CN113274555B

CN113274555B - Artificial ventricle with bionic spiral orientation microstructure and preparation method thereof

Info

Publication number: CN113274555B
Application number: CN202110598810.1A
Authority: CN
Inventors: 熊卓; 方永聪; 张婷; 鲁冰川
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2021-05-31
Filing date: 2021-05-31
Publication date: 2022-05-03
Anticipated expiration: 2041-05-31
Also published as: CN113274555A

Abstract

The invention discloses an artificial ventricle with a bionic spiral orientation microstructure and a preparation method thereof. The method comprises the following steps: s1, alternately extruding the myocardial fiber yarn carrying the myocardial cells and the hollow vascular fiber yarn carrying the angiogenesis cells by adopting a coaxial nozzle A and a coaxial nozzle B in the calcium ion cross-linking liquid, and collecting by a rotating ellipsoidal collector to realize 3D printing; meanwhile, the coaxial nozzles A and B are transversely moved in a reciprocating manner, and the deflection angle is changed layer by layer to obtain a fiber arrangement structure with spiral orientation; and S2, demolding from the ellipsoidal collector to obtain the artificial ventricle after the 3D printing is finished, and performing secondary crosslinking. The invention provides a new research model for researching myocardial regeneration and function regulation, the constructed heart physiology or pathology model is closer to the real state of a human body, the model is expected to replace an animal model in the aspects of heart disease research, drug screening and the like, and the invention has important guiding significance for developing a novel treatment strategy for repairing damaged myocardium.

Description

Artificial ventricle with bionic spiral orientation microstructure and preparation method thereof

Technical Field

The invention relates to an artificial ventricle with a bionic spiral orientation microstructure and a preparation method thereof, belonging to the technical field of tissue engineering and biological manufacturing.

Background

Heart disease has been the first killer of human health. At present, the clinically effective method for treating heart failure is heart transplantation, which can prolong the life of patients, but has problems such as immune rejection, limited donor sources and postoperative complications. The tissue engineering myocardium constructed in vitro is used as a substitute treatment means for heart diseases, and has urgent clinical requirements and wide application prospects. However, the heart is a relatively complex human visceral soft tissue in both structure and function compared to other tissues/organs, making the reconstruction of myocardial tissue in vitro particularly complex and technically difficult due to its non-regenerative and unique structural features. This is mainly reflected in the following aspects:

1) the myocardial tissue has the structural characteristics of directional ordered arrangement and complex spiral winding, so that the heart obtains higher blood pumping efficiency: the myocardial cells are connected with each other through intercalated disks to form myocardial fibers, the myocardial fibers are arranged in an oriented and ordered manner, and the included angles between the fibers and the equator are different according to different positions, so that the myocardial fibers are spirally tightened in a towel twisting manner during the beating of the heart. Further, Torrent-Guasp and colleagues found by anatomical analysis that the myocardium of the entire heart was spirally wound from a continuous strip of myocardial tissue (Torrent-Guasp F. the structure and function of the heart. Revista Epanola De Cardiology, 1998,51(2): 91-102.).

2) Myocardial tissue has an abundant vascular network, ensuring the supply of oxygen and nutrients: the heart has a complex blood supply system, coronary arteriovenous vessels are distributed on the outer surface of the heart, continuously branch off to the myocardium and are connected with a dense capillary network (the density is about 2500/mm)²). The distance between the capillary and the cell is not more than 100 μm, and the capillary is mostly distributed parallel to the direction of the myocardial fiber, so that the material exchange is more sufficient (Fleischer, S., D.N. Tavakol and G.Vunjaku Novakovic. from organisms to capsules: advanced to engineering human vascular tissue, advanced Functional Materials 2020.30(37): p.1910811).

With the rapid development of decades, a lot of new technologies including cell-gel casting technology, cell sheet technology, organoid technology, biological 3D printing and the like are developed for the in vitro construction of engineered myocardial tissues, so that the in vitro constructed functional myocardial tissues gradually develop from simple 3D strips, myocardial patches and other structures to ventricular and cardiac models with chamber structures (Li RA, Keung W, Cashman TJ, et al. However, current artificial ventricular constructions also focus primarily on macroscopic chamber structures, making it difficult to reproduce the spiral-oriented features and microvascular networks on a microscale, such that the pulsatile function of the ventricles is far from that of normal ventricles.

