CN100340220C - Method for planting esoderma/endothelial cell on inner surface of artificial blood vessel - Google Patents
Method for planting esoderma/endothelial cell on inner surface of artificial blood vessel Download PDFInfo
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- CN100340220C CN100340220C CNB2004100218756A CN200410021875A CN100340220C CN 100340220 C CN100340220 C CN 100340220C CN B2004100218756 A CNB2004100218756 A CN B2004100218756A CN 200410021875 A CN200410021875 A CN 200410021875A CN 100340220 C CN100340220 C CN 100340220C
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Abstract
The present invention provides a method for planting esoderma/endothelial cells on the inner surface of an artificial blood vessel. Endothelial cells are bonded on the inner wall of an artificial blood vessel via the action of the shearing stress of a pulsatory and staged-increasing fluid outside a body by adopting the method of biomechanics, and the endothelial cells bonded on the inner wall of the artificial blood vessel adapt to the action of the shearing stress of the fluid in advance so as to have good capacity for bearing the action of the shearing stress of a blood stream after the blood vessel is transplanted in a body. The endothelial cells can be planted on the surface of artificial blood vessel made of various materials by utilizing the method, comprising artificial blood vessels prepared from synthetic biological materials and natural biological materials.
Description
Technical field
The present invention relates to a kind of method at artificial blood vessel's inner surface plantation endothelium/endothelioid cells.
Background technology
Cardiovascular system diseases is one of disease that sickness rate is the highest in the world, also is the present prevailing cause of death, and its main treatment means is exactly a blood vessel grafting.Each is only will surpass 570,000 coronary artery bypass vascular transplant in the U.S., the enforcement of vascular bypass operation also needs a large amount of peripheral vascular transplanting in addition.Caused thus the small-bore (＜6mm) wilderness demand of blood vessel graft.
And the purpose that intravascular tissue engineering is learned is exactly in the ideal blood vessel graft of external structure, transplants the no immunological rejection in back, can keep the unobstructed for a long time of grafting vessel chamber.Cardiovascular surgery needs the blood vessel graft of various diameter as patching material.At present artificial blood vessel's graft of diameter＞6mm has been widely used in clinically, and higher because of blocking rate small-caliber artificial blood vessel graft wilderness demand the time, clinical practice obviously is restricted.Therefore cause the focus of present intravascular tissue engineering to be to prepare the little blood vessel of caliber less than 6mm.The reason of little blood vessel transplantation failure is mainly thrombosis in acute stage; Being mainly smooth muscle cell formation of hypertrophy and anastomosis pannus in the graft tube chamber in chronic phase causes tube chamber to block.For engineering blood vessel, the human body self blood vessel has good antithrombotic and forms ability because the barrier action of vascular endothelial cell is arranged.Therefore, making artificial blood vessel's material success endothelialization is the hope place of improving the small-caliber artificial blood vessel patency rate.
1, at artificial blood vessel's inner surface inoculation endothelium/endothelioid cells
By research to vascular endothelial cell (VEC), find that VEC has the height metabolic activity and endocrine function is arranged, it can synthesize and discharge multiple physiologically effective substance, many physiological activities there is regulating action, keep the active balance of body physiological, such as adjusting to aspects such as vascular smooth muscle tension force, microvascular permeability, biologically active pdgf, blood coagulation, anticoagulation and fibrinolytic systems.The VEC surface equally has negative charge with hemocyte, can stop the deposition of hemocyte on the artificial blood tube chamber, so the plantation of VEC has antiplatelet aggregation, prevents blood coagulation and thrombotic effect.
At first proposed the kind method for planting of endotheliocyte by Mansfield in 1970, numerous in recent years researcheres also in the performance of attempting improving by the endotheliocyte lining graft, makes graft can have better bionical ability in vivo.1998, ShinokaT etc. designed and produced external from body pulmonary artery grafting vessel.Take out the carotid artery of giving birth to back 20 days sheep, adopt the piece of tissue cultural method to obtain mixed population of cells, use fluorescent marker method again and separate smooth muscle cell (SMCs) and endotheliocyte (ECs), successively plant on absorbable material polylactic acid-common glycolic (PGLA), use the former sheep main pulmonary artery of vascular replacement of external structure after 7 days.After transplanting for 10～12 weeks, the sign that does not have obvious thrombosis and calcification, the beginning (the Shinoka T of novel blood vessel transplantation thing research has been started in this work, Tim AD, etal.Creation of viable pulmonary artery autograftsthrough tissue engineering.J Thorac Cardiovasc Surg.1998, the 115:536-546. exercise question is: the pulmonary artery autograft that adopts the Method of Tissue Engineering exploitation to live).
