CN106075596B - Preparation technology of three-layer artificial blood vessel - Google Patents
Preparation technology of three-layer artificial blood vessel Download PDFInfo
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- CN106075596B CN106075596B CN201610589001.3A CN201610589001A CN106075596B CN 106075596 B CN106075596 B CN 106075596B CN 201610589001 A CN201610589001 A CN 201610589001A CN 106075596 B CN106075596 B CN 106075596B
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Classifications
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- 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
- 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
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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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- A—HUMAN NECESSITIES
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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Abstract
本发明提供了一种三层(内层，中层和外层)人工血管的制备方法，内层是由墨水打印法制备的光滑致密薄层构成，能够抑制血浆蛋白和血小板粘附，防止急性血栓形成，同时提供轴向的力学支持。中层是由湿法纺丝或熔融纺丝制备的取向微米纤维缠绕构成，其主要作用是引导组织细胞向取向纤维空隙内部生长，实现细胞外基质取向沉积排布，同时取向微米纤维能够提供径向力学支持。外层由粗的聚合物纤维缠绕并与中层紧密粘接在一起，主要是在人工血管弯曲时起到防止打折的作用。本发明制备的人工血管能够显著提高通畅率，而且能够利用植入部位微环境实现拟天然重塑再生，在冠脉搭桥、血液透析、脑和外周血管替换方面具有良好的应用前景。The invention provides a preparation method of a three-layer (inner layer, middle layer and outer layer) artificial blood vessel. The inner layer is composed of a smooth and dense thin layer prepared by an ink printing method, which can inhibit the adhesion of plasma proteins and platelets and prevent acute thrombosis. form, while providing axial mechanical support. The middle layer is composed of oriented microfiber entanglement prepared by wet spinning or melt spinning. Its main function is to guide tissue cells to grow inside the oriented fiber voids to achieve oriented deposition and arrangement of extracellular matrix. At the same time, the oriented microfibers can provide radial Mechanical support. The outer layer is entangled with thick polymer fibers and tightly bonded with the middle layer, mainly to prevent folding when the artificial blood vessel is bent. The artificial blood vessel prepared by the invention can significantly improve the patency rate, and can realize pseudo-natural remodeling and regeneration by utilizing the microenvironment of the implantation site, and has good application prospects in coronary bypass, hemodialysis, and replacement of brain and peripheral blood vessels.
Description
Technical Field
The invention belongs to the field of tissue engineering, and particularly relates to a preparation method of a three-layer artificial blood vessel.
Background
Vascular disease is the most lethal disease worldwide, and the disease often occurs due to angiostenosis orThe blockage results in reduced blood flow and nutrient deficiency, which results in tissue or organ damage, often manifested as coronary heart disease, cerebrovascular disease, peripheral arterial disease, and deep vein thrombosis. According to the world health organization, the number of deaths worldwide from cardiovascular related diseases per year will increase to 2330 ten thousand by 2030. Vascular graft surgery remains the conventional means for treating such diseases, and such surgery is preferred to collect and use autologous blood vessels of a patient, such as the great saphenous vein, the bilateral internal thoracic artery, the radial artery and the like. However, some patients can only select small-bore artificial blood vessels for replacement because autologous blood vessels are already collected or complicated vascular lesions are generated. In addition, small-caliber artificial blood vessels are also needed for construction of hemodialysis arteriovenous fistula, traumatic arterial injury, peripheral aneurysm and the like. Currently, polyethylene terephthalate
Expanded polytetrafluoroethylene (Gore-
) And the large-caliber (the inner diameter is more than 6mm) artificial blood vessel prepared from materials such as polyurethane and the like has higher long-term patency rate after transplantation, and is widely applied to clinic. However, small-bore blood vessels prepared from such non-degradable materials have low patency in clinical applications, and although researchers modify them, such as grafting heparin, to improve their anticoagulant properties, the problem has not been solved yet. Therefore, the development of new small-bore artificial blood vessels (inner diameter < 6mm) is increasingly paid attention by scientists at home and abroad.
