A kind of artificial blood vessel and preparation method thereof
Technical field
The invention belongs to engineering blood vessel fields, more particularly, to a kind of artificial blood vessel and preparation method thereof.
Background technique
Currently, autologous vein such as great saphenous vein, internal mammary artery are clinically for coronary artery bypass surgery and other are small
Calibre bypass surgery bridge blood vessel the most main.U.S. Food and Drug Administration (FDA) approval at present carries out clinical
Two acellular matrix pipeline small-caliber vasculars of test are respectively:
(1) Nicolas L ' Heureux etc. will be free of the acellular matrix engineering blood vessel of synthetic material for the first time
Applied to clinic, it is 100% (n=3) that big animal rhesus macaque aorta transplantation, which tests 6 weeks patency rates, and the arteriovenous fistula I phase faces
It is 78% (7/9) that bed, which tests 1 month patency rate, and 6 months patency rates are 60% (5/8).Nicolas L ' is although Heureux etc. makes
Standby acellular matrix pipeline self out, but preparation process is complicated, prepares month root canal road time 7-9, 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. plants people's corpse vascular smooth muscle cells in vitro in degradable poly lactic acid (PGA)
Guan Shang is formed after PGA degradation then external dynamic cultivation 8 weeks in bioreactor and is rich in cell and extracellular matrix (collagen
Fiber) tubular tissue, finally take off cell and formed with collagenous fibres engineering blood vessel as main component (containing a small amount of remaining
PGA), i.e. allogeneic acellular matrix pipeline, big 6 months patency rates 100% (3/ of animal baboon arteriovenous fistula transplantation experiments
3), FDA ratifies patency rate in phase ii clinical trial June 63%, and patency rate is 28%, 18%, 15% respectively within 12,18,24 months.Due to
It is that there are immunclogical response of transplantation for allograft, and preparation process is complicated, preparation time at least two moon, easily leads to blood
Bolt formation, endometrial hyperplasia, patency rate is bad, and has propagation such as AIDS, hepatitis B using corpse vascular smooth muscle cells
The disease risks such as virus.
Due to structural pipe wall densification, peripheral cell not can enter tube wall and participates in reconstructing above two acellular matrix pipeline,
Tube wall extracellular matrix components cannot effectively update, and cannot especially synthesize the elastic fibers for maintaining ductus arteriosus wall elasticity, serious shadow
Ring its mechanical performance and patency rate.
The differences such as Charles Sparks, Gordon Campbell, Yasuhide Nakayama and Rotmans Joris
The not de- cell auto blood vessel graft of research group's preparation returns artery in implant, and there are mechanical property weaknesses, thrombosis, tube wall
Hyperplasia causes vascular patency low, while use is difficult to realize to face to the virose biomaterial of body in preparation process
Bed conversion.
Summary of the invention
Aiming at the above defects or improvement requirements of the prior art, the present invention provides a kind of artificial blood vessel and its preparation sides
Method, its object is to obtain a kind of artificial blood by the way that autologous tissue's pipeline is heparin modified by de- cell and covalent bond
Pipe, the artificial blood vessel poor mechanical property, anticoagulant functions weakness and immune response for thus solving the prior art cause blood vessel logical
The low technical problem of smooth rate.
To achieve the above object, according to one aspect of the present invention, a kind of artificial blood vessel is provided, including by autogenous cell
The tube wall that epimatrix is constituted, the tube wall are covalently bonded with anticoagulant molecule.
Preferably, the anticoagulant molecule is heparin, and the heparin content is every milligram of 5~10 microgram of artificial blood vessel.
Preferably, the artificial blood vessel pipe thickness is 124.9~690.5 microns, and preferred thickness is 400~650 microns.
Preferably, the tube wall burst pressure is 3157 ± 216mmHg, and suture tension is 3.94 ± 0.46N, maximum tension
Stress is 2.41 ± 0.22MPa, and maximum tension strain is 30.63 ± 2.74%.
