Transfer mold and method for manufacturing transfer mold

Abstract

To provide a transfer mold capable of transferring a surface structure formed by a biomimetic curved surface on a surface of a biocompatible material and stable and safe in terms of material, as well as a transfer mold manufacturing method capable of efficiently manufacturing such a transfer mold.SOLUTION: A transfer mold for transferring a surface structure to an object has a stainless steel substrate and a transfer surface structure formed on a surface of the stainless steel substrate. The transfer surface structure has a concave curve surface in an internal direction toward an interior of the stainless steel substrate, a convex curve surface in an external direction opposite to the internal direction, or a combination thereof, when viewed along a normal direction of the surface of the stainless steel substrate. At least a portion of the concave curve surface or at least a portion of the convex curve surface has a shoulder where a curvature of a curve surface changes.SELECTED DRAWING: Figure 5A

Description

[Technical Field]

[0001] The present invention relates to a transfer mold and a method for manufacturing a transfer mold. [Background technology]

[0002] Using biocompatible materials, a variety of medical devices and materials have been developed, including cell culture substrates, scaffolding materials for regenerative medicine (e.g., materials for regenerating cartilage, bone, vertebrae, nucleus pulposus, ligament, corneal stroma, skin, oral mucosa, gingiva, periodontal ligament, blood vessels, nerves, and liver tissue), transplant materials, wound dressing materials, bone filling agents, hemostatic materials, adhesion prevention materials, and drug delivery carriers.

[0003] In order to induce or enhance interactions between cells or tissues and biocompatible materials, microstructures such as irregularities, holes, and micropatterns may be formed on the surface of the biocompatible material. The surface structure is formed by contacting the surface of the biocompatible material with a mold having a structure complementary to the structure to be imparted to the biocompatible material, and transferring the complementary structure of the mold to the surface of the biocompatible material. The application of inkjet technology and lithography technology to the formation of the complementary structure of the mold has been investigated (see, for example, JP 2016-533244 A). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2016-533244 Summary of the Invention [Problem to be solved by the invention]

[0005] The above-mentioned technology allows for the formation of simple (hemispherical) or linear micropatterns on the surface of biocompatible materials by transferring the complementary structure of a mold. However, human tissues and organs are not composed of simple spheres or straight lines, but rather of complex combinations of curved and curved surfaces. For example, while the surface of biocompatible materials used in regenerative medical products that comes into contact with cells is naturally curved, the interface with the epithelial cell layer in scaffolds such as cultured skin is often flat or has linear irregularities. In such cases, the cultured tissue may not adhere to the scaffold, resulting in floating or peeling, making it difficult to obtain a cultured tissue suitable for practical use.

[0006] Furthermore, molds for transferring surface structures to biocompatible materials require low toxicity, ease of processing, strength, durability, and other properties as their constituent materials. However, developing low-toxicity materials poses many challenges. Furthermore, increasing strength and durability reduces processability. Conventional materials have not yet achieved a balanced and sufficient level of these properties.

[0007] The object of the present invention is to provide a transfer mold that can transfer a surface structure formed with a biomimetic curved surface to the surface of a biocompatible material, and that is stable and safe in terms of material, and a method for manufacturing a transfer mold that can efficiently manufacture such a transfer mold. [Means for solving the problem]

[0008] The present inventors have conducted extensive research to solve the above problems and have found that the above object can be achieved by employing the following configuration, thereby completing the present invention.

[0009] In one embodiment, the present invention provides A transfer mold for transferring a surface structure to a transfer target, the transfer mold has a stainless steel substrate and a transfer surface structure formed on a surface of the stainless steel substrate; the surface structure for transfer has, when viewed along a normal direction to the surface of the stainless steel substrate, a concave curved surface that is concave in an inward direction toward the inside of the stainless steel substrate, a convex curved surface that is convex in an outward direction opposite to the inward direction, or a combination thereof; The transfer mold has a shoulder portion formed on at least a part of the concave curved surface or at least a part of the convex curved surface, where the curvature of the curved surface changes.

[0010] In another embodiment, the present invention provides The method for manufacturing the transfer mold includes: A process in which a laser is irradiated onto the surface of a stainless steel substrate to form a surface structure for transfer. The present invention relates to a method for manufacturing a transfer mold, comprising: [Effects of the Invention]

[0011] In this transfer mold, the transfer surface structure formed on the surface of a stainless steel substrate has a concave curved surface that is concave inward, a convex curved surface that is convex outward, or a combination of these. Furthermore, in this transfer mold, not only are these simple curved surfaces formed, but at least a portion of the concave curved surface or at least a portion of the convex curved surface has a shoulder portion where the curvature of the curved surface changes. Therefore, simply by contacting the transfer surface structure of the transfer mold with the surface of the object to be transferred, a complex biomimetic curved surface can be imparted to the object to be transferred. Furthermore, because stainless steel is used as the constituent material of the transfer mold, it can exhibit high levels of low toxicity, ease of processing, strength, durability, etc.

