CN111000661B - Composite artificial blood vessel and preparation method thereof - Google Patents
Composite artificial blood vessel and preparation method thereof Download PDFInfo
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- CN111000661B CN111000661B CN202010092160.9A CN202010092160A CN111000661B CN 111000661 B CN111000661 B CN 111000661B CN 202010092160 A CN202010092160 A CN 202010092160A CN 111000661 B CN111000661 B CN 111000661B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/04—Hollow or tubular parts of organs, e.g. bladders, tracheae, bronchi or bile ducts
- A61F2/06—Blood vessels
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/0077—Special surfaces of prostheses, e.g. for improving ingrowth
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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- A—HUMAN NECESSITIES
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/24—Collagen
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- A—HUMAN NECESSITIES
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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
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/507—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials for artificial blood vessels
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- A—HUMAN NECESSITIES
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/58—Materials at least partially resorbable by the body
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- B32B3/00—Layered products comprising a layer with external or internal discontinuities or unevennesses, or a layer of non-planar form; Layered products having particular features of form
- B32B3/26—Layered products comprising a layer with external or internal discontinuities or unevennesses, or a layer of non-planar form; Layered products having particular features of form characterised by a particular shape of the outline of the cross-section of a continuous layer; characterised by a layer with cavities or internal voids ; characterised by an apertured layer
- B32B3/266—Layered products comprising a layer with external or internal discontinuities or unevennesses, or a layer of non-planar form; Layered products having particular features of form characterised by a particular shape of the outline of the cross-section of a continuous layer; characterised by a layer with cavities or internal voids ; characterised by an apertured layer characterised by an apertured layer, the apertures going through the whole thickness of the layer, e.g. expanded metal, perforated layer, slit layer regular cells B32B3/12
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- B32B37/06—Methods or apparatus for laminating, e.g. by curing or by ultrasonic bonding characterised by the heating method
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- B32B37/00—Methods or apparatus for laminating, e.g. by curing or by ultrasonic bonding
- B32B37/10—Methods or apparatus for laminating, e.g. by curing or by ultrasonic bonding characterised by the pressing technique, e.g. using action of vacuum or fluid pressure
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B32B5/00—Layered products characterised by the non- homogeneity or physical structure, i.e. comprising a fibrous, filamentary, particulate or foam layer; Layered products characterised by having a layer differing constitutionally or physically in different parts
- B32B5/02—Layered products characterised by the non- homogeneity or physical structure, i.e. comprising a fibrous, filamentary, particulate or foam layer; Layered products characterised by having a layer differing constitutionally or physically in different parts characterised by structural features of a fibrous or filamentary layer
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- B32—LAYERED PRODUCTS
- B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
- B32B5/00—Layered products characterised by the non- homogeneity or physical structure, i.e. comprising a fibrous, filamentary, particulate or foam layer; Layered products characterised by having a layer differing constitutionally or physically in different parts
- B32B5/22—Layered products characterised by the non- homogeneity or physical structure, i.e. comprising a fibrous, filamentary, particulate or foam layer; Layered products characterised by having a layer differing constitutionally or physically in different parts characterised by the presence of two or more layers which are next to each other and are fibrous, filamentary, formed of particles or foamed
- B32B5/24—Layered products characterised by the non- homogeneity or physical structure, i.e. comprising a fibrous, filamentary, particulate or foam layer; Layered products characterised by having a layer differing constitutionally or physically in different parts characterised by the presence of two or more layers which are next to each other and are fibrous, filamentary, formed of particles or foamed one layer being a fibrous or filamentary layer
- B32B5/26—Layered products characterised by the non- homogeneity or physical structure, i.e. comprising a fibrous, filamentary, particulate or foam layer; Layered products characterised by having a layer differing constitutionally or physically in different parts characterised by the presence of two or more layers which are next to each other and are fibrous, filamentary, formed of particles or foamed one layer being a fibrous or filamentary layer another layer next to it also being fibrous or filamentary
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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
- D01D5/00—Formation of filaments, threads, or the like
- D01D5/0007—Electro-spinning
- D01D5/0015—Electro-spinning characterised by the initial state of the material
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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
- D01D5/00—Formation of filaments, threads, or the like
- D01D5/0007—Electro-spinning
- D01D5/0015—Electro-spinning characterised by the initial state of the material
- D01D5/003—Electro-spinning characterised by the initial state of the material the material being a polymer solution or dispersion
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- D—TEXTILES; PAPER
- D04—BRAIDING; LACE-MAKING; KNITTING; TRIMMINGS; NON-WOVEN FABRICS
- D04H—MAKING TEXTILE FABRICS, e.g. FROM FIBRES OR FILAMENTARY MATERIAL; FABRICS MADE BY SUCH PROCESSES OR APPARATUS, e.g. FELTS, NON-WOVEN FABRICS; COTTON-WOOL; WADDING ; NON-WOVEN FABRICS FROM STAPLE FIBRES, FILAMENTS OR YARNS, BONDED WITH AT LEAST ONE WEB-LIKE MATERIAL DURING THEIR CONSOLIDATION
