Method for producing collagen sponge and collagen sponge produced by said method

Abstract

To provide: a production method of a collagen sponge, which creates an environment which accelerates cell migration; and a collagen sponge produced thereby.SOLUTION: The production method of the present invention comprises: dissolving a purified granular atelocollagen in distilled water under an acidic condition to prepare a first aqueous collagen solution which retains a triple helical structure characteristic of a collagen molecule; subjecting an aqueous collagen solution prepared separately by the above method to a heat treatment under a specified condition to prepare a second aqueous collagen solution having the triple helical structure changed to a random structure; mixing the first aqueous collagen solution and the second aqueous collagen solution in a specified ratio and adjusting the pH to a specified value to prepare a transparent mixed aqueous solution; dispensing this transparent mixed aqueous solution in a vessel having a desired shape and freeze vacuum drying the same to produce a collagen sponge having a desired shape; and irradiating the collagen sponge with UV rays under a specified condition to introduce intramolecular crosslinking to a desired position.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for producing a collagen sponge used in the regenerative treatment of tissue damage in the fields of oral surgery and plastic surgery, and to a collagen sponge produced by said method. [Background technology]

[0002] Patent Document 1 (JP Patent Publication No. 2004-222993) discloses that a space for tissue regeneration is maintained within the oral cavity for a predetermined period of time and decomposed in the body after tissue regeneration, that necessary growth factors, growth differentiation factors, etc. are supplied to the wound surface at an appropriate time, and that a suitable support material is provided that contributes to the growth of desired cells.

[0003] Furthermore, Patent Document 1 discloses a dental material that is made of regenerated atelocollagen having a layer therein that contains at least one kind of autologous tissue regeneration supporting substance.

[0004] Patent document 1 also discloses that the layer containing the self-tissue regeneration supporting substance is a layer made of gelatin, and has a sponge layer made of regenerated atelocollagen on both the top and bottom of the layer, or that the layer containing the self-tissue regeneration supporting substance is a gel layer made of regenerated atelocollagen, and has a sponge layer made of regenerated atelocollagen on either the top or bottom of the gel layer.

[0005] The sponge layer made of regenerated atelocollagen refers to a porous, sheet-like collagen body obtained by placing a homogenized acidic collagen solution in a suitable mold, freeze-drying it, and then cross-linking it by dry heat dehydration under vacuum.

[0006] Patent Document 2 (JP Patent Publication No. 2006-296896) relates to a collagen thin film sheet obtained by neutralizing an acidic collagen aqueous solution containing a physiologically active substance that induces cell regeneration to form a collagen hydrogel, and then naturally drying it at 4 to 10° C. The document discloses that the collagen thin film sheet plays the role of an artificial extracellular matrix when applied to a living body, and induces the regeneration of autologous tissue while being gradually degraded and absorbed in the living body, and that when the collagen thin film sheet is an autologous tissue regeneration-inducing artificial skin / mucosa, applying it to a defective part of the skin / mucosa induces the regeneration of autologous tissue and restores the function of the skin / mucosa.

[0007] Patent Document 3 (JP Patent Publication No. 2012-515035) relates to a multilayer sheet structure based on a purified collagen barrier sheet material derived from natural collagen-containing tissues contained in animal tissues (pleura, mesentery, pericardium, dura mater, intestine, etc.). This barrier sheet material consists of an outer smooth barrier layer and an inner fibrous layer, and discloses that a collagen sponge layer is attached to the surface of the fibrous layer by applying a collagen slurry to the surface and vacuum freeze-drying it.

[0008] Patent Document 4 (JP Patent Publication No. 2013-31730) relates to a novel collagen structure used for promoting nerve tissue regeneration and healing of soft tissue defects in the body, and a tissue regeneration component comprising the same. The tissue regeneration component has a thin film multilocular structure made of atelocollagen on the inside of a tubular biodegradable support, and discloses that an acidic aqueous solution (pH 3) of atelocollagen is freeze-dried in vacuum, and then dry-heat dehydration treatment is performed under vacuum to introduce crosslinks between collagen molecules.