[0012] According to this method for manufacturing a transfer mold, a transfer surface structure can be formed simply by irradiating a stainless steel substrate with a laser, without going through any complicated steps. Therefore, it is possible to efficiently manufacture a transfer mold having a complex biomimetic transfer surface structure that could not be achieved using conventional inkjet or lithography techniques. [Brief explanation of the drawings]

[0013] [Figure 1]FIG. 1 is a perspective view schematically illustrating a transfer mold according to one embodiment of the present invention. [Figure 2] FIG. 2 is a partially enlarged cross-sectional view taken along the line AA in FIG. [Figure 3A] FIG. 2 is a partially enlarged cross-sectional view schematically illustrating an example of a concave curved surface of the transfer surface structure. [Figure 3B] FIG. 2 is a partially enlarged cross-sectional view schematically illustrating an example of a convex curved surface of a transfer surface structure. [Figure 4A] 1 shows an enlarged view of a portion of a micropattern designed for forming a surface structure for transfer. [Figure 4B] 4B shows a wide-angle view of the design pattern of FIG. 4A. [Figure 4C] 4B shows a cross-sectional view of the design pattern of FIG. 4A taken along line A1-B1. [Figure 5A] 1 shows an SEM photograph (100x magnification) of a plan view of the transfer surface structure of the transfer mold of Example 1. [Figure 5B] 1 shows an SEM photograph (300x magnification) in which a part of the transfer surface structure of the transfer mold of Example 1 is enlarged. [Figure 5C] 1 shows a shape profile when the fine protrusions in the transfer surface structure of the transfer mold of Example 1 are scanned obliquely. [Figure 6] 1 shows an SEM photograph (100x magnification) of a transfer mold having fine protrusions in the shape of square pillars in Comparative Example 1. [Figure 7] 1 shows an SEM photograph (narrow field (left) 300x magnification; wide field (right) 100x magnification) of the surface structure of a fibrous collagen gel obtained using the transfer mold of Example 1. [Figure 8] 1 shows an SEM photograph (narrow field of view (left) 300x magnification; wide field of view (right) 30x magnification) of the surface structure of a fibrous collagen gel obtained using the transfer mold of Comparative Example 1. [Figure 9] 1 is a tissue image showing a cross section of a cultured tissue stained with HE, obtained from a fibrous collagen gel that has been transferred using a transfer mold in Example 1. [Figure 10] 1 is a tissue image showing a cross section of a cultured tissue stained with HE, obtained from a fibrous collagen gel that had been transferred using a transfer mold in Comparative Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0014] A transfer mold and a method for manufacturing a transfer mold according to one embodiment of the present invention will be described below with reference to the drawings. The present invention is not limited to these embodiments. In some or all of the drawings, parts not necessary for the explanation are omitted, and some parts are illustrated enlarged or reduced to facilitate the explanation. Terms indicating positional relationships such as up and down, referred to with reference to the drawings, are used merely to facilitate the explanation and are not intended to limit the configuration of the present invention in any way.

[0015] <Transfer mold> Fig. 1 is a perspective view schematically showing a transfer mold according to one embodiment of the present invention. Fig. 2 is a partially enlarged cross-sectional view taken along line AA in Fig. 1. Fig. 3A is a partially enlarged cross-sectional view schematically showing an example of a concave curved surface of the transfer surface structure, and Fig. 3B is a partially enlarged cross-sectional view schematically showing an example of a convex curved surface of the transfer surface structure.

[0016] The transfer mold 1 is a transfer mold for transferring a surface structure to a transfer target object. The transfer method will be described later. The transfer mold 1 has a stainless steel substrate 2 and a transfer surface structure 4 formed on a surface 3 of the stainless steel substrate 2. In the transfer mold 1 shown in FIG. 1, the transfer surface structure 4 is formed on a portion of the surface 3 of the stainless steel substrate 2 (a portion of the upper surface of the stainless steel substrate 2 in FIG. 1), but this is not limited thereto, and the transfer surface structure 4 may be formed on the entire surface 3 of the stainless steel substrate 2.

