CN106267369A - A kind of artificial blood vessel and preparation method thereof - Google Patents
A kind of artificial blood vessel and preparation method thereof Download PDFInfo
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- CN106267369A CN106267369A CN201610638232.9A CN201610638232A CN106267369A CN 106267369 A CN106267369 A CN 106267369A CN 201610638232 A CN201610638232 A CN 201610638232A CN 106267369 A CN106267369 A CN 106267369A
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
- A61L27/3679—Hollow organs, e.g. bladder, esophagus, urether, uterus, intestine
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/507—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials for artificial blood vessels
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- A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
- A61L33/0005—Use of materials characterised by their function or physical properties
- A61L33/0011—Anticoagulant, e.g. heparin, platelet aggregation inhibitor, fibrinolytic agent, other than enzymes, attached to the substrate
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- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
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Abstract
The invention discloses a kind of artificial blood vessel and preparation method thereof.The present invention is by using 3 [(3-gallbladder amido propyl) diethylamine] propane sulfonic acid reagent chemistry method for removing cells to slough the cell component of pipeline in autologous tissue's pipeline, then heparin is attached to the surface of autologous acellular matrix pipeline, it is thus achieved that artificial blood vessel in covalently bound mode.Artificial blood vessel prepared by this method is without immunogenicity, manufacturing cycle is short, expense is low, and different inner diameters can be prepared as, possesses good anticoagulant functions, without complicated external preparation process, patency rate is high, and cell can be efficiently entering tube wall and participate in reconstruct, it is completely superior to conventional allogeneic or xenogenesis acellular matrix pipeline, there is good potential applicability in clinical practice.
Description
Technical field
The invention belongs to engineering blood vessel field, more particularly, to a kind of artificial blood vessel and preparation method thereof.
Background technology
At present, autologous vein such as great saphenous vein, IMA is clinically for coronary artery bypass surgery with other are little
The bridge blood vessel that calibre bypass surgery is main.U.S. food Drug Administration (FDA) approval at present carries out clinic
Test two acellular matrix pipeline small-caliber vasculars respectively:
(1) Nicolas L ' Heureux etc. are first by the acellular matrix engineering blood vessel without synthetic material
Being applied to clinic, big animal Rhesus Macacus aorta transplantation 6 weeks patency rates of experiment are 100% (n=3), and the arteriovenous fistula I phase faces
Bed 1 month patency rate of experiment is 78% (7/9), and within 6 months, patency rate is 60% (5/8).Nicolas L ' is although Heureux etc. make
For going out autologous acellular matrix pipeline, but preparation process is complicated, prepares 7-9 month pipeline time, spends 1.5 ten thousand U.S. dollars, far
Phase patency rate is undesirable, is difficult to be applied to clinic.
(2) Laura Niklason etc. plant external for people's corpse vascular smooth muscle cell in degradable poly lactic acid (PGA)
Guan Shang, then external dynamic cultivation 8 weeks in bioreactor, formed rich in cell and extracellular matrix (collagen after PGA degraded
Fiber) tubular tissue, finally take off cell and form engineering blood vessel with collagen fiber as main component (containing a small amount of remaining
PGA), i.e. allogeneic acellular matrix pipeline, big animal 6 months patency rates 100% (3/ of baboon arteriovenous fistula transplantation experiments
3), FDA ratifies phase ii clinical trial patency rate in June 63%, and within 12,18,24 months, patency rate is 28%, 18%, 15% respectively.Due to
It is that allograft exists immunclogical response of transplantation, and preparation process is complicated, preparation time at least 2 months, is easily caused blood
Bolt is formed, neointimal hyperplasia, and patency rate is the best, and uses corpse vascular smooth muscle cell to have propagation such as acquired immune deficiency syndrome (AIDS), hepatitis B
The disease risks such as virus.
Above two acellular matrix pipeline is fine and close due to structural pipe wall, and peripheral cell can not enter tube wall and participate in reconstruct,
Tube wall extracellular matrix components can not effectively update, and can not synthesize the elastic fibers maintaining ductus arteriosus wall elastic, serious shadow especially
Ring its mechanical performance and patency rate.
The differences such as Charles Sparks, Gordon Campbell, Yasuhide Nakayama and Rotmans Joris
Research group preparation does not takes off cell auto blood vessel graft and returns tremulous pulse in implant, there is mechanical property weakness, thrombosis, tube wall
Hyperplasia causes vascular patency low, uses biomaterial virose to body simultaneously, be difficulty with facing in preparation process
Bed converts.
Summary of the invention
For disadvantages described above or the Improvement requirement of prior art, the invention provides a kind of artificial blood vessel and preparation side thereof
Method, its object is to by autologous tissue's pipeline is heparin modified through de-cell and covalent bond, it is thus achieved that a kind of artificial blood
Pipe, thus solves the artificial blood vessel poor mechanical property of prior art, anticoagulant functions weakness and immunoreation and causes blood vessel to lead to
The technical problem that smooth rate is low.