- D04H1/00—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres
- D04H1/70—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres
- D04H1/72—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres the fibres being randomly arranged
- D04H1/728—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres the fibres being randomly arranged by electro-spinning
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- A—HUMAN NECESSITIES
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- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/0077—Special surfaces of prostheses, e.g. for improving ingrowth
- A61F2002/0081—Special surfaces of prostheses, e.g. for improving ingrowth directly machined on the prosthetic surface, e.g. holes, grooves
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2210/00—Particular material properties of prostheses classified in groups A61F2/00 - A61F2/26 or A61F2/82 or A61F9/00 or A61F11/00 or subgroups thereof
- A61F2210/0004—Particular material properties of prostheses classified in groups A61F2/00 - A61F2/26 or A61F2/82 or A61F9/00 or A61F11/00 or subgroups thereof bioabsorbable
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- A61F2210/0076—Particular material properties of prostheses classified in groups A61F2/00 - A61F2/26 or A61F2/82 or A61F9/00 or A61F11/00 or subgroups thereof multilayered, e.g. laminated structures
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- A61F2250/0014—Special features of prostheses classified in groups A61F2/00 - A61F2/26 or A61F2/82 or A61F9/00 or A61F11/00 or subgroups thereof having different values of a given property or geometrical feature, e.g. mechanical property or material property, at different locations within the same prosthesis
- A61F2250/0023—Special features of prostheses classified in groups A61F2/00 - A61F2/26 or A61F2/82 or A61F9/00 or A61F11/00 or subgroups thereof having different values of a given property or geometrical feature, e.g. mechanical property or material property, at different locations within the same prosthesis differing in porosity
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- B32B2307/51—Elastic
Abstract
The invention relates to a composite artificial blood vessel and a preparation method for preparing the artificial blood vessel, wherein the artificial blood vessel is of a double-layer tubular structure and comprises a first nanofiber layer arranged on an inner layer and a second nanofiber layer arranged on an outer layer, the first nanofiber layer is in fit connection with the second nanofiber layer, the first nanofiber layer is formed by rolling one layer, two layers or multiple layers of nanofiber membranes, the second nanofiber layer is of an integrated structure, microporous structures are arranged on the first nanofiber layer and the second nanofiber layer, and the artificial blood vessel enables the degradable nanofiber small-caliber artificial blood vessel to realize three-dimensional infiltration of autologous cells and regeneration of new tissues before the fibers are degraded in a large amount, so that the blood vessel still has good strength and elasticity when the fibers are degraded in a large amount.
Description
Technical Field
The invention relates to an artificial blood vessel, in particular to a composite artificial blood vessel and a preparation method thereof.
Background
The vascular system includes the heart, blood vessels and blood. The heart delivers fresh blood to various parts of the body through large and small blood vessels for exchange of nutrients and waste products in the body to ensure proper functioning of the organs. However, atherosclerosis, hemangioma, external force wound and other causes various arterial and venous vascular defects, and repair treatment such as replacement, bypass and the like of the corresponding lesion vascular parts is required to be carried out by adopting vascular substitutes with different diameters through surgical operations. The vascular substitutes generally comprise autologous blood vessels, xenogeneic blood vessels, allogeneic blood vessels and artificial blood vessels, and a large amount of artificial blood vessels are clinically needed to repair diseased blood vessels because the autologous blood vessels are often limited by factors such as age of patients, existing vascular diseases and the like and the source problems of the xenogeneic blood vessels and the allogeneic blood vessels.
Electrospinning technology was proposed for the preparation of nanofiber materials as early as 1930 s, but has not been used for a relatively mature period of time until recently. Compared with the traditional spinning technology, the electrostatic spinning technology can obtain finer fibers, and can effectively control the diameter of the fibers and the shape of textiles, and besides, the nanofiber material prepared by the electrostatic spinning technology has larger specific surface area and higher porosity, can simulate the microstructure of extracellular matrixes, and has good biocompatibility. There are many studies on the preparation of degradable nanofiber small caliber artificial blood vessels (diameter 2-5 mm) by electrospinning method, and it is expected to regenerate autologous lesion blood vessels by using the degradable artificial blood vessels. However, the average pore size of electrospun nanofiber materials is generally less than 2 microns, while the diameters of smooth muscle cells and fibroblasts are 10-50 microns, and before the fibers are degraded and broken, the smooth muscle cells and the fibroblasts can only infiltrate into the vessel wall in small amounts, and it is difficult to infiltrate and generate new vascular tissues in large amounts, which limits the speed of vascular regeneration. After the artificial blood vessel is implanted into a body, the mechanical properties of the artificial blood vessel depend on the mechanical properties of nanofibers in the wall of the blood vessel and the mechanical properties of the neogenesis tissues, when the nanofibers begin to degrade in a large amount and break, the fibers basically lose the strength and the elasticity of the fibers, and if no neogenesis tissues with certain mechanical properties are generated, the strength and the elasticity of the artificial blood vessel are basically lost. When the artificial blood vessel does not have strength and elasticity, the vessel wall collapse occurs due to external pressure or the vessel wall rupture occurs due to pressure which cannot bear blood flow, thereby failing the revascularization operation.