[0017] The stainless steel substrate 2 is a component that constitutes the main body of the transfer mold 1 and is made of stainless steel. The shape of the stainless steel substrate 2 is typically a plate as shown in FIG. 1 , but is not limited to this. As described below, forming a transfer surface structure 4 on the surface 3 of the stainless steel substrate 2 simply requires irradiating the surface 3 of the stainless steel substrate 2 with a laser, so laser processing can be performed regardless of the shape of the stainless steel substrate 2. Other shapes of the stainless steel substrate 2 can be any shape, such as a cube, rectangular parallelepiped, linear, rod-like, spherical, or cylindrical, as long as they can be transferred to an object to be transferred. An appropriate shape can be adopted depending on the method for transferring the surface structure to the object to be transferred.

[0018] The stainless steel is not particularly limited, and may be any of martensitic stainless steel, ferritic stainless steel, austenitic stainless steel, austenitic-ferritic stainless steel, precipitation hardening stainless steel, or a combination thereof.

[0019] As shown in FIG. 2, when viewed along the normal direction z of the surface 3 of the stainless steel substrate 2, the transfer surface structure 4 is oriented in an inward direction D in a concave curved surface 41, which is concave in the internal direction D in The opposite external direction D out 2, the transfer surface structure 4 has a combination of a concave curved surface 41 and a convex curved surface 42, but is not limited to this and may have only a concave curved surface 51 as shown in FIG. 3A, or a convex curved surface 52 as shown in FIG. 3B. Furthermore, a portion of the transfer surface structure 4 formed on the surface 3 of the stainless steel substrate 2 may have only the concave curved surfaces 41, 51, and the remaining portion of the transfer surface structure 4 may have only the convex curved surfaces 42, 52.

[0020] In the transfer surface structure 4, the concave curved surfaces 41, 51 are preferably part of a plurality of micropores having a predetermined depth formed on the surface 3 of the stainless steel substrate 2. The convex curved surfaces 42, 52 are preferably part of a plurality of microprotrusions having a predetermined height formed on the surface 3 of the stainless steel substrate 2. In particular, the convex curved surfaces are preferably part of a plurality of microprotrusions having a predetermined height formed on the surface 3 of the stainless steel substrate 2. out It is preferable that the surface of at least a part of the dome-shaped projection is a convex portion.

[0021] In the surface structure 4 for transfer, the micropores and / or microprotrusions are preferably formed in the form of triangular lattice points or square lattice points. The depth and formation pitch of the micropores and the height and formation pitch of the microprotrusions can be appropriately set depending on the target surface structure (micropattern) to be transferred to the transfer target object. The depth and formation pitch of the micropores may be the same or different from each other, and the height and formation pitch of the microprotrusions may also be the same or different from each other. The lower limit of the formation pitch of the micropores and / or microprotrusions (the distance between the lattice points) is preferably 50 μm, more preferably 100 μm, even more preferably 150 μm, and particularly preferably 200 μm. The upper limit of the formation pitch is preferably 800 μm, more preferably 500 μm, even more preferably 300 μm, and particularly preferably 250 μm.

[0022] 2, 3A, and 3B, in addition to the concave curved surfaces 41, 51 and the convex curved surfaces 42, 52, shoulder portions 41s, 42s, 51s, and 52s at which the curvature of the curved surfaces changes are formed on at least a portion of the concave curved surfaces 41, 51 or at least a portion of the convex curved surfaces 42, 52. By providing the transfer surface structure 4 with such shoulder portions 41s, 42s, 51s, and 52s, it is possible to impart to the transfer surface structure 4 a complex biomimetic shape that cannot be obtained with a simple spherical, flat, or curved surface.

[0023] The shoulder portions 41s, 42s, 51s, and 52s are portions where the curvature of the curved surface on which they are formed changes, and extend from the outer surface of the curved surface (including both concave and convex curved surfaces) in the outward direction D outIn detail, for example, when focusing on the convex curved surface 42 shown in FIG. 2, it is assumed that the convex curved surface 42 is continuously and monotonously connected from one end (for example, the top) to the other end (for example, the bottom) in the normal direction z to the surface of the stainless steel substrate 2, and the angle of the convex curved surface 42 is the angle of the convex curved surface 42 in the outward direction D out The protrusion caused by the displacement of the convex curved surface 42 toward the surface of the stainless steel substrate 2 is the shoulder portion 42s. Similarly, the concave curved surface 41 is also assumed to be continuously and monotonically connected from one end to the other end in the normal direction z to the surface of the stainless steel substrate 2 (the plane including the dashed line connecting both ends of the base of the shoulder portion 41s in FIG. 2). out The protrusion caused by the displacement of the concave curved surface 41 toward the center is the shoulder portion 41s.

