THREE-DIMENSIONAL STRUCTURE FOR CARDIAC MUSCULAR TISSUE REGENERATION AND MANUFACTURING METHOD THEREFOR

Abstract

The present invention provides a preparation method of a three-dimensional construct for regenerating a cardiac muscle tissue comprising; a step of forming a three-dimensional construct by printing and crosslinking the first bioprinting composition comprising a tissue engineering construct forming solution containing decellularized extracellular matrix and a crosslinking agent, and cardiac progenitor cells, and the second bioprinting composition comprising the tissue engineering construct forming solution, mesenchymal stem cells and a vascular endothelial growth factor, to arrange the first bioprint layer and the second bioprint layer alternately; and a step of obtaining a crosslink-gelated three-dimensional construct by thermally gelating the crosslinked three-dimensional construct, and a three-dimensional construct for regenerating a cardiac muscle tissue, and the preparation method according to the present invention not only equally positions the cardiac progenitor cells in the construct but also implements a vascular network composed of vascular cells in the construct, so that the viability of cells can be maintained for a long time and the cell transfer efficiency into the myocardium can be significantly improved.

Claims

1. A method of preparing a three-dimensional construct for regenerating a cardiac muscle tissue comprising, (a) forming a three-dimensional construct by printing a first bioprinting composition comprising a tissue engineering construct forming solution containing decellularized extracellular matrix and cardiac progenitor cells, and a second bioprinting composition comprising the tissue engineering construct forming solution, mesenchymal stem cells and a vascular endothelial growth factor, to arrange the first bioprint layer and the second bioprint layer alternately; and (b) carrying out the thermal gelation for the three-dimensional construct, wherein the (a) step is performed at a temperature at which no thermal gelation occurs, and the (b) step is performed at a temperature at which thermal gelation occurs.

2. The preparation method of claim 1, wherein the decellularized extracellular matrix is derived from a cardiac tissue.

3. The preparation method of claim 1, wherein the decellularized extracellular matrix of the first bioprinting composition or the second bioprinting composition is contained in an amount of 1 to 4% by weight based on the total weight of the first bioprinting composition or the second bioprinting composition.

4. (canceled)

5. The preparation method of claim 1, wherein the cardiac progenitor cells are contained at a range of 10.sup.5 to 10.sup.8 cells/ml in the first bioprinting composition, or the mesenchymal stem cells are contained at a range of 10.sup.5 to 10.sup.8 cells/ml in the second bioprinting composition.

6. (canceled)

7. The preparation method of claim 1, wherein the vascular endothelial growth factor is contained in a range of 50 to 1000 ng/ml in the second bioprinting composition.

8. The preparation method of claim 1, wherein the tissue engineering construct forming solution has a pH of 6.5 to 7.5, and further comprises an acid and a protease.

9. (canceled)

10. The preparation method of claim 8, wherein the tissue engineering construct forming solution, further comprises; one or more kinds of acids selected from the group consisting of acetic acid and hydrochloric acid; one or more kinds of proteases selected from the group consisting of pepsin and matrix metalloproteinase (MMP); and a pH control agent.

11. The preparation method of claim 1, wherein the first bioprinting composition and the second bioprinting composition independently, further comprise, one or more kinds of cells selected from the group consisting of endothelial progenitor cells, endothelial cells and cardiomyocytes; and one or more kinds of growth factors selected from the growth factor group consisting of fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), angiopoietin-1, transforming growth factor beta (TGF-), erythropoietin (EPO), stem cell factor (SCF), epidermal growth factor (EGF) and colony stimulating factor (CSF).

12. The preparation method of claim 11, wherein the first bioprinting composition and the second bioprinting composition independently, further comprise, one or more kinds of enzymes selected from the enzyme group consisting of matrix metalloproteinase (MMP) and tissue inhibitor matrix metalloproteinase (TIMP).

13. The preparation method of claim 1, wherein the first bioprinting composition and the second bioprinting composition independently, have a viscosity at a shear rate of 1 s.sup.1 measured at 15 C. in the range of 1 to 30 Pa.Math.S.

14. The preparation method of claim 1, wherein the first bioprinting composition further comprises the crosslinking agent.

15. The preparation method of claim 14, wherein the crosslinking agent is comprised independently in the first bioprinting composition and the second bioprinting composition in an amount of 0.001 to 0.1% by weight, based on the total weight of each composition.

16. The preparation method of claim 1, wherein the method further comprise a crosslinking reaction at a temperature of below 15 C.

17. (canceled)

18. The preparation method of claim 1, wherein the thermal gelation is performed at 25 C. to 37 C.

19. The preparation method of claim 1, wherein the three-dimensional construct for regenerating a cardiac muscle tissue has a thickness of 50 to 1000 m.

