Collagen-rich organic composition and production method thereof

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

Collagen-rich organic composition and method for producing same

[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 (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) 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) 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.

[0002] Various collagens (actually, "collagen-rich organic compositions" would be more accurate, but they are often simply referred to as "collagen") have been developed. Most of these are atelocollagens produced by extraction from soft tissues in living organisms using enzymatic treatment, in which the telopeptides present at the N-terminus and C-terminus of the collagen amino acid sequence are cleaved, and poorly soluble collagen fibers are removed during a purification process. Collagens produced by extraction 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, while the quality of undenatured collagen derived from soft tissues (collagen with a triple helix structure due to the retention of telopeptides) is evaluated based on how highly pure collagen is produced by setting purification conditions such as pH. However, when producing collagen from hard tissues, 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 tissues, undenatured collagen, or products utilizing them.

[0004] Most collagen contained in biological tissues is type I collagen. Type I collagen has both physiological and non-physiological crosslinks, with pyridinoline crosslinks known as physiological crosslinking molecules and crosslinks derived from advanced glycation end products (AGEs), such as pentosidine, known as non-physiological crosslinking molecules (Non-Patent Document 1). Pyridinoline crosslinks are physiological, enzymatic crosslinks formed by the action of lysyl oxidase, and are regular crosslinking 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 (AGEs crosslinks)) involving blood glucose. When blood glucose is heated by body temperature, they are formed between arginines and lysines scattered throughout the amino acid sequence of collagen. Therefore, they are crosslinks formed randomly between adjacent arginines and lysines in the amino acid sequence of collagen, and are a factor in disrupting the physiological three-dimensional structure of collagen, 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, since the insoluble collagen fibers resulting from pentosidine crosslinks are eliminated during the purification process, atelocollagen contains almost no pentosidine crosslinks.

[0005] It has been 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 (AGEs 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).

[0006] M. Saito, CLINICIAN Vol. 554, 1141-1146, 2006M. Saito, THE BONE Vol. 21, No. 1, 53-58, 2007Sell, D. R. et al. ,V. M. , J. Biol. Chem. Vol. 284, 21597-21602, 1989A. Uchiyama et al. , J. Biochem. Vol. 110, 714-718, 1991M. TAKAHASHI et al. , Arthritis Rheum. Vol. 37, No. 5, 724-728, May 1994. H. HOSHINO et al. , Atherosclerosis Vol. 112, 39-46, 1995

[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 and 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, removal of 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. Meanwhile, 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 content to pyridinoline content per unit collagen (pentosidine content / pyridinoline content) in diseases, but 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 such as bone and cartilage, and soft tissues such as tendons and ligaments, the amount of pyridinoline crosslink molecules contained in the collagen tends to be large, and therefore the amount of pentosidine crosslink molecules tends to be large (M. Takahashi et al., Anal Biochem Vol. 232, 158-62, 1995). However, because these tissues are metabolically active, the pyridinoline crosslink molecules and pentosidine crosslink molecules contained therein are also metabolized, and therefore do not reflect the amount of pyridinoline crosslink molecules and pentosidine crosslink molecules accumulated throughout the lifetime of the mammalian host. 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 alkali 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 a tooth or bone extracted from a mammal, 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) of collagen using a predetermined method, and calculating a weight ratio of 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). To provide a biomaterial made from a 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 identifying a tooth or bone extracted from a mammal as a raw material for a biomaterial, a biomaterial or product having collagen as a main component, and a method for identifying a potential disease, the method comprising a step of selecting the tooth or bone as a raw material for a biomaterial, a biomaterial or product having collagen as a main component when the PYD or the PYD and the PpP are predetermined values. The method comprises the steps of: obtaining PYD, which is a quantitative value of the amount of pyridinoline (ng / mg) contained in collagen contained in teeth, and PEN, which is a quantitative value of the amount of pentosidine (ng / mg) contained in collagen contained in teeth of a mammal to be evaluated for disease risk and / or whether the mammal is potentially healthy; and calculating a weight ratio value of PpP (amount of pyridinoline (ng / mg) / amount of pentosidine (ng / mg)) from the obtained PYD and the obtained PEN; and and a step of obtaining R-PYD, which is a reference quantitative value of the amount of pyridinoline contained (ng / mg), and R-PEN, which is a reference quantitative value of the amount of pentosidine contained (ng / mg), of collagen contained in the teeth of one or more mammals, and calculating a reference weight ratio value of R-PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (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, 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.

