CN101361988B - Preparation method of blood vessel support or cardiac valve surface coating with good biocompatibility - Google Patents
Preparation method of blood vessel support or cardiac valve surface coating with good biocompatibility Download PDFInfo
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- CN101361988B CN101361988B CN2008100460512A CN200810046051A CN101361988B CN 101361988 B CN101361988 B CN 101361988B CN 2008100460512 A CN2008100460512 A CN 2008100460512A CN 200810046051 A CN200810046051 A CN 200810046051A CN 101361988 B CN101361988 B CN 101361988B
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Abstract
The invention discloses a preparation method of a surface coating of intravascular stents or cardiac valves with biocompatibility, which includes the steps: A. the preparation of a coating solvent: heparin is dissolved in deionized water, the pH value thereof is adjusted to 7 to 8 and then an endothelial cell growth factor is added to form a heparin/growth factor solution; collagens are dissolved in acetum to form a collagen solution; B. dip-coating: the intravascular stents or the cardiac valves are immersed in the heparin/growth factor solution and a layer of heparin/growth factor is dipped and coated on the surface of the intravascular stents or the cardiac valves, and then the intravascular stents or the cardiac valves are taken out for being cleaned by the deionized water and being dried by nitrogen gas; and the intravascular stents or the cardiac valves are immersed in the collagen solution, coated with a layer of collagen, and taken out for being cleaned by the deionized water and being dried by nitrogen gas; the steps of dip-coating by the use of the heparin/growth factor and the collagen are repeated in sequence for 1 time to 60 times; finally the step of dip-coating by the use of the heparin/growth factor is carried out again to obtain the surface coating with excellent biocompatibility of the intravascular stents or the cardiac valves. The intravascular stents or the cardiac valves prepared have excellent biocompatibility and low preparation cost.
Description
Affiliated technical field
The present invention relates to the bioid method of modifying on intravascular stent and heart valve materials surface.
Background technology
The cardiovascular disease incidence rate is the trend that rises year by year, and atherosclerosis causes that luminal stenosis or obstruction are the main causes that causes ischemic heart desease (coronary heart disease); Cardiac valve disease etc. is to be main pathological changes owing to valve disorder that reason congenital or posteriority causes causes the cardiac flow obstacle.
The latest study proves, endotheliocyte is extremely important for the vascular function performance: 1) complete endothelial layer can be secreted the hyper-proliferative that the nitric oxide equimolecular suppresses smooth muscle, the endothelial layer mechanism of secretion imbalance of damage causes the hyper-proliferative of smooth muscle and then causes narrow generation in the blood vessel; 2) complete endothelial layer can be secreted antiplatelet substance, the generation of anticoagulant, and the imbalance of the endothelial layer mechanism of secretion of damage, and the endothelial layer damage location will promote hematoblastic sticking, and then cause the generation of blood coagulation.After intravascular stent and heart valve materials implant thus,, narrow generation in blood coagulation and the blood vessel will be reduced if its surface can realize fast that endotheliocyte covers.
The intravascular stent implantation is satisfactory to treatment coronary heart disease clinical effectiveness, but support implantation back blood coagulation problem and restenosis problem all influence the long-term effect of treatment.Along with going deep into that mechanism is familiar with, there is the support of medication coat to be developed fast in surface applied, especially coating suppresses the thunderous handkerchief mycin of medicine of cell proliferation, the support of paclitaxel has been obtained effect preferably.But still there are some problems: the prolongation of implanting the time along with coating stent of medicine, when medicine suppresses the propagation (main cause that restenosis takes place) of smooth muscle cell, the propagation that has also suppressed endotheliocyte, rack surface can't form complete endothelial layer, and rack surface anticoagulation function is poor; And final drug release totally has the anticoagulant effect and suppresses the still not formation of complete endothelial layer of smooth muscle cell proliferation, and the probability that restenosis takes place in the intravascular stent after this significantly increases.
China is annual owing to the valve pathological changes seriously needs to implement the patient that artificial heart valve is replaced operation at present, about 200,000 examples, and these patient's majorities are between twenty and fifty, change lobe as can not in time performing the operation, and will cause immeasurable loss to society.Cardiac valve prosthesis commonly used at present is divided into mechanical valve prosthesis and biovalve two big classes.The former adopts metal material to make, because of its preferably durability be widely applied, but use patient's postoperative of mechanical prosthetic valve must take anticoagulant such as warfarin for a long time, anti-tampon.Bioprosthetic valve is because problem of aging may need the replacement that undergos surgery once more after certain after surgery year.If can make mechanical valve prosthesis surface form the complete endothelial layer of one deck, will the probability of blood coagulation be reduced greatly, promptly can reduce or no longer take anticoagulant, also need change valve hardly simultaneously, reduce patient's medical expense greatly.