Disclosure of Invention
The invention provides a composite artificial blood vessel, which aims to solve the problem that smooth muscle cells and fibroblasts are difficult to infiltrate in a large amount before fibers are degraded in a large amount and break in the conventional electrostatic spinning nanofiber artificial blood vessel, and the artificial blood vessel enables the degradable nanofiber small-caliber artificial blood vessel to realize three-dimensional infiltration of autologous cells and regeneration of new tissues before the fibers are degraded in a large amount, so that the blood vessel still has better strength and elasticity when the fibers are degraded in a large amount.
The technical scheme adopted by the invention is as follows: the composite artificial blood vessel is of a double-layer tubular structure and comprises a first nanofiber layer arranged on an inner layer and a second nanofiber layer arranged on an outer layer, wherein the first nanofiber layer is in fit connection with the second nanofiber layer, the first nanofiber layer is formed by rolling one layer, two layers or multiple layers of nanofiber membranes, the second nanofiber layer is of an integrated structure, and microporous structures are arranged on the first nanofiber layer and the second nanofiber layer;
the thickness of the first nanofiber layer is 50-100 micrometers, the average pore diameter of the microporous structure is 0.6-1.2 micrometers, and the average diameter of the fibers of the first nanofiber layer is 200-1000 nanometers;
the thickness of the second nanofiber layer is 200-600 microns, the average pore diameter of the microporous structure is 20-100 microns, and the average diameter of the fibers of the second nanofiber layer is 500-1400 nanometers.
Further, the thickness of the nanofiber membrane forming the first nanofiber layer is 5-50 microns.
Further, the first nanofiber layer is a collagen and caprolactone lactate copolymer nanofiber layer. Collagen has good biocompatibility and good hydrophilicity. Good biocompatibility, can reduce rejection reaction when the artificial blood vessel is used; good hydrophilicity, favorability for adhesion of endothelial cells, rapid endothelialization of artificial blood vessels, and reduced thrombosis. The lactic acid caprolactone copolymer is a degradable polymer synthetic material, has good biocompatibility and mechanical property, and can enable the nanofiber layer 1 to have good strength and elasticity.
Further, the second nanofiber layer is a caprolactone lactate copolymer nanofiber layer. The lactic acid caprolactone copolymer is a degradable high molecular synthetic material, has good biocompatibility and mechanical property, and can enable the nanofiber layer 2 to have good strength and elasticity.
Further, the diameter of the artificial blood vessel is 2-5mm, and the wall thickness of the artificial blood vessel is 250-700 microns.
Further, the second nanofiber layer is prepared by pouring a nanofiber solution with the concentration of 30% -50% into a mold.
The invention also provides a preparation method of the composite artificial blood vessel, which comprises the following steps of
Preparing a first spinning solution, and preparing a first nanofiber membrane by using the first spinning solution through an electrostatic spinning method;
winding the first nanofiber membranes with the set layer number on a mould rod to form a hollow tube shape serving as a first nanofiber layer, and bonding the first nanofiber membranes of each layer by hot pressing;
preparing a second spinning solution, and preparing a second nanofiber membrane by an electrostatic spinning method by using the second spinning solution;
cutting the second nanofiber membrane, placing the cut second nanofiber membrane into deionized water, and uniformly dispersing fibers in the deionized water by using a high-speed refiner to obtain nanofiber solution;
placing a hollow tubular first nanofiber layer in a die cavity, wherein a gap with a set distance is arranged between the outer wall of the first nanofiber layer and the inner wall of the die cavity, pouring the nanofiber solution in the die cavity, and filling the nanofiber solution in the gap between the outer wall of the first nanofiber layer and the inner wall of the die cavity;
forming a second nanofiber layer after the nanofiber solution is molded in the mold cavity, wherein the second nanofiber layer is connected with the first nanofiber layer to form an artificial blood vessel;
and (5) after demolding, carrying out cross-linking treatment on the artificial blood vessel.
Further, the first spinning solution is a spinning solution obtained by blending collagen/caprolactone lactate copolymer, the second spinning solution is a caprolactone lactate copolymer spinning solution, and the solvents of the first spinning solution and the second spinning solution are hexafluoroisopropanol.
Further, the mass ratio of the collagen/caprolactone lactate copolymer is 50:50 or 25:75, the concentration of the first spinning solution is 6% -12%, the concentration of the second spinning solution is 6% -12%, and the concentration of the nanofiber solution is 30% -50%.
