CN111938866A

CN111938866A - Special-shaped blood vessel model or artificial blood vessel and preparation method thereof

Info

Publication number: CN111938866A
Application number: CN202010383438.8A
Authority: CN
Inventors: 万烨; 张�杰; 俞中平
Original assignee: Lingbo Biotechnology Hangzhou Co ltd
Current assignee: Lingbo Biotechnology Hangzhou Co ltd
Priority date: 2020-05-08
Filing date: 2020-05-08
Publication date: 2020-11-17

Abstract

The invention belongs to the technical field of medical instruments, and particularly relates to a special-shaped blood vessel model or an artificial blood vessel and a preparation method thereof. A method for preparing a special-shaped blood vessel model or an artificial blood vessel comprises the following steps: acquiring three-dimensional size data of a natural blood vessel to be researched or replaced, and constructing a corresponding bionic blood vessel 3D mould; then preparing a polymer bracket on the bionic 3D mould; depositing cells and extracellular matrix on the vascular stent. Has the advantages that: the method can simulate natural special-shaped blood vessels in vitro, and is helpful for obtaining more accurate and meaningful research data; the blood vessel of the invention has enough mechanical strength, is favorable for the quasi-natural regeneration of blood vessel tissues, and can be used for the treatment and research of blood vessel diseases.

Description

Special-shaped blood vessel model or artificial blood vessel and preparation method thereof

Technical Field

The invention belongs to the technical field of medical appliances, and particularly relates to a special-shaped blood vessel model or an artificial blood vessel and a preparation method thereof.

Background

The cardiovascular and cerebrovascular diseases are most commonly cerebral apoplexy and myocardial infarction, and the morbidity and mortality are highest in the whole world. The root cause is angiopathy mainly including atherosclerosis, and the harm is reflected in brain and heart and ischemic necrosis of other organs, such as eye, liver, kidney, lower limb, etc. Therefore, the research on the occurrence and development mechanism of vascular diseases and the development of drugs for preventing and treating atherosclerosis are always the most important subjects in the field of cardiovascular and cerebrovascular research.

Many factors causing vascular diseases, such as hyperlipidemia, hypertension, hyperglycemia, drugs, etc., induce damage and functional changes of vascular endothelial cells and smooth muscle cells, and then cause vascular lesions. Therefore, in the research of vascular biology and vascular physiology, a large number of in vitro cell experiments are needed to observe and verify how various factors induce the damage and functional change of vascular endothelial cells and smooth muscle cells. Currently, these cell experiments rely mainly on simple two-dimensional cell culture on petri dishes, and individual laboratories have conducted studies using Flow cells (Flow chambers) to culture vascular endothelial cells or smooth muscle cells.

Considering the environments of space, pressure, flow mechanics, contraction and relaxation and the like of vascular cells under natural conditions, only by developing vascular cell experiments under the condition of simulating the environment of blood vessels, more meaningful vascular biology and vascular physiology information can be obtained, so that vascular disease mechanisms and prevention strategies can be more accurately researched. Therefore, the prepared bionic active artificial blood vessel is used for an in vitro model for vascular biology and physiology research, and has great application value. The natural blood vessels have various shapes and have special-shaped structures such as branches and bends, and when blood pulsates and flows in the natural blood vessels, the tube walls can generate stress action with various complex functions, so that the flow chamber cannot simulate. In addition, the common clinical cardiovascular bypass, head and neck bypass, hemodialysis arteriovenous fistula and the like adopt a blood vessel side suture mode (bridging). The blood flow dynamics at the branching or bridging position of the blood vessel is not uniform, and vascular diseases such as intimal hyperplasia or thrombus occur at the position. Therefore, when the in vitro cell experiment is used for researching the vascular lesion mechanism, the artificial blood vessel with a special-shaped structure is particularly needed. To the best of our knowledge, there is currently no technique and product for the preparation of models of blood vessels for experimental studies, in particular models of blood vessels without active, abnormal structures.

