CN101073677A - Endothelio-aneurysm stand and its production - Google Patents
Endothelio-aneurysm stand and its production Download PDFInfo
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- CN101073677A CN101073677A CN 200610026651 CN200610026651A CN101073677A CN 101073677 A CN101073677 A CN 101073677A CN 200610026651 CN200610026651 CN 200610026651 CN 200610026651 A CN200610026651 A CN 200610026651A CN 101073677 A CN101073677 A CN 101073677A
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Abstract
The invention is concerned with a kind of vascular endothelial cell aneurysm bracket and its produce method, belonging to biology medical treatment material field. This method takes It adheres the amplified vascular endothelial cell of blood vessel gathered from endoderm of body vena on the surface of metal aneurysm bracket with innocuous macromolecule biomaterial coating with innocuous macromoleculem, biocompatibility and biodegradable characters. It speeds the producetion and cover of rebirth endodermis to the neck of tumour after setting up the bracket, reduces the recrudescence of aneurism and avoids the straitness of artery with tumour, enhances the cure rate to aneurism, especially to intracranial aneurysm, and reduces the thrombosis inside of artery. It overcomes the shortages of stainless steel bracket without the problem of immunogenicity and ethic. This mature technology with low cost is benefit to enhance and drive the development of new-style stuff to endovascular treatment and the interventional therapy of intracranial aneurysm.
Description
Technical field
The invention belongs to biologic medical material field, relate to a kind of novel aneurysm support, be specifically related to a kind of Endothelio-aneurysm stand and preparation method thereof.
Technical background
Intracranial aneurysm is the main reason that causes acute spontaneous subarachnoid hemorrhage, also is one of major reason of human sudden death.Interventional therapy has become one of important means of intracranial aneurysm treatment at present in the blood vessel.Some intractable aneurysms particularly, as fusiformis or interlayer aneurysm, wide carotid aneurysm, vertebra basilar artery aneurysm etc., direct big, the weak effect of surgical operation therapy difficulty, and often accompany severe complication, endovascular treatment then presents the superiority of safety, Wicresoft.Past is used electrolysis taking off property turn (GDC) treatment intracranial aneurysm and is generally accepted by this area during the decade.Yet because that turn is treated aneurysmal history is limited, clinical sure although short term efficacy obtains, long-term efficacy still remains further to be observed.Hayakawa etc. check behind 455 aneurysm GDC thromboembolisms and find the not filling fully of 178 (39%) individual aneurysms, have the tumor neck residual.Its medium-and-large-sized (11-25mm) or epimegetic (＞25mm) aneurysm and wide neck (＞4mm) small-sized (4-10mm) aneurysm the probability of recurrence occurs far above narrow neck small aneurysms.Bavinzski (Bavinzski G, et al.JNeurosurg.1999; 91 (2): 284-93) find also in the postmortem of grade behind intracranial aneurysm GDC thromboembolism that the new intima of the aneurysm eck that the tumor neck is bigger covers less.Thereby Geremia etc. will prop up and be placed near the aneurysm tumor neck in the parent artery, disturb normal blood flow to impel thrombosis in the tumor by the network structure of support, tissue fibering, and then inaccessible tumor chamber.New situation has been opened up in the intractable aneurysmal treatment of intracranial that is applied as of support technology.Yet also there is defective in the application of support technology in aneurysm.At first, support can bring out the thrombosis of parent artery as metallic foreign body; Secondly, the zest effect of metal rack can cause vascellum endometrial hyperplasia, causes narrow or inaccessible (Levy EI, the et al.Neurosurgery.2002 of parent artery; 51 (5): 1280-5).These problems have limited the extensive use of support in clinical.This area research is thought for intracranial epimegetic and wide carotid aneurysm, how to set up generation and the covering that the newborn endothelium of tumor eck is accelerated in the back at support, is to reduce the aneurysm recurrence and avoid the narrow key issue of parent artery.