[0009] Patent Document 5 (JP Patent Publication No. 2013-508066) relates to a composition formed by activating platelet-rich plasma with a calcium chloride solution and a type I collagen solution. It discloses that a collagen solution is left to stand at room temperature to convert it into fibrous collagen, an opaque collagen solution is prepared, and this is mixed with platelet-rich plasma mixed with a calcium chloride solution to produce a gel, and that transplanting this gel into the lesion site promotes tissue regeneration. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] JP 2004-222993 A [Patent Document 2] JP 2006-296896 A [Patent Document 3] Special Publication No. 2012-515035 [Patent Document 4] JP 2013-31730 A [Patent Document 5] Special Publication No. 2013-508066 Summary of the Invention [Problem to be solved by the invention]

[0011] Patent Document 1 relates to a method for producing a composite material in which an atelocollagen sponge layer is combined on either or both sides of an intermediate layer (a gelatin globule aggregate layer or an atelocollagen gel layer) containing an active ingredient. This atelocollagen sponge layer is produced by homogenizing an acidic collagen aqueous solution (pH 3) under ice cooling to incorporate air bubbles, placing it in a mold, and freeze-drying it in a vacuum to produce a sponge, which is then subjected to a dry heat dehydration treatment under vacuum to introduce crosslinks between collagen molecules.

[0012] Patent Document 2 relates to a method for producing a collagen thin film sheet by neutralizing an acidic aqueous solution of collagen containing a physiologically active substance, heating the resulting collagen hydrogel to 37° C., and naturally drying the resulting collagen hydrogel at 4 to 10° C. This hydrogel sheet does not have crosslinks introduced between collagen molecules.

[0013] Patent Document 3 relates to a method for producing a composite material in which a collagen sponge is bonded to one side of a collagen barrier sheet derived from natural collagen-containing tissue. A collagen slurry is used to produce the collagen sponge. This slurry is a suspension of fine collagen particles in water, and is different from an aqueous solution in which collagen molecules are completely dissolved in water. This slurry is applied to the collagen barrier sheet and freeze-dried in a vacuum, and crosslinking is introduced using chemical reagents, dry heat dehydration crosslinking, or ultraviolet light irradiation.

[0014] Patent Document 4 relates to a method for producing a composite material for nerve regeneration, incorporating a collagen thin film multilocular structure inside a tubular biodegradable support. An acidic aqueous solution (pH 3) of atelocollagen is freeze-dried in vacuum to form a thin film multilocular structure, and cross-links are introduced between collagen molecules by dry heat dehydration treatment. The thin film multilocular structure is characterized by a novel structure that differs from colloidal, gel-like, and fibrous structures.

[0015] Patent Document 5 relates to a method for producing a gel for tissue regeneration obtained by mixing platelet-rich plasma, a calcium chloride solution, and a solution of fibrous collagen I. This utilizes the property of platelet-rich plasma becoming a gel when in contact with fibrous collagen.

[0016] Conventional techniques that apply collagen can be classified into (i) a method of producing a sponge by freeze-drying an acidic collagen aqueous solution (pH 3) and (ii) a method of producing a sponge by freeze-drying a neutral collagen aqueous solution (pH 7). The former produces a sponge from a state in which collagen molecules are uniformly dissolved. The latter produces a sponge from a solution in which collagen molecules are aggregated and form a colloid. Therefore, the state of collagen molecules in the produced sponge is significantly different. The former is non-fibrous collagen in which collagen molecules are dispersed, and the latter is fibrous collagen in which collagen molecules are aggregated. Incidentally, collagen is in a transparent aqueous solution state in the acidic range (pH 3), but becomes colloidal or gel-like at pH 4 or higher, and further becomes fibrous and precipitates in the neutral range (pH 7).

[0017] The common point in the conventional technologies is the concept that collagen derived from animals processed into various shapes is applied to the site of tissue damage in humans to provide a "scaffold" for new tissue, which is then decomposed and absorbed. This is a design based on medical material engineering.

[0018] In contrast to the above-mentioned conventional techniques, the present invention has two molecular design features. The first feature is to mix an aqueous solution of atelocollagen that retains the original triple helical structure of collagen with an aqueous solution of heat-denatured atelocollagen in which the triple helical structure has been changed to a random structure, adjust the pH under specified conditions to prepare a transparent mixed aqueous solution, and freeze-dry in vacuum to produce a collagen sponge of the desired shape. The second feature is to produce a collagen sponge in which collagen molecules are continuously eluted by irradiating with ultraviolet light under specified conditions to introduce crosslinks between collagen molecules.

