JP6570120B2 - Transparent sheet-like collagen molding - Google Patents

Description

  The present invention relates to a sheet-like collagen molded body having transparency.

  Collagen is an important protein that accounts for 30% of protein in the body and has functions such as skeletal support and cell adhesion. For example, bone, cartilage, ligament / tendon, corneal stroma, skin, liver, muscle, etc. Exist in different organizations. Molded products made from this collagen as a raw material are cell culture substrates and scaffold materials for regenerative medicine (eg, cartilage, bone, spine, nucleus pulposus, ligament, corneal stroma, skin, blood vessels, nerves, liver tissue) Regenerative materials), transplantation materials (wound dressing materials, bone filling materials, hemostatic materials, anti-adhesion materials, etc.), drug delivery carriers, hemostatic materials, and the like have been developed.

  Most of the collagen in the living body is composed of collagen fibrils (also referred to as collagen fibers or collagen fibrils) to form various tissues. The direction of collagen fibrils in tissues ranges from highly disordered like skin to highly oriented like ligaments and tendons. Here, collagen fibrils are those in which collagen molecules are regularly aligned and associated. Its characteristic is that it has a horizontal stripe of D periodicity. The interval between the D periodic horizontal stripes is generally set to 67 nm. Here, the collagen molecule is a molecule in which a triple helical structure is formed by three polypeptide chains. Furthermore, as a characteristic of collagen in a living body, collagen molecules are cross-linked to increase stability. In ligaments, tendons, and the like, collagen fibers are gathered to form collagen fibers, and higher order fiber bundles are formed (for example, Non-Patent Document 1). The corneal stroma is known to have a laminated structure in which collagen fibrils are regularly oriented.

  As a method for solubilizing collagen from a biological tissue to obtain a solubilized collagen solution, a method of solubilizing with an enzyme, a method of extracting with a dilute acid, a method of solubilizing with an alkali, and the like are widely known. Unless otherwise specified below, the term “solubilized collagen solution” refers to a collagen solution solubilized by any treatment method. FIG. 1 shows a schematic diagram of a solubilized collagen solution. When solubilized collagen is dissolved, it is generally considered that collagen molecules are present separately in the solution.

  When a suitable buffer solution is added to the solubilized collagen solution and the solubilized collagen solution has an appropriate ionic strength and pH, refibrillated (also referred to as fibrosis) collagen fibrils (hereinafter referred to as “refibrillated collagen fibrils”). It is known that it can be obtained (for example, Patent Document 1). FIG. 2 is a schematic diagram of refibrillated collagen fibrils. FIG. 3 is an enlarged schematic view of refibrillated collagen fibrils. Similar to collagen fibrils existing in a living body, collagen molecules are associated and a structure having a D period (about 67 nm) is reproduced.

  By the way, as described above, the refibrillated collagen fibril obtained by adding an appropriate buffer solution to the solubilized collagen solution and refibrillating is not ordered in the direction, that is, non-oriented. It is known to be.

  In the collagen field, orientation at the level of collagen molecules, orientation at the level of refibrillated collagen fibrils, orientation at the level of collagen fibers, and so on. It is known that there is.

  FIG. 4 is a schematic diagram showing orientation at the level of collagen molecules. In FIG. 4, the collagen molecules present in pieces are merely oriented in a regular manner. In this state, refibrillated collagen fibrils having a D cycle formed by the association of collagen molecules are not seen. Collagen in this state can be said to be in an amorphous state. Moreover, what dried this becomes transparent.

  So far, various alignment techniques in the field of collagen have been developed. For example, regarding the strong magnetic field, which is a well-known orientation technique, citing paragraph 0004 of Patent Document 2, “A technique using a strong magnetic field has been reported as a technique for achieving three-dimensional orientation of gel-constituting fine fibers. (Non-Patent Document 1) However, the technique has incomplete fiber orientation. ” The “gel-constituting fine fibers” here are presumed to refer to collagen molecules.

  Patent Documents 3 and 4 disclose a method for producing a collagen fiber by electrospinning a collagen solution containing hexafluoroisopropanol.

  Patent Document 5 discloses a technique in which a solubilized collagen solution is spin-coated and a collagen gel layer in which collagen molecules are oriented is laminated.

  In Patent Document 6, a solubilized collagen solution added with a chemical crosslinking agent is cast in the casting direction and then pressed in the thickness direction of the gel layer, and the casting direction is different from the lower layer. A method for producing a laminated collagen gel comprising an upper layer formed in the same manner is disclosed. Similar reports have been made in Non-Patent Document 2 and the like.

  Patent Documents 7 to 9 disclose techniques for forming a filamentous shape by discharging collagen from a nozzle.

  Patent Document 7 discloses a method of obtaining a “collagen fiber” by discharging a solubilized collagen solution from a spinning nozzle into a concentrated salt aqueous solution.