Research thinks that the purpose of support endothelial cell is to make aneurysm tumor neck place to be sealed by endotheliocyte, rebuilds blood vessel wall.For the infull person of thromboembolism, can make tumor chamber and blood circulation isolated; For the aneurysm of complete thromboembolism, can alleviate the impact of blood flow to thrombosis in turn or the tumor, reduce logical again incidence rate.Endotheliocyte on the support, help alleviating with repairing and treating in the blood vessel endothelium that damages, can avoid metal rack directly to contact with blood flow, reduce interference to blood flow; Endotheliocyte can also be secreted nitric oxide (NO), prostacyclin (PGI simultaneously
2) the isoreactivity material, reduce vasospasm and narrow incidence rate.Therefore realize endothelial cell at rack surface, thereby the exposed metal/bare metal area that can reduce support can alleviate the foreign body inflammatory reaction that causes after the implantation, the excretory active substance of endotheliocyte helps support to form the smooth surface with anticoagulant characteristic simultaneously.The prospect of support endothelial cell is very tempting, but still has the problem of many reality to have to be solved.As: the effect of support endothelial cell depends on the endotheliocyte number of plantation, the anti-blood flow scouring capability of adherent cell etc., and what wherein at first will solve is that endotheliocyte comes source problem.The allosome endotheliocyte should not be applied to clinical because of relating to immunological rejection; Transgenic immortalization endothelial cell line has potential tumorigenesis danger.At present in the In vitro culture of the human vascular endothelial that carries out, comparatively ripe with the vascular endothelial cell cultural method of umbilical vein and child's foreskin, the obvious all unlikely endothelial cell that is applied to support of these two kinds of methods.Although vascular cell has been obtained very big progress biology, the acquisition of endotheliocyte is still convenient inadequately.Thereby seeking new endotheliocyte acquisition approach is one of key issue of setting up on the endothelialization support that can supply clinical practice.
Select suitable coating material, forming the rack surface that is fit to endothelial cell growth is the another key issue of setting up the endothelialization support.After in the endothelialization support intravasation, its endotheliocyte that adheres to must possess anti-blood flow scouring capability, and barish coating material can not cause platelet aggregation and thrombosis takes place simultaneously.Fibronectin of Cai Yonging (Fibronectin) and poly-D-lysine (poly-L-lysine) adhesion were limited in the past, existed simultaneously to promote thrombotic hidden danger.
Summary of the invention
The objective of the invention is to improve the performance of existing metal material aneurysm support, overcome the defective of prior art, a kind of Endothelio-aneurysm stand is provided.The present invention will be from the body vascular endothelial cell to the backbone metal finishing, can set up generation and the covering that the newborn endothelium of tumor eck is accelerated in the back at support, reduce the aneurysm recurrence and avoid the narrow of parent artery, improve the particularly cure rate of intracranial aneurysm of aneurysm, reduce the thrombotic chance of intra-arterial.
Another object of the present invention provides the preparation method of described Endothelio-aneurysm stand.
The present invention has at first solved the source of endotheliocyte, employing is from the vascular endothelial cell of autogenous vein endodermis collection amplification, and with this cell adhesion in through biocompatibility, biodegradable, the rustless steel aneurysm rack surface that nontoxic polymeric biomaterial coating is handled, make from the body Endothelio-aneurysm stand, be used for the interventional therapy of aneurysmal endovascular treatment, particularly intracranial aneurysm.
Polymeric biomaterial of the present invention is selected from poly-(lactic acid-aminoacid) copolymer (number of patent application 01132179.2 of heparin bonding; International application no PCT/CN01/01622), its good biocompatibility, degradable and nontoxic.
The present invention prepares by following method, may further comprise the steps:
1, cultivates from the body vascular endothelial cell
Get the experimental dog jugular vein, vessel segment is put into 1% collagenase (Sigma), digestion in 0.125% pancreatin (Sigma), and sucking-off cell suspension, centrifugal, go supernatant, use the 80%DMEM20% hyclone, 20ng/mlVEGF culture fluid re-suspended cell is with 1 * 10
6The cell suspension of cell density is planted in the culture dish CO that scribbles poly-D-lysine
2Cultivate in the incubator, go down to posterity.
2, preparation coating material and stainless steel stent face coat
With lactide or lactone and contain the morpholine diketone derivant (derivative ofmorpholine-dione) of multifunctional amino acid, wherein both molar ratio examples were controlled at 99: 1～1: 99, were catalyst with the stannous octoate, carried out polymerization; Resulting polymers Pd/C is that catalyst hydrogenation or employing HBr/HAc are after catalyst is sloughed blocking group; the Biodegradable polyester that must have the reactable side group; again polymer behind the deprotection and heparin are dissolved among the mixed solvent THF/H2O; with the Dcci is that catalyst reacts, and obtains the medicine macromolecular material of fully biodegradable.
The Biodegradable polymer material of preparation is dissolved in the chloroform, obtains the solution of concentration from 10%wt～0.01%wt, spray to rack surface, dosage is drained from 10～1000ug, and the sterilization back is stand-by.
Described lactide or lactone comprise the L-lactide, the DL-lactide, and Acetic acid, hydroxy-, bimol. cyclic ester or caprolactone etc. are monomer similarly.
