JPH0312169A - Artificial blood vessel - Google Patents

Abstract

PURPOSE:To provide the artificial blood vessel which is used for rebuilding of the blood circulation in a small-diameter blood vessel and vein blood vessel by treating the wall of the blood vessel of a mammal with org. acid or guanidine hydrochloride, then culturing the smooth muscle cells and endothelio cells. CONSTITUTION:The blood vessel treated with the org. acid, such as formic acid, or the guanidine hydrochloride rebuilds the blood vessel wall. The smooth muscle cells and endothelio cells are cultured for this purpose. The rabbit smooth muscle cells, swine smooth muscle cells, bovine smooth muscle cells, canine smooth muscle cells, etc., are sued for the smooth muscle cells. The swine endothelio cells, canine endothelio cells, etc., are used for the endothelio cells. The culture is executed by a method of washing the blood vessel wall, which is treated with the sterilized formic acid, etc., with distilled water and physiological salt soln. and is permeated in a Dulbecco disintegration Eagle medium and is then cultured in the suspension of the swine smooth muscle cells or after the suspension (c) of the swine smooth muscle cells is injected at about 0.2ml/graft by an injection needle of 27G to the vessel wall, the cells are cultured. The concn. of the aq. formic acid to be used is 70 to 95%, more preferably 80 to 90% and the treatment time is one day to one week. The treating temp. is <=5 deg.C, more preferably <=4 deg.C.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is directed to processing artificial blood vessels, particularly mammalian blood vessels, and producing artificial blood vessels that are expected to have the same measures and functions as blood vessels in living bodies. The present invention relates to a method for doing so and an artificial blood vessel obtained therefrom.

[Conventional technology]

Many researchers have researched the materials and structure of artificial blood vessels,
Furthermore, surface modification is being studied, and materials made of Dacron (registered trademark) and Teflon (registered trademark) are being studied, and expanded polytetrafluoroethylene is used especially for small-caliber blood vessels in cases where autologous veins cannot be used. (EPTFE) vessels are mainly used. Recently, polyurethane has been attracting attention as a new material with antithrombotic properties and flexibility (Matsumoto Susushi 9, Organs Organs, 17:610
, 1988, and Yasushi Tamura et al., Artificial Organs, 17:61.
4, 1988).

In addition, even if existing materials are used, collagen coatings [ScottS, M
), et al., Journal of Cardiovascular Surgery (J, Cardiovasc, Surg, ) 28:4
98.1987) and albumin soaking (McGree (McGree)
gree G, S,) et al. American Surgeon (Ame
r, Surgeon,) 53:695°19g? ) and other artificial blood vessels have also been developed. Furthermore, endothelial cells for artificial blood vessels are planted using a culture system (se
Herring et al. [Herring N. et al.
(Surg, ) 84:498, 1987) and Graham et al.
98, 1980), many studies have been conducted since then. There is also the idea that it is better to use biologically derived blood vessel-binding fibers as a material than to use artificial materials that do not originally exist in the body, and vascular substitutes made from human chain veins cross-linked with glutaraldehyde are often used. [Dardic 11. et al., Ann, Surg. 183
:252゜1976, and Yuihiro Sasashima 1-day foreign journal, 85
:65, 1984).

Furthermore, in 1979, Jones cultivated rat smooth muscle cells on a dish to produce sufficient extracellular matrix, and then cultured bovine endothelial cells in the smooth muscle cell layer. planting on top
) created a type of vascular wall model and demonstrated the possibility of remodeling the vascular wall in culture [Jones P, A.: Proceedings of the National Academy of Sciences (P.
roc, Natl, Acad, Sci,), 76:1
882°1979). Wineberg
) created a blood vessel model using collagen gel [Weinberg + C, B, Science: 231:397, 1]
986), but it is difficult to apply it to a blood vessel substitute due to its strength and elasticity.