For achieving the above object, according to one aspect of the present invention, it is provided that a kind of artificial blood vessel, including by autogenous cell
The tube wall that epimatrix is constituted, described tube wall is covalently bonded with anticoagulant molecule.
Preferably, described anticoagulant molecule is heparin, and described heparin content is every milligram of artificial blood vessel 5～10 microgram.
Preferably, described artificial blood vessel pipe thickness is 124.9～690.5 microns, and preferred thickness is 400～650 microns.
Preferably, described tube wall burst pressure is 3157 ± 216mmHg, and stitching tension force is 3.94 ± 0.46N, maximum tension
Stress is 2.41 ± 0.22MPa, and maximum tension strain is 30.63 ± 2.74%.
According to another aspect of the present invention, it is provided that the preparation method of a kind of artificial blood vessel, comprise the steps:
(1) de-cell processes: autologous tissue's pipeline is used 3-[(3-gallbladder amido propyl)-diethylamine]-propane sulfonic acid reagent
Chemistry method for removing cells sloughs the cell component of pipeline, obtains autologous acellular matrix pipeline；
(2) covalent bond anticoagulant molecule: by anticoagulant molecule with covalently bound mode be attached to that step (1) obtains autologous
The surface of acellular matrix pipeline, it is thus achieved that artificial blood vessel.
Preferably, described step (1) comprises the steps:
(1-1) configuration of de-cell reagent: by 3-[(3-gallbladder amido propyl)-diethylamine]-propane sulfonic acid, EDTA.Na2,
NaCl, NaOH and sterile deionized water are prepared as 500mL solution, its molar concentration be respectively 6～10mmol/L, 20～
30mmol/L, 0.10～0.15mmol/L and 0.8～1.2mol/L, be de-cell reagent, wherein 3-[(3-gallbladder amide third
Base)-diethylamine]-propane sulfonic acid molar concentration preferably 8mmol/L, EDTA.Na2 molar concentration preferably 25mmol/L, NaCl rub
Your concentration is preferably 0.12mmol/L, NaOH molar concentration and is preferably 1mol/L；
(1-2) de-cell processes: described autologous tissue pipeline is put into the de-cell reagent configured equipped with step (1-1)
In, process 2～3h, the most every 2～3h the most described de-cell reagents of replacing, altogether replacing 4～6 times under room temperature, preferably 5 times,
Obtain autologous acellular matrix pipeline.
Preferably, described step (2) comprises the steps:
(2-1) preparation of heparin grafting agent
A () uses the MES buffer of 0.5mol/L containing 30～50mg/ml EDC and the Sulfo-of 10～30mg/ml
NHS solution, obtains solution A；
B () uses the heparin solution of the MES buffer 40～80mg/ml of 0.5mol/L to obtain B solution；
C (), by described solution A and B solution equal-volume mix homogeneously, incubated at room temperature 30～60 minutes, then uses hydroxide
Sodium solution regulation solution ph, to 7.0, finally filters with micro-filter, and obtaining clear liquid is aseptic heparin grafting agent.
(2-2) the autologous acellular matrix pipeline that step (1) obtains is placed in the container equipped with described heparin grafting agent
In, concussion processes 3～5 hours, i.e. obtains described artificial blood vessel.
Preferably, in described step (2-1) heparin grafting agent, the concentration of EDC is preferably 40mg/ml, described Sulfo-
The concentration of NHS is preferably 20mg/ml, and the concentration of described heparin is preferably 60mg/ml.
Preferably, in described step (2-1), the pH value of MES buffer is 5.5.
Preferably, the micro-filter aperture of (c) step in described step (2-1) is 0.2 micron.
In general, by the contemplated above technical scheme of the present invention compared with prior art, it is possible to show under acquirement
Benefit effect.
(1) present invention utilizes foreign body reaction principle, and non-degradable Teflon pipe nontoxic, internal is placed in subcutaneous preparing
Autologous tissue's pipeline, and prepare the artificial blood vessel of the present invention, organizational structure and mechanical property with it for raw material close to normal
Tremulous pulse, need not consider immunological rejection when autologous vein substitute, and without propagating such as acquired immune deficiency syndrome (AIDS), hepatitis B virus
Deng disease risks.
(2) present invention use CHAPS+EDTA method for removing cells is relatively mild and the structure of extracellular matrix and mechanics
Performance preserves preferably, and de-cell effect is good, is processed by de-cell and makes the permission of autologous acellular matrix structural pipe wall the thinnest
Born of the same parents enter tube wall and participate in tube wall reconstruct, are effectively synthesized elastic fibers and collagen fiber, and after improving internal reconstruct, mechanical property is with logical
Smooth rate, and close to normal autologous artery structure and mechanical property.
