CN111454467A - Smearing type biodegradable external vascular stent and preparation method thereof - Google Patents
Smearing type biodegradable external vascular stent and preparation method thereof Download PDFInfo
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- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
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- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
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- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
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- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
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Abstract
The invention discloses a smearing type biodegradable external vascular stent and a preparation method thereof. The hydrogel composite solution comprises the following components: 10-20 wt% of gelatin; 5-20 wt% of a biological enzyme; 0.1-1 wt% of bacterial cellulose; the balance being solvent. The hydrogel composite aqueous solution has high viscosity and high gelling speed, can be coated on the outer wall of a blood vessel in a smearing mode, greatly reduces the operation difficulty, and is suitable for complex conditions in clinical transplantation operations. The extravascular stent material is derived from bio-based polymers, has excellent biocompatibility, is safe and nontoxic in degradation products, and does not have potential risks of anaphylactic reaction or toxic reaction. The hydrogel extravascular stent loaded with the medicine for inhibiting the endothelial hyperplasia not only has the function of external support of a transplanted vein, but also can prevent and treat excessive proliferation of the vein intima through the medicine.
Description
Technical Field
The invention relates to an external vascular stent and a preparation method thereof, in particular to a smearing type biodegradable external vascular stent and a preparation method thereof, belonging to the field of tissue engineering.
Background
Atherosclerosis is one of the major diseases that threaten human health and cause death in the population today in the world. Coronary artery bypass graft surgery is the primary treatment for patients with advanced coronary heart disease. Although arterial blood vessels have their own advantages as graft vessel material, autologous saphenous vein remains the most commonly used graft vessel material necessary for most patients. However, after operation, the vein-bridge blood vessel is suddenly subjected to high arterial pressure, the hemodynamics is changed, the tension of the tube wall is obviously increased, the bridge blood vessel is excessively expanded to cause the damage of the tube wall, under the participation of a plurality of factors, the smooth muscle cells of the tunica media are proliferated and migrate to the tunica intima through the elastic layer in the blood vessel, and simultaneously, excessive extracellular matrix is generated to cause the formation of a new tunica intima and the thickening of the tube wall, and then atheromatous plaque is gradually formed on the basis of the factors, so that the vein-bridge blood vessel is.
The extravascular stent aims to limit the expansion of veins, maintain the consistency of the inner diameters of a transplanted vein and a target vessel, stabilize the hemodynamics, reduce the turbulence, reduce the incidence of thrombus and relieve the intimal-media hyperplasia of the blood vessels; the blood vessel external support can stimulate the generation of blood vessel adventitia inflammation, promote the generation of adventitia nourishing blood vessel, relieve the injury of blood vessel anoxia and the like, promote the migration of hyperplastic smooth muscle cells to the outer membrane, promote the generation of related cell factors and growth factors, relieve intimal hyperplasia and prevent blood vessel atherosclerosis. The material for manufacturing the external vascular stent is continuously developed into a metal material and a biodegradable high polymer material from early Teflon fiber, but the currently researched external vascular stent has a single form, is mostly used for preparing formed tubular objects, and is difficult to face more complicated conditions in practical clinical application, which undoubtedly limits the clinical application of the external vascular stent.
Collagen, one of the major structural proteins of the extracellular matrix, can be crosslinked to form a gel under physiological conditions, but its use is limited by its high cost and poor mechanical strength.
Disclosure of Invention
Aiming at the problems that the existing external vascular stent has single form and complex preparation process and can not adapt to complex clinical situations, the invention provides a coating type degradable artificial external vascular stent which can inhibit the endothelial proliferation of a transplanted vein and improve the long-term patency rate of the transplanted vein. The spreadable biodegradable outer blood vessel stent can load drugs for inhibiting vascular endothelial hyperplasia by utilizing the excellent biocompatibility of gelatin, and the outer stent is used for wrapping a vein graft to release the drugs through stent degradation, so that the aim of inhibiting vascular intimal hyperplasia is fulfilled; the mechanical support provided by the stent material is combined, so that the vein distortion is prevented, the hemodynamics is stabilized, the turbulence is reduced, and the purpose of preventing the restenosis of the transplanted vein is achieved.