In addition, there is a great demand for artificial blood vessels in the treatment of cardiovascular diseases or peripheral vascular diseases, and currently, the artificial blood vessels used clinically are mainly polyethylene terephthalate

Expanded polytetrafluoroethylene

And non-degradable polymer materials such as polyurethane, which has good effect in the replacement operation of large-caliber blood vessels, but has small caliber (diameter)<6mm), the artificial blood vessel made of the polymer material is easy to have acute thrombosis or stenosis and other complications after being implanted into a body, and further causes the transplantation failure due to the slow blood flow speed. In recent years, more and more degradable polymer artificial blood vessels appear, are prepared by adopting methods such as electrostatic spinning, 3D printing, melt spinning or weaving and the like, and show that the degradable polymer artificial blood vessels are superior to the degradable polymer artificial blood vessels

And

biocompatibility of artificial blood vessels and vascular tissue regeneration, however, a simple vascular material lacks tissue regeneration activity, resulting in slow endothelial formation, difficulty in smooth muscle regeneration, and the presence of calcification and advanced restenosis. In addition, the blood vessel replacement therapy faces increasingly complex graft requirements, and in the polymer artificial blood vessel research reported at present, the preparation technology of the artificial blood vessel with the special-shaped structure can solve the clinical indications with various structures. Therefore, the active artificial blood vessel and the abnormal artificial blood vessel of the inventionCan also be used for treating vascular diseases.

Disclosure of Invention

The invention aims to solve the technical problem of providing a special-shaped artificial blood vessel or blood vessel model and a preparation method thereof, and the special-shaped artificial blood vessel or blood vessel model with a similar special-shaped structure is designed and prepared according to the special-shaped structure and characteristics of the natural blood vessel to be researched or replaced, such as shape, pipe diameter change, curvature change, bifurcation, hemodynamics and the like. Solves the problems that the regular blood vessels can not carry out the research on the biology, physiology and hydrodynamics of the blood vessels of specific parts and can not transplant and treat diseased blood vessels with complex structures in the prior art.

The invention discloses a preparation method of a special-shaped blood vessel model or an artificial blood vessel, which comprises the following steps:

step 1, utilizing computer software to acquire three-dimensional data of the size of a blood vessel of a specific part of an animal or human body, wherein the data acquisition method comprises at least one of CT scanning, Doppler ultrasonic imaging, nuclear magnetic imaging and angiography. The bionic blood vessel 3D mold is constructed by utilizing the acquired three-dimensional data of the blood vessel size and adopting materials such as medical stainless steel, polypropylene (PP), silica gel and the like through methods such as 3D printing, casting, cutting, laser etching and the like.

And 2, preparing the porous and reticular polymer scaffold on the blood vessel mould in the step 1. The material is selected from one or a mixture of a plurality of Polycaprolactone (PCL), polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA), p-dioxanone (PDO) and poly L-lactide-caprolactone (PLCL) in any proportion, and the preparation method adopts at least one of electrostatic spinning, melt spinning, fiber weaving and 3D printing. The vascular mold and the reticulated polymer scaffold are combined to be called a vascular complex.

And 3, removing the blood vessel mold from the blood vessel complex prepared in the step 2, wherein the blood vessel mold is removed by at least one of drawing and taking out, mechanical disassembling, dissolving and taking out, and melting and taking out to obtain a blood vessel model or an artificial blood vessel A.

And 4, performing flow culture on the cells A prepared in the step 3 in an in vitro bioreactor, and removing the cells after the cells are fully proliferated and generate extracellular matrix to obtain an active blood vessel model or an artificial blood vessel B.

And 5, embedding the vascular complex prepared in the step 2 under the skin of an animal or human, taking out the vascular complex from the skin of the animal or human after sufficient cellularization and extracellular matrix generation, removing the vascular mould, and removing cells to obtain an active vascular model or an artificial blood vessel C.

Further, the porous, reticulated polymer scaffold prepared as described in step 2, wherein the structural parameters include fiber diameter, pore size, wall thickness, fiber orientation, and inter-fiber cross-over angle. The diameter of the fiber is 5-100 μm, the crossing angle between the fibers is 30-150 degrees, and the thickness of the fiber support is 100-1000 μm.

