CN109701080B

CN109701080B - 4-axis 3D printing tubular medical stent and preparation method thereof

Info

Publication number: CN109701080B
Application number: CN201910093166.5A
Authority: CN
Inventors: 游正伟; 雷东; 叶晓峰; 赵强
Original assignee: Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd; Donghua University
Current assignee: Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd; Donghua University
Priority date: 2018-07-27
Filing date: 2019-01-30
Publication date: 2021-06-29
Anticipated expiration: 2039-01-30
Also published as: CN109529122A; CN109701080A

Abstract

The invention relates to a 4-axis 3D printing tubular medical stent and a preparation method and application thereof. The invention solves the problem that the porous tubular stent is difficult to directly construct by extrusion type, particularly Fused Deposition Modeling (FDM)3D printing, the prepared medical stent has an accurate and controllable multistage pore structure in structure, the method is simple and quick, is suitable for various materials, and has good application prospect in repairing tubular tissues and organs including blood vessels, trachea, esophagus, urethra and the like.

Description

4-axis 3D printing tubular medical stent and preparation method thereof

Technical Field

The invention belongs to the field of medical stents, and particularly relates to a 4-axis 3D printing tubular medical stent and a preparation method thereof.

Background

Tissue engineering, as a interdisciplinary field, comprehensively applies the basic principles of engineering and life science, relates to the cross fusion of a series of disciplines such as clinical medicine, biological materials, cell biology, biological engineering and the like, and aims to research and develop a substitute with physiological functions for repairing, maintaining or improving damaged tissues of human bodies. Tissues or organs with a large number of tubular structures in a human body, such as blood vessels, trachea, esophagus, intestinal tract, urethra and the like, play an important role in maintaining normal physiological activities of the human body, and the pathological changes of the tubular tissues can cause great damage to the human body and even death. Worldwide, cardiovascular disease is one of the leading causes of death, second only to cancer. The main cause of the disease is myocardial infarction caused by insufficient nutrition supply of myocardial tissues due to unsmooth blood flow caused by coronary artery blockage. Trachea and esophagus have to be removed and reconstructed by removing the trachea due to the influence of tumor, physicochemical and other factors on the trachea function, and the problem of difficult direct anastomosis after ultralimit removal exists in surgical removal treatment. Therefore, a large number of tubular stents are urgently needed clinically for coronary artery bypass surgery as an artificial blood vessel, and for replacement and anastomosis of a defect site of an artificial trachea or an artificial esophagus.

The ideal tubular tissue engineering scaffold should have a micro environment suitable for cell growth and differentiation; has an open, interconnected microporous structure to facilitate cellular nutrient diffusion and metabolite release; has proper mechanical strength and tightness to replace the physiological function necessary for tubular tissues; has controllable biodegradability, requires that the degradation rate is matched with the tissue regeneration rate, and the biological scaffold is gradually degraded while the tissue is regenerated and finally metabolized and discharged out of the body. However, most of the artificial tubular grafts can be actually applied to clinical applications, and most of the artificial tubular grafts are prepared by using inert polymer materials such as terylene, silica gel and polytetrafluoroethylene and mainly preparing a tubular structure by a fiber weaving method. However, such grafts lack biological activity and are chronically implanted in the body as a replacement for foreign bodies; the method for processing the fiber into the tubular shape is complex, and the fiber is required to be prepared firstly and then woven and molded on a special die; the selectivity requirement on the material is high, and the method is only suitable for partial materials. Therefore, it is necessary to develop an open method that can be applied to various biomaterials and can obtain tubular medical stents with various sizes rapidly and precisely in a controllable manner.

The 3D printing technology (also known as 3D rapid prototyping technology or RP) is used as a novel digital prototyping technology, and the three-dimensional structure can be rapidly manufactured through an additive manufacturing mode of precisely accumulating materials. The medical device has the advantages of meeting the requirements of individual customization of patients, accurate medical treatment and the like in the medical field, and shows great application prospect. The current 3D printing technology is basically based on the movement of a spray head and a platform in three directions of X/Y/Z, and a three-dimensional structure is manufactured by stacking materials under the action of gravity. Such structures generally have a physical size and porous structure corresponding to a lesion model and are well suited for orthopedic and orthopedic applications. However, since the tubular stent has special requirements for a thin-wall porous structure, and the stacking and forming principle of 3D printing requires the use of a support structure, it is difficult to directly prepare the tubular stent by using an extrusion-molded 3D printing method.

Generally speaking, the amount of human tubular organ tissue defect and repair is large, and most of the existing artificial tubular grafts are inert long-term substitutes and lack biological activity. The material and the support forming method are two key elements for preparing the tubular support, and the 3D printing technology has great defects in printing the thin-wall porous tubular support at present.

