CN111714706A

CN111714706A - Vascular stent capable of promoting vascular cell proliferation and secreting extracellular matrix, preparation method of vascular stent and active artificial blood vessel

Info

Publication number: CN111714706A
Application number: CN202010383406.8A
Authority: CN
Inventors: 张�杰; 王恺; 万烨
Original assignee: Lingbo Biotechnology Hangzhou Co ltd
Current assignee: Lingbo Biotechnology Hangzhou Co ltd
Priority date: 2020-05-08
Filing date: 2020-05-08
Publication date: 2020-09-29
Anticipated expiration: 2040-05-08
Also published as: CN111714706B

Abstract

The invention belongs to the technical field of tissue engineering, and particularly relates to a vascular stent capable of promoting vascular cell proliferation and secreting extracellular matrix, which is characterized by comprising a tubular polymer fibrous stent added with active factors; the active factors comprise at least one of IGF-1, VEGF, PDGF and bFGF growth factors, and/or at least one of polypeptides corresponding to IGF-1, VEGF, PDGF and bFGF growth factors, and/or at least one of RGD short peptides and NO donor molecules; the tubular polymer fiber scaffold is made of degradable polymer materials. The invention has the beneficial effects that: the blood vessel bracket modified by activity and function can obviously improve the proliferation rate of cells and the rate of secreting extracellular matrix in the process of constructing the extracellular matrix artificial blood vessel by in vitro tissue engineering, and greatly shorten the in vitro cell culture period; in addition, the tissue regeneration activity of the blood vessel material is also obviously improved, and the requirement of clinical treatment is met.

Description

Vascular stent capable of promoting vascular cell proliferation and secreting extracellular matrix, preparation method of vascular stent and active artificial blood vessel

Technical Field

The invention belongs to the technical field of tissue engineering, and particularly relates to a vascular stent capable of promoting vascular cell proliferation and secreting extracellular matrix, a preparation method of the vascular stent and an active artificial blood vessel.

Background

Vascular disease is the most fatal disease worldwide, and the disease is frequently caused by reduced blood flow and nutrient deficiency due to stenosis or blockage of blood vessels, so that tissues or organs are damaged, and the diseases are usually manifested by coronary heart disease, cerebrovascular disease and peripheral artery disease. According to the world health organization, the number of deaths worldwide from cardiovascular related diseases per year will increase to 2330 ten thousand by 2030. Vascular graft surgery remains the conventional means for treating such diseases, and the use of autologous blood vessels (such as the great saphenous vein, the bilateral internal thoracic arteries, the radial artery, and the like) of a patient remains the gold standard for current vascular graft surgery. However, because the patient has complicated cardiovascular diseases or autologous blood vessels are collected, the length and the caliber of the collected autologous blood vessels are not matched, and the like, the healthy blood vessels meeting the transplantation requirements are difficult to find, and therefore, only artificial blood vessels can be selected for replacement.

Currently, polyethylene terephthalate

Expanded polytetrafluoroethylene (Gore-

) And large caliber (inner diameter) made of polyurethane and other materials>6mm) has higher long-term patency rate after artificial blood vessel transplantation, and is widely applied to clinic. But for small bore vascular prostheses (inner diameter)<6mm) and no ideal product is clinically available for coronary artery bypass of the heart, cerebrovascular replacement and peripheral blood vessel replacement below the knee. Therefore, the development of new biodegradable active small-caliber artificial blood vessels is increasingly valued by scientists all over the world.

People use Polycaprolactone (PCL), polycaprolactone-lactide (PLCL), degradable Polyurethane (PU), polysebacic acid glyceride (PGS), polylactic acid (PLA), polyglycolic acid ester (PGA), polylactic acid-glycolic acid copolymer (PLGA), Polydioxanone (PDS), polyethylene glycol (PEO), gelatin, collagen and other degradable materials to prepare the small-caliber artificial blood vessel in sequence. However, the small-caliber artificial blood vessel prepared by simply using the materials lacks bioactivity, the regeneration of the blood vessel is slow, and the long-term patency is not ideal.

It has been found that the main reason for the poor patency of small-bore artificial blood vessels is the lack of physiologically functional vascular endothelial coverage of the vessel lumen, and therefore researchers have long attempted to construct endothelialized tissue engineered vessels by implanting endothelial cells in vitro, but these efforts have been unsuccessful. The reason is that the conditions under which endothelialization is constructed in vitro and the environment in vivo are very different, and the endothelial monolayer formed lacks the natural tissue microenvironment, in particular lacks the support of smooth muscle. Therefore, the endothelial function of the endothelial tissue engineering blood vessel is weak, and endothelial shedding is easy to occur after the endothelial tissue engineering blood vessel is implanted into a blood circulation system, so that thrombosis and restenosis occur in the implanted blood vessel.

In recent years, researchers have changed the research strategy, and used traditional tissue engineering means to plant cells on a vascular stent, and culture the vascular stent in an in vitro flow bioreactor for about 8 weeks, so that the vascular stent is fully cellularized, the cells secrete a large amount of extracellular matrix, the stent generates sufficient mechanical strength and active extracellular matrix, and finally the cells are removed to obtain the active artificial blood vessel, and phase III clinical experiments have been completed in the United states.

However, these tissue engineering techniques all use simple polymer vascular stents, do not undergo any functional modification or drug addition, do not interfere with the proliferation and secretion of cells except for mechanical stimulation, and therefore require more than 8 weeks for the culture process in the in vitro bioreactor. The longer the process, the higher the cost and the greater the chance of bacterial contamination.

In addition, most studies have used adult vascular smooth muscle cells and fibroblasts, which have limited ability to subculture and secrete extracellular matrix, and in particular, over a period of weeks of in vitro expansion, the growth and viability of the cells are more significantly reduced.