Further, after the nanofiber solution is poured, freezing the mold with the first nanofiber layer and the nanofiber solution at a low temperature for 1-6 hours, and then freeze-drying;
the crosslinked artificial blood vessel is soaked in purified water for 5-10 min, and then dried in a blast drying oven at 40-55deg.C for 30-120 min to obtain the final composite artificial blood vessel.
Further, the more detailed preparation method is as follows: comprising the following steps:
a. preparing a spinning solution of collagen/PLCL blend, wherein the mass ratio of the collagen/PLCL is 50:50 or 25:75, the concentration of the spinning solution is 6% -12%, and the solvent is hexafluoroisopropanol;
b. c, preparing a nanofiber membrane with the thickness of 5-50 micrometers by using the spinning solution prepared in the step a through an electrostatic spinning method, wherein the spinning voltage is 8-20kV;
c. winding a nanofiber membrane with a certain number of layers on a stainless steel rod according to the thickness of the nanofiber layer 1 by using the nanofiber membrane prepared in the step b, and carrying out hot-pressing compounding at 45-55 ℃ to prepare the nanofiber layer 1;
d. preparing PLCL spinning solution, wherein the concentration of the spinning solution is 6% -12%, and the solvent is hexafluoroisopropanol;
e. d, preparing a PLCL nanofiber membrane by using the spinning solution prepared in the step d through an electrostatic spinning method, cutting the PLCL nanofiber membrane, uniformly dispersing fibers in deionized water by using a high-speed refiner, and preparing a nanofiber solution with the concentration of 30% -50%, wherein the nanofiber solution is subjected to vacuum degassing treatment before use;
f. and c, sleeving the first nanofiber layer prepared in the step c on an inner core of a blood vessel mold, sleeving an outer mold of the blood vessel mold to wrap the first nanofiber layer, and pouring the nanofiber solution prepared in the step e into a gap between the first nanofiber layer and the outer mold of the blood vessel mold. Standing and fixing the vascular preparation device, placing the vascular preparation device in a low-temperature refrigerator at the temperature of minus 80 ℃, freezing for 1-6 hours, and then transferring the vascular preparation device into a freeze dryer for freeze drying;
g. removing the freeze-dried artificial blood vessel from a blood vessel mold, and performing cross-linking treatment on the artificial blood vessel by using a genipin solution, wherein the concentration of the genipin solution is 0.5-2.0%, the dissolution temperature of the genipin solution is 37 ℃, the solvent is a blend of water and ethanol, and the volume ratio of the water to the ethanol is 80:20. The artificial blood vessel is soaked into genipin solution for crosslinking, the crosslinking time is 0.5h-24h, and the crosslinking temperature is 37 ℃. Genipin is a natural cross-linking agent, is mainly extracted from gardenia as a raw material, has cytotoxicity of 1/10000 of that of glutaraldehyde which is a traditional cross-linking agent, and has the advantages of low toxicity and good cell compatibility as well as high cross-linking efficiency similar to that of a traditional artificially synthesized chemical cross-linking agent. After genipin is used for crosslinking, the biodegradability and mechanical property of the collagen material can be improved to a certain extent;
h. soaking the artificial blood vessel prepared in the step g in purified water for 5-10 minutes, and then drying the artificial blood vessel in a forced air drying oven at the drying temperature of 40-55 ℃ for 30-120 minutes.
The beneficial effects of the invention include:
1. compared with the common nanofiber material prepared by the electrostatic spinning technology, the aperture of the second nanofiber layer is much larger, the aperture of the second nanofiber layer is in the same order of magnitude with the diameters of smooth muscle cells and fibroblasts, three-dimensional infiltration of autologous cells and regeneration of new tissues are facilitated before the fibers begin to degrade in a large amount, so that the blood vessel still has good strength and elasticity when the fibers degrade in a large amount, and the success rate of blood vessel regeneration operation is improved.
2. Good biocompatibility, can reduce rejection reaction when the artificial blood vessel is used; good hydrophilicity, favorability for adhesion of endothelial cells, rapid endothelialization of artificial blood vessels, and reduced thrombosis. The lactic acid caprolactone copolymer is a degradable high molecular synthetic material, has good biocompatibility and mechanical property, and can enable the first nanofiber layer to have good strength and elasticity. The lactic acid caprolactone copolymer is a degradable high molecular synthetic material, has good biocompatibility and mechanical property, and can enable the nanofiber layer 2 to have good strength and elasticity.
3. The composite artificial blood vessel can realize three-dimensional ingrowth of autologous cells and regeneration of new tissues before the fiber begins to degrade in a large amount, so that the blood vessel still has better strength and elasticity when the fiber degrades in a large amount, and the success rate of the blood vessel regeneration operation is improved. The composite artificial blood vessel is expected to be widely applied in the field of blood vessel regeneration. The method is simple to operate, good in repeatability and high in economic benefit.