[0019] The sustained elution of collagen molecules makes it possible to create an environment that promotes cell migration. There are two factors that promote tissue regeneration. The first factor is physical: the elution of collagen molecules from the sponge creates gaps within the sponge that allow cells to invade. The second factor is biological: the collagen molecules eluted from the sponge diffuse around the wound site and act as a "chemotactic factor" that attracts surrounding fibroblasts to the wound site.

[0020] The present invention is characterized by adding the basic concept of wound healing to the concept of biomedical material engineering. When tissue is damaged, collagen, the main matrix component of tissue, is decomposed and removed from the damaged area as low molecular weight polypeptides. Some of these diffuse to the periphery of the damaged area and attract surrounding fibroblasts to the damaged area. In other words, the collagen-derived polypeptides act as "chemotactic factors." Due to this biological action, fibroblasts that have gathered at the damaged area produce the patient's own collagen to form new tissue. It is important to design the applied collagen sponge so that it is decomposed and absorbed at the time when the formation of this new tissue is completed.

[0021] Regarding the above collagen-derived polypeptide, the thermally denatured random collagen molecules are immediately degraded by the protease present at the site of tissue damage to become polypeptides. Meanwhile, the triple helical collagen molecules are partially degraded by collagenase present at the site of tissue damage, the triple helical structure is destroyed to become random, and the triple helical structure is further degraded by the protease to become polypeptides. Therefore, the thermally denatured random collagen molecules and the collagen molecules that retain the triple helical structure are eluted from the sponge, making it possible to continuously supply the degraded polypeptides to the area around the site of tissue damage. [Means for solving the problem]

[0022] Therefore, the present invention provides a method for producing a molecularly designed collagen sponge, comprising the steps of: Step 1: Purified granular atelocollagen is dissolved under acidic conditions to prepare a first collagen aqueous solution that retains the original triple helical structure of collagen; Step 2: A second collagen aqueous solution is prepared by converting the collagen aqueous solution prepared in the same manner as above from a triple helical structure to a random structure by heat treatment; Step 3: mixing the first collagen aqueous solution and the second collagen aqueous solution in a predetermined ratio and adjusting the pH to a predetermined value to prepare a transparent mixed aqueous solution; Step 4: Dispense the pH-adjusted transparent mixed aqueous solution into a container with a desired shape, and freeze-dry it in vacuum to produce a collagen sponge with a desired shape; Step 5: The collagen sponge produced in step 4 is irradiated with ultraviolet light under specified conditions to introduce crosslinks between collagen molecules, thereby designing the collagen molecules so that they are dissolved continuously when the sponge is applied to the site of tissue damage.

[0023] The atelocollagen of the present invention is extracted by enzymatic treatment under acidic conditions from type I collagen, which is found in large quantities in the skin of pigs, cows, and other animals. It retains a triple helix structure in which three polypeptide chains are intertwined, and is in a purified granular form after removal of the antigenic determinants called telopeptides present at both ends of the molecule during the enzymatic treatment.

[0024] The granular atelocollagen produced by the above method dissolves in water under acidic conditions, for example, at pH 3 or less. At pH 3 or less, the binding force between the positively charged side chains of the collagen molecules and the surrounding water molecules increases, allowing water solubility to be maintained. Therefore, the first collagen aqueous solution prepared in step 1 is characterized in that the collagen molecules are dispersed and dissolved in the aqueous solution while maintaining their triple helical structure. Incidentally, at pH 3 or more, the charged state of the side chains becomes weak, and the binding force with the surrounding water molecules decreases, so that the collagen molecules with the triple helical structure aggregate side by side and precipitate. This forms a so-called fibrous aggregate.

[0025] The second collagen aqueous solution prepared in step 2 is a thermally denatured collagen aqueous solution obtained by heating an acidic aqueous solution of atelocollagen at a temperature of, for example, 40°C or higher. Heat treatment changes the collagen molecules from a triple helix structure to a random structure. Collagen molecules with a random structure cannot aggregate side-by-side due to mutual steric hindrance, so they do not precipitate even in the neutral pH range. Thus, the second collagen aqueous solution is characterized in that collagen molecules are dispersed and dissolved in the aqueous solution in a random structure state.

