CN111714706B

CN111714706B - 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: CN111714706B
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: 2023-04-28
Anticipated expiration: 2040-05-08
Also published as: CN111714706A

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 fiber stent added with active factors; the active factors comprise at least one of IGF-1 and VEGF, PDGF, bFGF growth factors, and/or at least one of polypeptides corresponding to IGF-1 and VEGF, PDGF, bFGF growth factors, and/or at least one of RGD short peptide and NO donor molecule; the tubular polymer fiber scaffold is made of degradable polymer materials. The invention has the beneficial effects that: the vascular stent with modified activity and function can obviously improve the proliferation rate of cells and the secretion rate of extracellular matrix in the process of constructing an extracellular matrix artificial blood vessel by in vitro tissue engineering, and greatly shortens the time between in vitro cell culture; in addition, the tissue regeneration activity of the vascular material is also obviously improved, and the requirements of clinical treatment are 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 in particular 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 diseases are diseases with highest global mortality, which occur frequently due to reduced blood flow and nutrient deficiency caused by stenosis or blockage of blood vessels, and thus damage of tissues or organs, which are often manifested as coronary heart disease, cerebrovascular disease, peripheral arterial disease. The world health organization predicts that the number of people worldwide who die from cardiovascular-related diseases by 2030 will increase to 2330 ten thousand each year. Vascular grafting surgery is still a common means of treating such diseases, and the use of patient autologous blood vessels (e.g., great saphenous vein, bilateral internal thoracic arteries, radial arteries, etc.) remains the gold standard for current vascular grafting procedures. However, because the patient suffers from complicated cardiovascular diseases or autologous blood vessels are collected, the length and caliber of the autologous blood vessels which can be collected are not matched, and the like, it is difficult to find healthy blood vessels which meet the transplanting requirements, and therefore, only artificial blood vessels can be selected to replace.

At present, polyethylene terephthalate

Expanded polytetrafluoroethylene (Gore->

) And polyurethane and the like>6 mm) has higher long-term patency rate after artificial blood vessel transplantation, and has been widely applied to clinic. But for small-caliber artificial blood vessels (inner diameter<6 mm), no ideal product is clinically available for heart coronary bypass, cerebral vessel replacement and peripheral vessel replacement below the knee. Thus, openThe new biodegradable active small-caliber artificial blood vessel is increasingly valued by scientists worldwide.

The small-caliber artificial blood vessel is prepared by using degradable materials such as Polycaprolactone (PCL), polycaprolactone-lactide (PLCL), degradable Polyurethane (PU), polysebacic Glyceride (PGS), polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid-glycolic acid copolymer (PLGA), polydioxanone (PDS), polyethylene glycol (PEO), gelatin, collagen and the like in sequence. However, the small-caliber artificial blood vessel prepared by using the materials only lacks biological activity, has slow regeneration of the blood vessel and is not ideal in long-term smoothness.

It has been found that the main reason for the low patency of small-caliber vascular prostheses is the lack of vascular endothelial coverage of the lumen of the vessel with physiological functions, and thus researchers have long tried to construct endothelialized tissue engineering vessels by in vitro engrafting of endothelial cells, however these efforts have not been successful. The reason is that the conditions for in vitro construction of endothelialization are very different from the in vivo environment, and that the endothelial monolayer formed lacks the natural tissue microenvironment, particularly the support of smooth muscle. Therefore, the endotheliosis tissue engineering blood vessel has weak endothelial function, and after being implanted into a blood circulation system, the endothelial is easy to fall off, so that the implanted blood vessel is subjected to thrombosis and restenosis.

Researchers have changed the research strategy in recent years, adopt the traditional tissue engineering means, plant the cell on the vascular stent, culture in the flow bioreactor of the vitro for about 8 weeks, make the vascular stent fully cellular, the cell secretes a large amount of extracellular matrix, make the stent produce sufficient mechanical strength and extracellular matrix with activity, remove the cell finally, get the active artificial blood vessel, have already finished the clinical experiment of stage III in the United states.

However, these tissue engineering techniques all use simple polymer vascular scaffolds, do not perform any functional modification or add drugs, do not perform any intervention on proliferation and secretion of cells except mechanical stimulation, and thus, the culture process in an in vitro bioreactor requires more than 8 weeks. The longer the process, the higher the cost and the greater the probability of bacterial contamination.

In addition, most studies employ adult vascular smooth muscle cells and fibroblasts, which have limited ability to grow passaged and secrete extracellular matrix, particularly with in vitro expansion over several weeks, with more significant reduction in cell growth and viability.