[Problem to be solved by the invention]

As mentioned above, in order to develop artificial blood vessels with excellent thrombogenicity, moderate elasticity, and good affinity for cells, we investigated the material, structure, and surface modification of artificial blood vessels.

In addition, hybrid artificial blood vessels using culture systems, etc.
A lot of research is being done. However, nothing beyond autologous veins has yet been developed. In addition, living blood vessel wall cells are not present in blood vessels made of biomaterials cross-linked with glutaraldehyde, polyepoxy compounds, etc., and cell affinity is not necessarily good.

Furthermore, regarding the creation of a blood vessel wall model similar to a natural blood vessel, there has been no example of creating a complete blood vessel wall model in vitro.

Therefore, it is desired to develop a fully satisfactory artificial blood vessel for use in revascularization of small-caliber blood vessels and venous blood vessels.

[Means to solve the problem]

The present inventors believe that the most ideal artificial blood vessel is a substitute blood vessel that has a structure and function almost equivalent to that of a natural blood vessel, in which blood vessel connecting fibers and blood vessel wall cells exist, so we developed a new biological substitute using a culture system. Aiming to create a blood vessel, we created a blood vessel wall model with a structure roughly equivalent to that of a natural blood vessel in vitro (
As a result of an attempt to create a blood vessel in vitro, it was discovered that by treating the walls of mammalian blood vessels with a specific solution and then culturing them with specific cells, vessels with walls similar to those of natural blood vessels could be created. Heading 1 This invention has been achieved.

That is, the present invention provides a method for lining an artificial blood vessel, which comprises treating the wall of a mammalian blood vessel with formic acid, and then culturing smooth muscle cells and endothelial cells to reconstruct the blood vessel wall, and the method described above. This invention relates to an artificial blood vessel made by the method.

As the mammalian blood vessel, the aorta of mammals including pigs, dogs, cows, rabbits, etc. can be used.

In terms of artificial blood vessels, biological blood vessel substitutes that have almost the same structure and function as natural blood vessels are considered to be the most ideal, and have a basic skeleton of collagen and elastin and smooth muscle cells and endothelial cells. Therefore, it is necessary to use a substitute blood vessel that can transform into the autologous blood vessel itself, and an elastic plate structure of elastin is considered to be necessary to obtain appropriate elasticity and strength. It is not easy to artificially create the extracellular matrix that forms the basic skeleton of the blood vessel wall, and it is convenient to use natural blood vessels, which requires removing the antigenicity of the foreign blood vessels. In addition, a treatment method is required that produces a matrix that has cell affinity. Therefore, we investigated a treatment method that would remove as much of the cellular components of the foreign blood vessels as possible, as well as proteins and sugars other than elastin and collagen, and as a result, treatment with a formic acid aqueous solution was found to be appropriate.

The concentration of the aqueous formic acid solution used is 70-95%, preferably 80-90%, and the treatment time is 1 day to 1 week.

The treatment temperature is suitably below 5°C, preferably below 4°C. Under certain conditions, formic acid can remove most of the components other than elastin in blood vessels.

It is known that the basic structure of the elastic plate is left almost in its original form, but
, M.A., Arch Path, 65:519,195
8), the present inventors have found that collagen remains to an extent that mechanical strength can be maintained if treated at 5° C. or lower.

Treatment with other solutions: For example, distilled water was thought to be able to swell cells and remove water-soluble sugars and proteins due to osmotic pressure, but it is difficult to apply this to the deep parts of blood vessel walls where connective fibers are dense. Triton X 100 (Rohm & Partner) is a nonionic surfactant that solubilizes biological membranes and was thought to be suitable for cell removal, but even this method was insufficient. Furthermore, guanidine hydrochloride is often used to fractionate collagen and elastin in blood vessel walls, and can be used in the same way as organic acids such as formic acid. Although the strength is slightly weakened by treatment with an organic acid such as formic acid or guanidine hydrochloride, this is not a disadvantage because the connecting fibers formed by blood vessel wall cells are strengthened by resynthesis. Blood vessels treated with organic acids such as formic acid or guanidine hydrochloride are then subjected to vessel wall reconstruction. Therefore, smooth muscle cells and endothelial cells are cultured.