Currently, scientists have developed various techniques for preparing various types of small-caliber artificial blood vessels, which can be roughly classified into three types: comprises the technology of preparing artificial blood vessels by taking synthetic materials and natural materials as matrixes, the natural blood vessel decellularization technology and the self-assembly technology. The artificial blood vessel prepared by using synthetic materials or natural materials can be implanted into a body through activity modification, and the artificial blood vessel which is similar to the natural artificial blood vessel can be regenerated in the body by using host remodeling potential, which is one of the current research hotspots in the field. Researchers have used various techniques such as electrospinning, particle leaching, phase separation, etc. to prepare vascular prostheses having pores, however, there is little consideration and control over the physical topology of the inner surface of the vascular prostheses in the preparation of vascular prostheses using these techniques. A great deal of basic research shows that the topological structure (such as smoothness and roughness) of the surface of the artificial blood vessel material can influence the adhesion of plasma protein and blood platelets, and can also influence the adhesion and proliferation of endothelial cells and smooth muscle cells, thereby influencing the patency of the artificial blood vessel. The degradable polymer artificial blood vessel has the problems that the degradable polymer artificial blood vessel is easy to deform and form aneurysm after being implanted into a body for a long time, and one of the main reasons is that the extracellular matrix of a new blood vessel cannot form pseudo-natural orientation arrangement after the material is degraded because the pore structure of the artificial blood vessel material is irregular, so that the blood pressure in the body cannot be endured for a long time. In addition, many of the artificial blood vessels prepared in the research form a dead fold after being bent at a certain angle, and the artificial blood vessels can block blood circulation after being implanted into a body.
In order to solve the above problems, we have designed and prepared a triple-layered artificial blood vessel composed of an inner layer, a middle layer and an outer layer. The inner layer is composed of a smooth and compact thin layer of 10-100 microns, the inner surface is smooth and compact, so that the adhesion of plasma proteins and blood platelets can be inhibited, meanwhile, the blood is prevented from permeating into the wall of the artificial blood vessel during implantation to cause acute thrombosis, and in addition, the compact inner layer can provide axial mechanical support. The middle layer is formed by winding spiral oriented micron fibers which are arranged at a certain angle and are 5-100 microns, the main function of the middle layer is to guide surrounding tissue cells to grow in an oriented mode towards the interior of the support, oriented deposition arrangement of extracellular matrix is achieved, and meanwhile the oriented micron fibers can provide radial mechanical support. The outer layer is wound by polymer fiber with the diameter of 50-500 microns and is tightly bonded with the middle layer, and the function of preventing the artificial blood vessel from being broken when the artificial blood vessel is bent is mainly achieved.
Disclosure of Invention
The invention is completed in three steps, and the first step is to prepare a compact and smooth thin layer of the artificial blood vessel by using an ink printing method. The specific method comprises preparing degradable polymer (including Polycaprolactone (PCL), Polylactide (PLA), poly (lactide-co-glycolic acid) (PLGA), polyglycolic acid (PGA), Polyhydroxyalkanoate (PHA) poly (lactide-caprolactone) copolymer (PLCL), poly (p-dioxanone) (PDS) and the like) or natural material (including fibroin protein, chitosan, gelatin, collagen and the like) solution with certain concentration (mass/volume fraction of 1-60%) by using chemical pure reagent (including tetrahydrofuran, dichloromethane, trichloromethane, acetic acid, acetone, trifluoroethanol, hexafluoroisopropanol and the like) as solvent, filling the dissolved synthetic or natural material solution into a syringe after complete dissolution, printing the polymer solution in the syringe onto a smooth metal receiving rod with controllable rotation speed and movement speed below by using a micro-injection pump, the flow rate is 0.1-50ml/h, the distance between the syringe needle and the receiving rod below is 0.1-5cm, the rotation speed of the receiving rod is 1-500rpm, the moving speed is 1mm-50mm/sec, the solvent component in the extruded polymer solution volatilizes, so that the polymer solution is solidified to form a film, and parameters such as the concentration of the polymer, the flow rate, the rotation speed of the receiving rod, the moving speed and the like are adjusted, so that the artificial blood vessel inner layer with controllable thickness (5-200 microns) is prepared.
