Collagen-rich organic composition and method for producing same

Abstract

[Problem] To provide: a collagen-rich organic composition; a biomaterial using the collagen-rich organic composition as a starting material; a biomaterial or other products containing collagen as a main ingredient; a method for producing the collagen-rich organic composition; a method for differentiating a tooth or a bone extracted from a mammal as a starting material for a biomaterial or a biomaterial or other products containing collagen as a main ingredient; and a method for assessing the potential risk of a disease in a mammal and / or whether the mammal is potentially in good health or not with the use of PYD and PYD and PpP as indicators. [Solution] PYD>100, and PYD>100 and PpP>740.

Description

[Technical Field]

[0001] The present invention relates to a collagen-rich organic composition produced from teeth or bones extracted from mammals, a biomaterial made from the collagen-rich organic composition as a raw material, a biomaterial or other product having collagen as a main component, a method for producing the collagen-rich organic composition, a method for distinguishing teeth or bones extracted from mammals as a raw material for biomaterials, biomaterials or other products having collagen as a main component, and a method for using a quantitative value of the pyridinoline content (ng / mg) of collagen contained in mammalian teeth, or a quantitative value of the pyridinoline content (ng / mg) of collagen contained in mammalian teeth, and a weight ratio calculated from the quantitative value of the pyridinoline content (ng / mg) of collagen contained in mammalian teeth and the quantitative value of the pentosidine content (ng / mg) (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) as an index for assessing the potential risk of disease in mammals and / or whether the mammal is potentially healthy. [Background technology]

[0002] Various collagens have been developed (although the more accurate term would be "collagen-rich organic compositions," they are commonly referred to simply as "collagen"). Most of these are atelocollagens, which are extracted from soft tissues in living organisms using enzymatic treatments. The telopeptides present at the N- and C-termini of the collagen's amino acid sequence are cleaved, and the poorly soluble collagen fibers are removed during the purification process. Collagens extracted from hard tissues such as bones and teeth, such as demineralized freeze-dried bone allograft (DFDBA) and demineralized dentin matrix (DDM), have also been developed. These collagens are produced by demineralization and are primarily used as biomaterials.

[0003] The quality of atelocollagen is evaluated based on the fact that telopeptides, which are said to have antigenicity, are cleaved by enzymatic treatment. The quality of undenatured collagen derived from soft tissue (collagen with a triple helix structure due to the retention of telopeptides) is evaluated based on the purity of the collagen produced, determined by the setting of purification conditions such as pH. However, when producing collagen from hard tissue, decalcification, i.e., acid treatment, is required to remove minerals and extract the collagen, which limits the collagen yield and requires a lot of effort. Therefore, almost all collagen products currently being developed are atelocollagen derived from soft tissue, undenatured collagen, or products utilizing them.

[0004] Most collagen contained in biological tissues is type I collagen (type I collagen). Type I collagen has both physiological and non-physiological cross-links, with pyridinoline cross-linking molecules known as physiological cross-linking molecules and cross-linking molecules derived from advanced glycation end products (AGEs), such as pentosidine, known as non-physiological cross-linking molecules (Non-Patent Document 1). Pyridinoline cross-links are physiological / enzymatic cross-links formed by the action of lysyl oxidase, and are regular cross-linking molecules at specific sites in the amino acid sequence of collagen, contributing to improved elasticity and strength of collagen fibers. On the other hand, pentosidine crosslinks are non-physiological, non-enzymatic crosslinks (aging crosslinks (AGE crosslinks)) that are mediated by blood glucose. When blood glucose is heated by body temperature, they are formed between arginines and lysines scattered throughout the collagen amino acid sequence. Therefore, they are crosslinks formed randomly between adjacent arginines and lysines in the collagen amino acid sequence, disrupting the physiological three-dimensional structure of collagen and resulting in a decrease in the elasticity and strength of collagen fibers. As mentioned above, atelocollagen contains almost no pyridinoline crosslinks because the telopeptides are cleaved by enzymatic treatment. Furthermore, the purification process removes the insoluble collagen fibers resulting from pentosidine crosslinks, resulting in a collagen that contains almost no pentosidine crosslinks.

[0005] It has been previously known that an increase in pyridinoline crosslink molecules in collagen increases the strength and elasticity of the collagen (Non-Patent Document 2), and that pentosidine crosslinks are aging crosslinks (AGE crosslinks) (Non-Patent Document 3). It has also been reported that the amount of pentosidine per unit collagen of human articular cartilage increases linearly with age, and that the ratio of pentosidine to pyridinoline per unit collagen (pentosidine content / pyridinoline content) increases at an accelerated rate with age (Non-Patent Documents 4 and 5). It has also been reported that the ratio of pentosidine to pyridinoline per unit collagen (pentosidine content / pyridinoline content) increases significantly in non-calcified lesions of dystrophic aortic calcification (Non-Patent Document 6). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] M. Saito,CLINICIAN Vol.554,1141-1146,2006 [Non-patent document 2] M.Saito,THE BONE Vol.21,No.1,53-58,2007 [Non-patent document 3] Sell,DRet al.,VM,J.Biol.Chem.Vol.284,21597-21602,1989 [Non-patent document 4] A. Uchiyama et al., J. Biochem. Vol. 110, 714-718, 1991 [Non-Patent Document 5] M.TAKAHASHI et al.,Arthritis Rheum.Vol.37,No.5,724-728,May 1994. [Non-patent document 6] H.HOSHINO et al.,Atherosclerosis Vol.112,39-46,1995 Summary of the Invention [Problem to be solved by the invention]

[0007] The quality of the above-mentioned collagen (collagen-rich organic composition) is generally evaluated based on the purity and characteristics of the collagen in the collagen-rich organic composition, and is not evaluated based on any molecular-level indicator of collagen, i.e., any indicator of the amino acid sequence level of collagen. Therefore, it is difficult to say that the quality of the collagen itself has been properly evaluated. Furthermore, the quality of collagen has not been evaluated based on the amount of pyridinoline contained, which contributes to improving collagen quality, such as improving the elasticity and strength of collagen fibers, or the amount of pentosidine contained, which is a factor that disrupts the physiological three-dimensional structure of collagen, resulting in a decrease in the elasticity and strength of collagen fibers and ultimately contributing to a decrease in collagen quality, or the weight ratio of the amount of pentosidine contained to the amount of pyridinoline contained and the amount of pentosidine contained.

[0008] Furthermore, up until now, no collagen-rich organic composition or collagen-based biomaterial has been found that can be evaluated as containing essentially high-quality collagen based on some collagen molecular level, i.e., some collagen amino acid sequence level, indicator. Furthermore, no attempt has been made to provide a collagen-rich organic composition or collagen-based biomaterial that can be evaluated as containing essentially high-quality collagen based on some collagen molecular level, i.e., some collagen amino acid sequence level, based on collagen-derived collagen, which, as mentioned above, contains residual telopeptides, limits collagen yield, and is laborious to extract, particularly from hard tissues, such as teeth, from which collagen extraction is difficult and the yield is limited, or bone, which contains a large amount of lipids and therefore requires a great deal of labor to extract collagen.

[0009] Because the telopeptide region of collagen is believed to be highly antigenic, some collagen products utilize atelocollagen that has been enzymatically treated to remove the telopeptides. However, removing the telopeptides also results in the loss of pyridinoline crosslinks, resulting in a loss of collagen's inherent flexibility and toughness. Furthermore, there is no clear evidence that telopeptide removal reduces antigenicity. Therefore, removing telopeptides does not provide any benefit to immunogenicity in the medical application of collagen. On the other hand, the incidence of allergies to bovine type I collagen is 2-4%, which is low compared to the incidence of nickel hypersensitivity (10-15%) and latex allergy (approximately 6%) (Lynn AK et al., J Biomed Mater Res B Appl Biomater. 2004 Nov. 15;71(2):343-354). It has been reported that type I collagen does not induce an immune response or exhibit antigenicity (Courtenay JS et al., Nature, 283, 14, 1980, 666-668; David E et al., J. Exp. Med. 1977, 46; 857-868).

[0010] Furthermore, the above-mentioned Non-Patent Documents 1 to 6 certainly make various references to pyridinoline crosslink molecules and pentosidine crosslink molecules in collagen, and also discuss the ratio of pentosidine to pyridinoline content per unit collagen (pentosidine content / pyridinoline content) in diseases. However, none of these documents specifically address collagen derived from teeth, from which collagen extraction is difficult. Furthermore, in tissues that require physiological strength, such as hard tissues like bone and cartilage, and soft tissues like tendons and ligaments, the amount of pyridinoline crosslink molecules contained in collagen and, therefore, the amount of pentosidine crosslink molecules tend to be high (M. Takahashi et al., Anal Biochem Vol. 232, 158-62, 1995). However, because these tissues undergo metabolism, the pyridinoline and pentosidine crosslink molecules contained therein are also metabolized, and therefore, the amount of pyridinoline and pentosidine crosslink molecules accumulated in the mammalian host over its lifetime. In contrast, teeth, which are hard tissues, are the only hard tissues that are not incorporated into the metabolic cycle, and therefore the pyridinoline crosslink molecules and pentosidine crosslink molecules contained in the collagen contained therein are not metabolized, so the amount of pyridinoline crosslink molecules and pentosidine crosslink molecules accumulated in the mammalian host for almost the entire lifetime is reflected. Therefore, in order to accurately evaluate the potential disease risk in mammals and whether the mammal is potentially healthy or not, it is clear that tooth-derived collagen (collagen-rich organic composition), in which pyridinoline crosslink molecules and pentosidine crosslink molecules accumulated in the mammalian host for the entire lifetime, should be used as an indicator. Furthermore, the collagen-rich organic composition and biomaterials containing collagen as a main component according to the present invention are produced by undergoing a strong alkaline treatment, which is not normally performed on proteins including collagen, at temperatures higher than room temperature, followed by a decalcification treatment, i.e., an acid treatment. As a result, the amount of endotoxin contained is significantly reduced, and the compositions are essentially endotoxin-free, or are endotoxin-free, or the endotoxin they contain is substantially inactivated, or the endotoxin they contain is inactivated; however, the above-mentioned Non-Patent Documents 1 to 6 do not state or even suggest such a thing.

[0011] The present invention provides a collagen-rich organic composition produced from teeth or bones extracted from mammals, which contains a predetermined PYD or a predetermined PYD and a predetermined PYD and a predetermined PPP, by quantifying the amount of pyridinoline (ng / mg) and the amount of pentosidine (ng / mg) contained in collagen using a predetermined method, and calculating a weight ratio PpP (amount of pyridinoline (ng / mg) / amount of pentosidine (ng / mg)) from the PYD, which is the quantitative value of the amount of pyridinoline (ng / mg), and the PEN, which is the quantitative value of the amount of pentosidine (ng / mg) contained. The present invention provides a biomaterial made from a collagen-rich organic composition as a raw material, a biomaterial having collagen as a main component, or other product, and a method for producing the collagen-rich organic composition. The invention also provides a method for distinguishing teeth or bones extracted from mammals and used as a raw material for biomaterials, biomaterials having collagen as a main component, or other products, which comprises a step of selecting the tooth or bone as a raw material for biomaterials, biomaterials having collagen as a main component, or other products when the PYD or the PYD and the PpP are predetermined values. The invention also provides a method for distinguishing teeth or bones extracted from mammals and used as a raw material for biomaterials, biomaterials having collagen as a main component, or products, which comprises a step of obtaining PYD, which is a quantitative value of the amount of pyridinoline (ng / mg) contained in collagen, and PEN, which is a quantitative value of the amount of pentosidine (ng / mg) contained in collagen, from the teeth of mammals to be evaluated for potential disease risk and / or whether the mammal is potentially healthy, and calculating the weight ratio PpP (amount of pyridinoline (ng / mg) / amount of pentosidine (ng / mg)) from the obtained PYD and the obtained PEN. The invention also provides a method for distinguishing teeth or bones extracted from mammals and used as a raw material for biomaterials, biomaterials having collagen as a main component, or products when the PYD or the PYD and the PpP are predetermined values. and a step of obtaining R-PYD, which is a reference quantitative value of the amount of pyridinoline (ng / mg) contained in collagen contained in the teeth of one or more mammals, and R-PEN, which is a reference quantitative value of the amount of pentosidine (ng / mg) contained in collagen contained in the teeth of one or more mammals, and calculating R-PpP, which is a reference weight ratio value (amount of pyridinoline (ng / mg) / amount of pentosidine (ng / mg)), from the obtained R-PYD and the obtained R-PEN; and comparing the PYD and the R-PYD, or comparing the PYD and the R-PYD, andThe object of the present invention is to provide a method using a quantitative value of the amount of pyridinoline (ng / mg) contained in collagen contained in mammalian teeth, or a quantitative value of the amount of pyridinoline (ng / mg) contained in collagen contained in mammalian teeth, and a weight ratio (amount of pyridinoline (ng / mg) / amount of pentosidine (ng / mg)) calculated from the quantitative value of the amount of pyridinoline (ng / mg) contained in collagen contained in mammalian teeth and the quantitative value of the amount of pentosidine (ng / mg) contained in collagen contained in mammalian teeth, as an index for assessing the potential risk of disease in a mammal and / or whether the mammal is potentially healthy, the method comprising a step of comparing the PpP and the R-PpP. [Means for solving the problem]

[0012] As a result of extensive research into solving the above-mentioned problems, the present inventors have found that, for a collagen-rich organic composition obtained by treating pulverized material of teeth or bones extracted from mammals with a strong alkali at a temperature higher than room temperature and lower than the boiling point of water, and then decalcifying the pulverized material that has been treated with a strong alkali under negative pressure, and for a biomaterial, biomaterial or other product containing collagen as a main component, when PYD, which is a quantitative value of the amount of pyridinoline contained per unit collagen (ng / mg), and PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)), which is a weight ratio calculated from the amounts of pyridinoline contained (ng / mg) and pentosidine contained (ng / mg) per unit collagen, are predetermined values, the collagen contained has a three-dimensional structure that imparts excellent elasticity and strength, and is of high quality. The present inventors have found that, when the PYD or the PYD and the PpP are predetermined values, it is possible to distinguish teeth or bones extracted from mammals that are used as raw materials for biomaterials, biomaterials whose main component is collagen, or other products, and that by comparing the above-mentioned PYD or PYD and PpP for collagen contained in the teeth of a mammal that is the subject of an assessment of the potential risk of disease and / or the potential health of the mammal with collagen contained in the teeth of one or more mammals selected from disease-free mammals, diseased mammals, healthy mammals, and unhealthy mammals, it is possible to assess the potential risk of disease in a mammal and / or the potential health of a mammal, and have completed the following inventions.