[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 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 (ng / mg) per unit collagen, and PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)), which is a weight ratio calculated from the amount of pyridinoline contained (ng / mg) and the amount of pentosidine contained (ng / mg) per unit collagen, are predetermined values, the contained collagen has a three-dimensional structure that imparts excellent elasticity and strength, and is of high quality. The inventors have found that, when the PYD or the PYD and the Ppp are at predetermined values, it is possible to distinguish teeth or bones extracted from mammals that are to be used as raw materials for biomaterials, biomaterials containing collagen as a main component, or other products, and that by comparing the collagen contained in the teeth of a mammal that is to be evaluated for its potential disease risk and / or its potential healthiness with the 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 evaluate the potential disease risk in a mammal and / or its potential healthiness, 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) the PYD, which is the quantitative value of the amount of pyridinoline (ng / mg) contained in the collagen contained in the collagen-rich organic composition, 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 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.

[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) A method for producing an organic composition with a high collagen content according to any one of (1) to (3), comprising the steps of: pulverizing a tooth or bone extracted from a mammal to obtain a pulverized material; treating the pulverized material with a strong alkali; and decalcifying the pulverized material treated with the strong alkali under negative pressure conditions.

[0018] (6) The manufacturing method described in (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.

[0019] (7) A step of quantifying the amount of pyridinoline (ng / mg) contained in collagen contained in a tooth or bone extracted from a mammal; A step of quantifying the amount of pentosidine (ng / mg) contained in collagen contained in a tooth or bone extracted from the mammal; A step of calculating a weight ratio PpP (amount of pyridinoline (ng / mg) / amount of pentosidine (ng / mg)) from PYD, which is the value of the quantified amount of pyridinoline (ng / mg), and PEN, which is the value of the quantified amount of pentosidine (ng / mg); and a step of 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), or the PpP, which is the calculated weight ratio (amount of pyridinoline content (ng / mg) / amount of pentosidine content (ng / mg)), is a predetermined value.

[0020] (8) A process of crushing a tooth or bone extracted from a mammal to obtain a crushed product; a process of treating the crushed product with a strong alkali; a process of decalcifying the crushed product treated with the strong alkali under negative pressure conditions to obtain a collagen-rich organic composition; a process of quantifying the amount of pyridinoline (ng / mg) contained in the collagen contained in the collagen-rich organic composition; a process of quantifying the amount of pentosidine (ng / mg) contained in the collagen contained in the collagen-rich organic composition; and a process of calculating a weight ratio PpP (amount of pyridinoline (ng / mg) / amount of pentosidine (ng / mg)) from PYD, which is the value of the quantified amount of pyridinoline (ng / mg), and PEN, which is the value of the quantified amount of pentosidine (ng / mg). and a step of 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), or the PpP, which is the calculated weight ratio (amount of pyridinoline content (ng / mg) / amount of pentosidine content (ng / mg)), is a predetermined value.

[0021] (9) The method according to (7) or (8), wherein the step of quantifying the amount of pyridinoline (ng / mg) contained in the collagen contained in the obtained collagen-rich organic composition and the step of quantifying the amount of pentosidine (ng / mg) contained in 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 using a quantitative value of the amount of pyridinoline contained in collagen contained in the teeth of a mammal, or a quantitative value of the amount of pyridinoline contained in collagen contained in the teeth of a mammal, and a weight ratio calculated from the quantitative value of the amount of pyridinoline contained in collagen contained in the teeth of a mammal, as an index for assessing the potential risk of a disease in a mammal and / or whether the mammal is potentially healthy, comprising the steps of: quantifying the amount of pyridinoline (ng / mg) contained in collagen contained in the teeth of a mammal that is to be assessed for the potential risk of a disease and / or whether the mammal is potentially healthy, to obtain a quantitative value of PYD; quantifying the amount of pentosidine (ng / mg) contained in collagen contained in the teeth of the mammal that is to be assessed for the potential risk of a disease and / or whether the mammal is potentially healthy, to obtain a quantitative value of PEN; and calculating a weight ratio value of PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)) from the obtained PYD and the obtained PEN. The method comprises the steps of: quantifying the amount of pyridinoline (ng / mg) contained in collagen contained in the teeth of one or more mammals selected from non-diseased mammals, diseased mammals, healthy mammals, and unhealthy mammals, to obtain a reference quantitative value of R-PYD; quantifying the amount of pentosidine (ng / mg) contained in collagen contained in the teeth of one or more mammals selected from non-diseased mammals, diseased mammals, healthy mammals, and unhealthy mammals, to obtain a reference quantitative value of R-PEN; calculating the weight ratio reference value of R-PpP (amount of pyridinoline (ng / mg) / amount of pentosidine (ng / mg)) from the obtained R-PYD and R-PEN; and comparing the PYD and the R-PYD, or comparing the PYD and the R-PYD and the PpP and the R-PpP.

[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 R-PEN as a reference quantitative value, and the step of comparing the PYD and the R-PYD, or comparing the PYD and the R-PYD, and comparing 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.

[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 then the pulverized material that has been subjected to the strong alkali treatment 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. Furthermore, the present invention can be used to distinguish 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.