The second step of the invention patent is the preparation method of the artificial blood vessel with the oriented micron fiber in the middle layer. The fiber can be processed by wet spinning or melt spinning, the receiving rod with the smooth and compact inner layer prepared in the first step is arranged on a wet spinning instrument, polymer solution with a certain concentration is added into an injector, the injector is arranged on an injection pump, and parameters such as the propelling speed of the injection pump, the rotating speed of the receiving rod, the moving speed and the like are adjusted to regulate and control the diameter of the middle-layer micrometer fibers and the angle between the fibers, so that the middle-layer oriented fiber with the diameter of 5-100 micrometers is prepared. Meanwhile, the receiving rod with the smooth and compact inner layer prepared in the first step can be arranged on a melt spinning instrument, the polymer is added into a constant-temperature heating cylinder, after the polymer is melted by heating, the diameter of the middle micrometer fiber and the angle between the fibers are regulated and controlled by adjusting parameters such as the speed of a pushing piston of the cylinder, the thickness of a needle head, the rotating speed of the receiving rod, the transverse moving speed and the like, so that the middle layer of the artificial blood vessel with the oriented fiber of 10-100 micrometers in diameter is prepared.
The third step of the present invention is to prepare the anti-folding outer layer of the artificial blood vessel. The method specifically comprises the steps of installing the receiving rod with the middle layer and the inner layer prepared in the second step on a melt spinning instrument, adding a polymer into a constant-temperature heating cylinder, heating to melt the polymer, and adjusting parameters such as the heating temperature of the cylinder, the piston propelling speed, the thickness of a needle head, the rotating speed of the receiving rod, the moving speed and the like to regulate and control the diameter of the micrometer fibers of the outer layer and the angle between the fibers so as to prepare the artificial blood vessel outer layer with the diameter of 50-500 micrometers and oriented fibers. The outer layer and the middle layer are tightly bonded by adjusting the temperature of the heating charging barrel.
Compared with the existing degradable polymer small-caliber artificial blood vessel, the degradable polymer small-caliber artificial blood vessel has the following advantages: 1. the artificial blood vessel has an ultrathin, compact and smooth inner surface, can obviously reduce the adhesion of platelets and proteins compared with the rough inner surface of a nano or micro fiber artificial blood vessel prepared by an electrostatic spinning technology commonly used in the existing research, and meanwhile, the compact inner layer can prevent blood from permeating into the wall of the artificial blood vessel, reduce the activation of the platelets and the formation of acute thrombus, and can enhance the axial mechanics of the artificial blood vessel; 2. because the middle layer of the artificial blood vessel has a spiral orientation micron fiber structure, the orientation micron fiber has the function of enhancing the radial mechanics of the artificial blood vessel, meanwhile, tissue cells around the implanted blood vessel part can rapidly migrate to the pores of the orientation micron fiber and secrete extracellular matrixes such as collagen and elastin, the secreted extracellular matrixes are arranged in a circumferential orientation mode, the radial mechanics of the artificial blood vessel after being implanted in vivo can be enhanced, and the orientation micron fiber and the orientation extracellular matrixes can jointly act to enable the artificial blood vessel to resist blood pressure for a long time after being implanted in vivo, so that the formation of aneurysm is avoided; 3, the outer layer of the artificial blood vessel is formed by spirally winding coarse polymer fibers, and the fibers of the outer layer are tightly adhered to the middle layer, so that the artificial blood vessel can be prevented from being broken when being bent; 4. the preparation process of the artificial blood vessel has strong controllability, can regulate and control the thickness of the inner layer film, the diameters of the middle layer fiber and the outer layer fiber, the angle between the fibers, the bonding degree and the like, and can also regulate and control the diameter of the artificial blood vessel.