Drawings
FIG. 1 is a schematic cross-sectional view of a blood vessel;
fig. 2 is a schematic diagram of a vascular processing mold.
In the figure, 1, a first nanofiber layer, 2, a second nanofiber layer, 3, an inner core, 4 and an outer mold.
Detailed Description
The invention will be explained in further detail below with reference to the drawings and the specific embodiments, but it should be understood that the scope of protection of the invention is not limited to the specific embodiments.
As shown in fig. 1, a composite artificial blood vessel in the invention is a double-layer tubular structure, which comprises a first nanofiber layer 1 arranged on an inner layer and a second nanofiber layer 2 arranged on an outer layer, wherein the first nanofiber layer 1 is in fit connection with the second nanofiber layer 2, the first nanofiber layer 1 is formed by rolling one layer, two layers or multiple layers of nanofiber membranes, the second nanofiber layer 2 is of an integrated structure, and microporous structures are arranged on the first nanofiber layer 1 and the second nanofiber layer 2;
the thickness of the first nanofiber layer 1 is 50-100 micrometers, the average pore diameter of the microporous structure is 0.6-1.2 micrometers, and the average diameter of the fibers of the first nanofiber layer 1 is 200-1000 nanometers; the second nanofiber layer 2 has a thickness of 200-600 microns, the average pore size of the microporous structure is 20-100 microns, and the average diameter of the fibers of the second nanofiber layer 2 is 500-1400 nanometers.
The thickness of the nanofiber membrane forming the first nanofiber layer 1 is 5-50 microns. The first nanofiber layer 1 is a collagen and caprolactone lactate copolymer nanofiber layer. Collagen has good biocompatibility and good hydrophilicity. Good biocompatibility, can reduce rejection reaction when the artificial blood vessel is used; good hydrophilicity, favorability for adhesion of endothelial cells, rapid endothelialization of artificial blood vessels, and reduced thrombosis. The lactic acid caprolactone copolymer is a degradable high molecular synthetic material, has good biocompatibility and mechanical property, and can enable the first nanofiber layer 1 to have good strength and elasticity.
The second nanofiber layer 2 is a caprolactone lactate copolymer nanofiber layer. The lactic acid caprolactone copolymer is a degradable high molecular synthetic material, has good biocompatibility and mechanical property, and can enable the nanofiber layer 2 to have good strength and elasticity. The second nanofiber layer 2 is prepared by pouring a nanofiber solution with the concentration of 30% -50% into a die.
The diameter of the artificial blood vessel is 2-5mm, and the wall thickness of the artificial blood vessel is 250-700 microns.
The preparation method of the artificial blood vessel comprises the following steps: comprising the following steps:
a. preparing a spinning solution of collagen/PLCL blend, wherein the mass ratio of the collagen/PLCL is 50:50 or 25:75, the concentration of the spinning solution is 6% -12%, and the solvent is hexafluoroisopropanol;
b. c, preparing a nanofiber membrane with the thickness of 5-50 micrometers by using the spinning solution prepared in the step a through an electrostatic spinning method, wherein the spinning voltage is 8-20kV;
c. winding a nanofiber membrane with a certain layer number on a stainless steel rod according to the thickness of the first nanofiber layer 1 by using the nanofiber membrane prepared in the step b, and carrying out hot-pressing compounding at 45-55 ℃ to prepare the nanofiber layer 1;
d. preparing PLCL spinning solution, wherein the concentration of the spinning solution is 6% -12%, and the solvent is hexafluoroisopropanol;
e. d, preparing a PLCL nanofiber membrane by using the spinning solution prepared in the step d through an electrostatic spinning method, cutting the PLCL nanofiber membrane, uniformly dispersing fibers in deionized water by using a high-speed refiner, and preparing a nanofiber solution with the concentration of 30% -50%, wherein the nanofiber solution is subjected to vacuum degassing treatment before use;
f. and c, sleeving the first nanofiber layer 1 prepared in the step c on an inner core 3 of a blood vessel mold, sleeving an outer mold 4 of the blood vessel mold to wrap the first nanofiber layer 1, and pouring the nanofiber solution prepared in the step e into a gap between the first nanofiber layer 1 and the outer mold 4 of the blood vessel mold. Standing and fixing the vascular preparation device, placing the vascular preparation device in a low-temperature refrigerator at the temperature of minus 80 ℃, freezing for 1-6 hours, and then transferring the vascular preparation device into a freeze dryer for freeze drying;
g. removing the freeze-dried artificial blood vessel from a blood vessel mold, and performing cross-linking treatment on the artificial blood vessel by using a genipin solution, wherein the concentration of the genipin solution is 0.5-2.0%, the dissolution temperature of the genipin solution is 37 ℃, the solvent is a blend of water and ethanol, and the volume ratio of the water to the ethanol is 80:20. The artificial blood vessel is soaked into genipin solution for crosslinking, the crosslinking time is 0.5h-24h, and the crosslinking temperature is 37 ℃. Genipin is a natural cross-linking agent, is mainly extracted from gardenia as a raw material, has cytotoxicity of 1/10000 of that of glutaraldehyde which is a traditional cross-linking agent, and has the advantages of low toxicity and good cell compatibility as well as high cross-linking efficiency similar to that of a traditional artificially synthesized chemical cross-linking agent. After genipin is used for crosslinking, the biodegradability and mechanical property of the collagen material can be improved to a certain extent;
h. soaking the artificial blood vessel prepared in the step g in purified water for 5-10 minutes, and then drying the artificial blood vessel in a forced air drying oven at the drying temperature of 40-55 ℃ for 30-120 minutes.