Recently successively relevant for transplant the report that promotes that the damage endothelium is repaired from body EC.Conte etc. use rabbit femoral artery balloon injury model, behind the blood flow of blocking-up femoral artery two ends, with the injecting endothelium from body jugular vein EC and strip off vessel segment of transfection beta-galactosidase gene, find to transplant and effectively to suppress hypercholesterolemia rabbit sacculus shaping postoperative restenosis from body jugular vein EC.Darcin etc. utilization Canis familiaris L. femoral artery balloon injury model will be transplanted to endothelium from body jugular vein EC and strip off vessel segment, and experimental result shows that this kind method also can obviously improve the long-term patency rates of injured blood vessel.
Though improved the thrombosis problem to a certain extent behind the engineering blood vessel graft endothelialization, still do not solved the long-term patency rate problem of small-caliber vascular.Discover for bore to have caused endotheliocyte adhesion on graft not enough, thereby produced all lower consequence of long-term patency rate, therefore aspect lining technology, also will further improve less than the low groundwater increment of the blood vessel graft EC of 6mm.Just point out on expanded PTFE (ePTFE) timbering material, to plant the failure that endotheliocyte makes the support endothelialization as far back as Herring in 1994, be associated with the formation of neointimal hyperplasia and inner cavity surface thrombosis, and point out that its possible reason is exactly the disappearance of the endothelial layer of material inner cavity surface after timbering material implants.Bhat VD etc. thinks that its mechanism may be exactly the adhesion that intravital blood flow has weakened the cell that implants, and makes the endothelial denudation of implantation, and the adhesive capacity that therefore how to improve inoculation back endotheliocyte becomes urgent problem again.
2, improve the adhesiveness of endothelium/endothelioid cells on blood vessel wall
Can the artificial blood vessel endothelialization, depends on the ability that repopulating cell adheres on artificial blood vessel's wall, grows and expand.Can pass through the high-density planting endotheliocyte on the one hand, form the firm fused cell of one deck at the blood vessel graft inner surface.After will guaranteeing then that on the other hand graft implants, repopulating cell still can be attached on the tube wall under the blood flow effect.Researcher finds that the external VEC that adheres on the graft is easy to come off in the shear stress field in vivo, it is insecure to find that promptly endotheliocyte adheres to, the at a specified future date unobstructed and thrombotic problem of engineering blood vessel still is not resolved, proposed the method for various raising EC thus, mainly contained at the graft surface adhesion:
(1) pre-lining cell's epimatrix (ECM) albumen on artificial blood vessel's wall: as fibronectin (fibronectin, FN), Fibrinogen (fibrinogen, FG), vitreous body connects albumen (vitronectin, VN), laminin (laminin, La) and collagen protein (collagen, CL) etc.Arginine-glycine-aspartic acid acid-serine (RGDs) of arranging in FN, FG and the VN structure is main cell adhesion determinant.RGDs has the specific recognition function, can with protein family of striding film on the endothelial cell membrane---integrin family (Integrinsfamily, IF) specific bond, thereby can adhere to endotheliocyte.Given this, some research groups are directly attached on artificial blood vessel's wall to be modified with the RGD peptide chain, perhaps fixedly comprises the polypeptide of RGD on artificial blood vessel's wall, can promote the adhesion of endotheliocyte.
(2) utilize biotin (biotin) to increase the adhesion of endotheliocyte with the specific bond power of avidin (avidin): endothelial cells cultured is handled through biotin, and in the artificial blood vessel wall liner with avidin, utilize their special affinity, can obviously strengthen the adhesion of endotheliocyte on artificial blood vessel's wall and the ability of anti-shearing stress.
(3) utilize the effect of cell growth factor, improve the adhesion and the energy for growth of endotheliocyte: as utilize fibroblast growth factor (fibroblast growth factor, FGF) with heparin the high affinity site is arranged, and the slow release of heparin can suppress the characteristics of smooth muscle proliferation, in artificial blood vessel's wall of the pre-lining of heparin-albumin, add FGF, also help the growth and the adhesion of endotheliocyte.
(4) change the charging property of implanting endotheliocyte: because endotheliocyte is electronegative, and artificial blood vessel's wall (as ePTFE) is also electronegative, has repulsive interaction each other.Can utilize electrostatic interaction to make the positive electricity of short time on the endotheliocyte band of implantation or few electronegative, thereby improve the adhesive ability of endotheliocyte on blood vessel wall and the ability of anti-blood flow shearing stress.But negative charge has increased thrombotic probability again when reducing.
(5) applied molecular biology technology: the development of modern molecular biology technique may be modified by the pair cell gene people.Therefore, in the endotheliocyte of inoculation, import hyperamization enlargement of pipe, antithrombotic genetic fragment, just be hopeful fundamentally to improve artificial blood vessel's patency rate.
(6) endotheliocyte and smooth muscle cell unite cultivation: smooth muscle cell (SMC) is the main cell component of blood vessel wall.This kind cell can produce the volume extracellular matrix components, and SMC influences the form of endotheliocyte by direct or indirect mode, thereby helps adhering to of endotheliocyte.