[0013] (1) A collagen-rich organic composition produced from teeth or bones extracted from a mammal, which is the following (i) or (i) and (ii): (i) PYD, which is a quantitative value of the amount of pyridinoline (ng / mg) contained in collagen contained in the collagen-rich organic composition, as determined by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pair reagents, is PYD>100; (ii) The PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)) is the weight ratio of PYD, which is the quantitative value of the amount of pyridinoline contained in the collagen contained in the high-collagen organic composition, to PEN, which is the quantitative value of the amount of pentosidine contained in the collagen contained in the high-collagen organic composition, as determined by high-performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pair reagents, and is PpP>740.

[0014] (2) The collagen-rich organic composition according to (1), which is substantially endotoxin-free, or contains no endotoxin, or the endotoxin it contains is substantially inactivated, or the endotoxin has been inactivated.

[0015] (3) The collagen-rich organic composition according to (1), wherein the mammal is one or more mammals selected from the group consisting of cows, pigs, horses, sheep, deer, dogs, cats and humans.

[0016] (4) A biomaterial, a biomaterial containing collagen as a main component, or other product made from the collagen-rich organic composition according to any one of (1) to (3).

[0017] (5) crushing the tooth or bone extracted from the mammal to obtain a crushed material; A step of treating the obtained pulverized product with a strong alkali; a step of decalcifying the strongly alkali-treated pulverized material under negative pressure conditions; A method for producing a collagen-rich organic composition according to any one of (1) to (3).

[0018] (6) The manufacturing method according to (5), wherein the step of treating the obtained pulverized material with a strong alkali is a step of treating the obtained pulverized material with a strong alkali under temperature conditions that are higher than room temperature and lower than the boiling point of water.

[0019] (7) quantifying the amount of pyridinoline (ng / mg) contained in collagen contained in a tooth or bone extracted from a mammal; Quantifying the amount of pentosidine (ng / mg) contained in collagen contained in the tooth or bone extracted from the mammal; a step of calculating a weight ratio PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)) from PYD, which is the value of the quantified amount of pyridinoline contained (ng / mg), and PEN, which is the value of the quantified amount of pentosidine contained (ng / mg); selecting the tooth or bone extracted from the mammal as a biomaterial, a collagen-based biomaterial, or a raw material for other products when the determined value of PYD, which is the value of the determined pyridinoline content (ng / mg), or the calculated weight ratio value of PpP (amount of pyridinoline content (ng / mg) / amount of pentosidine content (ng / mg)), is a predetermined value; 1. A method for identifying teeth or bones extracted from mammals as raw materials for biomaterials, collagen-based biomaterials or other products, comprising:

[0020] (8) crushing the tooth or bone extracted from the mammal to obtain a crushed material; A step of treating the obtained pulverized product with a strong alkali; a step of decalcifying the pulverized product treated with strong alkali under negative pressure conditions to obtain an organic composition containing a high amount of collagen; a step of quantifying the amount of pyridinoline (ng / mg) contained in the collagen contained in the obtained collagen-rich organic composition; Quantifying the amount of pentosidine (ng / mg) contained in the collagen contained in the obtained collagen-rich organic composition; a step of calculating a weight ratio PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)) from PYD, which is the value of the quantified amount of pyridinoline contained (ng / mg), and PEN, which is the value of the quantified amount of pentosidine contained (ng / mg); selecting the tooth or bone extracted from the mammal as a biomaterial, a collagen-based biomaterial, or a raw material for other products when the determined value of PYD, which is the value of the determined pyridinoline content (ng / mg), or the calculated weight ratio value of PpP (amount of pyridinoline content (ng / mg) / amount of pentosidine content (ng / mg)), is a predetermined value; 1. A method for identifying teeth or bones extracted from mammals as raw materials for biomaterials, collagen-based biomaterials or other products, comprising:

[0021] (9) The method according to (7) or (8), wherein the step of quantifying the pyridinoline content (ng / mg) of the collagen contained in the obtained collagen-rich organic composition and the step of quantifying the pentosidine content (ng / mg) of the collagen contained in the obtained collagen-rich organic composition are carried out by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pair reagents, and the predetermined values ​​are PYD>100 or PYD>100 and PpP>740, respectively.

[0022] (10) A method for assessing the potential risk of a disease in a mammal and / or the potential health of the mammal, comprising using a quantitative value of the amount of pyridinoline contained in collagen contained in the teeth of the mammal, or a quantitative value of the amount of pyridinoline contained in collagen contained in the teeth of the mammal, and a weight ratio calculated from the quantitative value of the amount of pyridinoline contained in collagen contained in the teeth of the mammal and the quantitative value of the amount of pentosidine contained, A step of quantifying the amount of pyridinoline (ng / mg) contained in collagen contained in the teeth of a mammal that is a target for evaluation of the potential risk of disease and / or whether the mammal is potentially healthy, to obtain a quantitative value of PYD; a step of quantifying the amount of pentosidine (ng / mg) contained in collagen contained in teeth of the mammal to be evaluated to obtain a quantitative value PEN; A step of calculating a weight ratio PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)) from the obtained PYD and the obtained PEN; Quantifying the amount of pyridinoline (ng / mg) contained in collagen contained in the teeth of one or more mammals selected from a mammal not affected by a disease, a mammal affected by a disease, a healthy mammal, and an unhealthy mammal, to obtain a reference quantitative value R-PYD; Quantifying the amount of pentosidine (ng / mg) contained in collagen contained in the teeth of one or more mammals selected from a mammal not affected by a disease, a mammal affected by a disease, a healthy mammal, and an unhealthy mammal, to obtain a reference quantitative value R-PEN; calculating a reference value of weight ratio R-PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)) from the obtained R-PYD and the obtained R-PEN; comparing the PYD and the R-PYD, or comparing the PYD and the R-PYD and the PpP and the R-PpP; A method comprising:

[0023] (11) The step of quantifying the amount of pyridinoline (ng / mg) contained to obtain the reference quantitative value R-PYD and the step of quantifying the amount of pentosidine (ng / mg) contained to obtain the reference quantitative value R-PEN are carried out by a high performance liquid chromatograph (HPLC-Flu) equipped with a fluorescence detector using heptafluorobutyric acid and formic acid as ion pair reagents. The method according to (10), wherein the step of quantifying the amount of tocidine (ng / mg) to obtain a reference quantitative value R-PEN, and the step of comparing the PYD and the R-PYD, or comparing the PYD and the R-PYD and comparing the PpP and the R-PpP, is a step of confirming whether the PYD is PYD>100, or whether the PYD is PYD>100 and whether the PpP is PpP>740. [Effects of the Invention]

[0024] According to the present invention, a pulverized material of teeth or bones extracted from mammals is subjected to a strong alkali treatment (preferably under predetermined conditions, such as at a temperature higher than room temperature), and the pulverized material thus obtained is decalcified under negative pressure conditions. This gives the collagen contained therein a three-dimensional structure that confers excellent elasticity and strength, and the resulting high-quality collagen-rich organic composition and biomaterials, biomaterials containing collagen as a main component, or other products can be provided. The present invention also provides a method for identifying teeth or bones extracted from mammals as raw materials for biomaterials, biomaterials containing collagen as a main component, or other products. Furthermore, as an index for assessing the potential risk of a disease in a mammal and / or whether the mammal is potentially healthy, the quantitative value of the amount of pyridinoline (ng / mg) contained in collagen contained in the teeth of a mammal, or the quantitative value of the amount of pyridinoline (ng / mg) contained in collagen contained in the teeth of a mammal, and the weight ratio calculated from the quantitative value of the amount of pyridinoline (ng / mg) contained in collagen contained in the teeth of a mammal and the quantitative value of the amount of pentosidine (ng / mg) contained in collagen contained in the teeth of a mammal (amount of pyridinoline (ng / mg) / amount of pentosidine (ng / mg)) can be used. [Brief explanation of the drawings]

[0025] [Figure 1] This is a graph showing the distribution of PYD, which is the quantitative value of the amount of pyridinoline (ng / mg) contained in collagen contained in an organic composition with a high content of collagen derived from dentin from the teeth of 18 humans, 36 cattle (including 19 bovine specified risk materials (SRM) and 17 Tokachi Young Cattle (registered trademark in Japan, early fattened (14-month-old) male Holsteins)), and 8 domestic pigs (6 months old) (62 samples in total). The collagen was quantified by high-performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pairing reagents. [Figure 2]This graph shows the distribution of PYD, the quantitative value of pyridinoline content (ng / mg), in dentin-derived collagen-rich organic compositions (n=62) from 18 human, 36 bovine (including 19 bovine SRM and 17 Tokachi Young Beef (registered trademark)), and 8 domestic pig teeth (62 total samples), Teruplag (n=3, bovine dermis-derived atelocollagen, telopeptide-free processing), high-grade gelatin (n=3, porcine acid processing, telopeptide-preserving processing, low-endotoxin collagen), Tokachi Young Beef (registered trademark) gingiva (n=3), and Tokachi Young Beef (registered trademark) muscle (n=3), as determined by high-performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pairing reagents. The dashed line indicates the PYD threshold. [Figure 3] This is a graph showing the distribution of PEN, which is the quantitative value of the pentosidine content (ng / mg), of collagen contained in an organic composition with a high collagen content derived from dentin from the teeth of 18 humans, 36 cows (including 19 bovine SRMs and 17 Tokachi Young Beef (registered Japanese trademark)), and 8 domestic pigs (62 cases in total), as quantified by high-performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pairing reagents. [Figure 4] This graph shows the distribution of PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)), which is the weight ratio calculated from PYD, which is the quantitative value of the amount of pyridinoline contained (ng / mg), and PEN, which is the quantitative value of the amount of pentosidine contained (ng / mg), for collagen contained in an organic composition with a high content of collagen derived from dentin from the teeth of 18 humans, 36 cows (including 19 bovine SRMs and 17 Tokachi Young Beef (a registered Japanese trademark)), and 8 domestic pigs (62 samples in total). The collagen was quantified by high-performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pairing reagents. [Figure 5]This is a graph showing the distribution of endotoxin values ​​(endotoxin level; EU / mg) quantified by endotoxin testing (LAL method) for collagen contained in an organic composition with a high collagen content derived from the dentin of teeth of six examples of Tokachi Young Beef (registered trademark of Japan), three examples of gelatin derived from pigskin, and three examples of high-grade gelatin. DETAILED DESCRIPTION OF THE INVENTION

[0026] Hereinafter, detailed descriptions will be given of a collagen-rich organic composition produced from teeth or bones extracted from mammals, a biomaterial using the collagen-rich organic composition as a raw material, a biomaterial or other product mainly composed of collagen, a method for producing the collagen-rich organic composition, a method for distinguishing teeth or bones extracted from mammals as raw materials for biomaterials, biomaterials or other products mainly composed of collagen, and a method for using a quantitative value of the pyridinoline content (ng / mg) of collagen contained in the teeth of a mammal, or a quantitative value of the pyridinoline content (ng / mg) of collagen contained in the teeth of a mammal, and a weight ratio calculated from the quantitative value of the pyridinoline content (ng / mg) and the quantitative value of the pentosidine content (ng / mg) of collagen contained in the teeth of a mammal (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) as an index for assessing the potential disease risk of a mammal and / or the potential health of a mammal. In this specification, a numerical range expressed using "to" or "from" means a range that includes the numerical values ​​written before and after "to" as the lower and upper limits.