(3) the endothelialization stainless steel stent is integrated
Adopt 1 * 10
7-5 * 10
7High titre cell suspension drop on the intravascular stent, make evenly to be attached on the support back and to immerse culture fluid, CO
2Cultivate in the incubator 24 hours standby.
The invention provides the endotheliocyte number of the plantation of decision support endothelial cell effect, guaranteed the anti-blood flow scouring capability of adherent cell.
The present invention has all carried out the detection of corresponding physicochemical property and biological property in each step of body endothelialization stainless steel stent preparation, the result shows and adheres to specification.Table 1 is the physics and chemistry and the biological characteristic of coating material.Table 2 is biological properties of endothelialization support.
Table 1
| Project | The result |
| The equal molecular mass of relative number (GPC records) structure ( 1H-NMR records) heparin body weight content | 2,000～400,000 lactide or the lactone and the molar ratio of morpholine diketone derivant that contains multifunctional amino acid from 99: 1～1: 99 from 1%wt～90%wt |
Table 2
| Project | The result | Conclusion |
| The test of hemolytic test cell toxicity test Intradermal irritant test Intradermal sensitization test (STT) platelet deposition | Hemolysis rate does not have Intradermal sensitivity response platelet deposition speed less than 0 grade of no Intradermal irritant reaction of 5% toxic reaction and is not more than reference group | Qualified qualified |
Place extracorporeal blood flow to wash away instrument above-mentioned integrated endothelialization stainless steel stent, adopt the 70dyne/cm2 shearing stress to wash away 24 hours, observed result shows: the rack surface endothelial cell morphology does not have significant change, and the adherent cell quantity of counting every 1mm length rack surface do not have obvious minimizing, and 0,12,24 hour cell numbers are respectively: 305.3 ± 22.54; 294.7 ± 20.45; 288.6 ± 30.97.
The used experiment reagent of the present invention, instrument and laboratory animal are commercial.
The present invention adopts from the body vascular endothelial cell and modifies rustless steel aneurysm rack surface, has overcome many defectives of rustless steel bare bracket, has following major advantage.
1, anti-parent artery thrombosis.
The rack surface of endothelialization has avoided metal directly to contact with blood, produces the thrombosis effect after the minimizing inflammatory reaction, reduces the narrow incidence rate of parent artery; The heparin of bonding slowly discharges after support arrives treatment position in the coating material, has stoped the thrombosis for the treatment of in the early stage parent artery.
2, promote the formation of treatment artery tumor tumor cervical region position endothelial layer.
By support bring into from the body endotheliocyte in tumor cervical region position the grid arrangement along support, this arrangement architecture has significantly reduced the distance that endotheliocyte will be creeped, the thrombosis surface that can loosen in tumor cervical region position forms endothelial layer, cures aneurysmal purpose thereby really reach.
3, gather, do not have immunogenicity easily, do not have ethical issues from the body endotheliocyte.
Human in the body endotheliocyte can the endodermis at great saphenous vein, gather amplification and get.Avoided needing the rejection considered and ethical issues etc. from the use of body endotheliocyte such as huve cell, immortalization endothelial cell line etc.
4, technical method mature and feasible involved in the present invention, cost is in tolerance interval.
Description of drawings
Fig. 1: dog vascular endothelial cell form (amplification: 200).
Fig. 2: A, B are the form of endothelial cell support under scanning electron microscope.
The specific embodiment
(1) cultivates from the body vascular endothelial cell
Get experimental dog one side jugular vein, the unpleasantness tissue of peeling off around the vein for aseptic time, use D-hank ' s liquid to wash blood vessel repeatedly, endovascular blood is rinsed well, with surgical sutures one of blood vessel is tied, blood vessel turns up, surgical sutures ties the blood vessel other end vessel segment is put into 1% collagenase (Sigma), 37 ℃ of digestion is 20 minutes in 0.125% pancreatin (Sigma), stops digestion sucking-off cell suspension with serum, centrifugal 1500 rev/mins 5 minutes, go supernatant, use the 80%DMEM20% hyclone, 20ng/mlVEGF culture fluid re-suspended cell is with 1 * 10
6The cell suspension of cell density is planted and put into 37 ℃ of 5%CO in the culture dish that scribbles poly-D-lysine
2Cultivate in the incubator, treat to carry out passage after cell is paved with at the bottom of the ware.
