Collagen-containing material and method for producing the same

Abstract

The objective of the present invention is to provide a sterilized scaffolding material with sufficient strength. [Solution] The above problem can be solved by the present invention, a sterilized collagen-containing material of biological tissue or cultured cell structure, characterized in that the ratio of endothermic heat per dry mass measured by differential scanning calorimetry between 40°C and 60°C is 48.0% or more.

Description

Technical Field

[0001] The present invention relates to a collagen-containing material and a method for producing the same. According to the present invention, it is possible to provide an in-vivo scaffold material having excellent strength.

Background Art

[0002] In the case of defects or injuries in living tissues, treatments such as transplantation of autologous or heterologous tissues or cultured constructs into the living body are performed. Such grafts serve as scaffold materials for the regeneration of living tissues, and cells and tissues are formed using the graft as a scaffold. For example, as scaffold materials, matrix scaffold materials containing collagen and the like can be mentioned, and they are designed to form an optimal three-dimensional structure for cell or tissue growth. (Patent Documents 1 and 2)

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] Scaffold materials need to have sufficient strength for maintaining their form during in-vivo transplantation surgery and in the living body after transplantation. However, the strength of sterilized scaffold materials sometimes decreases. Therefore, an object of the present invention is to provide a sterilized scaffold material having sufficient strength.

Means for Solving the Problems

[0005] The inventors diligently researched sterilization scaffolding materials with sufficient strength and, surprisingly, discovered that sterilization scaffolding materials possess excellent strength when the amount of heat absorbed by differential scanning calorimetry is at a specific value. This invention is based on these findings. Therefore, the present invention is [1] A collagen-containing material that is sterile from biological tissue or cultured cell structure, characterized in that the ratio of endothermic heat per dry mass measured by differential scanning calorimetry between 40°C and 60°C is 48.0% or more. [2] The collagen-containing material according to [1], wherein the sterilization is performed by irradiation. [3] The collagen-containing material described in [1] or [2], wherein the collagen-containing material has been decellularized. [4] A method for producing a collagen-containing material, comprising a sterilization step of sterilizing a living tissue or cultured cell structure with radiation at an oxygen concentration of 5% or less. [5] A method for producing a collagen-containing material according to [4], further comprising a heating step of heating the biological tissue or cultured cell structure after the sterilization step, [6] A method for producing a collagen-containing material according to [5], wherein the heating step is performed at an oxygen concentration of 5% or less, and [7] A method for producing a collagen-containing material according to any one of [4] to [6], wherein the biological tissue or cultured cell structure is decellularized. Regarding. [Effects of the Invention]

[0006] The collagen-containing material of the present invention provides a bio-scaffold material with excellent strength. [Brief explanation of the drawing]

[0007] [Figure 1] These are charts of differential scanning calorific value (DSC) for Example 1(A), Example 2(B), Example 3(C), Example 4(D), Example 5(E), and Example 6(F). [Figure 2]These are charts of differential scanning calorific value (DSC) for Comparative Example 1(A), Comparative Example 2(B), and Comparative Example 3(C). [Figure 3] This chart shows the heat absorption chart and an example of the analysis of the divided peaks in Example 4. [Figure 4] This graph shows the ratio of differential scanning heat quantities at 40°C to 60°C (A) and above 60°C (B) for Comparative Examples 1-2 and Examples 1-6. [Modes for carrying out the invention]

[0008] [1] Collagen-containing materials The collagen-containing material of the present invention is a sterilized collagen-containing material derived from biological tissue or cultured cell structures, wherein the endothermic ratio of the endothermic amount per dry mass measured by differential scanning calorimetry between 40°C and 60°C is 48.0% or more. The collagen-containing material of the present invention is manufactured from biological tissue or cultured cell structures. Alternatively, the collagen-containing material of the present invention may be a collagen structure containing collagen extracted from biological tissue or the like. Examples of collagen structures containing extracted collagen include collagen sponges and collagen sheets.

[0009] 《Biologically derived tissue》 The biologically derived tissues are not limited to those containing collagen, but include, for example, the liver, kidneys, ureters, bladder, urethra, tongue, tonsils, esophagus, stomach, small intestine, large intestine, anus, pancreas, heart, blood vessels, spleen, lungs, brain, bone, spinal cord, cartilage, testes, uterus, fallopian tubes, ovaries, placenta, cornea, skeletal muscle, tendons, nerves, and skin. As biologically derived tissues, cartilage, bone, liver, kidneys, heart, pericardium, aorta, skin, submucosal tissue of the small intestine, lungs, brain, internal thoracic artery, or spinal cord are preferred due to their high tissue regeneration effect, and more preferably the pericardium, internal thoracic artery, liver, cartilage, skin, submucosal tissue of the small intestine, or spinal cord.

[0010] The aforementioned living tissue is not particularly limited as long as it is a living tissue derived from a vertebrate, but living tissue derived from mammals or birds is preferred because it causes fewer rejection reactions, and living tissue derived from livestock mammals, livestock birds, or humans is even more preferred because it is easily available. Examples of livestock mammals include cattle, horses, camels, llamas, donkeys, yaks, sheep, pigs, goats, deer, alpacas, dogs, raccoons, weasels, foxes, cats, rabbits, hamsters, guinea pigs, rats, mice, squirrels, and raccoons. Examples of livestock birds include parakeets, parrots, chickens, ducks, turkeys, geese, guinea fowl, pheasants, ostriches, quail, and emus. Among these, living tissue from cattle, pigs, rabbits, and humans is preferred due to its stable availability.

[0011] 《Cultured cell structure》 The cultured cell structure used in the collagen-containing material of the present invention is not limited to containing collagen, but may be any structure formed by the binding of multiple cultured cells. For example, it may be a cultured cell structure formed by further culturing cell aggregates (spheroids) and cell aggregates (spheroids). In this specification, the cell aggregates (spheroids) may also be referred to as cultured cell structures, and therefore, both cell aggregates (spheroids) and cultured cell structures obtained by further culturing cell aggregates (spheroids) may be referred to as cultured cell structures. Furthermore, the cultured cell structure used in the collagen-containing material of the present invention may be decellularized and sterilized to form a collagen-containing material (decellularized cultured cell structure), as described later.

[0012] (cultured cell aggregate) Cultured cell aggregates are not particularly limited as long as they are cell aggregates obtained by in vitro culture. Specifically, they are those in which cells adhere to each other and maintain a three-dimensional aggregate shape by producing extracellular matrix (ECM) outside the cell during three-dimensional culture, and spheroids can be cited as an example. The size of the cultured cell aggregate is not particularly limited. However, in the case of spheroids, when observed with a scanning electron microscope (SEM), the shortest length is 80 μm or more, preferably 90 μm or more, more preferably 95 μm or more, and even more preferably 100 μm or more. The longest length is not limited either, but it is 600 μm or less, preferably 500 μm or less, more preferably 300 μm or less. The above upper and lower limits can be combined to form the range of the size of the cultured cell aggregate. The three-dimensional cell culture method for producing the cultured cell aggregate (spheroid) is not limited, but examples include the non-adhesive surface culture method, the hanging drop culture method, or the rotary culture method.

