycaprolactone to the hexafluoroisopropanol solution is 5:2:1: 60; the outer layer dope was the same as in example 3; a two-layer composite was formed by the same coaxial electrospinning as in example 3, followed by the same soaking, washing treatments, and freeze-drying.
The scaffold composite material prepared in the comparative example 2 has the fiber diameter distribution of 595-1250 nm, wider diameter distribution, 1040nm average diameter, 53.7% porosity, rough surface and 7.34% elongation.
Comparative example 3:
(1) fibroin was prepared as in example 3;
(2) electrostatic spinning: the inner layer spinning solution is the same as that of the example 3, the chitosan component is not contained in the outer layer spinning solution, a double-layer composite material is formed through coaxial electrostatic spinning which is the same as that of the example 3, and then the same soaking, washing and freeze drying are carried out.
The scaffold composite material prepared in comparative example 2 has the fiber diameter distribution of 460-990 nm, the diameter distribution is wide, the average diameter is about 730nm, the porosity is 57.4%, the surface is rough, and the elongation is 13.53%.
Comparative example 4:
a two-layer composite material was prepared as in example 3, and the organic solvent was removed with ammonia water, and then the two-layer composite material was directly washed and freeze-dried without being soaked in warm water.
The supporting force of the composite material is measured according to radial load test methods of balloon expansion and self-expanding vascular stents (YYT 1660-2019) and the iris method of ASTM F3067-14.
The results of the holding force measurements are shown in the table below.
According to the invention, the fibroin is dissolved by adopting a mixed solution of acetone and acetic acid as a solvent, polylactic acid and polycaprolactone are added, and polycaprolactone, chitosan and recombinant human collagen are dissolved by adopting a trifluoroacetic acid solution, and the solvent and auxiliary components cooperate with each other, so that the mechanical property of the composite material is improved, the compatibility of the material is improved, and the brittleness of the fibroin is reduced. From the comparative example 4 and the scaffold composite material prepared by the invention, it can be seen that the crystallinity of the film is increased by the warm water soaking post-treatment, and meanwhile, polycaprolactone in the material is melted, and the contact interface of the inner layer fiber and the outer layer fiber is bonded, so that the structural stability is enhanced, and the mechanical property of the material is improved.
Structural stability testing
The scaffold composite material prepared by the invention and the scaffold composite materials prepared by the comparative examples 1, 2, 3 and 4 are circularly infused by adopting the same tissue-mimicking liquid, the flow rate of the tissue-mimicking liquid is controlled to be 0.02mL/s and gradually increased to 0.1mL/s, and the formed shear stress is about 0.036N/m 2 And maintaining the flow rate, observing the material condition after 20 days, and finding that the scaffold composite materials prepared in comparative examples 1 and 2 have serious damage in structure, detect a large amount of precipitates formed by component shedding in the tissue-imitating liquid, have serious deformation in comparative example 3, have a very small amount of shedding precipitates in the tissue-imitating liquid, have slight deformation in comparative example 4, and have obvious layering phenomenon in the inner and outer layer structures, but no precipitate is generated in the tissue-imitating liquid.
The data show that the mechanical property of the scaffold composite material prepared by the invention is obviously improved, the brittleness of the fibroin is reduced, and the prepared composite material has high structural stability and is not easy to generate the phenomena of layering, falling off and the like. The comparative example 1 shows that the fibroin extracted by the sodium bicarbonate ethanol solution has lower brittleness, which is beneficial to improving the mechanical property of the material prepared by electrostatic spinning; known by comparative example 2, the structural stability of fibroin strengthens, polylactic acid has effectively improved the bonding strength between fibroin and polycaprolactone in the mixed organic solvent of acetone and acetic acid, the porosity and the fibre diameter homogeneity and the dispersibility of material have been improved simultaneously, thereby improve the mechanical properties of material, comparative example 3 can know, chitosan is to improving the cohesion between polycaprolactone and the recombinant human collagen, it has apparent effect to improve mechanical properties, can be clear and definite through comparative example 4, after organic solvent was got rid of to the aqueous ammonia, adopt warm water soaking again to handle, can show the bonding of reinforcing ectonexine fibre, and improve fibrous crystallinity, thereby the mechanical properties of reinforcing material.
Claims (7)
1. A medical blood vessel stent material is characterized in that: the polymer medical stent material is prepared by sequentially adding fibroin, polylactic acid and polycaprolactone into a mixed solution consisting of acetone and acetic acid according to a mass ratio of 4-6.5: 3-4.8, and stirring at a speed of 400-500 rpm for 30-40 min at a temperature of 30-50 ℃ to prepare an inner layer spinning solution; dissolving chitosan, polycaprolactone and recombinant human collagen in a trifluoroacetic acid solution with the mass concentration of 0.5-2% at 40-50 ℃ to prepare an outer-layer spinning solution, soaking the outer-layer spinning solution in ammonia water at normal temperature, then heating the soaked outer-layer spinning solution in purified water at 50-60 ℃ for 2-3 hours, washing, and freeze-drying to obtain a double-layer composite material, wherein the fiber diameter of the material is 130-160 nm, and the average value is about 144 nm; the mass ratio of the fibroin powder to the mixed solution of polylactic acid, polycaprolactone and acetone acetic acid in the inner layer spinning solution is 1-10: 1-5: 0.1-2: 40-73; the mass ratio of the chitosan to the polycaprolactone to the recombinant human collagen to the trifluoroacetic acid solution in the outer-layer spinning solution is 1-8: 8-12: 15-22: 80-120; the electrostatic spinning is characterized in that the positive voltage is 18-25 KV, the negative voltage is 4-6 KV, the rotating speed is 120-150 rpm, the temperature is 37-45 ℃, the humidity is 20-35%, the extrusion speed of the inner layer spinning solution is 2-10 mL/h, the extrusion speed of the outer layer spinning solution is 7-18 mL/h, and the receiving distance is 15-20 cm.