As the smooth muscle cells, rabbit smooth muscle cells, pig smooth muscle cells, bovine smooth muscle cells, large smooth muscle cells, etc. are used.

Further, as the endothelial cells, porcine endothelial cells, large endothelial cells, etc. are used.

As for the culture method, the sterilized blood vessel wall treated with formic acid, etc. was washed with distilled water and physiological saline, and then infiltrated into Dulbecco's modified Eagle's medium (DMEM) (containing 1004 g/ml of fibronectin, etc., which is used as a cell adhesion factor). After that, (1) porcine smooth muscle cell suspension (approximately I
(2) porcine smooth muscle cell suspension (approximately I x 10'-Gcells/ml);
After injecting approximately 0.2 ml/graft with a 27G injection needle, the cells are cultured. The culture time is one week or more, preferably one month or more.

Rabbit smooth muscle cells and the like are also cultured in the same manner as the pig smooth muscle cells.

In addition to smooth muscle cell culture, endothelial cells are cultured to reconstruct the blood vessel wall. This endothelial cell culture is carried out by smooth muscle cell culture, or by 0.5 mg/ml of the blood vessel wall after the same injection culture.
Fibronectin/DME
Immersion in cell adhesion substances such as M (room temperature to 37°C, 1 to several days)
After that, add pig endothelial cell suspension (approximately 1 x 10' cells/
ml).

As described above, the vascular wall was reconstructed in vitro using a mammalian blood vessel treated with formic acid. Culturing with smooth muscle cells is to create a state in which a large number of cells are scattered between elastic lamina, like a natural blood vessel, and culturing with endothelial cells is for intimal reconstruction. It takes a long time (more than 4 weeks) for smooth muscle cells to naturally invade the deep intramural layer from outside the wall.
It takes.

[Effect]

By treating mammalian blood vessels with organic acids or guanidine hydrochloride and culturing smooth muscle cells and endothelial cells, it is possible to create a vascular wall model similar to natural blood vessels with vascular connective tissue and vascular wall cells in vitro. It has been shown. This method makes it possible to create biological blood vessel substitutes that have almost the same structure and function as natural blood vessels. In other words, after treating a foreign blood vessel or a cadaver blood vessel, the blood vessel wall is reconstructed using blood vessel wall cells cultured and grown from a portion of the patient's own blood vessel, resulting in a living body substitute blood vessel with antithrombotic properties and elasticity, which can be used after transplantation. are decomposed, absorbed, and resynthesized by one's own cells, and we can expect blood vessels that can eventually transform into autologous blood vessels themselves.

〔Example〕

The present invention will be further explained with reference to examples below, but the present invention is not limited to these examples.

The materials used and various measurements were as follows: 1. Method for collecting and culturing vascular wall cells (endothelial cells and smooth muscle cells) Endothelial cells were collected from the intima of the porcine thoracic aorta by a scraping method.
Dulbecco's modified Eagle's medium (D
MEM). Smooth muscle cells were isolated and cultured from the media using the explant (6xplant) method. Rabbit smooth muscle cells were also collected and cultured from the thoracic aorta.

2% cells of 4 passages were used in each experiment.

2. Test method for tearing strength of various treated blood vessel walls A nylon thread with a 5-0 needle was passed through the walls of various treated blood vessels with a width of about 6 m, and a load was applied in one direction to measure the amount of load at the time of tearing.

3. Quantification of protein fraction in treated blood vessel walls The dry weight of each treated blood vessel before and after treatment was measured, and for the formic acid treatment group and the guanidine hydrochloride treatment group, the total protein content, collagen content, and elastin content were simply quantified.

(1) Quantification of collagen Hydroxyproline in the water-soluble fraction fractionated by the method shown in Table 4 and autoclaved (120°C, 12 hours, 2 times) was determined by the Woessner method (Woessner method).
Arch.