[0024] 3A, even when the transfer surface structure 4 has only the concave curved surface 51, the concave curved surface 51 has a shoulder portion 51s. out 3B, when the transfer surface structure 4 has only the convex curved surface 52, the convex curved surface 52 also has a shoulder 52s. When the convex curved surface 52 is assumed to be continuously and monotonically connected, the convex curved surface 52 is displaced in the outward direction D. out The shoulder 52s is formed by the displacement toward the center.

[0025] In the transfer mold 1, shoulders 41s, 42s where the curvature of the curved surface changes need only be formed on at least a portion of the concave curved surface 41 or at least a portion of the convex curved surface 42, and shoulders do not have to be formed on all of the concave curved surfaces or convex curved surfaces. Furthermore, when shoulders are formed on a curved surface, the number of shoulders on one curved surface is not limited to one, as shown in Figures 2, 3A, and 3B, and may be two, three, or more.

[0026] The shoulder is not necessarily in the outward direction D in the cross section shown in FIG. outThe shape does not need to be convex and have an extreme value. As long as the protrusions are formed by deviating from a curved surface (hereinafter also referred to as a "virtual curved surface") that is assumed to be continuously and monotonically connected, those protrusions are included in the shoulder portion. For example, the lower limit of the ratio of the height of the shoulder portion (the maximum amount of deviation from the virtual curved surface of the curved surface that gives the protrusion) to the height of the fine protrusions is preferably 1%, more preferably 3%, and even more preferably 5%. The upper limit of the above ratio is preferably 30%, more preferably 25%, and even more preferably 20%.

[0027] When the stainless steel substrate 2 is viewed in plan view (when the stainless steel substrate 2 shown in Figure 1 is viewed from above), the shoulder portion 42s may be formed around the entire outer surface (convex curved surface 42) of the fine protrusion portion, or may be formed on only a part of the outer surface.

[0028] Although the maximum height Rz of the surface structure 4 for transfer is not particularly limited, the lower limit is preferably 10 μm, more preferably 20 μm, even more preferably 30 μm, and particularly preferably 50 μm. The upper limit of the maximum height Rz is preferably 500 μm, more preferably 300 μm, even more preferably 200 μm, and particularly preferably 150 μm. By setting the maximum height Rz of the surface structure 4 for transfer within the above range, the surface structure can be efficiently transferred to the transfer target object. The maximum height Rz can be measured in accordance with JIS B 0601-2001.

[0029] The surface 3 of the transfer mold 1 may be subjected to an appropriate surface treatment such as hydrophilic treatment, hydrophobic treatment, hardening treatment, or mold release treatment.

[0030] <<Manufacturing method of transfer mold>> The method for manufacturing a transfer mold according to this embodiment includes a step of irradiating a surface of a stainless steel substrate with a laser to form a transfer surface structure. This embodiment employs laser irradiation of the surface of the stainless steel substrate to form the transfer surface structure, thereby enabling efficient and simple manufacture of a transfer mold having a transfer surface structure with a shoulder formed on a curved surface. Conventional inkjet and lithography techniques can form concave or convex curved surfaces in a transfer surface structure by integral processing using droplet jetting or etching after micropattern formation, but shoulders on curved surfaces are difficult to form even with these techniques. The formation of shoulders on the curved surface of a transfer surface structure can be achieved by appropriately controlling the type of laser, laser irradiation conditions (output, pulse width, repetition frequency, scanning speed), the hardness and crystallinity of the stainless steel substrate, the temperature of the stainless steel substrate during laser irradiation, processing time, etc.

[0031] Specifically, data for the desired transfer surface structure can be input into a laser irradiation device, and the surface 3 of the stainless steel substrate 2 can be irradiated with a laser while scanning it. The stainless steel substrate 2 can be placed on an xyz stage, and the stainless steel substrate 2 can be moved three-dimensionally. For example, when forming micro-protrusions that give a convex curved surface in a square lattice pattern, multiple grooves can be formed in a square lattice pattern by laser irradiation. A surface structure for transfer of a desired shape can be obtained by repeating laser irradiation as necessary.

[0032] The processing by laser irradiation may be photothermal processing or ablation processing, but ablation processing is preferred from the viewpoint of minimizing damage to the stainless steel substrate surface and suppressing debris generation.

[0033] The laser to be irradiated is not particularly limited as long as it can process the stainless steel substrate, but a picosecond laser is preferred from the viewpoints of processing speed, shape controllability, processing accuracy, and the possibility of ablation processing.