(3) the heparin covalent conjunctive tissue engineered blood vessels that the present invention uses Sulfo-NHS/EDC to mediate is the thinnest in vivo
Cellular toxicity, has good biocompatibility, good anticoagulant functions, and patency rate is high.
(4) the artificial blood vessel manufacturing cycle of the present invention is short, expense is low, and can be prepared as different inner diameters, is completely superior in the past
Acellular matrix pipeline, has good potential applicability in clinical practice.
Accompanying drawing explanation
Fig. 1 is autologous tissue's pipe burst pressure testing result before and after the de-cell described in embodiment 4；
Fig. 2 is autologous tissue's pipeline suture tensile strength testing result before and after the de-cell described in embodiment 4；
Fig. 3 is autologous tissue's pipeline maximum tensile stress testing result before and after the de-cell described in embodiment 4；
Fig. 4 is autologous tissue's pipeline maximum tension strain detecting result before and after the de-cell described in embodiment 4；
Fig. 5 is 1 month result of ultrasonography after the artificial vascular graft described in embodiment 5, and A is that artificial blood vessel transplants side
Carotid artery, B is offside Normal Cervical tremulous pulse；
Fig. 6 is 2 months result of ultrasonography after the artificial vascular graft described in embodiment 5, and A is that artificial blood vessel transplants side
Carotid artery, B is offside Normal Cervical tremulous pulse；
Fig. 7 is 1 month CT angiography result after the artificial vascular graft described in embodiment 5；
Fig. 8 is to carry out DAPI coloration result after the section of autologous tissue's pipeline before and after the de-cell described in embodiment 6, and A is de-
Before cell, after B is for de-cell；
Fig. 9 is the autologous tissue's pipeline specimen dna detection by quantitative result before and after the de-cell described in embodiment 6；
Figure 10 is that the toluidine blue described in embodiment 7 carries out determining of heparin grafting to the acellular matrix pipeline of grafting heparin
Property and Detection of Stability, A is the complete autologous tissue pipeline of covalent bond heparin, and B is that the longitudinal direction of covalent bond heparin is cut open
Autologous tissue's pipeline.
Detailed description of the invention
In order to make the purpose of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, right
The present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, and
It is not used in the restriction present invention.If additionally, technical characteristic involved in each embodiment of invention described below
The conflict of not constituting each other just can be mutually combined.
The present invention utilizes foreign body reaction principle, non-degradable Teflon pipe nontoxic, internal is placed in subcutaneous tissue, is allowed to shape
Become tubular tissue.Then after tubular tissue is carried out de-cell and heparinization moditied processing, it is thus achieved that autogenous cell epimatrix pipeline,
Artificial blood vessel the most of the present invention, concrete scheme is divided into following a few step:
(A) by tubulose Teflon (external diameter 1/16 inch, about 1.59mm), it is placed in subcutaneous tissue 2～takes out, to obtaining after 5 weeks
The autologous tissue's pipeline obtained carries out mechanics properties testing and histology, and wherein 4,5 weeks autologous tissue's pipelines have optimal force
Learn performance and histological structure group, for saving preparation time, use 4 weeks autologous pipelines to carry out at de-cell and covalent bond heparin
Reason.
(B) use chemistry method for removing cells (CHAPS reagent), slough autologous tissue's pipeline cell component, obtain autologous de-
Cellular matrix pipeline.
(C) use Sulfo-NHS/EDC that heparin covalent is attached to autologous acellular matrix pipeline, it is thus achieved that to have anticoagulation
The autogenous cell epimatrix pipeline of activity, this most described artificial blood vessel.
The artificial blood vessel preparation method of the present invention specifically comprises the following steps that
(1) preparation of autologous tissue's pipeline
Diameter is cut to appropriate length with Teflon (Teflon) pipe needing the vessel diameter substituted to match, 75%
Alcohol disinfecting 30 minutes, is placed in subcutaneous tissue, takes out after 2～5 weeks, preferably 4 weeks by subcutaneous Teflon pipe together with holding newborn group
Knit taking-up, extract Teflon pipe, it is thus achieved that autologous tissue's pipeline.4 weeks corresponding autologous tissue's pipelines and final artificial blood vessel
Product thickness is most suitable, and corresponding artificial blood vessel properties of product are optimal.