[0025] FIG. 1 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 human samples, 36 bovine samples (including 19 bovine specified risk materials (SRM) samples and 17 Tokachi young cattle (registered trademark in Japan, early fattened (14-month-old) male Holstein) samples), and 8 domestic pigs (6-month-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-pair reagents. This graph shows the distribution of PYD, the quantitative value of pyridinoline content (ng / mg), in collagen (n=62) contained in a dentin-derived collagen-rich organic composition from teeth of 18 human samples, 36 bovine samples (including 19 bovine SRM samples and 17 Tokachi Young Beef (registered trademark) samples), and 8 domestic pig samples (62 samples in total), Teruplag (n=3, bovine dermis-derived atelocollagen, telopeptide-free processing), high-grade gelatin (n=3, porcine acid processing, telopeptide-preserving processing method, 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 in the figure indicates the PYD threshold. 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 teeth of 18 human samples, 36 bovine samples (including 19 bovine SRM samples and 17 Tokachi Young Beef (registered Japanese trademark) samples), and 8 domestic pig samples (62 samples in total), as quantified by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pair reagents.This is a graph showing the distribution of values ​​for 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), as quantified by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion pair reagents, for collagen contained in an organic composition with a high content of collagen derived from dentin of teeth from 18 human samples, 36 bovine samples (including 19 bovine SRM samples and 17 Tokachi Young Beef (registered Japanese trademark) samples), and 8 domestic pig samples (62 samples in total). 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 Wakaushi (registered trademark of Japan), three examples of pigskin-derived gelatin, and three examples of high-grade gelatin.

[0026] The following provides detailed descriptions of the collagen-rich organic composition produced from teeth or bones extracted from mammals, biomaterials using the collagen-rich organic composition as a raw material, biomaterials or other products containing 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 raw materials for biomaterials, biomaterials or other products containing collagen as a main component, and a method for using the quantitative value of the pyridinoline content (ng / mg) of collagen contained in the teeth of mammals, or the quantitative value of the pyridinoline content (ng / mg) of collagen contained in the teeth of mammals, and a weight ratio (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) 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 mammals, as indicators for assessing the potential risk of disease in mammals and / or the potential health of mammals. In this specification, a numerical range expressed using "to" or "from" means a range that includes the numerical values ​​written before and after "to" or "from" 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 the quantitative value of the pyridinoline content (ng / mg) of collagen contained in the collagen-rich organic composition, as determined by high performance liquid chromatography (HPLC-Flu) with a fluorescence detector using heptafluorobutyric acid and formic acid as ion-pair reagents, is PYD>100; (ii) PpP, which is the weight ratio of PYD, which is the quantitative value of the pyridinoline content (ng / mg) of collagen contained in the collagen-rich organic composition, to PEN, which is the quantitative value of the pentosidine content (ng / mg), as determined by high performance liquid chromatography (HPLC-Flu) with a fluorescence detector using heptafluorobutyric acid and formic acid as ion-pair reagents, (amount of pyridinoline content (ng / mg) / amount of pentosidine content (ng / mg)), 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 tissue by detecting, for example, BMP-2, BMP-4, BMP-7, and osteocalcin, and can be determined to be derived from teeth by detecting dentin sialoprotein (DSP), dentin glycoprotein (DGP), and dentin phosphoprotein (DPP).

[0031] Generally, a "high-collagen organic composition" refers to an organic composition containing a high proportion of collagen in the entire organic composition, and atelocollagen, demineralized freeze-dried bone (DFDBA), and demineralized dentin matrix (DDM) are generally considered to be "high-collagen organic compositions." In the present invention, a "high-collagen organic composition" refers to an organic composition containing collagen in the entire organic composition, 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 high-collagen organic composition include the above-mentioned DSP, DGP, DPP, etc.

[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, respectively. 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) (pyridinoline amount (ng / mg) / pentosidine amount (ng / mg)) is calculated.

[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 described in detail below.

[0035] It is no exaggeration to say that pyridinoline crosslinks, which are physiological enzymatic crosslinking molecules, 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. The pyridinoline crosslinking molecules formed between the two lysine residues in the telopeptides located at the N- and C-termini of the collagen amino acid sequence are crosslinking molecules between collagen fibers, and therefore contribute greatly 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 a tooth or bone extracted from a mammal that is the raw material for the collagen-rich organic composition, is quantitatively measured as PYD, which is the amount of pyridinoline (ng / mg).The higher the PYD value, the better the quality of the collagen contained in the tooth or bone extracted from the mammal, and a collagen-rich organic composition produced from such teeth or bones extracted from a mammal can be said to be an excellent organic composition.

