CN100340220C - Method for planting esoderma/endothelial cell on inner surface of artificial blood vessel - Google Patents

Abstract

The present invention provides a method for planting endothelial/endothelial-like cells on the inner surface of an artificial blood vessel. By adopting a biomechanical method, the endothelial cells can better adhere to the artificial blood vessel through the action of pulsating and stepwise increased fluid shear stress in vitro. The inner wall of the artificial blood vessel enables the endothelial cells adhering to the inner wall of the artificial blood vessel to adapt to the action of fluid shear stress in advance, so that it has a better ability to withstand the action of blood flow shear stress after the blood vessel is transplanted into the body. The method can be used to plant endothelial cells on the surface of artificial blood vessels from various sources, including synthetic biological materials and natural biological materials, to prepare artificial blood vessels.

Description

A method for planting endothelial/endothelial-like cells on the inner surface of artificial blood vessels

technical field

The invention relates to a method for planting endothelial/endothelial-like cells on the inner surface of artificial blood vessels.

Background technique

Cardiovascular system disease is one of the diseases with the highest incidence rate in the world, and it is also the leading cause of death at present. The main treatment method is vascular transplantation. Every year in the United States alone, more than 570,000 coronary artery bypass grafts are performed. In addition, the implementation of bypass surgery also requires a large number of peripheral vascular grafts. This has resulted in a significant demand for small caliber (<6 mm) vascular grafts.

The purpose of vascular tissue engineering is to construct an ideal vascular graft in vitro, without immune rejection after transplantation, and to maintain the long-term patency of the grafted vessel lumen. Cardiovascular surgery requires vascular grafts of various diameters as repair materials. At present, artificial vascular grafts with a diameter > 6 mm have been widely used in clinical practice, while small-diameter artificial vascular grafts are in great demand and have a high blocking rate, so their clinical application is obviously limited. Therefore, the current focus of vascular tissue engineering is to prepare small blood vessels with a diameter of less than 6mm. The reasons for the failure of small vessel grafts are mainly thrombosis in the acute phase; in the chronic phase, the proliferation of smooth muscle cells into the lumen of the graft and the formation of pannus at the anastomosis lead to lumen obstruction. Compared with tissue-engineered blood vessels, human blood vessels have good anti-thrombosis ability due to the barrier effect of vascular endothelial cells. Therefore, the successful endothelialization of artificial blood vessel materials is the hope of improving the patency rate of small-caliber artificial blood vessels.

1. Inoculation of endothelial/endothelial-like cells on the inner surface of artificial blood vessels

Through the study of vascular endothelial cells (VEC), it is found that VEC has high metabolic activity and endocrine function. It can synthesize and release a variety of physiologically effective substances, regulate many physiological activities, and maintain the balance of physiological activities in the body, such as Regulation of vascular smooth muscle tension, microvascular permeability, platelet activity, coagulation, anticoagulant and fibrinolytic system. The surface of VEC has the same negative charge as blood cells, which can prevent the deposition of blood cells on the lumen of artificial blood vessels. Therefore, the planting of VEC has the effect of anti-platelet aggregation, preventing blood coagulation and thrombus formation.

In 1970, Mansfield first proposed the implantation method of endothelial cells. In recent years, many researchers are also trying to improve the performance of grafts through the lining of endothelial cells, so that the grafts can have better bionic capabilities in vivo. In 1998, ShinokaT et al. designed and produced in vitro autologous pulmonary artery grafts. The carotid artery of a 20-day-old sheep was taken out, and the mixed cell community was obtained by tissue block culture method, and then the smooth muscle cells (SMCs) and endothelial cells (ECs) were separated by the fluorescent labeling method, and planted in the absorbable material polylactic acid-coethanol successively acid (PGLA), the original sheep main pulmonary artery was replaced with an in vitro constructed vessel after 7 days. After 10-12 weeks of transplantation, there were no obvious signs of thrombus formation and calcification. This work created a precedent for the study of new vascular grafts (Shinoka T, Tim AD, et al. Creation of viable pulmonary artery autografts through tissue engineering. J Thorac Cardiovasc Surg .1998, 115:536-546. Title: Development of Living Pulmonary Artery Autografts Using Tissue Engineering Approaches).