(2) de-cell processes
(2-1) configuration of de-cell reagent: 3-[(3-gallbladder amido propyl)-diethylamine]-propane sulfonic acid (is called for short CHAPS examination
Agent), EDTA.Na2, NaCl, NaOH and sterile deionized water be prepared as 500mL solution, its molar concentration be respectively 6～
10mmol/L, 20～30mmol/L, 0.10～0.15mmol/L and 0.8～1.2mol/L, be de-cell reagent, wherein
CHAPS reagent molar concentration is preferably the molar concentration of 8mmol/L, EDTA.Na2 and is preferably the mole dense of 25mmol/L, NaCl
Degree is preferably the molar concentration of 0.12mmol/L, NaOH and is preferably 1mol/L；
(2-2) de-cell processes: described autologous tissue pipeline is put into the de-cell reagent configured equipped with step (1-1)
In, process 2～3h, the most every 2～3h the most described de-cell reagents of replacing under room temperature, change 4～6 times altogether, preferably 5 times, obtain
Take from body acellular matrix pipeline.
(2-3) washing step: autologous acellular matrix pipeline step (1-2) obtained uses aseptic PBS solution abundant
Washing, with the de-cell reagent that eluting is remaining.
(3) covalent bond anticoagulant molecule, the preferred heparin of anticoagulant molecule in the present invention:
(3-1) configuration of heparin grafting agent
A () uses MES buffer (pH value the is 5.5) preparation of 0.5mol/L containing 30～50mg/ml EDC (carbodiimide)
Sulfo-NHS (N-hydroxy-succinamide) solution with 10～30mg/ml, obtains solution A, and wherein the concentration of EDC is preferably
The concentration of 40mg/ml, Sulfo-NHS is preferably 20mg/ml；
B () uses the heparin solution of the MES buffer 40～80mg/ml of 0.5mol/L to obtain B solution, heparin concentration
It is preferably 60mg/ml；
C (), by described solution A and B solution equal-volume mix homogeneously, incubated at room temperature 30～60 minutes, then uses hydroxide
Sodium solution regulation solution ph, to 7.0, finally filters with the micro-filter of 0.2 micron, and obtaining clear liquid is the grafting examination of aseptic heparin
Agent.
(3-2) the autologous acellular matrix pipeline that step (1) obtains is placed in the container equipped with described heparin grafting agent
In, concussion processes 3～5 hours, i.e. obtains described artificial blood vessel.
CHAPS is a kind of amphion detergent, has the performance of nonionic and ionic detergent, including breaking simultaneously
Bad fat-fat, fat-protein-interacting or dissolving bag are starched and nucleus, and it goes cytosis the gentleest, extracellular matrix
Structural deterioration is little, and EDTA is the infiltrative reagent of a kind of regulation, by changing concentration, can be configured to hypotonic or hypertonicity
Solution, its effect mainly causes cell to crack, can not remove cell or cell component, the de-cell material used by this experiment
Material is the autologous tissue's pipeline prepared by foreign body reaction principle, and organizational structure and mechanical property compared with normal arteries are close,
And its objective is for autologous vein substitute, and immunological rejection need not be considered, CHAPS+EDTA method for removing cells is the warmest
With and the structure of extracellular matrix and mechanical property preserve preferably.
Carboxyl on heparin can be activated by Sulfo-NHS/EDC becomes succinimide ester, and succinimide ester is then
With the amino generation covalent bond on collagen protein surface, heparin relies on covalent bond to be anchored on collagen surface the most at last.Sulfo-
The heparin covalent conjunctive tissue engineered blood vessels of NHS/EDC mediation is not have cytotoxicity in vivo, and has good biofacies
Capacitive.
It is below embodiment:
Embodiment 1
(1) preparation of autologous tissue's pipeline
Diameter is cut to appropriate length with Teflon (Teflon) pipe needing the vessel diameter substituted to match, 75%
Alcohol disinfecting 30 minutes, is placed in subcutaneous tissue, is taken out together with holding cambium by subcutaneous Teflon pipe, extract after 4 weeks
Teflon manages, it is thus achieved that autologous tissue's pipeline, and its thickness is 324.1 ± 57.4 microns, and (n=6, n are the number detecting sample, below
The implication of n is identical).
(2) de-cell processes
(2-1) configuration of de-cell reagent
According to the formula of upper table, the reagent precisely weighed is added in clean wide mouth glass bottle, shaking table vibrates 2～
3h, makes reagent fully dissolve, and now solution becomes clarification, is prepared as de-cell reagent, saves backup in 4 DEG C.
(2-2) autologous tissue's pipeline takes off cell
Putting in sterile centrifugation tube by autologous tissue's pipeline, often pipe adds 3-[(the 3-gallbladder amide third that 10ml configures
Base)-diethylamine]-propanesulfonic acid solutions, it is placed on shaking table, room temperature treatment 3h, then every 3h changes a 3-[(3-gallbladder amide third
Base)-diethylamine]-propanesulfonic acid solutions, totally 5 times.After de-cell completes, use aseptic PBS solution 10ml instead, be positioned on shaking table, wash
Wash 30min, in accordance with the law repeated washing 5 times, with eluting remnants 3-[(3-gallbladder amido propyl)-diethylamine]-propane sulfonic acid reagent, it is to avoid
Cytotoxicity.Finally the de-cell pipeline handled well is inserted normal saline 4 DEG C to save backup.