The invention claims a hydrogel composite solution, which comprises the following components:
10-20 wt% of gelatin;
5-20 wt% of a biological enzyme;
0.1-1 wt% of bacterial cellulose;
the balance being solvent.
In addition, the invention also claims a hydrogel composite solution containing gelatin; alternatively, the first and second electrodes may be,
the gelatin is applied to preparing hydrogel composite solution or preparing degradable vascular stents or degradable extravascular stents.
Specifically, the hydrogel composite solution is the hydrogel composite solution;
the biological enzyme is glutamine transaminase; which catalyse the covalent cross-linking of proteins (or polypeptides);
the solvent is sterile PBS solution. The sterile PBS solution contains KH2PO4NaCl and Na2HPO4The pH value is 7.2-7.4, and the product is stored at 15-30 ℃.
The hydrogel composite solution also comprises a medicament; the medicine can be added according to different conditions of patients;
the medicine is a medicine for inhibiting endothelial hyperplasia; more specifically at least one selected from paclitaxel, berberine and rapamycin;
the concentration of the drug in the hydrogel composite solution is 1-15 wt%.
Specifically, in the hydrogel composite solution, the content of the biological enzyme is 10-20 wt%; in particular 16.7 percent;
the content of the gelatin is 15 wt%;
the content of the bacterial cellulose is 0.6 wt%;
the content of the medicine is 1-10 or 1-5 wt%;
the hydrogel composite solution can be any one of hydrogel composite solutions a-c composed of the following components:
in terms of mass percentage, the weight percentage of the alloy is,
the hydrogel composite solution a consists of 10.0 percent of gelatin, 0.6 percent of bacterial cellulose, 1.0 percent of medicine for inhibiting endothelial hyperplasia, 16.7 percent of glutamine transaminase and 71.7 percent of sterile PBS solution.
The hydrogel composite solution b is composed of 15.0% of gelatin, 0.6% of bacterial cellulose, 1.0% of a medicine for inhibiting endothelial hyperplasia, 16.7% of glutamine transaminase and 66.7% of sterile PBS solution.
The hydrogel composite solution c consists of 20.0 percent of gelatin, 0.6 percent of bacterial cellulose, 1.0 percent of medicine for inhibiting endothelial hyperplasia, 16.7 percent of glutamine transaminase and 61.7 percent of sterile PBS solution.
The method for preparing the hydrogel composite solution comprises the following steps:
and respectively carrying out ultraviolet irradiation on the gelatin, the biological enzyme solution, the bacterial cellulose and the medicine, heating and uniformly mixing the components except the biological enzyme and the solvent, and then adding the biological enzyme to obtain the composition.
In the step 1) of the above method, the intensity of the ultraviolet irradiation is 30w/cm2(ii) a The ultraviolet irradiation time is 12-24 hours;
in the step of heating and uniformly mixing, the heating temperature is 30-35 ℃; the time is 30min-60 min.
The invention provides a method for preparing an external vascular stent, which comprises the following steps: and coating the hydrogel composite solution on the outer surface of a target object, and curing to obtain the hydrogel composite solution.
In the curing step of the method, the time is less than 60 s;
the target object is an artificial blood vessel or a natural blood vessel. When the target object is an artificial blood vessel, the target object can be anastomosed first and then smeared and cured.
In addition, the invention also claims the extravascular stent prepared by the method.
The coated degradable intravascular stent prepared by the invention has good biocompatibility, adjustable physical and chemical properties and can realize normal-temperature drug embedding.
The invention has the following remarkable beneficial effects:
1. the extravascular stent material is based on a bio-based polymer, has excellent biocompatibility, can be well compounded with cells, has safe and nontoxic degradation products, and does not have potential risks of anaphylactic reaction or toxic reaction.
2. The hydrogel composite aqueous solution has high viscosity and high gelling speed, can be coated on the outer wall of a blood vessel in a smearing mode, greatly reduces the operation difficulty, and is suitable for complex conditions in clinical transplantation operations.
3. The hydrogel extravascular stent containing the medicine for inhibiting the endothelial hyperplasia not only has the function of external support of a transplanted vein, but also can prevent and treat excessive proliferation of the vein intima through the medicine.