Other side according to the invention provides a kind of preparation method of artificial blood vessel, includes the following steps:
(1) it takes off cell processing: autologous tissue's pipeline is used into 3- [(3- gallbladder amido propyl)-diethylamine]-propane sulfonic acid reagent
Chemical method for removing cells sloughs the cell component of pipeline, obtains self acellular matrix pipeline;
(2) anticoagulant molecule the anticoagulant molecule of covalent bond: is integrated to the self of step (1) acquisition in a manner of covalently bound
The surface of acellular matrix pipeline obtains artificial blood vessel.
Preferably, the step (1) includes the following steps:
(1-1) take off cell reagent configuration: by 3- [(3- gallbladder amido propyl)-diethylamine]-propane sulfonic acid, EDTA.Na2,
NaCl, NaOH and aseptic deionized water are prepared into 500mL solution, molar concentration be respectively 6~10mmol/L, 20~
30mmol/L, 0.10~0.15mmol/L and 0.8~1.2mol/L as take off cell reagent, wherein 3- [(3- gallbladder amide third
Base)-diethylamine]-propane sulfonic acid molar concentration is preferably that 8mmol/L, EDTA.Na2 molar concentration are preferably that 25mmol/L, NaCl rub
Your concentration is preferably 0.12mmol/L, and NaOH molar concentration is preferably 1mol/L;
(1-2) takes off cell processing: autologous tissue's pipeline is put into equipped with the configured de- cell reagent of step (1-1)
In, 2~3h is handled at room temperature, then every 2~3h replaces the primary de- cell reagent, altogether replacement 4~6 times, and preferably 5 times,
Obtain self acellular matrix pipeline.
Preferably, the step (2) includes the following steps:
The preparation of (2-1) heparin grafting agent
(a) contain the Sulfo- of 30~50mg/ml EDC and 10~30mg/ml using the MES buffer of 0.5mol/L
NHS solution, obtains solution A;
(b) B solution is obtained using the heparin solution of 40~80mg/ml of MES buffer of 0.5mol/L;
(c) solution A and B solution are uniformly mixed in equal volume, are incubated for 30~60 minutes at room temperature, then use hydroxide
Sodium solution adjusts solution ph to 7.0, is finally filtered with micro-filter, obtaining clear liquid is sterile heparin grafting agent.
The self acellular matrix pipeline that (2-2) obtains step (1) is placed in the container equipped with the heparin grafting agent
In, concussion handles 3~5 hours to get the artificial blood vessel is arrived.
Preferably, the concentration of EDC is preferably 40mg/ml in step (2-1) the heparin grafting agent, the Sulfo-
The concentration of NHS is preferably 20mg/ml, and the concentration of the heparin is preferably 60mg/ml.
Preferably, the pH value of MES buffer is 5.5 in the step (2-1).
Preferably, the micro-filter aperture of (c) step in the step (2-1) is 0.2 micron.
In general, through the invention it is contemplated above technical scheme is compared with the prior art, can obtain down and show
Beneficial effect.
(1) present invention utilizes foreign body reaction principle, and nontoxic, internal non-degradable Teflon pipe is placed in and is subcutaneously prepared
Autologous tissue's pipeline, and artificial blood vessel of the invention, institutional framework and mechanical property are prepared as raw material close to normally using it
Artery is used to not have to consider when autologous vein substitute immunological rejection, and without propagation such as AIDS, hepatitis type B virus
Equal disease risks.
(2) the CHAPS+EDTA method for removing cells that uses of the present invention is relatively mild and the structure and mechanics of extracellular matrix
Performance saves preferably, and de- cell effect is good, is handled by de- cell so that self acellular matrix structural pipe wall allows surrounding thin
Born of the same parents enter tube wall and participate in tube wall reconstruct, are effectively synthesized elastic fibers and collagenous fibres, improve after reconstruct in vivo mechanical property and logical
Smooth rate, and close to normal autologous artery structure and mechanical property.