Though researcheres have adopted the whole bag of tricks to improve the adhesiveness of endotheliocyte, after blood vessel graft implants, coming off of endothelial layer are taken place easily still in secular blood flow, thereby cause the formation of thrombosis.Through long-term exploration, people recognize that gradually endotheliocyte is the natural instrumentality that keeps blood vessel stability.Behind blood vessel injury, the endothelial cell monolayer of blood vessel has antithrombotic and forms and the outgrowth effect of smooth muscle cell.People such as Shannon L point out that the engineering blood vessel graft of success must guarantee to resist blood flow shearing force and thrombotic endothelial cell monolayer (Shannon L, Niklason MLE..Requirements for growing tissue-engineered vascular grafts.Cardiovascular Pathology.2003, the 12:59-64. exercise question is: to the demand of engineering blood vessel graft alive).
3, hydrodynamics environment is to the influence of vascular endothelial cell:
Existing known, the a great problem of tissue engineering vessel is that blood vessel is in a natural machine power environment, it comprises: the shearing force (shear stress) that blood flow produces through the tangent line effect of endodermis, the annular tube wall stretching stress (stretch stress) that produces and the normal stress of hydrostatic pressing generation of contracting that relax.Shearing force is to the formation and the structure important influence of form, propagation, polarity and the substrate of vascular endothelial cell.
(1) shearing force is to the influence of endotheliocyte (EC) form: a long time ago, people just notice that blood flow under the condition in vivo can influence form and the orientation of ECs.Be spindle shape at unidirectional stable high shear stress area E Cs, the cell major axis is consistent with the shearing stress direction.And in low shearing stress or blood resides zone, ECs is rounded, and the arrangement of cell does not have orientation.Experiment in vitro find human umbilical vein endothelial cell (HUVECs) by 38,75dyne/cm
2Hydrodynamic shear effect after 12 hours, endotheliocyte is elongated, major axis is consistent with flow field direction, and the elongation of endotheliocyte, degree of orientation and shearing stress size, length action time relevant (Chen Huaiqing, medical bio mechanics .2003,18 (supplementary issues): 3-4.).Nearest research has also proved still to be that blood flow shear action endotheliocyte is with stimulating endothelial cell delivery of prostacyclin (Prostacyclin, PGI external no matter in vivo
2) and EDRF (Endothelium-derived relaxing factor, EDRF).The static polygonal endotheliocyte that presents directly becomes elongated gradually and is oriented to flow direction under the blood flow shear action.The form of endotheliocyte is rebuild (the chamber face presents fairshaped feature) because of the form that the effect of shearing stress takes place, and helps to reduce the traction action of blood flow to endotheliocyte, thereby keeps the integrity of endothelial cell monolayer structure.
(2) shearing force is to the influence of endotheliocyte adhesion: studies show that the shearing force condition can improve its effect adhesive capacity of cell down, under flox condition, shearing force loads on the stock support of endotheliocyte lining gradually, and experiment finds that the effect of shearing force has promoted to adhere at the bottom of the reconstruction of cytoskeleton network and the cell based.Internal diameter is that (polyurethane, Pu) blood vessel has been planted EC, passes through 1-2dyn/cm for the polyurethane of 1.5mm
2Shear action 3 days, and then use 25dyn/cm
2Shearing force continuation effect 3 days again, experiment back researcher is found to have preserved more complete endothelial layer, and directly uses 25dyn/cm
2Shear action after have only (the Ott MJ that retains of a spot of endotheliocyte, Ballermann BJ.Shear stress-conditioned, endothelial cell-seeded vascular grafts:Improved cell adherence in response to invitro shear stress.Surgery.1995,117:334-339. exercise question is: the inoculation of shear stress domestication the blood vessel graft of endotheliocyte: the effect of external shear stress has increased cell adhesion) (Dardik A, Liu A, Ballermann B.Chronic in vitro shear stress stimulates endothelial cell retention on prostheticvascular grafts and reduces subsequent in vito neointimal thickness.Journal ofVascular Surgery.1999,29 (1): the 157-167. exercise question is: secular external shear stress effect has increased the retention rate of endotheliocyte on artificial blood vessel's graft, has reduced the thickness of new intima in the body afterwards).Internal diameter is the sheep carotid artery (Photofix) of 4mm, and the effect of the shear stress of controlling by researcheres makes that at the value of shearing initial value be about 0.25dyn/cm
2, final is about 1.2dyn/cm
2The time, be 6 hours action time, the reservation area of endotheliocyte on Photofix is 50% (Carnagey J, Anderson DH, Ranieri J, et al.Rapid endothelialization ofphotofix natural biomaterial vascular grafts.J Biomed Mater Res Part B:ApplBiomater.2003, the 65B:.171-179. exercise question is: the natural artificial blood vessel's of photofix quick endothelialization).Though the former value of shearing taked has certain randomness in these researchs, and the number of the value of shearing that adopts is very limited, only comprised 1 or 25dyn/cm
2, the value of shearing that the latter chooses is very little, has than big-difference with the normal value of shearing of human body, can improve the mobilization force of cell tolerance (opposing) physiological level but their result has hinted the shearing force that increases gradually.What take in the present invention is the shear stress of the increase of staged (stepwise), more emphasize the increase process gradually of shear stress, and the value of shearing that relates to is to be in the suffered value of shearing scope of normal human's vascular endothelial cell, result of study also confirms, adopt the inventive method to make to be seeded in the Human umbilical vein endothelial cells on the Wistar rat carotid artery of removing endotheliocyte to increase, finally up to 26dyn/cm by staged
2Shear stress effect after 24 hours, it keeps area is more than 80%.Li Yuquan in 2002 etc. have reported 40dyn/cm
2The stably stratified flow shearing stress under, with fibronectin (fibronectin among the ECM of smooth muscle associating endothelial cells cultured, Fn) and laminin (laminin, content Ln) increases, and has caused the adhesive capacity of endotheliocyte and anti-current body shear stress ability to increase.People such as Philippe use polyethyleneterephthalate (polyethyleneterephthalate in experiment, PET) blood vessel (internal diameter 6mm), and at inner cavity surface coating bovine collagen and low-density heparin, adopt the pulsation filling system to cultivate and stick to cell on the graft, experimental result shows that also the shearing force of secular perfusion and high level (equating with the blood flow shearing force) extremely helps planting the retaining on graft of endotheliocyte.