Example 1
Preparing a spinning solution of collagen/PLCL (caprolactone lactate copolymer) blend, wherein the mass ratio of the collagen/PLCL is 50:50, the concentration of the spinning solution is 10% w/v, and the solvent is hexafluoroisopropanol. The nanofiber membrane with the thickness of 10 micrometers is prepared by an electrostatic spinning method, the spinning voltage is 12kV, the prepared nanofiber membrane is wound on a stainless steel rod with the thickness of 3.0mm for 7 layers, the first nanofiber layer 1 is prepared by hot-pressing compounding at the temperature of 50 ℃, and the thickness of the first nanofiber layer 1 is 56 micrometers.
A PLCL spinning solution was prepared, the concentration of the spinning solution was 8%, and the solvent was hexafluoroisopropanol. The PLCL nanofiber membrane is prepared by an electrostatic spinning method, then the PLCL nanofiber membrane is sheared, the PLCL nanofiber membrane is placed in deionized water, the fibers are uniformly dispersed by a high-speed refiner, a nanofiber solution with the concentration of 35% is prepared, and vacuum degassing treatment is needed before the nanofiber solution is used.
And sleeving the prepared first nanofiber layer 1 on an inner core 3 of a vascular processing die, sleeving an outer die 4 of the vascular processing die, enabling the outer die 4 to wrap the first nanofiber layer 1, and pouring a 35% nanofiber solution into a gap between the first nanofiber layer 1 and the outer die 4 of the vascular processing die. The vessel preparation device was stationary and placed in a low temperature refrigerator at-80 ℃ for 3 hours of freeze treatment, followed by transfer to a freeze dryer for freeze drying.
Removing the freeze-dried artificial blood vessel from the blood vessel mold, and performing cross-linking treatment on the artificial blood vessel by using a genipin solution, wherein the concentration of the genipin solution is 1.0%, the dissolution temperature of the genipin solution is 37 ℃, the solvent is a blend of water and ethanol, and the volume ratio of the water to the ethanol is 80:20. The artificial blood vessel is soaked into genipin solution for crosslinking, the crosslinking time is 3 hours, and the crosslinking temperature is 37 ℃. The crosslinked artificial blood vessel is soaked in purified water for 5-10 min, and then is dried in a blast drying oven with the temperature of 50 ℃ and the drying time of 60 min.
The inner diameter of the prepared composite human hematopoietic tube is 3.0mm, the tube wall thickness is 350 microns, the thickness of the first nanofiber layer 1 is 56 microns, and the thickness of the second nanofiber layer 2 is 294 microns. The pore size of the first nanofiber layer 1 was 0.8 microns and the pore size of the second nanofiber layer 2 was 81 microns.
Example 2
The spinning solution of the collagen/PLCL blend is prepared, the mass ratio of the collagen/PLCL is 25:75, the concentration of the spinning solution is 9% w/v, and the solvent is hexafluoroisopropanol. The nanofiber membrane with the thickness of 10 micrometers is prepared by an electrostatic spinning method, the spinning voltage is 12kV, the prepared nanofiber membrane is wound on a stainless steel rod with the thickness of 4.0mm for 8 layers, the first nanofiber layer 1 is prepared by hot-pressing compounding at 50 ℃, and the thickness of the first nanofiber layer 1 is measured to be 62 micrometers by a laser calliper.
A PLCL spinning solution was prepared, the concentration of the spinning solution was 8%, and the solvent was hexafluoroisopropanol. The PLCL nanofiber membrane is prepared by an electrostatic spinning method, then the PLCL nanofiber membrane is sheared, the PLCL nanofiber membrane is placed in deionized water, the fibers are uniformly dispersed by a high-speed refiner, a nanofiber solution with the concentration of 40% is prepared, and vacuum degassing treatment is needed before the nanofiber solution is used.