20. A three-dimensional construct for regenerating a cardiac muscle tissue, wherein the three-dimensional construct for regenerating a cardiac muscle tissue is formed by performing thermal gelation for a first bioprint layer and a second bioprint layer which are prepared by printing a first bioprinting composition and a second bioprinting composition, wherein the first bioprinting composition comprises a tissue engineering construct forming solution containing decellularized extracellular matrix, and cardiac progenitor cells, wherein the second bioprinting composition comprises the tissue engineering construct forming solution, mesenchymal stem cells and a vascular endothelial growth factor.

21. The three-dimensional construct for regenerating a cardiac muscle tissue of claim 20, wherein the first bioprint layer, the second bioprint layer or both are in a form of fibers, tapes, or fabrics.

22. The three-dimensional construct for regenerating a cardiac muscle tissue of claim 20, wherein the first bioprint layer and the second bioprint layer are independently in a form of unidirectional fibers, and the bioprint layer and the second bioprint layer are laminated so as to have a crossing angle with each other.

23. The three-dimensional construct for regenerating a cardiac muscle tissue of claim 20, wherein the three-dimensional construct for regenerating a cardiac muscle tissue has a thickness of 50 to 1000 m.

24. The three-dimensional construct for regenerating a cardiac muscle tissue of claim 20, wherein the three-dimensional construct for regenerating a cardiac muscle tissue has a modulus of 1 to 100 kPa at 1 rad/s.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0081] FIG. 1 is a photograph of the decellularized extracellular matrix (hdECM) obtained from a cardiac tissue according to one example of the present invention.

[0082] FIG. 2 is an optical microscope photograph and a tissue staining photograph of the decellularized extracellular matrix (hdECM) obtained from a cardiac tissue according to one example of the present invention

[0083] FIG. 3 is a mimetic diagram of the three-dimensional construct prepared according to one example of the present invention.

[0084] FIG. 4 is photographs showing a shape of the prepared three-dimensional construct using PCL framework according to one example of the present invention.

[0085] FIG. 5 is an echocardiography m-mode photograph taken before and 8 weeks after construct transplantation, after transplanting the three-dimensional construct prepared according to one example of the present invention into a rat myocardial infarction model.

[0086] FIG. 6 is a photography showing cardiac function measured before, 4 weeks and 8 weeks after construct transplantation, after transplanting the three-dimensional construct prepared according to one example of the present invention into a rat myocardial infarction model, in numerical form.

[0087] FIG. 7 is the result of performing a histological analysis by sacrificing after 8 weeks, after transplanting the three-dimensional construct prepared according to one example of the present invention, and is a photograph observing the degree of fibrosis of the myocardial tissue and the change of myocardial wall thickness by performing masson's trichrome staining.

[0088] FIG. 8 is a photograph showing the regeneration degree of functional myocardium and the regeneration degree of blood vessel of the myocardial infarction region by immunofluorescence staining according to one example of the present invention.

[0089] FIG. 9 is a photograph showing cardiac progenitor cells uniformly positioned in the three-dimensional construct for regenerating a cardiac muscle tissue prepared according to one example of the present invention.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0090] Hereinafter, the present invention will be described in more detail by the following examples. However, the examples are given for illustrating the present invention only, and the scope of the present invention is not limited by the examples.

Preparation Example 1: Preparation of the First Bioprinting Composition

[0091] 1-1: Preparation of Decellularized Extracellular Matrix

[0092] Decellularized extracellular matrix was prepared according to a method disclosed in Falguni Pati, et al., Nat Commun. 5, 3935 (2014) using porcine cardiac tissues (hereinafter, hdECM). The prepared hdECM was finally lyophilized and kept frozen before used. The optical microscope photograph and tissue staining photograph were shown in FIG. 2.

[0093] 1-2: Preparation of Pre-Gel Form of a Tissue Engineering Construct Forming Solution

[0094] Liquid nitrogen was poured to the obtained lyophilized hdECM and it was crushed with mortar and a pestle. After the obtained hdECM powder (330 mg) was added to 0.5M acetic acid aqueous solution (10 ml), and pepsin (33 mg) (P7125, Sigma-Aldrich) was added, it was stirred for 48 hrs at a room temperature. Maintaining the temperature of the obtained solution below 10 C., riboflavin (2 mg) was added and 10 NaOH solution which was cooled below 10 C. was added, thereby controlling pH as approximately pH 7. The obtained pre-gel form of a tissue engineering construct forming solution was refrigerated at approximately 4 C.