(2) face coat of the preparation of coating material and stainless steel stent
With lactide or lactone and the morpholine diketone derivant that contains multifunctional amino acid is catalyst with the stannous octoate, carries out polymerization; Wherein both molar ratio examples are controlled to be 99: 1; Reaction temperature is 50-200 ℃, and the response time is 0.5-46 hour; Resulting polymers Pd/C is catalyst hydrogenation 24～180 hours, obtains having the Biodegradable polyester of reactable side group, and reaction temperature is 10-35 ℃, and the response time is 8-80h; Polymer behind the above-mentioned deprotection and heparin being dissolved among the mixed solvent THF/H2O, is that catalyst reacts with the Dcci again, promptly gets the medicine macromolecule of fully biodegradable.
The Biodegradable polymer material of preparation is dissolved in the chloroform, obtains the solution of concentration from 10%wt～0.01%wt, gained solution adopts computer-controlled spraying coating process to spray to rack surface, and dosage is 10ug, drains then, and the sterilization back is stand-by.
Described lactide or lactone can be selected the L-lactide for use, the DL-lactide, and Acetic acid, hydroxy-, bimol. cyclic ester or caprolactone etc. are monomer similarly.
(3) the endothelialization stainless steel stent is integrated
With 1 * 10
7High titre cell suspension drop in lentamente on the intravascular stent and runing rest, the visible vessels endotheliocyte is attached on the support back uniformly and immerses in the culture fluid and put into 37 ℃ of 5%CO under inverted microscope
2Cultivation is prepared to carry out body and is implanted into support in the incubator after 24 hours.
The present invention has all carried out the detection of corresponding physicochemical property and biological property in each step of body endothelialization stainless steel stent preparation, the result shows and adheres to specification.Table 1 is the physics and chemistry and the biological characteristic of coating material.
Table 2 is biological properties of endothelialization support.
Table 1
| Project | The result |
| The equal molecular mass of relative number (GPC records) structure ( 1H-NMR records) heparin body weight content | 2,000～400,000 lactide or lactone and the molar ratio that contains the morpholine diketone derivant of multifunctional amino acid are: 99: 1～1: 99 from 1%wt～90%wt |
Table 2
| Project | The result | Conclusion |
| The test of hemolytic test cell toxicity test Intradermal irritant test Intradermal sensitization test (STT) platelet deposition | Hemolysis rate does not have Intradermal sensitivity response platelet deposition speed less than 0 grade of no Intradermal irritant reaction of 5% toxic reaction and is not more than reference group | Qualified qualified |
Embodiment 2
1, cultivates from the body vascular endothelial cell with embodiment 1.
2, preparation coating material and stainless steel stent face coat
With lactide or lactone and contain the morpholine diketone derivant of multifunctional amino acid, wherein both molar ratio examples are 1: 99, are catalyst with the stannous octoate, carry out polymerization; Reaction temperature is 50～200 ℃, and the response time is 0.5～46 hour, and the molar ratio that obtains the lactide or the lactone of polymer and contain the morpholine diketone derivant of multifunctional amino acid is 1: 99; Resulting polymers HBr/HAc is after catalyst is sloughed blocking group, must have the Biodegradable polyester of reactable side group, and reaction temperature is 10-35 ℃, and the response time is 8-80h; Polymer behind the deprotection and heparin being dissolved among the mixed solvent THF/H2O, is that catalyst reacts with the Dcci again, promptly gets the medicine macromolecular material of fully biodegradable.
The Biodegradable polymer material of preparation is dissolved in the chloroform, obtains the solution of concentration from 10%wt～0.01%wt, spray to rack surface with the programme controlled spraying coating process of computer, dosage is 1000ug.Drain, the sterilization back is stand-by.
Described lactide or lactone can be selected the L-lactide for use, the DL-lactide, and Acetic acid, hydroxy-, bimol. cyclic ester or caprolactone etc. are monomer similarly.
(3) the endothelialization stainless steel stent is integrated
Adopt 5 * 10
7High titre cell suspension drop on the intravascular stent slowly, and runing rest makes evenly to be attached on the support back and to immerse in the culture fluid and put into 37 ℃ of 5%CO
2Cultivate in the incubator and prepare after 24 hours to use.Carry out body and be implanted into support.
Support of the present invention has carried out the detection of corresponding physicochemical property and biological property, and the result shows and adheres to specification.
The aneurysmal experiment of embodiment 3 endothelialization support interventional therapys
Evenly to be compressed in the microtubular balloon surface from the body Endothelio-aneurysm stand standby with above-mentioned.