[0026] The transparent mixed aqueous solution prepared in step 3 is a mixture of the first and second collagen aqueous solutions in a predetermined ratio, in which collagen molecules with triple helical structure and collagen molecules with random structure are dispersed and dissolved. The pH value at which precipitation begins in this transparent mixed aqueous solution varies depending on the mixing ratio of the first and second collagen aqueous solutions. In the case of a mixed aqueous solution in which the composition ratio of the first and second collagen aqueous solutions is 1:2, precipitation begins to occur at a pH of 5.5 or higher. In the case of mixed aqueous solutions in which the composition ratio is 1:1 or 2:1, precipitation begins to occur at a pH of 5 or higher. Therefore, it is important to set the pH value of the mixed aqueous solution to 4.5, at which no precipitation occurs.

[0027] In step 4, the pH-adjusted transparent mixed aqueous solution is dispensed into a container of a desired shape and freeze-dried to produce sheet-type and cylindrical collagen sponges, which are characterized by containing collagen molecules with triple helical structure and collagen molecules with random structure.

[0028] Furthermore, in step 5, the collagen sponge produced in step 4 is irradiated with ultraviolet light under predetermined conditions. Specifically, both sides or one side of the collagen sponge is irradiated with ultraviolet light for a predetermined period of time. Since intermolecular crosslinks are introduced only in the areas reached by ultraviolet light, the degree of crosslinking of the collagen sponge can be adjusted.

[0029] In the above-mentioned manufacturing method, the collagen sponge irradiated on both sides with UV light is characterized in that the collagen molecules in the outer layer of both sides of the collagen sponge are sufficiently cross-linked, but the collagen molecules in the middle layer where UV light does not reach are not cross-linked. When this collagen sponge irradiated on both sides with UV light is applied to a wound, the collagen molecules in the middle layer where cross-linking is insufficient are eluted first, followed by the collagen molecules in the outer layer where cross-linking is sufficient due to enzymatic degradation. Such sustained elution of collagen molecules creates an environment that promotes cell migration.

[0030] The sheet-type collagen sponge produced by the present invention can be used as a palate mucosa protective material or a gingival protective material in the field of oral surgery. In cases of gingival recession, a surgical method is to transplant mucosa harvested from the palate onto the incised gingival wound. This is the so-called "palatal mucosa free grafting procedure." In this case, the collagen sponge is applied to the palate defect after the mucosa has been harvested. This is the method of use as a palate mucosa protective material. On the other hand, it is also possible to apply the collagen sponge to the incised gingival wound. This is the method of use as a gingival protective material.

[0031] When using a sheet-type collagen sponge as a palatal mucosa protective material, it is required to function as a wound dressing for about two weeks, so it is desirable for the uncrosslinked collagen molecules to be slowly eluted, and the UV exposure time is extended to increase the degree of intermolecular crosslinking of the collagen sponge. Because the size of the palatal mucosa defect is small, epithelialization from the surrounding area is possible in parallel with the formation of granulation tissue.

[0032] Furthermore, when using the sheet-type collagen sponge as a substitute for a palate mucosa graft, i.e., as a gingival protective material, blood vessels and gingival fibroblasts can invade the sponge from the surrounding gingiva and form granulation tissue in about one week, so it is desirable for the uncrosslinked collagen molecules to be rapidly eluted, and the UV exposure time is shortened so that the degree of intermolecular crosslinking of the collagen sponge is low. Because the size of the gingival defect site is small, epithelialization from the surrounding area is possible in parallel with the formation of granulation tissue.

[0033] Furthermore, the sheet-type collagen sponge can be used as an intraoral hemostatic material. In implant surgery, which has become a common treatment in recent years, there is a process in which the gums are incised to expose the alveolar bone, and an excellent hemostatic material is required. When used as a hemostatic material, only one side (upper side) of the sheet-type collagen sponge is irradiated with ultraviolet light. This is characterized in that the collagen molecules on the upper surface of the collagen sponge are sufficiently cross-linked, while the collagen molecules on the lower surface (wound side) are not cross-linked. When this sheet-side collagen sponge is applied to an incision wound in the gums as a hemostatic material, the uncross-linked collagen molecules dissolve in the plasma and exudate, become a paste, and adhere to the wound surface. At the same time, a fibrin network is formed in the gaps left by the elution of the uncross-linked collagen molecules, providing an environment for wound healing.