Recently, it has been reported that autologous EC transplantation can promote the repair of damaged endothelium. Conte et al. used a rabbit femoral artery balloon injury model, blocked the blood flow at both ends of the femoral artery, injected autologous jugular vein EC transfected with β-galactosidase gene into the endothelial stripped vessel segment, and found that autologous jugular vein EC transplantation can effectively Inhibition of restenosis after balloon angioplasty in hypercholesterolemic rabbits. Darcin et al. used a dog femoral artery balloon injury model to transplant autologous jugular vein EC to the endothelialized vessel segment. The experimental results showed that this method could also significantly improve the long-term patency of the injured vessel.

Although the endothelialization of tissue-engineered vascular grafts has improved the thrombosis problem to a certain extent, the problem of long-term patency of small-caliber vessels has not yet been resolved. Studies have found that the low perfusion of EC for vascular grafts with a caliber of less than 6mm leads to insufficient adhesion of endothelial cells on the graft, resulting in a low long-term patency rate, so further improvement is needed in the lining technology . As early as 1994, Herring pointed out that the failure of planting endothelial cells on the expanded polytetrafluoroethylene (ePTFE) scaffold material to endothelialize the scaffold was associated with intimal hyperplasia and the formation of lumen thrombus, and pointed out the possible reasons It is the absence of the endothelial cell layer on the inner surface of the material after the scaffold material is implanted in the body. Bhat VD et al. believe that the mechanism may be that the blood flow in the body weakens the adhesion of implanted cells and makes the implanted endothelial cells fall off. Therefore, how to improve the adhesion of endothelial cells after inoculation has become an urgent problem to be solved.

2. Improve the adhesion of endothelial/endothelial-like cells on the vessel wall

Whether the artificial blood vessel can be endothelialized depends on the ability of the implanted cells to adhere, grow and expand on the wall of the artificial blood vessel. On the one hand, endothelial cells can be planted at a high density to form a solid layer of fusion cells on the inner surface of the vascular graft. On the other hand, it is necessary to ensure that after the graft is implanted in the body, the planting cells can still adhere to the vessel wall under the action of blood flow. The researchers found that the VEC adhered to the graft in vitro is easy to fall off in the shear stress field in vivo, that is, the adhesion of endothelial cells is found to be weak, and the problems of long-term patency and thrombus formation of tissue engineered blood vessels have not been resolved. Various methods have been proposed to improve the adhesion of EC on the graft surface, the main ones are:

(1) Pre-line extracellular matrix (ECM) proteins on the artificial blood vessel wall: such as fibronectin (FN), fibrinogen (fibrinogen, FG), vitronectin (VN), laminin ( laminin, La) and collagen (collagen, CL), etc. Arginine-glycine-aspartate-serine (RGDs) arranged in the structures of FN, FG and VN are major cell adhesion determinants. RGDs have a specific recognition function, and can specifically combine with a transmembrane protein family on the endothelial cell membrane - Integrins family (IF), so that they can adhere to endothelial cells. In view of this, some research groups directly attach RGD peptide chains to the artificial blood vessel wall for modification, or immobilize RGD-containing polypeptides on the artificial blood vessel wall, which can promote the adhesion of endothelial cells.

(2) Use the specific binding force of biotin and avidin to increase the adhesion of endothelial cells: the cultured endothelial cells are treated with biotin, and the inner wall of the artificial blood vessel is lined with avidin , using their specific affinity, can obviously enhance the adhesion of endothelial cells on the wall of artificial blood vessels and the ability to resist shear stress.

(3) Use the action of cell growth factors to improve the adhesion and growth ability of endothelial cells: for example, using fibroblast growth factor (fibroblast growth factor, FGF) has a high affinity site with heparin, and the slow release of heparin can inhibit Characterized by smooth muscle proliferation, the addition of FGF to heparin-albumin-prelined artificial vessel walls also facilitated endothelial cell growth and adhesion.

(4) Change the electrification of the implanted endothelial cells: because the endothelial cells are negatively charged, and the artificial blood vessel wall (such as ePTFE) is also negatively charged, they have mutual repulsion. Electrostatic action can be used to charge the implanted endothelial cells positively or less negatively for a short period of time, thereby improving the adhesion ability of endothelial cells on the blood vessel wall and the ability to resist blood flow shear stress. However, the reduction of negative charge increases the probability of thrombus formation.