Coronary artery bypass grafting is one of the most effective methods for treating coronary heart disease at present, and the operation repairs or rebuilds the blood circulation of obstructed coronary artery through vascular grafting, improves myocardial ischemia and improves the life quality of patients. The great saphenous vein is the most commonly used graft in clinic, but compared with artery grafts such as an internal mammary artery, a radial artery and the like, the long-term patency rate of the vein graft is lower, and the main reasons are early implantation thrombosis, middle and later stage intimal hyperplasia and atheromatous plaque formation. This limits the clinical use of intravenous plants. At present, various methods for preventing the restenosis of the transplanted vein are clinically used, such as anticoagulation, antiplatelet drugs and statins, and no-touch technology is used for obtaining the vein in the operation. Notably, researchers have been constantly exploring and studying the clinical utility of extravascular stents over the past few decades. Initially, extravascular stents were designed to limit venous expansion, maintain the consistency of the internal diameters of the graft vein and the target vessel, stabilize hemodynamics, reduce turbulence, and prevent thrombosis. Later researches show that the use of the external vascular stent can prevent thrombosis, reduce intimal hyperplasia of transplanted veins, improve long-term patency rate and ensure that the external vascular stent is widely applied and developed. The material for manufacturing the stent is continuously changed into a metal material and a biodegradable high polymer material from the early Teflon fiber. However, in recent years, there is no breakthrough progress in extravascular stents, and even studies have reported that extravascular stents have no significant effect on preventing restenosis of graft veins, which undoubtedly limits the clinical application of extravascular stents. Rapamycin is a macrolide antibiotic that inhibits smooth muscle cell proliferation, intimal hyperplasia in blood vessels, and venous restenosis by binding to the mTOR receptor. Aspirin is a non-steroidal anti-inflammatory drug, inhibits platelet aggregation by inactivating cyclooxygenase, and is the most commonly used anti-platelet drug in clinical practice. A biodegradable outer vascular stent of PCL-rapamycin is manufactured by using a 3D printing technology, the outer stent is used for wrapping a vein graft, and a medicament is released through stent degradation, so that the aim of inhibiting intimal hyperplasia of a blood vessel is fulfilled; the mechanical support provided by the stent material is combined, so that the vein distortion is prevented, the hemodynamics is stabilized, the turbulence is reduced, and the purpose of preventing the restenosis of the transplanted vein is achieved.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a 4-axis 3D printing tubular medical stent and a preparation method thereof, which solve the fundamental problem that the thin-wall porous tubular stent is difficult to prepare by extrusion type 3D printing, and the prepared medical stent has an accurately controllable multistage pore structure, excellent elasticity and sewability in mechanical properties, simple and rapid method, suitability for various biological materials and good application prospect.

The invention provides a 4-axis 3D printing tubular medical stent, which comprises nanofibers and a 4-axis 3D printing tubular spring stent, wherein the nanofibers are coated on the surface of the stent to form a double-layer structure; the multistage pore structure comprises pores formed by interweaving linear materials in the tubular spring support, micropores or nanopores which are uniformly distributed in the linear materials and are communicated with one another, and a nano pore structure formed by depositing, wrapping and interweaving nano fibers.

The invention also provides a preparation method of the 4-axis 3D printing tubular medical stent, which comprises the following steps:

(1) mixing a gel material, a thermoplastic material or a thermosetting material and a filling material according to the mass ratio of 1: 0.5-3, taking the composite material obtained by mixing as an ink material, and extruding the ink material into a linear shape by 3D printing; the rotating device is used as a receiving platform, and the rotating device and the receiving platform work cooperatively to form a 4-axis forming system of X/Y/Z/Rotation, so that a tubular support with a spring structure is obtained;

(2) curing and crosslinking the tubular support with the spring structure, and then removing the filling material to obtain the tubular spring support with the micropore or nanopore structure;

(3) and (3) placing the tubular spring support obtained in the step (2) on a rotating device or directly placing the tubular support with the spring structure obtained in the step (1) on the rotating device without the step (2) to serve as a receiving platform for receiving the nano-fibers formed through electrostatic spinning, and enabling the nano-fibers to be coated on the surface of the support, so that the 4-axis 3D printing tubular medical support is obtained.

The gel material in the step (1) is polyacrylamide, sodium alginate, collagen, gelatin or fibroin; the thermoplastic material is polycaprolactone, polylactic acid-glycolic acid copolymer or thermoplastic polyurethane; the thermosetting material is polysebacic acid glyceride, thermosetting polyurethane or epoxy resin.

The filling material in the step (1) is salt particles, graphene, carbon nano tubes, bioglass, silicon dioxide, inorganic ceramic materials, nano clay, nano short fibers (such as carbon fiber short fibers), biological factors or drugs.

The mixing mode in the step (1) is a solvent mixing method or a heating method.