  Patent Document 8 discloses two methods as a method for producing a re-fibrillated collagen gel using a solubilized collagen solution of type I collagen derived from a mouse tail. The first method is to form a deposited collagen solution by moving the injection needle in the direction opposite to the discharge direction while discharging the solubilized collagen solution from the injection needle, and then immersing it in 10 times the concentration of PBS. Thus, a refibrillated collagen gel is prepared. In the second method, an acidic solubilized collagen solution is discharged from a syringe needle in a 10-fold concentration of PBS to prepare a refibrillated collagen gel. The injection needle is moved in the direction opposite to the discharge direction.

  Patent Document 9 discloses a method similar to the second method of Patent Document 8, that is, a method for producing a string-like refibrillated collagen gel by discharging a solubilized collagen solution in PBS. . Patent Document 9 also discloses a method of obtaining a sheet-like collagen gel by arranging a plurality of the collagen gels in a flat shape and then drying them.

  Non-Patent Document 5 discloses a collagen matrix in which collagen microfibrils having a D period are arranged in a single layer (thickness of 3 nm or less) with orientation. In the method, the layered crystal of muscovite is cleaved to expose a flat surface, and then a drop of buffer solution is placed thereon, and a buffer solution containing collagen molecules is injected into the buffer solution. .

Republished Patent No. WO2012 / 070679 International Publication No. WO2007 / 066715 JP 2007-138364 A JP-T-2004-523484 JP 2010-148691 A Japanese Patent No. 5525523 JP-A-8-35193 Japanese Patent No. 5669760 Republished Patent No. WO2012 / 114707

Kazuo Watanabe, Journal of the Japan Society of Photography, Vol.61 No.2 (1998) p.72-76 “Structure and Properties of Collagen” Mitsuru Akashi, Research on Development and Clinical Application of Regenerative Medicine Technology for All Layers of Cornea 2007 General Review / Shared Research Report (2008) p.9-12 “Study on Development and Clinical Application of Regenerative Medicine Technology for All Layers of Cornea” Creation of oriented laminated collagen gel for the purpose of substantial regeneration " Toshiyuki Ikoma et al., Material Integration vol.23 No.22 (2010) p.6-11 “Basic Structure of Collagen” Ikuo Miyata et al., Textiles and Industry, Vol.39, No.11 (1983) p.427-435 “Application of Fiber Materials to Biotechnology” David A. Cisneros et al., Small Vol. 3, Issue 6, pages 956-963, 2007 "Creating Ultrathin Nanoscopic Collagen Matrices For Biological And Biotechnological Applications"

  The present invention develops a sheet-like collagen molded body having transparency and composed of a single-layer sheet or a multi-layer laminated sheet, and each layer is composed of refibrillated collagen fibrils oriented with regularity. Is an issue.

  By the way, although it is known that refibrillated collagen fibrils are non-oriented or have a low orientation degree, white color is known, but even if the orientation degree of refibrillated collagen fibrils is high, the density of collagen is low. In some cases, the present inventors have obtained the knowledge that transparency is improved as the density increases and the density increases.

  Here, the result considered about the orientation and transparency in the said illustrated technique and literature is described below. Moreover, what could be judged also about the level (collagen molecule, refibrillated collagen fibril, or collagen fiber) which gave the orientation was described.

  FIG. 3A of Patent Document 3 shows an electron micrograph of “collagen fiber”, but it cannot be said that the orientation is high. Further, it is described that the average value of the fiber diameter of “collagen fiber” is about 5 μm (paragraph 0043). On the other hand, page 9 of Non-Patent Document 3 describes that the thickness of collagen fibrils (referred to as “collagen fibers” in Non-Patent Document 3) is about 100 nm. It is reasonable to consider that the “collagen fiber” of a) is not a refibrillated collagen fibril but an aggregate thereof.

  In Patent Document 4, an SEM image of “fibers” of electrospun collagen having a fiber diameter of 390 nm ± 290 nm is shown in FIG. 10, but no orientation is observed in these “fibers”.

  Collagen fibers obtained by strong magnetic field orientation and electrospinning are difficult to obtain high transparency because the density of refibrillated collagen fibrils is not high. For example, it is described that the collagen fiber obtained in Example 1 of Patent Document 3 is white (paragraph 0035), and that the collagen mat obtained in Example 8 of Patent Document 4 is also white. (Paragraph 0222).

  Since Patent Document 5 does not have a step of fibrosis of the solubilized collagen solution, it is not a technique related to the orientation of refibrillated collagen fibrils but a technique related to orientation at the collagen molecule level.

  Patent Document 6 describes in paragraph 0025 that a chemical cross-linking agent is added in a state where fiber formation is promoted by adjusting the pH of the solubilized collagen solution. However, in Example 1, after the pig skin-derived Type I atelocollagen was dissolved in an aqueous solution of pH 4.0, the pH of the solubilized collagen solution was adjusted to 3.5 to 4.0 using NaOH, and a chemical crosslinking agent ( EDC) and carboxyl group activator (NHS) are added. From this series of procedures, the raw material atelocollagen is not at least alkali-solubilized collagen (the pH increases if alkali-soluble collagen is dissolved in pH 4.0 aqueous solution, so adjust to pH 3.5-4.0 with NaOH. It does not match the above procedure) and is supposed to be enzyme-solubilized collagen. In the case of enzyme-solubilized collagen, pH 3.5 to 4.0 is the pH of the collagen dissolution region, and therefore cannot be refibrillated (fibrotic).

