CN110507860A - A kind of method of combination process preparation in situ tissue engineered blood vessels - Google Patents
A kind of method of combination process preparation in situ tissue engineered blood vessels Download PDFInfo
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Abstract
The present invention relates to a kind of methods of combination process preparation in situ tissue engineered blood vessels.A kind of three layers of composite vascular stent similar to native blood vessels structure, in situ tissue engineering are prepared to wet spinning technology in conjunction with rapid shaping, electrostatic spinning；The tubular bracket with axial notch that its internal layer is prepared using 3D printing and electrostatic spinning technique；Middle layer has circumferential array fiber tubular bracket using wet spinning preparation；The tubular bracket that outer layer is made of the Random fiber for coating internal layer and middle layer prepared by electrostatic spinning.Intravascular stent prepared by the present invention passes through test tube of hepari, also it is loaded with vascular endothelial growth factor, the infection chance that cell can be raised in host, reduce in vitro culture realizes the preparation of in situ tissue engineered blood vessels, has broad prospects in engineering blood vessel field.
Description
Technical field
The present invention relates to biomedical tissue field of engineering technology, in particular to a kind of combination process prepares in situ tissue work
The method of journey blood vessel.
Background technique
With the progress faster of aging of population, many diseases related with human vas, such as atherosclerosis, hat
Heart trouble etc. is in high-incidence trend.When human body native blood vessel due to factors such as disease, wounds and cannot normally to human body blood supply when, just
Damaged blood vessels should be treated using surgical operations such as repairing, endovascular stent or autogenous vein graft arts.In general, hidden quiet
Arteries and veins is considered as good autologous vein substitute during small-diameter intravascular is rebuild, but due to some patientss self-disease or other originals
Because not being available autogenous vein, so using engineering blood vessel as substitute becomes the heat of research for tissue blood vessel reparation
Point.
Currently with high molecular material, preparation artificial blood vessel has obtained very big progress, such as polyester dacron, e-PTFE
The intravascular stent of the materials such as ethylene preparation has been commercialized；These stock supports are in high blood flow, major diameter (being greater than 5~6mm) blood vessel
It is very extensive in substitute application.However the intravascular stent of these materials synthesis is not able to satisfy wanting for small-diameter intravascular bracket
It asks.Therefore autogenous cell is introduced into material, such as inner cavity surface is covered with endothelial cell, so that it is promising for playing biological action
One of method.The application of organizational project is divided into two kinds of situations, one is by cell pre-vaccination on bracket, by culture maturation
After implant, referred to as in vitro tissue engineering；Another kind is to be implanted directly into bracket to induce own cells and tissue growth, is promoted
It is shaped into cambium, also referred to as in situ tissue engineering.
Normal blood vessels are the labyrinths being made of three layers of concentric coat.Inner membrance is located at vascular wall Cavity surface, thin by single layer endothelium
Born of the same parents' covering；Most of smooth muscle cells are located at the middle film of blood vessel, and the contraction and diastole of Flexible element make arterial contraction or expansion,
With this regulating blood flow；Outer layer, that is, outer membrane of blood vessel, the loose fiber net being made of fibroblast are constituted.Existing artificial blood vessel
It is mostly single layer structure made of electrostatic spinning, and unilayered vessels easily leak, the disorderly arranged and blood vessel of electrospun fibers
Bracket from the disassembly etc. on rotating collection stick be all presently, there are the problem of.Patent CN106668944A discloses three layers of one kind again
Close small-caliber vascular stent and preparation method thereof；Patent CN109259889A discloses a kind of combination process and prepares bionical blood vessel branch
The method of frame；CN103599568A discloses a kind of preparation method of double-load multilayer small-diameter vascular stent material；Patent
CN102764171B discloses a kind of electrostatic spinning composite vascular stent and preparation method thereof.Ideal prosthetic vascular graft answers
Energy antithrombus formation, inflammation and neointimal hyperplasia, such grafting vessel can remain long-term unobstructed in vivo.Therefore by other
The surface treatment of synthetic vessel graft is to reinforce the attaching of endothelial cell.When biomaterial surface has specific parallel groove
When structure, cell sticking on the surface of the material can be enhanced.Therefore the material selection of engineering blood vessel need with preparation process
Development and improvement.
