JP7769849B2 - Valved Graft Formation Substrate - Google Patents

Description

The present invention relates to a valved graft formation substrate that is placed in an environment where biological tissue material is present to form connective tissue on the substrate surface and form a valved graft having multiple valve leaflets that bulge radially inward from a conduit.

There is a great deal of research being conducted into regenerative medicine, which uses artificial materials and cells to revive cells, tissues, and organs lost due to illness or accident.

Normally, the body has a self-defense mechanism that attempts to expel a foreign object, such as a thorn, that penetrates shallowly within the body. It is also known that when a foreign object penetrates deep within the body, fibroblasts gradually gather around the object, forming a connective tissue capsule made primarily of fibroblasts and collagen to cover the object and isolate it within the body. Several techniques have been reported that utilize this latter self-defense mechanism to form connective tissue from living cells, by implanting a foreign material into the body (see Patent Documents 1 to 3).

Furthermore, Patent Document 4 discloses an artificial valve formation substrate in which a substrate is placed inside a living body or the like to form connective tissue on the substrate surface, thereby forming an artificial valve having multiple valve leaflets that bulge radially inward from an outer tube portion.

Japanese Patent Application Laid-Open No. 2007-312821 Japanese Patent Application Laid-Open No. 2008-237896 JP 2010-094476 A WO2016/098877A1 (Claim 1)

However, while the base material described in Patent Document 4 can form valve leaflets (cusps) of the desired shape and thickness in the valve leaflet formation space, the connective tissue formed around the base material body and cover member serves as the outer tubular portion (conduit), and it is not possible to form the outer tubular portion (conduit) to the desired thickness.

The present invention aims to provide a valved graft formation substrate that can form a conduit to a desired thickness.

To achieve the above objective, the valved graft formation substrate of the present invention is placed in an environment containing biological tissue material to form connective tissue on the substrate surface and form a valved graft with multiple valve leaflets bulging radially inward from a conduit. It comprises a cylindrical substrate body and an auxiliary substrate detachably attached to the end of the substrate body. Furthermore, the substrate body and auxiliary substrate are provided with a main body-side valve leaflet formation surface and an auxiliary-side valve leaflet formation surface, respectively, that face each other at a distance corresponding to the thickness of the valve leaflets. The space between these main body-side valve leaflet formation surfaces and auxiliary-side valve leaflet formation surfaces serves as a valve leaflet formation space where connective tissue infiltrates through inlet openings at the outer edges to form the valve leaflets of the valved graft. Furthermore, a cylindrical cover substrate is provided on the outer periphery of the substrate body and auxiliary substrate. The outer periphery of the substrate body and auxiliary substrate face the inner periphery of the cover substrate at a distance corresponding to the thickness of the conduit. The space between the outer periphery of the substrate body and auxiliary substrate and the inner periphery of the cover substrate serves as a conduit formation space where connective tissue infiltrates to form the conduit of the valved graft.

With the above configuration, a tubular cover substrate is provided on the outer periphery of the base material body and auxiliary substrate to form a conduit-forming space, allowing connective tissue to penetrate into this conduit-forming space and form a conduit of the desired thickness. Furthermore, because the conduit is formed between the base material body and auxiliary substrate and the cover substrate, its strength and other qualities can be improved compared to simply forming the conduit on the outer periphery of the base material body and auxiliary substrate.

In addition, since the base material body and auxiliary base material have a main body-side valve leaflet formation surface and an auxiliary-side valve leaflet formation surface, connective tissue can be allowed to infiltrate the valve leaflet formation space formed between these surfaces through an intrusion opening on the outer edge, forming the valve leaflets, while a connection between the conduit and the end of the valve leaflet can be formed at the intrusion opening.

This allows the creation of a valved graft in which the ends of the valve leaflets are supported by high-quality conduits of the desired thickness, and the leaflets can open and close the flow path by bending radially under fluid pressure.

Here, "connective tissue" generally refers to tissue composed primarily of collagen that is formed in vivo. However, in this specification and claims, the term also includes tissue equivalent to connective tissue formed in vivo when formed in an ex vivo environment.