[0027] The collagen-rich organic composition according to the present invention is a collagen-rich organic composition produced from teeth or bones extracted from a mammal, and is a collagen-rich organic composition that satisfies the following (i) or (i) and (ii): (i) PYD, which is a quantitative value of the amount of pyridinoline (ng / mg) contained in collagen contained in the collagen-rich organic composition, as determined by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pair reagents, is PYD>100; (ii) The PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)) value, which is the weight ratio of PYD, which is the quantitative value of the amount of pyridinoline contained (ng / mg) in the collagen contained in the collagen-rich organic composition, to PEN, which is the quantitative value of the amount of pentosidine contained (ng / mg), as determined by high-performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pair reagents, is PpP>740.

[0028] In the present invention, the term "mammal" refers to an animal that basically reproduces sexually, with many extant species being viviparous and raising its young by milk. The term is not particularly limited as long as it is such an animal. Examples of such animals include, but are not limited to, humans; apes such as orangutans, gorillas, chimpanzees, bonobos, and monkeys belonging to the Gibbon family; monkeys other than those belonging to the Gibbon family; large mammals such as cows, horses, pigs, wild boars, sheep, goats, serows, bears, sea lions, seals, walruses, sea lions, fur seals, and whales; and small mammals such as dogs, cats, rabbits, mice, squirrels, weasels, raccoons, and mongooses. Preferred mammals include cows, pigs, horses, sheep, deer, dogs, cats, and humans; more preferred mammals include cows, pigs, and horses; and most preferred mammals include cows and pigs.

[0029] "Collagen" is one of the proteins that form the framework of various tissues in living organisms, mainly constituting various organs, blood vessels, nerves, skin, ligaments, tendons, bones, cartilage, and tooth dentin of vertebrates, and is the main component of the extracellular matrix of multicellular animals. In the present invention, "collagen" mainly refers to type I collagen, and in this specification, organic compositions with a high collagen content may be simply referred to as "collagen." Furthermore, the "collagen" of the present invention is collagen in teeth or bones, and since teeth and bones are tissues that are subjected to strong loads such as body weight and occlusal force, tooth or bone collagen is required to have high strength enough to support the tissues.

[0030] Hard tissue collagen can be determined to be derived from hard tissues by detecting, for example, BMP-2, BMP-4, BMP-7, and osteocalcin, and tooth collagen can be determined to be derived from dentin by detecting dentin sialoprotein (DSP), dentin glycoprotein (DGP), and dentin phosphoprotein (DPP).

[0031] Generally, a "collagen-rich organic composition" refers to an organic composition containing a high proportion of collagen in the overall organic composition, and atelocollagen, demineralized freeze-dried bone (DFDBA), and demineralized dentin matrix (DDM) are examples of "collagen-rich organic compositions." In the present invention, a "collagen-rich organic composition" refers to a commonly used collagen-rich organic composition in which the collagen content of the overall organic composition is at least 60%, preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, and most preferably 95% or more. In the present invention, examples of organic components other than collagen contained in the collagen-rich organic composition include the above-mentioned DSP, DGP, and DPP.

[0032] In general, the term "biomaterial" refers to a material used in the body or in contact with biological components such as proteins and cells. A "collagen-based biomaterial" refers to a biomaterial in which collagen is the primary component. Decalcified freeze-dried bone (DFDBA) and decalcified dentin matrix (DDM) are examples of "collagen-based biomaterials." In the present invention, a "collagen-based biomaterial" refers to a biomaterial in which collagen accounts for at least 40% of the total biomaterial, preferably 45% or more, more preferably 50% or more, even more preferably 55% or more, even more preferably 60% or more, and most preferably 65% ​​or more. Examples of organic components other than collagen contained in the biomaterial include the aforementioned DSP, DGP, and DPP.

[0033] In the present invention, the pyridinoline and pentosidine contained in the collagen contained in the collagen-rich organic composition are pyridinoline, which constitutes a pyridinoline cross-linking molecule, a physiological cross-linking molecule in collagen, and pentosidine, which constitutes a pentosidine cross-linking molecule, a non-physiological cross-linking molecule in collagen. In the present invention, the amount of pyridinoline (ng / mg) contained in the collagen contained in the collagen-rich organic composition and the amount of pentosidine (ng / mg) contained in the collagen contained in the collagen-rich organic composition are each quantified, and the weight ratio of PYD, which is the quantified value of the pyridinoline amount (ng / mg), to PEN, which is the quantified value of the pentosidine amount (ng / mg), is calculated (the amount of pyridinoline (ng / mg) / the amount of pentosidine (ng / mg)).

[0034] When the PYD or PYD and PpP of the collagen-rich organic composition according to the present invention are within the above-mentioned predetermined values, the collagen contained therein has excellent elasticity and strength, and can be said to be of good quality. The reasons for this will be explained in detail below.

[0035] It is no exaggeration to say that pyridinoline crosslinks, a physiological enzymatic crosslinking molecule, were primarily designed to improve the fundamental strength of hard tissues. As mentioned above, they are crosslinking molecules formed by the action of the enzyme lysyl oxidase between two lysine residues in the telopeptides located at the N- and C-termini of the collagen amino acid sequence, and one lysine residue in the collagen amino acid sequence excluding both termini. Because pyridinoline crosslinks formed between two lysine residues in the telopeptides located at the N- and C-termini of the collagen amino acid sequence act as crosslinking molecules between collagen fibers, they can be said to contribute significantly to maintaining the three-dimensional structure of tropocollagen (the entire collagen sequence), which has a triple helix structure.

[0036] Therefore, when the pyridinoline cross-linking molecules increase in collagen contained in biological tissues, the quality of the collagen contained in the biological tissues becomes flexible and tenacious, the strength and calcification of the collagen increase, and it can also be a factor that improves the activity of the cells that make up the biological tissues, i.e., it can be a factor that makes it easier to maintain individual homeostasis by increasing the activity of tissue stem cells involved in tissue repair. Therefore, the amount of pyridinoline contained is higher in collagen derived from teeth and bones than in collagen from soft tissues such as skin, and collagen derived from teeth and bones is flexible, strong, and highly elastic.

[0037] The collagen contained in a collagen-rich organic composition derived from such pyridinoline crosslinking molecules, i.e., the collagen contained in teeth or bones extracted from mammals that serve as the raw material for the collagen-rich organic composition, is quantitatively measured as the amount of pyridinoline (ng / mg).The higher the PYD value, the better the quality of the collagen contained in the teeth or bones extracted from mammals, and a collagen-rich organic composition produced from teeth or bones extracted from such mammals can be said to be an excellent organic composition.

[0038] As shown in Figure 1, the PYDs, which are the quantitative values ​​of the pyridinoline content (ng / mg) of collagen contained in the dentin-derived collagen-rich organic composition of teeth (62 samples in total), which are examples of hard tissues, were obtained from 18 human mammals, 36 livestock cattle (including 19 bovine SRMs (Hayakita Plant, Hokkaido Livestock Corporation), 17 Tokachi Young Cattle (registered trademark in Japan, early fattened (14-month-old) male Holsteins; Tokachi Shimizu Town Agricultural Cooperative Association), and 8 domestic pigs (6-month-old; Hayakita Plant, Hokkaido Livestock Corporation). All of these PYDs are within the scope of the PYDs of the present invention. Here, "SRM" refers to specified risk materials (SRMs). In Japan, the tonsils and distal ileum (part of the small intestine) of all ages, and the head (excluding tongue and cheek meat), vertebral column, and spinal cord of animals over 30 months of age are designated as specified risk materials (Food Safety Commission, Cabinet Office).

[0039] On the other hand, as shown in Figure 2, the PYD, which is the quantitative value of the pyridinoline content (ng / mg), of soft tissue-derived Teruplag (n=3, bovine dermis-derived atelocollagen, telopeptide-free processing; Olympus Terumo Biomaterials Co., Ltd.), high-grade gelatin (n=3, porcine acid processing, telopeptide-preserving processing method, low-endotoxin collagen; Nippi Co., Ltd.), gingiva of Tokachi Young Beef (registered trademark of Japan) (n=3; Tokachi Shimizu Town Agricultural Cooperative), and muscle of Tokachi Young Beef (registered trademark of Japan) (n=3; Tokachi Shimizu Town Agricultural Cooperative) was all below 50.

[0040] From the above, the PYD value according to the present invention can be determined as PYD>100, which is the lower limit of the PYD of collagen contained in the collagen-rich organic composition derived from tooth dentin, which is hard tissue, of 18 human mammals, 36 cattle, and 8 domestic pigs, which are mammalian livestock.

[0041] In other words, in the present invention, when the quantitative value PYD of the pyridinoline content (ng / mg) of the collagen contained in the collagen contained in the collagen-rich organic composition, i.e., the collagen contained in the tooth or bone extracted from a mammal, which is the raw material for the collagen-rich organic composition, is PYD>100, it can be inferred that the collagen contained in the collagen-rich organic composition is not collagen derived from biological tissues of a mammal other than the tooth or bone.

[0042] On the other hand, crosslinks by advanced glycation end products (AGEs), typified by the non-physiological crosslinking molecule pentosidine, are pathological crosslinks (aging crosslinks (AGE crosslinks)) caused by blood glucose. As mentioned above, these crosslinks are formed at arginine and lysine residues scattered throughout the collagen amino acid sequence when blood glucose is heated by body temperature. When arginine and lysine residues are close to each other in the collagen amino acid sequence, pentosidine crosslinks are formed nonspecifically (randomly), which can lead to entanglement of collagen fibers, causing a loss of collagen fiber regularity and disrupting the three-dimensional structure of tropocollagen. Therefore, an increase in pentosidine crosslinks in the collagen amino acid sequence leads to a decrease in the flexibility, strength, and elasticity of collagen fibers. Because the formation of these pathological crosslinks is caused by blood glucose, their accumulation is thought to increase depending on the host's dietary habits (carbohydrate intake). Furthermore, because carbohydrates, the raw material for pentosidine cross-linking molecules, are supplied via the bloodstream, collagen, which is the support base for tissues throughout the body, is cross-linked evenly and uniformly, regardless of the location of the tissue. In other words, excessive sugar intake can be said to uniformly reduce the quality of collagen, which is the support base for the body, regardless of whether it is hard or soft tissue.

[0043] Furthermore, when pentosidine crosslinks increase in collagen, the collagen in the tissue becomes brittle and chalky, due to the fact that these crosslinks are glycation crosslinks formed by the Maillard reaction. Pentosidine crosslinks are also AGEs, and the collagen in the tissue exhibits a reduced strength and mineralization. The late products of the Maillard reaction are called advanced glycation end products (AGEs). Collagen from mammals with a high carbohydrate intake and consistently high blood glucose levels contains more pentosidine crosslinks than that from mammals of the same age (months) with a well-balanced diet. Therefore, aging crosslinks are more prevalent in collagen from mammals with consistently high blood glucose levels. The resulting pentosidine crosslinks disrupt the regular arrangement of collagen fibers, reducing collagen flexibility and making it stiff and brittle. They also inhibit collagen metabolism, resulting in a deterioration of the collagen quality throughout the body and a loss of tissue flexibility. Collagen is the main component of the supporting tissues that support not only all hard tissues such as bones and teeth, but also all soft tissues, including nerves and blood vessels. Therefore, a decline in collagen quality can lead to a deterioration in the quality of hard and soft tissues throughout the body. For example, when bone quality declines due to bone glycation, bones become brittle and osteoporosis occurs. In addition, when blood vessel quality declines due to glycation of blood vessel walls, arteries harden, leading to arteriosclerosis, which causes tissue damage such as elevated blood pressure. The same is true for the three major complications of diabetes: (1) diabetic retinopathy (which can worsen and lead to blindness), (2) diabetic nephropathy (which can worsen and lead to dialysis), and (3) diabetic neuropathy (which can worsen and lead to, for example, necrosis of the fingers and toes due to impaired circulation).The causes of these complications are said to be (1) impaired circulation in the retina, (2) capillary damage in the glomeruli of the kidneys, which are the blood filtering system, and (3) impaired blood flow in the extremities due to capillary neuropathy and arteriosclerosis.It can be said that the deterioration of collagen quality due to glycation is a major factor in all of these.

[0044] In addition, an increase in advanced glycation end products (AGEs), such as pentosidine crosslinks, facilitates binding of AGEs to RAGE, an AGE receptor present in cells composing tissues throughout the body, creating an in vivo environment favorable for RAGE signaling activation. This promotes inflammation-related NF-κB signaling, and chronic inflammation-inducing AGEs, such as pentosidine crosslinks, act as chronic inflammation triggers in overly glycated tissues. This mechanism is thought to exacerbate chronic inflammatory diseases, such as inflammatory bowel disease (IBD) and connective tissue diseases. As described above, collagen glycation gradually damages the body in various ways, unconsciously reducing quality of life (QOL) and potentially becoming a major factor in preventing healthy longevity. It has been reported that both pyridinoline and pentosidine crosslinks in collagen contained in human biological tissues increase with age (Shimizu, "Aging-related non-enzymatic glycation modification of dentin collagen protein", Osaka University Knowledge Archive. 2015; Walters C. et al., Calcif. Tissue. Int. Vol. 35, 401-405, 1983).