Based on the above-mentioned prior art, it is contemplated that structure function, cell growth conditions and the biomaterial of native blood vessels are special
Property, a kind of in situ tissue engineered blood vessels bracket of three-decker is prepared, there is development prospect in engineering blood vessel field.
Summary of the invention
In order to solve prior art problem, it is an object of the invention to overcome deficiency existing for prior art, beaten in conjunction with 3D
A kind of method that print technology proposes combination process preparation in situ tissue engineered blood vessels, prepares blood using the compound method of kinds of processes
Pipe holder makes pattern layers in bracket, can not only make up using limitation brought by single technique, but also being capable of preferably mould
The structure of quasi- native blood vessels, intravascular stent can raise cell, guidance cell arrangement growth in vivo, realize in situ tissue engineering
The preparation of intravascular stent makes the artificial blood vessel of preparation closer to the biological property and mechanical performance of native blood vessels, in vascular tissue
Engineering field has development prospect.
Purpose is created to reach foregoing invention, the present invention adopts the following technical solutions:
A kind of method of combination process preparation in situ tissue engineered blood vessels, comprising the following steps:
A. 3D printing water soluble polymer material A prepares intravascular stent and collects stick: its structure is cylindrical empty core structure, internal diameter
2.5~4mm, 3~4.5mm of outer diameter, and cylindrical outer surface designs a plurality of equal in width ridge, collects stick and uses water-soluble polymer material
Expect that A passes through FDM printing-forming.
B. engineering blood vessel internal layer: poly (l-lactic acid) (PLLA) and polycaprolactone (PCL) is prepared using electrostatic spinning process
It is dissolved in hexafluoroisopropanol (HFIP) by ultrasonic treatment until being completely dissolved, then with 1:1 with 19% and 5% concentration respectively
It is added in syringe after ratio uniform mixing.Polymer solution is conveyed with the flow velocity of 0.8~1.2ml/h using syringe pump, by a
On the rotary shaft as electrostatic spinning collecting platform, rotary shaft rotating speed is set as the collection stick installation printed in step: 800~
1000r/min；In syringe needle and stick both ends application voltage is collected, the nano-scale fiber silk winding of electrospinning is received in collection
Internal layer on stick, as intravascular stent.
C. the middle layer of fiber circumferential array is prepared using wet spinning technology: prepared PCL/PLLA solution is filled
Enter in syringe, the injection needle immerses in the coagulating bath of ethyl alcohol, with the drip of syringe pump driving solution with 1~2ml/h's
Flow velocity is expressed into coagulating bath.Fiber is collected into 6min on the stainless steel of rotation with 800~1000r/min.Then second is used
Alcohol washs stainless steel 3 times of fiber package, dries two days in vacuum desiccator then to remove residual solvent.Again by wet process
The winding on the inner layer outer surface for the intravascular stent that step b preparation is completed of the fiber of spinning is in circumferential array structure, and formation has
The intravascular stent middle layer of fiber circumferential array structure.
D. electrospinning intravascular stent outer layer: polymer material B is completely dissolved in solvent A with 10~12% w/v, is packed into
10ml syringe, then the electrospinning one layer of polymeric material B outside the intravascular stent middle layer that step c preparation is completed, electrospinning item
Part are as follows: voltage is 10~12kv, and injection speed is 1~3mL/h, and the revolving speed for collecting stick is controlled in 800-1000r/min, and syringe needle arrives
Collecting the distance between stick is 10~15cm, and electrospinning time is 2~3h, and final acquisition cladding internal layer completely polymerize with middle layer
B layers of object material.
