JPH01170467A - Blood vessel substitute material - Google Patents

Abstract

PURPOSE:To obtain a blood vessel substitute material excellent in bio- compatibility, by bonding a cell adhesive substance to the inner surface of an artificial blood vessel using extremely fine fibers at least in a part of the inner surface thereof and further bonding a vascular endothelial cell thereto in vitro. CONSTITUTION:In an artificial blood vessel used in a blood substitute material, extremely fine fibers of 0.5 denier or less are used at least in a part of the inner surface of said blood vessel and a cell adhesive substance such as collagen, gelatin or fibrin is applied to said inner surface. Subsequently, an endothelial cell is cultured in vitro to be bonded to the artificial blood vessel. Whereupon, the endothelial starts secretion and a basement membrane component such as collagen is strongly interlaced with the extremely fine fibers and, therefore, the endothelial cell becomes hard to release by blood flow. By this method, the blood substitute material excellent in bio-compatibility and generating no thromboembolism is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a blood vessel substitute material with excellent biocompatibility.

[Prior Art] Among blood vessel substitute materials such as artificial blood vessels and blood vessel patches, for example, artificial blood vessels with an inner diameter of 8 mm or more have been clinically proven and have been developed for practical use, although there are some problems. However, no material has been developed that is satisfactory in terms of handleability and biocompatibility. In particular, there is an extremely strong demand for the development of small-diameter artificial blood vessels with an inner diameter of 6 mm or less, but no one that can be put to practical use has yet been developed.

Therefore, there is a strong demand for artificial blood vessels with excellent characteristics that are closer to those of living organisms, and the development of thinner artificial blood vessels is a current concern of those concerned, and the development of this is eagerly desired.

As a means of developing such blood vessel substitute materials, many attempts have been made to essentially suppress thrombus formation by creating artificial blood vessels using polymeric materials having antithrombotic properties. However, there has yet to be a case in which thrombotic occlusion can be completely suppressed over a long period of time.

On the other hand, vascular endothelial cells that line the lumen of blood vessels serve as ideal antithrombotic surfaces. Vascular endothelial cells secrete prostacyclin and plasminogen activator, and also have thrombomodulin and heparan sulfate on their surface. In this way, vascular endothelial cells use various mechanisms to cause platelets to adhere to blood vessel walls and to prevent blood from coagulating within blood vessels. For this reason, attempts have been made to create a narrow-diameter artificial blood vessel that is free from occlusion by thrombi by attaching vascular endothelial cells to the lumen of the artificial blood vessel. - To give an example, Tan1y et al. pasted or embedded fibrin into the inner surface and voids of an artificial blood vessel with a length of 10 mm and a diameter of 4 mm, which was made from ordinary polyester fibers. When this artificial blood vessel is replaced with a dog's blood vessel, the rate of occlusion of the artificial blood vessel due to blood clots (patency rate) is higher when endothelial cells are attached.

However, when endothelial cells are implanted into conventional artificial blood vessels, the implanted artificial blood vessels are hard and easily fray.
Furthermore, since anastomosis and suturing are difficult and the sewing margin cannot be made small, blood clots adhere to the vicinity of the anastomosis, causing occlusion. Furthermore, because endothelial cells adhere poorly to the artificial base material, stable formation of an endothelial cell layer has been difficult, such as peeling off due to blood flow after implantation.

[Problems to be Solved by the Invention] An object of the present invention is to provide a blood vessel substitute material that does not have the above-mentioned drawbacks, is easy to handle, and has excellent biocompatibility, and is particularly applicable to small diameter artificial blood vessels. Another object of the present invention is to provide a material that is less likely to be blocked by blood clots.

[Means for Solving the Problems] An object of the present invention is to attach a cell-adhesive substance to the inner surface of an artificial blood vessel using ultrafine fibers of 0.5 denier or less on at least a portion of the inner surface, and to This can be achieved by using a vascular substitute material to which endothelial cells are attached in vitro.

The artificial blood vessel used in the blood vessel substitute forest material of the present invention must be made of an artificial blood vessel using ultrafine fibers of 0°5 denier or less on at least a portion of its inner surface. By making ultra-fine fibers, the ultra-fine fibers of the artificial blood vessel are tightly intertwined with basement membrane components such as collagen that are secreted by endothelial cells and grown on top of them.
Blood flow makes it difficult for endothelial cells to detach. Furthermore, after transplantation into a living body, the invasion of vascular smooth muscle cells and fibroblasts into the fiber interstices increases due to the use of ultrafine fibers, which further stabilizes the implanted endothelial cell layer. Furthermore, by forming the fibers into microfibers, not only the biocompatibility but also flexibility, anastomotic properties, and suturing properties are significantly improved.