Summary of the invention
Purpose of the present invention is exactly to propose a kind of preparation method with intravascular stent or cardiac valve surface coating of biocompatibility, and intravascular stent that this method makes or cardiac valve surface have good biocompatibility, and preparation cost is low.
The present invention realizes above goal of the invention, and the technical scheme that is adopted is that a kind of preparation method with intravascular stent or cardiac valve surface coating of biocompatibility the steps include:
The preparation of A, coating solution is dissolved in deionized water with heparin, and its pH value is adjusted to 7-8, and adds endothelial cell growth factor (ECGF), makes heparin/growth factor solution; With the acetum of collagenolysis, make collagen solution in 0.2mol/L;
B, dip-coating: intravascular stent or cardiac valve are immersed in 5-20min in heparin/growth factor solution, and dip-coating one deck heparin/somatomedin in its surface takes out the back washed with de-ionized water, nitrogen dries up; Again it is immersed in collagen solution 5-20min, dip-coating one deck collagen, washed with de-ionized water, nitrogen dry up after taking out; Repeat above heparin/somatomedin and collagen dip-coating step 1-60 time; Carry out above heparin/somatomedin dip-coating step at last once more, that is, manufactured goods are preserved under cryogenic conditions.
Compared with prior art, the invention has the beneficial effects as follows:
Somatomedin has good activity in pH value is the heparin solution of 7-8, and form highly active heparin/growth factor solution, the support or the valve that are immersed in wherein will be at the electronegative heparin/somatomedin of its surperficial dip-coating one deck, again it is immersed in collagen solution, because collagen is positively charged, to be combined on electronegative heparin/somatomedin layer by charge effect, form the collagenic coating of one deck positively charged again.The electronegative heparin of dip-coating/somatomedin coating on the collagenic coating of positive charge again then can obtain heparin/somatomedin and collagen two-layer composite more than 1 by the alternate repetition dip-coating.Its advantage is both to have made coating in conjunction with firm, thickness can reach 1500nm, make the coating composite construction possess the collagen of anticoagulant heparin, short endothelialization simultaneously again, induce CD34 in the peripheral blood or CD133 positive cell (stem cell a kind of) somatomedin in addition, thereby make support or valve surface that the present invention makes possess the excellent anticoagulation and the function of short endothelialization on the one hand simultaneously to the endotheliocyte directed differentiation; Be coated with the layer multi-layer dip-coating on the other hand, functional materials content is many, can not realize quick endothelialization before the hyper-proliferative at smooth muscle cell after support implants, the regulation of blood vessels function of performance body self, the effectively generation of anticoagulant and restenosis; The valve then endothelialization fast of rear surface that implants guarantees the realization of long-term anticoagulation function.Experiment confirm, the valve surface heparin after the modification of the present invention is with 1-3 μ g/ (cm
2* day) steadily discharge, heparin was still contained in the rear surface in 3 months.In a word, support and the valve surface after the modification of the present invention has excellent biocompatibility.
The concentration of above-mentioned collagen solution is 0.5-5mg/ml, and the heparin concentration of heparin solution is 0.5-25mg/ml, and the endothelial cell growth factor (ECGF) concentration in the heparin solution is 5-50ng/ml.Such concentration range, its dip-coating is effective, and each material binding capacity is moderate, combination is firm.
Last 2 heparin in the dip-coating in above-mentioned B step/somatomedin dip-coating operation, employed heparin solution also adds CD34 antibody or CD133 antibody, and the addition of CD34 antibody or CD133 antibody and the volume ratio of heparin solution are 1: 20-1: 200.
Because CD34 in the blood or CD133 antibody positive cell have the potentiality that are divided into endotheliocyte and proliferation activity far above endotheliocyte (belonging to terminally differentiated cells), effectively promote the reparation after the damage of blood vessel endothelium layer.Therefore this patent adds a selected amount of CD34 antibody or CD133 antibody in heparin solution, make CD34 antibody or CD133 antibodies in intravascular stent or valvular face coat, the easier implant site that concentrates on of CD34 or CD133 positive cell is played a role, can further strengthen short endothelialization function.Experiment showed, that intravascular stent that the present invention makes or cardiac valve surface coating increase 50%-150% to the amount of sticking of the CD34 positive or CD133 positive cell.