[0038] As shown in FIG. 1 , the PYD, which is the quantitative value of the amount of pyridinoline (ng / mg) contained in the collagen contained in the dentin-derived collagen-rich organic composition of teeth (62 examples in total), which are examples of hard tissues, are from 18 human mammals, 36 cattle mammals and livestock animals (including 19 SRM cattle (Hayakita Plant, Hokkaido Livestock Corporation), 17 Tokachi Young Cattle (registered trademark of Japan, early fattened (14-month-old) male Holstein; Tokachi Shimizu Town Agricultural Cooperative)), and 8 domestic pigs (6-month-old; Hayakita Plant, Hokkaido Livestock Corporation). All of these PYDs fall within the range of the PYD according to the present invention. Here, "SRM" stands for specified risk materials, and in Japan, the specified risk materials are the tonsils and distal ileum (part of the small intestine) of animals of all ages, as well as the head (excluding tongue and cheek meat), spine, and spinal cord of animals over 30 months of age (Food Safety Commission of the 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 Corporation), 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 50 or less.

[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 cows, and 8 domestic pigs, which are mammals and 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 caused by advanced glycation end products (AGEs), typified by pentosidine crosslink molecules, which are non-physiological crosslinking molecules, are pathological crosslinks (aging crosslinks (AGE crosslinks)) resulting from blood glucose. As mentioned above, these crosslinks are formed between arginine and lysine scattered throughout the amino acid sequence of collagen when blood glucose is heated by body temperature. When arginine and lysine are close to each other in the amino acid sequence of collagen, pentosidine crosslinks are formed nonspecifically (randomly), which can lead to entanglement of collagen fibers, causing the collagen fibers to lose their regularity in arrangement and disrupting the three-dimensional structure of tropocollagen. Therefore, an increase in pentosidine crosslinks in the amino acid sequence of collagen 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 said to increase depending on the host's dietary habits (carbohydrate intake habits). 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 leads to a uniform decline in the quality of collagen, which is the support base for the body, regardless of whether it is hard or soft tissue.

[0043] In addition, when the pentosidine crosslinking molecules increase in collagen, because the pentosidine crosslinking molecules are glycation crosslinking molecules produced by the Maillard reaction, the collagen contained in the living tissue will become brittle and chalk-like, and the strength and calcification degree of the collagen contained in the living tissue will decrease.The late products of the Maillard reaction are called advanced glycation end products (AGEs), and the pentosidine crosslinking molecules are AGEs.The collagen of mammals whose blood sugar level is constantly high due to a dietary habit with a high frequency of carbohydrate intake contains more pentosidine crosslinking molecules than the collagen of mammals of the same age (months) with a well-balanced dietary habit.Therefore, it can be said that the collagen of mammals whose blood sugar level is constantly high is undergoing aging crosslinking.The generated pentosidine crosslinking molecules disrupt the regular arrangement order of collagen fibers, impairing the flexibility of collagen, making it hard and brittle, and inhibiting collagen metabolism, which results in the deterioration of the quality of the collagen that constitutes the whole body and causing the 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 lead to blindness if it worsens), (2) diabetic nephropathy (which can lead to dialysis if it worsens), and (3) diabetic neuropathy (which can lead to, for example, necrosis of the fingers and toes due to impaired circulation if it worsens).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 devices, 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 crosslink molecules, facilitates binding of AGEs to RAGE, the AGE receptor present in cells that make up tissues throughout the body, creating an in vivo environment that favors signal activation. This promotes inflammation-related NF-κB signaling, and AGEs that induce chronic inflammation, such as pentosidine crosslink molecules, function as chronic inflammation trigger factors in overly glycated tissues. This mechanism is thought to exacerbate chronic inflammatory diseases, such as inflammatory bowel disease (IBD) and collagen diseases. As described above, collagen glycation gradually damages the body in various ways, unwittingly reducing quality of life (QOL) and potentially becoming a major factor in preventing healthy longevity. It has been reported that both pyridinoline crosslink molecules and pentosidine crosslink molecules in collagen contained in human biological tissues increase with age (Shimizu, "Aging-Related Nonenzymatic Glycation of Dentin Collagen Proteins," 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 a tooth or bone extracted from a mammal, which is 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 tooth or bone extracted from the mammal, and a collagen-rich organic composition produced from such teeth or bones extracted from a mammal 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 that are fed a consistent quality of food 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 evident in Figure 3, so as a standard indicator for the amount of pentosidine contained (ng / mg), humans should not be used as an indicator, but rather healthy livestock animals that live 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 organic composition with a high content of collagen derived from tooth dentin, which is an example of hard tissue, for 44 livestock animals (including 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)), can be set to 0 < PEN < 0.4, based on the upper limit value for the 44 livestock animals.