Based on the above problems in the prior art, there is a need for an actively modified vascular stent, which is added with bioactive substances or immobilized functional molecules, and can stimulate and enhance the proliferation and extracellular matrix secretion of cells during in vitro culture of cells, thereby significantly shortening the in vitro culture time. Furthermore, certain blood vessel related growth factors or polypeptides are added into the vascular stent, so that the phenotype and the function of vascular cells are maintained in the process of culturing vascular smooth muscle and endothelial cells, the reconstruction of vascular tissues is promoted, and finally, after cell removal treatment, the obtained extracellular matrix active artificial blood vessel has a bionic tissue structure and active components.

Disclosure of Invention

The technical problem to be solved by the invention is to provide the vascular stent capable of promoting vascular cell proliferation and secreting extracellular matrix, the preparation method of the vascular stent and the active artificial blood vessel, so that the obtained product can meet the requirements of important vascular treatments such as vascular bypass and the like, and the treatment effects of myocardial infarction, head and neck vascular diseases and severe occlusion of lower limb arteries are greatly improved.

The invention discloses a vascular stent capable of promoting vascular cell proliferation and secreting extracellular matrix, which comprises a tubular polymer fibrous stent added with active factors; the active factors comprise at least one of IGF-1, VEGF, PDGF and bFGF growth factors, and/or at least one of polypeptides corresponding to IGF-1, VEGF, PDGF and bFGF growth factors, and/or at least one of RGD short peptides and NO donor molecules; the tubular polymer fiber scaffold is made of degradable polymer materials.

Further, the tubular polymer fiber scaffold is a structure with porous side walls.

Further, the degradable polymer material is prepared from one or a mixture of more of Polycaprolactone (PCL), poly (lactide-caprolactone) copolymer (PLCL), Polyurethane (PU), poly-sebacic acid glyceride (PGS), poly-dioxanone (PDS), polyglycolic acid (PGA), Polylactide (PLA), poly (lactide-glycolic acid) copolymer (PLGA), Polyhydroxyalkanoate (PHA), polyethylene glycol (PEO), gelatin and collagen in any proportion.

Preferably, the tubular polymer fiber scaffold has an inner diameter of 2-10mm, a wall thickness of 200-1000 μm and a length of 5-40 cm.

Further, the tubular polymer fiber scaffold is of a single-layer or double-layer structure.

Preferably, when the tubular polymer fiber scaffold adopts a double-layer structure, the inner layer is melt-spun fibers arranged circumferentially, the fiber diameter is 10-60 μm, and the bionic arrangement of vascular smooth muscle cells and regeneration of mesodermal tissues are facilitated; the outer layer is the electrostatic spinning fiber with disorder arrangement, and the fiber diameter is 1-10 μm. This double layer structure helps to maintain the tensile strength of the stent and prevent leakage.

The invention also discloses a preparation method of the vascular stent capable of promoting vascular cell proliferation and secreting extracellular matrix, which comprises the following steps:

preparing a tubular polymer scaffold, the preparation method of the tubular polymer fiber scaffold comprising: at least one of melt spinning, 3D printing, electrostatic spinning, weaving and casting;

adding an active factor in a manner that comprises: adding into polymer solution, adsorbing on the surface of the stent, self-assembling or grafting and fixing by chemical bonds.

The added active factors can improve the activity of the artificial blood vessel and is beneficial to realizing reconstruction and regeneration after blood vessel transplantation.

The invention also discloses an active artificial blood vessel, which is prepared by the following method:

firstly, inoculating smooth muscle cells, uniformly distributing the cells in fibrous interstices of the whole vascular stent as much as possible, and completely attaching and stretching the inoculated smooth muscle cells after culturing for 1-6 days;

then, endothelial cells are inoculated on the inner surface of the lumen, the mixture is placed in a flow culture bioreactor after standing culture for 2 to 3 days, the flow culture bioreactor is connected with a circulating pipeline, the flow rate, the shearing force and the pressure of the liquid are gradually increased to the condition equivalent to the physiological level, and the culture is continued for 1 to 8 weeks, thus obtaining the active artificial blood vessel.

Furthermore, the vascular endothelial cells and the smooth muscle cells are derived from human iPS, and the vascular endothelial cells and the smooth muscle cells are prepared by induced differentiation. The preparation of iPS and the directional induction and differentiation of iPS to vascular endothelial cells and smooth muscle cells follow the instructions of the commercial kit.

Further, the preparation method of the active artificial blood vessel also comprises the following steps: the mild decellularization treatment is carried out on the active artificial blood vessel after the endothelial cells are inoculated and cultured, so that the thorough removal of the cells is ensured, the active extracellular matrix is retained to the maximum extent, and the original structure of the internal polymer fiber is not damaged.

Further, the method of decellularization treatment comprises: SDS method or liquid nitrogen freeze-thaw method.

Preferably, the SDS method is performed as follows: soaking the tissue engineering blood vessel in 1% SDThe S solution was put on a shaker at room temperature for 12 hours with slow shaking, followed by washing with sterile physiological saline to remove SDS, and then it was put into a sterile mixed solution of DNase and RNase in a volume of 40ml of an enzyme solution buffered with 0.2mol/L MgCl₂，0.2mol/L CaCl₂And 0.1mol/L Tris-HCl with pH of 6.4 and ultrapure water; the concentration of DNase was 50U/ml and the concentration of RNase was 1U/ml, the mixture was shaken on a shaker at room temperature for 24 hours, followed by washing the residual DNase and RNase with sterile physiological saline, and finally the resulting material was placed in sterile PBS and stored at 4 ℃ for further use.