Ultrafine polymers for artificial blood vessels used in the present invention include polyester, polyurethane, polyphenylene sulfide, polysulfone, polyether, polyamide, polyolefin, polytetrafluoroethylene, polycarbonate, polyacetal, polyglycolic acid, chitin, chitosan, cellulose, cellulose ester, These include cellulose ethers and copolymers thereof. Among these, polyesters such as polyethylene terephthalate, polybutylene terephthalate, or copolymers thereof into which hydrophilic groups such as sulfone groups, carboxyl groups, and acrylamide groups are introduced are particularly preferred.

In addition, in imparting hydrophilicity to ultrafine fibers, a treatment using a high-voltage electric field such as plasma treatment is also effective as a physical means.

The manufacturing and processing methods for ultrafine fibers have already been published in the US.
P3,5.31.368:USP3,350.488:
As seen in Japanese Patent Publication No. 61-4546, there is a method of forming multi-component fibers and removing or exfoliating the components to form ultra-fine fibers. This ultra-fine treatment may be performed in advance to form an artificial blood vessel, but it is also possible to perform the ultra-fine treatment after forming an artificial blood vessel. Of course, it is also possible to directly use ultra-m fibers without going through such a method.

Artificial blood vessels can be formed by direct methods such as weaving, knitting, braiding, and non-woven fabrics according to conventional methods.

In the present invention, improvement of resistance to fading is also an extremely important issue. For this reason, for example, as a means for improving the tube formation, it is possible to improve the structure by devising the structure such as twist weaving, warp knitting, torsion lace, etc. In addition, as a post-processing method, such a tube is heated by a laser etc.
There is a method of partially and intermittently fusing the fibers to each other to the extent that flexibility and strength are not impaired. Because it is an ultra-fine fiber, it is easy to partially melt even with a slight amount of heat, and it is possible to adhere the fibers to each other, that is, to prevent them from fraying, without losing flexibility.

Another method is to apply a thin beam of high pressure flow. More specifically, by blowing a thin, high-speed water stream or air onto the tube, the fibers can be made to intertwine to prevent fraying. At this time, by using ultrafine fibers, the effect of intertwining the fibers with each other is further promoted. In order to achieve even greater effectiveness, it is possible to lightly treat with another polymer solution or emulsion.

Light treatment with segmented polyurethane, for example, can stop fraying without interfering with the effect of the abrupt microfibers. By appropriately utilizing such means, prevention of fraying can be effectively achieved.

The characteristics of using microfibers for artificial blood vessels are that biological cell formation occurs quickly and well even in tissues with relatively low water permeability, and even better even under high water permeability, and the water permeability can be varied widely depending on the purpose. You can take it.

The vascular endothelial cells used in the present invention include rabbit, dog, pig,
Cells derived from various animals such as mice, monkeys, and guinea pigs can be used, but if the purpose is to ultimately transplant them into humans, it is necessary to use cells derived from humans. Most preferably, it is used.

These cells are derived from arteries, blood vessels, capillaries, etc., for example, "Development of Heart and Blood Vessel Research Methods," edited by Noribe Ebashi, p.

It can be collected by the same method as Nos. 27-37.

In humans, it is common to use those derived from the greater saphenous vein or the capillaries of adipose tissue.

After separation, these endothelial cells are attached to the artificial blood vessel described in this application and used as a blood vessel substitute, but it is preferable to use them for transplantation after culturing them in vitro in an appropriate medium. It is also effective to culture the cells in vitro in a tissue culture flask, for example, using a tissue culture flask, according to a conventional method, before attaching the cells to the substrate, and then attach the cells to the artificial blood vessel.

In order to stably attach endothelial cells to an artificial blood vessel, it is necessary to coat the inner surface of the artificial blood vessel with a cell-adhesive substance such as collagen, gelatin, fibronectin, vitronectin, laminin, or fibrin in advance. In order to make this coating more reliable, 1) glow discharge treatment and/or plasma treatment of the artificial blood vessel, 2)
It is particularly effective to crosslink the coated cell adhesion substance by chemical means, and 3) immobilize the cell adhesion factor to the lumen of the artificial blood vessel.