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

Disclosure of Invention

The invention aims to solve the technical problem of providing a vascular stent capable of promoting vascular cell proliferation and secretion of extracellular matrix, a preparation method of the vascular stent and an active artificial blood vessel, so that the obtained product can meet the requirements of important vascular treatment such as vascular bypass and the like, and the treatment effects of myocardial infarction, head and neck vascular lesions and serious 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 fiber stent added with active factors; the active factors comprise at least one of IGF-1 and VEGF, PDGF, bFGF growth factors, and/or at least one of polypeptides corresponding to IGF-1 and VEGF, PDGF, bFGF growth factors, and/or at least one of RGD short peptide and NO donor molecule; the tubular polymer fiber scaffold is made of degradable polymer materials.

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

Further, the degradable polymer material adopts one or a mixture of several of Polycaprolactone (PCL), poly (lactide-caprolactone) copolymer (PLCL), polyurethane (PU), polysebacic Glyceride (PGS), polydioxanone (PDS), polyglycolic acid (PGA), polylactide (PLA), poly (lactide-glycollic 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 5-40cm.

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 spinning fiber which is arranged circumferentially, and the fiber diameter is 10-60 mu m, thereby being beneficial to bionic arrangement of vascular smooth muscle cells and regeneration of medium membrane tissues; the outer layer is of unordered electrostatic spinning fiber with the diameter of 1-10 mu m. This bilayer structure helps to maintain the tensile strength of the stent and prevents 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, electrospinning, braiding, and casting;

adding active factors, wherein the adding mode of the active factors comprises the following steps: adding in polymer solution, adsorbing on the surface of the bracket, self-assembling or grafting and fixing by chemical bond.

The added active factors can improve the activity of the artificial blood vessel, and are beneficial to realizing reconstruction and regeneration after the 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 fiber gaps of the whole vascular stent as much as possible, and fully attaching and stretching the inoculated smooth muscle cells after culturing for 1-6 days;

and inoculating endothelial cells on the inner surface of the lumen, standing for 2-3 days, placing in a flow culture bioreactor, connecting with a circulation pipeline, gradually increasing the flow rate, shearing force and pressure of the liquid to the condition equivalent to the physiological level, and continuously culturing for 1-8 weeks to obtain the active artificial blood vessel.

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

Further, the preparation method of the active artificial blood vessel further comprises the following steps: the activated artificial blood vessel after grafting endothelial cells and culturing is subjected to mild decellularization treatment, so that thorough removal of cells is ensured, meanwhile, the activated extracellular matrix is reserved to the greatest extent, and the original structure of the internal polymer fiber is not damaged.

Further, the method of decellularizing treatment comprises: SDS method or liquid nitrogen freeze thawing method.

Preferably, the SDS method is operated as follows: soaking tissue engineering blood vessel in 1% SDS solution, placing on shaking table, slowly shaking 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 system is 40ml, and the buffer solution is composed of 0.2mol/L MgCl ₂ ，0.2mol/L CaCl ₂ And 0.1mol/L Tris-HCl with pH of 6.4 and ultrapure water; DNase concentration is 50U/ml, RNase concentration is 1U/ml, shake on shaker at room temperature for 24h, then rinse residual DNase and RNase with sterile physiological saline, and finally place the obtained material in sterile PBS, and store at 4deg.C for 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 and head and neck vascular bypass operation of patients; can also be used for preparing materials for the operations such as autologous heart bypass, head and neck vascular bypass and the like of patients after the active artificial blood vessel is prepared.

The invention has the beneficial effects that:

1. in the past, vascular cells are planted on a simple polymer vascular stent, and even if the polymer stent comprises PLGA, PGA, PU, PCL and the like, natural polymers such as gelatin and collagen can not provide an optimal tissue microenvironment for the vascular cells, so that the proliferation, secretion, tissue regeneration and reconstruction of the vascular cells are not facilitated. However, the vascular stent constructed by the invention is assembled with various growth factors and vascular active substances, including IGF-1, PDGF, bFGF, RGD and the like, so that the compatibility of materials to vascular cells can be obviously improved, especially the proliferation of cells and the secretion of extracellular matrixes are obviously promoted, the culture time in an in-vitro bioreactor can be greatly shortened, the extracellular matrix structure similar to a natural blood vessel can be obtained in a shorter time, the production cost is saved, and the risk of bacterial contamination in the culture process is reduced. In addition, the added vascular active substances are also helpful for maintaining the phenotype and the function of vascular cells, inducing the generation of a large amount of active substances for promoting the regeneration of blood vessels, remaining in extracellular matrixes and greatly improving the tissue regeneration activity of artificial blood vessels.