The 3D printing in the step (1) comprises the following process parameters: the temperature of the extrusion cavity and the temperature of the nozzle are 0-250 ℃ (preferably 100-200 ℃), the XY axis movement speed is set to be 0.1-20 mm/s, the T axis extrusion speed is 0.008-0.1mm/s, the rotation speed of the receiving device is 5-150 r/min, the diameter of the receiver is 2-20 mm, and the cross section of the receiver is circular, elliptical, triangular or quadrilateral.

The curing crosslinking in the step (2) is thermal crosslinking, photocrosslinking or other crosslinking.

The pore size distribution of the tubular spring support in the step (2) is 10 nanometers to 100 micrometers.

The electrostatic spinning process parameters in the step (3) are as follows: polymer solution concentration: 6-15% (w/v), high voltage electric field: 8-15kV, liquid feeding rate: 0.6-1.5mL/h, rotation speed of the rotating device: 20-1500 r/min.

The diameter of the extruded linear material in the step (1) can be controlled by parameters such as the diameter of a nozzle, the extrusion pressure and the like; the moving speed of the nozzle printing and the rotating speed of the receiving platform can be adjusted and matched to control the interweaving density and the pore size of the fibrous materials; the thickness of the tube wall can be controlled by the printing time; the pipe diameter size can be controlled by the size of the receiver; the tube cross-sectional shape can be controlled by the receiver shape.

The curing degree in the step (2) can be controlled through curing conditions and curing time so as to obtain the thermosetting tubular spring support with different mechanical characteristics and performances.

The micropores or nanopores in step (2) are uniformly distributed in the linear strip material, and the size depends on the size specification of the removable filling material. The particle size of the filler is generally selected from 10nm to 50 μm.

The electrostatic spinning nano-fiber in the step (3) can be uniformly and densely coated on the tubular spring support to form a tubular support with a double-layer structure; the material, the appearance, the diameter and the nano-microporous structure of the outer-layer nano-fiber can be adjusted through spinning parameters; the thickness of the outer layer nanofiber is controlled by the spinning time; the orientation of the outer layer nanofibers is controlled by adjusting the receiver rotation speed.

The invention also provides application of the 4-axis 3D printing tubular medical stent, and the tubular medical stent is used as a tracheal regeneration biological stent by combining related medicines or factors.

The invention also provides application of the 4-axis 3D printing tubular medical stent, which is used as an extravascular stent of a vein graft in coronary artery bypass by combining antiplatelet or anticoagulant medicaments. Can limit vein expansion, maintain the consistency of the inner diameters of the transplanted vein and the target vessel, reduce intimal hyperplasia of the transplanted vein, generate stable hemodynamics, reduce turbulence, prevent thrombosis and improve long-term patency rate.

The principle of the invention is illustrated by taking PGS as the material of the inner layer biological spring, salt particles as the filling material and collagen as the outer layer nano-fiber material as an example:

crushing salt particles by using a crusher, screening the salt particles within a certain size range by using a screen, mixing the salt particles and the PGS prepolymer according to different proportions, and comprehensively selecting the most appropriate mixing proportion to meet various requirements of 3D printing of the PGS composite material by investigating the printability of the composite material in an actual 3D fusion printing experiment, including the extrusion property, the stability of an initial form and the shape retention property in a subsequent high-temperature curing process. The mixture is loaded in a needle cylinder for 3D printing, ideal printing parameters are adjusted, continuous and uniform fiber extrusion (good extrusion property) is required, and good initial form stability is achieved after printing is completed.

For the PGS tubular biological spring of the inner layer, a multi-level pore structure is designed. The printing speed of 3D printing and the rotary receiving platform are jointly controlled and cooperated. The PGS prepolymer composite material is extruded in a linear shape and deposited on a rotating receiving shaft in a uniform spiral shape, and the PGS prepolymer composite material is in a fiber interweaving porous structure after reciprocating twice. And after further curing, a stable chemical crosslinking structure is formed, stable binding points are formed among the interwoven fibers, and micropores which are uniformly distributed and communicated with each other are formed in the bracket after salt particles are dissolved in water and removed. The contour form of the molded biological spring can be controlled by using receivers with different shapes and specifications; the diameter of the extruded strands can be adjusted by using different specification nozzles; the interweaving density can be controlled by conveniently adjusting the ratio of the material extrusion linear velocity to the receiving shaft rotation linear velocity or the reciprocating times of printing; the porosity and pore size of micropores distributed in the material can be controlled by adjusting the salt particle use ratio and size. Finally, the tubular biological spring with high control degree on characteristics of interweaving density, interweaving pores and micropores distributed in lines is obtained.