[0045] The collagen contained in a collagen-rich organic composition derived from such pentosidine crosslinking molecules, i.e., the quantitative value of the pentosidine content (ng / mg) of collagen contained in teeth or bones extracted from mammals, which are the raw material for the collagen-rich organic composition, is PEN. The smaller the value, the better the quality of the collagen contained in the teeth or bones extracted from mammals, and a collagen-rich organic composition produced from teeth or bones extracted from such mammals can be said to be an excellent organic composition.

[0046] On the other hand, in the case of humans, there is a strong tendency for them to eat a variety of foods as they grow, and therefore, compared to healthy livestock animals whose diets are managed to maintain a consistent quality throughout their lives, there is a clear tendency for pentosidine crosslinking molecules to accumulate more, and as a result, the quality of collagen tends to be inferior.This tendency is also reflected in Figure 3, so humans should not be used as a standard indicator for the amount of pentosidine contained (ng / mg), but rather healthy livestock animals living with standard sugar intake habits should be used as an indicator.

[0047] Therefore, as shown in Figure 3, the PEN, which is the quantitative value of the pentosidine content (ng / mg) of collagen contained in the collagen-rich organic composition derived from tooth dentin, which is an example of hard tissue, of 44 livestock animals (19 examples of bovine SRM (Hayakita Plant, Hokkaido Livestock Corporation), 17 examples of Tokachi young cattle (registered trademark of Japan; Tokachi Shimizu Town Agricultural Cooperative), and 8 examples of domestic pigs (Hayakita Plant, Hokkaido Livestock Corporation)), was 0.015 to 0.015, the upper limit of the 44 livestock animals. <PEN<0.4とすることができる。

[0048] In the present invention, the term "livestock animals" refers to mammals whose feed is managed to have a certain quality, and suitable livestock animals include cattle, pigs, horses, sheep, goats and deer.

[0049] Furthermore, in the present invention, the weight ratio PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)) between PYD, which is the quantitative value of the amount of pyridinoline (ng / mg) contained in the collagen contained in the collagen-rich organic composition, derived from pyridinoline crosslink molecules, i.e., the amount of pyridinoline (ng / mg) contained in the tooth or bone extracted from a mammal, which is the raw material for the collagen-rich organic composition, and PEN, which is the quantitative value of the amount of pentosidine (ng / mg) contained in the collagen contained in the collagen-rich organic composition, derived from pentosidine crosslink molecules, is a predetermined value. This means that, based on the properties of the pyridinoline crosslinks and pentosidine crosslinks described above, the collagen contained in the collagen-rich organic composition or the biomaterial containing collagen as the main component is of high quality, being flexible, strong, and highly elastic, while maintaining the regular arrangement and three-dimensional structure of collagen fibers. However, in this case, it is assumed that the PYD is a predetermined value, i.e., that the collagen contained in the collagen-rich organic composition or biomaterial containing collagen as its main component is not collagen derived from biological tissues other than teeth or bones.

[0050] In the present invention, the amount of pyridinoline (ng / mg) and the amount of pentosidine (ng / mg) contained in the collagen contained in the collagen-rich organic composition, i.e., the collagen contained in the tooth or bone extracted from a mammal, which is the raw material for the collagen-rich organic composition, can be quantified using a high-performance liquid chromatograph with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pair reagents. Furthermore, in the collagen-rich organic composition according to the present invention, PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)) is the weight ratio of PYD, which is a quantitative value of the amount of pyridinoline contained (ng / mg), to PEN, which is a quantitative value of the amount of pentosidine contained (ng / mg), measured by high-performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pair reagents. Since livestock animals are used as an indicator for PEN, as shown in Figure 4, PpP can be determined to be PpP>740, based on the lower limit of 44 livestock animals.

[0051] The preparation of the sample to be used for quantifying the amount of pyridinoline (ng / mg) and the amount of pentosidine (ng / mg) contained can be performed using conventional preparation means known in the art, but can generally be performed, for example, as follows.

[0052] (1) The dried collagen powder produced by the method for producing an organic composition with a high collagen content according to the present invention is weighed into a test tube and swelled in a small amount of 6N hydrochloric acid under degassing. (2) 6N hydrochloric acid is added to the dried collagen, the mixture is degassed, and then the mixture is sealed. (3) Hydrolysis is carried out by heating at 110°C for 24 hours. (4) Cool the sample to room temperature. (5) Excess hydrochloric acid is removed by drying under reduced pressure while heating to obtain a collagen hydrolysate. (6) The obtained collagen hydrolysate is solubilized in a 10% aqueous methanol solution. (7) Centrifuge at 12,000 rpm for 5 minutes at room temperature, and use the supernatant as a sample. (8) By adding a known amount of a standard substance to the sample, the extraction rate and calibration curve can be determined, and the collagen content can be corrected appropriately.

[0053] The amount of pyridinoline (ng / mg) and the amount of pentosidine (ng / mg) contained can also be quantified by a high performance liquid chromatograph equipped with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pairing reagents using conventional quantitative means known in the art, but can generally be performed, for example, as follows.

[0054] (1) The mobile phases are ultrapure water containing 0.05% heptafluorobutyric acid (mobile phase A) and a 1:1 mixture of methanol and ethanol containing 0.05% formic acid (mobile phase B), and are delivered in a gradient configuration for detection. (2) Specific pumping conditions are: detection column temperature 40°C, flow rate 0.5 mL / min, starting with a ratio of 90% mobile phase A and 10% mobile phase B, and pumping for 0.5 minutes. (3) After that, the mobile phase B is increased to 22% over 1 minute to create a concentration gradient, and then the solution is pumped for 7.5 minutes. (4) Next, the mobile phase B is increased to 80% over 1 minute, and then pumped for another 1 minute. (5) Finally, after pumping at a ratio of 10% mobile phase A and 90% mobile phase B, equilibrate at a ratio of 90% mobile phase A and 10% mobile phase B for 4 minutes. (6) Steps (1) to (5) are performed for a total of 15 minutes per sample. The separation column is a Cadenza CD-C18, 150 mm long and 3 mm in inner diameter, and a fluorescence detector is used for detection. Monitoring is performed at an excitation wavelength of 295 nm and an emission wavelength of 395 nm for the first 8 minutes, and at an excitation wavelength of 325 nm and an emission wavelength of 385 nm for the last 7 minutes. By these monitoring steps, pyridinoline and pentosidine are detected at a retention time of approximately 6.5 minutes and 9.1 minutes, respectively.

[0055] Next, the collagen-rich organic composition of the present invention is produced by subjecting it to a strong alkali treatment, which is not normally performed on proteins including collagen, at a temperature higher than room temperature, followed by a decalcification treatment, i.e., an acid treatment. As a result, as shown in FIG. 6, the amount of endotoxin contained therein is significantly reduced, or the activity of the endotoxin contained therein is significantly reduced, and the composition is essentially endotoxin-free, or does not contain endotoxin, or the contained endotoxin is substantially inactivated, or the endotoxin is inactivated.

[0056] A "reduced" amount or activity is typically a "statistically significant" amount or activity and can include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease in the amount or activity produced in the absence of the composition (in the absence of an agent or compound) or by a control composition, including all integers therebetween.

[0057] "Substantially free of endotoxin" generally refers to the presence of at most trace amounts of endotoxin (e.g., amounts that have no clinically adverse physiological effects on a subject), preferably undetectable amounts. Endotoxins are toxins associated with certain bacteria, typically gram-negative bacteria, but endotoxins can also be found in gram-positive bacteria such as Listeria monocytogenes. The most prominent endotoxins are lipopolysaccharides (LPS) or lipooligosaccharides (LOS), found in the outer membrane of various gram-negative bacteria, and are central pathogenic characteristics in the ability of these bacteria to cause disease. Small amounts of endotoxin in humans can cause fever, a drop in blood pressure, and activation of inflammation and blood coagulation, among other adverse physiological effects. Therefore, it is often desirable to remove most or all traces of endotoxin from the collagen-rich organic compositions and collagen-based biomaterials of the present invention, because even small amounts can cause adverse effects in humans.

[0058] Endotoxin can be detected using conventional techniques known in the art. For example, the Limulus Amebocyte Lysate Assay (LAL), which utilizes blood from horseshoe crabs, is a highly sensitive assay for detecting the presence of endotoxin. In this test, very low levels of lipopolysaccharide (LPS) can cause detectable coagulation of the Limulus lysate due to a powerful enzyme cascade that amplifies this reaction. Endotoxin can also be quantified by enzyme-linked immunosorbent assay (ELISA). To be substantially free of endotoxin, the endotoxin levels are about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.017, 0.018, 0.019, 0.02, 0.021, 0.022, 0.023, 0.024, 0.025, 0.026, 0.027, 0.028, 0.029, 0.030, 0.031, 0.032, 0.033, 0.034, 0.035, 0.036, 0.037, 0.038, 0.039, 0.040, 0.041, 0.042, 0.043, 0.044, 0.045, 0.046, 0.047, 0.048, 0.049, 0.050, 0.051, 0.052, 0.053, 0.054, 0.055, 0.056, 0.057, 0.058, 0.059, 0.060, 0.061, 0.062, 0.063, 0.064, 0.065, 0.066, 0.067, 0.068, 0.069, 0.070, 0.07 26, 0.027, 0.028, 0.029, 0.03, 0.031, 0.032, 0.033, 0.034, 0.035, 0.036, 0.037, 0.038, 0.039, 0.04, 0.041, 0.042, 0.043, 0.044, 0.045, 0.046, 0.047, 0.048, 0.049, 0.05, 0.051, 0.052, 0.053, 0.054, 0.055, 0.056, 0.05 7, 0.058, 0.059, 0.06, 0.061, 0.062, 0.063, 0.064, 0.065, 0.066, 0.067, 0.068, 0.069, 0.07, 0.071, 0.072, 0.073, 0.074, 0.075, 0.076, 0.077, 0.078, 0.079, 0.08, 0.081, 0.082, 0.083, 0.084, 0.085, 0.086, 0.087, 0.088 ,0.089,0.09,0.091,0.092,0.093,0.094,0.095,0.096,0.097,0.098,0.099,0.1,0.11,0.12,0.13,0.14,0.15,0.16,0.17,0.18,0.19,0.2,0.21,0.22,0.23,0.24,0.25,0.26,0.27,0.28,0.29,0.3,0.31,0.32,0.33,0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69 9, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 1.10, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.20, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29 ... 6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6 0.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10 EU / mg protein or less.

[0059] On the other hand, "substantially inactivated" means that the toxicity of the endotoxin is reduced as measured by any standard assay, and means that it can be determined that the endotoxin is at least 95% inactivated, preferably 98% inactivated, and more preferably 100% inactivated.

[0060] The biomaterials made from the collagen-rich organic composition according to the present invention as a raw material, the biomaterials having collagen as a main component, or other products are not particularly limited as long as they are made from the collagen-rich organic composition according to the present invention as a raw material, the biomaterials having collagen as a main component, or other products.

[0061] In order to directly identify the collagen-rich organic composition according to the present invention based on its structure and / or properties, it is necessary to prepare or produce a large number of collagen-rich organic compositions according to the present invention and attempt to identify them based on their structure or properties using various methods. However, such tests require even a competent person skilled in the art to conduct many tests, which requires a great deal of time and involves a large economic expenditure (see the Patent and Utility Model Examination Handbook, Part II, Chapter 2, Requirements for the Description of the Claims, "2205 Determination of 'Impossible or Impractical Circumstances' in the Examination When a Claim for an Invention of a Product Represents a Manufacturing Method for That Product," page 16, bottom line to page 17, lines 1-4). In other words, in order to directly identify the collagen-rich organic composition according to the present invention by its structure or properties, a person skilled in the art would need time and expense that would not be economically justifiable at the time of filing the application. Requiring such identification work would be unprofitable for applicants who are seeking the earliest possible application date under the first-to-file principle, given the rapid advances in technology and fierce international competition for patent acquisition (Supreme Court, 2012 (Judgment) No. 1204, p. 10, or Supreme Court, 2012 (Judgment) No. 2658, p. 10-11, etc.).

[0062] Next, a method for producing a collagen-rich organic composition according to the present invention will be described. Regarding the method for producing a collagen-rich organic composition according to the present invention, any components that are the same as or equivalent to those of the collagen-rich organic composition produced from teeth or bones extracted from mammals according to the present invention, biomaterials made from the collagen-rich organic composition as a raw material, biomaterials containing collagen as a main component, and other products will not be described again.

[0063] The method for producing a collagen-rich organic composition according to the present invention comprises the steps of: (i) a step of crushing a tooth or bone extracted from a mammal to obtain a crushed material (crushed material preparation step); (ii) a step of treating the pulverized product obtained in the pulverized product preparation step (i) with a strong alkali (strong alkali treatment step); (iii) a step of decalcifying the strongly alkali-treated pulverized material obtained in the strong alkali treatment step (ii) under negative pressure conditions (negative pressure decalcification step); The method comprises the steps (i) to (iii) above.

[0064] In the method for producing a collagen-rich organic composition according to the present invention, the pulverized product preparation step (i) is a step in which teeth or bones extracted from a mammal are pulverized using a conventional pulverizing means known in the art, such as a pulverizer, to obtain a pulverized product. In the present invention, the particle size of the pulverized product is preferably 0.05 to 3 mm, more preferably 0.1 to 2 mm.