Furthermore, "biological tissue materials" refer to substances necessary for forming desired biological tissues, and include, for example, animal cells such as fibroblasts, smooth muscle cells, endothelial cells, stem cells, ES cells, and iPS cells, various proteins (collagen, elastin), sugars such as hyaluronic acid, and various physiologically active substances present in the body, such as cell growth factors and cytokines. This "biological tissue material" includes those derived from mammals such as humans, dogs, cows, pigs, goats, and sheep, as well as birds, fish, and other animals, as well as equivalent artificial materials.

In addition, "an environment in which a biological tissue material exists" refers to an artificial environment containing a biological tissue material inside the body of an animal (such as a mammal, such as a human, dog, cow, pig, goat, or sheep, as well as a bird, fish, or other animal) (for example, implanted subcutaneously in the limbs, shoulder, back, or abdomen, or in the abdominal cavity) or outside the animal's body.

Furthermore, the auxiliary base material may be formed with a valve tip hole that connects the portion of the valve leaflet formation space corresponding to the tip of the valve leaflet with the outside of the base material.

With this configuration, the leaflet formation space is connected to the outside of the base material via the valve tip hole. This allows connective tissue to invade the leaflet formation space from outside the base material via the conduit formation space and the intrusion port, and also allows connective tissue to invade the leaflet formation space directly from outside the base material via the valve tip hole, thereby shortening the time required to form the valve leaflets.

In other words, because the cover substrate is provided on the outer periphery of the substrate body and auxiliary substrate, the connective tissue must first enter the conduit formation space inside the cover substrate and then enter the valve leaflet formation space through the entry port, which tends to lengthen the time required for valve leaflet formation. In contrast, by forming a valve tip hole in the auxiliary substrate and connecting the valve leaflet formation space with the outside of the substrate, the connective tissue can also enter through the valve tip hole, preventing delays in valve leaflet formation.

Furthermore, the valve leaflet formation space may be set to the shape of the valve leaflets when they are closed, and a common valve tip hole may be formed that connects the areas corresponding to the tips of multiple valve leaflets with the outside of the base material.

With this configuration, the leaflet formation spaces are set to the shape of the valve leaflets when they are closed, allowing the tips of multiple leaflet formation spaces to be brought closer together and the tip holes formed at the tips of each leaflet formation space to be integrated and common. This allows the opening of the tip holes to be larger outside the base material, making it easier for connective tissue to penetrate into the tip holes from outside the base material, more reliably forming high-quality leaflets and shortening the time required to form the leaflets.

Furthermore, by setting the leaflet formation space to the shape of the valve leaflets when they are closed, the valve leaflets can be formed in a closed state, which reduces the possibility of backflow occurring in a valved graft implanted in the body due to delayed closure of the valve leaflets, compared to when the valve leaflets are formed in an open state.

Furthermore, the auxiliary base material may be formed with promotion holes that connect the valve cusp formation space with the conduit formation space and promote the infiltration of connective tissue into the valve cusp formation space, and a cutting slit may be formed in the area from the end face of the auxiliary base material to the promotion hole to cut the connective tissue formed inside the promotion hole.

With this configuration, promotion holes are formed in the auxiliary base material, which promotes the infiltration of connective tissue from the conduit formation space on the outside of the auxiliary base material to the valve leaflet formation space on the inside. In this case, the connective tissue formed inside the promotion holes connects the conduit and valve leaflets of the valved graft across the auxiliary base material, but since cutting slits are formed in the auxiliary base material, the connective tissue formed inside the promotion holes can be easily cut. This allows the valved graft to be formed efficiently and removed from the valved graft formation base material without being damaged.

As described above, according to the present invention, a tubular cover substrate is provided on the outer periphery of the substrate body and auxiliary substrate to form a conduit formation space, and connective tissue is allowed to infiltrate this conduit formation space to form a conduit. This allows the conduit of the valved graft to be formed to the desired thickness and improves its quality, such as its strength.

Furthermore, by infiltrating connective tissue from the conduit formation space through the entry opening at the outer edge into the leaflet formation space between the main body side leaflet formation surface and the auxiliary side leaflet formation surface, it is possible to form the leaflets of the valved graft in the leaflet formation space while forming a connection between the conduit and the end of the valve leaflet at the entry opening.

1 is a perspective view of a valved graft forming substrate according to the present invention; 1 is a perspective view of a base body and an auxiliary base material with a cover base material removed; 3 is a perspective view of FIG. 2 viewed from the opposite side. A perspective view of the base body (a) is a plan view of the substrate body, and (b) is a cross-sectional view taken along the line A-A. FIG. 1 is a diagram illustrating a procedure for forming a valved graft using a valved graft formation substrate. 1A and 1B are perspective views of a valved graft, showing a closed state and an open state, respectively.