Detailed Description
Example 1: preparation of Polycaprolactone (PCL) three-layer artificial blood vessel
Preparing an artificial blood vessel inner layer: 2.0g of PCL with the number average molecular weight of 80000 was weighed into 10ml of chloroform, and dissolved overnight with stirring at room temperature to prepare a PCL solution with a concentration fraction of 20% (mass/volume). The artificial intravascular layer was prepared using a direct printing method in a room temperature fume hood. A stainless steel receiving rod with a diameter of 2.0mm was mounted on the printer. The PCL solution was drawn into a syringe, which was mounted on a syringe pump, and the syringe needle was placed 2mm above the metal receiving rod. Setting the propelling speed of a syringe pump to be 2ml/h, the rotating speed of the receiving rod to be 100rpm, the transverse moving speed to be 0.2mm/sec and the printing time to be 2min, and drying the receiving rod with the inner layer of the artificial blood vessel in vacuum after the preparation is finished.
Preparing an artificial blood vessel middle layer: the artificial media was prepared in a room temperature fume hood using wet spinning. Specifically, a receiving rod with a smooth and compact inner layer is arranged on a wet spinning instrument, 20% of PCL spinning solution is sucked into an injector, the injector is arranged on an injection pump, and the needle head of the injector is arranged at a position 1cm away from the receiving rod in a spinning coagulation bath. Setting the speed of the injection pump at 4ml/h, the rotation speed of the receiving rod at 1000rpm, the moving speed at 1mm/sec, and the spinning time at 15min, and removing the coagulation bath and the spinning solution solvent after completion.
Preparing an artificial blood vessel outer layer: the outer layer of the artificial blood vessel is prepared by a melt spinning method. Specifically, a receiving rod with an inner layer and a middle layer is arranged on a melt spinning instrument, 20.0g of PCL is added into a constant-temperature heating charging barrel, the temperature is increased to 100 ℃ to fully melt the PCL, the speed of a propelling piston of the charging barrel is set to be 10ml/h, the rotating speed of the receiving rod is 400rpm, the moving speed is 1mm/sec, and the time is 2 min. And after the outer layer spinning is finished, taking down the three layers of artificial blood vessels for later use.
Example 2: preparation of poly L-lactide-caprolactone (PLCL) three-layer artificial blood vessel
Preparing an artificial blood vessel inner layer: 1.5g of PLCL was weighed into 10ml of tetrahydrofuran solvent and dissolved overnight with stirring at room temperature to prepare a PLCL solution having a concentration fraction of 15% (mass/volume). The artificial intravascular layer was prepared using a direct printing method in a room temperature fume hood. A stainless steel receiving rod with a diameter of 3.0mm was mounted on the printer. Sucking the PLCL solution into a syringe, then installing the syringe on a syringe pump, placing a syringe needle at a position 3.0mm above a metal receiving rod, setting the propelling speed of the syringe pump to be 3ml/h, the rotating speed of the receiving rod to be 200rpm, the transverse moving speed to be 0.4mm/sec, and the printing time to be 1min, and after the preparation is finished, carrying out vacuum drying on the receiving rod with the inner layer of the artificial blood vessel.
Preparing an artificial blood vessel middle layer: the artificial media was prepared in a room temperature hood using melt spinning. The receiving rod with the smooth and dense inner layer was mounted on a melt spinning machine, 20.0g of PLCL was added to a constant temperature heating cylinder, the temperature was raised to 240 ℃ to melt the PLCL, and then the cylinder advancing piston speed was set to 1ml/h, the receiving rod rotational speed was 200rpm, the movement speed was 0.5mm/sec, and the time was 10 min.
Preparing an artificial blood vessel outer layer: the outer layer of the artificial blood vessel was prepared in a room temperature hood using melt spinning. Specifically, a receiving rod with an inner layer and a middle layer is arranged on a melt spinning instrument, 20.0g of PLCL is added into a constant-temperature heating cylinder, the temperature is increased to 240 ℃ to fully melt the PLCL, the speed of a pushing piston of the cylinder is set to be 12ml/h, the rotating speed of the receiving rod is 200rpm, the moving speed is 2.0mm/sec, and the time is 1 min. And after the outer layer spinning is finished, taking down the three layers of artificial blood vessels for later use.