(5) Application of molecular biology technology: The development of modern molecular biology technology makes it possible to modify cell genes. Therefore, it is hoped to fundamentally improve the patency of artificial blood vessels by introducing gene fragments that induce vasodilation and antithrombosis into the inoculated endothelial cells.

(6) Joint culture of endothelial cells and smooth muscle cells: smooth muscle cells (SMC) are the main cell components of the vessel wall. Such cells can produce a large amount of extracellular matrix components, and SMC directly or indirectly affects the morphology of endothelial cells, thereby facilitating the attachment of endothelial cells.

Although researchers have used various methods to improve the adhesion of endothelial cells, after vascular grafts are implanted in vivo, the endothelial cell layer is still prone to shedding in the long-term blood flow field, resulting in the formation of thrombus. After long-term exploration, people have gradually realized that endothelial cells are natural regulators of blood vessel stability. After vascular injury, the endothelial cell monolayer of blood vessels has antithrombotic and smooth muscle cell proliferation effects. Shannon L et al pointed out that successful tissue-engineered vascular grafts must ensure a monolayer of endothelial cells that can resist blood flow shear force and thrombus formation (Shannon L, Niklason MLE..Requirements for growing tissue-engineered vascular grafts. Cardiovascular Pathology .2003, 12:59-64. Title: The Need for Living Tissue-Engineered Vascular Grafts).

3. The influence of hydrodynamic environment on vascular endothelial cells:

It is now known that a major problem in constructing tissue engineered blood vessels is that the blood vessels are in a natural mechanical dynamic environment, which includes: the shear stress generated by the tangential action of the blood flowing through the endothelial layer, the shrinkage and contraction of the annular vessel wall The stretch stress (stretch stress) and the normal stress caused by hydrostatic pressure. Shear stress has an important effect on the morphology, proliferation, polarity and matrix composition and structure of vascular endothelial cells.

(1) The effect of shear stress on the morphology of endothelial cells (ECs): It has long been noticed that blood flow can affect the morphology and orientation of ECs under in vivo conditions. In the unidirectional stable high shear stress region, the ECs were long fusiform, and the long axis of the cells was consistent with the direction of the shear stress. In contrast, in areas of low shear stress or blood stagnation, ECs were round and the arrangement of cells was not oriented. In vitro experiments found that after human umbilical vein vascular endothelial cells (HUVECs) were subjected to fluid shear force of 38 and 75 dyne/ ^cm2 for 12 hours, the endothelial cells were elongated, and the long axis was consistent with the direction of the flow field, and the elongation of endothelial cells, The degree of orientation is related to the magnitude of the shear stress and the duration of the action (Chen Huaiqing, Medical Biomechanics. 2003, 18 (Supplement): 3-4.). Recent studies have also demonstrated that blood flow shears endothelial cells, both in vivo and in vitro, and stimulates endothelial cells to release prostacyclin (PGI ₂ ) and endothelium-derived relaxing factor (EDRF) . The static polygonal endothelial cells become elongated and oriented in the direction of flow directly under the shear force of blood flow. The morphological reconstruction of endothelial cells due to the action of shear stress (the cavity surface presents a streamlined feature) helps to reduce the pulling effect of blood flow on endothelial cells, thereby maintaining the integrity of the monolayer structure of endothelial cells.