Furthermore, the vascular stent capable of promoting vascular cell proliferation and secreting extracellular matrix can be directly used as a material for autologous heart bypass surgery and head and neck vascular bypass surgery of patients; can also be prepared into active artificial blood vessels and then used as materials for operations such as autologous heart bypass, head and neck blood vessel bypass and the like of patients.

The invention has the beneficial effects that:

1. in the past, vascular cells were implanted on simple polymer vascular stents, such as PLGA, PGA, PU, PCL, etc., even natural macromolecules such as gelatin and collagen, which do not provide an optimum tissue microenvironment for vascular cells, and are not conducive to proliferation, secretion, tissue regeneration and remodeling of vascular cells. However, the vascular stent constructed by the invention is assembled with various growth factors and vasoactive substances, including IGF-1, PDGF, bFGF, RGD and the like, so that the compatibility of the material to vascular cells can be obviously improved, particularly, the cell proliferation and the secretion of extracellular matrix are obviously promoted, the culture time in an in vitro bioreactor can be greatly shortened, an extracellular matrix structure similar to a natural blood vessel can be obtained in a short time, the production cost is saved, and the contamination risk in the culture process is reduced. In addition, the added vasoactive substances also help to maintain the phenotype and the function of vascular cells, induce and generate a large amount of vasoregeneration promoting active substances, remain in extracellular matrix, and greatly improve the tissue regeneration activity of the artificial blood vessel.

2. The vascular cells are inoculated in the vascular stent by a tissue engineering means, the tissue engineering blood vessel constructed by the flow bioreactor has tissue structures of endothelial cells (intima) and smooth muscle cells (media), the extracellular matrix similar to a natural blood vessel is reserved after the cells are removed, and the rapid regeneration of the blood vessel is favorably realized after the tissue engineering blood vessel is implanted into a body.

3. Endothelial cells and smooth muscle cells used in the construction of tissue-engineered blood vessels in the past are derived from human umbilical veins and arteries, respectively, are adult cells, and have limited capacity for in vitro passage and growth. However, iPS has unlimited proliferation capacity and multi-directional differentiation potential, can induce differentiation into vascular endothelial cells and smooth muscle cells, has proliferation capacity and cell activity greatly superior to those of adult vascular cells, and secretes vasoactive substances in extracellular matrix higher than those of artificial blood vessels prepared by the adult cells. The tissue engineering blood vessel constructed by the human stem cells has better compatibility and safety. Human-derived stem cells present no risk of being animal-derived. After being implanted into a body, the active artificial blood vessel containing the extracellular matrix can rapidly attract the immigration, proliferation and differentiation of cells, and rapidly regenerate vascular endothelium and smooth muscle. Whereas the normal pure polymer material artificial blood vessels lack these activities.

4. The active artificial blood vessel obtained after removing the cells reserves the extracellular matrix of endothelial cells and smooth muscle cells, obviously improves the activity of the initial vascular stent, and after being transplanted into a body, the vascular regeneration and the vascular function are obviously superior to those of the artificial blood vessel made of a pure polymer material.

5. The vascular stent is divided into two layers of structures, the inner layer is the melt spinning fiber arranged in the circumferential direction, the aperture is larger, and the vascular stent is beneficial to the proliferation, the bionic arrangement and the regeneration of mesodermal tissue of vascular smooth muscle cells; the outer layer is the electrostatic spinning fiber which is arranged in disorder, the aperture is small, and the tensile strength of the intravascular stent is kept and the leakage is prevented.

Drawings

FIG. 1 shows a structural diagram of a Nap-FFG-IGF-1 molecule;

FIG. 2 shows a structure diagram of the Nap-FFGRGD molecule;

FIG. 3 shows a structural diagram of a Nap-FFGGG-NO molecule;

FIG. 4 is a graph showing the results of adhesion and proliferation tests of human umbilical vein endothelial cells on the surfaces of the materials of example 3, comparative group 3-1 and comparative group 3-2;

FIG. 5 is a graph showing the results of the spreading, adhesion and proliferation tests of human umbilical vein endothelial cells on the surface of the material of example 4 and comparative group 4-1 at different times;

FIG. 6 is a graph showing the results of NO release detection of the material of example 5;

FIG. 7 an infrared spectrum of the material of example 6 (PCL/CS-NO) with PCL;

FIG. 8 is a representation of VEGF-HGFI modified PCL electrospun blood vessels in example 8;

FIG. 9 is a graph of the effect of VEGF-HGFI modification on endothelial cell proliferation and function as tested in example 8.

Detailed Description

The following embodiments of the present invention will be further described with reference to the drawings and examples, which are only used to more clearly illustrate technical embodiments of the present invention and should not be taken as limiting the scope of the present invention.

The technical embodiment adopted by the invention is as follows:

example 1

An IGF-1 polypeptide self-assembly modified PCL vascular scaffold capable of promoting vascular cell proliferation and secreting extracellular matrix is prepared by the following steps:

1. processing a bracket:

(1) the inner layer of the bracket is prepared by a melt spinning method, and the method comprises the following specific operation steps: weighing 5.0g of PCL with molecular weight of 80000, placing in a closed stainless steel syringe wrapped by a hot melt device, and heating at 100 deg.C for 1 h; connecting a stainless steel pipe (receiving rod) with the diameter of 2mm with a rotating motor, wherein the distance between a syringe needle and the receiving rod is 5mm, the flow rate of a PCL melt is 0.5mL/h, the rotating speed of the receiving rod is 300r/min, the translation speed of a receiver is 10mm/s, the spinning time is 5min, the angle of the prepared fiber is 50 degrees, the diameter of the fiber is 60 mu m, and the wall thickness of the prepared inner layer fiber is 300 mu m;