(3) the heparin covalent combination engineering blood vessel that the present invention is mediated using Sulfo-NHS/EDC is in vivo without thin
Cellular toxicity has good biocompatibility, good anticoagulant functions, patency rate height.
(4) artificial blood vessel short preparation period of the invention, expense are low, and can be prepared into different inner diameters, are completely superior to previous
Acellular matrix pipeline has good potential applicability in clinical practice.
Detailed description of the invention
Fig. 1 is autologous tissue's pipe burst pressure testing result before and after de- cell as described in example 4;
Fig. 2 is autologous tissue's pipeline suture tensile strength testing result before and after de- cell as described in example 4;
Fig. 3 is autologous tissue's pipeline maximum tensile stress testing result before and after de- cell as described in example 4;
Fig. 4 is autologous tissue's pipeline maximum tension strain detecting result before and after de- cell as described in example 4;
Fig. 5 is 1 month result of ultrasonography after artificial vascular graft described in embodiment 5, and A is that artificial blood vessel transplants side
Arteria carotis, B are the normal arteria carotis in opposite side;
Fig. 6 is 2 months result of ultrasonography after artificial vascular graft described in embodiment 5, and A is that artificial blood vessel transplants side
Arteria carotis, B are the normal arteria carotis in opposite side;
Fig. 7 is 1 month CT angiography result after artificial vascular graft described in embodiment 5;
Fig. 8 is progress DAPI coloration result after de- cell front and back autologous tissue's pipeline slice described in embodiment 6, and A is de-
Before cell, B is after taking off cell;
Fig. 9 is autologous tissue's pipeline specimen dna quantitative detection result before and after de- cell described in embodiment 6;
Figure 10 is that toluidine blue described in embodiment 7 carries out determining for heparin grafting to the acellular matrix pipeline of grafting heparin
Property and Detection of Stability, A be covalent bond heparin complete autologous tissue's pipeline, B be covalent bond heparin longitudinal direction split
Autologous tissue's pipeline.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.As long as in addition, technical characteristic involved in the various embodiments of the present invention described below
Not constituting a conflict with each other can be combined with each other.
The present invention utilizes foreign body reaction principle, and nontoxic, non-degradable in vivo Teflon pipe is placed in subcutaneous tissue, is allowed to shape
At tubular tissue.Then after carrying out de- cell and test tube of hepari moditied processing to tubular tissue, obtained from extracellular matrix pipeline,
Artificial blood vessel i.e. of the present invention, concrete scheme are divided into following a few steps:
(A) it by tubulose Teflon (1/16 inch of outer diameter, about 1.59mm), is taken out after being placed in subcutaneous tissue 2~5 weeks, to obtaining
The autologous tissue's pipeline obtained carries out mechanics properties testing and histology, wherein 4,5 weeks autologous tissue's pipelines have optimal force
Performance and histological structure group are learned, to save preparation time, is carried out at de- cell and covalent bond heparin using 4 weeks self pipelines
Reason.
(B) using chemical method for removing cells (CHAPS reagent), autologous tissue's pipeline cell component is sloughed, is obtained self de-
Cellular matrix pipeline.
(C) heparin covalent is integrated to by self acellular matrix pipeline using Sulfo-NHS/EDC, obtaining has anticoagulation
Active autogenous cell epimatrix pipeline, this i.e. described artificial blood vessel.
Specific step is as follows for artificial blood vessel preparation method of the invention:
(1) preparation of autologous tissue's pipeline
Diameter and Teflon (Teflon) pipe for needing the vessel diameter that substitutes to match are cut to appropriate length, 75%
Alcohol disinfecting 30 minutes, it is placed in subcutaneous tissue, is taken out after 2~5 weeks, preferably 4 weeks by subcutaneous Teflon pipe together with the newborn group of wrapping
Taking-up is knitted, abstraction Teflon pipe obtains 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 best.