(2) The effect of shear stress on the adhesion of endothelial cells: studies have shown that shear stress conditions can improve the adhesion of cells under its action. Under flow conditions, shear stress is gradually loaded on the material scaffold of the endothelial cell lining, It was found that the effect of shear force promoted the reconstruction of the cell scaffold network and cell-substrate adhesion. ECs were planted in polyurethane (Pu) blood vessels with an inner diameter of 1.5 mm, and subjected to a shear force of 1-2 dyn/cm ² for 3 days, and then a shear force of 25 dyn/cm ² for another 3 days. After the experiment The researchers found that a relatively complete endothelial cell layer was preserved, and only a small amount of endothelial cells remained after direct application of a shear force of 25dyn/cm ² (Ott MJ, Ballermann BJ.Shear stress-conditioned, endothelial cell-seeded vascular grafts : Improved cell adhesion in response to invitro shear stress.Surgery.1995, 117:334-339. Title: Shear stress acclimated vascular grafts inoculated with endothelial cells: The effect of in vitro shear stress increases cell adhesion) (Dardik A, Liu A, Ballermann B. Chronic in vitro shear stress stimulates endothelial cell retention on prosthetic vascular grafts and reduces subsequent in vito neointimal thickness. Journal of Vascular Surgery. 1999, 29(1): 157-167. Stress increases retention of endothelial cells on artificial vascular grafts and subsequently reduces neointimal thickness in vivo). The sheep carotid artery (Photofix) with an inner diameter of 4mm, through the action of the shear stress controlled by the researchers, makes the initial value of the shear stress value about 0.25dyn/cm ² and the final value of about 1.2dyn/cm ² , the action time is At 6 hours, 50% of the endothelial cells remained on Photofix (Carnagey J, Anderson DH, Ranieri J, et al. Rapid endothelialization of photofix natural biomaterial vascular grafts. J Biomed Mater Res Part B: Appl Biomater. 2003, 65B: .171 -179. The title is: rapid endothelialization of photofix natural artificial blood vessels). Although the shear stress values adopted by the former in these studies are somewhat random, and the number of shear stress values adopted is very limited, including only 1 or 25 dyn/cm ² Normal shear stress values vary widely, but their results suggest that gradually increasing shear stress can increase cells' tolerance (resistance) to physiological levels of flow forces. What take in the present invention is the increasing shear stress of stepwise (stepwise), more emphasizes the gradual increase process of shear stress, and the shear stress value involved is in the range of the shear stress value suffered by normal human vascular endothelial cells, research The result has also confirmed that adopting the method of the present invention makes the human umbilical vein endothelial cells inoculated on the carotid artery of Wistar rats in which endothelial cells are removed pass through a stepwise increase, finally up to 26dyn/ ^cm After the shear stress acts for 24 hours, its retained area It is more than 80%. In 2002, Li Yuquan et al. reported that under a steady laminar shear stress of 40 dyn/cm ² , the contents of fibronectin (Fn) and laminin (laminin, Ln) in the ECM of endothelial cells co-cultured with smooth muscle increased. This results in an increase in the adhesion and fluid shear stress resistance of endothelial cells. Philippe et al. used polyethylene terephthalate (polyethyleneterephthalate, PET) blood vessels (inner diameter 6 mm) in their experiments, and coated bovine collagen and low-density heparin on the inner surface, and cultured the adherent blood vessels using a pulsating perfusion system. For cells on grafts, the experimental results also show that long-term perfusion and high-level shear stress (equal to blood flow shear stress) are very conducive to the retention of planted endothelial cells on grafts.

(3) The effect of shear stress on the formation of stress fibers in endothelial cells: The rearrangement of EC induced by shear stress is closely related to the change of EC cytoskeleton. The cytoskeleton is composed of microfilaments, microtubules, and intermediate fibers, among which actin microfilaments (mainly composed of fibrous actin and F-actin) have been studied more. In the experiment, Chen Huaiqing not only found that the endothelial cells were elongated by the fluid shear force, which was consistent with the direction of the flow field, but also observed that the arrangement direction of F-actin in the cells was also consistent with the direction of the shear stress. As early as 1984, Franke et al. have found that under the action of 2dyne/cm ² shear force, the cultured monolayer HUVEC formed stress fibers in the center of the cells after 2-3 hours, but after increasing the stress time, the stress fibers were not obvious. increase, the shape of the cells remained polygonal. Dewey et al. also found that stress fibers appeared in endothelial cells after 8dyne/cm ² shear force for 24 hours, and the cell morphology did not change at this time. Our research found that in the flow state, after the cultured monolayer endothelial cells were exposed to 10dyne/cm ² for 24 hours, stress fibers formed by microfilaments appeared in the center of the cells. This result also shows that shear stress can induce the formation of stress fibers in endothelial cells role. In these studies, although the stress fibers have been formed, but only increased in number, and have not yet formed thick microfilaments, which may be related to the low level of shear stress. We found that the central Very significant bundle-like stress fibers appeared, and the length and thickness of microfilaments increased significantly. These experiments demonstrate that the degree of stress fiber formation and increase depends on the magnitude and duration of the shear force, and that high levels and relatively long periods of force are required for dynamic culture of endothelial cells during tissue building.