Below, we will explain the embodiments of the valved graft formation substrate according to the present invention using the drawings.

As shown in Figures 1 to 5, the valved graft formation substrate 1 is placed in an environment where biological tissue material is present to form connective tissue 2 on the substrate surface and form a valved graft 5, for example, used as a heart valve graft, having multiple valve leaflets 4 bulging radially inward from a conduit 3. It comprises a columnar substrate main body 6, an auxiliary substrate 7 that is detachably attached to the end of the substrate main body 6, and a tubular cover substrate 8 that is provided on the outer periphery of the substrate main body 6 and auxiliary substrate 7.

Furthermore, the base material body 6 and auxiliary base material 7 are each provided with a main body-side leaflet formation surface 9 and an auxiliary side leaflet formation surface 10, which face each other with a gap corresponding to the thickness of the valve leaflet. The space between the two leaflet formation surfaces 9, 10 forms a leaflet formation space 12 where connective tissue 2 infiltrates from an intrusion port 11 on the outer edge to form the leaflets 4 of the valved graft 5. The outer peripheral surfaces of the base material body 6 and auxiliary base material 7 face the inner peripheral surface of the cover base material 8 with a gap corresponding to the conduit thickness, and the space between these surfaces forms a conduit formation space 13 where connective tissue 2 infiltrates to form the conduit 3 of the valved graft 5.

The base body 6 is cylindrical and made of a polymeric material such as acrylic resin, with three body-side leaflet-forming surfaces 9 formed at its ends, separated by a central Y-shaped partition wall 14. Each body-side leaflet-forming surface 9 consists of the wall surface of the partition wall 14 and a curved surface that slopes from its lower edge toward the center axis of the base and expands radially outward, and is shaped to correspond to the upstream surface of the valve leaflet 4 in the closed state.

The body-side leaflet-forming surface 9 can be set by interpolation using five variables, such as the valve diameter (D), valve closure angle (θ), valve center length (L), commissure height (H), and commissure-to-commissure distance (a), using what is known as non-uniform rational B-spline (NURBS) interpolation. Here, the valve diameter (D) corresponds to the outer diameter of the base material 6; the valve closure angle (θ) is, for example, 20° and corresponds to the inclination angle of the outer edge of the body-side leaflet-forming surface 9 relative to the cross section of the base material; the valve center length (L) is, for example, 13.5 mm and corresponds to the length from the lower edge of the partition wall 14 to the outer edge of the body-side leaflet-forming surface 9; the commissure height (H) is, for example, 14.2 mm and corresponds to the height in the axial direction of the base material from the outer edge of the body-side leaflet-forming surface 9 to the tip of the partition wall 14; and the commissure-to-commissure distance (a) corresponds to the thickness of the partition wall 14.

Notches 15 are formed on the radially outer edge of the partition wall 14, connecting adjacent leaflet formation spaces 12 with a space of sufficient thickness to form commissure portions 16 in the valved graft 5 that are sufficiently thick and have a predetermined strength.

The auxiliary base material 7 is cylindrical and made of a polymeric material such as acrylic resin. The inner surface of a Y-shaped slit 18 formed in the area from the base material's central end face to the end plate 17 on the outer edge of the base material, and the central end face of the base material divided into three by the slit 18, form the auxiliary side cusp-forming surface 10. By inserting a partition wall 14 into the slit 18 and fitting its wall tip 19 into a fitting groove 20 formed in the end plate 17, the auxiliary base material 7 is attached to the end of the base material main body 6, and the auxiliary side cusp-forming surface 10 faces the main body side cusp-forming surface 9 to form the cusp-forming space 12.

The distance from the main body side leaflet forming surface 9 to the auxiliary side leaflet forming surface 10 is set to a range of, for example, 0.2 mm to 2.0 mm for each position on the surface, allowing the leaflets 4 to be formed with the desired leaflet thickness distribution, taking into account, for example, the blood pressure resistance and hemodynamics.

The width of the slit 18 is equal to the sum of the thickness of each side end of the two adjacent valve leaflet formation spaces 12 and the thickness of the partition wall 14. The width of the commissure portion 16 of the valved graft 5 is set to a sufficient size corresponding to the width of this slit 18, thereby forming a commissure portion 16 with the required strength.