(2) cell processing is taken off
(2-1) takes off the configuration of cell reagent: by 3- [(3- gallbladder amido propyl)-diethylamine]-propane sulfonic acid, (abbreviation CHAPS is tried
Agent), EDTA.Na2, NaCl, NaOH and aseptic deionized water be prepared into 500mL solution, molar concentration is respectively 6~
10mmol/L, 20~30mmol/L, 0.10~0.15mmol/L and 0.8~1.2mol/L, as de- cell reagent, wherein
CHAPS reagent molar concentration is preferably 8mmol/L, and the molar concentration of EDTA.Na2 is preferably 25mmol/L, and NaCl's is mole dense
Degree is preferably that the molar concentration of 0.12mmol/L, NaOH are preferably 1mol/L;
(2-2) takes off cell processing: autologous tissue's pipeline is put into equipped with the configured de- cell reagent of step (1-1)
In, 2~3h is handled at room temperature, and then every 2~3h replaces the primary de- cell reagent, replaces 4~6 times, preferably 5 times, obtains altogether
It is derived from body acellular matrix pipeline.
(2-3) washing step: the self acellular matrix pipeline that step (1-2) is obtained is abundant using sterile PBS solution
Washing, to elute remaining de- cell reagent.
(3) the anticoagulant molecule of covalent bond, the present invention in the anticoagulant preferred heparin of molecule:
The configuration of (3-1) heparin grafting agent
(a) the MES buffer (pH value 5.5) of 0.5mol/L is used to prepare (carbodiimide) containing 30~50mg/ml EDC
With Sulfo-NHS (n-hydroxysuccinimide) solution of 10~30mg/ml, solution A is obtained, wherein the concentration of EDC is preferably
The concentration of 40mg/ml, Sulfo-NHS are preferably 20mg/ml;
(b) B solution, heparin concentration are obtained using the heparin solution of 40~80mg/ml of MES buffer of 0.5mol/L
Preferably 60mg/ml;
(c) solution A and B solution are uniformly mixed in equal volume, are incubated for 30~60 minutes at room temperature, then use hydroxide
Sodium solution adjusts solution ph to 7.0, is finally filtered with 0.2 micron of micro-filter, obtaining clear liquid is the grafting examination of sterile heparin
Agent.
The self acellular matrix pipeline that (3-2) obtains step (1) is placed in the container equipped with the heparin grafting agent
In, concussion handles 3~5 hours to get the artificial blood vessel is arrived.
CHAPS is a kind of amphoteric ion detergent, while having non-ionic and ionic detergent performance, including broken
Bad rouge-rouge, rouge-protein-interacting or dissolution packet slurry and nucleus, go cytosis more mild, extracellular matrix
Structure destructiveness is small, EDTA be it is a kind of adjust infiltrative reagent, by changing concentration, can be configured to hypotonic or hypertonicity
Solution, effect mainly lead to cell cracking, can not remove cell or cell component, this experiment de- cell material used
Material is the autologous tissue's pipeline prepared by foreign body reaction principle, and institutional framework and the more normal arteries of mechanical property are close,
And the purpose is to be used for autologous vein substitute, do not have to consider that immunological rejection, CHAPS+EDTA method for removing cells are relatively warm
With and extracellular matrix structure and mechanical property save it is preferable.
Sulfo-NHS/EDC, which can activate the carboxyl on heparin, becomes succinimide ester, and succinimide ester is then
Covalent bond occurs with the amino on collagen surface, heparin is finally anchored on collagen surface by covalent bond.Sulfo-
The heparin covalent combination engineering blood vessel that NHS/EDC is mediated is no cytotoxicity in vivo, and has good biofacies
Capacitive.
The following are embodiments:
Embodiment 1
(1) preparation of autologous tissue's pipeline
Diameter and Teflon (Teflon) pipe for needing the vessel diameter that substitutes to match are cut to appropriate length, 75%
Alcohol disinfecting 30 minutes, it is placed in subcutaneous tissue, is taken out subcutaneous Teflon pipe together with wrapping cambium after 4 weeks, abstraction
Teflon pipe, obtains autologous tissue's pipeline, and with a thickness of 324.1 ± 57.4 microns, (n=6, n are the number for detecting sample, below
The meaning of n is identical).