[0034] The large-sized sheet-type collagen sponge, which is irradiated with ultraviolet light only on one side, can be used as a hemostatic material and wound dressing material in the field of plastic surgery. In emergency treatment of facial trauma, the function of hemostatic material and the function of wound dressing material are required. Furthermore, in burn treatment, split-thickness skin harvested from a healthy area is grafted onto the wound surface after excision of a severely burned area. The so-called "autologous split-thickness skin graft" is performed. The protective material used on the wound surface after harvesting the split-thickness skin is required to function as a hemostatic material and a wound dressing material. Incidentally, the "autologous split-thickness skin graft" performed in the field of plastic surgery requires a common treatment material to the "palatal mucosa free graft" performed in the field of oral surgery.

[0035] Furthermore, the cylindrical collagen sponge produced by the above-mentioned production method is characterized in that, by irradiating both sides with ultraviolet light, the collagen molecules in the outer layer surrounding the collagen sponge are sufficiently cross-linked, while the collagen molecules in the central portion are not cross-linked, and is used as a filling material for tooth extraction sockets.

[0036] The cylindrical collagen sponge used as the filling material for tooth extraction sockets requires a certain degree of strength, and therefore requires that a high-concentration collagen mixed aqueous solution be freeze-dried in vacuum.

[0037] When a cylindrical collagen sponge is used as a filling material for tooth extraction sockets, blood vessels and gingival fibroblasts invade the blood clot from the surrounding gingiva to form granulation tissue. The small size of the gingival defect allows epithelialization from the surrounding area. In this case, it is important to maintain a space for cell migration within the sponge that has merged with the blood clot. Effect of the Invention

[0038] According to the present invention, a transparent mixed aqueous solution containing atelocollagen molecules that maintain a triple helical structure and atelocollagen molecules that have been changed to a random structure is characterized in that it does not precipitate even at pH 4.5. In addition, a collagen sponge produced by freeze-vacuum drying of the transparent mixed aqueous solution can control the state of intermolecular crosslinking in the collagen sponge by changing the ultraviolet irradiation conditions. This has the characteristic that the desired elution state of collagen molecules can be achieved. [Brief description of the drawings]

[0039] [Figure 1] FIG. 1 is an explanatory diagram showing the production method of the present invention. [Diagram 2] FIG. 2 is an explanatory diagram of the first aqueous collagen solution. [Diagram 3] FIG. 3 is an explanatory diagram of the second aqueous collagen solution. [Figure 4] FIG. 4 is an explanatory diagram of the mixed aqueous solution. [Diagram 5] FIG. 5(a) is an explanatory diagram showing an example of a sheet-shaped collagen sponge, and FIG. 5(b) is an explanatory diagram showing the elution of collagen molecules. [Figure 6] FIG. 6(a) is an explanatory diagram showing another example of a sheet-shaped collagen sponge, and FIG. 6(b) is an explanatory diagram showing the elution of collagen molecules. [Figure 7] FIG. 7 is an explanatory diagram showing the elution of collagen molecules from a cylindrical collagen sponge. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

[0041] The method for producing a collagen sponge of the present invention is, for example, as shown in Figure 1. In step 1, purified granular atelocollagen is dissolved under acidic conditions to prepare a first collagen aqueous solution (1). This first collagen aqueous solution (1) is a transparent aqueous solution prepared under conditions of pH 3 or less, for example pH 2.8, and the collagen molecules (2) retain their original triple helix structure as shown in Figure 2. Note that (3) in Figure 2 indicates water molecules.

[0042] In step 2, the collagen aqueous solution prepared in the same manner as in step 1 is heated at a specified temperature, for example 60°C, for 1 hour to change the triple helical structure of the collagen molecules to a random structure, preparing a second collagen aqueous solution (4) as thermally denatured collagen molecules (Figure 3). Here, the randomly structured collagen molecules (5) cannot aggregate side-by-side due to mutual steric hindrance, and therefore have the characteristic of not precipitating even when the pH value is changed from the acidic to neutral range.

[0043] In step 3, the first collagen aqueous solution (1) and the second collagen aqueous solution (4) are mixed in a predetermined ratio to prepare a transparent mixed aqueous solution (6), and an aqueous sodium hydroxide solution is added dropwise with stirring to adjust the pH to a predetermined value, for example, pH 4.5.

[0044] In step 4, the transparent mixed aqueous solution (6) whose pH has been adjusted in step 3 is dispensed into a container (7) or (8) having a desired shape, and then freeze-dried in vacuum to produce a collagen sponge (9) or (10) having the desired shape. Note that (7) is a container for a sheet-type collagen sponge, and (8) is a container for a cylindrical collagen sponge.