[0034] The irradiation conditions of the picosecond laser may be set to conditions suitable for forming a shoulder portion on the curved surface of the transfer surface structure. The lower limit of the output is preferably 10 mW, more preferably 50 mW, and even more preferably 90 mW. The upper limit of the output is preferably 150 mW, but may also be 130 W or 110 mW. The lower limit of the pulse width is preferably 1 ps, more preferably 3 ps, even more preferably 5 ps, and particularly preferably 10 ps. The upper limit of the pulse width is preferably 30 ps, ​​more preferably 25 ps, and even more preferably 20 ps. The lower limit of the repetition frequency is preferably 1 kHz, more preferably 10 kHz, and even more preferably 20 kHz. The upper limit of the repetition frequency is preferably 100 kHz, more preferably 80 kHz, and even more preferably 50 kHz.

[0035] After the laser irradiation, various treatments such as cleaning, drying, surface treatment, etc. can be performed. By simply performing the laser irradiation and any post-processing, a transfer mold having a biomimetic, complex surface structure for transfer can be efficiently and easily produced.

[0036] <Transfer Method> A method for transferring a surface structure to a transfer target using a transfer mold includes at least a step of contacting the transfer target surface structure of the transfer mold with the transfer target. This transfers a surface structure complementary to the transfer target surface structure of the transfer mold to the transfer target. The manner of contact can be selected appropriately depending on the material and properties of the transfer target. When the transfer target is a solid, transfer can be achieved by contacting the transfer mold and the transfer target, preferably under pressure. If necessary, contact or pressing can be performed while heated to a temperature above the softening point of the transfer target. When the transfer target is a liquid or semi-solid that can be solidified (including gelation), transfer can be achieved by performing a solidification treatment (e.g., heating, radiation exposure, etc.) while the transfer mold and the transfer target are in contact.

[0037] The transfer target material is preferably a scaffold material containing one or more selected from the group consisting of collagen, gelatin, hyaluronic acid, fibrin, fibrinogen, alginate, agarose, chitosan, chitin, cellulose, pectin, starch, laminin, gluten, casein, albumin, vitronectin, tenascin, entactin (night gene), heparan sulfate proteoglycan (perlecan), poly(acrylic acid) and its derivatives, poly(ethylene oxide) and its copolymers, poly(vinyl alcohol), polyphosphazene, and Matrigel. The microenvironment (niche) in which stem cells reside is three-dimensional, and an environment with a mechanical gradient (unevenness) is considered physiologically similar to human tissues / organs. For example, if the uneven (papilla-like) micropattern at the human epithelium / connective tissue interface could be reproduced on the scaffold material, physiological cellular responses more similar to those in vivo could be obtained, potentially facilitating applications and development in regenerative medicine. The transfer mold has a biomimetic, complex surface structure for transfer, making it possible to reproduce a microstructure similar to that of human tissue as described above on the scaffold material to be transferred, which may contribute to applications in regenerative medicine, etc.

[0038] A method for transferring a surface structure when using a collagen gel as a scaffold material will be described below. A suitable embodiment of this method is to produce a fibrous collagen gel having a predetermined surface structure on its surface by contacting a gel-forming collagen aqueous solution with a transfer mold, degassing any remaining gaps as necessary, and then maintaining the contact state for a predetermined period of time.

[0039] The type of collagen used as the raw material for the fibrous collagen gel is not particularly limited, but type I collagen, which is abundant in the body, is preferred, and atelocollagen, from which the antigenic determinant telopeptide has been removed, is more preferred. Collagen derived from biological sources such as mammals, seafood, birds, and reptiles can usually be used, but collagen derived from seafood, which does not share viruses with humans, is preferred. Collagen derived from fish is particularly preferred, and examples of the collagen to be collected include scales and skin. The advantage of scales is that they contain fewer impurities, such as lipids that cause a fishy odor, and therefore produce collagen with high purity.

[0040] In this specification, "collagen" refers to collagen molecules having a triple helical structure and aggregates or assemblies of these collagen molecules, and does not include thermally denatured collagen (gelatin) or collagen peptides in which the triple helical structure has been broken.

[0041] Known methods for solubilizing collagen contained in biological tissues to obtain solubilized collagen aqueous solutions include solubilization treatment with enzymes, extraction treatment with dilute acids, solubilization treatment with alkalis, etc. In this specification, unless otherwise specified, the term "solubilized collagen aqueous solution" refers to an aqueous collagen solution solubilized by any treatment method.