And sleeving the prepared first nanofiber layer 1 on an inner core 3 of a vascular processing die, sleeving an outer die 4 of the vascular processing die, enabling the outer die 4 to wrap the first nanofiber layer 1, and pouring 40% nanofiber solution into a gap between the first nanofiber layer 1 and the outer die 4 of the vascular processing die. The vessel preparation device was stationary and placed in a low temperature refrigerator at-80 ℃ for 3 hours of freeze treatment, followed by transfer to a freeze dryer for freeze drying.
Removing the freeze-dried artificial blood vessel from the blood vessel mold, and performing cross-linking treatment on the artificial blood vessel by using a genipin solution, wherein the concentration of the genipin solution is 1.0%, the dissolution temperature of the genipin solution is 37 ℃, the solvent is a blend of water and ethanol, and the volume ratio of the water to the ethanol is 80:20. The artificial blood vessel is soaked into genipin solution for crosslinking, the crosslinking time is 3 hours, and the crosslinking temperature is 37 ℃. The crosslinked artificial blood vessel is soaked in purified water for 5-10 min, and then is dried in a blast drying oven with the temperature of 50 ℃ and the drying time of 60 min.
The inner diameter of the prepared composite human hematopoietic tube is 4.0mm, the tube wall thickness is 450 micrometers, the thickness of the first nanofiber layer 1 is 62 micrometers, and the thickness of the second nanofiber layer 2 is 388 micrometers. The pore size of the first nanofiber layer 1 was 0.7 microns and the pore size of the second nanofiber layer 2 was 59 microns.
Example 3
The spinning solution of the collagen/PLCL blend is prepared, the mass ratio of the collagen/PLCL is 25:75, the concentration of the spinning solution is 9% w/v, and the solvent is hexafluoroisopropanol. The nanofiber membrane with the thickness of 6 microns is prepared by an electrostatic spinning method, the spinning voltage is 12kV, the prepared nanofiber membrane is wound on a 2.5mm stainless steel rod for 15 layers, the first nanofiber layer 1 is prepared by hot-pressing compounding at 50 ℃, and the thickness of the first nanofiber layer 1 is 78 microns measured by a laser calliper.
A PLCL spinning solution was prepared, the concentration of the spinning solution was 6%, and the solvent was hexafluoroisopropanol. Preparing a PLCL nanofiber membrane by an electrostatic spinning method, cutting the PLCL nanofiber membrane, uniformly dispersing fibers in deionized water by using a high-speed refiner, and preparing a nanofiber solution with the concentration of 50%, wherein the nanofiber solution needs to be subjected to vacuum degassing treatment before use;
and sleeving the prepared first nanofiber layer 1 on an inner core 3 of a vascular processing die, sleeving an outer die 4 of the vascular processing die, enabling the outer die 4 to wrap the first nanofiber layer 1, and pouring 50% nanofiber solution into a gap between the first nanofiber layer 1 and the outer die 4 of the vascular processing die. The vessel preparation device was stationary and placed in a low temperature refrigerator at-80 ℃ for 3 hours of freeze treatment, followed by transfer to a freeze dryer for freeze drying.
Removing the freeze-dried artificial blood vessel from the blood vessel mold, and performing cross-linking treatment on the artificial blood vessel by using a genipin solution, wherein the concentration of the genipin solution is 1.0%, the dissolution temperature of the genipin solution is 37 ℃, the solvent is a blend of water and ethanol, and the volume ratio of the water to the ethanol is 80:20. The artificial blood vessel is soaked into genipin solution for crosslinking, the crosslinking time is 3 hours, and the crosslinking temperature is 37 ℃. The crosslinked artificial blood vessel is soaked in purified water for 5-10 min, and then is dried in a blast drying oven with the temperature of 50 ℃ and the drying time of 60 min.
The inner diameter of the prepared composite human hematopoietic tube is 2.5mm, the tube wall thickness is 260 micrometers, the thickness of the first nanofiber layer 1 is 78 micrometers, and the thickness of the second nanofiber layer 2 is 182 micrometers. The pore size of the first nanofiber layer 1 was 0.7 microns and the pore size of the second nanofiber layer 2 was 29 microns.
Referring to fig. 2, the vascular processing mold mentioned in the above embodiments 1-3 includes an inner core 3 and an outer mold 4, the outer mold 4 is a hollow cylindrical structure, the inner core 3 is inserted into the hollow cavity of the outer mold 4, the hollow cavity of the outer mold 4 is coaxial with the inner core 3, the first nanofiber layer 1 is sleeved on the inner core 3, then the inner core 3 is placed in the hollow cavity of the outer mold 4, a gap is provided between the first nanofiber layer 1 and the inner wall of the hollow cavity, and the nanofiber solution is poured into the gap for molding.
The above is only a preferred embodiment of the present invention, and the present invention is not limited to the contents of the embodiment. Various changes and modifications within the technical scope of the present invention will be apparent to those skilled in the art, and any changes and modifications are intended to be within the scope of the present invention.