  Non-Patent Document 4 describes that when a solubilized collagen solution is exposed to NaCl at a concentration of 5% or more, it is precipitated as an amorphous aggregate having no ordered arrangement. In the “collagen fiber”, collagen molecules are present in an amorphous form, and it is presumed that almost no collagen fibrils are present.

  FIG. 4 of Patent Document 8 is an atomic force microscope image of the collagen gel obtained by the first method. Although FIG. 4 is unclear, it does not appear to have orientation.

  8 and 9 of Patent Document 8 are atomic force microscope images of the collagen gel obtained by the second method. In FIG. 8 and FIG. 9, no orientation is observed. Therefore, it is presumed that the string-like refibrillated collagen gel of Patent Document 9 obtained by the same method as the second method of Patent Document 8 does not show orientation at the collagen fibril level. In addition, the sheet-shaped collagen material obtained by the method described in Example 2 of Patent Document 9 is obtained by drying a plurality of string-like collagen gels arranged in a plane, and is assembled with logs. It is shaped like a ridge and is considered to have no substantially flatness.

  Since the collagen matrix described in Non-Patent Document 5 is a single layer of collagen microfibrils arranged side by side, it may not be able to withstand mechanical loads such as handling with tweezers. It can be easily imagined, and therefore it cannot be expected to exhibit the function as a sheet. In addition, since the density is low, it is presumed that there is no transparency.

The present invention is as follows.
[1] A single-layer sheet or a laminated sheet composed of a plurality of layers, and a molded body having upper and lower surfaces that are substantially flat and having transparency, each layer of the single-layer sheet or the laminated sheet being substantially A transparent sheet-like collagen molded article characterized by comprising refibrillated collagen fibrils oriented with regularity.
[2] The transparent sheet-like collagen molded article according to [1], wherein the degree of orientation is in the range of 0.5 to 1.
However, the degree of orientation described above is obtained when a 10,000-fold scanning electron microscope image in an arbitrary layer of the transparent sheet-like collagen molded body is analyzed with image analysis software “A Image-kun (registered trademark)” manufactured by Asahi Kasei Engineering Corporation. This is a value calculated by the half width method.
[3] The transparent sheet-like collagen molded article according to the above [1] or [2], wherein the visible light transmittance measured in the thickness direction of the molded article is in the range of 50 to 80%.
[4] The transparent sheet-like collagen molded article according to any one of the above [1] to [3], wherein the density of collagen is in the range of 0.7 to 1.4 g / cm ³ .
[5] The transparent sheet-like collagen molded article according to any one of the above [1] to [4], wherein the molded article is subjected to a crosslinking treatment by a physical crosslinking method or a chemical crosslinking method.
[6] The method for producing a transparent sheet-like collagen molded article according to any one of [1] to [5], comprising the following steps.
A first step of obtaining a sheet-like material by discharging the solubilized collagen solution and spreading it into a sheet, or discharging the solubilized collagen solution into a sheet.
A second step in which the sheet-like material obtained in the first step is re-fibrillated by contacting with a physiological isotonic solution or a buffer solution to obtain a re-fibrillated sheet-like material.
A third step in which the refibrillated sheet-like material obtained in the second step is pressed in the thickness direction to be consolidated.
[7] The method for producing a transparent sheet-like collagen molded article according to the above [6], wherein the first step of obtaining a sheet-like material includes the following steps a and b.
Step a: discharging the solubilized collagen solution and spreading it into a sheet, or discharging the solubilized collagen solution into a sheet, and obtaining a sheet A.
Step b: the next step (i) or (ii).
(i) A step of producing a sheet-like material composed of a laminated sheet by discharging a solubilized collagen solution onto the sheet-like material A and spreading it into a sheet shape or discharging the solubilized collagen solution into a sheet shape.
(ii) A sheet-like material B was obtained by discharging the solubilized collagen solution to an arbitrary place other than on the sheet-like material A and spreading it in a sheet shape or discharging the solubilized collagen solution into a sheet shape. Then, the process of producing the sheet-like object which adheres the sheet-like object A and the sheet-like object B, and consists of laminated sheets.
However, in the case of producing a laminated sheet of three or more layers based on the two-layer laminated sheet obtained by carrying out the steps a to b once, the “sheet” in the step b (i) or b (ii) In place of the “form A”, the “sheet-like article made of the laminated sheet obtained in the previous step b” is applied, and the step b is repeated a predetermined number of times.
Further, as an optional treatment, a sheet-like material obtained in step a is dried at a temperature lower than the denaturation temperature of collagen, or a sheet-like material comprising a laminated sheet obtained in step b (i) or (ii). Treatment of drying below the denaturation temperature of collagen.
[8] In the first step, the method of discharging the solubilized collagen solution is a case where the solubilized collagen solution is discharged onto a planar discharge surface, and the operations at the time of discharge are the following (1) to (3) The method for producing a transparent sheet-like collagen molded article according to the above [6] or [7].
(1) The discharge surface is fixed, and the discharge port is moved in the direction opposite to the discharge direction along the discharge surface.
(2) The discharge port is fixed and the discharge surface is moved in the same direction as the discharge direction.
(3) The ejection port is moved along the ejection surface in the direction opposite to the ejection direction, and the ejection surface is moved in the same direction as the ejection direction.
[9] The transparent sheet-like collagen molding according to any one of the above [6] to [8], which is further subjected to a crosslinking treatment by a physical crosslinking method or a chemical crosslinking method after the third step. Body manufacturing method.
[10] The method for producing a transparent sheet-like collagen molded article according to any one of [6] to [9], wherein the physiological isotonic solution or buffer is an aqueous solution of an inorganic salt.
[11] A medical material using the transparent sheet-like collagen molded article according to any one of [1] to [5].