[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 tooth or bone extracted from a mammal that serves as 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, i.e., the tooth or bone extracted from a mammal that serves as the raw material for the collagen-rich organic composition, 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 the 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 equipped 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 index for PEN, as shown in FIG. 4, PpP can be made greater than 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) Dried collagen powder produced by the method for producing a collagen-rich organic composition according to the present invention is weighed into a test tube and swelled with a small amount of 6N hydrochloric acid under degassing. (2) 6N hydrochloric acid is added to the dried collagen, and the tube is then degassed and sealed. (3) Hydrolysis is carried out by heating at 110°C overnight. (4) The sample is cooled 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) The sample is centrifuged at 12,000 rpm for 5 minutes at room temperature, and the supernatant is used as the sample. (8) A known amount of a standard substance is added to the sample to determine the extraction rate and calibration curve, allowing the collagen content to be appropriately corrected.

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

[0054] (1) Detection was performed using a gradient configuration of 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). (2) Specific pumping conditions included a detection column temperature of 40°C, a flow rate of 0.5 mL / min, and a starting ratio of 90% mobile phase A and 10% mobile phase B for 0.5 minutes. (3) After that, the concentration of mobile phase B was increased to 22% over 1 minute to create a concentration gradient, and the pumping continued for 7.5 minutes. (4) Next, the concentration of mobile phase B was increased to 80% over 1 minute, and the pumping continued for another 1 minute. (5) Finally, the pumping continued at a ratio of 10% mobile phase A and 90% mobile phase B, followed by equilibration for 4 minutes at a ratio of 90% mobile phase A and 10% mobile phase B. (6) Steps (1) to (5) were performed for a total of 15 minutes per sample. The separation column used was a Cadenza CD-C18, 150 mm long and 3 mm in 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 a fluorescence wavelength of 395 nm, and for the last 7 minutes at an excitation wavelength of 325 nm and a fluorescence wavelength of 385 nm. By these monitoring steps, 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.

[0055] Next, the collagen-rich organic composition according to the present invention is produced by undergoing a strong alkali treatment, which is not normally performed on proteins including collagen, under temperature conditions 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 is significantly reduced, or the activity of the endotoxin contained is significantly reduced, so that 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 or elicited by 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 an undetectable amount of endotoxin. Endotoxins are toxins associated with certain bacteria, typically gram-negative bacteria, although endotoxins can also be found in gram-positive bacteria such as Listeria monocytogenes. The most common endotoxin is lipopolysaccharide (LPS) or lipooligosaccharide (LOS), found in the outer membrane of various gram-negative bacteria, and represents a central pathogenic characteristic 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 of the present invention and collagen-based biomaterials of the present invention, since even small amounts can cause harmful effects in humans.

[0058] Endotoxin can be detected using conventional techniques known in the art. For example, the Limulus Amebocyte Lysate Assay (LAL) method, which utilizes the blood of 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 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 level may be 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, 0.03, 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, 0.0 57, 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, 0.08 8, 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, 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, 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, It may be 6.2, 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 or less of protein.

[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 inactivated by at least 95%, preferably by 98%, and more preferably by 100%.

[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 bottom line of page 16 to lines 1-4 of page 17 of 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 Recites a Manufacturing Method for That Product" in the Patent and Utility Model Examination Handbook). That is, 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 unreasonable 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 Case 2012 (Judgment) 1204, p. 10, or Supreme Court Case 2012 (Judgment) 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 an organic composition with a high collagen content according to the present invention comprises the following steps (i) to (iii): (i) a step of pulverizing a tooth or bone extracted from a mammal to obtain a pulverized material (pulverized material preparation step); (ii) a step of treating the pulverized material obtained in the pulverized material preparation step (i) with a strong alkali (strong alkali treatment step); and (iii) a step of decalcifying the strong alkali-treated pulverized material obtained in the strong alkali treatment step (ii) under negative pressure conditions (negative pressure decalcification step).

[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, any conventional strongly basic aqueous solution known in the art can be used, and examples of such strongly basic aqueous solutions include aqueous sodium hydroxide, aqueous potassium hydroxide, aqueous calcium hydroxide, and aqueous sodium carbonate.

[0066] The strong alkali treatment step (ii) may also be a step of subjecting the pulverized material obtained in the pulverized material preparation step (i) to a strong alkali treatment 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 a step of performing a strong alkali treatment under conditions of 50 to 90°C, more preferably a step of performing a strong alkali treatment under conditions of 60 to 80°C, even more preferably a step of performing a strong alkali treatment under conditions of 65 to 75°C, and most preferably a step of performing a strong alkali treatment under conditions 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, and specifically, it is a step including a step of demineralizing the pulverized material under reduced pressure. By performing the demineralization step under reduced pressure, gases (mainly CO) generated during the treatment can be reduced. ２ ), and the air trapped therein 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 (iii) under negative pressure in the present invention may include a step of demineralizing treatment under reduced pressure, and for example, a step of demineralizing treatment under normal pressure may be combined with a step of demineralizing treatment under reduced pressure. The demineralization treatment may be carried out through one to several steps, including conventional demineralization treatment steps known in the art. For example, the demineralizing solution used may be phosphoric acid, hydrochloric acid, nitric acid, sulfuric acid, formic acid, ethylenediaminetetraacetic acid (EDTA), or solutions such as ethanol and hydrochloric acid, or ethanol and an aqueous EDTA solution, 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) a step of quantifying the amount of pyridinoline (ng / mg) contained in collagen contained in a tooth or bone extracted from a mammal (a step of quantifying the amount of pyridinoline contained); (v) a step of quantifying the amount of pentosidine (ng / mg) contained in collagen contained in a tooth or bone extracted from the mammal (a step of quantifying the amount of pentosidine contained); and (vi) a step of calculating a weight ratio value PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)) from PYD, which is the value of the amount of pyridinoline contained (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 contained (ng / mg) quantified in the step (v) of quantifying the amount of pentosidine contained (a step of calculating a weight ratio). (vii) a step of 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 (tooth / bone selection step) when PYD, which is the value of the amount of pyridinoline contained (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 contained (ng / mg) quantified in the step (iv) of quantifying the amount of pyridinoline contained, and 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; and the method comprises the steps (iv) to (vii) above.