In addition, some researchers have simulated the helically oriented microstructured features of the heart by stacking oriented fibers or sheet scaffolds one on top of the other and seeding cardiomyocytes (Wu, Y., et al., International oven aligned connected negative nanofiber yann/hydrogel composite scaffold for engineered 3D cardiac isotope ACS Nano,2017.11(6): p.5646-5659.); however, these studies are limited to myocardial thin slice tissue (only 5-10 cells thick) and have not been reproducible on a macroscopic scale on the ventricles.

Therefore, the current myocardial tissue in-vitro reconstruction has a key technical bottleneck that the micro-scale spiral oriented microstructure and the macro-scale cavity structure are difficult to be considered at the same time, and needs to be realized by innovative breakthroughs of a forming process.

Disclosure of Invention

The invention aims to provide a novel artificial ventricle with a bionic spiral oriented microstructure and a preparation method thereof, the artificial ventricle structure prepared by the invention simulates the spiral oriented microstructure and the blood vessel distribution of myocardial fiber bundles of human heart physiology, provides a foundation for researching the physiological structure and the function reconstruction mechanism of the heart, can also provide an important technical means for functional tissue reconstruction breaking through the technical bottleneck in the field of myocardial tissue engineering, and has important scientific significance and clinical application value in the aspects of myocardial infarction repair, heart disease research and the like.

The invention provides a preparation method of an artificial ventricle with a bionic spiral orientation microstructure, which comprises the following steps:

s1, alternately extruding the myocardial fiber filaments carrying the myocardial cells and the hollow vascular fiber filaments carrying the angiogenic cells by a coaxial nozzle A and a coaxial nozzle B in the calcium ion cross-linking liquid, and collecting by a rotating ellipsoidal collector to realize 3D printing; meanwhile, the coaxial spray head A and the coaxial spray head B are transversely moved in a reciprocating manner, and the deflection angle is changed layer by layer to obtain a fiber arrangement structure with spiral orientation;

and S2, demolding from the ellipsoidal collector to obtain the artificial ventricle after the 3D printing is finished, and performing secondary crosslinking.

In the preparation method, the myocardial fiber filaments carrying the myocardial cells are obtained by cross-linking a calcium chloride solution and a biological ink A carrying the myocardial cells, wherein the calcium chloride solution is used as an external phase, and the biological ink A is used as an internal phase;

the hollow vascular fiber filament carrying the angiogenic cells is obtained by cross-linking a calcium chloride solution and biological ink B carrying the angiogenic cells, wherein the calcium chloride solution is used as an internal phase, and the biological ink B is used as an external phase;

the biological ink A and the biological ink B are both sodium alginate aqueous solution or a mixture of sodium alginate and at least one of natural polymer hydrogel and synthetic polymer hydrogel, namely the cellosilk is obtained by utilizing the instantaneous crosslinking characteristic of sodium alginate and calcium ions.

In the above preparation method, the natural polymer hydrogel may be at least one of gelatin, collagen, chitosan, silk fibroin, hyaluronic acid, fibrinogen, chondroitin sulfate, albumin, and their methacrylated products (such as methacrylated gelatin (GelMA), methacrylated sodium alginate (AlgMA), etc.);

the synthetic polymer hydrogel material is at least one of polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyethylene glycol diacrylate (PEGDA), polyethylene oxide (PEO), Polyacrylamide (PAM), polyacrylic acid (PAA), polyphosphazene PAMPS, poly N-isopropylacrylamide (PNIPAAm) hydrogels and methacrylic acylation products thereof (such as concave-arm polyethylene glycol acrylate (4-arm-PEG-AC), methacrylic acylated polyvinyl alcohol (PVAMA) and the like).