  Since the transparent sheet-like collagen molded body of the present invention has transparency, it can be suitably used especially for applications requiring transparency.

It is a schematic diagram of a solubilized collagen solution. It is a schematic diagram of a refibrillated collagen fibril. It is the schematic diagram which expanded the refibrillated collagen fibril. It is a schematic diagram which shows the orientation of a collagen molecule. 2 is a scanning electron microscope image (10,000 times) of a molded article for evaluation in Example 1. FIG. 2 is a scanning electron microscope image (10,000 times) of an evaluation molded body in Comparative Example 1. FIG. 6 is a scanning electron microscope image (10,000 times) of a molded article for evaluation in Comparative Example 2. FIG. It is the photograph which showed the difference in transparency of the molded object of Example 1 and Comparative Example 1 by the transparency of the character.

(Transparent sheet-like collagen molding)
Hereinafter, the transparent sheet-like collagen molded body of the present invention (hereinafter referred to as “the molded body of the present invention”) will be described in detail.
The molded body of the present invention is composed of a single-layer sheet or a multi-layer laminated sheet, and is a molded body having upper and lower surfaces that are substantially flat and having transparency. Each layer is constituted by refibrillated collagen fibrils oriented with substantially regularity.

  Here, “substantially flat” means that there is no undulation, but it may include the case where sufficiently small unevenness is formed with respect to the length of one side of the upper surface or the lower surface of the molded body. means.

  Further, “oriented with substantially regularity” means a state in which most of the refibrillated collagen fibrils are arranged substantially in parallel when each layer of the molded article of the present invention is observed as a whole. It goes without saying that some refibrillated collagen fibrils are allowed to be present irregularly. Here, “substantially parallel” is intended to include not only completely parallel but also recognized as substantially parallel.

  An example of the method for measuring the degree of orientation of the molded body of the present invention is that a 10,000 times scanning electron microscope image on the upper surface or the lower surface of an arbitrary layer of the molded body of the present invention is image analysis software “A image” manufactured by Asahi Kasei Engineering Corporation. "Kun (registered trademark)" and the degree of orientation is calculated by the half-width method. The maximum value of the degree of orientation is 1, and a larger value indicates that the orientation is in a certain direction. The preferred range of the molded article of the present invention is in the range of 0.5-1. If it is in the said range, it can be said that it is in the state which orientated with substantially regularity. In addition, when it is difficult to measure the degree of orientation of the upper surface or the lower surface in an arbitrary layer, analysis may be performed on a cross section where the orientation can be evaluated.

  The transparency of the molded product of the present invention means at least translucent transparency. When an example of a suitable range of transparency is indicated by a numerical value, it is preferable that the visible light transmittance measured in the thickness direction of the molded article of the present invention is 50% or more. The higher the visible light transmittance, the better the range of 50 to 100% can be exemplified, but there is no particular problem as long as it is in the range of 50 to 80%.

  When the molded article of the present invention is composed of a laminated sheet having a plurality of layers, the orientation direction of the refibrillated collagen fibrils in the upper layer or the lower layer overlapping the arbitrary layer is the same. It may be different or different. For application to applications such as a corneal material or a material that requires strength due to a suturing treatment, it is also a preferable aspect that the orientation directions of the overlapping layers are orthogonal.

  The molded product of the present invention may be a gel or a dried product. Moreover, when it is immersed and crosslinked, it is good also as the state hold | maintained in the immersion liquid. The shape of the molded body of the present invention is not particularly limited, and examples thereof include a flat plate shape and a dome shape.

The molded body of the present invention exhibits transparency due to the high density of collagen in addition to high orientation, and the density may be in the range of 0.7 to 1.4 g / cm ³ , for example. preferable. When the density is less than 0.7 g / cm ³ , it becomes difficult to obtain sufficient transparency. Moreover, the thickness of the molded article of the present invention may be set by adjusting the degree of lamination as appropriate according to the application, but it is, for example, in the range of 0.01 to 2 mm.

  As one form of the molded body of the present invention, a product subjected to a crosslinking treatment by a physical crosslinking method or a chemical crosslinking method may be used. Depending on the type and degree of the crosslinking treatment, functions such as high strength and high water resistance can be imparted. Details of the crosslinking treatment method will be described later.