[0013] (Cultured cell construct) The cultured cell construct is a cell construct obtained by culturing in vitro. The cultured cell construct is not particularly limited as long as the cells are adhered by the cells producing extracellular matrix (ECM) extracellularly and maintain a three-dimensional shape. Examples include a cell block in which a plurality of spheroids are combined. The shape of the cultured cell structure is not particularly limited, and examples include sheet-like, tubular, or block-like shapes. The size of the cultured cell structure is also not particularly limited, but in the case of a cell block, when observed with a scanning electron microscope (SEM), the shortest length is 90 μm or more, preferably 100 μm or more, more preferably 150 μm or more, and even more preferably 200 μm or more. The longest length is not limited either, but is 2100 μm or less, preferably 2000 μm or less, more preferably 1700 μm or less, even more preferably 1400 μm or less, even more preferably 1200 μm or less, and most preferably 1000 μm or less. The upper and lower limits can be combined to define the size range of the cultured cell aggregate. The strength of the cultured cell structure is not particularly limited as long as it maintains a certain three-dimensional shape, but in the wet state, it preferably has a strength that allows handling with tweezers and suturing. This facilitates the production of decellularized cell structures according to the intended applications such as grafts and cell therapy. The cultured cell structure can be obtained by further culturing a plurality of cultured cell aggregates. Specific production methods include, for example, the net mold method, the cell sheet method, the bioprinting method, or the grid method.

[0014] (Cultured cells) Cells used in the cultured cell structure in the present invention include hepatocytes, stellate cells, Kupffer cells, vascular endothelial cells, endothelial cells, endothelial cells, osteoclasts, periodontal ligament-derived cells, epidermal cells, tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, epithelial cells, mammary gland cells, pericytes, smooth muscle cells, cardiomyocytes, myocytes, renal cells, pancreatic islet cells, peripheral nerve cells, nerve cells, chondrocytes, or osteocytes. Examples of adherent cells with differentiation potential include embryonic stem cells (ES cells), embryonic germ cells (EG cells), germline stem cells (GS cells), induced pluripotent stem cells (iPS cells), mesenchymal stem cells, hematopoietic stem cells, neural stem cells, cardiomyocyte progenitor cells, vascular endothelial progenitor cells, neural progenitor cells, adipocyte progenitor cells, fibroblasts, cutaneous fibroblasts, skeletal muscle myoblasts, osteoblasts, or odontoblasts. More specifically, examples include SH-SY5Y and SK-N-SH derived from human neuroblastoma, Neuro 2a and N1E-115 derived from mouse neuroblastoma, NTERA-2 derived from human pluripotent embryonic carcinoma, and PC12 derived from rat adrenal pheochromocytoma. The origin of the cells is the same as that of the aforementioned living organism-derived tissues.

[0015] Extracted collagen structure Collagen structures produced from collagen extracted from biological tissues or cultured cells include collagen sponges and collagen sheets. While the raw materials for collagen structures are not limited to biological tissues or cultured cells, skin, bones, or cartilage from fish, cattle, or pigs are often used because they yield large quantities of collagen. The manufacturing method for collagen structures is also not limited, but methods such as freeze-drying or distilled water treatment can be used. In freeze-drying, for example, an extracted collagen solution is frozen at around -40°C, and the collagen structure is obtained by freeze-drying. While not limited to these methods, the strength can be improved by thermal crosslinking or glutaraldehyde crosslinking. The distilled water treatment method involves dissolving purified collagen in distilled water under acidic conditions to obtain a collagen solution. By adjusting the prepared collagen solution into a specific structural shape, a collagen structure can be obtained.

[0016] "collagen" The collagen contained in the collagen-containing material of the present invention is not particularly limited, as long as the effects of the present invention are obtained. Collagen is an important protein that accounts for 30% of the proteins in living organisms and has functions such as skeletal support and cell adhesion. Collagen includes fibrous collagen, which forms collagen fibers, and non-fibrous collagen, which does not form fibers. Fibrous collagen is the collagen that is the main component of collagen fibers, and specifically includes type I collagen, type II collagen, type III collagen, type V collagen, or type XI collagen. Non-fibrous collagen is, for example, type IV collagen. Fibrous collagen consists of three collagen polypeptide chains that come together to form a triple helix structure (tropocollagen). This triple helix tropocollagen self-organizes, aligning with a 1 / 4 molecular length offset to form collagen fibrils. Furthermore, numerous collagen fibrils aggregate to form collagen fibers (collagen fiber bundles).

[0017] Type I collagen consists of three polypeptide chains, each with a molecular weight of approximately 100,000, which come together to form a "triple helix structure (tropocollagen)" with a molecular weight of approximately 300,000. This triple helix structure has the form of a single rigid rod, 300 nm long and 1.5 nm in diameter, and is called tropocollagen. This triple helix topocollagen self-assembles, aligning with a 1 / 4 molecular length offset to form collagen fibrils (collagen microfilaments). Furthermore, numerous collagen fibrils aggregate to form collagen fibers (collagen fiber bundles). In the corneal stroma and cortical bone, collagen fibers align in the same direction, forming overlapping layers, and these layers are stacked perpendicularly to each other, creating a plywood-like three-dimensional lamellar structure. Collagen with this plywood-like three-dimensional lamellar structure is transparent and has high strength. Furthermore, in type I collagen, Xaa in Gly-Xaa-Yaa is often proline or 3-hydroxyproline, and Yaa is often 4-hydroxyproline or hydroxylysine. Hydroxyproline is an amino acid unique to collagen and is not found in typical proteins. It is thought that the triple helix structure is stabilized by hydrogen bonding between the hydroxyl group of hydroxyproline and hydrated water.

[0018] The collagen content in the collagen-containing material of the present invention is not particularly limited as long as the effects of the present invention are obtained, but for example, the upper limit is 100% by weight or less, in some embodiments 95% by weight or less, in some embodiments 90% by weight or less, in some embodiments 85% by weight or less, in some embodiments 80% by weight or less, in some embodiments 70% by weight or less, in some embodiments 60% by weight or less, and in some embodiments 50% by weight or less. The lower limit of the collagen content is, for example, 30% by weight or more, in some embodiments 35% by weight or more, in some embodiments 40% by weight or more, in some embodiments 45% by weight or more, in some embodiments 50% by weight or more, in some embodiments 55% by weight or more, and in some embodiments 60% by weight or more. The upper and lower limits can be combined to define the range of collagen content.