In the preparation method, in the biological ink A and the biological ink B, the mass-volume concentration of the sodium alginate can be 10-100 mg/mL, and the mass-volume concentration of the natural polymer hydrogel and/or the synthetic polymer hydrogel is 1.0-100 mg/mL;

the bio-ink A and the bio-ink B are preferably a ternary system of sodium alginate/fibrinogen/Matrigel, wherein the concentration of the sodium alginate is preferably 20mg/mL, the concentration of the fibrinogen is preferably 10mg/mL, and the volume fraction of the Matrigel is preferably 10%.

In the above preparation method, the cardiomyocytes are at least one of primary cardiomyocytes, embryonic stem cells and cardiomyocytes derived from induced pluripotent stem cells by directed differentiation;

in the biological ink A, the density of the myocardial cells is 10⁶/mL～10⁸Per mL, e.g. 2X 10⁷/mL；

The diameter of the myocardial fiber filament is 50-200 μm.

In the above preparation method, the angiogenic cell is at least one of an endothelial cell or an endothelial cell and a smooth muscle cell, a pericyte and a fibroblast;

in the bio-ink B, the density of the angiogenesis cells is 10⁵/mL～10⁸Per mL, e.g. 5X 10⁶/mL；

The outer diameter of the hollow blood vessel fiber is 200-1000 μm, and the thickness is 20-200 μm.

In the above preparation method, in step S1, the ratio of the printing layers of the myocardial fiber filaments and the hollow vascular fiber filaments is 20: 1-1: 1;

the mass-volume concentration of the calcium ion cross-linking solution is 10-200 mg/mL.

In the above preparation method, in step S1, the ellipsoidal collector is designed by simulating the topology of the left ventricle of a human;

the material of the ellipsoidal collector is silicon rubber, stainless steel or aluminum alloy;

the number of the 3D printing layers is 3-100;

the extrusion speeds of the coaxial spray head A and the coaxial spray head B are both 0.1-10 mm³The distance of the transverse reciprocating movement is 5-100 mm, and the movement speed is 0.01-10.0 mm/s;

the deflection angle of each layer is 1-30 degrees, and the rotation speed of the collector is 0.1-100 rpm.

In the above preparation method, in step S2, the secondary crosslinking is crosslinking between sodium alginate and calcium ions or any one of the following crosslinking modes:

temperature crosslinking, enzymatic crosslinking, photo crosslinking, covalent crosslinking, and ionic crosslinking.

Reconstruction of the structure, function of alternative tissues/organs has been a key challenge in the field of regenerative medicine. The controllable and ordered assembly method of the cell-loaded gel fiber yarns, provided by the invention, can construct the artificial ventricle with the bionic spiral oriented microstructure, and has the following advantages and prominent effects compared with the existing myocardial in-vitro reconstruction technology:

(1) the wet spinning and rotating 3D printing composite strategy provided by the invention firstly reconstructs bionic spiral winding and blood vessel channel of cardiac myocardium in vitro on a cavity scale, and provides a new technical means for constructing complex cardiac tissue and ventricle in vitro. On one hand, compared with the conventional micro-extrusion 3D printing process, the wet spinning has milder forming conditions (such as small shearing loss), and can prepare thinner cell-loaded gel fiber yarns, which is beneficial to inducing the directional arrangement of myocardial cells in the fiber yarns, thereby enhancing the functions of the myocardial fiber yarn units and widening the application potential thereof; on the other hand, the fiber filaments are orderly assembled layer by using a rotary 3D printing process, so that the bionic spiral winding and the blood vessel channel of the heart myocardium can be reconstructed in vitro on the scale of the cavity, and meanwhile, reference is provided for constructing other tissues and organs with the characteristics of oriented structures.

(2) The functionalized ventricle with the spiral oriented microstructure and the vascularized channel provided by the invention provides a new research model for researching myocardial regeneration and function regulation. Compared with the commonly used in-vitro models such as 2D culture, myocardial microtissue, organoid and the like, the artificial ventricle reproduces the chamber structure and beating function of the heart in vitro, the constructed heart physiology or pathology model is closer to the real state of a human body, the model is expected to replace an animal model in the aspects of heart disease research, drug screening and the like, and has important guiding significance for developing a novel treatment strategy for repairing the damaged myocardium.