Preferably, the diameter of the fibers of the reticular fiber scaffold is 60 +/-5 μm, and the crossing angle between the fibers is 130 degrees. The reticular fiber scaffold enables fibers to be adhered to a certain degree through a heating method or an organic solvent swelling method.

The cell removing treatment steps in the step 4 and the step 5 comprise at least one of an SDS method and a liquid nitrogen freeze-thaw method.

Preferably, the SDS method comprises the steps of: soaking the cellularized vascular stent in 1% SDS solution, placing the vascular stent on a shaking table, shaking the vascular stent for 12 hours at room temperature, washing the SDS remained on the vascular stent with sterile physiological saline, and then placing the vascular stent in a sterile mixed solution of DNase and RNase (the enzyme liquid system is 40ml, and the buffer solution is 0.2mol/L MgCl)₂，0.2mol/L CaCl₂And 0.1mol/L Tris-HCl with pH of 6.4 and ultrapure water, wherein the concentration of DNase is 50U/ml, and the concentration of RNase is 1U/ml), shaking the mixture on a shaking table at room temperature for 24 hours, then washing residual DNase and RNase on the vascular stent by using sterile physiological saline, and finally placing the obtained product in sterile PBS for storage at 4 ℃.

Preferably, the liquid nitrogen freeze-thaw method comprises the following steps: the cellularized blood vessel scaffold is quickly frozen in liquid nitrogen for 20s, thawed at room temperature for 60s, repeated for 5 times, and then washed with sterile physiological saline for 4-5 times to wash away cell residues. The material is then placed in a sterile mixed DNase and RNase solution (enzyme)The liquid system is 40ml, and the buffer solution is composed of 0.2mol/L MgCl₂，0.2mol/L CaCl₂And 0.1mol/L Tris-HCl with pH of 6.4 and ultrapure water, wherein the concentration of DNase is 50U/ml and the concentration of RNase is 1U/ml), shaking the mixture on a shaking table at room temperature for 24 hours, then washing residual DNase and RNase on the vascular stent by using sterile physiological saline, and finally placing the prepared product in sterile PBS for storage at 4 ℃.

The invention also discloses a special-shaped blood vessel model or an artificial blood vessel, and the special-shaped blood vessel model or the artificial blood vessel is produced by using the preparation method of the special-shaped blood vessel model or the artificial blood vessel.

The invention has the beneficial effects that:

1. a heterotypic structure blood vessel model or artificial blood vessel has shape, diameter change, curvature change, bifurcation mechanics, irregular characteristics such as hemodynamics that match with natural blood vessel, have solved the problem that the structure, mechanical characteristic that the artificial blood vessel of existing regular shape causes because of the structure is homogeneous are unmatched with natural blood vessel, can be regarded as the blood vessel model to study the biological problem of blood vessel, study the pathological change mechanism of the blood vessel better, and carry on the vascular graft and treat;

2. the special-shaped blood vessel model or the artificial blood vessel constructed by the invention has activity, the activity is obtained by respectively inoculating adult cells or stem cells, performing flow culture in an in vitro bioreactor or subcutaneously implanting, both modes can generate extracellular matrix, and the polymer fiber scaffold is embedded. After the cells are removed, the components and the activity of the polymer fibers and the extracellular matrix are not affected.

3. The obtained special-shaped blood vessel model can be used for blood vessel cell culture, simulates the structure, mechanics and active tissue microenvironment of a natural blood vessel, can research the pathological mechanism of a diseased blood vessel with a specific part and a special shape, and obtains more valuable vascular biological information. Serving as a novel effective blood vessel model and research tool for cardiovascular scientific research.

4. Meanwhile, the tissue regeneration performance of the artificial blood vessel with the active extracellular matrix is obviously superior to that of the artificial blood vessel prepared by a simple material, and the artificial blood vessel can be used for clinical blood vessel transplantation treatment and obviously improves the treatment effect.