E. removal intravascular stent collects stick, is dried in vacuo to intravascular stent: the structure that above-mentioned preparation is completed is dismantled
Get off to be put into culture dish, deionized water is added in culture dish and submerges intravascular stent, the intravascular stent of internal layer is collected after 30min
Stick easy removal since the water soluble characteristic of its material gradually softens, the hollow structure for collecting stick can prevent polymer material A from meeting water
The size of intravascular stent is influenced after expansion, stick is collected in removal in the case where not generating damage to intravascular stent, by intravascular stent
After collection stick completely removes, bracket is put into vacuum oven 2 days, removes residual moisture and solvent.Finally obtaining internal layer has
Axial notch, the scaffold for vascular tissue engineering of middle layer circumferential array.
F. intravascular stent test tube of hepari: with 1- ethyl -3-(3- dimethylamino-propyl) carbodiimide hydrochloride (EDC)
It is crosslinking agent with n-Hydroxysulfosuccinimide (SULFO NHS), is interval with Diamino-polyethylene glycol (DI-NH2-PEG)
Object carries out heparin covalent connection to intravascular stent.By of short duration in sodium hydroxide (NaOH, 0.01N) hydrolysis 10 minutes, improve
The density of surface carbon oxygroup.The cleaning down bracket in phosphate buffered saline (PBS) (PBS), is then rinsed with deionized water.Then
Make the di-NH of the bracket sulfo group-NHS and 20mg/ml of EDC, 10mg/mL containing 20mg/ml at room temperature2The 2- of-PEG
Reaction 3 hours, bracket is rinsed in PBS, is then containing 20mg/ in (N- hydromorphone) ethanesulfonic acid (MES) buffer (pH5.5)
Reaction 3 in the MES buffer (pH5.5) of the unfraction heparin sodium salt of the sulfo group-NHS and 20mg/ml of EDC, 10mg/mL of ml
Hour.After heparin modified, bracket is thoroughly washed 30 minutes using 10mg/ml glycine in PBS, remaining reaction is quenched
Site.
G. the fixation of vascular endothelial growth factor (VEGF): VEGF is fixed on intravascular stent, is containing 0,1,2
16 hours culture brackets are placed on the oscillator under the conditions of 4 DEG C in the PBS solution of the recombinant human VEGF -165 of 44 μ g/ml.
After VEGF is fixed, repeat to rinse bracket 30 minutes in PBS.It is final to obtain grafting heparin and vascular endothelial growth factor
In situ tissue engineered blood vessels bracket.
In situ tissue engineered blood vessels bracket prepared by the present invention includes internal layer, middle layer and outer layer, and the internal layer is by 3D
Print carriage collects the axial notch pattern that stick is formed on static spinning membrane；The middle layer fiber is in circumferential array；Institute
The outer layer fiber stated is in disorderly arranged.
The present invention compared with prior art, has the advantages that following apparent:
1. intravascular stent prepared by the present invention collects stick using 3D printing water soluble polymer material and carries out receipts silk, gone in bracket
Except step is simple and feasible, intravascular stent internal layer will not be damaged, and pattern blood vessel inner layer, be conducive to endothelial cell
Stick and grow, axial notch is consistent with direction of flow of blood, can reduce the resistance of blood flow.
2. intravascular stent prepared by the present invention imitates the structure of native blood vessels middle layer smooth muscle cell circumferential array, adopt
The intravascular stent middle layer with circumferential array fiber is prepared with wet spinning technology, guidance smooth muscle cell is circumferentially grown.
3. intravascular stent outermost layer prepared by the present invention is wrapped up middle layer and internal layer using electrostatic spinning, anti-can stop blooding
Tracheal rupture guarantees the enough mechanical performances of intravascular stent.
4. intravascular stent prepared by the present invention imitates the structure and function of native blood vessels, in conjunction with 3D printing, electrostatic spinning with
Three layers of intravascular stent structure of one kind of wet spinning technology preparation.