[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 amount of pentosidine (ng / mg) contained in collagen contained in 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 the first embodiment, if the value of PYD, which is the value of the amount of pyridinoline contained (ng / mg) quantified in the step (iv) of quantifying the amount of pyridinoline contained, or the value of PYD, which is the value of the amount of pyridinoline contained (ng / mg) quantified in the step (iv) of quantifying the amount of pyridinoline contained, and the value of PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)) which is the weight ratio calculated in the step (vi) of calculating the weight ratio, 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 a tooth or bone extracted from a mammal. For example, when the step (iv) of quantifying the amount of pyridinoline and the step (v) of quantifying the amount of pentosidine are steps of quantifying the amount by 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 as long as the features of the present invention are not impaired.

[0076] Next, a second embodiment of the method for distinguishing 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. The method of this second embodiment includes: (i) a step of crushing the teeth or bones extracted from mammals to obtain a crushed product (crushed product preparation step); (ii) a step of treating the crushed product obtained in the crushed product preparation step (i) with strong alkali (strong alkali treatment step); (iii) a step of decalcifying the crushed product obtained in the strong alkali treatment step (ii) under negative pressure to obtain a collagen-rich organic composition (negative pressure decalcification 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 negative pressure decalcification step (iii) (pyridinoline content quantification step); and (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 negative pressure decalcification step (iii) (pentosidine content quantification step). (vi) a step of calculating a weight ratio value of PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)) from PYD, which is the value of the amount of pyridinoline contained (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 contained (ng / mg) quantified in the step (v) of quantifying the amount of pentosidine contained (weight ratio calculation step); (vii) a step of 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 PYD, which is the value of the amount of pyridinoline contained (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 contained (ng / mg) quantified in the step (iv) of quantifying the amount of pyridinoline contained, and 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).In addition, among the components of the method of this second embodiment, those components 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, biomaterials using the collagen-rich organic composition as a raw material, biomaterials and other products having 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 a raw material for biomaterials, biomaterials having collagen as a main component, or other products will be assigned the same symbols and will not be described again.

[0077] The pulverized material preparation step (i), the strong alkali treatment step (ii), and the decalcification step under negative pressure (iii), which are constituent elements of the method of the second embodiment, correspond to the pulverized material preparation step (i), the strong alkali treatment step (ii), and the decalcification step under negative pressure (iii), which are constituent elements of the method of producing a collagen-rich organic composition according to the present invention. Furthermore, the contained pyridinoline amount determination step (iv), the contained pentosidine amount determination step (v), the weight ratio calculation step (vi), and the tooth / bone selection step (vii), which are constituent elements of the method of the second embodiment, correspond to the contained pyridinoline amount determination step (iv), the contained pentosidine amount determination step (v), the weight ratio calculation step (vi), and the tooth / bone selection step (vii), which are 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 the 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, the method of 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, includes the following steps: (viii) quantifying the amount of pyridinoline contained in collagen (ng / mg) contained in the teeth of a mammal that is to be evaluated for the potential risk of a disease and / or whether the mammal is potentially healthy, to obtain the quantitative value PYD (quantification step of the amount of pyridinoline contained in the evaluation target); (ix) quantifying the amount of pentosidine contained in collagen (ng / mg) contained in the teeth of the mammal that is to be evaluated, to obtain the quantitative value PEN (quantification step of the amount of pentosidine contained in the evaluation target); (x) 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 contained (ng / mg) in collagen contained in the teeth of one or more mammals selected from non-diseased mammals, diseased mammals, healthy mammals, and unhealthy mammals to obtain a reference quantitative value of R-PYD (reference pyridinoline contained amount quantifying step); (xii) quantifying the amount of pentosidine contained (ng / mg) in collagen contained in the teeth of one or more mammals selected from non-diseased mammals, diseased mammals, healthy mammals, and unhealthy mammals to obtain a reference quantitative value of R-PEN (reference pentosidine contained amount quantifying step);(xiii) a step of calculating a reference weight ratio of R-PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)) from the R-PYD obtained in the reference pyridinoline-containing amount determination step (xi) and the R-PEN obtained in the reference pentosidine-containing 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, with the PpP calculated in the step (x) of calculating the weight ratio of the evaluation subject and the R-PpP calculated in the step (xiii) of calculating the weight ratio of the reference (PYD-weight ratio comparison step). The method 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 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 mammals and / or whether the mammal is potentially healthy, 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 will be assigned the same symbols and will not be described again.