Drawings

Fig. 1 is a schematic diagram of a coaxial nozzle for constructing a myocardial fiber filament carrying myocardial cells, wherein 1 is a calcium chloride solution, 2 is sodium alginate-based bio-ink carrying myocardial cells, and 3 is the cross-sectional shape of the myocardial fiber filament.

Fig. 2 is a schematic diagram of a coaxial nozzle for constructing a hollow vascular fiber filament carrying endothelial cells, wherein 4 is a calcium chloride solution, 5 is sodium alginate-based bio-ink carrying endothelial cells, and 6 is a cross-sectional shape of the hollow vascular fiber filament.

Fig. 3 is a schematic diagram of the artificial ventricle constructed by rotary 3D printing according to the present invention, in which 7 is a rotary collecting device, 8 is the artificial ventricle, and 9 is calcium ion cross-linked liquid.

Fig. 4 is a cross-section of an artificial ventricle constructed in accordance with the method of the present invention.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 in vitro construction of a Bionically vascularized myocardial Chamber

Human induced pluripotent stem cells are adopted for in vitro culture and induced differentiation to obtain the myocardial cells and vascular endothelial cells derived from the pluripotent stem cells.

A ternary material system of sodium alginate/fibrinogen/Matrigel is used, wherein the concentration of sodium alginate is 20mg/mL, the concentration of fibrinogen is 10mg/mL, and the volume fraction of Matrigel is 10%. Wherein, the instant crosslinking characteristic of sodium alginate and calcium ions is used as the first-stage crosslinking to ensure the rapid formation of the cellosilk structure; the mild crosslinking of fibrinogen and thrombin is used as the second-stage crosslinking to ensure the connection and fusion of the fibers; the Matrigel is used for improving the cell growth microenvironment by utilizing the characteristic that the Matrigel is rich in extracellular matrix proteins and growth factors so as to promote the growth, the spreading and the connection of myocardial cells and endothelial cells.

A stainless steel collector with a corresponding shape is designed by three-dimensional reconstruction of a human heart image and extraction of a topological structure of a left ventricle. Two different coaxial nozzle designs were used, in which the inner phase of coaxial nozzle A was cardiomyocytes (cell density 2X 10)⁷/mL) of sodium alginate/fibrinogen/Matrigel biological ink, and the external phase is 2% calcium chloride solutionLiquid (as shown in figure 1); the inner phase of the coaxial nozzle B is 2% calcium chloride solution, and the outer phase is endothelial cells (cell density is 5X 10)⁶mL) of sodium alginate/fibrinogen/Matrigel bio-ink (as shown in figure 2).

In a collecting tank containing 5% calcium chloride solution, using a double-nozzle to perform rotary 3D printing (rotating a stainless steel collector during printing), each 4 myocardial fiber layers are printed, wherein the diameter of the myocardial fiber filaments is 100 micrometers, then 1 hollow vascular fiber layer is printed, wherein the outer diameter of the hollow vascular fiber filaments is 250 micrometers, the thickness of the hollow vascular fiber filaments is 50 micrometers, and the steps are alternately repeated for 10 times to obtain a fiber layer with the thickness of 50 layers. The printing parameters of the myocardial fiber layer are: the extrusion speed was 0.2mm³The transverse moving speed of the spray head is 1mm/s, and the deflection angle of the spray head corresponding to each layer is 5 degrees; the printing parameters of the hollow blood vessel fiber layer are as follows: the extrusion speed was 0.5mm³The transverse moving speed of the spray head is 2mm/s, and the deflection angle of the spray head corresponding to each layer is 5 degrees (as shown in figure 3); the transverse reciprocating path of the two spray heads was 15mm, and the rotational speed of the collector was 10 rpm.

After printing was completed, the printed structure was demolded from a stainless steel collector to obtain an artificial ventricle structure (see FIG. 4), crosslinked using a 50U/mL thrombin solution to promote the interconnection between filaments, and then transferred to an incubator (37 ℃ C. and 5% CO)₂) And (4) incubating for 30min, so that the Matrigel in the fiber silk is subjected to temperature crosslinking.