The biological spring is placed on a metal rotary receiver, the gelatin solution is prepared into uniform nano fibers by utilizing the electrostatic spinning technology, and the uniform nano fibers are adsorbed and wrapped on the surface of the biological spring to form a compact outer-layer nano fiber structure. The micro-morphology of the fiber, such as the diameter, can be conveniently controlled by spinning parameters; the distribution of the fibers can be controlled by adjusting the rotation speed of the receiver (the orientation nano fibers can be obtained by high-speed rotation); the thickness of the fibre layer can be controlled by the spinning time. Treating the double-layer bracket obtained after spinning by using a glutaraldehyde solution to ensure that the gelatin on the outer layer is subjected to stable chemical crosslinking; the gelatin outer layer shrinks in size to help the inner and outer layers to be tightly bonded. Finally obtaining the 4-axis 3D printing tubular medical stent. The scaffold is then freeze-dried to remove water from the scaffold for ease of use and long-term storage.

The invention takes thermosetting poly-glycerol sebacate (PGS) biological elastomer and gelatin which are widely applied in the field of tissue engineering as an example, and forms a 4-axis printing system of X/Y/Z/Rotation by the cooperative work of a Fused Deposition Modeling (FDM)3D printing technology and a rotary receiving platform to prepare the porous tubular biological spring and realize the construction of the double-layer composite tubular bracket by combining an electrostatic spinning technology. The double-layer tubular scaffold with the inner layer of the 3D printing biological spring and the outer layer of the nano fiber is not reported at present.

Advantageous effects

(1) The invention solves the fundamental problem that the thin-wall porous tubular stent is difficult to prepare by extrusion type 3D printing, the prepared tubular medical stent has an accurate and controllable multi-stage pore structure, the method is simple and quick, is suitable for various biological materials, can be used for personalized customization of the required medical stent according to the data of CT and the like of a patient, can be used for preparing the tubular stent required by artificial blood vessels, tracheas, esophagus, urethra and other tissue engineering, and has good application prospect;

(2) on the basis of the previous research, the invention uses the 3D printing technology to manufacture the drug-loaded extravascular stent, and is used for preventing the thrombosis of the vein graft after the coronary artery bypass grafting, the intimal hyperplasia of the transplanted blood vessel, preventing the distortion of the blood vessel and improving the long-term patency rate of the vein graft.

Drawings

FIG. 1 is a schematic process flow diagram of an embodiment;

FIG. 2 is a schematic view of a double-layered structure and a multi-stage microporous structure of a tubular stent;

FIG. 3 is a diagram of a real object before and after cross-linking of a PGS tubular biologic spring and a double-layer tubular stent printed in 3D;

FIG. 4 is a surface electron micrograph of a PGS tubular bio-spring;

FIG. 5 is a cross-sectional electron micrograph of a PGS tubular bio-spring;

FIG. 6 is an electron microscope image of the outer gelatin nanofibers in the double-layered tubular scaffold;

FIG. 7 is a cyclic compression mechanical test of a double-layered tubular elastic stent;

FIG. 8 shows live-dead staining (a, b), apoptotic staining (c, d) and DNA quantification (e) of chondrocytes seeded in vitro on double-layered tubular scaffolds one and four days after culture;

FIG. 9 is a histological staining of a specimen section 8 weeks after in vitro implantation of chondrocytes from a double-layered tubular scaffold; wherein, a-b rebuilds a tubular cartilage object image, c-f respectively represent hematoxylin-eosin staining (HE), Safranin fast green staining (Safranin-O), massson and type II collagen staining;

FIG. 10 shows immunohistochemistry and evaluation of various characteristics of cartilage regeneration after two weeks of culture of double-layered tubular scaffolds implanted with cells in vitro and further implanted subcutaneously in nude mice for 12 weeks; wherein a-d are hematoxylin-eosin staining (HE), Safranin fast green staining (Safranin-O), massson and type II collagen staining respectively; e-j is the quantitative analysis comparison of the wet weight, thickness, Young's modulus, DNA content, GAG content and collagen content of the acellular scaffold implant, the loaded cell scaffold implant and the normal cartilage;

FIG. 11 shows a PCL tubular stent in different morphological structures; wherein, a and b are round tubular stents with different diameters, c and d are electron microscope images (surface and cross section views) of the tubular stents, e-h are model images, real images and electron microscope images (surface and cross section views) of the triangular tubular stents, and i-l are model images, real images and electron microscope images (surface and cross section views) of the hexagonal tubular stents;

figure 12 is a PCL tubular scaffold of different microstructures; wherein a is a 4-axis printed model diagram, b is a tubular support fiber interweaving model diagram, c is a tubular support object diagram with different interweaving densities, and d-g is an electron microscope diagram for obtaining tubular supports with different interweaving densities by controlling the rotating speed of a receiver under the condition of controlling other parameters to be unchanged (the rotating speeds are respectively 10r/min, 20r/min, 30r/min and 40 r/min);

FIG. 13 is a graph comparing theoretical model curves to experimental data; a is the comparison between an experimental curve for printing the spiral pitch of a single fiber and a theoretical curve, b is the comparison between an experimental curve for the parallel fiber pitch in an interweaving structure and the theoretical curve, c is the comparison between an experimental curve for the fiber diameter and the theoretical curve, and d is the comparison between an experimental curve for the fiber interweaving angle in the interweaving structure and the theoretical curve;

FIG. 14 is a schematic diagram of experimental operation of an extravascular stent animal;

fig. 15 is a graph of ultrasonic flow velocity measurements after an extravascular stent implantation.