5. intravascular stent internal layer prepared by the present invention and intermediate layer material are by adding low molecular weight in PLLA
PCL increases heparin molecule binding site.
6. the use test tube of hepari coating of intravascular stent prepared by the present invention, increases cellular infiltration into electrospinning graft,
To increase the generation of collagen and elastin laminin in graft wall.
7. intravascular stent prepared by the present invention is loaded with vascular endothelial growth factor (VEGF), can implant
The infection chance raised cell, reduce in vitro culture realizes the preparation of in situ tissue engineered blood vessels bracket.
Detailed description of the invention
Fig. 1 is the present invention in the model schematic for preparing intravascular stent collection stick.
Fig. 2 is that present invention collecting platform when collecting Electrospun equips schematic diagram.
Fig. 3 is the present invention in the equipment schematic diagram for preparing intravascular stent middle layer wet spinning.
Fig. 4 is the three-decker schematic diagram that the present invention prepares intravascular stent.
Fig. 5 is the process flow chart that the present invention prepares intravascular stent.
In Fig. 1 into Fig. 5:
1-collection stick, 2-rotary shafts, 3-motors,
4-stainless steel bars, 5-silk fibers, 6-coagulating baths,
7-outer layers, 8-middle layers, 9-internal layers.
Specific embodiment
Further explanation is made to above scheme below in conjunction with the drawings and specific embodiments, it is detailed that the present invention obtains preferred embodiment
It states as follows:
Embodiment 1:
As shown in figure 5, a kind of method of combination process preparation in situ tissue engineered blood vessels, comprising the following steps:
A. 3D printing water-soluble material prepares intravascular stent and collects stick: its structure is cylindrical empty core structure, interior as shown in Figure 1:
Diameter 3.5mm, outer diameter 4.5mm, and cylindrical outer surface designs 8 vallates, the size of bulge-structure is long 50mm, wide 0.4mm, height
0.2mm collects stick using water soluble polymer material polyvinyl alcohol and passes through FDM printing-forming.The nozzle temperature 160 of printer
DEG C, 60 DEG C of bed temperature, nozzle movement speed 20mm/s, and set 0.15mm for lift height.
B. engineering blood vessel internal layer is prepared using electrostatic spinning process, as shown in Figure 2: can be used for biology point to increase
Poly (l-lactic acid) (PLLA) and polycaprolactone (PCL) is passed through ultrasound respectively with the concentration of 19% and 5% by the quantity of the end group of son connection
Processing is dissolved in 1,1,1,3, in 3- hexafluoro -2- propyl alcohol (HFIP) until being completely dissolved, then to add after the mixing of 1:1 ratio uniform
It is added in syringe.Polymer solution is conveyed with the flow velocity of 1.2ml/h using syringe pump, the collection stick 1 printed in a step is pacified
Electrostatic spinning collecting platform is used as in rotary shaft 2,2 revolving speed of rotary shaft of motor 3 is set as: 1000r/min；In syringe
Syringe needle and collection 1 both ends of stick apply voltage, and the nano-scale fiber silk winding of electrospinning is received in and is collected on stick 1, as blood vessel branch
The internal layer of frame.
C. there is the blood vessel middle layer of circumferential orientation fiber using wet spinning technology preparation, as shown in Figure 3: will prepare
PCL/PLLA solution be fitted into syringe, the injection needle immerses in the coagulating bath 6 of ethyl alcohol, with syringe pump driving solution
Drip is expressed into coagulating bath 6 with the flow velocity of 2ml/h.Silk fiber 5 is collected on the stainless steel 4 of rotation with 1000r/min
6min.Then the stainless steel 4 wrapped up with ethanol washing fiber three times, then in vacuum desiccator dry two days it is residual to remove
Stay solvent.The fiber of wet spinning 5 is wound on the outer surface of artificial blood vessel bracket inner layer film again in circumferential array structure, shape
At the intravascular stent middle layer with fiber circumferential array structure.