4. The coated degradable intravascular stent provided by the invention can be used for limiting vein expansion in coronary bypass grafting operation, maintaining the consistency of the inner diameters of a transplanted vein and a target vessel, stabilizing the hemodynamics, reducing turbulence and preventing thrombosis, and has important application value.
Drawings
FIG. 1 is a shear-thinning characterization of the extravascular stent of the present invention;
FIG. 2 is a scanning electron microscope image of the porous structure of the extravascular stent of the invention;
FIG. 3 is a diagram showing the mechanical properties of the extravascular stent of the present invention;
fig. 4 is a schematic view of smearing the external vascular stent in vitro great saphenous vein of the invention.
FIG. 5 is a representation of the cytocompatibility of the extravascular scaffolds of the present invention.
Fig. 6 is a cytotoxicity test chart of the extravascular stent of the invention.
Fig. 7 is a schematic diagram of experimental operation of the extravascular stent animal of the invention.
Fig. 8 is a graph of ultrasonic flow velocity measurement after the implantation of the extravascular stent of the invention.
Detailed Description
The present invention is described below with reference to specific embodiments, but the present invention is not limited thereto, and any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. Wherein the bacterial cellulose is a product sold by Qihong science and technology Limited company, the mark is BC, the diameter is 50-100nm, the length is 10000-; the gelatin is a biological gelatin material which is purchased from Shanghai Aladdin Biotechnology limited, in particular G108395 gelatin used for microbiology, and the strength of the gelatin is 250G bloom; the transglutaminase used was purchased from Zhengzhou Morda chemical products Co.
In the quantitative tests in the following examples, three replicates were set up and the results averaged.
Example 1 preparation of extravascular stent hydrogel composite solution
(1) The raw materials used for the hydrogel composite solution in the embodiment are composed of the following components in percentage by mass: 10.0 percent of gelatin, 0.6 percent of bacterial cellulose, 1.0 percent of rapamycin which is a medicament for inhibiting endothelial hyperplasia, 16.7 percent of glutamine transaminase and 71.7 percent of sterile PBS solution.
(2) The preparation steps are as follows:
1) gelatin, bacterial cellulose and rapamycin which is a medicine for inhibiting endothelial hyperplasia are sequentially added into a square culture dish, and ultraviolet irradiation is carried out for 12-24 hours.
2) Adding the irradiated gelatin, bacterial cellulose, the endothelial proliferation inhibiting drug rapamycin in the step 1) and a sterile PBS solution into a Sichuan cattle bottle, and then magnetically stirring and heating the solution at the temperature of 30-35 ℃ for 1 hour to completely dissolve all the components to form a mixture (hydrogel liquid).
3) Adding transglutaminase to the suspension.
Example 2 preparation of extravascular stent hydrogel composite solution
(1) The raw materials used for the hydrogel composite solution in the embodiment are composed of the following components in percentage by mass: 15.0 percent of gelatin, 0.6 percent of bacterial cellulose, 1.0 percent of rapamycin which is a medicament for inhibiting endothelial hyperplasia, 16.7 percent of glutamine transaminase and 66.7 percent of sterile PBS solution.
(2) The preparation steps are as follows:
1) gelatin, bacterial cellulose and rapamycin which is a medicine for inhibiting endothelial hyperplasia are sequentially added into a square culture dish, and ultraviolet irradiation is carried out for 12-24 hours.
2) Adding the irradiated gelatin, bacterial cellulose, the endothelial proliferation inhibiting drug rapamycin in the step 1) and a sterile PBS solution into a Sichuan cattle bottle, and then magnetically stirring and heating the solution at the temperature of 30-35 ℃ for 1 hour to completely dissolve all the components to form a mixture (hydrogel liquid).
3) Adding transglutaminase to the suspension.
Example 3 preparation of extravascular stent hydrogel composite solution
(1) The raw materials used for the hydrogel composite solution in the embodiment are composed of the following components in percentage by mass: 20.0 percent of gelatin, 0.6 percent of bacterial cellulose, 1.0 percent of rapamycin which is a medicament for inhibiting endothelial hyperplasia, 16.7 percent of glutamine transaminase and 61.7 percent of sterile PBS solution.