A Y-shaped valve tip hole 21 is formed in the end plate 17 of the auxiliary base material 7, exposing the central portion of the wall tip 19 of the partition wall 14. This valve tip hole 21 is set to a width of, for example, 0.5 mm or more, so that the portion of the valve tip formation space 12 corresponding to the tips of the multiple valve leaflets 4 communicates with the outside of the base material via the common valve tip hole 21.

Facilitation holes 22 are formed on the peripheral surface of the auxiliary base material 7, connecting the cusp formation space 12 and the conduit formation space 13, to promote the infiltration of connective tissue 2 from the conduit formation space 13 into the cusp formation space 12. Furthermore, cutting slits 23 are formed in the end plate 17 extending from the promotion holes 22. When removing the valved graft 5, a scalpel or similar tool is inserted into the cutting slit 23 to cut the connective tissue 2 formed inside the promotion holes 22 and connecting the inside and outside of the auxiliary base material 7.

The cover substrate 8 is cylindrical and made of a polymeric material such as acrylic resin. A protrusion 25 protruding radially outward from the end plate 17 of the auxiliary substrate 7 fits into a recess 24 formed at its end, allowing the cover substrate 8 to be attached to the outer periphery of the substrate body 6 and auxiliary substrate 7 at a fixed interval of, for example, about 2 mm. This creates a conduit-forming space 13 between the outer periphery of the substrate body 6 and auxiliary substrate 7. The cover substrate 8 has intrusion holes 26 extending over almost the entire surface, allowing connective tissue 2 to easily infiltrate the conduit-forming space 13 from outside the substrate.

The material for the valved graft formation substrate 1 is preferably a resin that has the strength (hardness) to prevent significant deformation when implanted in a living body, is chemically stable, can withstand stresses such as sterilization, and has little or no leachable substances that irritate the living body. Examples of such resins include, but are not limited to, acrylic resins, as mentioned above.

Next, we will explain a method for producing a valved graft 5 using the valved graft formation substrate 1 described above.

As shown in Figure 6, this production method comprises an "installation step" in which the valved graft-forming substrate 1 is placed in an environment containing biological tissue material; a "formation step" in which connective tissue 2 is formed around the valved graft-forming substrate 1 and the connective tissue 2 is allowed to invade the valve leaflet formation space 12 and the conduit formation space 13; a "removal step" in which the valved graft-forming substrate 1 coated with connective tissue 2 is removed from the environment; and a "separation step" in which the connective tissue 2 is separated from the valved graft-forming substrate as a valved graft 5 comprising a conduit 3 and valve leaflets 4.

<Installation process>
After attaching the auxiliary substrate 7 to the end of the substrate body 6, the cover substrate 8 is attached to the outer periphery of the substrate body 6 and the auxiliary substrate 7 so as to fit onto the protrusions 25, thereby assembling the valved graft-forming substrate 1. Furthermore, the assembled valved graft-forming substrate 1 is placed in an environment where biological tissue material is present, such as under the skin of a goat ( FIG. 6( a) ).

Examples of environments in which biological tissue materials exist include the inside of an animal's body (e.g., implanted subcutaneously or intraperitoneally), or an artificial environment such as a solution in which the biological tissue material is suspended outside the animal's body. When the valved graft-forming substrate 1 is placed in an artificial environment, cell culture can be performed in a clean environment using known methods under various culture conditions. Examples of biological tissue materials that can be used include those derived from mammals such as goats, dogs, cows, pigs, rabbits, sheep, and humans, as well as those derived from birds, fish, and other animals, and artificial materials.

<Formation process>
After the installation step, by allowing a predetermined time, for example about three months, to pass, connective tissue 2 is formed around the valved graft-forming substrate 1, and the connective tissue 2 invades the conduit-forming space 13 through the end opening of the conduit-forming space 13 and the intrusion holes 26 in the cover substrate 8. Furthermore, the connective tissue 2 that has invaded the conduit-forming space 13 invades the valve leaflet-forming space 12 through the intrusion opening 11, the slit 18, and the promotion holes 22, and the connective tissue 2 outside the substrate invades directly into the valve leaflet-forming space 12 through the valve tip holes 21 ( FIG. 6( b) ).