(2) the outer layer of the bracket is prepared by adopting an electrostatic spinning method, and the specific operation steps are as follows: weighing 3g of PCL with the molecular weight of 80000, adding into 12mL of PCL with the volume ratio of 5:1, stirring and dissolving the mixture of chloroform and absolute methanol at room temperature overnight to prepare a PCL solution with the concentration fraction of 25 percent (m/v); placing the electrostatic spinning device in a fume hood, mounting the spinning bracket prepared above and a receiving rod on a rotating device, sucking the PCL solution into an injector, mounting the injector on an injection pump, enabling the distance between the needle head of the injector and the receiving rod to be 10cm, and applying 12kV voltage to a 21G metal needle head by using a high-voltage direct-current power supply. The advancing speed of the injection pump is set to be 8mL/h, the rotating speed of the receiving rod is set to be 100rpm, and the spinning time is set to be 4 min. After this operation, an electrospun fiber layer was formed outside the melt-spun scaffold, the fiber diameter being 5 μm and the thickness being 100. mu.m. The obtained bilayer PCL vascular stent: the inner diameter was 2mm and the total wall thickness was 400. mu.m.

2. IGF-1 polypeptide self-assembly modification:

the chemically modified IGF-1 polypeptide Nap-FFG-IGF-1 (molecular structure shown in figure 1) was dissolved in phosphate sodium chloride buffer (PBS) at 0.1% (w/v) at pH 7.4. And (3) soaking the prepared double-layer PCL vascular stent into a polypeptide solution, and standing overnight at room temperature.

Then obtaining the PCL vascular stent which is modified by IGF-1 polypeptide self-assembly and can promote vascular cell proliferation and secrete extracellular matrix.

Because PCL is a hydrophobic material, a longer incubation time is required to ensure that the polypeptide solution permeates into all scaffolds and a uniform self-assembly coating is formed on the surface of the fiber.

The identification and analysis of the polypeptide coating was verified using immunofluorescent staining, water contact angle, XPS, SEM or TEM.

Preparing an active artificial blood vessel:

the vascular stent is processed according to the following steps:

(1) preparing iPS cells:

taking human skin fibroblast to reprogram to prepare iPSCs, and mixing the plasmid carrying the reprogramming factor with 5 × 10⁵Human skin fibroblasts were mixed and the plasmid with the reprogramming factor was transferred into the cells by an electrotransfer system. The cells after electroporation were inoculated into a Matrigel-plated cell culture plate. The cells were cultured for about 30 days using a cell reprogramming medium (Beijing Saibei Biotech Co., Ltd.) to which a typical iPSCs clone appearedAfter cell clustering, selecting single clone and inoculating to obtain iPSCs.

(2) inducing differentiation of iPS into smooth muscle cells:

when the density of iPSCs reaches about 80%, cells are digested with dispase at 37 ℃ for 15 minutes to obtain Embryoid Bodies (EBs), and the EBs are planted in a six-well plate with low adhesion and containing mTeSR medium for culture. The following day the medium was changed to mTeSR medium and Embryoid Body (EBs) differentiation medium in a ratio of 1: 3. On the third day, the culture medium was replaced with EBs differentiation medium and cultured continuously for 3 days. On day 5, EBs were transferred to Gelatin (Gelatin) -coated six-well plates, and cultured again with EB differentiation medium for 5 days, with daily replacement with fresh EBs medium. After 5 days of culture, cells were digested with 0.25% trypsin, inoculated on a Matrigel-coated T75 flask, cultured with a smooth muscle cell growth medium (SmGM-2) for 7 days, and replaced with fresh medium every other day, to finally obtain vascular smooth muscle cells (iPSC-SMCs) induced by iPSCs.

(3) induction and differentiation of iPS into endothelial cells:

on day 0-1, inducing differentiation of human pluripotent stem cells with cell density of 95% or more using CDM3 differentiation medium supplemented with 4-8 μ M of GSK 3I; on day 2, the cells after the further induction culture were further induced to differentiate using CDM3 differentiation medium supplemented with 40-60ng/mL bFGF; on days 3-5, the cells after the above further induction culture were further induced to differentiate using CDM3 differentiation medium supplemented with 40-60ng/mL VEGF and 20-30ng/mLBMP 4; digesting the cells subjected to the induction culture in the last step on the 6 th day, and then continuously culturing the cells for 3-4 days by adopting an endothelial cell culture medium to obtain iPSCs (iPSC-ECs) induced and differentiated endothelial cells.

(4) Planting and culturing smooth muscle cells:

taking smooth muscle cell suspension, injecting into the lumen of the vascular stent from one end, standing for 5 minutes, placing the vascular stent on filter paper, uniformly rolling back and forth to suck dry the culture medium, repeating the operation for 6 times, and respectively performing 3 times at two ends of the blood vessel to ensure uniform planting. After the completion of the planting, the seeded blood vessels were placed in a six-well plate and allowed to stand for 10 minutes, followed by the addition of 10ml of medium. In that37℃，5％CO₂The culture box of (2) was left to stand for 24 hours, and rotated 90 ° every 4 hours to allow smooth muscle cells to adhere to the vascular stent. Use of

The TEB 500 bioreactor performs flow culture on artificial blood vessels. The culture environment is set to 37 ℃ and 5% CO₂And in the process of flow culture, the flow speed of the culture medium is controlled by a peristaltic pump, and the flow speed is slowly adjusted to a rated speed.