Biochem, Biophys, ), 93:440,
(1961) and converted into collagen amount (hydroxyproline amount x 10).

(2) Elastin quantification The insoluble fraction shown in Table 4 was taken as the amount of elastin.

(3) Quantification of total protein amount The total protein amount was defined as the sum of the protein amount measured by the Loigry method from the water-soluble fractions and autoclave soluble fractions shown in Table 4 and the elastin amount measured above. .

(1) Extracellular matrix (6xtra
In order to remove the antigenicity of foreign blood vessels and to create a matrix with cell affinity, the adventitia of the porcine aorta was removed and divided into strips of approximately 6 x 8 nn in size, followed by distillation. Water immersion (4℃, 72 hours), ■ Glutaraldehyde treatment (1%, 20℃, 48 hours), ■ Polyepoxy compound (DenacolEX 8
10. Nagase Kasei) treatment (5%, 20°C, 48 hours),
■Polyethylene glycol alkyl phenyl ether (
Triton X 100, manufactured by Rohm and Haas)
Treatment (1%, 4°C, 72 hours), ■ Guanidine hydrochloride treatment (5M, 4°C, 72 hours), ■ Formic acid treatment (88%, 4°C)
, 72 hours).

After each treatment, the specimens were thoroughly washed with distilled water and then stored immersed in 70% ethanol.

(2) Next, the various ethanol-immersed blood vessel walls were thoroughly washed with distilled water and physiological saline, and then treated with DMEM containing 100 μg/m 1 fibronectin.
(37° C., 24 hours), and then cultured in a rabbit smooth muscle cell suspension (approximately 5×10′ cells/m 1 ). Furthermore, various treated blood vessel walls and pig endothelial cell suspensions (approximately 4 x 10'
calls/ml) were cultured.

Rabbit smooth muscle cells were cultured on the treated pig aorta wall using the above method, and scanning electron microscopic observation after 2 days of culture revealed that the glutaraldehyde treated group Although cell adhesion was poor, it was good in other groups and became almost good after one week.

Figure 1 is a scanning electron microscope image (X400) after 2 days of culture of rabbit smooth muscle cells on various treated blood vessels, and the two surfaces treated with A-distilled water are almost completely covered with smooth muscle cells. B glue
taraldehyde treatment: slight adhesion of smooth muscle cells. CDenacol treatment: Many smooth muscle cells adhere. D. Formic acid treatment: indicates that the surface is completely covered with smooth muscle cells.

Furthermore, initial observation after 2 weeks of culture revealed that one to two layers of smooth muscle cells had been formed, except in the glutaraldehyde-treated group, but almost no cell invasion into the deep intramural layer was observed (Figure 3). ). Initial observation of the formic acid-treated blood vessel wall after 4 weeks of culture revealed that smooth muscle cells had invaded the deep intramural layer. However, it was rarely seen in other groups. In addition, the original cellular components within the blood vessel walls of the formic acid-treated group completely disappeared. However, in the other treatment groups, some cell components remained, especially in the distilled water treatment group, in large numbers (Figure 2).

Figure 2 shows electron microscopic images (X 400) after 2 weeks of culture of rabbit smooth muscle cells in various treated blood vessels, A: distilled water treatment, B:
glutaraldehyde treatment, CDenaco
D treated with formic acid (glutara
One to two layers of smooth muscle cells are observed except in ldehyde-treated vessels).

From the above results, it can be seen that treatment with formic acid is the best treatment method that completely removes the cellular components of the blood vessel wall and is suitable for cell adhesion and intrawall invasion.