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

3. Endothelial cells and smooth muscle cells used in the past for constructing tissue engineering blood vessels are derived from human umbilical veins and arteries respectively, are adult cells, and have limited capabilities for in vitro passage and growth. However, iPS has unlimited proliferation capacity and multidirectional differentiation potential, can be induced to differentiate into vascular endothelial cells and smooth muscle cells, and has proliferation capacity and cell viability superior to those of adult vascular cells, and the vascular active substances in the secreted extracellular matrix are also superior to 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 stem cells are not at risk of animal origin. After implantation in vivo, active vascular prostheses containing extracellular matrix can rapidly attract cellular migration, proliferation and differentiation, and rapidly regenerate vascular endothelium and smooth muscle. Whereas normal pure polymer material vascular prostheses lack these activities.

4. The active artificial blood vessel obtained after cell removal retains the extracellular matrix of endothelial cells and smooth muscle cells, the activity of the initial vascular stent is obviously improved, and after the vascular regeneration and vascular function are obviously superior to those of the artificial blood vessel made of pure polymer materials.

5. The vascular stent is divided into two layers, wherein the inner layer is melt spinning fiber which is arranged circumferentially, the aperture is large, and proliferation, bionic arrangement and medium membrane tissue regeneration of vascular smooth muscle cells are facilitated; the outer layer is of the electro-spinning fiber which is arranged in a disordered way, the aperture is smaller, and the stretching strength of the vascular stent is maintained and leakage is prevented.

Drawings

FIG. 1 is a diagram showing the molecular structure of Nap-FFG-IGF-1;

FIG. 2 is a diagram of the Nap-FFGRGD molecular structure;

FIG. 3 is a diagram showing the molecular structure of Nap-FFGGG-NO;

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, comparative group 4-1 at various times;

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

FIG. 7 IR spectra of example 6 material (PCL/CS-NO) and PCL;

FIG. 8 example 8VEGF-HGFI modified PCL electrospun angiogram;

FIG. 9 example 8 graphs of the effect of VEGF-HGFI modification on endothelial cell proliferation and function.

Detailed Description

The following description of the specific embodiments of the present invention will be further described with reference to the accompanying drawings and examples, which are only used to more clearly illustrate the technical examples of the present invention, and are not to be construed 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-assembled modified PCL vascular stent capable of promoting vascular cell proliferation and secretion of extracellular matrix, the preparation method comprises the following steps:

1. and (3) processing a bracket:

(1) The preparation method of the inner layer of the bracket by adopting the melt spinning method comprises the following specific operation steps: weighing 5.0g of PCL with molecular weight of 80000, placing in a sealed stainless steel syringe wrapped by a hot-melting device, and heating at 100deg.C for 1 hr; connecting a stainless steel tube (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 PCL melt is 0.5mL/h, the rotating speed of the receiving rod is 300r/min, the translation speed of the receiver is 10mm/s, the spinning time is 5min, and the prepared inner layer fiber with the fiber angle of 50 DEG, the fiber diameter of 60 mu m and the wall thickness of 300 mu m is prepared;

(2) The preparation method of the outer layer of the bracket by adopting the electrostatic spinning method comprises the following specific operation steps: 3g of PCL with molecular weight of 80000 were weighed and added to 12mL of a volume ratio of 5:1, stirring and dissolving the mixture of chloroform and absolute methanol at room temperature for overnight to obtain a PCL solution with the concentration fraction of 25% (m/v); placing an electrostatic spinning device in a fume hood, installing the spinning support prepared above on a rotating device together with a receiving rod, sucking PCL solution into a syringe, installing the syringe on a syringe pump, keeping the distance between a syringe needle and the receiving rod 10cm, and applying 12kV voltage on a 21G metal needle by using a high-voltage direct-current power supply. The syringe pump was set to 8mL/h, the receiving rod was rotated at 100rpm, and the spinning time was 4 minutes. After such operation, an electrospun fiber layer was formed outside the melt-spun stent, with a fiber diameter of 5 μm and a thickness of 100. Mu.m. The obtained double-layer PCL vascular stent comprises: the inner diameter was 2mm and the total wall thickness was 400. Mu.m.

2. Self-assembly modification of IGF-1 polypeptide:

chemically modified IGF-1 polypeptide Nap-FFG-IGF-1 (molecular structure shown in figure 1) was dissolved in Phosphate Buffered Saline (PBS) at a concentration of 0.1% (w/v), ph=7.4. The prepared double-layer PCL vascular stent is soaked in polypeptide solution and left at room temperature overnight.

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

Because PCL is a hydrophobic material, a longer incubation period is required to ensure that the polypeptide solution penetrates all of the scaffold, forming a uniform self-assembled coating on the fiber surface.

Identification and analysis of the polypeptide coating was verified using fluorescent immunostaining, water contact angle, XPS, SEM or TEM.