(3) covalent bond heparin
(3-1) covalent bond heparin reagent
Solution A formula
Solution A configures: added according to quantity by the reagent in a formula of liquid in clean 50ml centrifuge tube, vibration mixing, lucifuge, 4 DEG C
Overnight.
B solution configures: first add in the clean centrifuge tube of 50ml by 20ml MES-buffer, the most slowly by load weighted
Heparin adds in centrifuge tube, adds and rocks, be finally placed in shaker overnight, so that heparin fully dissolves.
Heparin grafting agent configures: next day, is mixed by a, b liquid equal-volume, incubated at room 30min, adjusts by 1M NaOH solution
Joint PH to 7, finally filters with 0.2 micron filter, is placed in 50ml sterile centrifugation tube, and lucifuge 4 DEG C saves backup.
(3-2) covalent bond heparin
The autologous acellular matrix pipeline handled well by de-cell is positioned in 6 orifice plates of numbering, and every hole sample adds and joins
The heparin grafting agent 5ml put, shaking table, lucifuge, 5h, i.e. obtain artificial blood vessel of the present invention, its thickness is 525 ±
125 microns (n=6), completes to be placed in normal saline solution 4 DEG C and saves backup.Vitro cytotoxicity testing inspection confirms nothing
Cytotoxicity.Embodiment 1 is preferred embodiment.
Embodiment 2
(1) preparation of autologous tissue's pipeline
Diameter is cut to appropriate length with Teflon (Teflon) pipe needing the vessel diameter substituted to match, 75%
Alcohol disinfecting 30 minutes, is placed in subcutaneous tissue, is taken out together with holding cambium by subcutaneous Teflon pipe, extract after 2 weeks
Teflon manages, it is thus achieved that autologous tissue's pipeline, its thickness is 154.3 ± 56.4 microns (n=6).
(2) de-cell processes
(2-1) configuration of de-cell reagent
According to the formula of upper table, the reagent precisely weighed is added in clean wide mouth glass bottle, shaking table vibrates 2～
3h, makes reagent fully dissolve, and now solution becomes clarification, is prepared as de-cell reagent, saves backup in 4 DEG C.
(2-2) autologous tissue's pipeline takes off cell
Putting in sterile centrifugation tube by autologous tissue's pipeline, often pipe adds 3-[(the 3-gallbladder amide third that 10ml configures
Base)-diethylamine]-propanesulfonic acid solutions, it is placed on shaking table, room temperature treatment 2h, then every 2h changes a 3-[(3-gallbladder amide third
Base)-diethylamine]-propanesulfonic acid solutions, totally 5 times.After de-cell completes, use aseptic PBS solution 10ml instead, be positioned on shaking table, wash
Wash 30min, in accordance with the law repeated washing 5 times, with eluting remnants 3-[(3-gallbladder amido propyl)-diethylamine]-propane sulfonic acid reagent, it is to avoid
Cytotoxicity.Finally the de-cell pipeline handled well is inserted normal saline 4 DEG C to save backup.
(3) covalent bond heparin
(3-1) covalent bond heparin reagent
Solution A formula
Solution A configures: added according to quantity by the reagent in a formula of liquid in clean 50ml centrifuge tube, vibration mixing, lucifuge, 4 DEG C
Overnight.
B solution configures: first add in the clean centrifuge tube of 50ml by 20ml MES-buffer, the most slowly by load weighted
Heparin adds in centrifuge tube, adds and rocks, be finally placed in shaker overnight, so that heparin fully dissolves.
Heparin grafting agent configures: next day, is mixed by a, b liquid equal-volume, incubated at room 50min, adjusts by 1M NaOH solution
Joint PH to 7, finally filters with 0.2 micron filter, is placed in 50ml sterile centrifugation tube, and lucifuge 4 DEG C saves backup.
(3-2) covalent bond heparin
The autologous acellular matrix pipeline handled well by de-cell is positioned in 6 orifice plates of numbering, and every hole sample adds and joins
The heparin grafting agent 5ml put, shaking table, lucifuge, 3h, i.e. obtain artificial blood vessel of the present invention, its thickness is 249.9 ±
125 microns (n=6), completes to be placed in normal saline solution 4 DEG C and saves backup.Vitro cytotoxicity testing inspection confirms nothing
Cytotoxicity.
Embodiment 3
(1) preparation of autologous tissue's pipeline
Diameter is cut to appropriate length with Teflon (Teflon) pipe needing the vessel diameter substituted to match, 75%
Alcohol disinfecting 30 minutes, is placed in subcutaneous tissue, is taken out together with holding cambium by subcutaneous Teflon pipe, extract after 5 weeks
Teflon manages, it is thus achieved that autologous tissue's pipeline, its thickness is 352.1 ± 43.2 microns (n=6).