(2) The preparation steps are as follows:
1) sequentially adding gelatin, bacterial cellulose and medicine for inhibiting endothelial hyperplasia into a square culture dish, and performing ultraviolet irradiation for 12-24 hours.
2) Adding the irradiated gelatin, bacterial cellulose, the endothelial proliferation inhibiting drug rapamycin in the step 1) and a sterile PBS solution into a Sichuan cattle bottle, and then magnetically stirring and heating the solution at the temperature of 30-35 ℃ for 1 hour to completely dissolve all the components to form a mixture (hydrogel liquid).
3) Adding transglutaminase to the suspension.
The shear thinning performance of the hydrogel composite solution obtained in the above-mentioned examples 1 to 3 of the present invention is shown in fig. 1, and the viscosity decreases with the increase of the shear rate.
Characterization of the scaffold structure was examined by SEM to observe scaffold pore distribution, as shown in figure 2. As can be seen from the figure, the scaffold has a three-dimensional porous structure of hydrogel, is beneficial to cell invasion, stimulates the adventitial reaction of a transplanted vein, and improves the adventitial ischemia phenomenon.
The hydrogel was cut into 1cm long strips and tested for tensile mechanical properties, as shown in figure 3. As can be seen from the figure, the hydrogel intravascular stent has enough strain and stress, and the maximum fracture stress can reach 0.1 MPa.
Example 4 in vitro smearing experiment of extravascular scaffolds
The isolated veins were fixed at both ends of the pump, and the hydrogel composite solution prepared in example 1 was uniformly applied to the veins for one week, as shown in fig. 4, and the hydrogel composite solution was cured (curing time was less than 60 seconds) on the outer wall of the veins to form a hydrogel extravascular stent.
Example 5 characterization of extracellular scaffold hydrogel composite solution cell compatibility
The extravascular stent hydrogel composite solution prepared in the example 1 is spread on a cell culture dish and polymerized under the condition of 37 ℃, L929 cells which are normally cultured are digested by pancreatin, centrifuged, and re-dispersed by using 9m L culture medium and 1m L serum to prepare cell suspension, the cell suspension is spread on the surface of the hydrogel for culturing, and as shown in figure 5, the cells can adhere to the surface of the hydrogel and propagate, thereby verifying that the extravascular stent material of the invention has excellent cell compatibility.
Example 6 detection of cytotoxicity of extravascular scaffold hydrogel composite solution
The extravascular stents prepared in the examples 1, 2 and 3 are immersed in 10m L Du's modified eagle's medium-high sugar (DMEM high sugar) culture solution for 24h under the condition of 37 ℃ to obtain leaching liquor, then the leaching liquor is diluted to obtain the leaching liquor with the concentration of 0.100g/m L, the leaching liquor with different concentrations is acted on L929 cells, and the cytotoxicity is respectively detected for 24h and 48 h.
As shown in FIG. 6, it is a bar chart of cell activities of L929 cells in different concentrations of leachate, the data in FIG. 6 shows that the cell activities are above 90%, demonstrating that the extravascular scaffold hydrogel composite solution provided by the present invention has no cytotoxicity.
Example 7 Experimental surgery on extravascular scaffolds
New Zealand white rabbits (2.2-2.5kg) and pentobarbital (30mg/kg) are used for intraperitoneal injection anesthesia, and the injection is performed in a supine position, and the skin is prepared, disinfected and paved conventionally. Making an operation incision along the center of the neck, making the incision about 5cm long, opening platysma, finding internal jugular vein and carotid artery, dissociating the right internal jugular vein and carotid artery, dissociating the internal jugular vein by about 3-4cm, dissociating the far end of the carotid artery to send out the internal carotid artery, dissociating the near end to the suprasternal fossa, carefully ligating small branches, and strictly stopping bleeding. After the whole body is heparinized by half amount (1mg/Kg) through veins, the two ends of an artery are blocked by a non-invasive vascular clamp, the ends of the veins and the artery are anastomosed by using an anastomosis wheel, the proximal end is anastomosed first, the distal end is anastomosed after bloodletting is finished, the hydrogel composite solution is smeared on the outer wall of the veins after confirming that an anastomotic opening is unobstructed and has no blood leakage, and a fixer is used for fixing for 30s, as shown in figure 6. Stopping bleeding carefully and suturing the wound. After vein transplantation, feeding was performed, and carotid artery ultrasonic examination was performed for one month as shown in fig. 7. As can be seen, the graft vein still maintains good patency. This demonstrates that the external stent can prevent restricted vein dilation, maintain consistency of the internal diameters of the graft vein and the target vessel, stabilize hemodynamics, reduce turbulence, and prevent thrombosis. The use of the external vascular stent can not only prevent thrombosis, but also reduce the intimal hyperplasia of the transplanted vein and improve the long-term patency rate.