(Production method)
One form of the manufacturing method of the molded object of this invention suitably includes the following processes.
(1) A first step of obtaining a sheet-like product by discharging the <1> solubilized collagen solution and spreading it into a sheet, or <2> discharging the solubilized collagen solution into a sheet.
(2) A second step in which the sheet-like material obtained in the first step is re-fibrillated by contacting with a physiological isotonic solution or buffer solution to obtain a re-fibrillated sheet-like material.
(3) A third step of pressing the refibrillated sheet-like material obtained in the second step in the thickness direction.
In addition, the manufacturing method of the said form has an advantage that a sheet-like molded object can be obtained with a simple manufacturing method.

(First step)
The solubilized collagen solution is preferably a solution in which water-soluble collagen having a triple helical structure is dissolved, and as the collagen, atelocollagen from which telopeptides which are antigenic determinants of collagen are removed is particularly preferable. The type of collagen is not particularly limited, but type I having a large amount in vivo is preferable. The solubilized collagen solution may contain peptides, amino acids, gelatin and the like, but it is preferable that they are excluded as much as possible.

  The solubilized collagen solution can be obtained by a known method from collagen-containing tissues of biological materials such as mammals, seafood, birds and reptiles. In particular, a solubilized collagen solution prepared from fish that does not have a virus common to humans is preferred. As the solubilized collagen solution derived from fish, those having a relatively high denaturation temperature are preferable from the viewpoint of applicability to various uses, and an example of such fish species is Oreochromis. Of the genus Oreochromis, tilapia, which is mainly cultivated for food from China to Southeast Asia and is readily available, is particularly preferred.

  Here, a method described in Japanese Patent Nos. 4863433 and 5692770 will be briefly introduced as a method of solubilizing collagen using fish scale as a raw material and using an enzyme. In this method, scales decalcified with an acid are treated with a protease such as pepsin to atelocollagen, and if necessary, a purification treatment is performed. For the purification treatment, for example, in addition to salting out, a method using activated carbon having a pH of 7 or less described in Japanese Patent No. 5522857 can be applied.

  The collagen concentration in the solubilized collagen solution is preferably a concentration at which at least the discharged solubilized collagen solution can maintain the discharged form, and the higher the concentration, the easier the refibrillated collagen fibrils are oriented with a regularity. Moreover, it is preferable to use a high-concentration solubilized collagen solution in order to increase the density of the molded article of the present invention. For example, the collagen concentration is preferably in the range of 0.3 to 40% by mass, and more preferably in the range of 1 to 20% by mass. The concentration can be increased by a known method, and examples thereof include methods such as centrifugation and dialysis. Alternatively, a solubilized collagen solution may be prepared by dissolving solid collagen such as a freeze-dried collagen product to a predetermined concentration.

  In any of the above methods <1> and <2>, the method for discharging the solubilized collagen solution is not particularly limited, and a discharge method suitable for each method may be applied as appropriate. It is particularly desirable to use methods that produce laminar flow in the process. In order to generate a laminar flow, it is preferable to appropriately set conditions such as a flow channel size and a flow rate according to conditions such as the concentration and viscosity of the solubilized collagen solution. For example, a method of passing a narrow part having a certain length at a slow speed is preferable, and examples of specific examples of the narrow part are a nozzle, an injection needle, and the like. It is preferable to set the shape of the discharge port as appropriate.

  When discharging the solubilized collagen solution to, for example, a planar discharge surface, (1) fixing the discharge surface and moving the discharge port in the direction opposite to the discharge direction along the discharge surface, (2) fixing the discharge port And (3) moving the discharge port in the direction opposite to the discharge direction along the discharge surface and moving the discharge surface in the same direction as the discharge direction, etc. Can be used. At this time, the discharge surface may be installed horizontally or may be installed obliquely as required. Moreover, it is preferable that the discharge port and the discharge surface are not separated too much. More preferably, the discharge port and the discharge surface are brought into contact with each other.

  The spreading method in the method <1> is not particularly limited as long as it can be spread and extended without impairing the orientation of the discharged solubilized collagen solution. For example, any one of the above (1) to (3) For example, a method may be used in which the solubilized collagen solution discharged by the above method is formed into a sheet using a roll, a plate, a rod, or the like. As a specific method, for example, an operation of pressing the plate-shaped spreading member from above the solubilized collagen solution with an appropriate force against the solubilized collagen solution discharged in a rod shape, Operation to push the spreading member in a direction perpendicular to the direction of the rod parallel to the surface where the rod exists, and to push the spreading member in the forward or backward direction with respect to the discharge direction of the solubilized collagen solution. A method of forming into a sheet by an operation such as the above can be exemplified. It is preferable to select a material that is difficult for collagen to adhere to the spreading member. Further, the adhesion of collagen may be prevented by applying a polymer compound such as PEG to the spreading member within a range that does not adversely affect the application.

  As a series of methods of discharging and spreading, for example, a method based on a rolling method of a metal material may be used in addition to the above. Specifically, it is a method in which the solubilized collagen solution is discharged vertically downward, dropped between two rolls having the opposite rotation directions installed horizontally, and stretched.

  The method of <2> is not particularly limited as long as the orientation of the solubilized collagen solution is not impaired, and using a known sheet forming apparatus is also one preferred embodiment.