[0019] Differential scanning heat quantity The endothermic amount per unit dry mass of the collagen-containing material of the present invention, as measured by differential scanning calorimetry, has an endothermic ratio of 48.0% or more between 40°C and 60°C. Alternatively, the endothermic amount per unit dry mass of the collagen-containing material of the present invention, as measured by differential scanning calorimetry, may have an endothermic ratio of 48.0% or more between 40°C and 60°C, and an endothermic ratio of 52.0% or less above 60°C. Differential scanning calorimetry (DSC) can obtain thermal properties such as glass transition temperature, melting point, and endothermic peaks. Specifically, by applying a constant heat to a reference material and a sample, and measuring the temperatures of both, the thermophysical properties of the sample are captured as a temperature difference, and endothermic and exothermic reactions due to changes in the sample's state are measured. Differential scanning calorimetry can analyze not only simple thermal changes in the sample's state, such as melting, but also phase transitions of the crystal structure, crystallization, and heat values ​​from peak areas. In this invention, differential scanning calorimetry can be used to obtain the ratio of the endothermic amounts of collagen-containing materials between 40°C and 60°C. Furthermore, in this invention, differential scanning calorimetry can be used to obtain the endothermic amounts of collagen-containing materials between 40°C and 60°C and the ratio of endothermic amounts above 60°C.

[0020] In the collagen-containing material of the present invention, the endothermic amount per dry mass measured by differential scanning calorimetry is such that the endothermic ratio between 40°C and 60°C is 48.0% or more, in some embodiments it is 50% or more, in some embodiments it is 55% or more, in some embodiments it is 60% or more, in some embodiments it is 65% or more, in some embodiments it is 70% or more, in some embodiments it is 75% or more, in some embodiments it is 78.0% or more, in some embodiments it is 79.0% or more, in some embodiments it is 80.0% or more, in some embodiments it is 81.0% or more, in some embodiments it is 82.0% or more, in some embodiments it is 83.0% or more, in some embodiments it is 84.0% or more, in some embodiments it is 85.0% or more, in some embodiments it is 86.0% or more, in some embodiments it is 87.0% or more, in some embodiments it is 88.0% or more, in some embodiments it is 89.0% or more, and in some embodiments it is 90.0% or more. Furthermore, the heat absorption ratio at temperatures above 60°C is 52.0 or less, in some embodiments it is 50% or less, in other embodiments it is 45% or less, in other embodiments it is 40% or less, in other embodiments it is 35% or less, in other embodiments it is 30% or less, in other embodiments it is 25% or less, in other embodiments it is 22.0% or less, in other embodiments it is 21.0% or less, in other embodiments it is 20.0% or less, in other embodiments it is 19.0% or less, in other embodiments it is 18.0% or less, in other embodiments it is 17.0% or less, in other embodiments it is 16.0% or less, in other embodiments it is 15.0% or less, in other embodiments it is 14.0% or less, in other embodiments it is 12.0% or less, in other embodiments it is 11.0% or less, and in other embodiments it is 10.0% or less. In the collagen-containing material of the present invention, the endothermic ratio per dry mass measured by differential scanning calorimetry is often 0% for temperatures below 40°C. However, it may be 5.0% or less, and in some embodiments it may be 4.0% or less, in other embodiments it may be 3.0% or less, in other embodiments it may be 2.0% or less, and in other embodiments it may be 1.0% or less. When the endothermic ratio below 40°C is 5.0% or less, it is often considered that the endothermic ratio above 60°C is reduced.

[0021] The collagen-containing material of the present invention, although not limited to these, has an endothermic peak between 43°C and 60°C as measured by differential scanning calorimetry. The lower limit of the endothermic peak is, but not limited to these, 43°C or higher, in some embodiments 44°C or higher, in some embodiments 45°C or higher, and in some embodiments 46°C or higher. The upper limit of the endothermic peak is, but not limited to these, 60°C or lower, in some embodiments 59°C or lower, in some embodiments 58°C or lower, in some embodiments 57°C or lower, in some embodiments 56°C or lower, in some embodiments 55°C or lower, in some embodiments 54°C or lower, in some embodiments 53°C or lower, in some embodiments 52°C or lower, in some embodiments 51°C or lower, and in some embodiments 50°C or lower. The upper and lower limits can be appropriately combined to determine the range of the endothermic peak.

[0022] 《Sterilization》 The collagen-containing material of the present invention is a sterilized collagen-containing material. Sterilization methods include ethylene oxide gas (EOG) sterilization or radiation sterilization, but radiation sterilization is preferred in order to obtain the effects of the present invention. Radiation sterilization is not limited to alpha rays, beta rays, gamma rays, neutron rays, electron beams, or X-rays, as long as the effects of the present invention are obtained, but gamma rays are particularly preferred. For example, gamma ray sterilization is a common method that can be performed by those skilled in the art and poses no particular problem. The gamma ray irradiation dose is 10-50 kGy, preferably 20-50 kGy, and the temperature is 15-35°C, preferably 20-30°C.

[0023] 《Decellularization》 The collagen-containing material of the present invention is not limited, but is preferably a decellularized collagen-containing material. Decellularization can be performed using conventionally known methods. The decellularization method is not particularly limited as long as the effects of the present invention are obtained, but examples include a method by high hydrostatic pressure treatment, a method by freeze-thaw treatment, a method using a surfactant, a method by ultrasonic treatment, a method using an enzyme, or a method by treatment with a hypertonic electrolyte solution, a method by physical stirring, a hypertonic / hypotonic solution method, a method by enzymatic treatment with proteolytic enzymes or nucleolytic enzymes, or a method by treatment with an alcohol solvent, and two or more of these may be combined. However, in order to efficiently obtain the collagen-containing material of the present invention and to exhibit the effects of the present invention, a method by high hydrostatic pressure treatment is preferred. Specifically, high hydrostatic pressure treatment can be performed by the method of "high hydrostatic pressure treatment" in the method for producing the collagen-containing material described later.

[0024] 《DNA content》 The collagen-containing material of the present invention is not limited to having a DNA content of 1.000% by mass or less per dry mass. The DNA content of the decellularized support composition is 0.900% by mass or less per dry mass, preferably 0.800% by mass or less, more preferably 0.700% by mass or less, even more preferably 0.600% by mass or less, even more preferably 0.500% by mass or less, even more preferably 0.400% by mass or less, even more preferably 0.300% by mass or less, and even more preferably 0.250% by mass or less. This makes it possible to obtain a decellularized support composition that is properly decellularized and exhibits less rejection reaction upon transplantation. DNA content can be measured by the PicoGreen method. A dried specimen of the decellularized support composition (hereinafter sometimes referred to as the sample) is immersed in a proteolytic enzyme solution to dissolve it, then treated with phenol / chloroform to remove the protein and recover the DNA. Alternatively, after removing the protein with phenol / chloroform, ethanol precipitation may be performed. The recovered DNA is fluorescently stained with PicoGreen (Life Technologies) and the fluorescence intensity is measured to quantify the DNA and calculate the DNA content (mass) of the sample. For quantification, a calibration curve created using the standard DNA attached to PicoGreen is used. The DNA ratio is calculated from the dry mass and DNA content of the sample according to the following formula. (DNA content per unit dry mass (hereinafter sometimes referred to as the decellularized DNA ratio) = (DNA content of the dried test specimen of the decellularized support composition) / (mass of the dried test specimen of the decellularized support composition)