[0065] In the method for producing a collagen-rich organic composition according to the present invention, the strong alkali treatment step (ii) is a step of stirring the pulverized material obtained in the pulverized material preparation step (i) in a strongly basic aqueous solution. As the strongly basic aqueous solution, a conventional strongly basic aqueous solution known in the art can be used, such as an aqueous sodium hydroxide solution, an aqueous potassium hydroxide solution, an aqueous calcium hydroxide solution, or an aqueous sodium carbonate solution.

[0066] The strong alkali treatment step (ii) may be a step of treating the pulverized material obtained in the pulverized material preparation step (i) with a strong alkali at a temperature higher than room temperature and lower than the boiling point of water. Specifically, the strong alkali treatment step (ii) in the present invention is preferably performed at a temperature of 50 to 90°C, more preferably at a temperature of 60 to 80°C, even more preferably at a temperature of 65 to 75°C, and most preferably at a temperature of 70°C.

[0067] In the method for producing a collagen-rich organic composition according to the present invention, the demineralization step under negative pressure (iii) is a step of demineralizing the strong alkali-treated pulverized material obtained in the strong alkali treatment step (ii) under negative pressure conditions, specifically, a step including a step of demineralizing while reducing pressure. By performing demineralization while reducing pressure, gases (mainly CO2) generated during the treatment and air trapped inside can be removed, allowing the demineralizing solution to quickly penetrate the pulverized material, resulting in a shorter demineralization time. This significant chemical effect allows demineralization to be completed quickly without raising the temperature, thereby indirectly protecting substances that are relatively sensitive to acid. The demineralization step under negative pressure (iii) of the present invention may include a step of demineralizing while reducing pressure, and may, for example, combine a step of demineralizing under normal pressure with a step of demineralizing while reducing pressure. Furthermore, decalcification can be carried out through one to several steps, including conventional decalcification steps known in the art. For example, the decalcification solution used may be phosphoric acid, hydrochloric acid, nitric acid, sulfuric acid, formic acid, ethylenediaminetetraacetic acid (EDTA), or a mixture of ethanol and hydrochloric acid, or an aqueous solution of ethanol and EDTA, which may be used alone or in combination.

[0068] The method for producing a collagen-rich organic composition according to the present invention may include not only the pulverized product preparation step (i), the strong alkali treatment step (ii), and the demineralization step under negative pressure (iii) described above, but also other steps within the scope of the present invention, such as a further pulverization step, drying step, vacuum drying step, washing step, heating step, cooling step, neutralization step, mixing step, and elution step.

[0069] Next, a first embodiment of a method for distinguishing teeth or bones extracted from mammals as raw materials for biomaterials, biomaterials mainly composed of collagen, or other products according to the present invention will be described. Note that, within the method of this first embodiment, components that are the same as or correspond to those of the collagen-rich organic composition produced from teeth or bones extracted from mammals according to the present invention, the biomaterials using the collagen-rich organic composition as a raw material, the collagen-based biomaterials and other products, and the method for producing the collagen-rich organic composition according to the present invention will be designated by the same reference numerals, and repeated explanations will be omitted.

[0070] The method of the first embodiment includes: (iv) quantifying the amount of pyridinoline (ng / mg) contained in collagen contained in a tooth or bone extracted from a mammal (quantification step of pyridinoline content); (v) quantifying the amount of pentosidine (ng / mg) contained in collagen contained in the tooth or bone extracted from the mammal (quantification step of the amount of pentosidine contained); (vi) A step of calculating the weight ratio PpP (amount of pyridinoline (ng / mg) / amount of pentosidine (ng / mg)) from PYD, which is the value of the amount of pyridinoline (ng / mg) quantified in the step (iv) of quantifying the amount of pyridinoline contained, and PEN, which is the value of the amount of pentosidine (ng / mg) quantified in the step (v) of quantifying the amount of pentosidine contained (ng / mg)), (vii) a step of selecting the tooth or bone extracted from the mammal as a biomaterial, a collagen-based biomaterial or a raw material for other products when PYD, which is the value of the amount of pyridinoline (ng / mg) quantified in the step (iv) of quantifying the amount of pyridinoline contained, or PYD, which is the value of the amount of pyridinoline (ng / mg) quantified in the step (iv) of quantifying the amount of pyridinoline contained, or PpP, which is the weight ratio value calculated in the step (vi) of calculating the weight ratio (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)), are predetermined values ​​(tooth / bone selection step); The method comprises the steps (iv) to (vii).

[0071] In the step (iv) of quantifying the amount of pyridinoline contained and the step (v) of quantifying the amount of pentosidine contained in the method of the first embodiment, the method for quantifying the amount of pyridinoline contained (ng / mg) and the amount of pentosidine contained (ng / mg) in collagen contained in teeth or bones extracted from mammals is not particularly limited as long as it is possible to quantify them. As such a quantification method, for example, a high-performance liquid chromatograph with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pair reagents can be used.

[0072] In the weight ratio calculation step (vi) of the method of the first embodiment, PYD, which is the quantitative value of the amount of pyridinoline (ng / mg) contained in collagen contained in the tooth or bone extracted from the mammal, as quantified in the pyridinoline amount quantification step (iv), is divided by PEN, which is the quantitative value of the pentosidine amount (ng / mg) contained in collagen contained in the tooth or bone extracted from the mammal, as quantified in the pentosidine amount quantification step (v), to calculate PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)), which is the weight ratio value of the quantitative value of the amount of pyridinoline contained (ng / mg) and the quantitative value of the amount of pentosidine contained (ng / mg).

[0073] In the tooth / bone selection step (vii) of the method of this first embodiment, if the value of PYD, which is the value of the amount of pyridinoline (ng / mg) quantified in the pyridinoline amount quantification step (iv), or the value of PYD, which is the value of the amount of pyridinoline (ng / mg) quantified in the pyridinoline amount quantification step (iv), and the value of PpP (amount of pyridinoline (ng / mg) / amount of pentosidine (ng / mg)) calculated in the weight ratio calculation step (vi), are predetermined values, the tooth or bone extracted from the mammal is selected as a biomaterial, a biomaterial whose main component is collagen, or a raw material for other products.

[0074] The "predetermined value" in the tooth / bone selection step (vii) is defined by a method for quantifying the amount of pyridinoline (ng / mg) and the amount of pentosidine (ng / mg) contained in collagen contained in teeth or bones extracted from mammals. For example, when the pyridinoline amount quantification step (iv) and the pentosidine amount quantification step (v) are quantification steps using high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pair reagents, the predetermined values ​​are PYD>100 or PYD>100 and PpP>740, respectively.

[0075] As with the method for producing an organic composition with a high collagen content according to the present invention, the method of the first embodiment may include not only the above-mentioned step (iv) of quantifying the amount of contained pyridinoline, step (v) of quantifying the amount of contained pentosidine, step (vi) of calculating the weight ratio, and step (vii) of selecting teeth and bones, but also other steps within the scope that do not impair the characteristics of the present invention.

[0076] Next, a second embodiment of the method for identifying teeth or bones extracted from mammals as raw materials for biomaterials, collagen-based biomaterials, or other products according to the present invention will be described. (i) a step of crushing a tooth or bone extracted from a mammal to obtain a crushed material (crushed material preparation step); (ii) a step of treating the pulverized product obtained in the pulverized product preparation step (i) with a strong alkali (strong alkali treatment step); (iii) a step of demineralizing the strongly alkali-treated pulverized material obtained in the strong alkali treatment step (ii) under negative pressure conditions to obtain a collagen-rich organic composition (negative pressure demineralization step); (iv) a step of quantifying the amount of pyridinoline (ng / mg) contained in the collagen contained in the collagen-rich organic composition obtained in the decalcification step under negative pressure (a step of quantifying the amount of pyridinoline contained); (v) a step of quantifying the amount of pentosidine (ng / mg) contained in the collagen contained in the collagen-rich organic composition obtained in the decalcification step under negative pressure (pentosidine amount quantification step); (vi) A step of calculating the weight ratio PpP (amount of pyridinoline (ng / mg) / amount of pentosidine (ng / mg)) from PYD, which is the value of the amount of pyridinoline (ng / mg) quantified in the step (iv) of quantifying the amount of pyridinoline contained, and PEN, which is the value of the amount of pentosidine (ng / mg) quantified in the step (v) of quantifying the amount of pentosidine contained (ng / mg)), (vii) a step of selecting the tooth or bone extracted from the mammal as a biomaterial, a collagen-based biomaterial or a raw material for other products when PYD, which is the value of the amount of pyridinoline (ng / mg) quantified in the step (iv) of quantifying the amount of pyridinoline contained, or PYD, which is the value of the amount of pyridinoline (ng / mg) quantified in the step (iv) of quantifying the amount of pyridinoline contained, or PpP, which is the weight ratio value calculated in the step (vi) of calculating the weight ratio (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)), are predetermined values ​​(tooth / bone selection step); The method of the second embodiment comprises the steps (i) to (vii) above. Among the components of the method of the second embodiment, those that are the same as or correspond to those of the first embodiment of the collagen-rich organic composition according to the present invention produced from teeth or bones extracted from mammals, the biomaterials made from the collagen-rich organic composition as a raw material, the biomaterials and other products mainly composed of collagen, the method for producing the collagen-rich organic composition according to the present invention, and the method for distinguishing teeth or bones extracted from mammals as raw materials for biomaterials, biomaterials mainly composed of collagen, or other products will be denoted by the same reference numerals, and will not be described again.

[0077] The constituent elements of the method of the second embodiment, namely, the pulverized material preparation step (i), the strong alkali treatment step (ii), and the decalcification step under negative pressure (iii), correspond to the constituent elements of the method of producing an organic composition with a high collagen content according to the present invention, namely, the pulverized material preparation step (i), the strong alkali treatment step (ii), and the decalcification step under negative pressure (iii). Furthermore, the constituent elements of the method of the second embodiment, namely, the quantification step (iv) of the amount of contained pyridinoline, the quantification step (v) of the amount of contained pentosidine, the weight ratio calculation step (vi), and the tooth / bone selection step (vii), correspond to the constituent elements of the first embodiment of the method of the present invention for distinguishing teeth or bones extracted from mammals as raw materials for biomaterials, biomaterials containing collagen as a main component, or other products.

[0078] In addition, like the first embodiment of the method for producing a collagen-rich organic composition according to the present invention and the method for distinguishing teeth or bones extracted from mammals as raw materials for biomaterials, biomaterials containing collagen as a main component, or other products according to the present invention, the method of this second embodiment may also include not only the above-mentioned pulverized material preparation step (i), strong alkali treatment step (ii), decalcification step under negative pressure (iii), quantification step of the amount of contained pyridinoline (iv), quantification step of the amount of contained pentosidine (v), weight ratio calculation step (vi), and tooth / bone selection step (vii), but also other steps within the scope that do not impair the characteristics of the present invention.

[0079] Next, a method of using the quantitative value of the amount of pyridinoline contained in collagen contained in the teeth of a mammal, or the quantitative value of the amount of pyridinoline contained in collagen contained in the teeth of a mammal, and the weight ratio (amount of pyridinoline contained / amount of pentosidine contained) calculated from the quantitative value of the amount of pyridinoline contained in collagen contained in the teeth of a mammal as an index for evaluating the potential risk of a disease in a mammal and / or whether the mammal is potentially healthy, according to the present invention, is as follows: (viii) quantifying the amount of pyridinoline (ng / mg) contained in collagen contained in the teeth of a mammal to be evaluated for potential disease risk and / or whether the mammal is potentially healthy, to obtain a quantitative value of PYD (quantification step of pyridinoline contained in the evaluation target); (ix) quantifying the amount of pentosidine (ng / mg) contained in collagen contained in the teeth of the mammal to be evaluated to obtain a quantitative value PEN (quantification step of the amount of pentosidine contained in the evaluation target); (x) a step of calculating the weight ratio PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)) from the PYD obtained in the step (viii) of quantifying the amount of pyridinoline contained in the evaluation target and the PEN obtained in the step (ix) of quantifying the amount of pentosidine contained in the evaluation target (evaluation target weight ratio calculation step); (xi) quantifying the amount of pyridinoline (ng / mg) contained in collagen contained in the teeth of one or more mammals selected from a mammal not affected by a disease, a mammal affected by a disease, a healthy mammal, and an unhealthy mammal, to obtain a reference quantitative value R-PYD (reference pyridinoline amount quantification step); (xii) quantifying the amount of pentosidine (ng / mg) contained in collagen contained in the teeth of one or more mammals selected from mammals not affected by a disease, mammals affected by a disease, healthy mammals, and unhealthy mammals, to obtain a reference quantitative value R-PEN (reference pentosidine amount quantification step); (xiii) A step of calculating the reference weight ratio R-PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)) from the R-PYD obtained in the reference pyridinoline amount determination step (xi) and the R-PEN obtained in the reference pentosidine amount determination step (xii) (reference weight ratio calculation step); (xiv) a step of comparing the PYD obtained in the step (viii) of quantifying the amount of pyridinoline contained in the evaluation subject with the R-PYD obtained in the step (xi) of quantifying the amount of pyridinoline contained in the reference, or a step of comparing the PYD obtained in the step (viii) of quantifying the amount of pyridinoline contained in the evaluation subject with the R-PYD obtained in the step (xi) of quantifying the amount of pyridinoline contained in the reference, as well as the PpP calculated in the step (x) of calculating the weight ratio of the evaluation subject with the R-PpP calculated in the step (xiii) of calculating the weight ratio of the reference (PYD-weight ratio comparison step); The process comprises the steps (viii) to (xiv).