[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 the quantitative value of the amount of pyridinoline contained in collagen contained in a mammal's teeth, or the quantitative value of the amount of pyridinoline contained in collagen contained in a 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 a mammal's teeth as an index for evaluating 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 according to the present invention, which uses the quantitative value of the amount of pyridinoline contained in collagen contained in a mammal's teeth, or the quantitative value of the amount of pyridinoline contained in collagen contained in a 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 a mammal's teeth as an index for evaluating the potential risk of a 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 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.

[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 evaluating 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 using a collagen extracted from a mammal as a raw material for the biomaterial according to the present invention, a biomaterial containing collagen as a main component, or other products. This 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 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 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.

[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 evaluating a 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 tooth or bone 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 "collagen contained in the teeth of one or more mammals selected from non-diseased mammals, diseased mammals, healthy mammals, and unhealthy mammals" different from the "collagen contained in the teeth of one or more mammals selected from non-diseased mammals, diseased mammals, healthy mammals, and unhealthy mammals" used in 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, 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) of 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 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 and the quantitative value of the amount of pentosidine contained, as an index for evaluating the potential risk of a disease 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 target 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, 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 and the quantitative value of the amount of pentosidine contained, as an index for evaluating 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 mammal that is not affected by a disease, a mammal that is affected by a disease, 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 features of the present invention.

[0094] The method of the present invention, which uses 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 as an index for assessing the potential risk of disease in mammals and / or whether the mammal is potentially healthy, is limited to "teeth" because the pyridinoline crosslinks and pentosidine crosslinks in type I collagen are both crosslinking reactions 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; and it is collagen contained in teeth (particularly dentin) that 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, whereas 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, the rationale for excluding bone is explained below. The values ​​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 are substances released into the blood and urine as collagen degradation products due to the activity of matrix metalloproteinases (MMPs), and contain pyridinoline cross-link molecules. This indicates that the pyridinoline and pentosidine cross-link molecules contained in bone-containing collagen are partially cleared by metabolism. In addition, when using extracted bone, bone is an organ that 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 for the amount of pyridinoline contained in the collagen contained in the mammal's teeth, or PYD, which is a quantitative value for 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 for the amount of pyridinoline contained in the collagen contained in the mammal's teeth, and PEN, which is a quantitative value for the amount of pentosidine contained, can be used.The method of the present invention using 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 using 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 is potentially healthy or not, 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, 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 a mammal 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.

[0099] Example 1 Measurement of pyridinoline and pentosidine amounts in collagen derived from human, bovine, and porcine teeth (mainly dentin) Using the following procedure, 18 human cases, 36 bovine cases (including 19 bovine SRM (Hayakita Plant, Hokkaido Livestock Corporation) and 17 Tokachi Young Beef (a registered Japanese trademark, early fattened (14-month-old) male Holstein; Tokachi Shimizu Town Agricultural Cooperative)) and domestic pigs (6-month-old; Hokkaido Livestock Corporation) were measured. 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] Hard tissue was pulverized to particle diameters of approximately 1 mm using a grinder. [1-2] The tissue was stirred in a 1% aqueous sodium carbonate solution at 70°C for several hours to remove any attached soft tissue. [1-3] Decalcification was performed. Specifically, the tissue was decalcified for several hours under reduced pressure using a decalcification solution containing 1M phosphoric acid and ethanol, and then replaced with a solution diluted several times and decalcified for several hours under reduced pressure. Subsequently, the tissue was completely decalcified by shaking for approximately one day at room temperature using the decalcification solution diluted 5 times. The tissue was then washed several times with 5% ethanol at room temperature. [1-4] The decalcified hard tissue was transferred to a mortar and mortar and ground in 70% ethanol to produce a fine powder. [1-5] The tissue was stirred in 100% ethanol, washed, degreased, and dehydrated. [1-6] The remaining ethanol in the collagen was completely removed by drying, yielding a dry collagen powder.

[0101] 2. Preparation of Analytical Samples [2-1] The dried collagen powder obtained in [1-6] was hydrolyzed. Specifically, the obtained dried collagen powder 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 sealed under degassing. The sealed test tube was then 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 10% aqueous methanol solution and centrifuged at 12,000 rpm for 5 minutes at room temperature. The supernatant was used as a sample. [2-3] A known amount of a standard substance was added to the sample to determine the extraction rate and calibration curve, and the collagen content was appropriately corrected.

