ISCHEMIC HEART DISEASE ANIMAL MODEL USING 3-DIMENSIONAL BIOPRINTED OCCLUDE, AND MANUFACTURING METHOD THEREFOR

Abstract

The present invention relates to an animal model with ischemic heart disease, by a 3-dimensional bioprinted occluder and method for producing the same.

Claims

1-8. (canceled)

9. A method for inducing myocardial infarction of an animal, comprising preparing a cylindrical hollow construct with a through-hole extended in a longitudinal direction, which has an outer diameter ranging 70% to 130% of an inner diameter of a target blood vessel and an inner diameter with a certain size allowing blood flow to pass through, by 3-dimensional printing of polymer melt; and inducing myocardial infarction by implanting the hollow construct inside the target blood vessel to partially block blood flow, wherein the target blood vessel is a blood vessel of an animal except for human.

10. The method for inducing myocardial infarction according to claim 9, wherein the target blood vessel is one or more selected from the group consisting of a left anterior descending artery, a right coronary artery and a left circumflex artery.

11. The method for inducing myocardial infarction according to claim 9, wherein the preparing a cylindrical hollow construct is preparing so that an inner diameter of the cylindrical hollow construct is 50% or less of the outer diameter of the cylindrical hollow construct.

12. The method for inducing myocardial infarction according to claim 9, wherein the preparing a cylindrical hollow construct is preparing a length of the cylindrical hollow construct to a length of 1.5 to 5 mm.

13. The method for inducing myocardial infarction according to claim 9, wherein the polymer is one or more selected from the group consisting of polycaprolactone (PCL), poly(lactate-co-glycolate) (PLGA), poly lactic acid (PLA), polyurethane (PU), poly(lactide-co-caprolactone) (PLCL), polydioxanone (PDO), polystyrene (PS), poly(ethylene glycol) (PEG), poly(vinyl acetate) (PVA), polypropylene glycol (PPG), polyacrylamide (PAAm), polyglycolic acid (PGA), polymethylmethacrylate (PMMA), polyhydroxybutyrate (PHB), and polyvinylpyrrolidone (PVP).

14. The method for inducing myocardial infarction according to claim 9, wherein the implanting is injecting the cylindrical hollow construct into a carotid artery to implant it into a left anterior descending artery.

15. The method for inducing myocardial infarction according to claim 9, wherein the method for inducing myocardial infarction does not comprise removing the cylindrical hollow construct.

16. The method for inducing myocardial infarction according to claim 9, wherein the method for inducing myocardial infarction induces myocardial infarction while maintaining the cylindrical hollow construct without removing it.

17. The method for inducing myocardial infarction according to claim 9, further comprising breeding an animal in which the cylindrical hollow construct is implanted.

18. The method for inducing myocardial infarction according to claim 9, wherein the survival rate of the animal in 5 weeks after implanting the cylindrical hollow construct is 70% or more.

19. The method for inducing myocardial infarction according to claim 9, wherein the cylindrical hollow construct is implanted to an animal to induce one or more of the following (1) to (6): (1) reduction of a left anterior descending artery diameter (2) reduction of ejection fraction (3) necrosis of cardiac tissue (4) abnormal morphological changes in cardiomyocytes (5) collagen accumulation inside myocardial tissue (6) induction of hypoxia of myocardial tissue

20. The method for inducing myocardial infarction according to claim 9, wherein the animal is one or more selected from the group consisting of pigs, monkeys, chimpanzees, sheep, goats, cows, horses, camels, dogs and cats.

21. An animal model with myocardial infarction except for human, comprising an implantable hollow construct for inducing myocardial infarction in a target blood vessel, wherein the implantable hollow construct for inducing myocardial infarction is a cylindrical hollow construct with a through-hole extended in a longitudinal direction, which has an outer diameter ranging 70% to 130% of an inner diameter of the target blood vessel and an inner diameter with a certain size.

22. The animal model according to claim 21, wherein the animal is one or more selected from the group consisting of pigs, monkeys, chimpanzees, sheep, goats, cows, horses, camels, dogs and cats.

23. The animal model according to claim 21, wherein the target blood vessel is one or more selected from the group consisting of a left anterior descending artery, a right coronary artery and a left circumflex artery.