To attach endothelial cells to the inner surface of an artificial blood vessel to which a cell-adhesive substance has been attached, a cell culture solution in which vascular endothelial cells are suspended in a medium such as M199 medium containing 15% Fe2 is injected into the artificial blood vessel. This can be done by bringing the cells into contact with the inner surface, or by culturing the cell culture solution within an artificial blood vessel.

[Example] Next, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to these.

Example 1 Using 0.07 denier polyester ultrafine fiber, plain weave and inner diameter 3rl! An artificial blood vessel with r+1 and a length of 6 cm was created. A high-pressure water stream was then applied to entangle the fibers with each other. It had a structure in which several split ultra-fine fibers crossed between the weaves. After this artificial blood vessel was subjected to plasma treatment in the presence of argon gas, a 0.5% purified aqueous atelocollagen solution was injected into the lumen and air-dried to coat the inside of the artificial blood vessel with collagen.

The vascular endothelial cells used were those derived from the external jugular vein of dogs. After the artery was aseptically removed and washed several times with phosphate buffered saline (PBS), PBS containing 0.05% trypsin was added, both ends were tied, and the artery was left at room temperature for 30 minutes. Floating cells were collected by centrifugation and added with 20% fetal bovine serum (Fe2
) and 1.5 μ/ml ECGF (manufactured by Coloborative) using M199 medium and cultured in a CO2 incubator at 37 degrees.

The cells were confirmed to be vascular endothelial cells by fluorescent staining with a fluorescently labeled factor-related antigen antibody. By culturing for 10 days, 1×10 5 vascular endothelial cells were obtained.

After the cells were treated with I~lipsin, they were suspended in the above-mentioned medium and injected into the lumen of an artificial blood vessel. Then, both ends were tied with sutures, and the roller bottle was gently rotated for 4 hours while maintaining the horizontal position in the roller bottle containing the culture medium.
Vascular endothelial cells were attached to the artificial blood vessel. By this operation, approximately 65% of the injected cells adhered to the artificial blood vessel. After opening both ends of the artificial blood vessel, endothelial cells attached to the artificial blood vessel were cultured for two days in a thermostatic chamber at 37 degrees while slowly rotating the bottle itself. When a part of the artificial blood vessel was cut open and stained with hematoxylin-eosin, it was confirmed that more than 50% of the inner surface of the artificial blood vessel was covered with endothelial cells. Using 5 dogs, 2 blood vessel substitutes were made from each dog's cells (10 in total)
It was created.

The cells from each of these two artificial blood vessels were collected and replaced with the femoral arteries of the same five dogs.

The patency rate after 30 days was 80%. In those that were patent, more than 70% of the inner surface was covered with vascular endothelial cells, and no red thrombus was observed at any location. During implantation, the anastomosis and suturing properties were good, the needle passed through easily, and there was no fraying despite the sewing margin of about 1.5 mm from the cut end.

Comparative Example Using the same artificial blood vessels as in Example, 1) one in which endothelial cells were not cultured after coating with collagen, 2) one in which endothelial cells were attached without coating with collagen,
and 3) artificial blood vessels made using the same 1.4 denier material were coated with collagen and cultured with endothelial cells, and 10 of each were replaced in five dogs. The patency rate after 30 days is 1 for both 1) and 2).
0%, 3) was 50%. Among the patency cases of 3), 2
In this example, the endothelial cell coverage was 60% or more, but in the other three cases, red blood clots were observed especially near the anastomosis, and the endothelial cell coverage was 20% or less. In addition, in case 3), the cut end frayed, making it difficult to perform anastomosis unless a seam allowance of 2.5 mm or more was taken.

[Effects of the Invention] The material of the present invention has excellent biocompatibility and is unlikely to cause thrombotic occlusion, so it is useful as a blood vessel substitute material, and is particularly an excellent material for use in small-diameter artificial blood vessels.

Claims (1)

[Claims] (1) A vascular substitute material, which is obtained by attaching a cell-adhesive substance to the inner surface of an artificial blood vessel using ultrafine fibers of 0.5 denier or less on at least a portion of the inner surface, and further adhering vascular endothelial cells in vitro.