[0025] 《Suture retention strength》 The collagen-containing material of the present invention exhibits a suture-holding strength of 3N or higher, although this is not limited to the present invention. The collagen-containing material of the present invention can be used as an in vivo scaffold material. That is, it may be implanted into the body by surgical procedures, etc., and it is preferable that it has a certain level of suture-holding strength. Accordingly, the suture-holding strength of the collagen-containing material of the present invention is, for example, 3N or higher, 4N or higher in some embodiments, 5N or higher in some embodiments, 6N or higher in some embodiments, 7N or higher in some embodiments, 8N or higher in some embodiments, 9N or higher in some embodiments, and 10N or higher in some embodiments. The upper limit of the suture-holding strength of the collagen-containing material of the present invention is not limited to the range in which it can be used in surgical procedures, etc. In other words, the effects of the present invention can be obtained as long as the suture retention strength is high, and therefore the present invention is not limited to this, but for example, it may be 150N or less, 100N or less in one embodiment, 50N or less in another embodiment, 40N or less in another embodiment, 30N or less in another embodiment, 25N or less in another embodiment, 20N or less in another embodiment, and 15N or less in another embodiment. The lower limit and upper limit can be combined as appropriate to set the range of suture retention strength. The suture retention strength can be determined in accordance with ISO 7198, as described in the examples below.

[0026] Burst strength The collagen-containing material of the present invention exhibits a burst strength of 30 N or more, but is not limited to this. The collagen-containing material of the present invention can be used as an in vivo scaffold material. That is, it is preferable for the scaffold material to have a certain level of burst strength or more. Accordingly, the burst strength of the collagen-containing material of the present invention is, for example, 30 N or more, 40 N or more in some embodiments, 50 N or more in some embodiments, 60 N or more in some embodiments, 70 N or more in some embodiments, 80 N or more in some embodiments, 90 N or more in some embodiments, 100 N or more in some embodiments, 110 N or more in some embodiments, 140 N or more in some embodiments, and 200 N or more in some embodiments. The upper limit of the suture burst strength of the collagen-containing material of the present invention is not limited to the range in which it can be used as an in vivo scaffold material. In other words, the effects of the present invention can be obtained as long as the stitch burst strength is high, and therefore it is not limited to that, but for example it may be 1500N or less, 1300N or less in one embodiment, 1000N or less in another embodiment, 800N or less in another embodiment, 600N or less in another embodiment, 500N or less in another embodiment, 400N or less in another embodiment, 300N or less in another embodiment, and 250N or less in another embodiment. The lower limit and upper limit can be combined as appropriate to determine the range of burst strength. The burst intensity can be determined in accordance with ISO 7198, as described in the examples below.

[0027] [2] Method for producing collagen-containing material The present invention provides a method for producing a collagen-containing material, which includes a sterilization step of irradiating a biological tissue or cultured cell structure with an oxygen concentration of 5% or less.

[0028] 《Sterilization process》 The biological tissue or cultured cell structure to be sterilized in the sterilization process is the biological tissue or cultured cell structure described in the section "[1] Collagen-containing material".

[0029] (Oxygen concentration) The oxygen concentration in the sterilization process is not particularly limited as long as the effects of the present invention are obtained, but for example it may be 5% by volume or less, 4% or less in some embodiments, 3% or less in some embodiments, 2% or less in some embodiments, 1% or less in some embodiments, or 0.5% or less in some embodiments. The oxygen concentration can be controlled by adjusting the oxygen concentration in the gas contained in the sterilization packaging material used during sterilization. For example, the oxygen concentration can be controlled by sealing a gas with an adjusted oxygen concentration together with the biological tissue or cultured cell structure in the sterilization packaging material that encloses the biological tissue or cultured cell structure. Furthermore, by sealing an oxygen absorber together with biological tissue or cultured cell structures in sterile packaging material, the oxygen concentration can be controlled to 5% or less. In other words, the oxygen absorber is not particularly limited as long as it can keep the oxygen concentration in the sterile packaging material below 5%, but for example, in one embodiment it may be 4% or less, in another 3% or less, in another 2% or less, in another 1% or less, and in another 0.5% or less.

[0030] The oxygen absorber is not particularly limited as long as the effects of the present invention are obtained, but it is preferable that it is non-toxic, does not generate other gases when absorbing oxygen, and does not generate other gases when irradiated with radiation. Specifically, it is a catalyst whose reaction rate is controlled by a catalyst mainly composed of an activated metal, such as iron, zinc, copper, and tin, and preferably mainly composed of activated iron oxide. Commercially available examples include Sansocut (trade name, manufactured by Nittetsu Fine Products Co., Ltd.), Ageless (trade name, manufactured by Mitsubishi Gas Chemical Co., Ltd.), Tamotsu (trade name, manufactured by Oji Duck Co., Ltd.), Wellpack (trade name, manufactured by Taisei Co., Ltd.), and A500-HS oxygen absorber (trade name, manufactured by I.S.O. Co., Ltd.). In addition to these, sugars, polysaccharides, vitamin C, L-ascorbic acid, erythorbic acid, activated carbon, chitin-based activated carbon, chitosan-based activated carbon, cellulose-based activated carbon, zeolite, carbon molecular sieve, silica gel, and activated alumina can also be used.

[0031] The aforementioned sterile packaging material is, but is not limited, preferably a non-permeable packaging material. In the present invention, a non-permeable packaging material is a material that does not easily allow oxygen to permeate. Specifically, the oxygen permeability coefficient at a temperature of 25°C, humidity of 50%, and atmospheric pressure is 5.0 × 10⁻⁶. 2 cc / m 2 • Preferably, the density is 25 μm / hour or less. More preferably, 1.0 × 10 3 cc / m 2 The air permeability is less than or equal to 25 μm / hour. Specific examples of non-permeable packaging materials include stretched nylon, polyester, polyvinylidene chloride, polyvinylidene chloride-coated stretched nylon, polyvinylidene chloride-coated polyester, polyvinyl chloride-coated polypropylene, polyvinyl alcohol, poly(ethylene-vinyl alcohol) copolymer, aluminum-deposited polyethylene, aluminum-deposited polyester, or silica-coated polyester. Furthermore, the sterilization packaging material is preferably resistant to radiation sterilization, blocks external light, and is impermeable to water vapor.