(2) cell processing is taken off
(2-1) takes off the configuration of cell reagent
According to the formula of upper table, the reagent precisely weighed is added in clean wide mouth glass bottle, on shaking table vibrate 2~
3h dissolves reagent sufficiently, and solution becomes clarification at this time, is prepared into de- cell reagent, saves backup in 4 DEG C.
(2-2) autologous tissue's pipeline takes off cell
Autologous tissue's pipeline is put into sterile centrifugation tube, the configured 3- of 10ml [(3- gallbladder amide third is added in every pipe
Base)-diethylamine]-propanesulfonic acid solutions, it is placed on shaking table, room temperature handles 3h, and then every 3h replaces a 3- [(3- gallbladder amide third
Base)-diethylamine]-propanesulfonic acid solutions, totally 5 times.After the completion of de- cell, uses sterile PBS solution 10ml instead, be placed on shaking table, wash
30min is washed, washes repeatedly 5 times, to elute remnants 3- [(3- gallbladder amido propyl)-diethylamine]-propane sulfonic acid reagent, avoids in accordance with the law
Cytotoxicity.The de- cell pipeline handled well is finally placed in 4 DEG C of physiological saline to save backup.
(3) covalent bond heparin
(3-1) covalent bond heparin reagent
Solution A formula
Solution A configuration: the reagent in a formula of liquid is added according to quantity in clean 50ml centrifuge tube, and oscillation mixes, and is protected from light, 4 DEG C
Overnight.
B solution configuration: first 20ml MES-buffer being added in the clean centrifuge tube of 50ml, then slowly will be load weighted
Heparin is added in centrifuge tube, shakes while being added, is finally placed in shaker overnight, so that heparin sufficiently dissolves.
The configuration of heparin grafting agent: next day mixes a, b liquid in equal volume, 30min is incubated at room temperature, with 1M NaOH solution tune
PH to 7 is saved, is finally filtered with 0.2 micron filter, is placed in 50ml sterile centrifugation tube, is protected from light 4 DEG C and saves backup.
(3-2) covalent bond heparin
The self acellular matrix pipeline that de- cell is handled well is placed in the 6 orifice plates of number, and every hole sample addition is matched
The heparin grafting agent 5ml set, shaking table, be protected from light, 5h is to get to artificial blood vessel of the present invention, with a thickness of 525 ±
125 microns (n=6), completion, which is placed in normal saline solution, to be saved backup for 4 DEG C.Vitro cytotoxicity test detection confirms nothing
Cytotoxicity.Embodiment 1 is preferred embodiment.
Embodiment 2
(1) preparation of autologous tissue's pipeline
Diameter and Teflon (Teflon) pipe for needing the vessel diameter that substitutes to match are cut to appropriate length, 75%
Alcohol disinfecting 30 minutes, it is placed in subcutaneous tissue, is taken out subcutaneous Teflon pipe together with wrapping cambium after 2 weeks, abstraction
Teflon pipe, obtains autologous tissue's pipeline, with a thickness of 154.3 ± 56.4 microns (n=6).
(2) cell processing is taken off
(2-1) takes off the configuration of cell reagent
According to the formula of upper table, the reagent precisely weighed is added in clean wide mouth glass bottle, on shaking table vibrate 2~
3h dissolves reagent sufficiently, and solution becomes clarification at this time, is prepared into de- cell reagent, saves backup in 4 DEG C.