(5) Endothelial cell planting and culturing:

and (3) taking off the artificial blood vessel stent cultured in the step, soaking the inner cavity of the blood vessel with 100 mu g/mL fibronectin solution, taking endothelial cell suspension, injecting the endothelial cell suspension into the inner cavity of the blood vessel stent from one end, standing for 5 minutes, putting the blood vessel stent on filter paper, uniformly rolling back and forth to absorb redundant culture medium, repeating the operation for 6 times, and respectively carrying out 3 times at two ends of the blood vessel to ensure uniform planting. After the planting is finished, the blood vessel with the seeded cells is placed in a six-well plate and stands still for 10min, and then 10ml of culture medium is added. At 37 ℃ 5% CO₂The culture box is kept still for 24 hours and rotates 90 degrees every 4 hours so as to lead the endothelial cells to be adhered to the vascular stent. Then the artificial blood vessel is connected into a cell flow culture bioreactor (

TEB 500), the culture environment was set at 37 ℃ and 5% CO₂And in the process of flow culture, the flow speed of the culture medium is controlled by a peristaltic pump, and the flow speed is slowly adjusted to a rated speed.

So as to obtain the active artificial blood vessel.

In this embodiment, the active artificial blood vessel may further be processed by decellularization to obtain an active artificial blood vessel having a bionic extracellular matrix structure.

The method comprises the following steps:

the activated artificial blood vessel prepared above was immersed in a 1% SDS solution, placed on a shaker, slowly shaken at room temperature for 12 hours, and then washed with a sterile physiological saline to remove SDS, and then placed in a sterile DNase and DNaseThe RNase mixture (40 ml for the enzyme solution, 0.2mol/L MgCl for the buffer solution)₂，0.2mol/L CaCl₂And 0.1mol/L Tris-HCl with pH of 6.4 and ultrapure water; DNase concentration of 50U/ml and RNase concentration of 1U/ml), shaking on a shaker at room temperature for 24 hours, followed by rinsing the residual DNase and RNase with sterile physiological saline, and finally placing the resulting material in sterile PBS and storing at 4 ℃ for later use.

The obtained active artificial blood vessel has good angiogenesis activity and can meet the requirement of clinical treatment.

Example 2

A PCL/collagen composite vascular stent capable of promoting vascular cell proliferation and secreting extracellular matrix for slowly releasing IGF-1 polypeptide is prepared by the following steps:

firstly, preparing the PCL/collagen composite vascular stent by adopting an electrostatic spinning technology. PCL was dissolved in a mixed solvent of chloroform and methanol (volume ratio 5: 1) at a concentration of 10% (w/v). Type I collagen was dissolved in hexafluoroisopropanol at a concentration of 8% (w/v). IGF-1 polypeptide was dissolved in deionized water at a concentration of 2 mg/mL. Adding the IGF-1 polypeptide solution into the collagen solution in a volume ratio of 4: 1, mixing uniformly.

The PCL solution and collagen/IGF-1 solution were loaded into two 10-mL syringes, respectively, with a 21-G needle. The two syringes are respectively arranged on the two injection pumps, and the rotating receiving rod is a stainless steel pipe with the diameter of 2-6 mm. The distance between the two syringe needles and the receiving rod is 15 cm.

The voltage of the PCL solution connection was 15kV and the syringe pump advancing speed was 2 mL/h. The voltage of the collagen solution connection was 12kV and the syringe pump advancing speed was 0.6 mL/h.

Thus obtaining the PCL/collagen composite vascular stent which slowly releases IGF-1 polypeptide and can promote vascular cell proliferation and secrete extracellular matrix.

The vascular scaffold thus prepared carries IGF-1 polypeptide, and as collagen is decomposed during cell culture and in vivo implantation, IGF-1 is gradually released to promote cell proliferation and angiogenesis.

Preparing an active artificial blood vessel:

the same procedure as in example 1 was followed.

Example 3

A PCL/gelatin composite vascular stent modified by VEGF growth factor and capable of promoting vascular cell proliferation and secreting extracellular matrix is prepared by the following steps:

firstly, preparing the PCL/gelatin composite vascular stent by adopting an electrostatic spinning technology. PCL was dissolved in a mixed solvent of chloroform and methanol (volume ratio 5: 1) at a concentration of 25% (w/v). Then, a gelatin solution was prepared, and gelatin was dissolved in hexafluoroisopropanol at a concentration of 6% (w/v). The two solutions are respectively added into two 10mL syringes, a 21G needle is used for installing the two syringes on two injection pumps, and the high-voltage power supplies for connecting the two syringes of PCL and gelatin are respectively PCL11kV and gelatin 17 kV. The distances between the two syringe needles and the receiving rod are respectively 25cm for PCL and 15cm for gelatin. The two materials are spun simultaneously to prepare the PCL/gelatin composite vascular stent. The solvent in the scaffold material was thoroughly evaporated and thoroughly dried in a vacuum desiccator.

Heparin and VEGF were first fixed according to the following steps: adding connecting reagents EDC and heparin into 50% ethanol water solution to make the concentrations of the connecting reagents EDC and the heparin be 30mM and 0.5mg/mL respectively, then soaking the vascular stent into the solution, placing the vascular stent in a zero-degree ice water bath, and reacting for 12 h. Then washed three times with distilled water. The mixture was allowed to stand at room temperature until dry. A VEGF growth factor solution in PBS was prepared at a concentration of 1. mu.g/mL, and the vascular stent was immersed in the VEGF solution, incubated overnight at 4 ℃ and finally washed with PBS.

Thus obtaining the PCL/gelatin composite vascular stent which is modified by the VEGF growth factor and can promote vascular cell proliferation and secrete extracellular matrix.

Preparing an active artificial blood vessel:

the same procedure as in example 1 was followed.

To further illustrate the beneficial effects of this example, the following experiment was specifically set up:

taking an artificial blood vessel stent made of PCL material with the same size as the blood vessel stent described in the embodiment 3 as a comparison group 3-1;

the artificial blood vessel stent made of heparin-modified PCL (HP-PCL) with the same size as the blood vessel stent described in the embodiment 3 is taken as a comparison group 3-2;

the adhesion and proliferation of human umbilical vein endothelial cells on the surfaces of PCL scaffolds (VEGF-HP-PCL) co-modified by VEGF growth factor and heparin described in comparative group 3-1, comparative group 3-2 and example 3 were tested.