[0080] In addition, among the methods of the present invention that use the quantitative value of the pyridinoline content of collagen contained in mammalian teeth, or the quantitative value of the pyridinoline content of collagen contained in mammalian teeth, and the weight ratio (pyridinoline content / pentosidine content) calculated from the quantitative value of the pyridinoline content of collagen contained in mammalian teeth as an index for evaluating the potential risk of disease in mammals and / or whether the mammal is potentially healthy, the same symbols are used to designate components that are the same as or equivalent to those of the first and second embodiments of the collagen-rich organic composition produced from teeth or bones extracted from mammals according to the present invention, the biomaterials made from the collagen-rich organic composition as a raw material, the biomaterials and other products containing collagen as a main component, the method for producing the collagen-rich organic composition according to the present invention, and the method for distinguishing teeth or bones extracted from mammals as raw materials for biomaterials, biomaterials and other products containing collagen as a main component, and the method for distinguishing teeth or bones extracted from mammals as raw materials for biomaterials, biomaterials and other products containing collagen as a main component, and the method for producing the collagen-rich organic composition according to the present invention ...

[0081] In the present invention, the term "potential risk of contracting a disease" does not refer to a state in which a person is infected with a disease, but rather refers to a state in which the host's defense response against the disease is at its maximum and the person has barely contracted the disease, and refers to a state in which the presence of the disease cannot be confirmed by subjective symptoms, objective findings, or conventional general test findings.

[0082] In addition, in the present invention, "potentially healthy" refers to a state that does not lead to an unhealthy state, and means a state in which it is hardly possible to confirm that one is unhealthy based on subjective, objective, or conventional general test findings. In the present invention, "potentially unhealthy" refers to a state that does not lead to a healthy state, and means a state in which it is hardly possible to confirm that one is healthy based on subjective, objective, or conventional general test findings.

[0083] In the method according to the present invention, which uses a quantitative value of the amount of pyridinoline contained in collagen contained in a mammal's teeth, or a quantitative value of the amount of pyridinoline contained in collagen contained in a mammal's teeth, and a weight ratio (amount of pyridinoline contained / amount of pentosidine contained) calculated from the quantitative value of the amount of pyridinoline contained in collagen contained in a mammal's teeth as an index for assessing the potential risk of a disease in a mammal and / or whether the mammal is potentially healthy, the step (viii) of quantifying the amount of pyridinoline contained in the evaluation target corresponds to the step (iv) of quantifying the amount of pyridinoline contained in the evaluation target in the first and second embodiments of the method according to the present invention for distinguishing teeth or bones extracted from mammals as raw materials for biomaterials, biomaterials containing collagen as a main component, or other products.

[0084] Furthermore, in the method according to the present invention, which uses the quantitative value of the amount of pyridinoline contained in collagen contained in the teeth of a mammal, or the quantitative value of the amount of pyridinoline contained in collagen contained in the teeth of a mammal, and the weight ratio (amount of pyridinoline contained / amount of pentosidine contained) calculated from the quantitative value of the amount of pyridinoline contained in collagen contained in the teeth of a mammal as an index for evaluating the potential risk of a disease in a mammal and / or whether the mammal is potentially healthy, the step (ix) of quantifying the amount of pentosidine contained in the evaluation target corresponds to the step (v) of quantifying the amount of pentosidine contained in the first and second embodiments of the method according to the present invention for distinguishing teeth or bones extracted from mammals as raw materials for biomaterials, biomaterials containing collagen as a main component, or other products.

[0085] Furthermore, in the method of the present invention, which uses the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, or the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, and the weight ratio (amount of pyridinoline contained / amount of pentosidine contained) calculated from the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth as an index for evaluating the potential risk of disease in a mammal and / or whether the mammal is potentially healthy, the evaluation target weight ratio calculation step (x) is a step corresponding to the weight ratio calculation step (vi) in the first and second embodiments of the method of the present invention for distinguishing teeth or bones extracted from mammals as raw materials for biomaterials, biomaterials containing collagen as a main component, or other products.

[0086] In addition, in the method according to the present invention, a quantitative value of the amount of pyridinoline contained in collagen contained in a mammal's tooth, or a quantitative value of the amount of pyridinoline contained in collagen contained in a mammal's tooth, and a weight ratio (amount of pyridinoline contained / amount of pentosidine contained) calculated from the quantitative value of the amount of pyridinoline contained in collagen contained in a mammal's tooth, are used as an index for assessing the potential risk of a disease in a mammal and / or whether the mammal is potentially healthy or not, the reference pyridinoline amount quantification step (xi) is carried out by using a collagen extracted from a mammal as a raw material for the biomaterial, collagen-based biomaterial or other products according to the present invention. This corresponds to the step (iv) of quantifying the amount of pyridinoline contained in the evaluation target tooth or bone in the first and second embodiments of the method for distinguishing between damaged teeth or bones, and the step (viii) of quantifying the amount of pyridinoline contained in the evaluation target in the method according to the present invention, which uses, as an index for assessing the potential disease risk in a mammal and / or whether the mammal is potentially healthy, the quantitative value of the amount of pyridinoline contained in collagen contained in the mammal's teeth, or the quantitative value of the amount of pyridinoline contained in collagen contained in the mammal's teeth, and the weight ratio (amount of pyridinoline contained / amount of pentosidine contained) calculated from the quantitative value of the amount of pyridinoline contained in collagen contained in the mammal's teeth and the quantitative value of the amount of pentosidine contained.

[0087] In addition, in the method according to the present invention, a quantitative value of the amount of pyridinoline contained in collagen contained in a mammal's tooth, or a quantitative value of the amount of pyridinoline contained in collagen contained in a mammal's tooth, and a weight ratio (amount of pyridinoline contained / amount of pentosidine contained) calculated from the quantitative value of the amount of pyridinoline contained in collagen contained in a mammal's tooth, are used as an index for assessing the potential risk of a disease in a mammal and / or whether the mammal is potentially healthy or not, the reference pentosidine amount quantification step (xii) is carried out by using a reference collagen extracted from a mammal as a raw material for the biomaterial, collagen-based biomaterial or other products according to the present invention. This corresponds to the step (v) of quantifying the amount of pentosidine contained in the evaluation target in the first and second embodiments of the method for distinguishing extracted teeth or bones, and the step (ix) of quantifying the amount of pentosidine contained in the evaluation target in the method according to the present invention, which uses, as an index for assessing the potential risk of disease in a mammal and / or whether the mammal is potentially healthy, the quantitative value of the amount of pyridinoline contained in collagen contained in the mammal's teeth, or the quantitative value of the amount of pyridinoline contained in collagen contained in the mammal's teeth, and the weight ratio (amount of pyridinoline contained / amount of pentosidine contained) calculated from the quantitative value of the amount of pyridinoline contained in collagen contained in the mammal's teeth and the quantitative value of the amount of pentosidine contained.

[0088] In addition, the "collagen contained in the teeth of one or more mammals selected from non-diseased mammals, diseased mammals, healthy mammals, and unhealthy mammals" in the reference-containing pentosidine amount quantification step (xii) may be a different "collagen contained in the teeth of one or more mammals selected from non-diseased mammals, diseased mammals, healthy mammals, and unhealthy mammals" from the reference-containing pyridinoline amount quantification step (xi), or may be the same "collagen contained in the teeth of one or more mammals selected from non-diseased mammals, diseased mammals, healthy mammals, and unhealthy mammals."

[0089] Furthermore, in any step, a "non-diseased mammal" includes a "self that is a mammal that is not diseased," a "diseased mammal" includes a "diseased mammal" and a "healthy mammal" includes a "healthy mammal" and an "unhealthy mammal" includes a "unhealthy mammal."

[0090] In addition, in the method according to the present invention, a quantitative value of the amount of pyridinoline contained in collagen contained in a mammal's tooth, or a quantitative value of the amount of pyridinoline contained in collagen contained in a mammal's tooth, and a weight ratio (amount of pyridinoline contained / amount of pentosidine contained) calculated from the quantitative value of the amount of pyridinoline contained in collagen contained in a mammal's tooth and the quantitative value of the amount of pentosidine contained therein, are used as an index for evaluating a potential risk of a disease in a mammal and / or whether the mammal is potentially healthy or not, the reference weight ratio calculation step (xiii) is carried out by using a mammalian tooth as a raw material for the biomaterial, collagen-based biomaterial or other products according to the present invention. This corresponds to the weight ratio calculation step (vi) in the first and second embodiments of the method for distinguishing teeth or bones extracted from objects, and the evaluation target weight ratio calculation step (x) in the method according to the present invention, which uses, as an index for assessing the potential risk of a disease in a mammal and / or whether the mammal is potentially healthy, the quantitative value of the amount of pyridinoline contained in collagen contained in a mammalian tooth, or the quantitative value of the amount of pyridinoline contained in collagen contained in a mammalian tooth, and the weight ratio (amount of pyridinoline contained / amount of pentosidine contained) calculated from the quantitative value of the amount of pyridinoline contained in collagen contained in a mammalian tooth and the quantitative value of the amount of pentosidine contained.

[0091] Furthermore, in the method according to the present invention, which uses a quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, or a quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, and a weight ratio (amount of pyridinoline contained / amount of pentosidine contained) calculated from the quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth and the quantitative value of the amount of pentosidine contained, as an index for evaluating a potential disease risk in a mammal and / or whether the mammal is potentially healthy, the PYD / weight ratio comparison step (xiv) is a step of comparing PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)), which is the weight ratio value calculated in the evaluation weight ratio calculation step (x), with R-PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)), which is the reference value for the weight ratio calculated in the reference weight ratio calculation step (xiii).

[0092] In the method according to the present invention, a quantitative value of the amount of pyridinoline contained in collagen contained in a mammal's teeth, or a quantitative value of the amount of pyridinoline contained in collagen contained in a mammal's teeth, and a weight ratio (amount of pyridinoline contained / amount of pentosidine contained) calculated from the quantitative value of the amount of pyridinoline contained in collagen contained in a mammal's teeth and the quantitative value of the amount of pentosidine contained, are used as an index for assessing the potential risk of a disease in a mammal and / or whether the mammal is potentially healthy. The step (viii) of quantifying the amount of pyridinoline contained in an evaluation target, the step (ix) of quantifying the amount of pentosidine contained in an evaluation target, the step (xi) of quantifying the amount of pyridinoline contained in a reference, and the step (xii) of quantifying the amount of pentosidine contained in a reference can be performed on extracted teeth of a mammal that is the subject of evaluation for potential disease risk assessment, a disease-free mammal, a diseased mammal, a healthy mammal, and an unhealthy mammal, and can also be performed in a manner such as cutting, contact, or non-contact.

[0093] In addition, a method of using the quantitative value of the pyridinoline content of collagen contained in mammalian teeth, or the quantitative value of the pyridinoline content of collagen contained in mammalian teeth, and a weight ratio (pyridinoline content / pentosidine content) calculated from the quantitative value of the pyridinoline content and the quantitative value of the pentosidine content of collagen contained in mammalian teeth as an index for evaluating the potential risk of disease in a mammal and / or whether the mammal is potentially healthy according to the present invention, can also be used in the method of producing a collagen-rich organic composition according to the present invention, and the biomaterial according to the present invention. As in the first and second embodiments of the method for distinguishing teeth or bones extracted from mammals as raw materials for biomaterials or other products whose main component is collagen, the method may include not only the above-mentioned step (viii) of quantifying the amount of pyridinoline contained in the evaluation target, step (ix) of quantifying the amount of pentosidine contained in the evaluation target, step (x) of calculating the weight ratio of the evaluation target, step (xi) of quantifying the amount of pyridinoline contained in the reference, step (xii) of quantifying the amount of pentosidine contained in the reference, step (xiii) of calculating the weight ratio of the reference, and step (xiv) of comparing the PYD-weight ratio, but also other steps within the scope that do not impair the characteristics of the present invention.

[0094] In the method of the present invention, the quantitative value of the pyridinoline content of collagen contained in mammalian teeth, or the quantitative value of the pyridinoline content of collagen contained in mammalian teeth, and the weight ratio (pyridinoline content / pentosidine content) calculated from the quantitative values ​​of the pyridinoline content and the pentosidine content of collagen contained in mammalian teeth are used as indicators for assessing the potential risk of disease in mammals and / or whether the mammal is potentially healthy. The reason for limiting this to "teeth" is that the pyridinoline crosslinks and pentosidine crosslinks in type I collagen are crosslinking reactions that are accelerated by factors such as internal heat due to body temperature and sugar supplied via the blood vessels, and are not localized but occur uniformly throughout the body. Among these, collagen contained in teeth (particularly dentin) is thought to most accurately reflect the accumulation of pyridinoline crosslink molecules and pentosidine crosslink molecules. Teeth are the only tissues in the body that are not part of the metabolic cycle, and once formed, they are not absorbed or broken down, so pyridinoline and pentosidine crosslinks accumulate in the collagen contained in teeth (especially dentin).