Tree SEM 5CORE: Cell occupation area ratio in scanning electron microscope observation field 0: Cell (-) 1: O ~ 102:10 ~ 403:40 ~
60 4:60~905:90~100%**R3MC
:Rabbit smooth muscle cells*0*PC:Pig endothelial cells Also, in scanning electron microscopy vA observation after 2 days of culture of pig endothelial cells,
Similar to rabbit smooth muscle cells, almost no adhesion of endothelial cells was observed in the glutaraldehyde-treated group. However, in other groups, the cells adhered and proliferated relatively well (Table 1), and the growth was almost good after the first week of culture (Figs. 3 and 4).

Figure 3 is a scanning electron microscopy image of porcine smooth muscle cells and endothelial cell culture in a formic acid-treated blood vessel.
x 400), B after 2 weeks of smooth muscle cell culture: covered with a complete smooth muscle cell layer (xloo), C after 1 week of endothelial cell culture: a complete endothelial cell layer has been formed (x
400), which indicates that.

In addition, Figure 4 shows various electron microscopic images, A: untreated blood vessel: the intimal side is covered with a single layer of endothelial cells, and the wall surface has many smooth muscle cells (X400), B: formic acid treatment Blood vessel: Cell components have completely disappeared, but the elastic lamina structure is preserved (X400), C smooth muscle cell seeding 4 weeks later: Smooth muscle cells have invaded deep into the wall (X40)
0), D Endothelial cells in the knee layer are formed after 1 week of endothelial cell seeding (X400), E Smooth muscle cells are injected and cultured for 2 weeks: Smooth muscle cells are scattered between the elastic lamina (
X400), F smooth muscle cell injection 1 week + endothelial cells see
3 days after ding: Although the intima is not completely in one layer, endothelial cells are attached, and there are a small number of smooth muscle cells scattered within the wall (x 200). be.

Furthermore, the tear strength was weak in the formic acid treated group and the guanidine hydrochloride treated group, and was stronger in the decanol or glutaraldehyde crosslinking treated group than the untreated group (Table 2).

Table 2: Rupture strength tree of various treated blood vessel walls SEN 5CORE (mean ± SD, n = 5) and
The dry weight measurement results after each treatment revealed the amount of loss of blood vessel wall components due to each treatment, and looking at the protein fractions of the formic acid treatment group and the guanidine hydrochloride treatment group, the amount of collagen decreased slightly, but the amount of elastin It was found that there was almost no change in the fraction before and after the treatment, and there was a large loss of other protein components (Table 3). This shows that the formic acid treatment or the guanidine hydrochloride treatment can leave the vascular wall structure mainly composed of elastin and collagen.

Table 3 Dry weight and remaining percentage of protein fraction of various treated blood vessel walls. Mean ± SD,
n=3) Large fl side blood vessel wall (dry weight) ↓ After cutting, add distilled water and homogenize ↓ Centrifugation → Supernatant...Water-soluble fraction ↓ Residue ↓ Add distilled water and autoclave at 120℃. 12 hours x 2 ↓ Centrifugation → Supernatant... Heat soluble fraction ↓ Residue... Insoluble fraction (elastin fraction) (1) Formic acid treatment of porcine aortic wall frozen preservation (-80℃) Outside of porcine aorta After removing the membrane, cut into pieces approximately 6 x 8 m+a in size and immerse in 88% formic acid (4°C, 7°C).
After 2 hours), distilled water and phosphate buffer (pH 8,
0) was sufficiently washed and neutralized. Furthermore, for sterilization, it was immersed and stored in 70% ethanol for 24 hours or more.

(2) Simple culture of porcine smooth muscle cells The sterilized formic acid-treated vascular wall was thoroughly washed with distilled water and saline,
After soaking (37°C, 24 hours) in porcine smooth muscle cell suspension (approximately I x 10" cells/ml)
) was cultured in

(3) Injection culture of porcine smooth muscle cells 2 injections of porcine smooth muscle cell suspension (approximately 1×105″-Gcells/ml) into the blood vessel wall treated in the same manner as above.
After injecting approximately 0.2 ml/graft with a 7G needle, culture was continued.