  Through the above first step, a sheet-like material having a single layer structure can be obtained. When the upper and lower surfaces of the obtained sheet-like material are not substantially flat, the shape is adjusted so as to be substantially flat.

In order to obtain a sheet-like material comprising a laminated sheet, it is preferable to apply a manufacturing method including the following ab steps in the first step.
Step a: A step of obtaining the sheet-like material A by discharging the solubilized collagen solution and spreading it in a sheet shape, or discharging the solubilized collagen solution in a sheet shape.
Step b: the next step (i) or (ii).
(i) A step of producing a sheet-like material composed of a laminated sheet by discharging a solubilized collagen solution onto the sheet-like material A and spreading it into a sheet shape or discharging the solubilized collagen solution into a sheet shape.
(ii) A sheet-like material B was obtained by discharging the solubilized collagen solution to an arbitrary place other than on the sheet-like material A and spreading it in a sheet shape or discharging the solubilized collagen solution into a sheet shape. Then, the process of producing the sheet-like object which adheres the sheet-like object A and the sheet-like object B, and consists of laminated sheets.

  Adhesion in the step b (ii) may be performed by superimposing the sheet-like material A and the sheet-like material B using the adhesiveness of the collagen gel itself. Moreover, you may make it adhere | attach using the adhesive agent etc. which do not have a trouble in a use.

  In the lamination, the directionality between the ejection direction of the previous layer and the ejection direction of the next layer is not particularly limited, and may be the same direction or different directions. Moreover, you may press moderately in order to raise the adhesiveness of layers.

  When producing a laminated sheet of three or more layers based on the two-layer laminated sheet obtained by carrying out the steps a and b once, the “sheet-like material” in the step b (i) or b (ii) Instead of “A”, it is preferable to apply “a sheet-like material made of a laminated sheet obtained in the previous step b” and repeat step b a predetermined number of times. For example, when the b (i) step is carried out following the a step in the production of the two-layer laminated sheet, the b (i) step may be carried out again in order to obtain a three-layer laminated sheet, (ii) You may implement a process. The b (i) step when the b (i) step is carried out again is as follows: “The sheet of the laminated sheet obtained in the previous b step is ejected with a solubilized collagen solution and spread into a sheet. Or a step of producing a sheet-like material made of a laminated sheet by discharging the solubilized collagen solution into a sheet.

  Further, as an optional treatment, a sheet-like material obtained in step a is dried at a temperature lower than the denaturation temperature of collagen, or a sheet-like material comprising a laminated sheet obtained in step b (i) or (ii). You may implement the process dried below the denaturation temperature of collagen. Here, a suitable method for the drying treatment is freeze-drying.

(Second step)
Next, in the second step, the sheet-like material obtained in the first step (including the sheet-like material composed of laminated sheets) is refibrillated by contacting with a physiological isotonic solution or buffer solution. A refibrillated sheet is obtained.

  Physiological isotonic fluid, as defined in the Biochemical Dictionary, 4th edition, means “extracellular fluids such as blood and tissue fluids, and fluids with the same osmotic pressure as intracellular fluids”. A good example is physiological saline, although those containing it are also known. Examples of the physiological buffer include phosphate buffer, Tris buffer, HEPES buffer, acetate buffer, carbonate buffer, and citrate buffer, and are physiological saline thereof. Examples include PBS, D-PBS, Tris buffered saline, HEPES buffered saline, and the like. In particular, an aqueous solution of an inorganic salt is preferably used as a physiological isotonic solution or buffer solution in order to facilitate refibrillation.

  The contact method between the sheet-like material and the physiological isotonic solution or buffer solution is not particularly limited as long as the collagen can be refibrillated. For example, the sheet-like material is immersed in a physiological isotonic solution or buffer solution. And a method of spraying a physiological isotonic solution or buffer solution on a sheet. The occurrence of refibrillation can be visually observed when the transparent or translucent sheet is clouded. It is desirable that conditions such as a contact method and a contact time are appropriately set, and the sheet-like material is sufficiently refibrillated until a white color is exhibited.

Here, an advantage when a solubilized collagen solution derived from fish is used as the solubilized collagen solution will be described.
Fish-derived solubilized collagen generally has the property of a faster refibrillation rate than mammalian-derived solubilized collagen, so when using a fish-derived solubilized collagen solution, the refibrillation process can be carried out in a short time. That is, the time until the transparent or translucent sheet-like material becomes white and turns white is short.
If refibrillation progresses slowly, it becomes susceptible to various factors (for example, molecular motion in the gel, temperature change, etc.) that lead to a decrease in the degree of orientation of refibrillated collagen fibrils. Therefore, it can be said that the use of a solubilized collagen solution derived from fish having a high refibrillation rate is a more preferable material selection.

(Third step)
In the third step, the refibrillated sheet-like material obtained in the second step is pressed in the thickness direction to be consolidated.