(4) Effect of shear stress on endothelial cell proliferation: Brian S. Conklin, MS, et al. Shear Stress Regulates Occludin and VEGF Expression in Porcine Arterial Endothelial Cells. Journal of Surgical Research. .The title is: Shear stress regulates the ^expression of transmembrane tight junction protein and vascular endothelial cell growth factor of porcine arterial endothelial cells ⁾ . The substance (taken from porcine carotid artery) provides pressure and flow state under physiological conditions, so that endothelial cells and smooth muscle cells can survive for a long time. Studies have demonstrated that low shear stress leads to proliferation of endothelial cells, up-regulates the expression of VEGF in endothelial cells, and VEGF also promotes proliferation of endothelial cells.

(5) The effect of shear stress on the physiological activity of endothelial cells: at the level of molecular biology, shear stress can up-regulate the mRNA levels of proto-oncogenes such as c-fos, c-jun, and c-myc, and their gene products act as transcriptional activators Factors or repressors bind to transcription sites on DNA promoters and regulate gene expression. At the same time, shear stress can regulate the mRNA levels of target genes such as endothelin-1, tissue plasminogen activator (tPA), platelet-derived growth factor (PDGF), and adhesion factors. The expression products of these genes play an important role in remodeling blood vessels and regulating the specific adsorption between cells and between cells and matrix. These conditions are quite different from those of endothelial cells cultured statically in vitro. High levels of shear stress also promote the release of cytokines from endothelial cells that inhibit coagulation, leukocyte migration, and smooth muscle cell proliferation, while low levels of shear stress have the opposite effect.

Contents of the invention

In order to promote the adhesion of endothelial cells seeded on the inner wall of artificial blood vessels and to provide the ability of endothelial cells to withstand the shear stress of blood flow after adhesion, the present invention proposes a method for planting endothelial/endothelial-like cells on the inner surface of artificial blood vessels, namely Using biomechanical methods, through the pulsating and stepwise increasing fluid shear stress in vitro, the endothelial cells can better adhere to the inner wall of the artificial blood vessel, so that the endothelial cells adhered to the inner wall of the artificial blood vessel can adapt to the fluid shear stress in advance The effect makes it have a better ability to withstand the shear stress of blood flow after the blood vessel is transplanted into the body.

In order to achieve the above object, the present invention adopts the following two methods: method one, after collecting the endothelial cell suspension, inoculate it on the inner surface of the artificial blood vessel, culture it statically until the cells become confluent on the inner surface of the artificial blood vessel, and then apply pulsation The fluid shear stress increases stepwise until reaching the shear stress value of the endothelial cells in vivo. During the inoculation process, the artificial blood vessel rotates around its central axis at a certain angular velocity. Method 2: after collecting the endothelial cell suspension, inoculate it on the inner surface of the artificial blood vessel, and start to apply pulsating and stepwise fluid shear stress until the endothelial cell is subjected to the shear stress value in vivo. During the inoculation process, the artificial blood vessel rotates around its central axis at a certain angular velocity.

The artificial blood vessel in the present invention can be synthetic biological material, as dacron (Dacron), expanded polytetrafluoroethylene (e-PTFE), polyurethane (polyurethane, Pu) etc., can be natural biological material, as small intestine submucosa , pericardium, fascia, blood vessels, or acellular matrix made of dermis.

The advantage of the method of the present invention is that by the action of the pulsating, stepwise increased fluid shear stress, compared with the prior art, the present invention puts more emphasis on the stepwise loading process of the fluid shear stress, and has the characteristics of a relatively uniform speed and gradual increase, After that, after reaching the required shear stress value in the body, maintain this value until the application such as animal experiments is stopped, and the shear stress is provided for a long time; the applied shear stress is pulsating, close to the physiological situation and the range of shear stress values involved in the present invention is in the endothelial cell tolerance range of normal human body, so that the method of the present invention prepares artificial blood vessels with potential clinical application value; Stress acclimation, it has better adhesion on artificial vascular materials, which other researchers did not mention. This causes the present invention to have greater advantages in the following aspects: 1. The cell morphology and arrangement of the endothelial/endothelial-like cells seeded on the inner surface of the artificial blood vessel are close to the situation in the body, that is, the cells are elongated and arranged in the direction of the fluid ; ②Increase the adhesion ability of endothelial cells on the surface of artificial blood vessels, so that after the artificial blood vessels are transplanted into the body, they can withstand the scouring effect of the blood flow shear stress in the body without falling off; ③Induce the stress of endothelial/endothelial-like cells The formation of fibers; ④ inhibit the excessive proliferation of endothelial/endothelial-like cells in the process of in vitro culture, so that the endothelial/endothelial-like cells inoculated on the inner surface of the artificial blood vessel in a confluent state are in the contraction phenotype in vivo; Endothelial/endothelial-like cells with a confluent inner surface have physiological activities similar to those in vivo. In this way, the effect of gradually acclimating endothelial cells and increasing their adhesion and physiological activity can be achieved.