[0032] Radiation sterilization The biological tissue or cultured cell structure enclosed in the sterile packaging material is sterilized by radiation. Radiation sterilization is a conventional method that can be performed by those skilled in the art and poses no particular problems. The radiation dose (e.g., gamma rays) is 10 to 50 kGy, preferably 20 to 50 kGy, and the temperature is 15 to 35°C, preferably 20 to 30°C.

[0033] 《Decellularization》 In the sterilization process, the biological tissue or cultured cell structure to be sterilized is, but is not limited to, a decellularized biological tissue or cultured cell structure. The decellularization method is not particularly limited, but examples include a method using high hydrostatic pressure, a freeze-thaw method, a method using a surfactant, a method using ultrasound, a method using enzymes, a method using a hypertonic electrolyte solution, a method using physical stirring, a hypertonic / hypotonic solution method, a method using enzymes such as proteolytic enzymes or nucleolytic enzymes, and a method using alcohol solvents. Two or more of these methods may be combined. However, in order to efficiently obtain the collagen-containing material of the present invention and to exert the effects of the present invention, a method using high hydrostatic pressure is preferred.

[0034] (High hydrostatic pressure treatment) The aforementioned high hydrostatic pressure treatment involves applying a hydrostatic pressure of 50 to 1500 MPa to a living tissue or cultured cell structure in a medium. From the viewpoint of ensuring sufficient decellularization, the applied hydrostatic pressure is preferably 50 MPa or higher, eliminating the need for a pressure vessel capable of withstanding the application, and requiring little energy. Furthermore, if the medium used for application is an aqueous medium, ice will form, and from the viewpoint of preventing damage to the cultured cell aggregates by the formed ice, a pressure of 1500 MPa or lower is preferred. The applied hydrostatic pressure is more preferably 80 to 1300 MPa, even more preferably 90 to 1200 MPa, even more preferably 95 to 1100 MPa, even more preferably 95 to 700 MPa, and most preferably 400 to 700 MPa, which exhibits decellularization, sterilization, and virus inactivation effects, as well as ease of application.

[0035] Examples of media used for applying hydrostatic pressure include culture media with serum added to the basal medium, serum-free media, water, physiological saline, water for injection, propylene glycol or its aqueous solution, glycerin or its aqueous solution, and sugar aqueous solutions. Examples of buffers include acetate buffer, phosphate buffer, citrate buffer, borate buffer, tartaric acid buffer, Tris buffer, HEPES buffer, and MES buffer. These media may also contain surfactants.

[0036] The temperature for high hydrostatic treatment is not particularly limited as long as it does not generate ice and does not cause heat damage to biological tissues or cultured cell structures. However, a temperature of 0 to 45°C is preferred, more preferably 4 to 37°C, and most preferably 15 to 36°C, as this allows for smooth decellation and minimizes impact on biological tissues or cultured cell structures. The duration of high hydrostatic treatment should not be too short, as this will not sufficiently destroy cells. Conversely, if the duration is too long, it will lead to energy waste. Therefore, the time for maintaining the target applied pressure during high hydrostatic treatment should be 1 to 120 minutes, more preferably 5 to 60 minutes, and even more preferably 7 to 30 minutes.

[0037] (Nucleic acid enzyme treatment) The aforementioned high-hydrostatic-pressure-treated biological tissue or cultured cell structure is preferably subjected to nucleolytic enzyme treatment. The nucleolytic enzyme removes nucleic acid components from the biological tissue or cultured cell structure to which hydrostatic pressure has been applied, and is not particularly limited, but examples include DNases derived from the pancreas, spleen, or Escherichia coli (e.g., DNase I, DNase II). Nucleolytic enzymes can be added to the medium used in the high-hydrostatic treatment (e.g., water, physiological saline, injection solution, or buffer solution) and allowed to act. The amount of enzyme to be added varies depending on the type of enzyme and the definition of the number of units (U), but can be appropriately set by those skilled in the art. For example, DNase I can be used at a concentration of 50 to 2000 U / mL. The treatment temperature also varies depending on the nucleolytic enzyme used, but can be set to a temperature of, for example, 1°C to 40°C. The treatment time is not particularly limited, but can be, for example, 1 to 120 hours (preferably 1 to 96 hours, more preferably 1 to 72 hours), with longer treatment times at low temperatures and shorter treatment times at high temperatures.

[0038] The bio-derived tissue or cultured cell structure that has been treated with high hydrostatic pressure is washed with a washing solution. The washing solution may be the same as or different from the medium used for high hydrostatic pressure treatment. The washing solution may contain an organic solvent or a chelating agent. Organic solvents can improve the efficiency of lipid removal, and chelating agents can prevent calcification when the bio-derived tissue or cultured cell structure of the present invention is applied to a diseased area by inactivating calcium ions and magnesium ions in the bio-derived tissue or cultured cell structure. As for the organic solvent, water-soluble organic solvents are preferred because they have a high lipid removal effect, and ethanol, isopropanol, acetone, and dimethyl sulfoxide are preferred. Examples of chelating agents include iminocarboxylic acid-based chelating agents or their salts, such as ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraminehexaacetic acid (TTHA), 1,3-propanediaminetetraacetic acid (PDTA), 1,3-diamino-2-hydroxypropanetetraacetic acid (DPTA-OH), hydroxyethyliminodiacetic acid (HIDA), dihydroxyethylglycine (DHEG), glycol etherdiaminetetraacetic acid (GEDTA), dicarboxymethylglutamic acid (CMGA), 3-hydroxy-2,2'-iminodisuccinic acid (HIDA), and dicarboxymethylaspartic acid (ASDA); and hydroxycarboxylic acid-based chelating agents or their salts, such as citric acid, tartaric acid, malic acid, and lactic acid. Examples of salts of these chelating agents include sodium salts and potassium salts. The washing temperature is not particularly limited as long as it does not cause damage to biological tissues or cultured cell structures due to heat, but 0 to 45°C is preferred, 1 to 40°C is more preferred, and 2 to 35°C is most preferred, as it provides good washing performance and has little impact on biological tissues or cultured cell structures. When washing, the washing solution may be shaken or stirred as needed.

[0039] 《Heating process》 The present invention provides a method for producing a collagen-containing material, which preferably further includes a heating step of heating the biological tissue or cultured cell structure after the sterilization step.