Above-mentioned intravascular stent or cardiac valve are immersed in it earlier in 60-80 ℃ the strong base solution of 0.5-5mol/L and activated in 3-24 hour before the dip-coating of carrying out B step; Take out, be immersed in again after the deionized water ultrasonic cleaning in the Poly-L-Lysine Solution of 0.1mg/ml-10mg/ml, soaked 1-12 hour, take out washed with de-ionized water, after nitrogen dries up, carry out the B dip-coating in step again.
Realize that by the activation processing in strong base solution before intravascular stent or the cardiac valve dip-coating support or cardiac valve surface have more negative charge like this, and realized surperficial charged amplification with the polylysine of a large amount of positive charges by absorption, making in the B dip-coating in step for the first time, the amount of bonded electronegative heparin/somatomedin increases 5%-20%.
The dip-coating in above-mentioned B step is carried out in ice-water bath, and the container that is about to fill heparin solution and collagen solution is positioned in the container that is mixed with ice cube and water.The dip-coating step has realized in the frozen water territory the active protection of each composition.
The present invention is described in further detail below in conjunction with drawings and Examples.
Description of drawings
Fig. 1 is that the cardiac valve behind the inventive method dip-coating face coat (modification) is simulated under the blood flow condition the hematoblastic electron scanning micrograph of surface adhesion at external flow cavity.
Fig. 2 is that unmodified cardiac valve is simulated under the blood flow condition the hematoblastic electron scanning micrograph of surface adhesion at external flow cavity.
Fig. 3 be intravascular stent surface after the inventive method modification under external static conditions, the adherent optical microscope picture of superficial cell.
Fig. 4 be unmodified intravascular stent under external static conditions, the adherent optical microscope picture of superficial cell.
The specific embodiment
Embodiment 1
A kind of preparation method with intravascular stent face coat of good biocompatibility the steps include:
The preparation of A, coating solution is dissolved in deionized water with heparin, and its pH value is adjusted to 7, and adds endothelial cell growth factor (ECGF), makes heparin/growth factor solution; Heparin concentration in this routine heparin/growth factor solution is that 0.5mg/ml, endothelial cell growth factor (ECGF) concentration are 5ng/ml.
With the acetum of collagenolysis in 0.2mo l/L, make collagen solution, the concentration of collagen solution is 0.5mg/ml.
Before carrying out the following B dip-coating in step, earlier intravascular stent is immersed in 60 ℃ the strong base solution of 0.5mol/L and activated in 3 hours; Take out, be immersed in again after the deionized water ultrasonic cleaning in the Poly-L-Lysine Solution of 0.1mg/ml, soaked 1 hour, take out washed with de-ionized water, after nitrogen dries up, carry out the B dip-coating in step again.
B, dip-coating: the container that fills heparin solution and collagen solution is positioned in the container that is mixed with ice cube and water.Intravascular stent is immersed in 5min in heparin/growth factor solution, and dip-coating one deck heparin/somatomedin in its surface takes out the back washed with de-ionized water, nitrogen dries up; Again it is immersed in collagen solution 5mi n, dip-coating one deck collagen, washed with de-ionized water, nitrogen dry up after taking out; Repeat above heparin/somatomedin dip-coating, that is, manufactured goods are preserved under cryogenic conditions.
In the heparin of above B step dip-coating/somatomedin operation, employed heparin solution also is added with CD34 antibody, and the addition of CD34 antibody and the volume ratio of heparin solution are 1: 200.
Embodiment 2
A kind of preparation method with cardiac valve surface coating of good biocompatibility the steps include:
The preparation of A, coating solution is dissolved in deionized water with heparin, and its pH value is adjusted to 8, and adds endothelial cell growth factor (ECGF), makes heparin/growth factor solution; Heparin concentration in heparin/growth factor solution is that 25mg/ml, endothelial cell growth factor (ECGF) concentration are 50ng/ml.
With the acetum of collagenolysis in 0.2mol/L, make collagen solution, the concentration of collagen solution is 5mg/ml.
Cardiac valve is immersed in it earlier in 80 ℃ the strong base solution of 5mol/L and activated in 24 hours before carrying out the following B dip-coating in step; Take out, be immersed in again after the deionized water ultrasonic cleaning in the Poly-L-Lysine Solution of 10mg/ml, soaked 12 hours, take out washed with de-ionized water, after nitrogen dries up, carry out the B dip-coating in step again.