Using the method of the present invention, the human umbilical vein endothelial cells inoculated on the carotid artery of Wistar rats from which endothelial cells have been removed are subjected to a stepwise increased shear stress of up to 26 dyn/ ^cm2 for 24 hours, and the retained area is more than 80%. However, under the same experimental conditions, after 2 hours with the action of 15 dyn/cm ² , obvious gaps appeared between the cells, and the retained area was less than 50%.

Figure 1 The human umbilical vein endothelial cells seeded on the carotid artery of Wistar rats with depleted endothelial cells were subjected to stepwise increasing shear stress. The shear stress started from 10dyn/cm ² and increased by 2dyn/cm ² every 3 hours, pulsating The frequency is 40Hz, and finally reaches as high as 26dyn/cm ² . The speed of blood vessel rotation around its central axis is 120° per hour. After 24 hours of treatment, it is silver-stained. The magnification of the photo under the Olymus microscope is 80 times. The darker line in the photo is the membrane of the cell stained with silver, so it shows the outline of the cell. It can be seen from the photo that the cells are closely arranged.

Fig. 2 The human umbilical vein endothelial cells inoculated on the carotid artery of Wistar rats from which the endothelial cells were removed were subjected to a shear stress of 15 dyn/cm ² for 2 hours and then silver-stained. The photos under the Olymus microscope, the magnification is 40 times. The darker color in the photo is the cells stained with silver. It can be seen from the photo that there are obvious gaps between the cells.

Description of drawings

Fig. 1 is a photomicrograph of human umbilical vein endothelial cells inoculated on the carotid artery of Wistar rats removing endothelial cells by this method;

Figure 2 is a photograph of human umbilical vein endothelial cells seeded on the carotid artery of Wistar rats from which endothelial cells have been removed, subjected to a shear stress of 15 dyn/cm ² for 2 hours and then stained with silver under an Olymus microscope.

Detailed ways

Example 1. Static inoculation on murine carotid artery vascular graft material with cell matrix removed, followed by a stepwise increase in fluid shear stress, while the vessel was rotated around its central axis to cultivate endothelial cells

Take the carotid artery of Wistar rats whose endothelial cells have been removed by freezing method, and inoculate rabbit primary arterial endothelial cells acclimated by a shear stress of 10 dyns/ ^cm2 in the intima. Wash the intima three times with PBS, digest the cultured primary rabbit endothelial cells with 0.1% trypsin, collect the cells, the cell concentration is about 1×10 ⁶ cells/cm ² , inoculate the collected cells into the vascular intima, 37 ℃, 5% CO ₂ incubator for about 3 days, until the cells have fused into a single layer, then, apply a stepwise increase of fluid shear stress to the inoculated endothelial cells for "acclimation", and the loading shear stress starts from 10dyns/cm ² , until 40dyns/cm ² , the step is to increase 5dyns/cm ² every 3h, the time required is about 12h, and then maintain the shear stress of 40dyns/cm ² , while loading the artificial blood vessel around it at an angular velocity of 90° every half hour The axis was rotated and cultured in a 37°C, 5% CO ₂ incubator. Thus, the vascular graft material of the invention was obtained.

Example 2. Inoculation on the mouse carotid artery vascular graft material with the cell matrix removed and simultaneously applying a stepwise increase in fluid shear stress, while the blood vessel was rotated around its central axis to cultivate endothelial cells

Take the carotid artery of Wistar rats whose endothelial cells were removed by freezing method, and inoculate rabbit primary arterial endothelial cells on the intima. The cultured primary rabbit endothelial cells were digested with 0.1% trypsin, and the cells were collected at a concentration of about 1×10 ⁶ cells/cm ² . The collected cells were inoculated into the intima of blood vessels, and at the same time, a stepwise increase of fluid shear was applied to the endothelial cells. Stress, the initial value of loading shear stress is about 5dyns/cm ² , the step is to increase 0.1dyns/cm ² every 1h, the required time is about 12h, and then change the step to increase 1.0dyns/cm ² every 1h, the loading time is about 12h , while loading, the artificial blood vessel was rotated around its central axis at an angular velocity of 90° every half hour, and cultured in a 37°C, 5% CO ₂ incubator. Thus, the vascular graft material of the invention was obtained.