Preparation of active artificial blood vessel:

the vascular stent is processed according to the following steps:

(1) iPS cell preparation:

human skin fibroblasts were reprogrammed to prepare iPSCs. The plasmids carrying reprogramming factors were combined with 5X 10 ⁵ Human skin fibroblasts were mixed and plasmids with reprogramming factors were transduced into cells by an electrotransformation system. The electrotransformed cells were inoculated into Matrigel-plated cell culture plates. The cells were cultured using a cell reprogramming medium (Beijing Seebeck Biotechnology Co., ltd.) for about 30 days, and after a typical iPSCs clone-like cell mass appeared, the monoclonal was selected and inoculated to obtain iPSCs cells.

(2) iPS induced differentiation to smooth muscle cells:

when the iPSCs reached a density of about 80%, cells were digested with a dispase at 37℃for 15 minutes to obtain Embryoid Bodies (EBs), which were grown in six well plates with low adhesion and containing mTESR medium. The following day the medium was replaced with mTESR medium and Embryoid Body (EBs) differentiation medium in a 1:3 ratio. The medium was replaced with EBs differentiation medium on the third day, and the culture was continued for 3 days. On day 5, EBs were transferred to six well plates coated with Gelatin (Gelatin), and cultured with EB differentiation medium for 5 days, with new EBs medium being changed daily. After 5 days of culture, cells were digested with 0.25% trypsin, inoculated onto Matrigel gel coated T75 flasks, cultured with smooth muscle cell growth medium (SmGM-2) for 7 days, and replaced with fresh medium every other day, finally iPSCs induced differentiated vascular smooth muscle cells (iPSC-SMCs) were obtained.

(3) iPS induction differentiation into endothelial cells:

on day 0-1, induced differentiation is carried out on the human pluripotent stem cells with the cell density reaching more than 95% by using CDM3 differentiation medium added with GSK3I of 4-8 mu M; on day 2, further inducing differentiation of the cells after the induction culture of the previous step by using CDM3 differentiation medium added with 40-60ng/mL bFGF; on days 3-5, further inducing differentiation of the cells after the induction culture of the previous step by using CDM3 differentiation medium added with 40-60ng/mL VEGF and 20-30ng/mL BMP 4; and (3) digesting the cells after the induction culture in the last step on the 6 th day, and then adopting an endothelial cell culture medium to continue to culture for 3-4 days, thus obtaining the iPSC-ECs.

(4) Smooth muscle cell planting and culture:

taking smooth muscle cell suspension, injecting the smooth muscle cell suspension into the inner cavity of the vascular stent from one end, standing for 5 minutes, uniformly rolling the vascular stent back and forth on filter paper to suck the culture medium, repeating the above operation for 6 times, and respectively carrying out 3 times at two ends of the blood vessel to ensure uniform planting. After the completion of the planting, the blood vessel seeded with cells was placed in a six-well plate for 10 minutes, followed by addition of 10ml of medium. At 37 ℃,5% CO ₂ Is left to stand for 24 hours, rotated 90 ° every 4 hours, to adhere smooth muscle cells to the vascular stent. Using

The TEB 500 bioreactor performs flow culture on the vascular prosthesis. The culture environment was set at 37℃and 5% CO ₂ In the flow culture process, the flow rate of the culture medium is controlled by a peristaltic pump, and the flow rate is slowly regulated to the rated rate.

(5) Endothelial cell planting and culturing:

taking down the artificial vascular stent cultured in the steps, soaking the inner cavity of a blood vessel with 100 mug/mL fibronectin solution, taking endothelial cell suspension, injecting the endothelial cell suspension into the inner cavity of the vascular stent from one end, standing for 5 minutes, uniformly rolling the vascular stent back and forth on filter paper to suck redundant culture medium, repeating the above operation for 6 times, and respectively carrying out 3 times at the two ends of the blood vessel to ensure uniform planting. Placing the blood vessel with the cells into a six-hole plate for standing for 10min after planting, and then adding10ml of medium was added. At 37 ℃,5% CO ₂ Is left to stand for 24 hours and rotated 90 ° every 4 hours to adhere endothelial cells to the vascular stent. Then the artificial blood vessel is connected to the cell flow culture bioreactor

TEB 500), the culture environment was set at 37℃and 5% CO ₂ In the flow culture process, the flow rate of the culture medium is controlled by a peristaltic pump, and the flow rate is slowly regulated to the rated rate.

Thus obtaining the active artificial blood vessel.

According to the embodiment, the active artificial blood vessel can be further subjected to cell removal treatment to obtain the active artificial blood vessel with the bionic extracellular matrix structure.