Detailed Description

The following examples are given to illustrate the technical examples of the present invention more clearly and should not be construed as limiting the scope of the present invention.

The technical embodiment adopted by the invention is as follows:

example 1

The embodiment uses a near-field direct writing system to prepare a porous artificial blood vessel stent with curvature change and a 'bell mouth' structure suture end, is used for a blood vessel model of a heart bypass surgery, and researches a mechanism of the bypass blood vessel in stenosis occurrence and development, and comprises the following steps:

CT imaging and angiography are carried out on a coronary artery bypass surgery part of a beagle dog, the part needing bypass and a needed blood vessel are accurately measured through software, a special-shaped artificial blood vessel three-dimensional model which has XYZ-axis three-dimensional curvature change and a 'horn mouth' structure and is used for controlling blood flow force at a suture end is constructed, and hemodynamic data acquisition is carried out on blood flow of the blood vessel;

according to the constructed three-dimensional model of the artificial blood vessel with three-dimensional curvature change, a special-shaped structure mould which has the diameter of 2.5mm, the curvature radius of 8cm and 13cm and is vertically bent and two conical structures at two ends is prepared by polypropylene (PP) through a casting technology;

according to the three-dimensional construction of a transplanted blood vessel and the hemodynamic data of a blood vessel transplantation part, a porous fiber scaffold which can be matched with the hemodynamics of the transplantation part is designed by utilizing computer software, and the porous fiber scaffold which is vertically bent, has the caliber of 2.5mm, the curvature radius of 8cm and 13cm respectively, the fiber diameter of 6 mu m and the wall thickness of 250 mu m and is provided with a horn mouth structure at two ends is prepared on a PP (polypropylene) mould by using a polymer PCL (polycaprolactone) through a near-field direct writing system;

the PP mould is detached and taken out, the human induced pluripotent stem cells (h-iPS) are planted on the fibrous scaffold, and the PP mould is rotated by 90 degrees every 4 hours in the planting process so as to ensure that the planted cells are uniformly distributed. The culture was left standing for 24 hours, and then continued for 5 days to proliferate the cells. After the proliferation culture was completed, the culture medium was replaced with a culture medium supplemented with a smooth muscle cell inducing factor, and the flow culture was continued for 7 days while gradually increasing the flow rate of the culture medium from 20mL/min to 200mL/min at an increasing rate of 30mL/min per day by means of a peristaltic pump.

After the culture process is finished, the cellularized fiber scaffold is subjected to decellularization treatment according to the following steps: quick freezing in liquid nitrogen for 20s, thawing at room temperature for 60s, repeating for 5 times, washing with sterile physiological saline for 4-5 times, and washing cell residue. Then, the material was put into a sterile mixed solution of DNase and RNase (enzyme solution system 40ml, buffer solution prepared from 0.2mol/L MgCl2, 0.2mol/L CaCl2, 0.1mol/L Tris-HCl with pH 6.4 and ultrapure water, DNase concentration 50U/ml, RNase concentration 1U/ml) on a shaker at room temperature for 24 hours, then DNase and RNase remained on the fiber scaffold were washed clean with sterile physiological saline, and finally the obtained product was put into sterile PBS and stored at 4 ℃.

The active blood vessel model with the diameter of 2.5mm, regular shape and no curvature change is prepared by adopting the process and is used as a reference material.

The obtained heterotypic structure active blood vessel model with curvature change is used as an in vitro blood vessel model of the beagle heart bypass, and the influence of the hemodynamics at the anastomotic site of the coronary artery bypass on the restenosis of the coronary artery graft is researched by using the active blood vessel model with regular shape and no curvature change as a contrast. The method comprises the following specific steps: respectively planting vascular smooth muscle cells on the two vascular models, rotating 90 degrees every 4 hours in the planting process, and after the planting is finished, placing the vascular stent at 37 ℃ and 5% CO₂The culture box is kept still for 24 hours to ensure that the inoculated cells are adhered to the vascular model bracket. The vessel model was then inoculated into a flow culture reactor and cultured for 7 days with shear stress and/or radial compressive stress simulating blood flow conditions at the site of cardiac bypass as required for the study. By analyzing and comparing cell proliferation, gene expression, protein expression and the like, the generation mechanism of vascular restenosis at a heart bypass part caused by hemodynamic stimulation is explored, and abnormal blood vessels and 'bell mouth' are evaluated "The suture mode has significance in the heart bypass operation.