D. electrospinning intravascular stent outer layer: poly- (Pfansteihl -6-caprolactone) PLCL material is completely dissolved in 10% w/v
In hexafluoroisopropanol HFIP, it is packed into 10ml syringe, then one strata of electrospinning (the L- cream outside the middle layer that step c preparation is completed
Acid -6-caprolactone), electrospinning condition are as follows: voltage 10kv, injection speed 2mL/h, the revolving speed for collecting stick are controlled in 800r/
Min, syringe needle are 10~15cm, electrospinning time 4h to the distance between stick is collected, and finally obtain cladding internal layer completely and centre
B layers of polymer material of layer.
E. water-soluble removal PVAC polyvinylalcohol collects stick, is then dried in vacuo to intravascular stent: in superclean bench
On, the structure that above-mentioned preparation is completed is disassembled and is put into culture dish, deionized water is added in culture dish and submerges blood vessel branch
Frame, the intravascular stent of internal layer is collected stick and is easily removed since the water soluble characteristic of its material gradually softens after 30min, collects the hollow of stick
Characteristic can prevent polymer material A from influencing the size of intravascular stent after water-swellable, not generate damage to intravascular stent
In the case of removal collect stick will intravascular stent collect stick intravascular stent in completely remove after, bracket is put into vacuum oven 2 days,
Remove residual moisture and solvent.Internal layer is obtained with axial notch, the intravascular stent of middle layer circumferential array.
F. intravascular stent test tube of hepari: with 1- ethyl -3-(3- dimethylamino-propyl) carbodiimide hydrochloride (EDC)
It is crosslinking agent with n-Hydroxysulfosuccinimide (sulfo-NHS), with Diamino-polyethylene glycol (di-NH2- PEG) it is interval
Object carries out heparin covalent connection to intravascular stent.By of short duration in sodium hydroxide (NaOH, 0.01N) hydrolysis 10 minutes, improve
The density of surface carbon oxygroup.The cleaning down bracket in phosphate buffered saline (PBS) (PBS), is then rinsed with deionized water.Then
Make the di-NH of the bracket sulfo group-NHS and 20mg/ml of EDC, 10mg/mL containing 20mg/ml at room temperature2The 2- of-PEG
Reaction 3 hours, bracket is rinsed in PBS, is then containing 20mg/ in (N- hydromorphone) ethanesulfonic acid (MES) buffer (pH5.5)
Reaction 3 in the MES buffer (pH5.5) of the unfraction heparin sodium salt of the sulfo group-NHS and 20mg/ml of EDC, 10mg/mL of ml
Hour.After heparin modified, bracket is thoroughly washed 30 minutes using 10mg/ml glycine in PBS, remaining reaction is quenched
Site.
G. the fixation of vascular endothelial growth factor (VEGF): VEGF is fixed on intravascular stent, is containing 0,1,2
16 hours culture brackets are placed on the oscillator under the conditions of 4 DEG C in the PBS solution of the recombinant human VEGF -165 of 4 μ g/ml.In
After VEGF is fixed, repeat to rinse bracket 30 minutes in PBS.Final grafting heparin and the vascular endothelial growth factor of obtaining
In situ tissue engineered blood vessels bracket (PLLA/PCL internal layer 9, the middle layer 8 of PLLA/PCL circumferential array, PLCL outer layer 7), such as Fig. 4
It is shown.