As a member used for pressing, it is preferable to use a member that can press the refibrillated sheet-like material evenly. For example, when a flat refibrillated sheet is pressed as it is, a flat member is used. When processing a flat refibrillated sheet material into a dome shape by pressing, the refibrillated sheet material is placed on a dome shape mold and a member having the same shape as the dome shape mold is used. .
As the material for the pressing member, it is preferable to select a material that does not easily adhere to collagen like the spreading member, and a polymer compound such as PEG is applied to the pressing member within a range that does not adversely affect the application. In this way, adhesion of collagen may be prevented.

  The degree of consolidation by pressing is carried out to the extent that at least the transparency of the refibrillated sheet can be obtained.

(Crosslinking treatment)
The molded product of the present invention can be further subjected to a crosslinking treatment as necessary. The crosslinking treatment is particularly effective when it is desired to increase the strength. For the crosslinking treatment, either a physical crosslinking method or a chemical crosslinking method may be used. Examples of the physical cross-linking method include γ-ray irradiation, electron beam irradiation, plasma irradiation, irradiation cross-linking method by UV irradiation, thermal cross-linking method and the like. Chemical crosslinking methods include glutaraldehyde, polyepoxy compounds (ethylene glycol diglycidyl ether, glycerol polyglycidyl ether, etc.), carbodiimide compounds (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, hydrochloride, etc.) And a crosslinking method using a crosslinking agent such as a reducing sugar (such as ribose).

  The use of the molded body of the present invention is, for example, a medical material. Specific examples include corneal material, cell culture substrate, regenerative medical scaffold material, muscle fiber regeneration scaffold, transplant material, cosmetic material, wound dressing material, nerve regeneration tube, artificial blood vessel, dialysis tube, DDS carrier, suture Examples thereof include yarns, nonwoven fabrics, and adhesion prevention materials.

  EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto. In Examples, “%” means “% by mass” unless otherwise specified.

[Example 1]
As a solubilized collagen solution, “Cell Campus FD-08G” (lyophilized product) manufactured by Taki Chemical Co., Ltd. manufactured from tilapia scales was dissolved in an HCl solution of pH 3, and then a collagen concentration of 13% and pH 4.5 The colorless and transparent solution prepared in (1) was used.

  The solubilized collagen solution was filled into a 15 ml syringe by aspiration. A cylinder having a length of about 2 cm was attached to the tip of the syringe, and the solubilized collagen solution was discharged on a flat polystyrene plate while moving in the direction opposite to the discharge direction. Subsequently, the sheet was spread using a plastic rod to obtain a sheet-like material having a thickness of about 1 mm. The plastic rod was pushed and moved in the discharge direction and the forward direction.

  Next, the polystyrene plate was gently immersed in D-PBS and allowed to stand overnight at room temperature to refibrillate the collagen. The obtained refibrillated sheet was white.

  Next, after replacing D-PBS with ethanol, using a plastic flat plate, the refibrillated sheet was pressed in the thickness direction until transparency was obtained, and dried under reduced pressure conditions. It was. The upper and lower surfaces of the obtained molded body were substantially flat.

[Comparative Example 1]
A re-fibrillated sheet-like product (molded product) having a white color was obtained in the same manner as in Example 1 except that the step of pressing and compacting using a plastic flat plate was not performed.

[Comparative Example 2]
As the solubilized collagen solution, a colorless transparent solution having a collagen concentration of 1% and a pH of 3.3 prepared in the same manner as in Example 1 except for the collagen concentration was used.

  Without moving the discharge port, the solubilized collagen solution was discharged so as to fill the entire 30 × 30 × 2.5 mm silicon mold, and then molded by pressing the flat plate from above.

  Next, the shaped collagen gel was gently dipped in D-PBS and allowed to stand overnight at room temperature to refibrillate the collagen. The obtained refibrillated sheet was white.

  Next, after replacing D-PBS with ethanol, using a plastic flat plate, the refibrillated sheet-like material was pressed in the thickness direction with sufficient pressing force in the same manner as in Example 1 to be consolidated. And dried. The upper and lower surfaces of the obtained molded body were substantially flat.

[Evaluation]
(Density measurement)
Each molded body obtained in the above Examples and Comparative Examples was molded into a 1 cm square. About this square molded object, the weight weighed precisely was divided by the volume, and the density was determined. In addition, as thickness when calculating | requiring a volume, thickness measured 5 points | pieces with the micrometer, and the average value was used. Incidentally, each thickness was 0.287 mm in Example 1, 0.956 mm in Comparative Example 1, and 0.059 mm in Comparative Example 2.

(Transparency evaluation)
Each molded body obtained in the above Examples and Comparative Examples passes through the quartz cell of the color / turbidity simultaneous measuring device (“COH-400” manufactured by Nippon Denshoku Industries Co., Ltd.) so that the transmitted light passes in the thickness direction. The total light transmittance was measured.

(Scanning electron microscope observation)
Each molded body obtained in the above Examples and Comparative Examples was observed with a scanning electron microscope (“JSM-6010LA” manufactured by JEOL Ltd.).
As a result, in Example 1 and Comparative Example 1, it was found that refibrillated collagen fibrils were oriented with high regularity substantially in parallel (Example 1: FIG. 5, Comparative Example 1: FIG. 6). On the other hand, in Comparative Example 2, it was found that refibrillated collagen fibrils were randomly oriented (FIG. 7). 5 to 7 is 10,000 times.