(2-2) autologous tissue's pipeline takes off cell
Autologous tissue's pipeline is put into sterile centrifugation tube, the configured 3- of 10ml [(3- gallbladder amide third is added in every pipe
Base)-diethylamine]-propanesulfonic acid solutions, it is placed on shaking table, room temperature handles 2h, and then every 2h replaces a 3- [(3- gallbladder amide third
Base)-diethylamine]-propanesulfonic acid solutions, totally 5 times.After the completion of de- cell, uses sterile PBS solution 10ml instead, be placed on shaking table, wash
30min is washed, washes repeatedly 5 times, to elute remnants 3- [(3- gallbladder amido propyl)-diethylamine]-propane sulfonic acid reagent, avoids in accordance with the law
Cytotoxicity.The de- cell pipeline handled well is finally placed in 4 DEG C of physiological saline to save backup.
(3) covalent bond heparin
(3-1) covalent bond heparin reagent
Solution A formula
Solution A configuration: the reagent in a formula of liquid is added according to quantity in clean 50ml centrifuge tube, and oscillation mixes, and is protected from light, 4 DEG C
Overnight.
B solution configuration: first 20ml MES-buffer being added in the clean centrifuge tube of 50ml, then slowly will be load weighted
Heparin is added in centrifuge tube, shakes while being added, is finally placed in shaker overnight, so that heparin sufficiently dissolves.
The configuration of heparin grafting agent: next day mixes a, b liquid in equal volume, 50min is incubated at room temperature, with 1M NaOH solution tune
PH to 7 is saved, is finally filtered with 0.2 micron filter, is placed in 50ml sterile centrifugation tube, is protected from light 4 DEG C and saves backup.
(3-2) covalent bond heparin
The self acellular matrix pipeline that de- cell is handled well is placed in the 6 orifice plates of number, and every hole sample addition is matched
The heparin grafting agent 5ml set, shaking table, be protected from light, 3h is to get to artificial blood vessel of the present invention, with a thickness of 249.9 ±
125 microns (n=6), completion, which is placed in normal saline solution, to be saved backup for 4 DEG C.Vitro cytotoxicity test detection confirms nothing
Cytotoxicity.
Embodiment 3
(1) preparation of autologous tissue's pipeline
Diameter and Teflon (Teflon) pipe for needing the vessel diameter that substitutes to match are cut to appropriate length, 75%
Alcohol disinfecting 30 minutes, it is placed in subcutaneous tissue, is taken out subcutaneous Teflon pipe together with wrapping cambium after 5 weeks, abstraction
Teflon pipe, obtains autologous tissue's pipeline, with a thickness of 352.1 ± 43.2 microns (n=6).
(2) cell processing is taken off
(2-1) takes off the configuration of cell reagent
According to the formula of upper table, the reagent precisely weighed is added in clean wide mouth glass bottle, on shaking table vibrate 2~
3h dissolves reagent sufficiently, and solution becomes clarification at this time, is prepared into de- cell reagent, saves backup in 4 DEG C.
(2-2) autologous tissue's pipeline takes off cell
Autologous tissue's pipeline is put into sterile centrifugation tube, the configured 3- of 10ml [(3- gallbladder amide third is added in every pipe
Base)-diethylamine]-propanesulfonic acid solutions, it is placed on shaking table, room temperature handles 2.5h, and then every 2.5h replaces a 3- [(3- gallbladder acyl
Amine propyl)-diethylamine]-propanesulfonic acid solutions, totally 5 times.After the completion of de- cell, uses sterile PBS solution 10ml instead, be placed in shaking table
On, 30min is washed, is washed repeatedly 5 times in accordance with the law, to elute remnants 3- [(3- gallbladder amido propyl)-diethylamine]-propane sulfonic acid reagent,
Avoid cytotoxicity.The de- cell pipeline handled well is finally placed in 4 DEG C of physiological saline to save backup.
(3) covalent bond heparin
(3-1) covalent bond heparin reagent
Solution A formula
Solution A configuration: the reagent in a formula of liquid is added according to quantity in clean 50ml centrifuge tube, and oscillation mixes, and is protected from light, 4 DEG C
Overnight.