B, dip-coating: the container that fills heparin solution and collagen solution is positioned in the container that is mixed with ice cube and water.Cardiac valve is immersed in 20min in heparin/growth factor solution, and dip-coating one deck heparin/somatomedin in its surface takes out the back washed with de-ionized water, nitrogen dries up; Again it is immersed in collagen solution 20min, dip-coating one deck collagen, washed with de-ionized water, nitrogen dry up after taking out; Repeat above heparin/somatomedin and collagen dip-coating step 60 time; Carry out above heparin/somatomedin dip-coating step at last once more, that is, manufactured goods are preserved under cryogenic conditions.
In last 2 heparin of above B step dip-coating/somatomedin dip-coating operation, employed heparin solution also is added with CD133 antibody, and the addition of CD133 antibody and the volume ratio of heparin solution are 1: 20.
Embodiment 3
A kind of preparation method with intravascular stent face coat of good biocompatibility the steps include:
The preparation of A, coating solution is dissolved in deionized water with heparin, and its pH value is adjusted to 7.5, and adds endothelial cell growth factor (ECGF), makes heparin/growth factor solution; Heparin concentration in heparin/growth factor solution is that 5mg/ml, endothelial cell growth factor (ECGF) concentration are 10ng/ml.
With the acetum of collagenolysis in 0.2mol/L, make collagen solution, the concentration of collagen solution is 1mg/ml.
Intravascular stent is immersed in it earlier in 70 ℃ the strong base solution of 3mol/L and activated in 10 hours before carrying out the following B dip-coating in step; Take out, be immersed in again after the deionized water ultrasonic cleaning in the Poly-L-Lysine Solution of 2mg/ml, soaked 4 hours, take out washed with de-ionized water, after nitrogen dries up, carry out the B dip-coating in step again.
B, dip-coating: the container that fills heparin solution and collagen solution is positioned in the container that is mixed with ice cube and water.Intravascular stent is immersed in 15min in heparin/growth factor solution, and dip-coating one deck heparin/somatomedin in its surface takes out the back washed with de-ionized water, nitrogen dries up; Again it is immersed in collagen solution 15min, dip-coating one deck collagen, washed with de-ionized water, nitrogen dry up after taking out; Repeat above heparin/somatomedin and collagen dip-coating step 20 time; Carry out above heparin/somatomedin dip-coating step at last once more, that is, manufactured goods are preserved under cryogenic conditions.
In last 2 heparin of B step dip-coating/somatomedin dip-coating operation, employed heparin solution also is added with CD34 antibody, and the addition of CD34 antibody and the volume ratio of heparin solution are 1: 50.
A kind of preparation method with cardiac valve surface coating of good biocompatibility the steps include:
The preparation of A, coating solution is dissolved in deionized water with heparin, and its pH value is adjusted to 7, and adds endothelial cell growth factor (ECGF), makes heparin/growth factor solution; Heparin concentration in heparin/growth factor solution is that 15mg/ml, endothelial cell growth factor (ECGF) concentration are 10ng/ml.
With the acetum of collagenolysis in 0.2mol/L, make collagen solution, the concentration of collagen solution is 1mg/ml.
B, dip-coating: the container that fills heparin solution and collagen solution is positioned in the container that is mixed with ice cube and water.Cardiac valve is immersed in 15min in heparin/growth factor solution, and dip-coating one deck heparin/somatomedin in its surface takes out the back washed with de-ionized water, nitrogen dries up; Again it is immersed in collagen solution 15min, dip-coating one deck collagen, washed with de-ionized water, nitrogen dry up after taking out; Repeat above heparin/somatomedin and collagen dip-coating step 40 time; Carry out above heparin/somatomedin dip-coating step at last once more, that is, manufactured goods are preserved under cryogenic conditions.
Following experimental result proves that intravascular stent or cardiac valve surface that the inventive method makes have good biocompatibility:
Test the ability of cardiac valve surface anticoagulant under the moving chamber simulation of the extracorporeal flow blood flow condition.
Cardiac valve is placed in the flat flow chamber, and the 50ml platelet rich plasma is fed into the flow cavity system, regulates the flow rate of liquid in the flow cavity, makes the shearing force size be about 1.0Pa, similar arteriolar shearing force.Flat flow chamber is at CO
2Incubator (37 ℃, 5%CO
2) hatch after 4 hours and take out sem observation.Fig. 1 is the hematoblastic electron scanning micrograph of cardiac valve surface adhesion after the modification of the present invention, and Fig. 2 is the hematoblastic electron scanning micrograph of unmodified cardiac valve surface adhesion.Cardiac valve surface adhesion platelet counts after the visible modification of the present invention of Fig. 1, Fig. 2 significantly reduces, and blood compatibility significantly improves.Intravascular stent carries out same experiment, and it comes to the same thing.