Claims (11)
1. A preparation method of a composite artificial blood vessel is characterized by comprising the following steps: the method comprises the following steps:
preparing a first spinning solution, and preparing a first nanofiber membrane by using the first spinning solution through an electrostatic spinning method;
winding the first nanofiber membranes with the set layer number on a mould rod to form a hollow tube shape serving as a first nanofiber layer, and bonding the first nanofiber membranes of each layer by hot pressing;
preparing a second spinning solution, and preparing a second nanofiber membrane by an electrostatic spinning method by using the second spinning solution;
cutting the second nanofiber membrane, placing the cut second nanofiber membrane into deionized water, and uniformly dispersing fibers in the deionized water by using a high-speed refiner to obtain nanofiber solution;
placing a hollow tubular first nanofiber layer in a die cavity, wherein a gap with a set distance is arranged between the outer wall of the first nanofiber layer and the inner wall of the die cavity, and pouring the nanofiber solution in the die cavity to fill the nanofiber solution in the gap between the outer wall of the first nanofiber layer and the inner wall of the die cavity;
forming a second nanofiber layer after the nanofiber solution is molded in the mold cavity, wherein the second nanofiber layer is connected with the first nanofiber layer to form an artificial blood vessel;
and (5) after demolding, carrying out cross-linking treatment on the artificial blood vessel.
2. The method for preparing the composite artificial blood vessel according to claim 1, wherein: the first spinning solution is a spinning solution obtained by blending collagen/caprolactone lactate copolymer, the second spinning solution is a caprolactone lactate copolymer spinning solution, and the solvents of the first spinning solution and the second spinning solution are hexafluoroisopropanol.
3. The method for preparing the composite artificial blood vessel according to claim 2, wherein: the mass ratio of the collagen/caprolactone lactate copolymer is 50:50 or 25:75, the concentration of the first spinning solution is 6% -12%, the concentration of the second spinning solution is 6% -12%, and the concentration of the nanofiber solution is 30% -50%.
4. The method for preparing the composite artificial blood vessel according to claim 1, wherein: freezing the mold with the first nanofiber layer and nanofiber solution at low temperature for 1-6 hours after the nanofiber solution is poured, and then freeze-drying;
the crosslinked artificial blood vessel is soaked in purified water for 5-10 min, and then dried in a blast drying oven at 40-55deg.C for 30-120 min to obtain the final composite artificial blood vessel.
5. A composite vascular prosthesis, characterized in that: is prepared by the method of any one of claims 1 to 4.
6. The composite vascular prosthesis of claim 5, wherein: the artificial blood vessel is of a double-layer tubular structure and comprises a first nanofiber layer arranged on an inner layer and a second nanofiber layer arranged on an outer layer, wherein the first nanofiber layer is in fit connection with the second nanofiber layer, the first nanofiber layer is formed by rolling one, two or more nanofiber membranes, the second nanofiber layer is of an integrated structure, and microporous structures are arranged on the first nanofiber layer and the second nanofiber layer;
the thickness of the first nanofiber layer is 50-100 micrometers, the average pore diameter of the microporous structure is 0.6-1.2 micrometers, and the average diameter of the fibers of the first nanofiber layer is 200-1000 nanometers;
the thickness of the second nanofiber layer is 200-600 microns, the average pore diameter of the microporous structure is 20-100 microns, and the average diameter of the fibers of the second nanofiber layer is 500-1400 nanometers.
7. The composite vascular prosthesis of claim 6, wherein: the thickness of the nanofiber membrane forming the first nanofiber layer is 5-50 microns.
8. The composite vascular prosthesis of claim 6, wherein: the first nanofiber layer is a collagen and caprolactone lactate copolymer nanofiber layer.
9. The composite vascular prosthesis of claim 6, wherein: the second nanofiber layer is a caprolactone lactate copolymer nanofiber layer.
10. The composite vascular prosthesis of claim 6, wherein: the diameter of the artificial blood vessel is 2-5mm, and the wall thickness of the artificial blood vessel is 250-700 microns.
11. The composite vascular prosthesis of claim 6, wherein: the second nanofiber layer is prepared by pouring nanofiber solution with the concentration of 30% -50% into a mold.
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| CN104921841A (en) * | 2015-04-10 | 2015-09-23 | 南开大学 | Method for manufacturing artificial blood vessels with double-layered structures and application of artificial blood vessels |
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| CN101214393A (en) * | 2007-12-28 | 2008-07-09 | 苏州大学 | Nano fibrous tissue engineering blood vessel and preparation thereof |
| CN104921841A (en) * | 2015-04-10 | 2015-09-23 | 南开大学 | Method for manufacturing artificial blood vessels with double-layered structures and application of artificial blood vessels |
| CN106110401A (en) * | 2016-06-30 | 2016-11-16 | 上海大学 | Micro-nano composite double layer dermal scaffold and preparation method thereof |
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