The artificial ventricle is put into a perfusion bioreactor to be cultured for 2 weeks, and the mature vascular structure is formed in the artificial ventricle by observing the integral synchronous pulsation of the artificial ventricle, and the myocardial fiber has directional arrangement of myocardial cells and a mature sarcomere structure, so that the function maturity of the artificial ventricle is indicated.

Claims

1. A preparation method of an artificial ventricle with a bionic spiral orientation microstructure comprises the following steps:

s1, alternately extruding the myocardial fiber yarn carrying the myocardial cells and the hollow vascular fiber yarn carrying the angiogenesis cells by adopting a coaxial nozzle A and a coaxial nozzle B in the calcium ion cross-linking liquid, and collecting by a rotating ellipsoidal collector to realize 3D printing; meanwhile, the coaxial spray head A and the coaxial spray head B are transversely moved in a reciprocating manner, and the deflection angle is changed layer by layer to obtain a fiber arrangement structure with spiral orientation;

2. The method of claim 1, wherein: the myocardial fiber wire carrying the myocardial cells is obtained by crosslinking a calcium chloride solution and the biological ink A carrying the myocardial cells;

the hollow vascular fiber filament carrying the angiogenic cells is obtained by crosslinking a calcium chloride solution and biological ink B carrying the angiogenic cells;

the biological ink A and the biological ink B are both sodium alginate aqueous solution or a mixture of sodium alginate and at least one of natural polymer hydrogel and synthetic polymer hydrogel.

3. The method of claim 2, wherein: the natural polymer hydrogel is at least one of gelatin, collagen, chitosan, silk fibroin, hyaluronic acid, fibrinogen, chondroitin sulfate, albumin and methacrylic acylation products thereof;

the synthetic polymer hydrogel material is at least one of polyethylene glycol, polypropylene alcohol, polyethylene glycol diacrylate, polyethylene oxide, polyacrylamide, polyacrylic acid, polyphosphazene, poly N-isopropyl acrylamide hydrogel and a methylacryloylation product thereof.

4. The production method according to claim 2 or 3, characterized in that: in the biological ink A and the biological ink B, the mass-volume concentration of the sodium alginate is 10-100 mg/mL, and the mass-volume concentration of the natural polymer hydrogel and/or the synthetic polymer hydrogel is 1.0-100 mg/mL.

5. The method of claim 4, wherein: the cardiac muscle cell is at least one of primary cardiac muscle cell, embryonic stem cell and cardiac muscle cell from induced pluripotent stem cell directional differentiation;

in the biological ink A, the density of the myocardial cells is 10⁶/mL～10⁸/mL；

The diameter of the myocardial fiber filament is 50-200 μm.

6. The method of claim 5, wherein: the angiogenesis cells are endothelial cells or mixed cells of the endothelial cells and at least one of smooth muscle cells, pericytes and fibroblasts;

in the bio-ink B, the density of the angiogenesis cells is 10⁵/mL～10⁸/mL；

7. The method of claim 6, wherein: in step S1, the ratio of the printing layers of the myocardial fiber filaments and the hollow vascular fiber filaments is 20: 1-1: 1;

8. The method of claim 7, wherein: in step S1, the ellipsoidal collector is obtained by simulating a topological structure design of a human left ventricle;

the ellipsoidal collector is made of silicon rubber, stainless steel or aluminum alloy;

the number of the 3D printing layers is 3-100;

the extrusion speeds of the coaxial nozzle A and the coaxial nozzle B are both 0.1-10 mm³The distance of the transverse reciprocating movement is 5-100 mm, and the transverse movement speed is 0.01-10.0 mm/s;

9. The method of claim 8, wherein: in step S2, the secondary crosslinking is crosslinking between sodium alginate and calcium ion or any one of the following crosslinking manners:

temperature crosslinking, enzymatic crosslinking, photo crosslinking, and covalent crosslinking.

10. An artificial ventricle prepared by the method of any one of claims 1 to 9.