The experimental results are shown in the attached figure 4:

a) laser confocal images of PI-labeled endothelial cells adhered to the control group 3-1 for 1 h;

b) laser confocal images of PI-labeled endothelial cells adhered to the control group 3-2 for 1 h;

c) confocal laser images of PI-labeled endothelial cells 1h after adhesion on example 3;

d) statistics of the average counts per field of view of adherent cells on the three materials;

e) proliferation of endothelial cells on different materials. P <0.01(vs PCL); each set of n-3, with a scale of 250 μm.

As can be seen from FIG. 4, example 3 significantly improved the adhesion and proliferation of Human Umbilical Vein Endothelial Cells (HUVEC) on the surface of the material, compared to comparative groups 3-1 and 3-2. The VEGF modified on the surface of the material can be combined with VEGF receptors on endothelial cells to induce the rapid adhesion of the endothelial cells, and in addition, the slow release of the VEGF from the material also contributes to the proliferation of the endothelial cells.

Example 4

A PCL vascular stent which is modified by RGD short peptide self-assembly and can promote vascular cell proliferation and secrete extracellular matrix is prepared by the following steps:

chemically modifying the RGD short peptide to obtain a molecule Nap-FFGRGD (the molecular structure is shown in figure 2) capable of self-assembling into a glue. The Nap-FFGRGD can be self-assembled into gel, and can also be self-assembled on the surface of the material to form a coating, so that RGD is fixed on the surface of the material, the affinity of the surface of the material to cells is improved, and the proliferation of the cells is promoted.

Nap-FFGRGD was dissolved in PBS at a concentration of 0.2mg/mL, pH 7.4. The procedure of stent fabrication was as in example 1, the bilayer PCL stent was immersed in Nap-FFGRGD solution, left overnight at 37 ℃, and then washed 3 times with PBS buffer. The Nap-FFGRGD forms a uniform self-assembled coating on the fiber surface.

Thus obtaining the PCL vascular stent which is self-assembled and modified by the RGD short peptide and can promote vascular cell proliferation and secrete extracellular matrix.

Preparing an active artificial blood vessel:

the same procedure as in example 1 was followed.

an artificial blood vessel stent made of PCL material and having the same size as the blood vessel stent described in the embodiment 4 is taken as a comparison group 4-1;

cytoskeleton of the control group 4-1 and the cytoskeleton of example 4 were stained with phalloidin, and the spreading of Human Umbilical Vein Endothelial Cells (HUVEC) on the surface of the scaffold was observed.

The experimental results are shown in figure 5:

a) the spread state of endothelial cells after 2 hours of adhesion on the control group 4-1;

b) endothelial cells were attached to example 4 in a spread state after 2 h;

c) the spread state of endothelial cells after 4 hours of adhesion on the control group 4-1;

d) endothelial cells were spread 4h after adhesion on example 4;

e) adhesion of endothelial cells to different materials;

e) proliferation of endothelial cells on different materials.

As shown in the attached FIG. 5, after 2h of culture, compared with HUVEC, the PCL surface is mostly spherical and not spread, and on the material surface modified by Nap-FFGRGD, the cells are flaky and have larger spread area; after 4h of culture, most cells on the surface of PCL-RGD present the morphology of mature endothelial cells, namely an elongated spindle shape, and the spreading degree of PCL group cells is far lower than that of RGD modified group. In addition, the Nap-FFGRGD modification also effectively improves the proliferation of HUVEC on the surface of the stent.

The results show that RGD modification provides a more ideal microenvironment for HUVEC growth, and effectively promotes the adhesion and growth of HUVEC on the surface of the PCL scaffold.

Example 5

A PCL vascular stent which is modified by self-assembly of NO donor molecules and can promote vascular cells to proliferate and secrete extracellular matrix is prepared by the following steps:

the NO donor molecule Nap-FFGGG-NO (the molecular structure is shown in figure 3) capable of self-assembling into gel is prepared by polypeptide synthesis reaction and reversed phase high performance liquid chromatography purification. Nap-FFGGG-NO was dissolved in PBS (pH 7.4) to prepare a 0.1 wt% solution. The PCL stent is processed in the same manner as in example 1. Immersing the PCL intravascular stent into Nap-FFGGG-NO solution, and standing for 2h at room temperature.

Nap-FFGGG-NO forms a submicron hydrogel coating on the surface of the PCL fiber, and the wet condition can be stable for one month. The Nap-FFGGG-NO coating modified blood vessel stent is usually stored in a dry state after being frozen and dried.

Thus obtaining the PCL vascular stent which is modified by the self-assembly of NO donor molecules and can promote the proliferation of vascular cells and secrete extracellular matrix.

The Nap-FFGGG-NO surface modification not only changes the hydrophobic PCL fiber surface into a hydrophilic surface, improves the affinity of cells, but also controls the released NO to promote the regeneration of blood vessels.

To demonstrate that the NO donor molecule Nap-FFGGG-NO is formed and can release NO during the reaction of this example, the following experiment was designed:

the blood scaffold material described in example 5, after catalyzing NO release, was divided into two groups:

a. DAF-FM was not added with NO probe; b. the NO probe DAF-FM was added.

And respectively collecting a macroscopic optical photo of the surface of the material and a corresponding fluorescence image thereof.

As a result, as shown in FIG. 6, the NO probe DAF-FM forms a Benzotriazole structure (Benzotriazole) capable of generating green fluorescence upon binding to NO. Under a fluorescence microscope, it can be observed that the PCL material surface after catalyzing NO release has significant green fluorescence (fig. 6 b).