24. The animal model according to claim 21, wherein the survival rate of the animal in 5 weeks after implanting the cylindrical hollow construct is 70% or more.

25. The animal model according to claim 21, wherein in the animal model, one or more of the following (1) to (6) are induced: (1) reduction of a left anterior descending artery diameter (2) reduction of ejection fraction (3) necrosis of cardiac tissue (4) abnormal morphological changes in cardiomyocytes (5) collagen accumulation inside myocardial tissue (6) induction of hypoxia of myocardial tissue

26. A method for preparation of an animal model with myocardial infarction except for human, comprising inducing myocardial infarction of the animal by implanting an implantable hollow construct for inducing myocardial infarction into inside of a target blood vessel of the animal, wherein the implantable hollow construct for inducing myocardial infarction is a cylindrical hollow construct with a through-hole extended in a longitudinal direction, which has an outer diameter ranging 70% to 130% of an inner diameter of the target blood vessel and an inner diameter with a certain size to allow blood flow to pass through.

27. A method of screening a substance for prevention or treatment of a myocardial infarction, comprising administering a candidate substance for prevention or treatment of the myocardial infarction to the animal model with myocardial infarction according to claim 21; and selecting the candidate substance as a substance for prevention or treatment of myocardial infarction, when myocardial infarction of the animal model is improved after administering the candidate substance.

28. The method of screening according to claim 27, wherein the improvement of myocardial infarction is due to one or more of the following (1) to (7): (1) maintenance or increase of diameter of a left anterior descending artery (2) maintenance or increase of ejection fraction (3) alleviation or delay of necrosis of cardiac tissue (4) alleviation or delay of abnormal morphological changes of cardiomyocytes (5) alleviation or delay of collagen accumulation inside myocardial tissue (6) alleviation or improvement of hypoxia of myocardial tissue (7) increase in survival rate compared to a control group not administered with the candidate substance.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0086] FIG. 1a is a drawing which shows the overall view of the 3-dimensional bioprinting system according to one embodiment of the present invention.

[0087] FIG. 1B is a drawing which shows the shapes and outer diameter and inner diameter of the hollow constructs for inducing myocardial infarction in various sizes prepared according to one embodiment of the present invention.

[0088] FIG. 1c is a schematic diagram which shows the process of injection of the hollow construct for inducing myocardial infarction placed in a coronary artery according to one embodiment of the present invention.

[0089] FIG. 1d is a drawing which shows the figure of the front portion and side portion of the hollow construct for inducing myocardial infarction according to one embodiment of the present invention.

[0090] a of FIG. 2 is a drawing which shows the initial cardiac angiography result of the animal model in which the hollow construct for inducing myocardial infarction according to one embodiment of the present invention is implanted, and b of FIG. 2 is a drawing which shows the cardiac angiography result after placing the hollow construct, and the position of the hollow construct is indicated by a white arrow, and c of FIG. 2 is a drawing which shows the cardiac angiography result which confirms partial blood flow blocking induced by the hollow construct.

[0091] FIG. 3a is a graph which shows the change in the coronary artery inner diameter by the hollow construct for inducing myocardial infarction according to one embodiment of the present invention.

[0092] FIG. 3b is a graph which shows the change in the ejection fraction of left ventricle by the hollow construct for inducing myocardial infarction according to one embodiment of the present invention.

[0093] FIG. 4 is a drawing which shows the result of observing the change in the left ventricle wall after in 6 weeks after inducing myocardial infarction according to one embodiment of the present invention through various biopsy methods (Bar: 100 μm), and a of FIG. 4 is a drawing which shows the TTC staining result, and b and e of FIG. 4 are drawings which show the H&E staining result, and c and f of FIG. 4 are drawings which show the picrosirius red staining result, and d and g of FIG. 4 are drawings which show the immunohistochemical staining result.

MODE FOR INVENTION

[0094] Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are intended to illustrate the present invention only, but the scope of the present invention is not limited by these examples.