Example 3: preparation of Polylactide (PLA) three-layer artificial blood vessel
Preparing an artificial blood vessel inner layer: 1.2g of PLA was weighed and added to 10ml of methylene chloride, and stirred and dissolved overnight at room temperature to prepare a PLA solution having a concentration fraction of 12% (mass/volume). The artificial intravascular layer was prepared using a direct printing method in a room temperature fume hood. A 4.0mm diameter stainless steel receiving rod was mounted on the printer. After the PLA solution is sucked into the syringe, the syringe is arranged on a syringe pump, the needle head of the syringe is arranged at a position 3.0mm above the metal receiving rod, the propelling speed of the syringe pump is set to be 3ml/h, the rotating speed of the receiving rod is 200rpm, the transverse moving speed is 0.4mm/sec, the printing time is 1min, and the receiving rod with the inner layer of the artificial blood vessel is dried in vacuum after the preparation is finished.
Preparing an artificial blood vessel middle layer: the artificial media was prepared in a room temperature hood using melt spinning. The receiving rod with the smooth and compact inner layer is installed on a melt spinning instrument, 10.0g of PLA is added into a constant-temperature heating cylinder, the temperature is increased to 260 ℃ to melt the PLA, the speed of a pushing piston of the cylinder is set to be 2ml/h, the rotating speed of the receiving rod is 100rpm, the moving speed is 0.7mm/sec, and the time is 12 min.
Preparing an artificial blood vessel outer layer: the outer layer of the artificial blood vessel was prepared in a room temperature hood using melt spinning. Specifically, a receiving rod with an inner layer and a middle layer is arranged on a melt spinning instrument, 20.0g of PLA is added into a constant-temperature heating cylinder, the temperature is increased to 260 ℃ to fully melt the PLA, the speed of a pushing piston of the cylinder is set to be 14ml/h, the rotating speed of the receiving rod is 600rpm, the moving speed is 3.0mm/sec, and the time is 1 min. And after the outer layer spinning is finished, taking down the three layers of artificial blood vessels for later use.
Claims (4)
1. A preparation method of a three-layer artificial blood vessel is characterized by comprising the following steps:
preparing an inner layer of an artificial blood vessel;
the inner layer of the artificial blood vessel is prepared by an ink printing method by taking biodegradable macromolecules as raw materials and is composed of a smooth compact thin layer of 10-100 microns;
step two, preparing an artificial blood vessel middle layer;
the middle layer is prepared at the outer side of the inner layer of the artificial blood vessel by one or more of electrostatic spinning, wet spinning, melt spinning or 3D printing technology and is formed by winding spiral oriented fibers which are arranged at a certain angle and have the diameter of 5-100 microns;
step three, preparing an artificial blood vessel outer layer;
the outer layer is prepared on the outer sides of the inner layer and the middle layer of the artificial blood vessel by a melt spinning method and a 3D printing method, and is wound by polymer fibers of 50-500 microns and tightly bonded with the middle layer.
2. The method for preparing a triple-layered artificial blood vessel according to claim 1, wherein the biodegradable polymer is one or more of polylactic acid (PLA), Polycaprolactone (PCL), poly-L-lactide-caprolactone (PLCL), Polyhydroxyalkanoate (PHA), polylactic-co-glycolic acid (PLGA), Polydioxanone (PDS), and Polyurethane (PU).