Embodiment 2:
The present embodiment is substantially the same manner as Example 1, is particular in that:
In the present embodiment, a kind of method of combination process preparation in situ tissue engineered blood vessels, is collected outside stick by intravascular stent
Portion, which prepares different in width and the ridge of quantity, leads to pattern layers in bracket, includes the following steps:
A. 3D printing water-soluble material prepares intravascular stent and collects stick: design intravascular stent collects stick: its structure is cylindrical empty
Core structure, internal diameter 4mm, outer diameter 5mm, and cylindrical outer surface design 6 vallates, and the size of bulge-structure is long 50mm, wide 0.5mm,
High 0.3mm collects stick using water soluble polymer material polyvinyl alcohol and passes through FDM printing-forming.The nozzle temperature of printer
160 DEG C, 60 DEG C of bed temperature, nozzle movement speed 20mm/s, and 0.15mm is set by lift height；
Remaining step is identical in embodiment 1.
Embodiment 2:
The present embodiment is substantially the same manner as Example 1, is particular in that:
In the present embodiment, a kind of method of combination process preparation in situ tissue engineered blood vessels, prepares multiple material for electrospinning
The composite vascular of outer layer, includes the following steps:
A. this step is same as Example 1；
B. this step is same as Example 1；
C. this step is same as Example 1；
D. electrospinning intravascular stent outer layer: polycaprolactone (PCL) material is completely dissolved in methylene chloride and DMF with 10% w/v, is filled
Enter 10ml syringe, then the one strata caprolactone of electrospinning outside the middle layer that step c preparation is completed, electrospinning condition are as follows: voltage
For 10kv, injection speed 2mL/h, the revolving speed control of stick is collected in 800r/min, syringe needle is 10 to the distance between stick is collected
~15cm, electrospinning time 4h, the final PCL layer for obtaining cladding internal layer and middle layer completely；
E. this step is same as Example 1；
F. this step is same as Example 1；
G. this step is same as Example 1.
Claims (4)
1. a kind of method of combination process preparation in situ tissue engineered blood vessels, which comprises the following steps:
A. 3D printing water soluble polymer material A prepares intravascular stent and collects stick: its structure is cylindrical empty core structure, internal diameter
2.5~4mm, 3~4.5mm of outer diameter, and cylindrical outer surface designs a plurality of equal in width ridge, the collection stick uses water-soluble polymer
Materials A passes through FDM printing-forming；
B. use electrostatic spinning process to prepare engineering blood vessel internal layer: poly (l-lactic acid) and polycaprolactone are respectively with 19% and 5%
Concentration is dissolved in hexafluoroisopropanol until being completely dissolved, then to be added to after the mixing of 1:1 ratio uniform by ultrasonic treatment
In syringe；Polymer solution is conveyed with the flow velocity of 0.8~1.2ml/h using syringe pump, the collection stick printed in a step is pacified
Dress is used as electrostatic spinning collecting platform on the rotary shaft, and rotary shaft rotating speed is set as: 800~1000r/min；In syringe needle
Apply voltage with stick both ends are collected, the nano-scale fiber silk winding of electrospinning is received in and is collected on stick, as in intravascular stent
Layer；
C. there is the middle layer of circumferential orientation fiber using wet spinning technology preparation: by prepared poly (l-lactic acid) and gathering oneself
Lactone mixed solution is fitted into syringe, and the injection needle immerses in the coagulating bath of ethyl alcohol, with the drop of syringe pump driving solution
Stream is expressed into coagulating bath with the flow velocity of 1~2ml/h；Fiber is collected on the stainless steel of rotation with 800~1000r/min
6min；Then then stainless steel 3 times wrapped up with ethanol washing fiber are remained for dry two days in vacuum desiccator with removing
Solvent；By the fiber of wet spinning, winding is in circumferential array on the inner layer outer surface for the intravascular stent that step b preparation is completed again
Structure forms the intravascular stent middle layer with fiber circumferential array structure；