Example 2

In the embodiment, a PU fiber scaffold with a bifurcation structure is prepared by biological weaving, a tissue engineering blood vessel is constructed to serve as a blood vessel model, the blood vessel of the bifurcation part of an abdominal aorta is simulated, and the influence of hydromechanics, material compliance and microenvironment on the physiology and function of the blood vessel is researched in vitro, which comprises the following steps:

imaging the abdominal aorta branch position of the beagle dog by angiography, determining the diameter (8-6-4mm, the main blood vessel 8mm, the primary branch blood vessel 6mm, the secondary branch blood vessel 4mm), the bifurcation angle (70 degrees) and the length (6-5-4cm, the main blood vessel 6cm, the primary branch blood vessel 5cm and the secondary branch blood vessel 4cm), constructing a three-dimensional structure of a blood vessel, and collecting and analyzing the hemodynamic data of the part;

the medical stainless steel is segmented by adopting a forging technology to prepare a die which meets the three-dimensional structure of the blood vessel required by transplantation, and the assembly of the die does not influence the separation from the subsequent reticular fibrous scaffold;

designing a reticular fibrous scaffold meeting the mechanical strength of the abdominal aorta transplantation by adopting computer software according to the three-dimensional structure of the blood vessel and the hemodynamics characteristics of the abdominal aorta constructed in the steps;

adopting a biological weaving technology, weaving a reticular fiber scaffold with the fiber diameter of 100 mu m, the tube diameter of 8-6-4mm, the bifurcation angle of 70 degrees and the length of 6-5-4cm by using PU polymer on an assembled abdominal aorta bifurcation mould to obtain a complex consisting of medical stainless steel and the reticular fiber scaffold;

embedding the complex under sheepskin, taking out after one month, disassembling and removing the stainless steel mould to obtain an artificial blood vessel material consisting of polymer fibers, cells and extracellular matrix;

the artificial blood vessel material is subjected to cell removal treatment, firstly placed in liquid nitrogen for quick freezing for 20s, unfrozen for 60s at room temperature, repeated for 5 times, and then washed with sterile normal saline for 4-5 times, and cell residues are washed clean. Then placing the artificial blood vessel material in a sterile DNase and RNase mixed solution (the system is the same as the above 2.1), shaking for 24h at room temperature on a shaking table, and then washing residual DNase and RNase on the vessel with sterile normal saline in an ultra-clean workbench to obtain a branched heterotype structure active blood vessel model;

the heterotypic active blood vessel model with bifurcation obtained in the above steps is used as an in vitro model for researching blood vessels at the abdominal aorta bifurcation part of beagle dog, and a conventional flow chamber model is used as a control. Planting vascular endothelial cells on the vascular model, rotating 90 degrees every 4 hours in the planting process, and after the planting is finished, placing the vascular stent at 37 ℃ and 5% CO₂The culture box is statically cultured for 24 hours, so that the inoculated cells are adhered to the vascular model bracket. In the control group, endothelial cells were seeded on cell-specific slides and placed in a flow cell. The vessel model and the flow cell were then simultaneously introduced into the flow culture reactor. Shear stress and/or radial compressive stress simulating the blood flow conditions at the abdominal aortic bifurcation sites of dogs were applied for 7 days as required for the study. After the culture is finished, the outline of the vascular endothelial cells is stained by a silver staining technology so as to observe the shape and orientation distribution of the endothelial cells. In addition, total proteins of cells cultured on the blood vessel model and the glass slide are respectively extracted, differential expression proteins are searched by a two-dimensional gel electrophoresis technology, and protein identification is carried out by a mass spectrometry technology. Analyzing the differential expression protein obtained in the steps, finding out the protein related to mechanical stimulation, adopting software such as IPA (insulin Pathway analysis) to analyze the function and signal path of the differential protein, and inspecting the influence of the hydrodynamic stimulation on the gene regulation and the protein expression of the vascular cells. The analysis result is compared with a conventional flow cell model, and the influence and the effect of the hemodynamics on the physiological function of the vascular endothelial cells under the bionic condition are researched.