The method comprises the following steps:

soaking the above obtained active artificial blood vessel in 1% SDS solution, standing on shaking table, slowly shaking at room temperature for 12 hr, washing with sterile physiological saline to remove SDS, and standing in a mixed solution of sterile DNase and RNase (enzyme solution system 40ml, buffer solution of 0.2mol/L MgCl) ₂ ，0.2mol/L CaCl ₂ And 0.1mol/L Tris-HCl with pH of 6.4 and ultrapure water; DNase concentration 50U/ml and RNase concentration 1U/ml), shaking on a shaker at room temperature for 24 hr, washing residual DNase and RNase with sterile physiological saline, and storing the obtained material in sterile PBS at 4deg.C.

The active artificial blood vessel thus obtained has excellent blood vessel regeneration activity and may be used in clinical treatment.

Example 2

A PCL/collagen composite vascular stent for slowly releasing IGF-1 polypeptide, which can promote vascular cell proliferation and secrete extracellular matrix, the preparation method comprises 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 2mg/mL. Adding IGF-1 polypeptide solution into collagen solution, wherein the volume ratio is 4:1, uniformly mixing.

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

The voltage of PCL solution connection is 15kV, and the pushing speed of the injection pump is 2mL/h. The voltage at which the collagen solution was connected was 12kV, and the syringe pump advancing speed was 0.6mL/h.

Thus obtaining the PCL/collagen composite vascular stent of the slow-release IGF-1 polypeptide, which can promote vascular cell proliferation and secrete extracellular matrix.

The vascular stent thus prepared carries IGF-1 polypeptide, and during cell culture and in vivo implantation, IGF-1 is gradually released along with collagen decomposition, thereby promoting cell proliferation and angiogenesis.

Preparation of active artificial blood vessel:

the procedure is as in example 1.

Example 3

A VEGF growth factor modified PCL/gelatin composite vascular stent capable of promoting vascular cell proliferation and secreting extracellular matrix, the preparation method comprising the steps of:

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). Gelatin solution was then prepared and gelatin was dissolved in hexafluoroisopropanol at a concentration of 6% (w/v). The two solutions were added into two 10mL syringes respectively, and the two syringes were connected to PCL11kV and gelatin 17kV respectively using 21G needles mounted on the two syringe pumps, respectively. The distance between the two syringe needles and the receiving rod is 25cm for PCL and 15cm for gelatin, respectively. The two materials are spun simultaneously to prepare the PCL/gelatin composite vascular stent. The solvents in the stent material are thoroughly volatilized and dried thoroughly in a vacuum dryer.

Heparin and VEGF were immobilized first according to the following steps: the connection reagent EDC and heparin are added into 50% ethanol water solution to make the concentration of the connection reagent EDC and heparin be 30mM and 0.5mg/mL respectively, then the vascular stent is soaked into the solution, and the vascular stent is placed into a zero-degree ice water bath for reaction for 12 hours. Then washed three times with distilled water. Left to dry at room temperature. A PBS solution of VEGF growth factor was prepared at a concentration of 1. Mu.g/mL, the vascular stent was immersed in the VEGF solution, incubated overnight at 4℃and finally washed with PBS.

The PCL/gelatin composite vascular stent modified by VEGF growth factor and capable of promoting vascular cell proliferation and secreting extracellular matrix can be obtained.

Preparation of active artificial blood vessel:

the procedure is as in example 1.

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

taking a PCL artificial vascular stent with the same size as the vascular stent described in the embodiment 3 as a comparison group 3-1;

taking a heparin-modified PCL artificial vascular stent (HP-PCL) with the same size as the vascular stent described in the embodiment 3 as a comparison group 3-2;

adhesion and proliferation of human umbilical vein endothelial cells on the surface of a PCL scaffold co-modified with VEGF growth factor and heparin (VEGF-HP-PCL) as described in comparative group 3-1, comparative group 3-2, example 3, respectively, were tested.

The experimental results are shown in fig. 4:

a) A laser confocal image of PI marked endothelial cells adhered on the comparative group 3-1 for 1h;

b) A laser confocal image of PI marked endothelial cells adhered on the comparative group 3-2 for 1h;

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

d) Average counts per field of adherent cells on three materials;

e) Proliferation of endothelial cells on different materials. * P <0.01 (vs PCL); each group n=3, scale 250 μm.

As can be seen from fig. 4, example 3 significantly improved adhesion and proliferation of Human Umbilical Vein Endothelial Cells (HUVECs) on the surface of the material relative to comparative group 3-1 and comparative group 3-2. The VEGF modified on the surface of the material can be combined with VEGF receptor on endothelial cells to induce the rapid adhesion of the endothelial cells, and in addition, the slow release of VEGF from the material also helps the proliferation of the endothelial cells.