[0042] When a fibrous agent such as a buffer solution is added to a solubilized collagen aqueous solution to adjust the solubilized collagen aqueous solution to an appropriate ionic strength and pH, the collagen molecules are oriented and assume a structure similar to collagen fibers in vivo, resulting in a collagen gel with a specific shape. This collagen gel is called a "fibrous collagen gel." Furthermore, in this specification, a solubilized collagen aqueous solution having an ionic strength and pH suitable for forming a fibrous collagen gel is called a "gel-forming collagen aqueous solution."

[0043] A gel-forming collagen aqueous solution can be obtained by providing a solubilized collagen aqueous solution with an ionic strength and pH suitable for forming a fibrous collagen gel. Specifically, the ionic strength and pH are adjusted by adding a fibrosing agent to the solubilized collagen aqueous solution. Preferred examples of fibrosing agents include saline, buffer solutions, buffered saline, acidic salt aqueous solutions, neutral salt aqueous solutions, and alkaline salt aqueous solutions. The pH of the aqueous solution is preferably set appropriately within a range of, for example, pH 3 to 10 depending on the type of collagen (acid-solubilized collagen, enzyme-solubilized collagen, alkali-solubilized collagen, etc.). For example, for enzyme-solubilized collagen, a buffer solution, buffered saline, neutral salt aqueous solution, etc., with a pH range of 6 to 8 is preferably used. Specific examples of buffer solutions and buffered saline solutions include phosphate buffer, Tris buffer, HEPES buffer, acetate buffer, carbonate buffer, citrate buffer, phosphate-buffered saline (PBS), Dulbecco's phosphate-buffered saline (D-PBS), Tris-buffered saline, and HEPES-buffered saline.

[0044] The following two methods can be used to bring the gel-forming collagen aqueous solution into contact with the transfer mold. (Contact method 1): A method in which a transfer mold is placed with the area having the transfer surface structure facing upward, and a gel-forming collagen aqueous solution is poured into the area. (Contact method 2): A method in which a transfer mold is placed on top of a gel-forming collagen aqueous solution contained in a specified container, with the area having the transfer surface structure facing downward. In the above contact method 1, when the transfer mold alone is not able to hold the gel-forming collagen aqueous solution, it is preferable to use a predetermined frame or container that surrounds the transfer mold.

[0045] When unintended residual gaps are formed during contact between the gel-forming collagen aqueous solution and the transfer mold, it is preferable to remove the air present in the residual gaps by degassing. Degassing is an effective measure when the gel-forming collagen aqueous solution is difficult to enter into the recesses of the transfer mold due to its viscosity, or when it is difficult to uniformly inject the gel-forming collagen aqueous solution into the recesses of the transfer mold. The degassing method is not particularly limited, and known methods using an aspirator, vacuum pump, vacuum dryer, etc. can be used. When degassing, it is desirable to take appropriate measures such as fixing the transfer mold or applying pressure to the gel-forming collagen aqueous solution.

[0046] The degree of removal of air present in the remaining gaps may be appropriately set so that the desired surface structure is imparted to the fibrous collagen gel, and it is not necessary to completely remove the air. In other words, it is not necessary to completely transfer the surface structure complementary to the surface structure for transfer of the transfer mold to the fibrous collagen gel.

[0047] From the time when the solubilized collagen aqueous solution is provided with an ionic strength and pH suitable for the formation of a fibrous collagen gel until the time when the fibrous collagen gel is formed, it is preferable to hold the solution at a temperature of, for example, 15 to 30°C (but below the denaturation temperature of collagen) for a certain period of time, for example, 6 to 24 hours.

[0048] In parallel with or after the gelation reaction, γ-ray irradiation, electron beam irradiation, U Crosslinking may be performed by irradiation with radiation such as ultraviolet (V) irradiation or plasma irradiation, or by ionizing radiation irradiation. Alternatively, chemical crosslinking may be performed using a carbodiimide crosslinking agent (e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride).

[0049] When a fibrous collagen gel is observed with a scanning electron microscope at a magnification of 10,000 times, for example, and numerous fibrous structures are found, the presence of fibrous collagen can be confirmed. Although it is generally not easy to confirm that fibrous collagen has a D period using a scanning electron microscope, if a D period is confirmed in even a part of the fibrous collagen, it is generally safe to conclude that the entire fibrous collagen has a D period. [Example]

[0050] The present invention will be described in detail below using examples, but the present invention is not limited to the following examples as long as it does not depart from the gist of the invention.