(3) shearing force influence that the endotheliocyte stress fiber is formed: the variation of the rearrangement of the EC that shearing force causes and EC cytoskeleton is closely related.Cytoskeleton is made up of microfilament, microtubule, median fiber, and wherein actin filament (mainly by F-actin, F-actin forms) research is more.Except being elongated of the endotheliocyte of finding the hydrodynamic shear effect, outside flow field direction was consistent, the orientation of also having observed F-actin in the cell simultaneously was also consistent with the shearing stress direction in experiment for Chen Huaiqing.As far back as 1984, Franke etc. just had been found that the monolayer HUVEC that will cultivate is at 2dyne/cm
2Under the shear action, cell central authorities have formed stress fiber after 2-3 hour, but increase stress after the time, and stress fiber does not have obvious increase, and the form of cell still keeps polygon.Dewey etc. also find 8dyne/cm
2Stress fiber occurred in the endotheliocyte behind the shear action 24h, the cellular morphology of this moment does not change yet.We discover that under flow regime, the monolayer endothelial cell of cultivation is through 10dyne/cm
2Behind the effect 24h, the stress fiber that microfilament forms appears in cell central authorities, and this result has also illustrated the effect that shearing force has the inducing endothelial cell stress fiber to form.In these researchs, though stress fiber forms, but the just increase of quantity, do not form thick microfilament as yet, this may be low relevant with the shearing stress level, we find that by the level and the long action time of increase shearing force the pencil stress fiber of highly significant has appearred in cell central authorities, and the length of microfilament, thickness etc. all have tangible increase.These description of tests the degree of the formation of stress fiber and increase depend on the size and the action time of shearing force, also having illustrated needs to carry out the dynamic cultivation of endotheliocyte with high level and relative long active force in the tissue construction process.
(4) shearing force is to the influence of endothelial cell proliferation: researcher (Brian S.Conklin, M.S, et al.Shear StressRegulates Occludin and VEGF Expression in Porcine Arterial Endothelial Cells.Journal of Surgical Research.2002, the 102:13-21. exercise question is: shear stress has been regulated the expression that film close connects albumen and vascular endothelial cell growth factor of striding of pig arterial endothelial cell) application blood vessel graft perfused culture system (15dyn/cm
2And 1.5dyn/cm
2) provide pressure and the flow regime under the physiological condition for extracorporeal blood vessel graft (taking from pig carotid artery), make endotheliocyte and smooth muscle cell obtain secular survival.Studies have shown that low-shearing force causes the propagation of endotheliocyte, raise the expression of endothelial cell VEGF, and VEGF has also promoted endothelial cell proliferation.
(5) shearing force is to the influence of endotheliocyte physiologically active: on the molecular biology level, shear stress can raise c-fos, c-jun, proto-oncogene mRNA levels such as c-myc, its gene outcome is incorporated into transcription site on the DNA promoter as activating transcription factor or inhibitive factor, and regulator gene is expressed.Simultaneously, target gene mRNA levels such as shear stress scalable endothelin-1, tissue plasminogen activator (tPA), platelet-derived growth factor (PDGF), adhesion factor.These expression of gene products are for reinventing blood vessel, and the specific adsorption of regulating between iuntercellular, cell and substrate plays an important role.Exist huge difference between the endotheliocyte of these situations and external static culture.High-caliber shearing force has also promoted endotheliocyte to discharge cytokine, and these factors can anticoagulant, the propagation of leukocytic migration and smooth muscle cell, and low-level shearing force has then played opposite effect.