Example 4

The preparation method of the RGD short peptide self-assembled modified PCL vascular stent capable of promoting vascular cell proliferation and secreting extracellular matrix comprises the following steps:

the RGD short peptide is subjected to chemical modification to obtain a molecule Nap-FFGRGD capable of self-assembling into gel (the molecular structure is shown in figure 2). Nap-FFGRGD can be self-assembled into gel or can be self-assembled on the surface of a material to form a coating, so that RGD is fixed on the surface of the material, the affinity of the surface of the material for 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. Vascular stent fabrication procedure the same procedure as in example 1 was followed by immersing the bilayer PCL vascular stent in Nap-FFGRGD solution, standing overnight at 37℃and then flushing the PCL stent 3 times with PBS buffer. 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 RGD short peptide and can promote vascular cell proliferation and secrete extracellular matrix.

Preparation of active artificial blood vessel:

the procedure is as in example 1.

taking a PCL artificial vascular stent with the same size as the vascular stent described in the embodiment 4 as a comparison group 4-1;

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

The experimental results are shown in fig. 5:

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

b) A spread state of endothelial cells after 2h of adhesion on example 4;

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

d) A spread state of endothelial cells after 4 hours of adhesion on example 4;

e) Adhesion of endothelial cells to different materials;

e) Proliferation of endothelial cells on different materials.

As can be seen from FIG. 5, after 2 hours of culture, compared with HUVEC, most of the cells on the surface of PCL are in a spherical shape and are not spread, and cells are in a sheet shape and have a larger spreading area on the surface of the material modified by Nap-FFGRGD; after 4h of culture, most cells on the PCL-RGD surface show the form of mature endothelial cells, namely, the form of an elongated spindle, and the spreading degree of cells in the PCL group is far lower than that of cells in the RGD modified group. In addition, nap-FFGRGD modification effectively improves the proliferation condition of HUVEC on the surface of the stent.

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

Example 5

A PCL vascular stent self-assembled and modified by NO donor molecules to promote vascular cell proliferation and secrete extracellular matrix, the method of preparation comprising the steps of:

the self-assembled gel-forming NO donor molecule Nap-FFGGG-NO (molecular structure is shown in figure 3) is prepared through polypeptide synthesis reaction and reversed-phase high performance liquid chromatography purification. Nap-FFGGG-NO was dissolved in PBS (pH=7.4) to prepare a solution having a concentration of 0.1 wt%. The procedure for PCL vascular stents was as in example 1. Immersing the PCL vascular stent into Nap-FFGGG-NO solution, and standing for 2h at room temperature.

Nap-FFGGG-NO forms a hydrogel coating with submicron thickness on the surface of PCL fiber, and the wet condition can be stabilized for one month. The Nap-FFGGG-NO coated vascular stents are typically stored in a dry state after lyophilization.

The PCL vascular stent which is self-assembled and modified by the NO donor molecule and can promote vascular cell proliferation and secrete extracellular matrix can be obtained.

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 can promote the regeneration of blood vessels by controlling the released NO.

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

after the blood stent material described in example 5 catalyzes the release of NO, it was divided into two groups:

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

And respectively collecting macroscopic optical pictures and corresponding fluorescent images of the material surface.

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. It was observed under a fluorescence microscope that the PCL material surface after catalytic NO release had significant green fluorescence (fig. 6 b).

Preparation of active artificial blood vessel:

the procedure is as in example 1.

Example 6

A PCL vascular stent for promoting vascular cell proliferation and secreting extracellular matrix, loaded with NO donor molecules, prepared by a method comprising the steps of:

the procedure for PCL vascular stents was as in example 1. In order to improve the hydrophilicity of the PCL electrostatic spinning bracket, the PCL electrostatic spinning bracket is firstly subjected to hydrolysis treatment. The PCL electrospun scaffold was rinsed in 50% ethanol solution while removing aluminum foil attached to the electrospun scaffold. The cleaned PCL electrospun scaffold was then immersed in an aqueous NaOH solution at ph=12 and hydrolyzed at 37 ℃ for 48h. The PCL electrospun scaffold was then rinsed 3 times, 1h each with deionized water to remove residual NaOH. The cleaned PCL electrospun scaffold was placed in a vacuum oven and dried at room temperature for 2 days. Cutting the hydrolyzed PCL electrospun stent into a round shape with the diameter of 1 cm. Then, 50. Mu.L of CS-NO aqueous solution with the concentration of 20mg/mL is uniformly coated on the surface of the electrospun scaffold by using a dripping coating method. After two days of drying at room temperature, a CS-NO coating was formed on the electrospun scaffold surface.

Thus obtaining the PCL vascular stent loaded with NO donor molecules and capable of promoting vascular cell proliferation and secreting extracellular matrix.