Select above-mentioned same experimental dog (15-20Kg) for use, set up aneurysm of common carotid artery model (tumor neck: 8mm after the external jugular vein collection; Tumor body 6 * 8 * 10mm
3).Three weeks after the modelling, the aneurysm of common carotid artery dog is fixed in digital subtraction angiography (DSA) bed behind general intravenous anesthesia, insert the 6F arterial sheath through right common femoral artery.After routine angiography shows aneurysm site and form, the 6F guiding catheter is placed year Carotid near-end of tumor.Be transported to aneurysm tumor neck position from the body Endothelio-aneurysm by microtubular, discharge support after balloon expandable puts in place.At once check DSA and show that backing positions is satisfied, eddy current produces in the aneurysm, part thrombosis, and the tumor body dwindles.Check DSA demonstration aneurysm is not developed after March.The aneurysm dissection is exsomatized, show that through immunohistochemical staining tumor cervical region position has endothelial layer to form.Matched group adopts naked stainless steel stent after identical approach treatment, and DSA shows that the aneurysm obturation is incomplete after March, and immunohistochemical staining shows that tumor cervical region position endothelial layer forms incomplete.
Claims (7)
1, a kind of Endothelio-aneurysm stand, it is characterized in that forming, attach from the body vascular endothelial cell on the described rustless steel aneurysm rack surface coating by the rustless steel aneurysm support of the degradable polymeric biomaterial of coating biology with from the body vascular endothelial cell.
2,, it is characterized in that describedly being selected from the autogenous vein endodermis from the body vascular endothelial cell by the described Endothelio-aneurysm stand of claim 1.
3, by the described Endothelio-aneurysm stand of claim 1, the polymeric biomaterial that it is characterized in that described rack surface coating is the polylactic acid-amino acid copolymer of heparin bonding.
4, press the preparation method of the Endothelio-aneurysm stand of claim 1, it is characterized in that may further comprise the steps,
(1) cultivates from the body vascular endothelial cell
Get experimental dog jugular vein vessel segment, put into 1% collagenase, digest in 0.125% pancreatin, sucking-off cell suspension, centrifugal, go supernatant, use the 80%DMEM20% hyclone, 20ng/mlVEGF culture fluid re-suspended cell is with 1 * 10
6The cell suspension of cell density is planted in the culture dish CO that scribbles poly-D-lysine
2Cultivate in the incubator, go down to posterity;
(2) preparation coating material and stainless steel stent face coat
With lactide or lactone and contain the morpholine diketone derivant of multifunctional amino acid, with the stannous octoate is catalyst, carry out polymerization, resulting polymers Pd/C is that catalyst hydrogenation or employing HBr/HAc are after catalyst is sloughed blocking group, the Biodegradable polyester that must have the reactable side group, polymer behind the deprotection and heparin being dissolved among the mixed solvent THF/H2O, is that catalyst reacts with the Dcci again, obtains the medicine macromolecular material of fully biodegradable;
The Biodegradable polymer material of preparation is dissolved in the chloroform, and the gained solution spraying is drained to rack surface, and the sterilization back is stand-by;
(3) the endothelialization stainless steel stent is integrated
Adopt 1 * 10
7-5 * 10
7Titre cell suspension drop on the intravascular stent, make evenly to be attached on the support back and to immerse culture fluid, CO
2Cultivate in the incubator 24 hours standby.
5, by the preparation method of claim 4, it is characterized in that described lactide of step 2 or lactone are selected from the L-lactide, the DL-lactide, Acetic acid, hydroxy-, bimol. cyclic ester or caprolactone etc. are monomer similarly.
6, by the preparation method of claim 4, the molar ratio example that it is characterized in that the lactide or the lactone of step 2 and contain the morpholine diketone derivant of multifunctional amino acid is 99: 1～1: 99.
7, by the preparation method of claim 4, it is characterized in that the gained solution concentration that is dissolved in the chloroform of step 2 is 10%wt～0.01%wt, the dosage that sprays to rack surface is 10～1000ug.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104726408A (en) * | 2015-03-30 | 2015-06-24 | 彭霞 | Fast extracting method of vascular endothelial cell |
| CN109055298A (en) * | 2018-08-10 | 2018-12-21 | 佛山科学技术学院 | A kind of isolated culture method of primary dog vascular endothelial cell |
-
2006
- 2006-05-17 CN CN 200610026651 patent/CN101073677A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104726408A (en) * | 2015-03-30 | 2015-06-24 | 彭霞 | Fast extracting method of vascular endothelial cell |
| CN104726408B (en) * | 2015-03-30 | 2018-04-27 | 彭霞 | The rapid extracting method of vascular endothelial cell |
| CN109055298A (en) * | 2018-08-10 | 2018-12-21 | 佛山科学技术学院 | A kind of isolated culture method of primary dog vascular endothelial cell |
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