Example 1: Production of Hollow Constructs for Inducing Myocardial Infarction

[0095] (1) Production of Hollow Constructs Having Various Outer Diameters and Inner Diameters

[0096] The hollow construct for inducing myocardial infarction according to one embodiment of the present invention can be produced by various materials according to the purpose, and in the present example, illustratively, a PCL (Polycaprolactone) polymer with excellent biocompatibility was used as a material.

[0097] The PCL polymer melt was injected at the polymer extrusion head at the top of the 3D bioprinting system capable of temperature and pressure control under the condition of the syringe internal temperature of 70° C. and pneumatic pressure of 550 kPa, and the PCL melt was extruded into a micro-size through a nozzle with a dimeter of 150 μm to prepare cylindrical hollow constructs having a through-hole in the center were prepared in various sizes. A nozzle transfer path for the cylindrical hollow constructs was produced using a G-code production program, and a continuous and stable hollow construct production process was established by giving a stage transfer rate of 60 mm/min during polymer extrusion (FIG. 1a). FIG. 1a is a drawing which shows the overall view of the 3-dimensional bioprinting system according to one embodiment of the present invention.

[0098] Myocardial infarction is disease that generally occurs when blood flow is blocked due to deformation inside the left anterior descending artery (LAD) and sufficient oxygen and nutrients are not received therefrom, and therefore, in order to imitate the environment where myocardial infarction occurs, a hole was formed in the center of the implant, and the internal shape of the coronary artery and the hollow construct form which minimizes damages from blood pressure were considered, and it was possible to customize with various sizes of outer diameters and inner diameters, so it was possible to block unexpected variables that might occur in an animal experiment (FIG. 1B). FIG. 1B is a drawing which shows the form and outer diameter and inner diameter of the hollow constructs with various sizes prepared according to one embodiment of the present invention. As shown in FIG. 1B, the length of the hollow construct was prepared as 3 mm, and the outer diameter and inner diameter of the hollow construct could be controlled up to a 0.1 mm unit. The conventional myocardial infarction model production method induced myocardial infarction by a method of temporarily blocking blood flow by inserting a catheter into an artery to induce myocardial damage and then removing the catheter again, but in the process of removing the catheter, unintentional physical damage to blood vessels or tissues may occur, which may lead to a decrease in the survival rate, and due to the nature of the implant to be inserted into a very thin blood vessel, even a light change in the size of the implant may affect induction of myocardial infarction. Therefore, the method for production according to one embodiment of the present invention could finely adjust the size of the implant for inducing myocardial infarction and be individual-specifically customized, and thus, could induce myocardial infarction with high stability.

[0099] (2) Production of Hollow Construct Customized to Target Blood Vessel

[0100] Seven three-month-old Yorkshire×Landrace F1 crossbred castrated male pigs were prepared, and the inner diameters of left anterior descending arteries were measured through quantitative coronary angiography. The measured inner diameters of the left anterior descending artery of the 7 pigs were shown in Table 1.

TABLE-US-00001 TABLE 1 Left anterior descending artery inner Implant outer Implant inner Sample NO. diameter (mm) diameter (mm) diameter (mm) 1 1.94 2.10 0.30 2 2.1 2.40 0.30 3 2.13 2.10 0.30 4 1.71 1.80 0.50 5 2.05 1.80 0.50 6 2.35 2.40 0.30 7 1.98 2.10 0.30 Standard 0.20 — — deviation

[0101] As shown in Table 1, considering that the standard deviation of the inner diameter of the left anterior descending artery of each individual was shown as about 0.20 mm, and the average inner diameter of the left anterior descending artery was very thin as about 2 mm, the deviation was very large, so it could be found that it was necessary to specifically prepare an implant for inducing myocardial infarction for each individual. Accordingly, the outer diameter and inner diameter of the implant was prepared suitable for the left anterior descending artery inner diameter of each individual shown in Table 1, and the outer diameter and inner diameter of the hollow construct specific to each individual were shown in Table 1. The outer diameter of the hollow construct was set to be 70% to 130% of the value based on the inner diameter of the left anterior descending artery of each individual, and the inner diameter was set to be 10% to 40% of the implant outer diameter considering the probability of death of each individual and appropriateness of myocardial induction that could be induced by partial blood flow blockage.