Detailed Description

The present invention will be further illustrated by the following examples, which are representative of the thermosetting polymer PGS and the thermoplastic polymer Polycaprolactone (PCL). It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

1. Preparation process

1.1 printing Material preparation

The mixing parameters of the PGS prepolymer (Pre-PGS) and the salt particles directly determine the extrudability, the initial form stability, the high-temperature curing shape retention property and the pore structure of the stent during printing, thereby indirectly determining the properties of the stent, including the mechanical property and the biodegradability. Dissolving 10g Pre-PGS in 10ml acetone to form a viscous solution; after NaCl is put into a grinder to be ground, salt particles with the diameter less than 38 mu m are obtained by a screen mesh; mixing salt granules and Pre-PGS according to the mass ratio of 1: 2, adding 20g of the acetone, uniformly mixing, standing until the acetone is slightly volatilized, placing in a vacuum oven at 30 ℃, and removing the acetone for 24 hours;

1.23D printing Pre-PGS fiber interwoven tubular Structure

The mixed material of Pre-PGS and NaCl particles was loaded into a cartridge. The extrusion chamber temperature and the nozzle temperature were set at 45 ℃. A23G needle was selected and the print length was 6 cm. The XY axis movement speed is set to be 0.1mm/s, and the T axis extrusion speed is set to be 0.008 mm/s. A stainless steel round bar with a diameter of 5mm was selected as the receiver to be mounted on a rotatable receiving platform. The distance between the spray head and the receiver is adjusted to be 3mm, and the rotation speed is 10 r/min. The composite material is extruded in a linear shape and deposited on the surface of a round bar, a spiral structure is printed on the surface of the round bar along with the movement of a spray head, and the spiral structure is printed twice (or for multiple times) in a reciprocating way, so that a porous fiber-interwoven tubular structure is formed.

1.3 curing crosslinking of Pre-PGS Bio-spring and extraction of filling Material

The tubular structure interwoven by the Pre-PGS fiber and the metal rod are placed in a vacuum oven at 100 ℃ and under the vacuum degree of 0.5bar for primary curing for 12 hours, and then the temperature is adjusted to 150 ℃ and the vacuum degree of 1bar for further curing for 24 hours. Thereby obtaining a stable chemically cross-linked structure while the positions where the fibers are interlaced with each other form stable binding sites. In order to remove salt particles and non-crosslinked polymer in the scaffold, the scaffold was immersed in a solution mixed with distilled water at a ratio of 1:3 at room temperature, changed every 4 hours, washed 3 times, frozen for 12 hours, and then freeze-dried in a freeze-dryer at-20 ℃ and 50Pa to obtain a porous tubular PGS bio-spring.

1.4 preparation of double-layer tubular scaffolds

A gelatin/hexafluoroisopropanol solution (12%, w/v) was prepared as an electrospinning solution, drawn into a 5ml syringe, and mounted on a micro syringe pump. The tubular PGS bio-spring is threaded into a metal receiver of corresponding gauge and mounted on a speed adjustable rotary receiving device. The method comprises the steps of forming uniform and continuous nano fibers under the action of 12KV high-voltage static electricity with the injection rate of 1ml/h, placing a rotary receiving device 15cm below an electrostatic spinning needle head, receiving the nano fibers in a rotating mode at the speed of 30r/min, depositing and wrapping the nano fibers on the surface of a PGS tubular biologic spring through electrostatic adsorption, and forming a densely-wrapped nano fiber layer on the outer layer after receiving for 2 hours. After the gelatin nano fiber is removed from the metal rod, the gelatin nano fiber is placed in 5% glutaraldehyde/ethanol & water solution for 5 minutes to enable the gelatin nano fiber on the outer layer to be crosslinked, and good water-soluble stability is obtained. And washing with 5 times of ethanol and 3 times of distilled water to remove residual glutaraldehyde and uncrosslinked gelatin. Freezing for 12 hours, and freeze-drying in a freeze dryer at-20 ℃ and 50Pa to obtain the 4-axis 3D printing tubular medical stent.

2. Characterization and detection

2.1 characterization and testing of morphological structures

Characterization of the scaffold structure was examined by SEM to see the morphology of the scaffold, whether deformation occurred, collapse, and pore distribution and overall structure.

2.2 mechanical Property testing and characterization

The double-layer tubular support is cut into a sample strip with the length of 1cm, the mechanical property of the double-layer tubular support in the circulating compression under the flat state is tested, the compression deformation is 50 percent, the speed is 20mm/min, and the circulating frequency is 10 times.