(Analysis of orientation)
For the analysis of the degree of orientation, each of the 10,000 times scanning electron microscope images was used, and the half-width method of image analysis software “A Image-kun (registered trademark)” manufactured by Asahi Kasei Engineering Co., Ltd. was used.

  Table 1 shows the results of the degree of orientation, density, and total light transmittance. From Comparative Example 1 and Comparative Example 2, it was found that if either one of the degree of orientation and the density was low, the total light transmittance was low. On the other hand, from Example 1, it was found that when both the degree of orientation and density were high, the total light transmittance was high.

  FIG. 8 is a photograph showing the difference in transparency between the molded body obtained in Example 1 and the molded body obtained in Comparative Example 1 by the transparency of characters. Also from this photograph, it can be seen that the molded body obtained in Example 1 has higher transparency than the molded body obtained in Comparative Example 1.

Claims (8)

A transparent sheet-like collagen molded product having the following features [1] to [4] .
[1] The molded body is composed of a single-layer sheet or a multi-layer laminate sheet, and each layer of the single-layer sheet or the laminate sheet is composed of refibrillated collagen fibrils, The refibrillated collagen fibrils are oriented with substantially regularity, and the degree of orientation is in the range of 0.5-1.
However, the degree of orientation is a half-width method when a 10,000 times scanning electron microscopic image in an arbitrary layer of the molded body is analyzed by image analysis software “A Image-kun (registered trademark)” manufactured by Asahi Kasei Engineering Corporation. Is a value calculated by.
[2] The upper and lower surfaces of the molded body are substantially flat.
[3] The visible light transmittance measured in the thickness direction of the molded body is 50% or more.
[4] The collagen density in the molded body is in the range of 0.7 to 1.4 g / cm ³ . The shaped body is a physical crosslinking method or a chemical crosslinking method by crosslinking treatment it is one that has been subjected claim 1 Symbol placement transparency sheet collagen molded body. The manufacturing method of the transparent sheet-like collagen molded object of Claim 1 or 2 including the following processes.
A first step of obtaining a sheet-like material by discharging the solubilized collagen solution and spreading it into a sheet, or discharging the solubilized collagen solution into a sheet.
A second step in which the sheet-like material obtained in the first step is re-fibrillated by contacting with a physiological isotonic solution or a buffer solution to obtain a re-fibrillated sheet-like material.
A third step in which the refibrillated sheet-like material obtained in the second step is pressed in the thickness direction to be consolidated. The method for producing a transparent sheet-like collagen molded article according to claim 3 , wherein the first step of obtaining a sheet-like product comprises the following steps a and b.
Step a: A step of obtaining the sheet-like material A by discharging the solubilized collagen solution and spreading it in a sheet shape, or discharging the solubilized collagen solution in a sheet shape.
Step b: the next step (i) or (ii).
(i) A step of producing a sheet-like material composed of a laminated sheet by discharging a solubilized collagen solution onto the sheet-like material A and spreading it into a sheet shape or discharging the solubilized collagen solution into a sheet shape.
(ii) A sheet-like material B was obtained by discharging the solubilized collagen solution to an arbitrary place other than on the sheet-like material A and spreading it in a sheet shape or discharging the solubilized collagen solution into a sheet shape. Then, the process of producing the sheet-like object which adheres the sheet-like object A and the sheet-like object B, and consists of laminated sheets.
However, in the case of producing a laminated sheet of three or more layers based on the two-layer laminated sheet obtained by carrying out the steps a to b once, the “sheet” in the step b (i) or b (ii) In place of the “form A”, the “sheet-like article made of the laminated sheet obtained in the previous step b” is applied, and the step b is repeated a predetermined number of times.
Further, as an optional treatment, a sheet-like material obtained in step a is dried at a temperature lower than the denaturation temperature of collagen, or a sheet-like material comprising a laminated sheet obtained in step b (i) or (ii). Treatment of drying below the denaturation temperature of collagen. In the first step, the method of discharging the solubilized collagen solution is a case of discharging the solubilized collagen solution to a planar discharge surface,
The method for producing a transparent sheet-like collagen molded article according to claim 3 or 4, wherein an operation during discharge is any of the following (1) to (3).
(1) The discharge surface is fixed, and the discharge port is moved in the direction opposite to the discharge direction along the discharge surface.
(2) The discharge port is fixed and the discharge surface is moved in the same direction as the discharge direction.
(3) The ejection port is moved along the ejection surface in the direction opposite to the ejection direction, and the ejection surface is moved in the same direction as the ejection direction. The method for producing a transparent sheet-like collagen molded article according to any one of claims 3 to 5 , further comprising a crosslinking treatment by a physical crosslinking method or a chemical crosslinking method after the third step. The method for producing a transparent sheet-like collagen molded article according to any one of claims 3 to 6 , wherein the physiological isotonic solution or buffer is an aqueous solution of an inorganic salt. The medical material using the transparent sheet-like collagen molded object of Claim 1 or 2 .

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