Preparing an active artificial blood vessel:

the same procedure as in example 1 was followed.

Example 6

A PCL vascular stent loaded with NO donor molecules and capable of promoting vascular cell proliferation and secreting extracellular matrix is prepared by the following steps:

the PCL stent is processed in the same manner as in example 1. In order to improve the hydrophilicity of the PCL electrospun scaffold, it is first subjected to a hydrolysis treatment. And (3) rinsing the PCL electrospun scaffold in a 50% ethanol solution, and simultaneously removing the aluminum foil attached to the electrospun scaffold. Then, the cleaned PCL electrospun scaffold is immersed in NaOH aqueous solution with the pH value of 12 and is subjected to hydrolysis treatment at 37 ℃ for 48 h. And then washing the PCL electrospinning bracket with deionized water for 3 times, wherein each time is 1h, so as to remove residual NaOH. And (3) placing the cleaned PCL electrospun scaffold in a vacuum drying oven, and drying for 2 days at room temperature. Cutting the PCL electrospun scaffold subjected to hydrolysis treatment into a circle with the diameter of 1 cm. And then, uniformly coating 50 mu L of CS-NO aqueous solution with the concentration of 20mg/mL on the surface of the electrospinning bracket by using a dripping coating method. After drying for two days at room temperature, a CS-NO coating is formed on the surface of the electrospinning bracket.

Thus obtaining the PCL vascular stent which is loaded with NO donor molecules and can promote vascular cell proliferation and secrete extracellular matrix.

In order to prove that the CS-NO coating is formed during the reaction process of this example, the intravascular stent material and PCL material described in this example are subjected to infrared detection, and the results are shown in fig. 7:

the infrared spectrograms of PCL and PCL/CS-NO show that the area of the PCL bracket surface loaded with CS-NO is 1363cm-¹Two characteristic absorption peaks belonging to the nitrogen enol group appear: 1363cm^-1The asymmetric stretching characteristic peak and 945cm of the O-N-O group at^-1The characteristic peak of planar symmetry stretching of N-N at (A) indicates that CS-NO is successfully loaded on PCL fibers.

Preparing an active artificial blood vessel:

the same procedure as in example 1 was followed.

Example 7

A PCL vascular scaffold which is modified by IGF-1 polypeptide and NO donor molecule gradient and can promote vascular cell proliferation and secrete extracellular matrix is prepared by the following steps:

(1) the method comprises the following specific operation steps of loading NO donor molecules on PCL fibers of an inner layer: PCL was dissolved in a mixed solvent of chloroform and anhydrous methanol at a volume ratio of 5:1 at a concentration of 15% (m/v). To this was added galactose group protected NO donor molecule at a concentration of 0.5% (m/v). The PCL solution was added to a 10mL syringe with a needle of 21G and mounted on a syringe pump, the receiving rod was a stainless steel tube with a diameter of 2mm, the distance between the syringe needle and the receiving rod was 15cm, the advancing speed of the PCL solution was 0.5mL/h, the voltage was 15kV, the inner layer spinning time was 4min, the inner layer fiber diameter was about 2 μm, and the inner layer thickness was about 50 μm.

(2) The outer layer PCL fiber loads IGF-1 polypeptide, and the specific operation steps are as follows: PCL was dissolved in a mixed solvent of chloroform and anhydrous methanol at a volume ratio of 5:1, at a concentration of 25% (m/v). IGF-1 polypeptide was added thereto at a concentration of 30. mu.g/ml; the PCL solution was added to a 10mL syringe with a 21G needle, mounted on a syringe pump, and spinning was continued on the basis of the former inner layer fiber. The distance between the syringe needle and the receiving rod is 10cm, the propelling speed of the PCL solution is 8mL/h, the voltage is 12kV, and the spinning time is 25 min. The diameter of the outer layer fiber is 6-8 μm, and the thickness is 400 μm.

Then PCL vascular stent which is modified by IGF-1 polypeptide and NO donor molecule in gradient and can promote vascular cell proliferation and secrete extracellular matrix can be obtained.

The obtained vascular stent is double-layer, has thinner inner diameter fiber, loads NO donor molecules, and is favorable for anticoagulation and promotion of endothelial formation. The outer layer fiber is thicker and the pore diameter is larger, thus being beneficial to cell immigration, and meanwhile, the IGF-1 is slowly released, thus promoting cell proliferation and secretion and being beneficial to the rapid regeneration of vascular smooth muscle.

Preparing an active artificial blood vessel:

the same procedure as in example 1 was followed.

Example 8

A PCL vascular scaffold which can promote vascular cell proliferation and secrete extracellular matrix and is self-assembled and modified by VEGF fusion protein is prepared by the following steps:

the fusion protein VEGF-HGFI is prepared by a molecular cloning technology, wherein VEGF is an active protein, and HGFI is an amphiphilic protein with self-assembly capability. VEGF-HGFI aqueous solution is prepared, and the concentration is 200 mug/mL. The PCL stent is processed in the same manner as in example 1. The vascular stent was immersed in VEGF-HGFI solution, incubated at 4 ℃ for 12h, and washed with PBS.

Thus obtaining the PCL vascular stent which is self-assembled and modified by the VEGF fusion protein and can promote vascular cell proliferation and secrete extracellular matrix.

Preparing an active artificial blood vessel:

the same procedure as in example 1 was followed.

the influence of the VEGF-HGFI fusion protein modification on the appearance of the vascular stent is tested:

an artificial blood vessel stent made of PCL material and having the same size as the blood vessel stent described in the embodiment 8 is taken as a comparison group 8-1;

the cytoskeleton morphology of comparative group 8-1(PCL) and example 8(VEGF-HGFI-PCL) was observed.