Test capturing endothelial ancestral cell under the two external static conditions, the endothelialization experiment of induced material surface.
Fig. 3, Fig. 4 are the experimental results of capturing endothelial ancestral cell under the external static conditions, and cell culture experiments in vitro shows, is adding 5 * 10
4When cells/ml CD34 positive cell was cultivated 1 day, Fig. 3 was that the intravascular stent superficial cell sticks the optical microscope picture after the modification, and Fig. 4 sticks the optical microscope picture for unmodified intravascular stent superficial cell.The adherent CD34 positive cell quantity of intravascular stent after the visible modification of the present invention of Fig. 3, Fig. 4 significantly increases, and cell compatibility significantly improves.Cardiac valve carries out same experiment, and it comes to the same thing.
Test the release result of heparin in intravascular stent after the modification under the moving chamber simulation of the three extracorporeal flows blood flow condition or the cardiac valve surface coating.
Intravascular stent after the modification of the present invention or cardiac valve are placed extracorporeal circulation apparatus, the NaCl solution of adding 0.9%, tremulous pulse environment in the flow velocity analogue body, keeping temperature is 37 ± 0.5 ℃, changed liquid respectively at 1,3,5,7,9,11 day, and take out the release of 5ml liquid by the colorimetric determination heparin and remain on 1-3 μ g/ (cm
2* day), and after one month, heparin is still contained on the surface.As seen, intravascular stent after the modification of the present invention and the heparin in the cardiac valve surface coating have long-term slow-release function.
Claims (5)
1. the preparation method with intravascular stent or cardiac valve surface coating of biocompatibility the steps include:
The preparation of A, coating solution is dissolved in deionized water with heparin, and its pH value is adjusted to 7-8, and adds endothelial cell growth factor (ECGF), makes heparin/growth factor solution; With the acetum of collagenolysis, make collagen solution in 0.2mol/L;
B, dip-coating: intravascular stent or cardiac valve are immersed in 5-20min in heparin/growth factor solution, and dip-coating one deck heparin/somatomedin in its surface takes out the back washed with de-ionized water, nitrogen dries up; Again it is immersed in collagen solution 5-20min, dip-coating one deck collagen, washed with de-ionized water, nitrogen dry up after taking out; Repeat above heparin/somatomedin and collagen dip-coating step 1-60 time; At last carry out above heparin/somatomedin dip-coating step once more, promptly; Manufactured goods are preserved under cryogenic conditions.
2. a kind of preparation method according to claim 1 with intravascular stent or cardiac valve surface coating of biocompatibility, it is characterized in that: the concentration of described collagen solution is 0.5-5mg/ml, and the heparin concentration in heparin/growth factor solution is that 0.5-25mg/ml, endothelial cell growth factor (ECGF) concentration are 5-50ng/ml.
3. a kind of preparation method according to claim 2 with intravascular stent or cardiac valve surface coating of biocompatibility, it is characterized in that: in last 2 heparin of described B step dip-coating/somatomedin dip-coating operation, employed heparin solution also adds CD34 or CD133 antibody-solutions, and the addition of this antibody-solutions and the volume ratio of heparin/growth factor solution are 1: 20-1: 200.
4. a kind of preparation method according to claim 1 with intravascular stent or cardiac valve surface coating of biocompatibility, it is characterized in that: described intravascular stent or cardiac valve are immersed in it earlier in 60-80 ℃ the strong base solution of 0.5-5mol/L and activated in 3-24 hour before the dip-coating of carrying out B step; Take out, be immersed in again after the deionized water ultrasonic cleaning in the Poly-L-Lysine Solution of 0.1mg/ml-10mg/ml, soaked 1-12 hour, take out washed with de-ionized water, after nitrogen dries up, carry out the B dip-coating in step again.
5. a kind of preparation method according to claim 1 with intravascular stent or cardiac valve surface coating of biocompatibility, it is characterized in that: the dip-coating in described B step is carried out in ice-water bath, and the container that is about to fill heparin solution and collagen solution is positioned in the container that is mixed with ice cube and water.
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Cited By (1)
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| CN109675106A (en) * | 2018-11-28 | 2019-04-26 | 曲阜师范大学 | A kind of electrospinning fibre bracket of load cells growth factor and preparation method thereof |
| CN111973313A (en) * | 2019-05-06 | 2020-11-24 | 深圳市罗湖区人民医院 | Small-caliber artificial blood vessel and preparation method thereof |
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