B solution configuration: first 20ml MES-buffer being added in the clean centrifuge tube of 50ml, then slowly will be load weighted
Heparin is added in centrifuge tube, shakes while being added, is finally placed in shaker overnight, so that heparin sufficiently dissolves.
The configuration of heparin grafting agent: next day mixes a, b liquid in equal volume, 60min is incubated at room temperature, with 1M NaOH solution tune
PH to 7 is saved, is finally filtered with 0.2 micron filter, is placed in 50ml sterile centrifugation tube, is protected from light 4 DEG C and saves backup.
(3-2) covalent bond heparin
The self acellular matrix pipeline that de- cell is handled well is placed in the 6 orifice plates of number, and every hole sample addition is matched
The heparin grafting agent 5ml set, shaking table, be protected from light, 4h is to get to the artificial blood vessel, micro- with a thickness of 570.5 ± 120
Rice (n=6), completion, which is placed in normal saline solution, to be saved backup for 4 DEG C.Vitro cytotoxicity test detection confirms acellular poison
Property.
Embodiment 4
By it is described in embodiment 1 carry out de- cell treated autologous tissue's pipeline (autogenous cell epimatrix pipeline) with not
Autologous tissue's pipeline of de- cell processing carries out burst pressure, suture tensile strength and maximum tensile stress and maximum drawing respectively
Stretching strain is detected and is compared respectively, and testing result shows the difference of Liang Zhong autologous tissue pipeline items test measured value not
With statistical significance, illustrate that the present invention handles the de- cell of autologous tissue's pipeline the mechanical property shadow to autologous tissue's pipeline
It rings less, specific test method and result are as follows:
(1) burst pressure (Burst pressure) detects
Burst pressure is completed by homemade pressure measuring system with precision pressure gauge.PBS buffering is filled up in pressure measuring system
The test serum pipeline that length is 5cm is connected to pressure measuring system, is fixed, be slowly increased in pressure measuring system with No. 7 silk threads by liquid
Pressure, until pressure drop, autogenous cell epimatrix pipeline cut occur, record maximum pressure value, this value is burst pressure
Power.
Not de- cell auto tissue tract burst pressure is 3696 ± 194mmHg (n=6), and burst pressure is 3157 after taking off cell
± 216mmHg (n=6), P > 0.05, difference do not have statistical significance.(Fig. 1)
(2) suture tensile strength (Suture retention strength) detects
Suture tensile strength (Suture retention strength) refers to power of the suture by tissue tearing when, herein
It is measured according to 7198 standard of ANSI/AAMI/ISO.Prepare 2cm long test serum pipeline, by 6-0Prolene linear slit in
One end of pipeline, back gauge 2mm, the 120 degree of angles in interval separately stitch one, stitch 3 needles in total, every suture is individually formed by knots tied one
Ring, by the seamless line end of pipeline and wherein 1 suture is separately fixed on universal tensile testing machine, is adjusted tensile speed and is
50mm/min, records maximum pull, successively fixes other 2 sutures and is tested, and 3 test results are averaged gained
It as a result is suture tensile strength.
Not de- cell auto tissue tract suture tension is 4.97 ± 0.55N (n=6), and suture tension is after de- cell
3.94 ± 0.46N (n=6), P > 0.05, difference do not have statistical significance.(Fig. 2)
(3) maximum tensile stress (Ultimate tensile strength, UTS) and maximum tension strain (ultimate
strain)
Test serum pipeline is cut into the annulus of 5mm wide, measure and records its thickness and diameter.Its both ends is hung over into hollow
On needle, then 2 bend needles are fixed on universal tensile testing machine, initial tensile length is to break 10% of length or so,
Adjusting tensile speed is 50mm/min, and maximum tensile stress and maximum tension strain are respectively maximum stress when annulus is pulled off
And strain.