The experimental results are shown in figure 8:

A) cross section of the vascular stent of the comparison group 8-1 and the example 8 (upper graph), lumen surface of the vascular stent of the comparison group 8-1 and the example 8 (lower graph);

B) SEM image of comparative group 8-1 (left image), SEM image of example 8 (right image);

C) the fluorescence pattern of the control group 8-1 blood vessel stent VEGF antibody staining (green) (left panel), and the fluorescence pattern of the example 8 blood vessel stent VEGF antibody staining (green) (right panel).

As shown in the attached figure 8, the loading of the amphipathic protein HGFI and the VEGF-HGFI fusion protein has no significant influence on the morphology of the fiber of the vascular stent, and VEGF effective loading can be observed on the surface of the fiber of the vascular stent through VEGF antibody staining and is uniformly distributed in the whole stent.

The influence of the VEGF-HGFI fusion protein modification on the proliferation and the function of endothelial cells is tested:

a blank Glass sheet was taken as a comparative group 8-2 (Glass); comparison was made with a Glass slide (VEGF-HGFI-Glass) coated with the VEGF-HGFI fusion protein described in example 8.

Fluorescence detection of NO release from endothelial cells was performed with DAF-FM probe for comparison 8-2(Glass) and example 8(VEGF-HGFI-Glass), respectively, and the results are shown in FIG. 9A.

Fluorescence detection of the uptake of DiI-ac-LDL by endothelial cells was performed for comparative group 8-2(Glass) and example 8(VEGF-HGFI-Glass), respectively, and the results of the experiment are shown in FIG. 9B.

As shown in figure 9, the modification of VEGF-HGFI fusion protein can effectively promote the adhesion and proliferation of endothelial cells, and significantly improve the NO release capacity (figure 9A) and the uptake capacity (figure 9B) of the endothelial cells to acetylated low-density lipoprotein, thereby enhancing the function of the endothelial cells growing on the modified surface.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A vascular stent capable of promoting vascular cell proliferation and extracellular matrix secretion, which is characterized by comprising a tubular polymer fiber stent added with active factors; the active factors comprise at least one of IGF-1, VEGF, PDGF and bFGF growth factors, and/or at least one of polypeptides corresponding to IGF-1, VEGF, PDGF and bFGF growth factors, and/or at least one of RGD short peptides and NO donor molecules; the tubular polymer fiber scaffold is made of degradable polymer materials.

2. The vascular stent capable of promoting vascular cell proliferation and extracellular matrix secretion according to claim 1, wherein the tubular polymer fiber stent is a structure with porous side walls.

3. The vascular stent capable of promoting proliferation and secretion of extracellular matrix of vascular cells according to claim 1, wherein the degradable polymer material is selected from Polycaprolactone (PCL), poly (lactide-caprolactone) copolymer (PLCL), Polyurethane (PU), poly-glycerol sebacate (PGS), poly-p-dioxanone (PDS), polyglycolic acid (PGA), Polylactide (PLA), poly (lactide-glycolic acid) copolymer (PLGA), Polyhydroxyalkanoate (PHA), polyethylene glycol (PEO), gelatin, collagen, or a mixture thereof in any ratio.

4. The vascular stent capable of promoting vascular cell proliferation and extracellular matrix secretion according to claim 1, wherein the tubular polymer fiber stent has a single-layer or double-layer structure.

5. The vascular stent capable of promoting vascular cell proliferation and extracellular matrix secretion according to claim 4, wherein when the tubular polymer fiber stent adopts a double-layer structure, the inner layer is circumferentially arranged melt-spun fibers, the fiber diameter is 10-60 μm, and the bionic arrangement of vascular smooth muscle cells and regeneration of mesodermal tissues are facilitated; the outer layer is the electrostatic spinning fiber with disorder arrangement, and the fiber diameter is 1-10 μm.

6. A method for preparing a vascular stent capable of promoting vascular cell proliferation and secreting extracellular matrix, which is characterized by comprising the following steps:

7. An active artificial blood vessel, which is characterized by being prepared by the following method:

firstly, inoculating smooth muscle cells, enabling the cells to be uniformly distributed in fiber gaps of the blood vessel support of any one of claims 1-5 as much as possible, and after culturing for 1-6 days, enabling the inoculated smooth muscle cells to be completely attached and stretched;

8. The active artificial blood vessel of claim 7, wherein the source of the vascular endothelial cells and the smooth muscle cells is human iPS, and the vascular endothelial cells and the smooth muscle cells are prepared by induced differentiation.

9. The active vascular prosthesis of claim 7, wherein the method of preparation further comprises the steps of: the mild decellularization treatment is carried out on the active artificial blood vessel after the endothelial cells are inoculated and cultured, so that the thorough removal of the cells is ensured, the active extracellular matrix is retained to the maximum extent, and the original structure of the internal polymer fiber is not damaged.

10. The active vascular prosthesis of claim 9, wherein the decellularization process comprises: SDS method or liquid nitrogen freeze-thaw method; the SDS method comprises the following steps: soaking tissue engineering blood vessel in 1% SDS solution, shaking slowly at room temperature for 12 hr, washing with sterile physiological saline to remove SDS, and placing in sterile mixed solution of DNase and RNase, wherein the enzyme solution is 40ml, and the buffer solution is composed of 0.2mol/L MgCl₂，0.2mol/L CaCl₂And 0.1mol/L Tris-HCl with pH of 6.4 and ultrapure water; the concentration of DNase was 50U/ml and the concentration of RNase was 1U/ml, the mixture was shaken on a shaker at room temperature for 24 hours, followed by washing the residual DNase and RNase with sterile physiological saline, and finally the resulting material was placed in sterile PBS and stored at 4 ℃ for further use.