Example 3:

the implementation case uses a reticular PLCL fiber scaffold to construct an active artificial blood vessel, selects the carotid artery of sheep as a transplantation part, and carries out the research of remodeling of the physiological functions of the blood vessel, and comprises the following steps:

the embodiment adopts melt spinning to prepare the PLCL fiber scaffold tissue engineering blood vessel with variable pipe diameter, and adopts sheep carotid artery bypass transplantation, which comprises the following steps:

performing Doppler ultrasonic imaging system, CT and nuclear magnetic resonance imaging detection on the carotid artery of the experimental animal sheep to obtain structural data of a blood vessel at a transplanted part, correcting the diameter of a natural blood vessel to be uniformly reduced from 4mm to 2.5mm by using a computer model, wherein the length of the natural blood vessel is 8cm, and detecting the hemodynamic data of the part by using a biosensor;

preparing a medical silica gel rod mould with the pipe diameter uniformly reduced to 2.5mm from 4mm and the length of 8cm by adopting a 3D printing method;

calculating the required strength of the PLCL melt spinning reticular fiber scaffold by using computer control software according to the measured pipe diameter change and length and the hemodynamic data of the transplantation part;

setting the melting temperature of a melt spinning machine to be 200 ℃, melting 2g of PLCL for 1h, spinning a reticular fiber support with the fiber diameter of 60 mu m, the fiber intersection angle of 50 degrees and the thickness of 400-350 mu m on a silica gel rod with the length of 8cm and the outer diameter of 4cm uniformly changed to 2.5cm by utilizing the control of computer programming software, and taking out a silica gel rod mould after the preparation is finished;

and (3) carrying out cell planting on the tubular fiber scaffold. Vascular smooth muscle cells were first seeded on scaffolds at 37 ℃ and 5% CO₂The culture chamber of (1) was statically cultured for 24 hours to allow smooth muscle cells to adhere to the vascular stent, and a flow culture was performed for 7 days while controlling the flow rate of the medium by a peristaltic pump and gradually increasing from 20mL/min to 200mL/min at an increasing rate of 30mL/min per day. Then, endothelial cells are planted on the inner cavity surface of the artificial blood vessel, and the endothelial cells are uniformly attached to the inner cavity surface through uniform rotation culture for 8 hours. Then continuing to culture for one week under the same flow culture parameters as the first week;

after the flow culture is finished, the obtained tissue blood vessel is subjected to decellularization treatment, and the steps are as follows: soaking the cellularized vascular stent in 1% SDS solution, placing the vascular stent on a shaking table, shaking the vascular stent for 12 hours at room temperature, washing the SDS remained on the vascular stent with sterile physiological saline, and then placing the vascular stent in a sterile mixed solution of DNase and RNase (the enzyme liquid system is 40ml, and the buffer solution is 0.2mol/L MgCl)₂，0.2mol/L CaCl₂Mixing with 0.1mol/L Tris-HCl with pH of 6.4 and ultrapure water, wherein the concentration of DNase is 50U/ml and the concentration of RNase is 1U/ml), shaking on a shaking table at room temperature for 24h, then washing residual DNase and RNase on the vascular stent with sterile physiological saline, and finally placing the obtained product in sterile PBS for storage at 4 ℃;

implanting the prepared active artificial blood vessel into the corresponding carotid artery of the sheep, and adopting a blood vessel bypass and end-to-end anastomosis mode to ensure that one end with the diameter of 4mm is connected with the proximal end and the other end with the diameter of 2.5mm is connected with the distal end. By analyzing in vitro monitoring hemodynamics, histology after material drawing and the like, evaluation research is carried out on the processes of in vivo vascular biology, pathological regeneration and reconstruction of the special-shaped artificial blood vessel, and the changes of hemodynamics, cytology and vessel wall physiology caused by different diameters of an anastomotic part are focused.