[0045] In step 5, the collagen sponge (9) or (10) produced in step 4 is irradiated with ultraviolet light under specified conditions. For example, collagen sponge (11) is obtained by irradiating both the upper and lower surfaces of a sheet-type collagen sponge with ultraviolet light for a specified period of time, and is used, for example, as a palate mucosa protective material or a gingival protective material. Collagen sponge (12) is obtained by irradiating only one surface (the upper surface) of a sheet-type collagen sponge with ultraviolet light for a specified period of time, and can be used, for example, as a hemostatic material in oral surgery. Furthermore, it can be used as a hemostatic material and wound dressing material in plastic surgery.

[0046] The collagen sponge (13) is obtained by irradiating ultraviolet light from both sides of a cylindrical collagen sponge for a predetermined period of time, and is used, for example, as a filling material for tooth extraction sockets. EXAMPLES

[0047] Granular purified atelocollagen derived from pigskin was dissolved in distilled water to prepare a first collagen aqueous solution (1) (1.2%, pH 2.8). Separately, a collagen aqueous solution was prepared in a similar manner and heated at 60°C for 1 hour to prepare a second thermally denatured collagen aqueous solution (4) (1.2%, pH 2.8).

[0048] The first collagen aqueous solution (1) and the second collagen aqueous solution (4) were mixed at a composition ratio of 2:1, and an aqueous sodium hydroxide solution was added dropwise while stirring to adjust the pH to 4.5. A predetermined amount of this transparent mixed aqueous solution (6) was dispensed into a sheet-type sponge container (7), placed in a refrigerator at 4°C for more than 6 hours, then frozen in an ultra-low temperature freezer at -85°C for a day and night, and then placed in a vacuum freeze dryer to produce a sheet-type collagen sponge (9). Both sides of this sheet-type collagen sponge were irradiated with a 15W ultraviolet lamp from a distance of 20 cm for 15 minutes to introduce crosslinks between collagen molecules, producing a sheet-type collagen sponge (11) with a high degree of intermolecular crosslinking in both outer layer portions.

[0049] This sheet-type collagen sponge was immersed in distilled water at 37°C for a certain period of time, then collected, frozen, and vacuum-dried to measure the weight of the resulting sponge, which was then compared with the weight of the sponge before immersion in distilled water to calculate the elution rate of collagen molecules. After immersion in water for 3 days, it showed a 20% weight loss. After immersion in water for 7 days, it showed a 24% weight loss. It was demonstrated that the elution rate of collagen molecules from a sheet-type collagen sponge with a high degree of intermolecular cross-linking is quite slow.

[0050] This sheet-type collagen sponge was soaked in distilled water for 10 minutes, then collected and placed on a mesh for 30 minutes to remove excess water, after which it was weighed and compared with the weight of the sponge before soaking in distilled water to calculate the water-absorbing capacity of the sheet-type collagen sponge. It was demonstrated that the sponge could absorb 46 times its own weight in water.

[0051] This sheet-type collagen sponge has an extremely high water absorption capacity, so when applied to a wound, it is able to fully absorb exudates containing various cell growth factors. Therefore, it is expected to provide an environment suitable for promoting wound healing. This sheet-type collagen sponge (11) is preferably used as a palate mucosa protective material.

[0052] As shown in Figures 5(a) and (b), for example, in the sheet-type collagen sponge (11) designed as a palatal mucosa protective material, the degree of cross-linking between collagen molecules due to ultraviolet light is high in the upper and lower outer layers 11A and 11B of the sheet-type collagen sponge (11), but the degree of cross-linking is low in the middle layer 11C because the ultraviolet light does not reach the middle layer 11C sufficiently. Therefore, when the sheet-type collagen sponge (11) is applied to the mucosal defect after collection of a palatal mucosa piece, the uncross-linked collagen molecules inside the sheet-type collagen sponge (11) can pass between the cross-linked collagen molecules and be eluted.