2.3 use of scaffolds for in vivo and in vitro tubular tracheal cartilage regeneration

Chondrocytes were obtained by digestion from the ear cartilage of New Zealand white rabbits (1.0X 108cells/ml) and seeded onto double-layered tubular scaffolds. After 1 day and 4 days of culture, the biocompatibility and the cell survival condition of the scaffold are evaluated through live and dead cell staining, an apoptosis test and DNA quantitative analysis. After 8 weeks of in vitro culture or 2 weeks of in vitro culture and then 12 weeks of subcutaneous implantation in nude mice, slice immunohistochemical test and related quantitative analysis are carried out, and the regeneration condition of tubular cartilage is comprehensively evaluated.

3. Results and analysis

3.1 printability of Pre-PGS fiber interlaced Structure

Experiments show that the ratio of the PGS prepolymer to the salt particles is 1: 2 the composite material obtained by mixing has good extrusion performance, can be continuously and uniformly extruded into a linear shape, and the salt particles can be used as filling materials to keep the stability of the printed lines of the material. The composite material lines can be attached to the surface of the metal rod, and are in a spiral structure along with the movement of the printing head along the direction of the central axis of the metal rod and the rotation of the metal rod; the lines are mutually stacked after repeated reciprocating to form an interwoven mesh structure (figure three). The structure can be controlled by adjusting the moving speed of the printing head and the rotating speed of the metal rod, the ratio of the moving speed of the printing head to the linear speed of the metal rod is smaller, the distance between lines is smaller, the interweaving density is larger, and the grid pores are smaller. Different diameter or different shaped receptacles may also be used.

3.2 curing and hierarchical pore Structure of PGS tubular spring

As can be seen from FIG. 3, the overall shape of the Pre-PGS fiber interwoven structure is not substantially changed before and after curing, because the supporting effect of the salt particles ensures that the original printed macroscopic tubular structure and microscopic line structure can be maintained under the high temperature condition. As can be seen from the surface electron microscope figure 4 of the PGS tubular spring, after curing, chemical crosslinking action is generated between the fibers which are interwoven with each other, so that stable binding sites are formed, and the integral structural stability of the bracket is improved; the diameter of the fiber is about 320 mu m and is matched with the inner diameter of the nozzle by 340 mu m; meanwhile, a large number of micropores are distributed on the surface of the fiber, so that the roughness and the surface area are increased, and the adhesion growth of cells is facilitated. As can be seen from the sectional electron micrograph of FIG. 5, the PGS spring maintains a good tubular structure with an inner diameter of about 5mm that substantially corresponds to the diameter of the metal rod used; meanwhile, a large number of micropore structures are uniformly distributed in the fiber, and the micropores are communicated with each other, so that a good substance exchange space is provided for the application of subsequent tissue engineering. The micropore structures distributed on the surface and in the stent are derived from the dissolution effect of salt particles as pore-forming agents, and the number and the size of micropores depend on the proportion and the size of the salt particles used in the preparation of the composite material.

3.3 PGS/gelatin double-layer elastic tubular Stent

As can be seen from the material object diagram of fig. 3 and the electron microscope diagram of fig. 6, a large amount of gelatin nanofibers formed by electrospinning are deposited and coated on the surface of the PGS spring to form a uniform and dense nanofiber layer. The diameter of the fiber is in the nanometer scale, the appearance is good, smooth and without beads. This dense nanofiber layer provides good perfusion to the scaffold, ensuring that it does not leak in the particular application. In addition, the nanofiber can better simulate natural extracellular matrix in morphological structure and has ultrahigh specific surface area, so that a good growth microenvironment can be provided for corresponding cells. As can be seen from the cyclic compression mechanics test chart of fig. 7, due to the dual functions of the inner PGS, the bio-elastomer, and the corresponding tubular spring structure, the finally prepared double-layered tubular medical stent has good elasticity and fatigue resistance, and can ensure that good compliance, matching property, and deformation recovery property can be maintained in a dynamic mechanical environment in vivo.

3.4 regeneration of tubular tracheal cartilage

It can be seen from fig. 8 that the double-layer elastic tubular scaffold has good biocompatibility, and chondrocytes have good survival state and obvious proliferation on the scaffold. As can be seen in FIG. 9, after 8 weeks of in vitro culture, a significant tubular cartilage structure was grown using the scaffold as a template. After staining with Safranin-O, massson and type II collagen, a distinct cartilage structure was seen. Also, as can be seen from fig. 10, after two weeks of in vitro cell culture, the obtained mature tubular cartilage was confirmed by staining after further culture implanted subcutaneously in nude mice. The tubular scaffold is directly implanted into the skin to be used as a negative control group, the normal cartilage is used as a positive control group, and the weight, the thickness, the Young modulus, the DNA content, the glycosaminoglycan content and the total collagen content of a sample are tested and quantitatively analyzed. It can be shown that the double-layer tubular scaffold can regenerate tubular mature cartilage and has better performance and result in various characteristics.