D. electrospinning intravascular stent outer layer: polymer material B is completely dissolved in solvent A with 10~12%w/v, is packed into 10ml note
Emitter, then the electrospinning one layer of polymeric material B outside the intravascular stent middle layer that step c preparation is completed, electrospinning condition are as follows: electricity
Pressure is 10~12kv, and injection speed is 1~3mL/h, collects the revolving speed control of stick in 800-1000r/min, syringe needle to collection stick
The distance between be 10~15cm, electrospinning time is 3~5h, the final polymer material for obtaining cladding internal layer and middle layer completely
B layers；
E. removal intravascular stent collects stick, is dried in vacuo to intravascular stent: the structure that above-mentioned preparation is completed is disassembled
Be put into culture dish, deionized water be added in culture dish and submerges intravascular stent, after 30min the intravascular stent of internal layer collect stick by
Gradually soften in the water soluble characteristic of its material and easily remove, the hollow structure for collecting stick can prevent polymer material A swollen in chance water
The size of intravascular stent is influenced after swollen, stick is collected in removal in the case where not generating damage to intravascular stent, and intravascular stent is received
After collection stick completely removes, bracket is put into vacuum oven 2 days, removes residual moisture and solvent；Internal layer is finally obtained with axis
To groove, the scaffold for vascular tissue engineering of middle layer circumferential array；
F. intravascular stent test tube of hepari: with 1- ethyl -3-(3- dimethylamino-propyl) carbodiimide hydrochloride and N- hydroxyl sulphur
Base succinimide is crosslinking agent, using Diamino-polyethylene glycol as spacer, carries out heparin covalent connection to intravascular stent；Pass through
Of short duration hydrolysis 10 minutes, improve the density of surface carbon oxygroup in sodium hydroxide；The cleaning down branch in phosphate buffered saline (PBS)
Then frame is rinsed with deionized water；Then make bracket at room temperature sulfo group-the NHS of EDC, 10mg/mL containing 20mg/ml and
The di-NH of 20mg/ml2The 2-(N- hydromorphone of-PEG) ethanesulfonic acid buffer, pH5.5, middle reaction 3 hours, the flushing branch in PBS
Frame, it is then slow in the MES of the unfraction heparin sodium salt of the sulfo group-NHS and 20mg/ml of EDC, 10mg/mL containing 20mg/ml
Fliud flushing, pH5.5, middle reaction 3 hours；After heparin modified, bracket 30 is thoroughly washed using 10mg/ml glycine in PBS
Minute, remaining reaction site is quenched；
G. the fixation of vascular endothelial growth factor: VEGF is fixed on intravascular stent, is containing 0,1,2 and 4 μ g/ml's
Place 16 hours culture brackets in the PBS solution of recombinant human VEGF -165 on the oscillator under the conditions of 4 DEG C；It is fixed in VEGF
Afterwards, it repeats to rinse bracket 30 minutes in PBS；The final in situ tissue for obtaining grafting heparin and vascular endothelial growth factor
Engineered blood vessels bracket.
2. the method for combination process preparation in situ tissue engineered blood vessels according to claim 1, which is characterized in that the step
In a, the polymer material A that production intravascular stent collects stick is water-soluble PVAC polyvinylalcohol.
3. the method for combination process preparation in situ tissue engineered blood vessels according to claim 1, which is characterized in that the step
In a, the exterior design that intravascular stent collects stick is a plurality of equal in width ridge, and the quantity of ridge is 4~8, the length and receipts of every vallate
Collection stick is isometric, and wide 0.3~0.9mm, high 0.2~0.5mm are shaped by FDM technology, and collecting stick outer shape leads to electrostatic spinning
Interior pattern layers, axial notch structure guide endothelial cell adhesion growth, reduce blood flow resistance effect.
4. the method for combination process preparation in situ tissue engineered blood vessels according to claim 1, which is characterized in that the step
In d, polymer material B is poly- (Pfansteihl -6-caprolactone) PLCL, thermoplastic polyurethane TPU, polycaprolactone (PCL) polyphosphazene polymer
Close one of object；Solvent A is methylene chloride, in N,N-dimethylformamide DMF, tetrahydrofuran THF, hexafluoroisopropanol HFIP
One or two.
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