[0053] Blood vessels and fibroblasts from the surrounding mucosa invade the sponge and begin to form granulation tissue. At the same time, collagen molecules with sufficient cross-linking are also eluted by enzymatic degradation. The continuous elution of collagen molecules provides an environment that promotes cell migration, and it is expected that epithelialization will be promoted by the proliferation and migration of mucosal epithelial cells from the surrounding area in parallel with the formation of granulation tissue. EXAMPLES

[0054] Example 2 shows a sheet-type collagen sponge (11) used as a gingival protective material. When used as a gingival protective material, it is desirable for the collagen molecules to be rapidly eluted. Both sides of the sheet-type collagen sponge were irradiated with a 15W ultraviolet lamp from a distance of 20cm for 5 minutes to introduce crosslinks between collagen molecules.

[0055] Using a method similar to that described above, the elution rate of collagen molecules from this sheet-type collagen sponge was calculated. Immersion in water for 3 hours showed an 11% weight loss. Immersion in water for 1 day showed a 34% weight loss. Immersion in water for 3 days showed a 49% weight loss. It was demonstrated that the elution rate of collagen molecules from a sheet-type collagen sponge with a low degree of intermolecular cross-linking is quite fast. This sheet-type collagen sponge (11) is desirable for use as a gingival protective material.

[0056] When the sheet-type collagen sponge (11) of Example 2 is used as a substitute for a palatal mucosa graft, i.e., as a gingival protective material, collagen molecules are eluted from the collagen sponge, and at the same time, blood vessels and gingival fibroblasts infiltrate into the sponge from the surrounding gingiva, initiating granulation tissue formation. EXAMPLES

[0057] In Example 3, a sheet-type collagen sponge was produced in the same manner as in Example 1. However, in order to increase the bubble size of the sponge, the collagen concentrations of the collagen aqueous solution (1) and the thermally denatured collagen aqueous solution (4) were set to 0.8%. The sponge produced in Example 3 is a sheet-type collagen sponge (12) characterized in that ultraviolet light was irradiated only on the upper surface side. The upper surface side of this sheet-type collagen sponge was irradiated with a 15 W ultraviolet lamp from a distance of 20 cm for 15 minutes to introduce crosslinks between collagen molecules.

[0058] Using the same method as above, the elution rate of collagen molecules from this sheet-type collagen sponge was calculated. When immersed in water for 3 hours, it showed a 19% weight loss. When immersed in water for 1 day, it showed a 42% weight loss. When immersed in water for 3 days, it showed a 50% weight loss. It was demonstrated that the initial elution rate of collagen molecules from the sheet-type collagen sponge in which intermolecular cross-linking was introduced only on the upper surface side was quite fast.

[0059] As shown in Figure 6, the collagen molecules on the upper surface (12A) of the sheet-type collagen sponge (12) have a high degree of cross-linking, while the collagen molecules on the lower surface (12B) are not cross-linked. This sheet-type collagen sponge (12) is preferably used as a hemostatic material for incised wounds in the gums, for facial injuries, and for wounds where autologous split-thickness skin has been harvested in burn treatment.

[0060] When the sheet-type collagen sponge (12) according to Example 3 is used as a hemostatic material, the uncrosslinked collagen molecules on the lower surface 12B dissolve in the plasma and exudate and become a paste when applied to the wound surface. At the same time, a fibrin network (15) is formed in the gaps left by the outflow of uncrosslinked collagen, providing an environment for wound healing following hemostasis. EXAMPLES

[0061] In Example 4, a collagen sponge was produced in the same manner as in Example 1. However, in order to increase the strength of the sponge, the collagen concentration of the collagen aqueous solution (1) and the thermally denatured collagen aqueous solution (4) was set to 2.0%. The transparent mixed aqueous solution was placed in a cylindrical collagen sponge container 8, placed in a refrigerator at 4°C for more than 6 hours, frozen in a freezer at -85°C for a day and night, and then placed in a vacuum freeze dryer to produce a cylindrical collagen sponge (10). Both sides of this cylindrical collagen sponge were irradiated with a 15W ultraviolet lamp from a distance of 20 cm for 15 minutes to introduce crosslinks between collagen molecules.

[0062] Using the same method as above, the elution rate of collagen molecules from this cylindrical collagen sponge was calculated. When immersed in water for 3 hours, it showed a 12% weight loss. When immersed in water for 1 day, it showed a 56% weight loss. When immersed in water for 3 days, it showed a 64% weight loss. It was demonstrated that the initial elution rate of collagen molecules from the cylindrical collagen sponge with intermolecular cross-linking introduced in the outer peripheral layer was quite fast. The tooth extraction socket is an environment with less exudate than palatal mucosa defect or gingival defect wound. Therefore, if the sponge decomposes slowly, there is a risk that it will remain and inhibit healing.