Example 4

In this embodiment, a 3D printing technology is used to prepare a PCL porous fibrous scaffold with bionics, cells are planted on the scaffold, an artificial vascular model of the superficial temporal artery of a human is established in vitro by a flow culture technology, the influence of hemodynamics on endothelial cells and smooth muscle cells and a vascular lesion mechanism are studied, and the PCL porous fibrous scaffold is used for drug screening and evaluation, and the steps are as follows:

carrying out CT imaging on the superficial temporal artery to obtain a three-dimensional structure of a blood vessel, preparing a corresponding blood vessel mould by taking medical silica gel as a material through fusion casting, printing and preparing a porous fiber scaffold with the fiber diameter of 10 mu m and the structure consistent with the superficial temporal artery on the mould by taking PCL as a material through a biological 3D printing technology, and taking out the silica gel mould after the preparation is finished;

vascular smooth muscle cells were seeded on the porous fibrous scaffold according to 3 x 10⁶Individual cell/cm²The tube body is inoculated on the porous bracket at the density, and the tube body is rotated by 90 degrees every 6 hours in the inoculation process. After inoculation, the flow culture was continued, and the cells were proliferated by continuing the culture for 5 days. After the proliferation culture was completed, the number of cells in the lumen was 1 x 10^{6 are}Individual cell/cm²Uniformly inoculating vascular endothelial cells, standing for 8 hr, performing flow culture for 7 daysThe flow rate of the medium was controlled by a peristaltic pump during the culture and gradually increased from 20mL/min to 200mL/min at an increasing rate of 30mL/min per day. Obtaining a superficial temporal artery tissue engineering blood vessel;

as a control, cells were seeded and cultured on commercial ePTFE artificial blood vessels using the same method described above; in addition, vascular smooth muscle cells were seeded on the glass slide of the flow cell, and vascular endothelial cells were seeded on top of the smooth muscle cell layer.

The three types of models simulating arterial blood vessels are used for in vitro experiments to study the biological and physiological problems of blood vessels. The three blood vessel models are respectively connected to a flow culture bioreactor, and are stimulated by the same flow mechanics, shearing force and pressure simulating the normal blood flow condition, and the growth, gene expression and protein expression rules of all groups of cells are analyzed and compared. Or simulating vascular injury, treating vascular cells by using LPS or TNF-alpha to cause cellular injury, adding 250mmol/L trapidil or 30 mu g/mL salvia miltiorrhiza extract and the like into a culture medium, applying drug treatment to the three simulated arterial vascular models, detecting the recovery of cellular functions, and screening and evaluating the pharmacological effects of the trapidil or the salvia miltiorrhiza extract and other drugs. Compared with commercial ePTFE blood vessels or flow chamber models, the bionic tissue engineering blood vessels can simulate the physiological environment of the blood vessels more truly and carry out more scientific in-vitro experimental research.

Example 5

The embodiment of the scheme adopts a wet spinning technology, a fiber support is constructed on a medical silicone tube by taking PLCL as a material, and the fiber support is embedded under the skin of a rabbit to prepare the active artificial blood vessel with extracellular matrix for the blood vessel transplantation treatment, and the implementation steps are as follows:

firstly, testing the natural carotid artery of an adult New Zealand rabbit by using a Doppler ultrasonic imaging system to obtain the blood vessel specification of a transplanted part, and preparing an inner diameter silicone tube by fusion casting according to the natural blood vessel specification;

a10% PLCL solution was extruded into the coagulation bath at a rate of 2.5ml/h, and a silicone tube was fitted over the stainless steel tube as a receiving rod to collect PLCL fibers at a rate of 1000 rpm. The prepared fibrous scaffolds were washed 3 times with ethanol and then dried in a vacuum desiccator for 2 days to remove residual solvent;