(4) Co-culture of smooth muscle cells and endothelial cells Pig smooth muscle cell injection After 1 to 2 weeks of culture, the blood vessel wall was immersed in 0.5■,/+1 fibronectin/DMEM (37°C, 10 minutes), then pig Endothelial cell suspension (approx. I x 105 cells)
/ml).

The results obtained are as follows.

(1) By treating the arterial wall with formic acid, it is possible to create a piece of connective tissue mainly composed of elastin and collagen, and no cellular components are seen at the beginning or by scanning electron microscopy, and the artery remains in its original shape. A close elastic lamina structure, collagen fibers, and elastic fiber network were observed (Fig. 3C, Fig. 4D).

(2) Smooth muscle cell culture on the formic acid-treated blood vessel wall After 1 to 2 weeks, the surface of the ffl tissue was completely covered with a smooth muscle cell layer, and there was only a small amount of cell invasion into the elastic lamina; After 4 weeks or more, cell invasion into the deep intramural layer was observed (Fig. 4C).

(3) Smooth muscle cells were injected into the formic acid-treated blood vessel wall. Light microscopic images after 1 to 2 weeks of culture showed that smooth muscle cells were scattered, but not evenly, between the elastic plates (Fig. 4E).

(4) In co-culture of smooth muscle cell-injected vascular walls and endothelial cells, the intimal surface was almost completely covered with an endothelial cell layer within several days (
Figure 3C2 Figure 4F). This had an elastin elastic plate structure containing smooth muscle cells, and the surface was covered with endothelial cells, and could be said to be a vascular wall model similar to natural blood vessels.

〔Effect of the invention〕

According to the present invention, an artificial blood vessel has a structure and function almost equivalent to a natural blood vessel containing vascular connective tissue and vascular wall cells, and can be used for revascularization of small-caliber blood vessels and venous blood vessels compared to conventional artificial blood vessels. can be expected to be created.

[Brief explanation of drawings]

Figure 1 is a scanning electron microscope image (X400) after 2 days of culture of rabbit smooth muscle cells in various treated blood vessels, and is a photograph showing the morphology of the organisms. Figure 1A is treated with distilled water, Figure 1B is Figure 1C is the one treated with glutaraldehyde, Figure 1 C is the one treated with decanol, and the first figure is treated with formic acid. Figure 2 is a light microscope image (x400) after 2 weeks of culture of rabbit smooth muscle cells in various treated blood vessels, showing the morphology of the organisms; Figure 2A is treated with distilled water, Figure 2B is treated with glutarium. The aldehyde treatment, Figure 2C is the decanol treatment, and the second figure is the formic acid treatment. Figure 3 is a scanning electron microscope image of porcine smooth muscle cells and endothelial cell culture in a blood vessel treated with formic acid, and is a photograph showing the morphology of the organisms; Figure 3A is the intimal surface of a blood vessel treated with formic acid; B shows the result after 2 weeks of smooth muscle cell culture, and FIG. 3C shows the result after 1 week of endothelial cell culture. Figure 4 shows various electron micrographs showing the morphology of living organisms. Figure 4A is an untreated blood vessel, Figure 4B is a formic acid treated blood vessel, and Figure 4C is a smooth muscle cell seeding ( seeding
) After 4 weeks, the fourth plot is endothelial cell seeding (see
ding) One week later, Figure 4E shows smooth muscle cell injection culture 2.
After a week, Figure 4F is 1 week after smooth muscle cell injection + 3 days after endothelial cell seeding.

Claims (3)

[Claims] (1) A method for producing an artificial blood vessel, which comprises treating the vascular wall of a mammalian blood vessel with an organic acid or guanidine hydrochloride, and then culturing smooth muscle cells and endothelial cells to reconstruct the vascular wall. (2) The method for producing an artificial blood vessel according to claim 1, wherein the mammalian blood vessel is an aortic blood vessel of a pig, dog, cow, or rabbit. (3) An artificial blood vessel produced by the method according to claim 1.