4. Conclusion

The embodiment provides a preparation method of a 4-axis 3D printing tubular medical stent, which solves the fundamental problem that thin-wall porous tubular stents are difficult to prepare by extrusion type 3D printing. The tubular bracket has an accurately controllable multistage pore structure in structure, has excellent elasticity and sewability in mechanical property, is simple and quick in method, is suitable for various biological materials, takes tracheal cartilage regeneration as an application example, proves that the tubular bracket can regenerate a mature tubular cartilage structure, and has good application prospect.

Example 2

1. Preparation process

1.13D printing Polycaprolactone (PCL) tubular stents of different macroscopic forms

PCL with 8 ten thousand molecular weight is preheated in a printer for 30 minutes at 200 ℃ to be fully melted and extruded into uniform material by a 22G needle head at 180 DEGAnd deposited on a rotating receiving device. The shape of the receiving device is controlled to obtain tubular stents with different morphological structures. Wherein the printer heating chamber has an inner diameter of (D)_e)18mm。

1.23D PCL tubular Stent with different microstructures

Under the above printing conditions, the diameter (D) is used_r)5.14cm cylindrical receiving device, maintaining extrusion Rate (V)_e) 0.03mm/min, and a nozzle moving speed of (V)_m)1mm/s, and printing was repeated 5 times. The rotation rate n of the receiving device is controlled to be 10, 20, 30 and 40min/r respectively.

1.33D printing blood vessel outer sleeve support

PCL and rapamycin are blended to be used as printing raw materials. A rotary receiving device with the outer diameter of 1.5mm is adopted, and a tubular drug-loaded tubular stent with the inner diameter of 1.5mm and the outer diameter of 2mm is printed out and used as a blood vessel jacket of abdominal aorta of a rat.

1.4 surgical methods

Male SD rats (250-300g), pentobarbital (30mg/kg) were used for intraperitoneal injection for anesthesia, in the supine position, prepared for skin by a conventional method, sterilized and draped. Taking a right anterior sternocleidomastoid anterior incision, cutting skin and subcutaneous tissue layer by layer, blunt-separating submaxillary gland, turning up, exposing and separating external jugular vein, wherein the length of the incision is about 3 cm. The length of the vein bridge is about 1.5cm, the two ends are ligated by 8-0 atraumatic suture, the lumen is flushed by heparin normal saline (containing heparin 12500U/L) until the wall is whitish and no mural thrombus is left, and the vein bridge is placed in the vein for preservation. A vascular sleeve (about 5mm in length) was prepared with a venous indwelling needle and a groove was scored about 1mm from the end to be ligated and secured. Muscle bundles were retracted between the ipsilateral sternocleidomastoid muscle and sternohyoid muscle, exposing the carotid sheath, leaving approximately 2cm of free common carotid artery. The two ends of the common carotid artery block blood flow by a non-invasive vascular clamp, the middle of the common carotid artery is cut off by 0.3-0.5 cm, and the lumen is flushed by heparin saline. 2 8-0 traction lines are sewn at each broken end of two sides of the blood vessel, the leading guide is led to pass through the artery at the heart end from the self-made blood vessel sleeve, the traction lines are slightly drawn, the artery is outwards overturned and sleeved outside the blood vessel sleeve under the assistance of toothless forceps, the blood vessel is fixed, the inverted vein far-end is inserted (the inversion aims to prevent the blood supply after the transplantation of a tiny vein valve in the vein blood vessel), and 8-0 nylon lines are tied at the groove. The vein is guided to pass through the outer blood vessel stent, the distal artery and the proximal vein are fixed by the same method, after the transplanted vein blood vessel is firmly tied and sleeved, the noninvasive blood clamp is slowly loosened to recover blood flow of a blood vessel bridge, the color of the blood vessel bridge turns red, and the blood flow is smooth, the filling is obvious, and the model is prompted to be successfully manufactured by pulsation.

2. Characterization and detection

2.1 characterization and testing of morphological structures

The characterization of the scaffold structure is detected by SEM, and the morphology of the scaffold is observed, whether deformation or collapse occurs is observed. Measurement of structural parameters of stents of different microstructures including helical spacing S, fiber spacing L, fiber diameter D_fThe fiber interlace angle 2 θ. And simultaneously modeling the printing process, and constructing a theoretical formula for the corresponding structural parameters according to the model.

2.2 ultrasonic testing

And (5) carrying out ultrasonic detection on carotid arteries at 1, 4 and 8W after vein transplantation, measuring the diameter and the blood flow direction of the blood bridge vessel and judging the smoothness.

2.3 morphological analysis

After vein transplantation, 8W of experimental animals are anesthetized again, the original incision is exposed, a vein bridge is separated, the vein bridge and artery parts at two ends are cut off together, the blood clots are washed by PBS after the materials are taken, and 4% paraformaldehyde is fixed for 24 hours for later use. HE. Masson staining, observing tissue morphology, measuring the inside and outside diameters of blood vessels, and the thickness of the intima and media of blood vessels.