[0095] 1-3: Preparation of a Printing Composition Containing Pre-Gel Form of a Tissue Engineering Construct Forming Solution and Cells

[0096] Just before conducting a printing process, 510.sup.6 cells/ml of cardiac progenitor cells were added to the pre-gel form of the tissue engineering construct forming solution, thereby preparing the first bioprinting composition. The cardiac progenitor cells were obtained from human cardiac muscle tissue-derived cardiac progenitor cells, Pusan National University School of Medicine, Department of Physiology.

Preparation Example 2: Preparation of the Second Bioprinting Composition

[0097] 2-1: Preparation of Decellularized Extracellular Matrix

[0098] Decellularized extracellular matrix was prepared according to a method disclosed in Falguni Pati, et al., Nat Commun. 5, 3935 (2014) using porcine cardiac tissues (hereinafter, hdECM). The prepared hdECM was finally lyophilized and kept frozen before used. The optical microscope photograph and tissue staining photograph were shown in FIG. 2.

[0099] 2-2: Preparation of Pre-Gel Form of a Tissue Engineering Construct Forming Solution

[0100] Liquid nitrogen was poured to the obtained lyophilized hdECM and it was crushed with mortar and a pestle. After the obtained hdECM powder (330 mg) was added to 0.5M acetic acid aqueous solution (10 ml), and pepsin (33 mg) (P7125, Sigma-Aldrich) was added, it was stirred for 48 hrs at a room temperature. Maintaining the temperature of the obtained solution below 10 C., riboflavin (2 mg) was added and 10 NaOH solution which was cooled below 10 C. was added, thereby controlling pH as approximately pH 7. The obtained pre-gel form of a tissue engineering construct forming solution was refrigerated at approximately 4 C.

[0101] 2-3: Preparation of a Printing Composition Containing Pre-Gel Form of a Tissue Engineering Construct Forming Solution and Cells

[0102] Just before conducting a printing process, 510.sup.6 cells/ml of mesenchymal stem cells and vascular endothelial growth factor (serial number: 293-VE-10, R&D systems company) (100 ng/ml) were added respectively to the pre-gel form of the tissue engineering construct forming solution, thereby preparing the second bioprinting composition. The mesenchymal stem cells were inferior turbinate-derived mesenchymal stem cells obtained from Catholic University of Korea, Department of Otolaryngology Medical Science.

Comparative Example 1: Preparation of a Three-Dimensional Construct for Tissue Engineering

[0103] A three-dimensional construct was fabricated according to the method disclosed in Falguni Pati, et al., Nat Commun. 5, 3935 (2014) using the first bioprinting composition obtained from the preparation example 1.

[0104] Specifically, polycaprolactone (PCL) framework was loaded on the syringe (the first syringe) of multi-head tissue and organ printing system (Jin-Hyung Shim et al., J. Micromech. Microeng. 22 085014 (2012)), and it was heated at approximately 80 C. to melt a polymer. The pre-gel form of first bioprinting composition obtained from the preparation example 1 was loaded on another syringe (the second syringe), and the temperature was maintained below approximately 10 C. A thin PCL framework having below approximately 100 um line width, approximately 300 um of gap and 120 um of thickness by putting approximately 600 kPa of pneumatic pressure to the first syringe, and after spraying the contents of the second syringe on the PCL framework, approximately 360 nm of UVA was radiated for 3 min, thereby crosslinking. Then, a layer-by-layer process through spraying the contents of the second syringe and the crosslinking was conducted, thereby forming a three-dimensional construct shape. The obtained three-dimensional construct shape was thermally gelated by putting into approximately 37 C. of incubator (humid incubator) and maintaining for 30 min, thereby preparing a three-dimensional construct (refer to CPC printed). The obtained three-dimensional construct has approximately 300 to 400 um thickness.

Example 1: Preparation of a Three-Dimensional Construct for Tissue Engineering

[0105] A three-dimensional construct was fabricated using the first bioprinting composition and the second bioprinting composition obtained from the preparation examples 1 and 2.

[0106] Specifically, polycaprolactone (PCL) framework was loaded on the syringe (the first syringe) of multi-head tissue and organ printing system (Jin-Hyung Shim et al., J. Micromech. Microeng. 22 085014 (2012)), and it was heated at approximately 80 C. to melt a polymer. The pre-gel form of the first bioprinting composition obtained from the preparation example 1 and the second bioprinting composition obtained from the preparation example 2 were loaded on another syringe (the second and third syringes, respectively), and the temperature was maintained below approximately 10 C. A thin PCL framework having below approximately 100 um line width, approximately 300 um of gap and 120 um of thickness by putting approximately 600 kPa of pneumatic pressure to the first syringe, and after spraying the contents of the second syringe on the PCL framework, approximately 360 nm of UVA was radiated for 3 min, thereby crosslinking. Then, a layer-by-layer process through spraying the contents of the second and third syringes and the crosslinking was conducted, thereby forming a three-dimensional construct shape.