[0095] Furthermore, collagen in teeth (especially dentin) is more insoluble than that in bone. Pepsin digestion in 0.01 N hydrochloric acid at 4°C for 72 hours solubilizes approximately 35% of the collagen in adult bovine bone, but only 5.6% of the collagen in adult bovine dentin. Furthermore, at pH 2, insoluble collagen from skin and Achilles tendon swells to 4–8 times its volume, while insoluble collagen in adult bovine bone swells to 1.2 times its volume. However, insoluble collagen in adult bovine dentin does not swell at all (Nagai Yutaka and Fujimoto Daizaburo, eds., Collagen Experimental Methods, Kodansha Scientific, pp. 21–22). Furthermore, dentin collagen has the highest content of pyridinoline crosslinks in living organisms. Therefore, it is reasonable to use PYD, which is a quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, or PYD, which is a quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, and PpP (amount of pyridinoline contained / amount of pentosidine contained), which is a weight ratio calculated from PYD, which is a quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, and PEN, which is a quantitative value of the amount of pentosidine contained, as indicators to evaluate the potential risk of disease in mammals and / or whether the mammal is potentially healthy.

[0096] On the other hand, bone was excluded from the study. The levels of type I collagen C-terminal telopeptide (ICTP) in blood (serum) and NTx (type I collagen cross-linked N-telopeptide) in urine are widely used as indicators of osteoporosis. However, these substances are released into the blood and urine as collagen degradation products due to the activity of matrix metalloproteinases (MMPs), and contain pyridinoline cross-linking molecules. This suggests that the pyridinoline and pentosidine cross-linking molecules contained in bone-containing collagen are partially cleared by metabolism. Furthermore, when using excised bone, bone contains a large amount of lipids derived from yellow bone marrow, making defatting difficult and purifying highly pure collagen extremely difficult.

[0097] Furthermore, if the collagen contained in the teeth of a mammal contains a high amount of pyridinoline and a low amount of pentosidine, the mammal will have collagen that improves its own homeostasis and is less likely to induce chronic inflammation, and can be evaluated as having a low risk of developing various potential diseases and can be determined to be potentially healthy. On the other hand, if the collagen contained in a mammal's teeth contains a low amount of pyridinoline and a high amount of pentosidine, the mammal will have collagen that reduces its own homeostasis and is prone to causing chronic inflammation, and can be evaluated as being at high risk of developing various potential diseases and can be determined to be potentially unhealthy.Therefore, as indicators for evaluating a mammal's potential risk of disease and / or whether a mammal is potentially healthy, PYD, which is a quantitative value of the amount of pyridinoline contained in the collagen contained in the mammal's teeth, or PYD, which is a quantitative value of the amount of pyridinoline contained in the collagen contained in the mammal's teeth, and PpP (amount of pyridinoline contained / amount of pentosidine contained), which is a weight ratio calculated from PYD, which is a quantitative value of the amount of pyridinoline contained in the collagen contained in the mammal's teeth, and PEN, which is a quantitative value of the amount of pentosidine contained, can be used.The method of the present invention uses PYD, which is a quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, or PYD, which is a quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, and PpP (amount of pyridinoline contained / amount of pentosidine contained), which is a weight ratio calculated from PYD, which is a quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, and PEN, which is a quantitative value of the amount of pentosidine contained, as an index for evaluating the potential risk of disease in a mammal, and the method of the present invention uses PYD, which is a quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, or PYD, which is a quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, and PpP (amount of pyridinoline contained / amount of pentosidine contained), which is a weight ratio calculated from PYD, which is a quantitative value of the amount of pyridinoline contained in collagen contained in mammalian teeth, and PEN, which is a quantitative value of the amount of pentosidine contained, as an index for evaluating whether a mammal of the present invention is potentially healthy, may be independent or related to each other.

[0098] The following examples illustrate the collagen-rich organic composition produced from teeth or bones extracted from mammals, biomaterials made from the collagen-rich organic composition, collagen-based biomaterials and other products, a method for producing the collagen-rich organic composition of the present invention, a method for identifying teeth or bones extracted from mammals as biomaterials, collagen-based biomaterials, and other products, and a method for assessing the potential risk of disease in mammals and / or the potential health of mammals using a quantitative value of the pyridinoline content of collagen in mammalian teeth, or a quantitative value of the pyridinoline content of collagen in mammalian teeth, and a weight ratio (pyridinoline content / pentosidine content) calculated from the quantitative values ​​of the pyridinoline content and the pentosidine content of collagen in mammalian teeth. The technical scope of the present invention is not limited to the embodiments illustrated by these examples. [Example]

[0099] Example 1: Measurement of pyridinoline and pentosidine contents in collagen derived from human, bovine, and porcine teeth (mainly dentin) Using the following procedure, 18 human cases, 36 cattle cases (including 19 cattle SRM (Hokkaido Livestock Corporation, Hayakita Plant) and 17 Tokachi Young Cattle (a registered Japanese trademark, early fattening (14 months old) male Holstein; Tokachi Shimizu Town Agricultural Cooperative)) and domestic pigs (6 months old; Hokkaido Livestock Corporation) were tested. The pyridinoline and pentosidine levels were measured in collagen (n=62) derived from teeth (mainly dentin) of eight specimens (Hayakita Plant), soft tissue-derived Teruplag (n=3, bovine dermis-derived atelocollagen, telopeptide-free processing; Olympus Terumo Biomaterials Co., Ltd.), high-grade gelatin (n=3, porcine acid processing, telopeptide-preserving processing method, low-endotoxin collagen; Nippi Co., Ltd.), gingiva of Tokachi young cattle (registered trademark of Japan) (n=3; Tokachi Shimizu Town Agricultural Cooperative), and muscle of Tokachi young cattle (registered trademark of Japan) (n=3; Tokachi Shimizu Town Agricultural Cooperative).

[0100] 1. Preparation of collagen powder [1-1] The hard tissue was crushed into particles of approximately 1 mm in diameter using a crusher. [1-2] The specimen was stirred in a 1% aqueous solution of sodium carbonate at 70°C for several hours to remove any attached soft tissue. [1-3] Decalcification treatment was performed. Specifically, decalcification treatment was performed for several hours under reduced pressure using a decalcification solution containing 1M phosphoric acid and ethanol, and then the solution was replaced with the same decalcification solution diluted several times, and decalcification treatment was performed for several hours under reduced pressure. Next, the same decalcification solution diluted five times was used, and the tissue was shaken at room temperature for about a day and a night to be completely decalcified. After that, the tissue was washed several times with 5% ethanol at room temperature. [1-4] The decalcified hard tissue was transferred to a mortar and ground into a fine powder in 70% ethanol. [1-5] The sample was stirred with 100% ethanol, washed, degreased, and dehydrated. [1-6] The ethanol remaining in the collagen was completely removed by drying to obtain a dry collagen powder.

[0101] 2. Preparation of Samples for Analysis The dried collagen powder obtained in [2-1] and [1-6] was hydrolyzed. Specifically, the dried collagen powder obtained was weighed into a test tube and thoroughly swollen with a small amount of 6N hydrochloric acid under degassing. An appropriate amount of 6N hydrochloric acid was then added to the dried collagen, and the test tube was welded and sealed under degassing. The sealed test tube was heated overnight at 110°C to perform hydrolysis, after which it was cooled to room temperature, cut, and dried under reduced pressure while heating to remove excess hydrochloric acid, yielding a collagen hydrolysate. [2-2] The obtained collagen hydrolysate was solubilized in a 10% aqueous methanol solution. The mixture was centrifuged at 12,000 rpm at room temperature for 5 minutes, and the supernatant was used as a sample. [2-3] By adding known amounts of standard substances to the samples, extraction rates and calibration curves were determined, and collagen content was corrected appropriately.

[0102] 3. Quantitative determination of the amount of pyridinoline and pentosidine contained and calculation of their weight ratio by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pairing reagents. The mobile phases were ultrapure water containing 0.05% heptafluorobutyric acid (mobile phase A) and a 1:1 mixture of methanol and ethanol containing 0.05% formic acid (mobile phase B). The mobile phases were pumped in a gradient configuration. Specifically, the detection column temperature was 40°C and the flow rate was 0.5 mL / min. The starting ratio was 90% mobile phase A and 10% mobile phase B, and the pumping was continued for 0.5 minutes. The concentration of mobile phase B was then increased to 22% over 1 minute, creating a gradient, and the pumping continued for 7.5 minutes. The concentration of mobile phase B was then increased to 80% over 1 minute and pumped for another 1 minute. Finally, the pumping was continued at a ratio of 10% mobile phase A and 90% mobile phase B, followed by a reduction in the concentration of mobile phase B to 10% and equilibration for 4 minutes at 90% mobile phase A and 10% mobile phase B. The detection was then continued for a total of 15 minutes per sample. The separation column used was a Cadenza CD-C18, 150 mm long and 3 mm inner diameter, and a fluorescence detector was used for detection. Monitoring was performed for the first 8 minutes at an excitation wavelength of 295 nm and an emission wavelength of 395 nm, and for the last 7 minutes at an excitation wavelength of 325 nm and an emission wavelength of 385 nm. By this monitoring, pyridinoline was detected at a retention time of approximately 6.5 minutes, and pentosidine was detected at a retention time of approximately 9.1 minutes. The weight ratio (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) was calculated from the amounts of pyridinoline and pentosidine obtained.

[0103] Although measurements were taken on permanent teeth in humans, taking into account the timing of dentin formation in permanent teeth, it can be assumed that dentin formation begins in tooth germs within the jawbone on average around age 10, and that glycation cross-link formation also begins along with this (Study on the timing of eruption of deciduous and permanent teeth in Japanese children II, Journal of the Japanese Society of Pediatric Dentistry, 57(3), 363-373, 2019, 363). The same can be said for the mandibular teeth of bovine SRM, starting on average after 18 months of age. Therefore, the formula shown in Table 1 below was used to convert age to months for selected teeth in humans and bovine SRM.

[0104] [Table 1] TIFF0007731175000001.tif121165

[0105] Table 2 below shows the quantitative values ​​of pyridinoline content (ng / mg) contained in the collagen-rich organic composition derived from dentin of 18 human teeth, PYD, and PEN, respectively, which were determined by high-performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pairing reagents. The values ​​are also shown in Table 2. The values ​​are the quantitative values ​​of pyridinoline content (ng / mg), pentosidine content (ng / mg), and pentosidine content (ng / mg), respectively. The values ​​are also shown in Table 2. The values ​​are the quantitative values ​​of pyridinoline content (ng / mg) and pentosidine content (ng / mg), and the weight ratio calculated from PYD and PEN.

[0106] [Table 2] TIFF0007731175000002.tif55165

[0107] Table 3 below shows the PYD, which is the quantitative value of the amount of pyridinoline (ng / mg) contained in the collagen-rich organic composition derived from the dentin of 19 bovine SRMs, the PEN, which is the quantitative value of the amount of pentosidine (ng / mg) contained, and the PPP (amount of pyridinoline (ng / mg) / amount of pentosidine (ng / mg)) calculated from the PYD, which is the quantitative value of the amount of pyridinoline (ng / mg), and the PEN, which is the quantitative value of the amount of pentosidine (ng / mg) contained.

[0108] [Table 3] TIFF0007731175000003.tif55165

[0109] The amounts of pyridinoline (PYD) and pentosidine (PEN) contained in the collagen-rich organic composition derived from the dentin of 17 Tokachi Young Beef (registered trademark of Japan) samples were quantified using a high-performance liquid chromatograph with a fluorescence detector (HPLC-Flu) with heptafluorobutyric acid and formic acid as ion-pairing reagents. The amounts were also shown in Table 4. The amounts of pyridinoline (PYD) and pentosidine (PEN) were quantitatively determined. The amounts of pyridinoline (PYD) and pentosidine (PEN) were quantitatively determined. The amounts of pyridinoline (PYD) and pentosidine (PEN) were also ... pentosidine (PYD) and pentosidine (PEN) were quantitatively determined. The amounts of pyridinoline (PYD) and pentosidine (PEN) were quantitatively determined. The amounts of pentosidine (PY

[0110] [Table 4] TIFF0007731175000004.tif55165

[0111] Table 5 below shows the quantitative values ​​of pyridinoline content (ng / mg) contained in the collagen-rich organic composition derived from the dentin of eight domestic pigs, PYD, PEN, and PPP (pyridinoline content (ng / mg) / pentosidine content (ng / mg)), which were determined by high-performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pairing reagents. The values ​​were also shown in Table 5. The values ​​were the quantitative values ​​of pyridinoline content (ng / mg) contained in the collagen-rich organic composition derived from the dentin of eight domestic pigs, PYD, PEN, and PPP (pyridinoline content (ng / mg) / pentosidine content (ng / mg)), which were calculated from the quantitative values ​​of pyridinoline content (ng / mg) and pentosidine content (ng / mg) contained in the collagen-rich organic composition.