Summary of the invention
In order to promote the endotheliocyte that is seeded in artificial blood vessel's inwall to adhere to, for the ability that adheres to the effect of back endotheliocyte tolerance blood flow shear stress is provided, the present invention proposes a kind of method at artificial blood vessel's inner surface plantation endothelium/endothelioid cells, promptly adopt the method for biomechanics, by external pulsation, the fluid shear stress effect that staged increases, make endotheliocyte stick to artificial blood vessel's inwall better, make the endotheliocyte that sticks on artificial blood vessel's inwall adapt to the effect of fluid shear stress in advance, the ability of better tolerance blood flow shear stress effect is arranged after making it in body is gone in blood vessel transplantation.
For achieving the above object, the present invention has adopted following two kinds of methods: method one, after the collection endotheliocyte becomes cell suspension, be seeded in artificial blood vessel's inner surface, static culture to cell after artificial blood vessel's inner surface becomes the fusion state, apply fluid shear stress pulsation, that staged increases, the suffered value of shearing of endotheliocyte when reaching at body.In seeded process, the artificial blood vessel rotates with certain angular velocity around its axis.Method two after the collection endotheliocyte becomes cell suspension, is seeded in artificial blood vessel's inner surface, begins to apply fluid shear stress pulsation, that staged increases simultaneously, the suffered value of shearing of endotheliocyte when reaching at body.In seeded process, the artificial blood vessel rotates with certain angular velocity around its axis.
Artificial blood vessel among the present invention can be for the synthesising biological material, as terylene (Dacron), expanded PTFE (e-PTFE), polyurethane (polyurethane, Pu) etc., can be for natural biologic material, the acellular matrix of making as submucous layer of small intestine, pericardium, fascia, blood vessel or corium etc.
The advantage of the inventive method is by the effect of the fluid shear stress of pulsation, staged increase, compared with prior art, the present invention more emphasizes the staged loading procedure of fluid shear stress, the characteristics that have more at the uniform velocity and increase gradually, afterwards, reaching after the value of shearing that body requires to bear, keeping this value, when carrying out application such as zoopery, just stop, shear stress provide the time longer; The shear stress that is applied is pulsed, and approaches physiological conditions; And the value of shearing scope that relates among the present invention is in normal human's endotheliocyte tolerance range, makes the inventive method prepare the artificial blood vessel like this and has potential clinical value; In addition, the endotheliocyte that the present invention adopts can be that it has better adhesiveness on artificial blood vessel's material through the domestication of fluid shear stress, and other researcheres of this point are not mentioned.Cause the present invention aspect following, to have bigger advantage like this: the situation when 1. making the cellular morphology of the endothelium/endothelioid cells that is seeded in artificial blood vessel's inner surface and arranging situation approach at body, i.e. cell elongation, following current body direction is arranged; 2. increase the adhesive capacity of endotheliocyte on the artificial blood vessel surface, thereby after making the artificial blood vessel in being implanted into body, can be through can standing souring at the blood flow shear stress of body, and unlikely coming off; 3. induce the formation of endothelium/endothelioid cells stress fiber; 4. the undue propagation that suppresses endothelium/endothelioid cells in the In vitro culture process makes the contraction phenotype when being seeded in endothelium/endothelioid cells that artificial blood vessel's inner surface is the fusion state and being in body; 5. make and be seeded in endothelium/endothelioid cells that artificial blood vessel's inner surface is the fusion state and have the physiologically active that is similar at body.Reach the effect that taming gradually of endotheliocyte is increased its adhesiveness and physiologically active like this.
Adopt the inventive method to make the Human umbilical vein endothelial cells on the Wistar rat carotid artery that is seeded in the removal endotheliocyte pass through finally up to 26dyn/cm
2The shear stress effect that increases of staged after 24 hours, it keeps area is more than 80%.And under same experiment condition, adopt 15dyn/cm
2Effect after 2 hours, tangible vacancy district appears between the cell, it keeps area less than 50%.
Fig. 1 is seeded in the shear stress effect that the Human umbilical vein endothelial cells on the Wistar rat carotid artery of removing endotheliocyte increases by staged, and shear stress is from 10dyn/cm
2Beginning, ladder are per 3 hours increase 2dyn/cm
2, ripple frequency is 40Hz, finally reaches up to 26dyn/cm
2, blood vessel is per hour 120 ° around the speed of its axis rotation, acts on and dying through silver after 24 hours, at the photo of Olymus microscopically, amplification is 80 times.Having in the photo than the dark colour line is that the film of cell is dyed by silver and forms, so its performance is the profile of cell, as can be seen, arranges closely between the cell from photo.
The Human umbilical vein endothelial cells that Fig. 2 is seeded on the Wistar rat carotid artery of removing endotheliocyte is passed through 15dyn/cm
2The shear stress effect dye through silver after 2 hours, at the photo of Olymus microscopically, amplification is 40 times.Having in the photo than dark colour is that cell is dyed by silver and forms, and as can be seen, tangible vacancy district is arranged between the cell from photo.