[0063] This cylindrical collagen sponge (13) is preferably used as a filling material for tooth extraction sockets. Figure 7 shows the molecular movement when the cylindrical collagen sponge (13) is used as a filling material for tooth extraction sockets.

[0064] When this cylindrical collagen sponge (13) is filled into an extraction socket, collagen molecules with insufficient cross-linking inside the sponge are eluted from both ends 13A and 13B of the cylindrical collagen sponge (13), and then collagen molecules with sufficient cross-linking in the outer layer surrounding the sponge are also eluted by enzymatic degradation. When the cylindrical collagen sponge (13) is used as a filling material for an extraction socket, blood vessels and gingival fibroblasts invade the sponge from the surrounding gingiva at the same time as the collagen molecules are eluted, and granulation tissue formation begins. A space for cell migration is maintained within the sponge that has merged with the blood clot.

[0065] As described above, according to the present invention, it is possible to continuously elute collagen molecules from the collagen sponge, and furthermore, to adjust the elution rate. The voids created by the elution of collagen molecules facilitate cell infiltration from the surroundings, and the eluted collagen molecules act as "chemotactic factors" that attract cells from the surroundings to the damaged area. These actions make it possible to provide an environment that promotes the regeneration of tissue damage. [Explanation of symbols]

[0066] 1. First collagen aqueous solution 2. Triple-helical structure of atelocollagen molecule 3 water molecules 4. Second collagen aqueous solution 5. Random structure of atelocollagen molecules 6. Clear mixed aqueous solution 7,8 container 9. Sheet-type collagen sponge 10 Cylindrical collagen sponge 11,12 Cross-linked collagen sheet sponge 13 Cross-linked cylindrical collagen sponge 14 Intermolecular crosslinking 15 Fibrin meshwork UV ultraviolet irradiation

Claims

1. A method for producing a collagen sponge for oral surgery and plastic surgery, comprising the steps of: Step 1: Purified granular atelocollagen is dissolved in distilled water under acidic conditions to prepare a first collagen aqueous solution that retains the triple helix structure specific to collagen molecules; Step 2: A second collagen aqueous solution is prepared by heat-treating the collagen aqueous solution prepared in the same manner as above to change the structure of the collagen molecule from a triple helix structure to a random structure; Step 3: Mixing the first collagen aqueous solution and the second collagen aqueous solution in a predetermined ratio and adjusting the pH to a predetermined value to prepare a transparent mixed aqueous solution; Step 4: Dispense the pH-adjusted transparent mixed aqueous solution into a container having a desired shape, and freeze-dry it to prepare a collagen sponge having a desired shape; Step 5: A method for producing a collagen sponge, comprising irradiating the collagen sponge produced in step 4 with ultraviolet light under predetermined conditions to introduce intermolecular crosslinks at desired sites.

2. 2. The method according to claim 1, wherein the container is a sheet type, and the collagen sponge is a sheet type collagen sponge.

3. 2. The method according to claim 1, wherein the container is cylindrical, and the collagen sponge is a cylindrical collagen sponge.

4. 3. The method for producing a sheet-type collagen sponge according to claim 2, characterized in that both the upper and lower surfaces of the sheet-type collagen sponge are irradiated with ultraviolet light for a predetermined period of time, so that the outer layer portions of both surfaces of the sheet-type collagen sponge have a high degree of intermolecular cross-linking, and the intermediate layer portion of the sheet-type collagen sponge has a low degree of intermolecular cross-linking.

5. 3. The method for producing a sheet-type collagen sponge according to claim 2, wherein ultraviolet light is irradiated only onto the upper surface of the sheet-type collagen sponge for a predetermined period of time, so that the upper layer of the sheet-type collagen sponge has a high degree of intermolecular cross-linking, and the lower layer of the sheet-type collagen sponge has no intermolecular cross-linking.

6. 4. The method for producing a cylindrical collagen sponge according to claim 3, characterized in that both sides of the cylindrical collagen sponge are irradiated with ultraviolet light for a predetermined period of time, so that the peripheral outer layer portion of the cylindrical collagen sponge has a high degree of intermolecular cross-linking, and the central portion of the cylindrical collagen sponge has a low degree of intermolecular cross-linking.

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