[0040] (Heating temperature) The heating temperature is not particularly limited as long as the effects of the present invention are obtained, but for example it may be 35°C or higher, 40°C or higher in some embodiments, 45°C or higher in some embodiments, 50°C or higher in some embodiments, 55°C or higher in some embodiments, 60°C or higher in some embodiments, 65°C or higher in some embodiments, 70°C or higher in some embodiments, 75°C or higher in some embodiments, 80°C or higher in some embodiments, 85°C or higher in some embodiments, 90°C or higher in some embodiments, 95°C or higher in some embodiments, and 100°C or higher in some embodiments. The upper limit of the heating temperature is not particularly limited as long as the effects of the present invention are obtained, but for example it is 140°C or less, 135°C or less in some embodiments, 130°C or less in some embodiments, 125°C or less in some embodiments, 120°C or less in some embodiments, 115°C or less in some embodiments, 110°C or less in some embodiments, 105°C or less in some embodiments, and 100°C or less in some embodiments. The lower limit and upper limit can be combined as appropriate to set the range of heating temperatures. There are no particular limitations on the heating time, but for example, the lower limit is 5 minutes or more, in some embodiments it is 10 minutes or more, in some embodiments it is 20 minutes or more, and in some embodiments it is 30 minutes or more. There are no limitations on the upper limit either, but for example it is 48 hours or less, in some embodiments it is 24 hours or less, in some embodiments it is 12 hours or less, in some embodiments it is 6 hours or less, in some embodiments it is 3 hours or less, and in some embodiments it is 2 hours or less. The lower limit and upper limit can be combined as appropriate to define the range of heating time.

[0041] Generally, the effects of the present invention can be efficiently obtained by increasing the heating time when the heating temperature is high, and increasing the heating time when the heating temperature is low. The heating method can also be any method that is not limited to the conventional methods in this art. For example, heating can be done using a forced-air constant-temperature dryer, a constant-temperature dryer, or a hot bath as the heating device.

[0042] (Oxygen concentration) The heating step is not limited, but is preferably carried out at an oxygen concentration of 5% or less. The oxygen concentration in the heating step is not particularly limited as long as the effects of the present invention are obtained, but for example it is 5% by volume or less, in some embodiments it is 4% or less, in some embodiments it is 3% or less, in some embodiments it is 2% or less, in some embodiments it is 1% or less, and in some embodiments it is 0.5% or less. To obtain a low oxygen concentration, it is preferable to perform heating in the sterile packaging material used for sterilization. This is because if the biological tissue or cultured cell structure is removed from the sterile packaging material used for sterilization, the biological tissue or cultured cell structure will be contaminated by the external environment. However, the present invention does not preclude the method of producing collagen-containing material in which the biological tissue or cultured cell structure is removed from the sterile packaging material after the sterilization process and the heating process is performed.

[0043] 《Action》 The reason why the collagen-containing material of the present invention possesses excellent strength has not been analyzed in detail, but it can be presumed as follows. However, the present invention is not limited by the following presumption. The collagen-containing material of the present invention has an endothermic ratio of 48.0% or more in the 40°C to 60°C range. Endothermic activity in the 60-70°C range in differential scanning calorimetry is thought to be due to thermal deformation of collagen (the main component of the extracellular matrix) in the tissue. Collagen in biological tissues is known to have a triple helix structure called a collagen helix. The inventors estimate that endothermic activity in the 60-70°C range in biological tissues and decellularized tissues is due to the cleavage of hydrogen bonds between collagen and collagen or between collagen and coordinated water in the triple helix structure of collagen, and that the amount of endothermic activity in decellularized tissue correlates not with the collagen content of the decellularized tissue, but with the content of collagen with a triple helix structure. Endothermic activity in the 60-70°C range is presumed to be due to collagen molecules that maintain their triple helix structure or three-dimensional structure. Furthermore, the endothermic activity in the 30-40°C range is presumed to be due to the decomposition of collagen molecules caused by ionization and excitation due to gamma ray irradiation, resulting in the cleavage of collagen fiber bonds, fragmentation of some triple helix structures, and a breakdown of the three-dimensional structure. On the other hand, the endothermic activity in the 40-60°C range, being a higher temperature range than that observed with gamma ray irradiation alone, is presumed to be due to the cross-linking of the fragmented triple helix structures, the reconstruction (recombination) of partial higher-order structures, and a state of stabilization. Based on the above, it is estimated that the collagen-containing material of the present invention has a major component with an endothermic ratio of 48.0% or more at 40°C to 60°C. [Examples]

[0044] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0045] Example 1 In this embodiment, a collagen-containing material was prepared by gamma ray irradiation under reduced oxygen concentration conditions. The bovine pericardium was cut open and separated into a sheet, and the fat was completely removed. Hereafter, this sheet-like bovine pericardium will simply be referred to as the "pericardial sheet." The obtained pericardial sheets were placed in a polyethylene bag with a high-hydrostatic treatment medium (water for injection with added phosphate buffer (PBS, 0.01 M, pH 7.4), the same applies hereafter), and subjected to high-hydrostatic treatment at 600 MPa for 10 minutes using a high-pressure treatment device for research and development (Kobe Steel, Ltd.: Dr.CHEF, the same applies hereafter). Pericardial sheets treated with high hydrostatic pressure were shaken at 4°C for 18 hours using the nuclease DNase I (100 U / mL). Subsequently, they were shaken for 1 hour in 80% ethanol heated to 4°C, washed with sterile water for injection at 4°C, and then freeze-dried. Furthermore, the freeze-dried sample was placed in an aluminum bag, and gamma-ray irradiation (25 kGy) was performed under conditions where the oxygen concentration was reduced (oxygen concentration of 1% or less) using an oxygen absorber (manufactured by Mitsubishi Gas Chemical Co., Ltd.; Z-200PKC, the same applies hereafter) to obtain the collagen-containing material of Example 1.

[0046] Example 2 In this embodiment, a collagen-containing material was prepared by performing gamma ray irradiation under reduced oxygen concentration conditions (oxygen concentration of 1% or less), followed by heat treatment under reduced oxygen concentration conditions (oxygen concentration of 1% or less). Specifically, after performing gamma ray irradiation (25 kGy) under the same conditions as in Example 1, the oxygen concentration was reduced using an oxygen scavenger (oxygen concentration of 1% or less), and the material was heat-treated at 60°C for 30 minutes using a forced-air constant-temperature drying oven (Tokyo Rikakikai Co., Ltd.; WFO-420, the same applies hereafter) to obtain the collagen-containing material of Example 2.

[0047] Example 3 In this embodiment, a collagen-containing material was prepared by performing gamma ray irradiation under reduced oxygen concentration conditions (oxygen concentration of 1% or less), followed by heat treatment under the heating conditions shown in Table 1 while the oxygen concentration was still reduced (oxygen concentration of 1% or less). Specifically, after performing gamma ray irradiation (25 kGy) under the same conditions as in Example 1, the oxygen concentration was reduced using an oxygen scavenger (oxygen concentration of 1% or less), and the material was heat-treated at 80°C for 30 minutes using a forced-air constant-temperature drying oven to obtain the collagen-containing material of Example 3.

[0048] Example 4 In this embodiment, after gamma ray irradiation was performed under reduced oxygen concentration conditions (oxygen concentration of 1% or less), collagen-containing materials were prepared under the heating conditions shown in Table 1, while the oxygen concentration was still reduced (oxygen concentration of 1% or less). Specifically, after performing gamma ray irradiation (25 kGy) under the same conditions as in Example 1, the oxygen concentration was reduced using an oxygen scavenger (oxygen concentration of 1% or less), and the material was heat-treated at 100°C for 30 minutes using a forced-air constant-temperature drying oven to obtain the collagen-containing material of Example 4.