Not de- cell auto tissue tract maximum tensile stress is 3.16 ± 0.30MPa (n=6), takes off maximum drawing after cell
Stretching stress is 2.41 ± 0.22MPa (n=6), and P > 0.05, difference is without statistical significance.(Fig. 3)
Not de- cell auto tissue tract maximum tension strain is 24.33 ± 2.15% (n=6), and maximum is drawn after taking off cell
Stretching strain is 30.63 ± 2.74% (n=6), and P > 0.05, difference is without statistical significance.(Fig. 4)
Embodiment 5
The artificial blood vessel that embodiment 1 is prepared carries out autogenous vessel graft experiment to miniature pig, and experiment completes 8 in total
The transplanting of miniature pig autogenous vessel graft object, wherein 5 follow-up 1 month, in addition 3 follow-up 2 months, put to death the ultrasound inspection that moves ahead
Look into, 1 month follow-up terminal put to death before in addition row CTA check that ultrasonic examination vascular patency, patency rate is 100% at 1 month
(5/5), 2 months when patency rate be 67% (2/3).
(1) miniature pig arteria carotis communis transplantation experiments: 1 month ultrasound of post-transplantation show blood vessel graft section without thrombosis,
Inner membrance is smooth, without atheromatous plaque, without obvious endometrial hyperplasia.(Fig. 5, wherein A is grafting vessel ultrasound, and B is super for opposite side normal blood vessels
Sound)
(2) miniature pig arteria carotis communis transplantation experiments: 2 months ultrasounds of post-transplantation 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 grafting vessel ultrasound, and B is that opposite side is normal
Vascular Ultrasonography)
CT angiography finds that blood vessel graft is unobstructed, and no narrow and tumor sample becomes, and matches with normal miniature pig arteria carotis communis
Preferably.(Fig. 7 is previous anastomotic at arrow meaning)
Embodiment 6
DAPI dyeing is carried out after autologous tissue's pipeline slice prepared by the embodiment 1 before and after de- cell, and (Fig. 8, A are de- thin
Before born of the same parents, B is after taking off cell), blue-fluorescence represents nucleus, and autologous tissue's pipeline is observed that many cells before de- cell
Core, autologous tissue's pipeline does not detect that blue-fluorescence exists after taking off cell, and nuclear targeting is negative, it was demonstrated that the removing of tube wall cell
Completely.
DNA quantitative detection is carried out to autologous tissue's pipeline sample before and after de- cell, specimen dna content is before de- cell
0.081 ± 0.010 μ g/ ㎎ (n=6), specimen dna content is 0.011 ± 0.003 μ g/ ㎎ (n=6), P < 0.05 after taking off cell,
Its difference has statistical significance.(Fig. 9)
Embodiment 7
Using toluidine blue to the acellular matrix pipeline that heparin is grafted in embodiment 1 carry out heparin grafting qualitative and
(Figure 10, A are complete autologous tissue's pipeline of covalent bond heparin to Detection of Stability, and B is what the longitudinal direction of covalent bond heparin was splitted
Autologous tissue's pipeline), the left side diagram A, B is Heparin-binding qualitative detection as a result, self acellular matrix pipeline pipe presentation is special
Property blue, indicate that heparin is successfully incorporated on self acellular matrix pipeline, be that Heparin-binding stability is examined on the right side of Figure 10 A, B
It surveys, i.e., elutes autologous tissue's pipeline of the grafting heparin on the left of Figure 10 A and B 12 hours in the shaking table of 37 C water baths, with
Detect the stability of heparin grafting.Self acellular matrix pipeline pipe color ratio left side is slightly shallow but does not disappear, indicates to pass through water-bath
After elution, the heparin of the non-stable bond of small part is washed away, and most of heparin is stablized in conjunction with self acellular matrix pipeline,
It is not eluted.Heparin-binding quantitative detection result: the self acellular matrix pipeline covalent bond heparin quality of unit mass is 8.2
± 0.9 μ g/mg (n=6).
As it will be easily appreciated by one skilled in the art that the foregoing is merely illustrative of the preferred embodiments of the present invention, not to
The limitation present invention, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should all include
Within protection scope of the present invention.