In this formation process, by providing the valve tip holes 21, connective tissue 2 is formed in the valve cusp formation spaces 12 in a relatively short time, not only through penetration via the conduit formation spaces 13, but also through direct penetration of connective tissue 2 from outside the base material. In particular, by connecting multiple valve cusp formation spaces 12 to the outside of the base material through a common valve tip hole 21, which enlarges the valve tip hole 21, further promoting penetration of connective tissue 2 into the valve cusp formation spaces 12. Connective tissue 2 is composed of fibroblasts and extracellular matrix such as collagen.

<Removal process>
After the formation process has been carried out for a predetermined period of time, and the connective tissue 2 has been sufficiently formed in the valve leaflet formation space 12 and the conduit formation space 13, a removal process is carried out in which the valved graft forming substrate 1 coated with the connective tissue 2 is removed from the environment in which the biological tissue material is present.

<Separation process>
The connective tissue 2 covering the outer surface of the cover substrate 8 is removed while cutting with a scalpel or the like between the connective tissue 2 formed inside the entry hole 26 of the cover substrate 8, and the connective tissue 2 at both ends of the valved graft-forming substrate 1 is also removed. Next, the cover substrate 8 is removed after deforming and peeling off the connective tissue 2 in the conduit formation space 13 with tweezers or the like so as to leave it on the substrate main body 6 and auxiliary substrate 7 (FIG. 6(c)).

When the cover substrate 8 is removed, the connective tissue 2 that makes up the conduit 3 is revealed, and the connective tissue 2 formed inside the entry holes 26 of the cover substrate 8 remains on its surface in the form of protrusions. However, even if there are protrusions on the outer surface of the conduit 3, they do not affect blood flow, so there is no need to remove them.

A scalpel or similar tool is inserted into the cutting slit 23 covered with connective tissue 2, and the connective tissue 2 formed inside the promotion holes 22 of the auxiliary base material 7 is cut to separate the connective tissue 2 inside and outside the auxiliary base material 7 that constitutes the conduit 3 and valve cusp 4. The auxiliary base material 7 is then removed while the connective tissue 2 is peeled off from the outer peripheral surface and auxiliary side valve cusp forming surface 10. The connective tissue 2 covering the wall tip 19 of the partition wall 14 of the base material main body 6 is then removed, and the connective tissue 2 inside the promotion holes 22 that remains as protrusions on the conduit 3 and valve cusp 4 is excised (Figure 6(d)).

The valved graft 5 is obtained by separating the connective tissue 2 from the substrate body 6 so that it peels off from the outer surface of the substrate body 6 and the body-side valve leaflet-forming surface 9 (Figure 6(e)).

As shown in Figure 7, the valved graft 5 produced using the valved graft formation substrate 1 is a cardiac valve graft that is implanted into a blood vessel such as the aorta emerging from the heart to impart valve function. The pressure of the fluid flowing inside causes multiple valve leaflets 4 that bulge radially inward from the conduit 3 to displace inward and outward, thereby opening and closing the flow path inside the conduit 3.

The valve leaflets 4 are connected to the conduit 3 via the slits 18 and the commissures 16 formed at the entry ports 11 in the valved graft-forming substrate 1, and the valve base. When the flow direction in the flow channel is from the distal end to the proximal end, the pressure of the fluid attempting to enter between the valve leaflets 4 and the conduit 3 causes the valve leaflets 4 to bend radially inward, closing the flow channel (Figure 7(a)). On the other hand, when the flow direction in the flow channel is from the proximal end to the distal end, the valve leaflets 4 bend radially outward due to the flow, opening the flow channel (Figure 7(b)).

The valved graft 5 produced using the valved graft formation substrate 1 has its valve leaflets 4 formed in a closed state, so the free state is closed, reducing the risk of backflow due to delayed closure.

The present invention is not limited to the above-described embodiments, and modifications can be made as appropriate within the scope of the present invention. For example, while the above embodiments have been described using a tricuspid valve as an example, valves with any number of cusps, such as bicuspid or quadricuspid valves, may also be used. Furthermore, the entry holes 26 in the cover substrate 8 and the promotion holes 22 and cutting slits 23 in the auxiliary substrate 7 may be omitted as appropriate. Furthermore, the valve leaflet holes do not have to be common, and each valve leaflet formation space may have one valve leaflet hole, and the valve leaflet holes may also be omitted.