[0049] Example 5 In this embodiment, a collagen-containing material was prepared by performing gamma ray irradiation under reduced oxygen concentration conditions (oxygen concentration of 1% or less), followed by heat treatment under the heating conditions shown in Table 1, while the oxygen concentration was still reduced (oxygen concentration of 1% or less). Specifically, after performing gamma ray irradiation (25 kGy) under the same conditions as in Example 1, the oxygen concentration was reduced using an oxygen scavenger (oxygen concentration of 1% or less), and the material was heat-treated at 105°C for 120 minutes using a forced-air constant-temperature drying oven to obtain the collagen-containing material of Example 5.

[0050] Example 6 In this embodiment, after gamma ray irradiation was performed under reduced oxygen concentration conditions (oxygen concentration of 1% or less), a collagen-containing material was prepared by heat treatment under the heating conditions shown in Table 1, while the oxygen concentration was still reduced (oxygen concentration of 1% or less). Specifically, after performing gamma ray irradiation (25 kGy) under the same conditions as in Example 1, the oxygen concentration was reduced using an oxygen scavenger (oxygen concentration of 1% or less), and the material was heat-treated at 105°C for 150 minutes using a forced-air constant-temperature drying oven to obtain the collagen-containing material of Example 6.

[0051] Comparative Example 1 In this comparative example, collagen-containing materials were prepared by gamma ray irradiation under conditions of high oxygen concentration (airborne oxygen concentration of 20-21%). Specifically, after freeze-drying under the same conditions as in Example 1, gamma irradiation (25 kGy) was performed without an oxygen absorber (at an atmospheric oxygen concentration of 20-21%) to obtain the collagen-containing material of Comparative Example 1.

[0052] Comparative Example 2 In this comparative example, a collagen-containing material was prepared by irradiating with gamma rays under conditions of high oxygen concentration (air oxygen concentration of 20-21%), followed by heat treatment. After freeze-drying under the same conditions as in Example 1, gamma irradiation (25 kGy) was performed without an oxygen absorber (at an atmospheric oxygen concentration of 20-21%). Subsequently, without an oxygen absorber enclosed (at an atmospheric oxygen concentration of 20-21%), the material was heated in a forced-air constant-temperature dryer at 105°C for 150 minutes to obtain the collagen-containing material of Comparative Example 2.

[0053] Comparative Example 3 In this manufacturing example, collagen-containing material was produced without gamma irradiation or heat treatment. Specifically, the pericardial sheet was first placed in a polyethylene bag with a high-hydrostatic treatment medium (water for injection with added phosphate buffer (PBS, 0.01M, pH 7.4)), and then subjected to high-hydrostatic treatment at 600 MPa for 10 minutes using a high-pressure treatment device for research and development (Kobe Steel, Ltd.: Dr.CHEF). Pericardial sheets treated with high hydrostatic pressure were shaken at 4°C for 18 hours using the nuclease DNase I (100 U / mL). Subsequently, they were shaken in 80% ethanol heated to 4°C for 1 hour and washed with sterile water for injection at 4°C. Subsequently, freeze-drying treatment was performed to obtain the collagen-containing material of Comparative Example 3.

[0054] Comparative Example 4 Unprocessed bovine pericardium was used as Comparative Example 4.

[0055] [Table 1]

[0056] The collagen-containing materials obtained in Examples 1-6 and Comparative Examples 1-4 were subjected to suture retention tests and burst strength tests as follows. Strength Test (Suture retention test) The suture retention strength test was performed according to ISO 7198 as follows. (1) Sampling and preparation of test specimens Freeze-dried decellularized tissue or material was immersed in physiological saline for at least 15 minutes. From the swollen decellularized tissue or material, rectangular specimens measuring 20-30 × 15-20 mm were taken. Subsequently, a thread with a diameter of 0.165 mm was passed through the specimen 2 mm from the end of the shorter side to form a loop (the ends of the thread were tied).

[0057] (2) Test Procedure In accordance with ISO 7198, the tensile test was performed as follows: The loop of thread passed through the test specimen was hooked onto a hook-type fixture installed on top of a mechanical testing machine (MCT2150, AND Corporation or EZ TEST EX-SX, SHIMAZU Corporation), and the lower part of the test specimen was clamped, securing both ends of the specimen symmetrically. The speed of the hook-type fixture was set to 200 mm / min. Furthermore, any tests in which the specimen became detached during the test were discarded, and the test was repeated until a total of six tests (three in the long axis direction and three in the short axis direction) of the original sample were performed correctly. The measured value was calculated as the maximum load [N] at fracture. The average of the six measurement results in the long axis direction and the short axis direction was used as the representative value for one specimen, and the average of the representative values ​​of 3 to 5 specimens was used as the representative value for each treatment group.

[0058] (Burst strength test) The burst intensity test was conducted according to ISO 7198 as follows: (1) Sampling and preparation of test specimens For freeze-dried decellularized tissue or material, immersion in physiological saline for 15 minutes or more was required to obtain 20-25 mm square test pieces from the swollen decellularized tissue or material. For the unprocessed bovine pericardium in Comparative Example 4, 20-25 mm square test pieces were taken directly.

[0059] (2) Test Procedure In accordance with ISO7198, the burst strength test was conducted as follows. Specifically, the test specimen was first fixed in a 20 mm square fixture on a mechanical testing machine (MCT2150, manufactured by AND Corporation, or MCT-2150W, manufactured by AND Corporation). Next, a 9.5 mm diameter probe was applied to the test specimen using a mechanical testing machine at a speed of 200 mm / min. Tests in which the specimen became detached during the test were discarded, and the test was repeated until five successful tests were performed. The measured value was calculated as the maximum load [N] at the time of fracture. The average of five measurement results was used as the representative value for one test specimen, and the average of the representative values ​​of 3 to 5 test specimens was used as the representative value for each treatment group.

[0060] Table 2 shows the suture retention strength and burst strength of the examples and comparative examples. [Table 2]

[0061] Example 1 showed higher strength compared to Comparative Examples 1 and 2. This is thought to be because the reduction in oxygen concentration during gamma-ray irradiation suppressed the cleavage and fragmentation of the triple helix structure caused by gamma-ray irradiation, thus maintaining relative strength. Examples 2-6 showed higher strength compared to Example 1. This is thought to be because, in Examples 2-6, the fractured triple helix structure was crosslinked after heat treatment following gamma ray irradiation under reduced oxygen concentration, resulting in a further increase in strength compared to Example 1. Furthermore, it was found that increasing the heating temperature and heating time promoted the increase in strength. This is thought to be because, with higher heating temperatures and heating times, radicals were consumed more quickly in the recombination of collagen molecules than in Example 1, resulting in higher strength values. Furthermore, the decellularized tissue of Comparative Example 3, which was not irradiated with gamma rays, maintained a strength comparable to that of the unprocessed bovine pericardium of Comparative Example 4.