3. A trilaminar vascular prosthesis produced by the method of any one of claims 1 or 2.
4. A triple layer vascular prosthesis according to claim 3, characterised by an internal diameter of 2 or 3 mm.
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Families Citing this family (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN115137881B (en) * | 2022-07-27 | 2023-08-25 | 天津大学温州安全(应急)研究院 | Three-layer biomimetic artificial blood vessel for antithrombotic and tissue regeneration promotion and preparation method thereof |
| CN115998953B (en) * | 2023-02-07 | 2024-07-19 | 苏州大学 | Double bionic orientation artificial small blood vessel and preparation method thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1791576A2 (en) * | 2004-06-14 | 2007-06-06 | Thoratec Corporation | A multilayer composite vascular access graft |
| CN101755081A (en) * | 2007-03-29 | 2010-06-23 | 纳幕尔杜邦公司 | Production of nanofibers by melt spinning process |
| CN103007344A (en) * | 2012-12-26 | 2013-04-03 | 东华大学 | Hollow heterotype bacteria cellulose artificial blood vessel stent material with gradient structure and preparation method thereof |
| CN103857416A (en) * | 2011-10-07 | 2014-06-11 | W.L.戈尔及同仁股份有限公司 | Piercable and Resealable Grafts |
| CN103861158A (en) * | 2012-12-11 | 2014-06-18 | 北京航空航天大学 | Preparation method of silk fibroin and sulfated silk fibroin composite tubular scaffold |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008540022A (en) * | 2005-05-17 | 2008-11-20 | ナイキャスト リミテッド | Implantable charged medical device |
| JP5010854B2 (en) * | 2006-05-18 | 2012-08-29 | 帝人株式会社 | Revascularization material |
| CN101584612B (en) * | 2009-06-12 | 2011-11-09 | 广州迈普再生医学科技有限公司 | Regeneration type artificial blood vessel based on in-situ self stem cell technology and preparation method thereof |
| CN102784015B (en) * | 2012-08-30 | 2015-06-03 | 广州迈普再生医学科技有限公司 | Artificial blood vessel loaded with pseudo-ginseng medicines, and preparation method and application for artificial blood vessel |
| CN103876859A (en) * | 2014-03-25 | 2014-06-25 | 南开大学 | Artificial blood vessel composed of micrometer fiber and provided with large-hole structure and preparation method and application thereof |
| CN104921841B (en) * | 2015-04-10 | 2017-03-29 | 南开大学 | A kind of preparation method of double-decker artificial blood vessel |
| CN105031735B (en) * | 2015-06-24 | 2018-01-19 | 东华大学 | A kind of three-layer composite structure small-caliber artificial blood vessel and preparation method thereof |
| CN104984405B (en) * | 2015-06-29 | 2017-10-24 | 上海大学 | The method that combination process prepares intravascular stent |
| CN105457101B (en) * | 2015-12-17 | 2019-04-09 | 华南理工大学 | A kind of preparation method of three-layer structure small-caliber vascular stent |
-
2016
- 2016-07-21 CN CN201610589001.3A patent/CN106075596B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1791576A2 (en) * | 2004-06-14 | 2007-06-06 | Thoratec Corporation | A multilayer composite vascular access graft |
| CN101755081A (en) * | 2007-03-29 | 2010-06-23 | 纳幕尔杜邦公司 | Production of nanofibers by melt spinning process |
| CN103857416A (en) * | 2011-10-07 | 2014-06-11 | W.L.戈尔及同仁股份有限公司 | Piercable and Resealable Grafts |
| CN103861158A (en) * | 2012-12-11 | 2014-06-18 | 北京航空航天大学 | Preparation method of silk fibroin and sulfated silk fibroin composite tubular scaffold |
| CN103007344A (en) * | 2012-12-26 | 2013-04-03 | 东华大学 | Hollow heterotype bacteria cellulose artificial blood vessel stent material with gradient structure and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| "3D Bioprinting of Vascularized,Heterogeneous Cell-Laden Tissue Constructs";David B. Kolesky et al;《Advanced Materials》;20140531;第26卷(第19期);第3124-3130页 * |
| "Bioresorbable elastomeric vascular tissue engineering scaffolds via melt spinning and electrospinning";Sangwon Chung et al;《Acta Biomaterialia》;20091211;第6卷;第1958-1967页 * |
| "喷墨打印技术同步打印细胞和生物支架材料及在组织工程中的应用";付明福 等;《中国组织工程研究与临床康复》;20111015;第15卷(第42期);第7892-7896页 * |
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