Example 3. Inoculation and simultaneous application of pulsating, step-wise increasing fluid shear stress on goat carotid artery graft material without cell matrix, while the vessel rotates around its central axis to cultivate endothelial cells

Take the goat carotid artery from which endothelial cells have been removed by enzymatic digestion, and inoculate the primary cultured human umbilical vein endothelial cells on the intima. , the cultured primary human umbilical vein endothelial cells were digested with 0.1% trypsin, and the cells were collected at a concentration of about 1×10 ⁶ cells/cm ² . The collected cells were inoculated into the intima of the blood vessel, and pulsating pressure was applied to the endothelial cells at the same time. 1. The fluid shear stress increases stepwise, the pulsation frequency is 80Hz, the initial value of the loaded shear stress is about 5dyns/cm ² , the step is to increase 0.3dyns/cm ² every 1h, the time required is about 12h, and then the step is changed to every Increase 10 dyns/cm ² every 1 hour, load for about 4 hours, and then maintain a shear stress of about 50 dyns/cm ² , load while rotating the artificial blood vessel around its central axis at an angular velocity of 20° per minute, at 37°C, 5% CO ₂ cultured in an incubator. Thus, the vascular graft material of the invention was obtained.

Example 4. Cultured endothelial cells statically seeded on expanded polytetrafluoroethylene (ePTFE) vascular material followed by application of pulsatile, step-wise increasing fluid shear stress while the vessel rotates about its central axis

After sterilizing the expanded polytetrafluoroethylene (ePTFE) vascular stent material, take a section (about 1.5mm in inner diameter, about 4-5cm in length) and inoculate rabbit primary arteries acclimated by fluid shear stress of 5-10dyns/ ^cm2 The endothelial cells were inoculated in the intima, and the inoculation method was as follows: the intima of the expanded polytetrafluoroethylene (ePTFE) vascular stent material was washed three times with PBS, and after pre-lined with fibronectin (FN), the cultured primary rabbit Endothelial cells were digested with 0.1% trypsin, and the cells were collected at a concentration of about 1×10 ⁶ cells/cm ² . The collected cells were inoculated into the intima of blood vessels, and cultured statically for about 3 days in a 5% CO ₂ incubator at 37°C. After the cells have fused into a single layer, apply pulsating, stepwise increasing fluid shear stress for "acclimation", the pulsating frequency is 150Hz, and the loading shear stress starts from 2dyns/cm ² to 20dyns/cm ² , and the step increases every 1h 1dyns/cm ² , the required time is about 18h, and then maintain a shear stress of 20dyns/cm ² , while loading, the artificial blood vessel is rotated around its central axis at an angular velocity of 300° per hour, in a 37°C, 5% CO ₂ incubator nourish. Thus, the vascular graft material of the invention was obtained.

Example 5. Seeding on polyurethane (polyurethane, Pu) vascular material and simultaneously applying pulsating, stepwise increased fluid shear stress, while the blood vessel rotates around its central axis to cultivate endothelial cells

After sterilizing the polyurethane (Pu) vascular material, take a section (about 2mm in inner diameter and about 4-5cm in length) and inoculate the intima with human primary arterial endothelial cells acclimated by fluid shear stress of 5-10dyns/ ^cm2 , the inoculation method was as follows: the intima of the polyurethane stent material was washed 3 times with PBS, and pre-lined with vitronectin (LN), the cultured primary human umbilical vein endothelial cells were digested with 0.1% trypsin, collected Cells, the cell concentration is about 1×10u ⁶ cells/cm ² , the collected cells are inoculated into the intima of the blood vessel, and at the same time, a pulsating, stepwise increasing fluid shear stress is applied to the endothelial cells, the pulsation frequency is 40Hz, and the initial shear stress is The value is about 2dyns/cm ² , the step is to increase 0.2dyns/cm ² every 1h, the time required is about 12h, and then change the step to increase 5dyns/cm ² every 1h, the loading time is about 5h, and then maintain about 35dyns/cm ² shear stress, while loading the artificial blood vessel at an angular velocity of 10° per minute around its central axis, culture it in a 37°C, 5% CO ² incubator. Thus, the vascular graft material of the invention was obtained.