[0107] The obtained three-dimensional construct shape was thermally gelated by putting into approximately 37 C. of incubator (humid incubator) and maintaining for 30 min, thereby preparing a three-dimensional construct for tissue engineering (refer to CPC/MSC printed). The obtained three-dimensional construct has approximately 300 to 400 um thickness, and its shape was same as FIG. 3.

Test Example 1. Measurement of Complex Modulus with or without Riboflavin Addition

[0108] After crosslinking the pre-gel form of solution obtained from the preparation example 1 by irradiating approximately 360 nm of UVA for 3 min, it was put into approximately 37 C. of incubator (humid incubator) and maintained for 30 min, thereby inducing a thermal gelation and forming a hydrogel (crosslinked hydrogel A). In addition, a pre-gel form of a tissue engineering construct forming solution prepared according to the same method as the preparation example 1 except using no riboflavin was put into approximately 37 C. of incubator (humid incubator) and maintained for 30 min, thereby inducing a thermal gelation and forming a hydrogel (hydrogel B).

[0109] Then, as to the obtained respective hydrogel, complex modulus at 1 rad/s frequency was measured and the result was as the following Table 1.

TABLE-US-00001 TABLE 1 modulus classification (n = 3, 1 rad/s) crosslinked hydrogel A 10.58 3.4 kPa hydorgel B 0.33 0.13 kPa

[0110] As can be seen from the result of Table 1, the crosslinked hydrogel A according to the present invention has 10.58 kPa modulus at 1 rad/s, and it was demonstrated that it has approximately over 30-fold strength enhancement by crosslinking than the hydrogel B where only gelation treatment was carried out without crosslinking.

Test Example 2

[0111] The chest of 4 to 5 week old rats (weight: 30030 g) was incised and permanent ligation of Left Anterior Descending artery (LAD) was performed, thereby inducing myocardial infarction.

[0112] Seven days later, the three-dimensional construct prepared in the comparative example 1 (CPC printed) and the three-dimensional construct prepared in the example 1 (CPC/MSC printed) were cut into 8 mm diameters, respectively, and after transplanted into myocardial infarction region, sutured (respectively, n=7). The result of confirming left ventricle diameter (LV diameter) by echocardiography at 4 and 8 weeks after transplantation was shown in FIG. 5, and the result of performing significance evaluation between experimental samples by the student T-test by calculating left ventricle ejection fraction (LVEF) and fractional shortening (FS) which show myocardial functions was shown in FIG. 6.

[0113] As can be seen in FIGS. 5 and 6, the left ventricular internal diameter was significantly reduced compared with the experimental group (control group) which had not been subjected to any treatment after 4 and 8 weeks after transplantation of the three-dimensional construct according to example 1. In addition, it can be observed that the left ventricular systolic function was significantly increased in the CPC/MSC group compared to the CPC group, and this shows that a cardiac remodeling was inhibited by the construct.

[0114] After harvesting the heart of rats at 8 weeks after transplantation and collecting tissues at the same location (approximately point from the apex of the heart in the base direction) per object (3 um thickness), Masson's trichrome staining was carried out. The heart muscle (red) and the scar tissue of the myocardial infarction region (blue) were discriminated using the staining method, and the result was shown in FIG. 7.

[0115] As can be seen from FIG. 7, the thickness of the inner wall of the left ventricle after 8 weeks of the three-dimensional construct transplantation prepared according to the present invention was kept thicker than the control group, and the formation of scar tissues of the myocardial infarction region was less. In addition, in contrast to the three-dimensional construct formed by only the first bioprinting composition (CPC printed) as in the comparative example 1, when the three-dimensional construct formed by the first bioprinting composition and the second bioprinting composition (CPC/MSC printed) as the example 1 was transplanted, the formation of scar tissues was more effectively inhibited, and the thickness of the inner wall of the left ventricle was not thinned and was maintained.

[0116] As can be seen from FIG. 8, the expression of (3-myosin heavy chain (MHC) representing contractibility of cardiac muscle tissue and cluster of differentiation 31 (CD31) representing angiogenesis on the myocardial infarction region at which the three-dimensional construct prepared according to the present invention was transplanted was significantly increased, and in particular, when the three-dimensional construct (CPC/MSC printed) (example 1) formed by the first bioprinting composition and the second bioprinting composition was transplanted, the effect was more maximized.

[0117] Thus, it was demonstrated that the three-dimensional construct prepared by the preparation method of the present invention can maintain the viability of cells for a long time by implementing a vascular network in the construct (that is, by implementing a microenvironment of the cardiac muscle tissue effectively), thereby significantly improving the cell transfer efficiency into the myocardium.