Claims (10)
1. A hydrogel composite solution comprises the following components:
10-20 wt% of gelatin;
5-20 wt% of a biological enzyme;
0.1-1 wt% of bacterial cellulose;
the balance being solvent.
2. A hydrogel composite solution containing gelatin; alternatively, the first and second electrodes may be,
the gelatin is applied to preparing hydrogel composite solution or preparing degradable vascular stents or degradable extravascular stents.
3. The hydrogel composite solution or use of claim 2, wherein: the hydrogel composite solution is the hydrogel composite solution of claim 1 or claim 4 or claim 5.
4. The hydrogel composite solution or use of any one of claims 1 to 3, wherein: the biological enzyme is glutamine transaminase;
the solvent is sterile PBS solution.
5. The hydrogel composite solution or use of any one of claims 1 to 4, wherein: the hydrogel composite solution also comprises a medicament;
the medicine is a medicine for inhibiting endothelial hyperplasia; more specifically at least one selected from paclitaxel, berberine and rapamycin;
the concentration of the drug in the hydrogel composite solution is 1-15 wt%.
6. A method of preparing the hydrogel composite solution of any one of claims 1-5, comprising:
and respectively carrying out ultraviolet irradiation on the gelatin, the biological enzyme solution, the bacterial cellulose and the medicine, heating and uniformly mixing the components except the biological enzyme and the solvent, and then adding the biological enzyme to obtain the composition.
7. The method of claim 6, wherein: in the step 1) of ultraviolet irradiation, the intensity of ultraviolet irradiation is 30w/cm2(ii) a The ultraviolet irradiation time is 12-24 hours;
in the step of heating and uniformly mixing, the heating temperature is 30-35 ℃; the time is 30min-60 min.
8. A method of preparing an extravascular stent, comprising: the hydrogel composite solution as claimed in any one of claims 1 to 5 is applied to the outer surface of a target object and cured.
9. The method of claim 8, wherein: in the curing step, the time is less than 60 s;
the target object is an artificial blood vessel or a natural blood vessel.
10. An extravascular stent prepared according to the method of claim 8 or 9.
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| CN110591126A (en) * | 2019-09-20 | 2019-12-20 | 西安交通大学 | Injectable magnetic hydrogel and preparation method thereof |
| CN110801532A (en) * | 2019-11-28 | 2020-02-18 | 中国人民大学 | Biological ink and preparation method thereof |
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2020
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| US5766584A (en) * | 1995-06-02 | 1998-06-16 | Massachusetts Institute Of Technology | Inhibition of vascular smooth muscle cell proliferation with implanted matrix containing vascular endothelial cells |
| CN1533813A (en) * | 2003-03-28 | 2004-10-06 | 微创医疗器械〔上海〕有限公司 | Medicinal coating layer supporting frame for preventing/treating renarrowing after inbellow arteria coronaria shaping operation |
| CN106540327A (en) * | 2016-12-06 | 2017-03-29 | 北京航空航天大学 | A kind of three layers of artificial blood vessel bracket of imitative nature blood vessel and preparation method thereof |
| CN110591126A (en) * | 2019-09-20 | 2019-12-20 | 西安交通大学 | Injectable magnetic hydrogel and preparation method thereof |
| CN110801532A (en) * | 2019-11-28 | 2020-02-18 | 中国人民大学 | Biological ink and preparation method thereof |
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