the fiber diameter of the bracket is 20 μm, a complex body formed by the fiber bracket and the medical silicone tube is tied by medical sutures at two ends, and is implanted under the back skin of a New Zealand rabbit for cultivation for one month;

taking out PLCL fibrous scaffold containing cells and extracellular matrix from subcutaneous part, removing peripheral connective tissue and silicone tube, and directly transplanting autologous rabbit carotid artery, or preparing acellular active artificial blood vessel for allogeneic rabbit carotid artery transplantation according to the above acellular treatment process. The patency of the blood vessel is inspected through Doppler ultrasound and angiography, and histological analysis is carried out after the material is taken, so as to evaluate the regeneration and remodeling of the tissue engineering blood vessel and the active artificial blood vessel in vivo.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A preparation method of a special-shaped blood vessel model or an artificial blood vessel is characterized by comprising the following steps:

step 1, three-dimensional data acquisition is carried out on blood vessels of specific parts of an animal or human body by using computer software, and the acquisition method comprises one or more of CT scanning, Doppler ultrasonic imaging, nuclear magnetic imaging and angiography; constructing a corresponding blood vessel 3D model according to the obtained three-dimensional data of the blood vessel size;

step 2, preparing a corresponding blood vessel mould by using a plurality of technologies according to the blood vessel 3D model in the step 1;

step 3, preparing a porous and reticular polymer scaffold on the blood vessel mould in the step 2; the vascular mold and the reticular polymer stent are combined to be called a vascular complex;

step 4, removing the blood vessel mould from the blood vessel complex prepared in the step 3 to obtain a blood vessel model or an artificial blood vessel A;

step 5, performing flow culture on the cells A prepared in the step 4 in an in vitro bioreactor, and removing the cells after the cells are fully proliferated and generate extracellular matrix to obtain an active blood vessel model or an artificial blood vessel B;

and 6, embedding the vascular complex prepared in the step 3 under the skin of an animal or human, taking out the vascular complex from the skin of the animal or human after sufficient cellularization and extracellular matrix generation, removing the vascular mould, and removing cells to obtain an active vascular model or an artificial blood vessel C.

2. The method for preparing the special-shaped blood vessel model or the artificial blood vessel according to claim 1, wherein the method for preparing the blood vessel mold in the step 2 comprises 3D printing, casting, cutting, laser etching and the like, and the blood vessel mold is made of medical stainless steel, polypropylene (PP), silica gel and the like; step 3, the preparation method of the reticular polymer stent comprises one or more of melt spinning, 3D printing, electrostatic spinning, weaving and casting, wherein the polymer comprises one or more of Polycaprolactone (PCL), Polyurethane (PU), polylactic acid (PLA), Polyhydroxyalkanoate (PHA), polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA), p-dioxanone (PDO) and poly L-lactide-caprolactone (PLCL) in any proportion; the method for removing the blood vessel mould in the steps 4 and 6 comprises one or more of mechanical disassembly, solvent dissolution, dragging and taking out and melting.

3. The method of claim 1, wherein the preparation parameters include porosity, pore size, wall thickness, fiber diameter and morphology of the porous reticulated polymer scaffold.

4. The shaped vessel model or artificial vessel according to claim 1, wherein the shape and size of the natural vessel are simulated, including a single, straight vessel with a regular shape and a shaped vessel with branches, bends, uneven calibre and the like.

5. The method for preparing a shaped blood vessel model or artificial blood vessel according to claim 1, wherein A, B, C prepared in steps 4, 5 and 6 can be used as blood vessel model in vitro experiment to study the biological problem of blood vessel, the mechanism of blood vessel disease, and the screening of blood vessel related drugs; A. b, C can be used as artificial blood vessel, or medical implant material to replace or repair diseased blood vessel.

6. A shaped blood vessel model or an artificial blood vessel produced by the method of any one of claims 1 to 5.