(2) de-cell processes
(2-1) configuration of de-cell reagent
According to the formula of upper table, the reagent precisely weighed is added in clean wide mouth glass bottle, shaking table vibrates 2～
3h, makes reagent fully dissolve, and now solution becomes clarification, is prepared as de-cell reagent, saves backup in 4 DEG C.
(2-2) autologous tissue's pipeline takes off cell
Putting in sterile centrifugation tube by autologous tissue's pipeline, often pipe adds 3-[(the 3-gallbladder amide third that 10ml configures
Base)-diethylamine]-propanesulfonic acid solutions, it is placed on shaking table, room temperature treatment 2.5h, then every 2.5h changes a 3-[(3-gallbladder acyl
Amine propyl group)-diethylamine]-propanesulfonic acid solutions, totally 5 times.After de-cell completes, use aseptic PBS solution 10ml instead, be positioned over shaking table
On, wash 30min, in accordance with the law repeated washing 5 times, with eluting remnants 3-[(3-gallbladder amido propyl)-diethylamine]-propane sulfonic acid reagent,
Avoid cytotoxicity.Finally the de-cell pipeline handled well is inserted normal saline 4 DEG C to save backup.
(3) covalent bond heparin
(3-1) covalent bond heparin reagent
Solution A formula
Solution A configures: added according to quantity by the reagent in a formula of liquid in clean 50ml centrifuge tube, vibration mixing, lucifuge, 4 DEG C
Overnight.
B solution configures: first add in the clean centrifuge tube of 50ml by 20ml MES-buffer, the most slowly by load weighted
Heparin adds in centrifuge tube, adds and rocks, be finally placed in shaker overnight, so that heparin fully dissolves.
Heparin grafting agent configures: next day, is mixed by a, b liquid equal-volume, incubated at room 60min, adjusts by 1M NaOH solution
Joint PH to 7, finally filters with 0.2 micron filter, is placed in 50ml sterile centrifugation tube, and lucifuge 4 DEG C saves backup.
(3-2) covalent bond heparin
The autologous acellular matrix pipeline handled well by de-cell is positioned in 6 orifice plates of numbering, and every hole sample adds and joins
The heparin grafting agent 5ml put, shaking table, lucifuge, 4h, i.e. obtain described artificial blood vessel, its thickness is 570.5 ± 120 micro-
Rice (n=6), completes to be placed in normal saline solution 4 DEG C and saves backup.Vitro cytotoxicity testing inspection confirms acellular poison
Property.
Embodiment 4
Autologous tissue's pipeline (autogenous cell epimatrix pipeline) after de-for carrying out described in embodiment 1 cell is processed with not
Autologous tissue's pipeline that de-cell processes carries out burst pressure, suture tensile strength and maximum tensile stress respectively and maximum is drawn
Stretching strain detects respectively and compares, and testing result shows the difference of Liang Zhong autologous tissue pipeline every test measured value the most not
There is statistical significance, illustrate that the present invention processes the mechanical property shadow to autologous tissue's pipeline to the de-cell of autologous tissue's pipeline
Ringing little, concrete method of testing and result are as follows:
(1) burst pressure (Burst pressure) detection
Burst pressure by homemade, complete with the pressure measuring system of precision pressure gauge.Pressure measuring system fills up PBS buffering
Liquid, is connected to pressure measuring system by the tissue tract to be measured of a length of 5cm, fixes with No. 7 silk threads, is slowly increased in pressure measuring system
Pressure, until pressure drop, there is cut, records maximum pressure value in autogenous cell epimatrix pipeline, and this value is burst pressure
Power.
Not taking off cell auto tissue tract burst pressure is 3696 ± 194mmHg (n=6), and after de-cell, burst pressure is 3157
± 216mmHg (n=6), P ＞ 0.05, difference does not have statistical significance.(Fig. 1)
(2) suture tensile strength (Suture retention strength) detection
Suture tensile strength (Suture retention strength) refer to suture by power during torn tissue, herein
It is measured according to ANSI/AAMI/ISO 7198 standard.Prepare 2cm length tissue tract to be measured, by 6-0Prolene linear slit in
One end of pipeline, back gauge is 2mm, is spaced 120 degree of angles and separately stitches one, altogether stitches 3 pins, every suture is individually formed by knots tied one
Ring, by the seamless line end of pipeline and wherein 1 suture be separately fixed on universal tensile testing machine, regulation draw speed is
50mm/min, records maximum pull, fixes other 2 sutures successively and tests, gained of 3 test results being averaged
Result is suture tensile strength.