Example 2: Implantation of Hollow Construct for Inducing Myocardial Infarction and Production of Animal Model

[0102] The hollow construct for inducing myocardial infarction specific to an individual prepared in Example 1 was fixed to the center of the guide wire at the top of the balloon expansion catheter and implanted so as to be placed from the carotid artery to the left anterior descending artery (LAD) of each individual (FIG. 1c and FIG. 1d). This implantation method could achieve a high survival rate after inducing myocardial infarction, by minimizing a surgical process and partially blocking generated blood flow. FIG. 1c is a schematic diagram which shows the process of injection of the hollow construct placed to a coronary artery, and FIG. 1d is a drawing which shows the figure of the front portion and side portion of the hollow construct.

Example 3: Confirmation of Myocardial Infarction Induction of Animal Model with Myocardial Infarction

[0103] (1) Observation of Blood Flow Inside Coronary Artery

[0104] In order to confirm whether the hollow construct prepared in Example 1 performs substantial blood flow blockage in the animal model with myocardial infarction, an angiocardiography test was performed before implantation of the hollow construct (a of FIG. 2), immediately after follow implantation of the hollow construct (b of FIG. 2) and in 5 weeks after implantation of the hollow construct (c of FIG. 2), respectively, and the blood flow inside the coronary artery was observed and shown in FIG. 2. As indicated by a white arrow in b of FIG. 2, the hollow construct used in the experiment was placed accurately on the position of the left anterior descending arteries of all pigs. As shown in c of FIG. 2, by inducing partial blood flow up to 5 weeks after implantation, stable myocardial infarction was successfully induced.

[0105] (2) Observation of Change in Heart Function

[0106] The change in heart function due to the hollow construct was observed through echocardiography and quantitative coronary angiography. Table 2 shows the change in the coronary artery inner diameter after implanting the hollow construct according to one embodiment of the present invention, and this was graphed and shown in FIG. 3a. As shown in Table 2 and FIG. 3a, the inner diameter of the left anterior descending artery was significantly reduced over time after applying the hollow construct. In addition, Table 3 shows the change in the ejection fraction of the left ventricle after implanting the hollow construct according to one embodiment of the present invention, and this was graphed and shown in FIG. 3b. As shown in Table 3 and FIG. 3b, the most important index for evaluating heart function, the ejection fraction (EF) was also reduced by about 30% in 6 weeks after implanting the hollow construct, showing the numerical value of severe myocardial infarction was shown, and quantitative suitability of the large animal-based myocardial infarction model produced by the 3-dimensional bioprinting technology according to one embodiment of the present invention was verified. In FIG. 3a and FIG. 3b, the baseline means the blood vessel inner diameter before implantation.

TABLE-US-00002 TABLE 2 Left anterior descending Left anterior descending artery inner artery inner diameter Classification diameter (mm) reduction rate (%) Baseline (before 2.04 ± 0.18 — implantation) Immediately after 1.61 ± 0.20 21.08 implantation In 6 weeks after 1.08 ± 0.05 47.06 implantation

TABLE-US-00003 TABLE 3 Ejection Ejection fraction Classification fraction (%) reduction rate (%) Baseline (before implantation) 67.98 ± 3.81 — In 1 week after implantation 52.40 ± 4.35 22.92 In 2 weeks after implantation 45.41 ± 4.45 33.20 In 6 weeks after implantation 39.09 ± 3.31 42.50

[0107] (3) Histopathological Test

[0108] Through a histopathological test, whether the method for inducing myocardial infarction according to one embodiment of the present invention successfully induced myocardial infarction was confirmed. The histopathological test is classified into 4 kinds of staining methods such as histological test of TTC (Triphenyltetrazolium chloride), H&E (Haematoxylin and Eosin), picrosirius red, and an immunohistochemical test of HIF-1 Alpha.

[0109] First, it was possible to observe the difference in the portion where myocardial infarction was induced compared to general tissues through TTC staining, and as shown in a of FIG. 4, as the staining around the wall of the left ventricle was different, it was confirmed that macroscopic necrosis occurred in the corresponding area. Specifically, the necrosis rate (%) was measured and averaged by calculating the volume of the TTC-stained myocardial infarction site compared to the total left ventricle volume of 5 myocardial infarction models prepared in Example 1, and as a result, it could be confirmed that macroscopic necrosis with a necrosis rate of about 33(%) or more occurred (Table 4).