[0112] [Table 5] TIFF0007731175000005.tif41165

[0113] Furthermore, Fig. 1 shows the distribution of PYD, which is the quantitative value of the amount of pyridinoline (ng / mg) contained in collagen contained in the dentin-derived collagen-rich organic composition of 62 tooth samples, consisting of 18 human samples, 36 bovine samples (including 19 bovine SRM samples and 17 Tokachi Young Beef (registered trademark of Japan)), and 8 domestic pig samples, measured by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pair reagents. Figure 2 shows the distribution of PYD, the quantitative value of the pyridinoline content (ng / mg), for collagen contained in collagen contained in the dentin-derived collagen-rich organic composition of eight teeth (n=62), soft tissue-derived Teruplag (n=3, derived from bovine dermis), high-grade gelatin (n=3), gingiva of Tokachi Young Cattle (registered trademark of Japan) (n=3), and collagen contained in muscle of Tokachi Young Cattle (registered trademark of Japan) (n=3). The PYD was quantified by high-performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pairing reagents.

[0114] As shown in Figure 1, the PYD, which is the quantitative value of the pyridinoline content (ng / mg) of collagen contained in the dentin-derived collagen-rich organic composition of teeth, which are an example of hard tissue, (62 cases in total) from 18 human mammals, 36 cattle mammals and livestock animals (including 19 bovine SRMs and 17 Tokachi Young Cattle (registered Japanese trademark)), and 8 domestic pigs, all fell within the range of the PYD according to the present invention.

[0115] On the other hand, as shown in Figure 2, the PYD, which is the quantitative value of the amount of pyridinoline contained (ng / mg), for soft tissue-derived Teruprag (n=3, derived from bovine dermis), high-grade gelatin (n=3), Tokachi Young Beef (registered trademark of Japan) gums (n=3), and Tokachi Young Beef (registered trademark of Japan) muscle (n=3) were all below 50.

[0116] Based on the above, the PYD value according to the present invention was determined to be PYD>100, based on the lower limit of the PYD of collagen contained in the collagen-rich organic composition derived from tooth dentin, which is hard tissue, of 18 human mammals, 36 cattle, and 8 domestic pigs, which are mammalian livestock.

[0117] Figure 3 shows the distribution of PEN, the quantitative value of the pentosidine content (ng / mg) of collagen contained in the dentin-derived collagen-rich organic composition from a total of 62 samples: 18 humans, 36 cows (19 of which were bovine SRM and 17 were Tokachi Young Beef (registered Japanese trademark)), and 8 domestic pigs. The collagen was quantified by high-performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pair reagents.

[0118] As shown in Figure 3, the PEN, which is the quantitative value of the pentosidine amount (ng / mg) contained in collagen contained in the collagen-rich organic composition derived from tooth dentin, which is an example of hard tissue, of 44 livestock animals (including 19 cases of bovine SRM, 17 cases of Tokachi young cattle (registered trademark of Japan), and 8 cases of domestic pigs), was 0.01 mg / kg, which is the upper limit value of the 44 livestock animals. <PEN<0.4とした。

[0119] Furthermore, collagen contained in the dentin-derived collagen-rich organic composition from 62 tooth samples (18 human samples, 36 cow samples (including 19 bovine SRM samples and 17 Tokachi Young Beef (registered Japanese trademark)) and 8 domestic pig samples) was quantified by high-performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pairing reagents. Figure 4 shows the distribution of PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)), which is the weight ratio calculated from PYD, which is the quantitative value of the amount of pyridinoline contained (ng / mg), and PEN, which is the quantitative value of the amount of pentosidine contained (ng / mg).

[0120] As shown in Figure 4, PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)), which is the weight ratio of PYD, which is the quantitative value of the amount of pyridinoline contained (ng / mg), to PEN, which is the quantitative value of the amount of pentosidine contained (ng / mg), was set to PpP > 740 because livestock animals are used as an indicator for PEN.

[0121] Example 2: Endotoxin test for collagen derived from bovine teeth (mainly dentin), gelatin derived from pigskin, and high-grade gelatin Endotoxin tests were performed on collagen derived from teeth (mainly dentin) of six Tokachi Young Cattle (registered trademark of Japan, early-fattened (14-month-old) male Holstein; Tokachi Shimizu Town Agricultural Cooperative), three samples of gelatin derived from porcine skin (gelatin from Merck), and three samples of high-grade gelatin. The endotoxin tests were performed by colorimetric method using LAL reagent prepared from Limulus amebocyte lysate using blood derived from horseshoe crab. The results are shown in Figure 5.

[0122] The collagen samples derived from teeth of six Tokachi young cattle (registered trademark of Japan) were prepared by the following method. (1) For collagen containing residual mineral components, the mineral components were completely removed by shaking the sample at 37°C for 2 days using a decalcifying solution containing 500 mM EDTA, while exchanging the decalcifying solution, and then the EDTA was removed by washing with ultrapure water. (2) The obtained collagen was completely dried to obtain dry collagen. (3) The dried collagen was completely decomposed using Proteinase K (Takara Bio Inc.) at 56°C. (4) Then, Proteinase K was inactivated by heating at 95°C for 5 minutes. (5) The mixture was centrifuged at 12,000 rpm at room temperature, and the supernatant was used as a collagen sample.

[0123] As shown in Figure 5, the endotoxin levels of the tooth-derived collagen from six Tokachi young cattle (registered trademark of Japan) samples were less than 0.125, the endotoxin levels of the three pigskin-derived gelatin samples were around 1.25, and the endotoxin levels of the three high-grade gelatin samples were less than 0.125. Considering that the endotoxin standard value for famotidine injection is 15 EU / mg, the endotoxin standard value for thiamine chloride hydrochloride injection is 6.0 EU / mg, the endotoxin standard value for injectable roxatidine acetate hydrochloride is 4.0 EU / mg, the endotoxin standard value for pyridoxine hydrochloride injection is 3.0 EU / mg, and the endotoxin standard value for morphine hydrochloride injection is 1.5 EU / mg, it can be said that not only are the endotoxin levels of the tooth-derived collagen from six Tokachi young cattle (registered trademark of Japan) samples and the endotoxin levels of the three high-grade gelatin samples excellent, but the endotoxin levels of the three pigskin-derived gelatin samples are also excellent.

[0124] That is, it has been revealed that the collagen-rich organic composition according to the present invention contains a significantly reduced amount of endotoxin, or the activity of the endotoxin contained therein is significantly reduced, and the composition is substantially endotoxin-free, or contains no endotoxin, or the endotoxin contained therein has been substantially inactivated, or the endotoxin has been inactivated.

Claims

1. A collagen-rich organic composition produced from teeth extracted from a mammal, which is the following (i) or (i) and (ii): (i) PYD, which is a quantitative value of the amount of pyridinoline (ng / mg) contained in collagen contained in the collagen-rich organic composition, measured by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pair reagents, is PYD>100; (ii) The weight ratio PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)) of PYD, which is a quantitative value of the amount of pyridinoline contained in the collagen contained in the high-collagen organic composition, to PEN, which is a quantitative value of the amount of pentosidine contained in the collagen contained in the high-collagen organic composition, as determined by high-performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pair reagents, is PpP>740.

2. 2. The collagen-rich organic composition according to claim 1, which is substantially endotoxin-free, or endotoxin-free, or the endotoxin it contains is substantially inactivated, or the endotoxin has been inactivated.

3. 2. The collagen-rich organic composition according to claim 1, wherein the mammal is one or more mammals selected from the group consisting of cows, pigs, horses, sheep, deer, dogs, cats and humans.

4. A biomaterial, a biomaterial containing collagen as a main component, or other product made from the collagen-rich organic composition according to any one of claims 1 to 3 as a raw material.

5. A step of crushing a tooth extracted from a mammal to obtain a crushed material; A step of treating the obtained pulverized product with a strong alkali; a step of decalcifying the strongly alkali-treated pulverized material under negative pressure conditions; 4. A method for producing the collagen-rich organic composition according to claim 1, comprising:

6. 6. The method according to claim 5, wherein the step of treating the obtained pulverized material with a strong alkali is a step of treating the obtained pulverized material with a strong alkali at a temperature higher than room temperature and lower than the boiling point of water.

7. Quantifying the amount of pyridinoline (ng / mg) contained in collagen contained in a tooth extracted from a mammal; Quantifying the amount of pentosidine (ng / mg) contained in collagen contained in the tooth extracted from the mammal; calculating a weight ratio PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)) from the PYD, which is the value of the quantified amount of pyridinoline contained (ng / mg), and the PEN, which is the value of the quantified amount of pentosidine contained (ng / mg); selecting the tooth extracted from the mammal as a biomaterial, a collagen-based biomaterial, or a raw material for other products when the PYD, which is the value of the quantified pyridinoline content (ng / mg), or the PYD, which is the value of the quantified pyridinoline content (ng / mg), and the calculated weight ratio PpP (amount of pyridinoline content (ng / mg) / amount of pentosidine content (ng / mg)), are predetermined values; A method for identifying teeth extracted from mammals as raw materials for biomaterials, collagen-based biomaterials or other products, comprising:

8. A step of crushing a tooth or bone extracted from a mammal to obtain a crushed material; A step of treating the obtained pulverized product with a strong alkali; a step of decalcifying the pulverized product treated with strong alkali under negative pressure conditions to obtain an organic composition containing a high amount of collagen; a step of quantifying the amount of pyridinoline (ng / mg) contained in the collagen contained in the obtained collagen-rich organic composition; Quantifying the amount of pentosidine (ng / mg) contained in the collagen contained in the obtained collagen-rich organic composition; calculating a weight ratio PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)) from the PYD, which is the value of the quantified amount of pyridinoline contained (ng / mg), and the PEN, which is the value of the quantified amount of pentosidine contained (ng / mg); selecting the tooth or bone extracted from the mammal as a biomaterial, a biomaterial containing collagen as a main component, or a raw material for other products when the PYD, which is the value of the quantified pyridinoline content (ng / mg), or the PYD, which is the value of the quantified pyridinoline content (ng / mg), and the calculated weight ratio PpP (amount of pyridinoline content (ng / mg) / amount of pentosidine content (ng / mg)), are predetermined values; 1. A method for identifying teeth or bones extracted from mammals as raw materials for biomaterials, collagen-based biomaterials or other products, comprising:

9. The method according to claim 7 or 8, wherein the steps of quantifying the pyridinoline content (ng / mg) of the collagen contained in the obtained collagen-rich organic composition and quantifying the pentosidine content (ng / mg) of the collagen contained in the obtained collagen-rich organic composition are carried out by high performance liquid chromatography (HPLC-Flu) with a fluorescence detector using heptafluorobutyric acid and formic acid as ion-pair reagents, and the predetermined values ​​are PYD > 100 or PYD > 100 and PpP > 740, respectively.

10. A method for assessing the potential risk of a mammal suffering from a disease and / or the potential health of the mammal, comprising using a quantitative value of the amount of pyridinoline contained in collagen contained in the teeth of the mammal, or a quantitative value of the amount of pyridinoline contained in collagen contained in the teeth of the mammal, and a weight ratio calculated from the quantitative value of the amount of pyridinoline contained in collagen contained in the teeth of the mammal and the quantitative value of the amount of pentosidine contained therein, A step of quantifying the amount of pyridinoline (ng / mg) contained in collagen contained in teeth of a mammal that is a target for evaluation of the potential risk of disease and / or the potential health of the mammal to obtain a quantitative value of PYD; a step of quantifying the amount of pentosidine (ng / mg) contained in collagen contained in teeth of the mammal to be evaluated to obtain a quantitative value PEN; calculating a weight ratio PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)) from the obtained PYD and the obtained PEN; Quantifying the amount of pyridinoline (ng / mg) contained in collagen contained in the teeth of one or more mammals selected from a mammal not affected by a disease, a mammal affected by a disease, a healthy mammal, and an unhealthy mammal, to obtain a reference quantitative value R-PYD; Quantifying the amount of pentosidine (ng / mg) contained in collagen contained in the teeth of one or more mammals selected from a mammal not affected by a disease, a mammal affected by a disease, a healthy mammal, and an unhealthy mammal, to obtain a reference quantitative value R-PEN; calculating a reference value of weight ratio R-PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)) from the obtained R-PYD and the obtained R-PEN; comparing the PYD and the R-PYD, or comparing the PYD and the R-PYD and the PpP and the R-PpP; A method comprising:

11. The step of quantifying the amount of pyridinoline (ng / mg) contained to obtain a reference quantitative value of R-PYD and the step of quantifying the amount of pentosidine (ng / mg) contained to obtain a reference quantitative value of R-PEN are carried out by a high performance liquid chromatograph (HPLC-Flu) equipped with a fluorescence detector using heptafluorobutyric acid and formic acid as ion pair reagents.

11. The method of claim 10, wherein the step of comparing the PYD and the R-PYD, or the step of comparing the PYD and the R-PYD and the PppP and the R-PppP, is a step of confirming whether the PYD satisfies PYD>100, or whether the PYD satisfies PYD>100 and whether the PppP satisfies Ppp>740.

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