2. A method for preparing the medical blood vessel stent material of claim 1, which is characterized in that: sequentially adding fibroin powder, polylactic acid and polycaprolactone into a mixed solution consisting of acetone and acetic acid according to a mass ratio of 4-6.5: 3-4.8, stirring at a speed of 400-500 rpm for 30-40 min at 30-50 ℃ to prepare an inner-layer spinning solution, dissolving chitosan, polycaprolactone and recombinant human collagen in a trifluoroacetic acid solution with a mass concentration of 0.5-2% at 40-50 ℃ to prepare an outer-layer spinning solution, performing coaxial electrostatic spinning to prepare a double-layer composite material, soaking in ammonia water at normal temperature, then placing in purified water, heating and soaking, washing, and freeze-drying; the mass ratio of the fibroin powder to the mixed liquid of polylactic acid, polycaprolactone and acetone acetic acid in the inner layer spinning solution is 1-10: 1-5: 0.1-2: 40-73; the mass ratio of the chitosan to the polycaprolactone to the recombinant human collagen to the trifluoroacetic acid solution in the outer-layer spinning solution is 1-8: 8-12: 15-22: 80-120; the extrusion speed of the electrostatic spinning inner layer spinning solution is 2-10 mL/h, the extrusion speed of the outer layer spinning solution is 7-18 mL/h, and the receiving distance is 15-20 cm.
3. The method for preparing the medical blood vessel stent material as claimed in claim 2, wherein: the method specifically comprises the steps of heating a sodium bicarbonate ethanol solution to 60-70 ℃, adding silkworm cocoons, keeping the temperature, continuously stirring for 30-50 min, filtering, washing with clear water, drying to obtain silk fibroin fibers, completely dissolving the silk fibroin fibers in a calcium chloride aqueous solution with the mass concentration of 40-55%, filtering, dialyzing, concentrating after dialysis to the concentration of 6-9 g/mL, centrifuging, filtering to obtain a purified silk protein liquid, and freeze-drying to obtain silk protein powder.
4. The method for preparing the medical blood vessel stent material according to claim 3, characterized in that: the mass concentration of the sodium bicarbonate ethanol solution is 0.3-1.5%, and the mass ratio of the silkworm cocoons to the sodium bicarbonate ethanol solution is 1-1.5: 10.
5. The method for preparing the medical blood vessel stent material as claimed in claim 4, wherein: the washing is to wash the inner layer of the double-layer composite material after soaking by sequentially adopting 75%, 50% and 15% of ethanol and purified water by mass fraction.
6. The method for preparing the medical blood vessel stent material as claimed in claim 5, wherein: and specifically, adding absolute ethyl alcohol into the filtrate after filtration, transferring the filtrate into a dialysis bag with the molecular weight of 8-14 kDa, dialyzing the filtrate for 72 hours with distilled water, and replacing the distilled water every 2-3 hours to obtain the salinized fibroin protein solution.
7. The preparation method of the medical intravascular stent material is characterized by comprising the following steps of:
step 1, preparing fibroin powder
Heating a sodium bicarbonate ethanol solution to 60-70 ℃, adding silkworm cocoons, keeping the temperature, continuously stirring for 30-50 min, filtering, washing with clear water, drying to obtain silk fibroin fibers, completely dissolving the silk fibroin fibers in a calcium chloride aqueous solution with the mass concentration of 40-55%, filtering, adding absolute ethanol into filtrate, transferring the filtrate into a dialysis bag with the molecular weight of 8-14 kDa, dialyzing with distilled water for 72 hours, replacing the distilled water every 2-3 hours to obtain a hydrolyzed silk fibroin solution, concentrating the solution after dialysis until the concentration is 6-9 g/mL, centrifuging, filtering to obtain a purified silk fibroin solution, and freeze-drying to obtain silk fibroin powder;
step 2, preparing the double-layer composite material
(1) Preparation of the spinning dope
Sequentially adding fibroin powder, polylactic acid and polycaprolactone into a mixed solution consisting of acetone and acetic acid according to a mass ratio of 4-6.5: 3-4.8, and stirring at a speed of 400-500 rpm for 30-40 min at 30-50 ℃ to prepare an inner layer spinning solution, wherein the mass ratio of the fibroin powder, the polylactic acid, the polycaprolactone and the acetone-acetic acid mixed solution is 1-10: 1-5: 0.1-2: 40-73; dissolving chitosan, polycaprolactone and recombinant human collagen in a trifluoroacetic acid solution with the mass concentration of 0.5-2% at 40-50 ℃ to prepare an outer-layer spinning solution, wherein the mass ratio of the chitosan to the polycaprolactone to the recombinant human collagen to the trifluoroacetic acid solution is 1-8: 8-12: 15-22: 80-120;
(2) electrostatic spinning
Under the parameter environment that the positive voltage is 18-25 KV, the negative voltage is 4-6 KV, the rotating speed is 120-150 rpm, the temperature is 37-45 ℃, and the humidity is 20-35%, the extrusion speed of the spinning solution at the inner layer is 2-10 mL/h, the extrusion speed of the spinning solution at the outer layer is 7-18 mL/h, the receiving distance is 15-20 cm, and the double-layer composite material is formed by spinning;
and 3, carrying out post-treatment, soaking the double-layer composite material prepared by spinning in ammonia water at normal temperature for 20min, then transferring to purified water, heating to 50-60 ℃, soaking for 2-3 h, then sequentially washing the inner layer of the double-layer composite material by using 75%, 50% and 15% of ethanol and purified water in mass concentration, and carrying out freeze drying.
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