Do not take off cell auto tissue tract sewing up tension force is 4.97 ± 0.55N (n=6), sews up tension force and be after de-cell
3.94 ± 0.46N (n=6), P ＞ 0.05, difference does not have statistical significance.(Fig. 2)
(3) maximum tensile stress (Ultimate tensile strength, UTS) and maximum tension strain (ultimate
strain)
Tissue tract to be measured is cut into the wide annulus of 5mm, measures and record its thickness and diameter.Its two ends are hung over hollow
On pin, being then fixed on universal tensile testing machine by 2 bend needles, incipient extension is a length of breaks about the 10% of length,
Regulation draw speed is 50mm/min, and maximum tensile stress and maximum tension strain are respectively maximum stress when annulus is pulled off
And strain.
Not taking off cell auto tissue tract maximum tensile stress is 3.16 ± 0.30MPa (n=6), and after de-cell, maximum is drawn
Stretching stress is 2.41 ± 0.22MPa (n=6), and P ＞ 0.05, difference does not have statistical significance.(Fig. 3)
Not taking off the strain of cell auto tissue tract maximum tension is 24.33 ± 2.15% (n=6), and after de-cell, maximum is drawn
Stretching strain is 30.63 ± 2.74% (n=6), P ＞ 0.05, and difference does not have statistical significance.(Fig. 4)
Embodiment 5
Artificial blood vessel embodiment 1 prepared carries out autogenous vessel graft experiment to miniature pig, and experiment completes 8 altogether
Miniature pig autogenous vessel graft thing is transplanted, and wherein follows up a case by regular visits to 1 month for 5, follows up a case by regular visits to 2 months for other 3, puts to death the ultrasonic inspection that moves ahead
Looking into, within 1 month, follow up a case by regular visits to additionally row CTA inspection before terminal is put to death, ultrasonic examination vascular patency, when 1 month, patency rate is 100%
(5/5), 2 months time patency rate be 67% (2/3).
(1) miniature pig common carotid artery transplantation experiments: post-transplantation 1 month is ultrasonic show blood vessel graft section without thrombosis,
Inner membrance is smooth, without atheromatous plaque, without obvious neointimal hyperplasia.(Fig. 5, wherein A is that grafting vessel is ultrasonic, and B is that offside normal blood vessels surpasses
Sound)
(2) miniature pig common carotid artery transplantation experiments: post-transplantation 2 months is ultrasonic show blood vessel graft section without thrombosis,
Inner membrance is smooth, and color Doppler shows that blood vessel graft blood flow is unobstructed.(Fig. 6, wherein A is that grafting vessel is ultrasonic, and B is that offside is normal
Vascular Ultrasonography)
CT angiography finds that blood vessel graft is unobstructed, becomes without narrow and tumor sample, mates with normal miniature pig common carotid artery
Preferably.(Fig. 7 is anastomotic stoma at arrow indication)
Embodiment 6
(Fig. 8, A are for de-thin to carry out DAPI dyeing after autologous tissue's pipeline section embodiment 1 before and after de-cell prepared
Before born of the same parents, after B is for de-cell), blue-fluorescence represents nucleus, and autologous tissue's pipeline before de-cell is it is observed that a lot of cell
Core, after de-cell, autologous tissue's pipeline is not detected by blue-fluorescence existence, and nuclear targeting is negative, it was demonstrated that tube wall cell removes
Completely.
Autologous tissue's pipeline specimen before and after de-cell is carried out DNA detection by quantitative, and before de-cell, specimen dna content is
0.081 ± 0.010 μ g/ (n=6), after de-cell, specimen dna content is 0.011 ± 0.003 μ g/ (n=6), P ＜ 0.05,
Its difference has statistical significance.(Fig. 9)
Embodiment 7
Utilize toluidine blue the acellular matrix pipeline being grafted heparin in embodiment 1 is carried out the qualitative of heparin grafting and
(Figure 10, A are the complete autologous tissue pipeline of covalent bond heparin to Detection of Stability, and B is that the longitudinal direction of covalent bond heparin is cut open
Autologous tissue's pipeline), it is illustrated that being Heparin-binding qualitative detection result on the left of A, B, autologous acellular matrix pipeline pipe presents special
Property blue, represent that heparin is successfully incorporated on autologous acellular matrix pipeline, for the inspection of Heparin-binding stability on the right side of Figure 10 A, B
Survey, will autologous tissue's pipeline of grafting heparin on the left of Figure 10 A and B at the shaking table eluting 12 hours of 37 C water bath, with
The stability of detection heparin grafting.Autologous acellular matrix pipeline pipe color is more shallow than left side but does not disappears, and represents through water-bath
After eluting, the heparin of the non-stable bond of small part is washed away, and major part heparin is combined stable with autologous acellular matrix pipeline,
It is not eluted.Heparin-binding detection by quantitative result: unit mass autologous acellular matrix pipeline covalent bond heparin quality is 8.2
± 0.9 μ g/mg (n=6).