1 Valved graft formation substrate 2 Connective tissue 3 Conduit 4 Valve cusp 5 Valved graft 6 Substrate body 7 Auxiliary substrate 8 Cover substrate 9 Main body side valve cusp formation surface 10 Auxiliary side valve cusp formation surface 11 Entry port 12 Valve cusp formation space 13 Conduit formation space 14 Partition wall 15 Notch 16 Commissure portion 17 End plate 18 Slit 19 Wall tip portion 20 Fitting groove 21 Valve cusp hole 22 Facilitation hole 23 Cutting slit 24 Recess 25 Protrusion 26 Entry hole D Valve diameter θ Valve closure angle L Valve center length H Commissure portion height a Distance between commissure portions

Claims (4)

A valved graft forming substrate for forming a valved graft having a plurality of valve leaflets bulging radially inward from a conduit by placing the substrate in an environment where a biological tissue material is present to form connective tissue on the surface of the substrate, the valved graft forming substrate comprising:
The device comprises a columnar substrate body and an auxiliary substrate detachably attached to an end of the substrate body,
a main body-side leaflet forming surface and an auxiliary-side leaflet forming surface are respectively set on the base body and the auxiliary base material, the main body-side leaflet forming surface facing each other with a gap corresponding to the thickness of the valve leaflet, and a leaflet forming space is defined between the main body-side leaflet forming surface and the auxiliary-side leaflet forming surface, where connective tissue is allowed to invade from an intrusion port on the outer edge to form the leaflets of the valved graft;
a cylindrical cover substrate is provided on the outer peripheral sides of the substrate body and auxiliary substrate, the outer peripheral surfaces of the substrate body and auxiliary substrate face the inner peripheral surface of the cover substrate at a distance corresponding to the thickness of the conduit, and the space between the outer peripheral surfaces of the substrate body and auxiliary substrate and the inner peripheral surface of the cover substrate forms a conduit formation space into which connective tissue is allowed to infiltrate to form the conduit of the valved graft ;
A valved graft formation substrate characterized in that the auxiliary substrate has a valve tip hole formed therein, which connects a portion of the valve leaflet formation space corresponding to the tip of the valve leaflet with the outside of the substrate . 2. The valved graft formation substrate according to claim 1, characterized in that the valve leaflet formation space is set to the shape of the valve leaflet in a closed state, and a common valve tip hole is formed that connects the areas corresponding to the tips of multiple valve leaflets with the outside of the substrate. 3. A valved graft formation substrate according to claim 1, characterized in that the auxiliary substrate has a promotion hole formed therein that connects the valve leaflet formation space with the conduit formation space and promotes the infiltration of connective tissue into the valve leaflet formation space, and a cutting slit is formed in the range from the end face of the auxiliary substrate to the promotion hole for cutting the connective tissue formed inside the promotion hole. A valved graft forming substrate for forming a valved graft having a plurality of valve leaflets bulging radially inward from a conduit by placing the substrate in an environment where a biological tissue material is present to form connective tissue on the surface of the substrate, the valved graft forming substrate comprising:
The device comprises a columnar substrate body and an auxiliary substrate detachably attached to an end of the substrate body,
a main body-side leaflet forming surface and an auxiliary-side leaflet forming surface are respectively set on the base body and the auxiliary base material, the main body-side leaflet forming surface facing each other with a gap corresponding to the thickness of the valve leaflet, and a leaflet forming space is defined between the main body-side leaflet forming surface and the auxiliary-side leaflet forming surface, where connective tissue is allowed to invade from an intrusion port on the outer edge to form the leaflets of the valved graft;
a cylindrical cover substrate is provided on the outer peripheral sides of the substrate body and auxiliary substrate, the outer peripheral surfaces of the substrate body and auxiliary substrate face the inner peripheral surface of the cover substrate at a distance corresponding to the thickness of the conduit, and the space between the outer peripheral surfaces of the substrate body and auxiliary substrate and the inner peripheral surface of the cover substrate forms a conduit formation space into which connective tissue is allowed to infiltrate to form the conduit of the valved graft;
A valved graft formation substrate characterized in that the auxiliary substrate has formed therein promotion holes that connect the valve cusp formation space with the conduit formation space and promote the infiltration of connective tissue into the valve cusp formation space, and a cutting slit is formed in the range from the end face of the auxiliary substrate to the promotion hole for cutting the connective tissue formed inside the promotion hole.