Example 6. Inoculation of goat carotid artery vascular graft material without cell matrix and simultaneous application of pulsating, step-wise increasing fluid shear stress, while the vessel rotates around its central axis to cultivate endothelial cells

Take the goat carotid artery from which endothelial cells have been removed by enzymatic digestion, and inoculate primary cultured human umbilical vein endothelial cells on the intima. Wash 3 times, digest the cultured primary human umbilical vein endothelial cells with 0.1% trypsin, collect the cells, the cell concentration is about 1×10 ⁶ cells/cm ² , inoculate the collected cells into the vascular intima, and at the same time, treat the endothelial cells Apply pulsating, step-wise increasing fluid shear stress, the pulsation frequency is 60Hz, the initial value of loading shear stress is about 10dyns/cm ² , the step is to increase 0.3dyns/cm ² every 1h, the time required is about 12h, and then change The step is to increase 3 dyns/cm ² every 1 h, the loading time is about 10 h, and then maintain a shear stress of about 45 dyns/cm ² , while loading, the artificial blood vessel is rotated around its central axis at an angular speed of 5° per minute, at 37°C, 5 %CO ₂ incubator. Thus, the vascular graft material of the invention was obtained.

Claims (9)

1. A method for planting endothelial/endothelial-like cells on the inner surface of artificial blood vessels. After collecting endothelial cells into a cell suspension, they are seeded on the inner surface of artificial blood vessels, cultured statically until the cells become confluent on the inner surface of artificial blood vessels, and then applied Fluid shear stress until it reaches the shear stress value of the endothelial cells in vivo; during the inoculation process, the artificial blood vessel rotates around its central axis at a certain angular velocity; it is characterized by a pulsating and stepwise increase in the applied fluid shear stress fluid shear stress. 2. A method for planting endothelial/endothelial-like cells on the inner surface of artificial blood vessels. After collecting endothelial cells into a cell suspension, they are seeded on the inner surface of artificial blood vessels. The value of the shear stress received; during the inoculation process, the artificial blood vessel rotates around its central axis at a certain angular velocity; it is characterized in that the applied fluid shear stress is a pulsating, stepwise increased fluid shear stress. 3. The method for planting endothelial/endothelial-like cells on the inner surface of artificial blood vessels according to claim 1 or 2, characterized in that: the range of the stepwise increased shear stress applied to the endothelial/endothelial-like cells seeded on the surface of artificial blood vessels is Greater than 0 and less than 50dyes/cm ² , the rate of stepwise increase in shear stress is greater than 0 and less than 10dyes/cm ² per hour. 4. The method for planting endothelial/endothelial-like cells on the inner surface of artificial blood vessels according to claim 1 or 2, characterized in that: the inoculated endothelial/endothelial-like cells can be derived from endothelial/endothelial-like cells that have been acclimated by fluid shear stress in advance . 5. The method according to claim 3, characterized in that the rate of stepwise increase of the shear stress is 0.1-1 dyes/cm ² per hour. 6. The method for planting endothelial/endothelial-like cells on the inner surface of artificial blood vessels according to claim 1 or 2, characterized in that pulsating fluid shear stress is applied to the endothelial/endothelial-like cells inoculated on the surface of artificial blood vessels, and the pulsating frequency is The range is greater than 0 and less than 150Hz. 7. The method for planting endothelial/endothelial-like cells on the inner surface of artificial blood vessels according to claim 6, characterized in that the pulsation frequency ranges from 40-80 Hz. 8. The method for planting endothelial/endothelial-like cells on the inner surface of an artificial blood vessel according to claim 1 or 2, characterized in that: during the inoculation process, the angular velocity range of the artificial blood vessel rotating around its central axis is greater than 0 per minute and less than 10°. 9. The method for planting endothelial/endothelial-like cells on the inner surface of artificial blood vessels according to claim 8, characterized in that: the rotation speed ranges from 100-200° per hour.

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