[0062] Differential scanning calorimetry was performed on the collagen-containing materials obtained in Examples 1-6 and Comparative Examples 1-4 as follows. Differential scanning calorimetry (Observation of endothermic charts) The samples for the examples and comparative examples were molded into 0.5 cm squares with a dry weight of 20-30 mg each to form test specimens. They were rehydrated with physiological saline (Otsuka Saline Injection), and a comparison group was set with physiological saline alone. The differential scanning calorimetry was measured using a differential scanning calorimetry analyzer (DKSH MicroDSC EVO) when the test specimens were heated at a heating rate of 2 K / min from 25 to 85°C.

[0063] (Calculation of heat absorption ratio) The differences between the examples and comparative examples were analyzed using the ratio of heat absorption. The area of ​​the peaks detected from each endothermic chart was calculated as the amount of heat absorbed by the test specimen. The denaturation temperature of the test specimen was determined using the onset value (the temperature at which a tangent line is drawn to the rising edge of the peak at the start of heat absorption).

[0064] The heat absorption charts for Examples 1, 2, 3, 4, and 6 are shown in Figure 1. The heat absorption charts for Comparative Examples 1 to 3 are shown in Figure 2.

[0065] (Calculation of the heat absorption ratio between the low-temperature and high-temperature sides) The obtained peak area was divided into two main peak areas. For the low temperature side (40°C to 60°C) and the high temperature side (above 60°C), the respective peak areas were calculated as the heat absorption ratio, and the ratio of the heat absorption amounts on the low temperature side and the high temperature side to the total heat absorption was calculated using the following formula. Here, the peak area represents the area enclosed by the tangent line and the heat absorption chart (shaded area in Figure 3). Figure 3 shows, from top to bottom, the heat absorption ratio for 30-40°C, the heat absorption ratio for 40-60°C, and the heat absorption ratio for above 60°C. Total heat absorbed = Peak area on the low-temperature side (40°C to 60°C) + Peak area on the high-temperature side (60°C to 70°C) Ratio of heat absorption on the low-temperature side (40°C to 60°C) = Peak area on the low-temperature side / Total heat absorption Ratio of heat absorption on the high-temperature side (60°C to 70°C) = Peak area on the high-temperature side / Total heat absorption

[0066] When comparing the endothermic ratio at low temperatures (40°C to 60°C) and high temperatures (60°C to 70°C) of the collagen-containing material of Example 6 and the collagen-containing material of Comparative Example 1, it was found that applying heat treatment after gamma ray irradiation reduced (degraded) the collagen originating from the low temperature side, while significantly increasing (degraded) the collagen originating from the high temperature side. (Figures 1E, 2A, and 4)

[0067] In Figure 1, the endothermic activity in the 60-70°C range during differential scanning calorimetry of collagen-containing materials is thought to be due to thermal deformation of collagen (the main component of the extracellular matrix) in the tissue.

[0068] Comparative Example 3 showed a denaturation temperature of 63.0°C, with significant endothermic activity in the 60-70°C range. This temperature range was similar to that of Comparative Example 4 (denaturation temperature 63.5°C), and it was found that there was no effect on the triple helix structure of collagen because gamma ray irradiation was not performed. Comparative Example 1 showed significantly greater endothermic effects in the 30-40°C range compared to Examples 1, 6, and Comparative Example 3. It was found that the triple helix structure of collagen was disrupted due to the ionization and excitation effects of gamma ray irradiation, as well as the oxidation reaction by oxygen, which occurred under conditions of high oxygen concentration. Compared to Comparative Example 1, Example 1 showed reduced endothermic activity in the 30-40°C range and observed endothermic activity in the 60-70°C range. It was found that gamma ray irradiation was performed with a reduced oxygen concentration, which suppressed the oxidation reaction by oxygen and prevented the breakdown of the triple helix structure due to gamma ray irradiation. Compared to Example 1, Example 6 showed even lower endothermic activity in the 30-40°C range and greater endothermic activity in the 40-60°C range. It was found that because gamma ray irradiation and heat treatment were performed with a reduced oxygen concentration, the breakdown of the triple helix structure due to gamma ray irradiation was suppressed, and the triple helix structure and fragmented collagen molecules that were cleaved by gamma ray irradiation were cross-linked, resulting in the reconstruction of a partial higher-order structure.

[0069] The amount of DNA in Examples 1 and 4, and Comparative Example 4 was measured. Calculation of the ratio of decellularized DNA The dry mass of the samples from Example 1, Example 4, and Comparative Example 4 was measured and used as the sample. After dissolving by immersion in a protease solution, the protein was removed by treatment with phenol / chloroform and the DNA was recovered. The recovered DNA was fluorescently stained with PicoGreen (Life Technologies) and its fluorescence intensity was measured to quantify the DNA. The DNA content per unit of dry mass of the sample was then calculated from the dry mass of the sample and the amount of DNA. A calibration curve created using the standard DNA included with PicoGreen was used for DNA quantification. The results are shown in Table 3. (Decellularized DNA ratio) = (DNA content of the dried sample) / (Dried mass of the sample)

[0070] [Table 3]

[0071] Based on the results of the decellularized DNA ratio, it was found that decellularization was successfully performed in Examples 1 and 4.

[0072] Calculation of oxygen concentration In Example 1, a probe for measuring oxygen saturation was inserted into an aluminum bag containing an oxygen absorber, and the oxygen concentration inside the aluminum bag after 24 hours was measured using an oxygen concentration meter (FireSting oxygen monitor, BAS Corporation). The results are shown in Table 4. [Table 4] [Industrial applicability]

[0073] The collagen-containing material of the present invention can be used as an in vivo scaffold material.

Claims

1. A collagen-containing material comprising a sterile tissue or cultured cell structure derived from a living organism, characterized in that the ratio of endothermic heat per dry mass measured by differential scanning calorimetry between 40°C and 60°C is 48.0% or more.

2. The collagen-containing material according to claim 1, wherein the sterilization is performed by radiation irradiation.

3. The collagen-containing material according to claim 1 or 2, wherein the collagen-containing material is decellularized.

4. A method for producing a triple-helix collagen-containing material, comprising a sterilization step of sterilizing a living tissue or cultured cell structure with radiation at an oxygen concentration of 5% or less.

5. A method for producing a collagen-containing material according to claim 4, further comprising a heating step of heating the biological tissue or cultured cell structure after the sterilization step.

6. The collagen-containing material according to claim 5, wherein the heating step is performed at an oxygen concentration of 5% or less. A method for manufacturing this product.

7. A method for producing a collagen-containing material according to any one of claims 4 to 6, wherein the biological tissue or cultured cell structure is decellularized.

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