[0051] <<Manufacturing of transfer molds>> Example 1 A stainless steel plate (SUS316L-H) measuring 40 mm long, 50 mm wide, and 0.4 mm thick was prepared as the stainless steel substrate. A 17 mm x 17 mm area near the center of one side of the stainless steel plate was irradiated with laser light to form a surface structure for transfer. The surface structure for transfer was designed to have 7,225 microprotrusions (85 x 85) approximately 130 μm high in a square lattice pattern with a spacing of 0.2 mm. Figure 4A shows a partially enlarged view of the designed micropattern, Figure 4B shows a wide-angle view of Figure 4A, and Figure 4C shows a cross-section of the designed pattern along line A1-B1. Laser irradiation was performed according to this design pattern. The laser was a picosecond pulse laser with a wavelength of 532 nm. The irradiation conditions were an output of 100 mW, a pulse width of 15 ps, a repetition rate of 20 kHz, and a scanning speed of 1 mm / s. Figure 5A shows a planar SEM photograph (100x magnification) of the obtained surface structure for transfer, and Figure 5B shows a partially enlarged SEM photograph (300x magnification) of the surface structure for transfer. Figure 5C shows the shape profile obtained when the micro-protrusions on the surface structure for transfer were scanned obliquely. As is clear from Figures 5A, 5B, and 5C, the obtained surface structure for transfer has dome-shaped protrusions that are convex outward as micro-protrusions, and shoulders are formed on the curved surfaces that correspond to the sides of the dome-shaped protrusions.

[0052] <Comparative Example 1> The same stainless steel plate as in Example 1 was prepared, and square pillar-shaped micro-projections (top surface dimensions 100 μm × 100 μm) were formed in a square lattice pattern using lithography. Figure 6 shows an SEM photograph (100x magnification) of the resulting transfer mold having square pillar-shaped micro-projections. It can be seen that no shoulders were formed in the transfer mold of Comparative Example 1.

[0053] <Transfer of surface structure to collagen gel> "Cellcampus FD-08G" (freeze-dried product) manufactured by Taki Chemical Co., Ltd., which was produced from tilapia scales, was dissolved in an HCl solution with a pH of 3, and the collagen concentration was then adjusted to 1.1% and the pH to 3, yielding a colorless, transparent aqueous solution of solubilized collagen. A silicon plate (2.5 mm thick) with a central hole was placed on top of the transfer mold of Example 1, which was placed so that the area having the transfer surface structure faced upward. The hole was circular with a diameter of 17 mm, and the silicon plate was placed so that the area having the transfer surface structure of the transfer mold was located in the center of the hole.

[0054] Next, 0.78 mL of a mixture of 9 parts by volume of the solubilized collagen aqueous solution and 1 part by volume of 10x Dulbecco's phosphate-buffered saline (D-PBS) was poured into the holes in the silicon plate. The mixture easily filled the recesses of the transfer surface structure of the transfer mold. After that, the mixture was kept at 25°C for 12 hours to obtain a fibrous collagen gel (0.78 g, collagen concentration 1% by mass). The fibrous collagen gel was easily removed from the transfer mold, and no deposits were found on the transfer mold.

[0055] When the surface of the fibrous collagen gel that had been in contact with the region of the transfer mold having the surface structure for transfer was examined using an SEM photograph, it was confirmed that the gel had a region in which a complex surface structure complementary to the surface structure for transfer had been formed. Figure 7 shows SEM photographs (narrow field of view (left) 300x; wide field of view (right) 100x) of the surface structure of the fibrous collagen gel obtained using the transfer mold of Example 1.

[0056] The surface structure was similarly transferred onto the surface of a fibrous collagen gel using the transfer mold of Comparative Example 1 instead of the transfer mold of Example 1. Fig. 8 shows SEM photographs (narrow field of view (left) 300x; wide field of view (right) 30x) of the surface structure of a fibrous collagen gel obtained using the transfer mold of Comparative Example 1.

[0057] Cell culture test Using each of the fibrous collagen gels (17 mm in diameter in plan view) obtained using the transfer molds of Example 1 and Comparative Example 1 as cell culture substrates (scaffolding materials), cell culture tests were conducted according to the following procedure. The thickness of each cell culture substrate was approximately 2.5 mm. Primary cultured cells derived from the oral mucosal epithelium of a patient who had visited the Department of Oral Surgery at Niigata University Medical and Dental Hospital were used. This experiment was approved by the Ethics Committee of the Niigata University School of Dentistry.