Description of drawings
Fig. 1 is the microphotograph that is seeded in the Human umbilical vein endothelial cells on the Wistar rat carotid artery of removing endotheliocyte by this method;
Fig. 2 is that the Human umbilical vein endothelial cells that is seeded on the Wistar rat carotid artery of removing endotheliocyte is passed through 15dyn/cm
2The shear stress effect dye through silver after 2 hours, at the photo of Olymus microscopically.
The specific embodiment
Example 1, static inoculation on the cytostromatic Mus carotid artery vascular graft materials of removal apply the fluid shear stress that staged increases afterwards, and blood vessel is around its axis rotating and culturing endotheliocyte simultaneously
The freezing method of learning from else's experience is removed the Wistar rat carotid artery of endotheliocyte, and inoculation is through 10dyns/cm
2The rabbit arterial endothelial cell of former generation of shear stress domestication in inner membrance, inoculation method is as follows: will remove the Mus carotid artery of endotheliocyte through freezing method, inner membrance cleans 3 times with PBS liquid, with former generation rabbit endotheliocyte 0.1% trypsinization of cultivating, collecting cell, cell concentration about 1 * 10
6Cells/cm
2, the cell inoculation of collecting is arrived tunica intima, 37 ℃, 5%CO
2About 3 days of incubator static culture treats that cell fusion becomes monolayer, and then, the fluid shear stress that the endotheliocyte of inoculating is applied the staged increase carries out " domestication ", loads shear stress from 10dyns/cm
2Beginning is until 40dyns/cm
2, ladder is to increase 5dyns/cm every 3h
2, the about 12h of required time keeps 40dyns/cm then
2Shear stress, load simultaneously with the artificial blood vessel with per half an hour 90 ° angular velocity around its axis rotation, at 37 ℃, 5%CO
2Cultivate in the incubator.Obtain the vascular graft of this invention thus.
Example 2, removing on the cytostromatic Mus carotid artery vascular graft materials inoculation and applying the fluid shear stress that staged increases simultaneously, blood vessel is around its axis rotating and culturing endotheliocyte simultaneously
The freezing method of learning from else's experience is removed the Wistar rat carotid artery of endotheliocyte, inoculation rabbit arterial endothelial cell of former generation is in inner membrance, inoculation method is as follows: will remove the Mus carotid artery of endotheliocyte through freezing method, inner membrance cleans 3 times with PBS liquid, with former generation rabbit endotheliocyte 0.1% trypsinization of cultivating, collecting cell, cell concentration about 1 * 10
6Cells/cm
2, the cell inoculation of collecting is arrived tunica intima, simultaneously endotheliocyte is applied the fluid shear stress that staged increases, load the shear stress initial value and be about 5dyns/cm
2, ladder is to increase 0.1dyns/cm every 1h
2, the about 12h of required time changes ladder again for increasing 1.0dyns/cm every 1h
2About 12h of load time, load simultaneously with the artificial blood vessel with per half an hour 90 ° angular velocity around its axis rotation, at 37 ℃, 5%CO
2Cultivate in the incubator.Obtain the vascular graft of this invention thus.
Example 3, removing on the cytostromatic goat carotid artery vascular graft materials inoculation and applying fluid shear stress pulsation, that staged increases simultaneously, blood vessel is around its axis rotating and culturing endotheliocyte simultaneously
The enzyme digestion of learning from else's experience is removed the goat carotid artery of endotheliocyte, inoculate the former foster Human umbilical vein endothelial cells of being commissioned to train in inner membrance, inoculation method is as follows: the enzyme digestion of learning from else's experience is removed the goat carotid artery of endotheliocyte, inner membrance cleans 3 times with PBS liquid, with former generation Human umbilical vein endothelial cells 0.1% trypsinization of cultivating, collecting cell, cell concentration about 1 * 10
6Cells/cm
2, the cell inoculation of collecting is arrived tunica intima, simultaneously endotheliocyte is applied fluid shear stress pulsation, that staged increases, ripple frequency is 80Hz, loads the shear stress initial value and is about 5dyns/cm
2, ladder is to increase 0.3dyns/cm every 1h
2, the about 12h of required time changes ladder again for increasing 10dyns/cm every 1h
2, about 4h of load time keeps about 50dyns/cm afterwards
2Shear stress, load simultaneously the artificial blood vessel with the angular velocity of 20 ° of per minutes around its axis rotation, at 37 ℃, 5%CO
2Cultivate in the incubator.Obtain the vascular graft of this invention thus.