As it will be easily appreciated by one skilled in the art that and the foregoing is only presently preferred embodiments of the present invention, not in order to
Limit the present invention, all any amendment, equivalent and improvement etc. made within the spirit and principles in the present invention, all should comprise
Within protection scope of the present invention.
Claims (10)
1. an artificial blood vessel, it is characterised in that include the tube wall being made up of autogenous cell epimatrix, described tube wall covalent bond
There is anticoagulant molecule.
2. artificial blood vessel as claimed in claim 1, it is characterised in that described anticoagulant molecule is heparin, and described heparin content is
Every milligram of artificial blood vessel 5～10 microgram.
3. artificial blood vessel as claimed in claim 1, it is characterised in that described artificial blood vessel pipe thickness is 124.9～690.5
Micron, preferred thickness is 400～650 microns.
4. artificial blood vessel as claimed in claim 1, it is characterised in that described tube wall burst pressure is 3157 ± 216mmHg, seam
Conjunction tension force is 3.94 ± 0.46N, and maximum tensile stress is 2.41 ± 0.22MPa, and maximum tension strain is 30.63 ± 2.74%.
5. the preparation method of an artificial blood vessel, it is characterised in that comprise the steps:
(1) de-cell processes: autologous tissue's pipeline uses 3-[(3-gallbladder amido propyl)-diethylamine]-propane sulfonic acid reagent chemistry
Method for removing cells sloughs the cell component of pipeline, obtains autologous acellular matrix pipeline；
(2) covalent bond anticoagulant molecule: by anticoagulant molecule with covalently bound mode be attached to that step (1) obtains autologous de-thin
The surface of cytoplasmic matrix pipeline, it is thus achieved that artificial blood vessel.
6. the preparation method of artificial blood vessel as claimed in claim 5, it is characterised in that described step (1) comprises the steps:
(1-1) configuration of de-cell reagent: by 3-[(3-gallbladder amido propyl)-diethylamine]-propane sulfonic acid, EDTA.Na2, NaCl,
NaOH and sterile deionized water are prepared as 500mL solution, its molar concentration be respectively 6～10mmol/L, 20～30mmol/L,
0.10～0.15mmol/L and 0.8～1.2mol/L, it is de-cell reagent, wherein 3-[(3-gallbladder amido propyl)-diethyl
Amine]-propane sulfonic acid molar concentration is preferably 8mmol/L, EDTA.Na2 molar concentration to be preferably 25mmol/L, NaCl molar concentration excellent
Elect 0.12mmol/L, NaOH molar concentration as and be preferably 1mol/L；
(1-2) de-cell processes: put into by described autologous tissue pipeline in the de-cell reagent configured equipped with step (1-1),
Process 2～3h, the most every 2～3h the most described de-cell reagents of replacing under room temperature, change altogether 4～6 times, preferably 5 times, obtain
Autologous acellular matrix pipeline.
7. the preparation method of artificial blood vessel as claimed in claim 5, it is characterised in that described step (2) comprises the steps:
(2-1) preparation of heparin grafting agent
A () uses the MES buffer of the 0.5mol/L Sulfo-NHS containing 30～50mg/ml EDC and 10～30mg/ml molten
Liquid, obtains solution A；
B () uses the heparin solution of the MES buffer 40～80mg/ml of 0.5mol/L to obtain B solution；
C (), by described solution A and B solution equal-volume mix homogeneously, incubated at room temperature 30～60 minutes, then uses sodium hydroxide molten
Liquid regulation solution ph, to 7.0, finally filters with micro-filter, and obtaining clear liquid is aseptic heparin grafting agent.
(2-2) the autologous acellular matrix pipeline that step (1) obtains is placed in the container equipped with described heparin grafting agent, shake
Swing process 3～5 hours, i.e. obtain described artificial blood vessel.
8. the preparation method of artificial blood vessel as claimed in claim 7, it is characterised in that described step (2-1) heparin grafting examination
In agent, the concentration of EDC is preferably 40mg/ml, and the concentration of described Sulfo-NHS is preferably 20mg/ml, and the concentration of described heparin is excellent
Elect 60mg/ml as.
9. the preparation method of artificial blood vessel as claimed in claim 3, it is characterised in that MES buffer in described step (2-1)
PH value be 5.5.
10. the preparation method of artificial blood vessel as claimed in claim 6, it is characterised in that (c) step in described step (2-1)
Rapid micro-filter aperture is 0.2 micron.
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| CN107496991A (en) * | 2017-08-17 | 2017-12-22 | 邱雪峰 | A kind of kit persistently to keep clear and method for making artificial blood vessel |
| WO2024021869A1 (en) * | 2022-07-29 | 2024-02-01 | 海迈医疗科技(苏州)有限公司 | In-vivo rapid recellularization tissue engineered blood vessel and preparation method therefor |
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