[0058] (Day 0) Each cell culture substrate was placed in a 12-well plate. For each fibrous collagen gel, the surface with the surface structure was placed facing up. Each well was coated with a mixture of 25 μL of 1 μg / μL type IV collagen solution and 500 μL of phosphate buffer, and then left to stand overnight at 4°C. (Day 1) The oral mucosal epithelial-derived primary cultured cells were mixed with Medium A (EpiLife (registered trademark) Thermo Fisher Scientific) supplemented with EDGS (EpiLife Defined Growth Supplements) and 1.2 mM Ca++ (high calcium) to prepare a cell suspension. 1 mL of the cell suspension was used to culture 1 × 10 6 After seeding the cells onto the surface of the fibrous collagen gel at 1.5 cells / well, 3.8 mL of Medium A was poured into the wells, bringing the total medium volume to 4.8 mL. After that, the cells were cultured in a submerged culture medium, with the medium changed daily until Day 4 of culture. (Day 4) The cells were then transferred to air-liquid interface culture, and the medium was changed every other day. The culture was continued until Day 11. (Day 11) Each cell culture substrate was removed and immersed in 4% paraformaldehyde overnight (4°C) to fix the resulting cultured tissue. The paraffin-embedded sections were then stained with hematoxylin and eosin (HE) using standard methods and subjected to morphological observation using an optical microscope.

[0059] (result) FIG. 9 is a histological image showing a cross section of HE-stained cultured tissue obtained from the fibrous collagen gel transferred using the transfer mold of Example 1. FIG. 10 is a histological image showing a cross section of HE-stained cultured tissue obtained from the fibrous collagen gel transferred using the transfer mold of Comparative Example 1. From FIG. 9, the formation of a continuous epithelial layer was observed over the entire surface of the cell culture substrate (collagen gel scaffold material). Furthermore, epithelial leg-like cells proliferated in the recesses of the cell culture substrate were also observed, which could be said to resemble the oral mucosal epithelial tissue of a living body having a papilla-like structure. Furthermore, Example 1 was more similar to the oral mucosal epithelial tissue of a living body. On the other hand, from FIG. 10, in the image of the cultured tissue of the fibrous collagen gel made using the transfer mold of Comparative Example 1, the applied square pillar-shaped microprojections were deformed, crushed, and flattened, dissimilar to the oral mucosal epithelial tissue of a living body. Furthermore, the formed cultured tissue was easily peeled off from the substrate (scaffold material). [Explanation of symbols]

[0060] 1 Transfer mold 2 Stainless steel substrate 3. Surface of stainless steel substrate 4. Surface structure for transfer 41, 51 Concave surface 41s, 51s Shoulder formed on a concave curved surface 42, 52 Convex curved surface 42s, 52s Shoulder formed into a convex curved surface Din Inward direction towards the inside of the stainless steel substrate D out Outward direction towards the outside of the stainless steel substrate z Normal direction to the surface of the stainless steel substrate

Claims

1. A transfer mold for transferring a surface structure to a transfer target, the transfer mold has a stainless steel substrate and a transfer surface structure formed on a surface of the stainless steel substrate; the surface structure for transfer has a convex curved surface that is convex in an outward direction opposite to an inward direction toward the inside of the stainless steel substrate when viewed along a normal direction to the surface of the stainless steel substrate, a shoulder portion is formed on at least a portion of the convex curved surface, the curvature of the curved surface changing; A transfer mold in which the maximum height Rz of the transfer surface structure is 10 μm or more and 500 μm or less.

2. The transfer mold according to claim 1 , wherein the convex curved surface is a surface of at least a part of the dome-shaped protrusion that is convex in the outward direction.

3. 3. The transfer mold according to claim 1 or 2, wherein the transfer target is a scaffold material comprising one or more selected from the group consisting of collagen, gelatin, fibrin, fibrinogen, alginic acid, agarose, chitosan, chitin, pectin, laminin, gluten, casein, albumin, vitronectin, tenascin, entactin (night gene), heparan sulfate proteoglycan (perlecan), poly(acrylic acid) and derivatives thereof, poly(ethylene oxide) and copolymers thereof, polyphosphazene, and Matrigel.

4. A method for manufacturing a transfer mold according to claim 1, A process in which a laser is irradiated onto the surface of a stainless steel substrate to form a surface structure for transfer. A method for manufacturing a transfer mold, comprising:

5. The method for manufacturing a transfer mold according to claim 4, wherein the laser is a picosecond laser.

6. 6. The method for manufacturing a transfer mold according to claim 5, wherein the picosecond laser irradiation conditions are an output of 10 mW to 150 mW, a pulse width of 1 ps to 30 ps, ​​and a repetition frequency of 1 kHz to 100 kHz.

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