Example 4, static inoculation on expanded PTFE (ePTFE) vascular grafts apply fluid shear stress pulsation, that staged increases afterwards, and blood vessel is around the endotheliocyte of its axis rotating and culturing simultaneously
After expanded PTFE (ePTFE) vascular stent material sterilization treatment, get one section (the about 1.5mm of internal diameter is about 4-5cm), inoculation is through 5-10dyns/cm
2The rabbit arterial endothelial cell of former generation of fluid shear stress domestication is in inner membrance, inoculation method is as follows: the inner membrance of expanded PTFE (ePTFE) vascular stent material is cleaned 3 times with PBS liquid, and after serving as a contrast fiber adhesion albumen (FN) in advance, with former generation rabbit endotheliocyte 0.1% trypsinization of cultivating, collecting cell, cell concentration about 1 * 10
6Cells/cm
2, the cell inoculation of collecting is arrived tunica intima, 37 ℃, 5%CO
2About 3 days of incubator static culture, treat that cell fusion becomes monolayer after, apply fluid shear stress pulsation, that staged increases and carry out " domestication ", ripple frequency is 150Hz, loading shear stress is from 2dyns/cm
2Beginning is until 20dyns/cm
2, ladder is to increase 1dyns/cm every 1h
2, the about 18h of required time keeps 20dyns/cm then
2Shear stress, load simultaneously the artificial blood vessel with 300 ° angular velocity per hour around its axis rotation, at 37 ℃, 5%CO
2Cultivate in the incubator.Obtain the vascular graft of this invention thus.
Example 5, (blood vessel is around its axis rotating and culturing endotheliocyte simultaneously for polyurethane, Pu) inoculation and apply fluid shear stress pulsation, that staged increases simultaneously on the vascular grafts at polyurethane
(polyurethane Pu) after the vascular grafts sterilization treatment, gets one section (the about 2mm of internal diameter is about 4-5cm), and inoculation is through 5-10dyns/cm with polyurethane
2People's arterial endothelial cell of former generation of fluid shear stress domestication is in inner membrance, inoculation method is as follows: will clean 3 times with PBS liquid through the inner membrance of polyurethane vascular stent material, and after serving as a contrast vitronectin (LN) in advance, with former generation Human umbilical vein endothelial cells 0.1% trypsinization of cultivating, collecting cell, the about 1 * 10u of cell concentration
6Cells/cm
2, the cell inoculation of collecting is arrived tunica intima, simultaneously endotheliocyte is applied fluid shear stress pulsation, that staged increases, ripple frequency is 40Hz, loads the shear stress initial value and is about 2dyns/cm
2, ladder is to increase 0.2dyns/cm every 1h
2, the about 12h of required time changes ladder again for increasing 5dyns/cm every 1h
2, about 5h of load time keeps about 35dyns/cm afterwards
2Shear stress, load simultaneously the artificial blood vessel with the angular velocity of 10 ° of per minutes around its axis rotation, at 37 ℃, 5%CO
2Cultivate in the incubator.Obtain the vascular graft of this invention thus.
Example 6, removing on the cytostromatic goat carotid artery vascular graft materials inoculation and applying fluid shear stress pulsation, that staged increases simultaneously, blood vessel is around its axis rotating and culturing endotheliocyte simultaneously
The enzyme digestion of learning from else's experience is removed the goat carotid artery of endotheliocyte, inoculate the former foster Human umbilical vein endothelial cells of being commissioned to train in inner membrance, inoculation method is as follows: will remove goat carotid artery vascular graft materials inner membrance PBS liquid cleaning 3 times of endotheliocyte through enzyme digestion, with former generation Human umbilical vein endothelial cells 0.1% trypsinization of cultivating, collecting cell, cell concentration about 1 * 10
6Cells/cm
2, the cell inoculation of collecting is arrived tunica intima, simultaneously endotheliocyte is applied fluid shear stress pulsation, that staged increases, ripple frequency is 60Hz, loads the shear stress initial value and is about 10dyns/cm
2, ladder is to increase 0.3dyns/cm every 1h
2, the about 12h of required time changes ladder again for increasing 3dyns/cm every 1h
2, about 10h of load time keeps about 45dyns/cm afterwards
2Shear stress, load simultaneously the artificial blood vessel with the angular velocity of 5 ° of per minutes around its axis rotation, at 37 ℃, 5%CO
2Cultivate in the incubator.Obtain the vascular graft of this invention thus.
Claims (9)
1, a kind of method at artificial blood vessel's inner surface plantation endothelium/endothelioid cells, after the collection endotheliocyte becomes cell suspension, be seeded in artificial blood vessel's inner surface, static culture to cell after artificial blood vessel's inner surface becomes the fusion state, apply fluid shear stress, the suffered value of shearing of endotheliocyte when reaching at body; In seeded process, the artificial blood vessel rotates with certain angular velocity around its axis; It is characterized in that the fluid shear stress that applies is fluid shear stress pulsation, that staged increases.
2, a kind of method at artificial blood vessel's inner surface plantation endothelium/endothelioid cells after the collection endotheliocyte becomes cell suspension, is seeded in artificial blood vessel's inner surface, begins to apply fluid shear stress simultaneously, the suffered value of shearing of endotheliocyte when reaching at body; In seeded process, the artificial blood vessel rotates with certain angular velocity around its axis; It is characterized in that the fluid shear stress that applies is fluid shear stress pulsation, that staged increases.