2.4 immunohistochemistry: PCNA, TGF-b1, Vimentin, alpha-SMA, and observing the degree of proliferation of vascular intimal cells. TUNEL observed apoptosis.

3. Results and analysis

3.1 control of morphology and microstructure of PCL controlled scaffolds

As can be seen in fig. 11a-i, the macroscopic morphology of the tubular stent is well matched to the rotating means. Tubular stents of different diameters can be obtained conveniently by this method. By using the receiving device with the anisotropic structure, various anisotropic tubular stents with uniform shapes can be quickly obtained, and the interfaces of the stents comprise triangles, hexagons and the like. From fig. 12a-g it can be seen that in the theoretical model of stent printing and the forming process, the extruded fibers are interwoven with each other to form a network structure. With increasing rotational speed, the weave density of the fiber mesh increases. The theoretical parameters of the corresponding structure are calculated according to the model as follows:

as can be seen in FIGS. 13a-D, the pitch of the helix (S), the pitch of the fiber (L), and the diameter of the fiber (D)_f) And the fiber interweaving angle 2 theta is inversely proportional to the rotating speed (n) of the receiving device, and the structural parameters of the experimental sample are highly fitted with a theoretical curve, so that the controllability and the reproducibility are good.

3.2 PCL tubular Stent for Small vessel sheath

As can be seen in FIG. 14, the PCL tubular stent can be used as a vascular sheath stent after vein transplantation. The stent has good flexibility and shows the matching property with blood vessels. As can be seen from fig. 15, the external stent can prevent the restriction of the vein expansion, maintain the consistency of the inner diameters of the graft vein and the target vessel, stabilize the hemodynamics, reduce the turbulence, and prevent the thrombus formation. The use of the external vascular stent can not only prevent thrombosis, but also reduce the intimal hyperplasia of the transplanted vein and improve the long-term patency rate.

4. Conclusion

The embodiment provides a preparation method of a 4-axis 3D printing tubular medical stent, and the stent can be loaded with various anticoagulation, anti-platelet and anti-intimal hyperplasia drugs. The stent can regulate and control the macroscopic morphology and the microstructure thereof, and shows the advantage of personalized customization. As an external stent, the stable reticular structure can effectively prevent the vasodilatation caused by mechanical mismatching after vein transplantation, and the combination treatment of the loaded medicine can prevent intimal hyperplasia, stabilize blood flow and maintain long-term smooth circulation rate.

Claims

1. The utility model provides a 4 medical support of tubulose 3D printing which characterized in that: the device comprises nanofibers and a 4-axis 3D printed tubular spring support, wherein the nanofibers are coated on the surface of the support to form a double-layer structure; the multistage pore structure comprises pores formed by interweaving linear materials in the tubular spring support, micropores or nanopores which are uniformly distributed in the linear materials and are communicated with one another, and a nano pore structure formed by depositing, wrapping and interweaving nano fibers.

2. A preparation method of a 4-axis 3D printing tubular medical stent comprises the following steps:

3. The preparation method of the 4-axis 3D printing tubular medical stent according to claim 2, wherein the method comprises the following steps: the gel material in the step (1) is polyacrylamide, sodium alginate, collagen, gelatin or fibroin; the thermoplastic material is polycaprolactone, polylactic acid-glycolic acid copolymer or thermoplastic polyurethane; the thermosetting material is polysebacic acid glyceride, thermosetting polyurethane or epoxy resin.

4. The preparation method of the 4-axis 3D printing tubular medical stent according to claim 2, wherein the method comprises the following steps: the filling material in the step (1) is salt particles, graphene, carbon nano tubes, bioglass, silicon dioxide, nano clay, nano short fibers or biological factors.

5. The preparation method of the 4-axis 3D printing tubular medical stent according to claim 2, wherein the method comprises the following steps: the mixing mode in the step (1) is a solvent mixing method or a heating method.

6. The preparation method of the 4-axis 3D printing tubular medical stent according to claim 2, wherein the method comprises the following steps: the 3D printing in the step (1) comprises the following process parameters: the temperature of the extrusion cavity and the temperature of the nozzle are 0-250 ℃, the XY axis movement speed is set to be 0.1-20 mm/s, the T axis extrusion speed is 0.008-0.1mm/s, the rotation speed of the receiving device is 5-150 r/min, the diameter of the receiver is 2-20 mm, and the cross section of the receiver is circular, oval, triangular or quadrilateral.

7. The preparation method of the 4-axis 3D printing tubular medical stent according to claim 2, wherein the method comprises the following steps: the curing crosslinking in the step (2) is thermal crosslinking or photocrosslinking.

8. The preparation method of the 4-axis 3D printing tubular medical stent according to claim 2, wherein the method comprises the following steps: the pore size distribution of the tubular spring support in the step (2) is 10 nanometers to 100 micrometers.