TABLE-US-00004 TABLE 4 Sample NO. Necrosis rate (%) 1 26.54 2 24.03 3 27.14 4 54.91 5 31.60 Average 32.84 ± 11.30

[0110] In addition, the result of performing H&E staining at the initial stage and in 6 weeks after implantation of hollow construct, respectively, was shown in b of FIG. 4 (initial stage of implantation) and e of FIG. 4 (in 6 weeks after implantation). As shown in e of FIG. 4, an abnormal morphological change of cardiomyocytes compared to normal tissues could be observed microscopically.

[0111] Furthermore, the result of performing picrosirius red staining at the initial stage and in 6 weeks after implantation of hollow construct, respectively, was shown in c of FIG. 4 (initial stage of implantation) and f of FIG. 4 (in 6 weeks after implantation). As shown in f of FIG. 4, accumulation of collagen inside the necrotic myocardial tissue could be observed through the picrosirius red staining, and loss of function of general myocardial tissues due to myocardial infarction induction could be confirmed.

[0112] Finally, the result of performing immunohistochemical staining at the initial stage and in 6 weeks after implantation of hollow construct, respectively, was shown in d of FIG. 4 (initial stage of implantation) and g of FIG. 4 (in 6 weeks after implantation). As shown in g of FIG. 4, expression of HIF-1 alpha after implantation of hollow construct was clearly observed, and therefore, it could be confirmed that hypoxia occurred due to myocardial infarction.

Example 4: Survival Rate Measurement

[0113] In order to confirm how successfully the method for producing an animal model with myocardial infarction according to one embodiment of the present invention produces an animal model with myocardial infarction, by a conventional method for producing an animal model with myocardial infarction as a comparative example, and the method for producing an animal model with myocardial infarction according to one embodiment of the present invention, animal models of 7 individuals, respectively, were prepared, and the survival rate after 5 weeks was measured and shown in Table 5. The myocardial infarction model prepared by the conventional method was prepared by a method of temporarily blocking blood flow inside a carotid artery using a generally used balloon catheter (Comparative example 1).

TABLE-US-00005 TABLE 5 Survival rate Classification after 5 weeks Example 2 90% Comparative Example 1 50%

[0114] As shown in Table 5, the method for production according to one embodiment of the present invention showed a significantly high survival rate, and there was a problem in that different from mice for experiments, the pigs had a characteristic of a high degree of deviation of the myocardial structure and function, and the trend of numerical values for the blood flow and inner diameter of the coronary artery of each porcine individual was various, so it was difficult to perform a stable animal experiment, but the present invention is a model for studying myocardial infarction by customizing the diameter and through-hole of a hollow construct for inducing myocardial infarction in a unit of several tens of micrometers, and could stably secure various data and could control quantitative numerical values of the data, and could place on not only left anterior descending arteries but also inside other blood vessels, and thus, it could successively imitate myocardial infarction.

Example 5: Change in Necrosis Rate and Ejection Rate Depending on Through-Hole Size of Hollow Construct

[0115] By comparing necrosis rate and ejection fraction values after producing hollow constructs having various through-hole sizes and implanting them, an effect of the through-hole size on inducing myocardial infarction was investigated. Specifically, after producing a hollow construct having a through-hole in a size of 24% of the hollow construct diameter (Example 5-1) and a hollow construct having a through-hole in a size of 14% of the hollow construct diameter (Example 5-2), they were implanted to 3-month-old Yorkshire×Landrace F1 crossbred castrated male pigs, respectively. In 28 days after implantation, by the substantially same method as (3) of Example 3, the necrosis rate and ejection fraction were measured and shown in Table 6.

TABLE-US-00006 TABLE 6 Classification Necrosis rate (%) Ejection fraction (%) Example 5-1 20 51 Example 5-2 30 39

[0116] As shown in Table 6, as the size of the through-hole was relatively small, a more severe ejection fraction was measured due to an increase in myocardial blood pressure received from the inside of the left anterior descending coronary artery, and an increase in myocardial necrosis caused thereby could be observed.