3, according to claim 1 or 2 described methods at artificial blood vessel's inner surface plantation endothelium/endothelioid cells, it is characterized in that: the shear stress scope that the staged that the endothelium/endothelioid cells that is seeded in the artificial blood vessel surface is applied increases is greater than 0, less than 50dyes/cm
2, the speed that the shear stress staged increases for per hour greater than 0, less than 10dyes/cm
2
4, according to claim 1 or 2 described methods at artificial blood vessel's inner surface plantation endothelium/endothelioid cells, it is characterized in that: the endothelium/endothelioid cells of inoculation can derive from the endothelium/endothelioid cells of taming through fluid shear stress in advance.
5, method according to claim 3 is characterized in that: the speed that the shear stress staged increases is 0.1-1dyes/cm per hour
2
6, according to claim 1 or 2 described methods at artificial blood vessel's inner surface plantation endothelium/endothelioid cells, it is characterized in that: the fluid shear stress that the endothelium/endothelioid cells that is seeded in the artificial blood vessel surface is applied pulsation, its ripple frequency scope is greater than 0, less than 150Hz.
7, according to the described method at artificial blood vessel's inner surface plantation endothelium/endothelioid cells of claim 6, it is characterized in that: the ripple frequency scope is 40-80Hz.
8, according to claim 1 or 2 described methods at artificial blood vessel's inner surface plantation endothelium/endothelioid cells, it is characterized in that: in seeded process, the artificial blood vessel is that per minute is greater than 0, less than 10 ° around its axis rotational angular scope.
9, the described according to Claim 8 method at artificial blood vessel's inner surface plantation endothelium/endothelioid cells, it is characterized in that: the velocity of rotation scope is per hour 100-200 °.
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| CN100431504C (en) * | 2005-05-09 | 2008-11-12 | 北京中孵友信医药科技有限公司 | Cell coated stent |
| US20160178491A1 (en) | 2014-12-22 | 2016-06-23 | Saint-Gobain Performance Plastics Corporation | Capture system of cells and methods |
| CN104758980B (en) * | 2015-02-03 | 2018-02-09 | 北京航空航天大学 | A kind of method that external structure has the intravascular cortex of anti-Osima jacoti, Osima excavata and anti-platelet aggregation function |
| CN105268025B (en) * | 2015-10-15 | 2018-07-03 | 盐城工业职业技术学院 | A kind of preparation method of silk-fibroin cell composite vascular stent |
| CN107456296B (en) * | 2016-09-14 | 2020-07-14 | 四川蓝光英诺生物科技股份有限公司 | Lumen tissue construct, method and device for producing lumen tissue construct |
| CN107411845A (en) * | 2016-09-14 | 2017-12-01 | 四川蓝光英诺生物科技股份有限公司 | Lumen organization's construct, lumen organization's structure preparation and its device |
| CN107308495A (en) * | 2017-07-06 | 2017-11-03 | 苏州期佰生物技术有限公司 | A kind of artificial blood vessel based on trees-Osima jacoti, Osima excavata and preparation method thereof |
| CN110327030A (en) * | 2019-04-16 | 2019-10-15 | 南方医科大学珠江医院 | Simulate experimental model and its application of coronary artery bridge vascular flow state |
| CN112608882B (en) * | 2021-01-14 | 2022-11-18 | 大连理工大学 | Method for manufacturing silica gel blood vessel model planted with endothelial cells |
| CN116617460B (en) * | 2023-05-29 | 2024-06-11 | 中国人民解放军陆军军医大学 | In-vitro endothelialized vascular implant, and preparation method and application thereof |
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| WO1993001843A1 (en) * | 1991-07-25 | 1993-02-04 | University Of Leicester | Preparing grafts for implantation |
| US5792603A (en) * | 1995-04-27 | 1998-08-11 | Advanced Tissue Sciences, Inc. | Apparatus and method for sterilizing, seeding, culturing, storing, shipping and testing tissue, synthetic or native, vascular grafts |
| US5843781A (en) * | 1993-04-28 | 1998-12-01 | The Johns Hopkins University School Of Medicine | Implantable prosthetic vascular device having an adherent cell monolayer produced under shear stress |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993001843A1 (en) * | 1991-07-25 | 1993-02-04 | University Of Leicester | Preparing grafts for implantation |
| US5843781A (en) * | 1993-04-28 | 1998-12-01 | The Johns Hopkins University School Of Medicine | Implantable prosthetic vascular device having an adherent cell monolayer produced under shear stress |
| US5792603A (en) * | 1995-04-27 | 1998-08-11 | Advanced Tissue Sciences, Inc. | Apparatus and method for sterilizing, seeding, culturing, storing, shipping and testing tissue, synthetic or native, vascular grafts |
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