To demonstrate that a CS-NO coating was formed during the reaction of this example, infrared detection was performed on the stent material and PCL material described in this example, and the results are shown in FIG. 7:

as can be seen from the infrared spectrograms of the PCL and the PCL/CS-NO, after the CS-NO is loaded on the surface of the PCL bracket, the PCL bracket is carried by 1363cm- ¹ Two characteristic absorption peaks belonging to the nitrogen enol group appear: 1363cm ^-1 Asymmetric stretching characteristic peak of O-N-N-O group at the position and 945cm ^-1 The N-N plane symmetry at this point stretches the characteristic peak, which indicates successful loading of CS-NO on the PCL fiber.

Preparation of active artificial blood vessel:

the procedure is as in example 1.

Example 7

An IGF-1 polypeptide and NO donor molecular gradient modified PCL vascular stent capable of promoting vascular cell proliferation and secretion of extracellular matrix, the preparation method thereof comprises the following steps:

(1) The inner layer PCL fiber is loaded with NO donor molecules, and the specific operation steps are as follows: PCL was dissolved in a mixed solvent of chloroform and absolute methanol at a volume ratio of 5:1 at a concentration of 15% (m/v). To this was added a galactose group-protected NO donor molecule at a concentration of 0.5% (m/v). The PCL solution was added to a 10mL syringe, the needle was 21G, the syringe was mounted on a syringe pump, the receiving rod was a stainless steel tube having 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 spinning time of the inner layer was 4 minutes, the diameter of the inner layer fiber was about 2. Mu.m, and the thickness of the inner layer was about 50. Mu.m.

(2) The outer PCL fiber is loaded with IGF-1 polypeptide, and the specific operation steps are as follows: PCL was dissolved in a mixed solvent of chloroform and absolute 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 needle of 21G, mounted on a syringe pump, and spinning was continued on the basis of the preceding inner layer fiber. The distance between the syringe needle and the receiving rod is 10cm, the advancing speed of the PCL solution is 8mL/h, the voltage is 12kV, and the spinning time is 25min. The diameter of the outer layer fiber is 6-8 μm, and the thickness is 400 μm.

Thus obtaining the PCL vascular stent which is modified by IGF-1 polypeptide and NO donor molecule gradient and can promote vascular cell proliferation and secrete extracellular matrix.

The vascular stent is double-layered, has thinner inner diameter fiber, loads NO donor molecules, and is favorable for anticoagulation and endothelial formation promotion. The outer layer fiber is thicker, the pore diameter is larger, the cell migration is facilitated, the IGF-1 is slowly released, the cell proliferation and secretion can be promoted, and the rapid regeneration of vascular smooth muscle is facilitated.

Preparation of active artificial blood vessel:

the procedure is as in example 1.

Example 8

A PCL vascular stent which is self-assembled and modified by a VEGF fusion protein and can promote vascular cell proliferation and secrete extracellular matrix, the preparation method of the PCL vascular stent comprises 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. An aqueous solution of VEGF-HGFI was prepared at a concentration of 200. Mu.g/mL. The procedure for PCL vascular stents was as in example 1. The vascular stents were immersed in VEGF-HGFI solution, incubated for 12h at 4℃and washed with PBS.

The PCL vascular stent which is self-assembled and modified by VEGF fusion protein and can promote vascular cell proliferation and secrete extracellular matrix can be obtained.

Preparation of active artificial blood vessel:

the procedure is as in example 1.

modification of VEGF-HGFI fusion protein effects on vascular stent appearance test:

taking a PCL artificial vascular stent with the same size as the vascular stent described in the embodiment 8 as a comparison group 8-1;

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

The experimental results are shown in fig. 8:

a) The cross-section of the vascular stent of the comparison group 8-1 and the vascular stent of the example 8 (upper diagram), and the inner cavity surface of the vascular stent of the comparison group 8-1 and the vascular stent of the example 8 (lower diagram);

b) Comparative group 8-1SEM images (left), example 8SEM images (right);

c) Comparative group 8-1 vascular stent VEGF antibody staining (green) fluorescence plot (left panel), example 8 vascular stent VEGF antibody staining (green) fluorescence plot (right panel).

As can be seen from fig. 8, the loading of amphiphilic proteins HGFI and VEGF-HGFI fusion proteins has no significant effect on the morphology of vascular stent fibers, and VEGF payloads can be observed on the surface of vascular stent fibers by VEGF antibody staining and uniformly distributed in the whole stent.

Modification of VEGF-HGFI fusion protein effects on endothelial cell proliferation and function assays:

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

The DAF-FM probe for fluorescence detection of endothelial cell NO release was performed on control group 8-2 (Glass), example 8 (VEGF-HGFI-Glass), respectively, and the experimental results are shown in FIG. 9A.

Fluorescence detection of endothelial cell uptake of DiI-ac-LDL was performed on control group 8-2 (Glass), example 8 (VEGF-HGFI-Glass), respectively, and the experimental results are shown in FIG. 9B.

As can be seen from fig. 9, the modification of the VEGF-HGFI fusion protein is effective in promoting adhesion and proliferation of endothelial cells, significantly improving NO release ability of endothelial cells (fig. 9A) and uptake ability of acetylated low density lipoprotein (fig. 9B), and enhancing the function of growing endothelial cells on the modified surface.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. A method for preparing a vascular stent capable of promoting proliferation of vascular cells and secreting extracellular matrix, comprising:

1) And (3) processing a bracket:

(1) The preparation method of the inner layer of the bracket by adopting the melt spinning method comprises the following specific operation steps: weighing 5.0. 5.0g PCL with molecular weight of 80000, placing in a sealed stainless steel syringe wrapped by a hot-melting device, and heating lh at 100deg.C; connecting a stainless steel tube with the diameter of 2mm with a rotating motor, wherein the distance between a syringe needle and a receiving rod is 5mm, the flow rate of PCL melt is 0.5mL/h, the rotating speed of the receiving rod is 300r/min, the translation speed of a receiver is l 0mm/s, the spinning time is 5min, and the prepared inner layer fiber has the fiber angle of 50 degrees, the fiber diameter of 60 mu m and the wall thickness of 300 mu m;

(2) The preparation method of the outer layer of the bracket by adopting the electrostatic spinning method comprises the following specific operation steps: weighing 3g PCL with molecular weight of 80000, adding into a mixed solvent of chloroform and absolute methanol with volume ratio of 12mL being 5:1, stirring at room temperature for dissolving overnight, and obtaining PCL solution with concentration fraction of 25% (m/v); placing an electrostatic spinning device in a fume hood, mounting the spinning support prepared above on a rotating device together with a receiving rod, sucking PCL solution into a syringe, mounting the syringe on a syringe pump, and applying 12kV voltage to a 21G metal needle by using a high-voltage direct-current power supply, wherein the distance between the syringe needle and the receiving rod is 10 cm; setting the propelling speed of the injection pump to 8mL/h, the rotating speed of the receiving rod to l00 rpm, and the spinning time to 4min; after the operation, forming a layer of electrostatic spinning fiber layer outside the melt spinning bracket, wherein the fiber diameter is 5 mu m, and the thickness is l00 mu m, thus obtaining the double-layer PCL vascular bracket; an inner diameter of 2mm and a total wall thickness of 400 μm;

2) Self-assembly modification of IGF-1 polypeptide:

dissolving the chemically modified IGF-1 polypeptide Nap-FFG-IGF-1 in Phosphate Buffered Saline (PBS) at a concentration of 0.1% (w/v), ph=7.4; soaking the prepared double-layer PCL vascular stent into a polypeptide solution, and standing at room temperature overnight to obtain the PCL vascular stent which is self-assembled and modified by IGF-1 polypeptide and can promote vascular cell proliferation and secretion of extracellular matrix.

2. An active vascular prosthesis characterized by being prepared by the following method:

firstly inoculating smooth muscle cells, uniformly distributing the cells in the fiber gaps of the whole vascular stent prepared in the claim 1 as much as possible, and fully attaching and stretching the inoculated smooth muscle cells after culturing for 1-6 days;

inoculating endothelial cells on the inner surface of the lumen, standing for 2-3 days, placing in a flow culture bioreactor, connecting with a circulation pipeline, gradually increasing the flow rate, shearing force and pressure of liquid to the condition equivalent to the physiological level, and continuously culturing for 1-8 weeks to obtain the active artificial blood vessel;

the endothelial cells and the smooth muscle cells are derived from human iPS, and vascular endothelial cells and smooth muscle cells are prepared through induced differentiation;

the preparation method also comprises the following steps: performing mild decellularization treatment on the cultured active artificial blood vessel inoculated with endothelial cells to ensure thorough removal of the cells, and simultaneously keeping active extracellular matrix to the greatest extent without damaging the original structure of the internal polymer fiber;

the method for cell removal treatment comprises the following steps: SDS method or liquid nitrogen freeze thawing method; the SDS method comprises the following steps: soaking tissue engineering blood vessel in 1% SDS solution, placing on shaking table, slowly shaking at room temperature for 12h, washing with sterile physiological saline to remove SDS, and placing in sterile mixed DNase and RNase solution, wherein the enzyme solution system is 40ml, and the buffer solution is composed of 0.2mol/L MgCl ₂ , 0.2 mol/L CaCl ₂ And 0.l mol/LpH Tris-HCl and ultrapure water with the mol/LpH of 6.4; DNase concentration is 50U/ml, RNase concentration is l U/ml, shaking at room temperature on shaking table for 24h, washing residual DNase and RNase with sterile physiological saline, and storing in sterile PBS at 4deg.CAnd (5) standby.