[0102] 3. Quantitation of the amount of pyridinoline and the amount of pentosidine contained and calculation of their weight ratio by high performance liquid chromatography (HPLC-Flu) with a fluorescence detector using heptafluorobutyric acid and formic acid as ion-pair reagents 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) were used as the mobile phase, and the solution was pumped in a gradient setting and detected. Specifically, the detection column temperature was 40 ° C., and the flow rate was 0.5 mL / min, starting with a ratio of 90% mobile phase A and 10% mobile phase B, and pumped for 0.5 minutes. Thereafter, mobile phase B was increased to 22% over 1 minute to apply a concentration gradient, and then pumped as is for 7.5 minutes. Subsequently, mobile phase B was increased to 80% over 1 minute, and then pumped for another 1 minute. Finally, the mobile phase A was pumped at a ratio of 10% mobile phase B to 90% mobile phase B, followed by reducing the mobile phase B to 10% and equilibrating for 4 minutes at a ratio of 90% mobile phase A to 10% mobile phase B. Subsequently, detection was performed for a total of 15 minutes per sample. A Cadenza CD-C18 separation column, 150 mm long and 3 mm inner diameter, was used, and a fluorescence detector was used for detection. Monitoring was performed at an excitation wavelength of 295 nm and a fluorescence wavelength of 395 nm for the first 8 minutes, and at an excitation wavelength of 325 nm and a fluorescence wavelength of 385 nm for the last 7 minutes. By this monitoring, pyridinoline content was detected at a retention time of approximately 6.5 minutes, and pentosidine content was detected at a retention time of approximately 9.1 minutes. From the obtained amounts of pyridinoline and pentosidine content, their weight ratio (amount of pyridinoline content (ng / mg) / amount of pentosidine content (ng / mg)) was calculated.

[0103] Although the measurement was performed on permanent teeth in humans, taking into account the period 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 accordingly (Study on the eruption period of deciduous and permanent teeth in Japanese children II, Journal of the Japanese Society of Pediatric Dentistry, 57(3), 363-373, 2019, 363). On the other hand, the same can be said for the mandibular teeth of bovine SRM, starting on average 18 months after birth. Therefore, the formula for converting age to age in months for certain teeth in humans and bovine SRM was used as shown in Table 1 below.

[0104] [Table 1]

[0105] Table 2 below shows 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 18 human teeth, PEN, which is the quantitative value of the amount of pentosidine (ng / mg) contained, as quantified by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pair reagents, and PpP, which is the weight ratio calculated from PYD, which is the quantitative value of the amount of pyridinoline (ng / mg), and PEN, which is the quantitative value of the amount of pentosidine (ng / mg) contained.

[0106] [Table 2]

[0107] Table 3 below shows 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 SRM samples, PEN, which is the quantitative value of the amount of pentosidine (ng / mg) contained, as quantified by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pair reagents, and PpP, which is the weight ratio calculated from PYD, which is the quantitative value of the amount of pyridinoline (ng / mg), and PEN, which is the quantitative value of the amount of pentosidine (ng / mg) contained.

[0108] [Table 3]

[0109] Table 4 below shows the amounts of pyridinoline (ng / mg) contained in the dentin-derived collagen-rich organic composition from 17 examples of Tokachi Young Beef (registered trademark of Japan), as determined by high performance liquid chromatography (HPLC-Flu) with a fluorescence detector using heptafluorobutyric acid and formic acid as ion-pair reagents. The amounts are shown as PYD, which is the quantitative value of the amount of pentosidine (ng / mg), PEN, which is the quantitative value of the amount of pyridinoline (ng / mg), and PpP (amount of pyridinoline (ng / mg) / amount of pentosidine (ng / mg)) calculated from PYD, which is the quantitative value of the amount of pyridinoline (ng / mg), and PEN, which is the quantitative value of the amount of pentosidine (ng / mg).

[0110] [Table 4]

[0111] Table 5 below shows the amounts of pyridinoline (ng / mg) contained in the collagen-rich organic composition derived from the dentin of eight domestic pigs, as quantified by high performance liquid chromatography (HPLC-Flu) with a fluorescence detector using heptafluorobutyric acid and formic acid as ion-pair reagents. PYD is the quantitative value of the amount of pyridinoline (ng / mg), PEN is the quantitative value of the amount of pentosidine (ng / mg), and PpP (amount of pyridinoline (ng / mg) / amount of pentosidine (ng / mg)) is the weight ratio calculated from PYD, the quantitative value of the amount of pyridinoline (ng / mg), and PEN, the quantitative value of the amount of pentosidine (ng / mg).

[0112] [Table 5]

[0113] Furthermore, the distribution of PYD, which is the quantitative value of the amount of pyridinoline contained (ng / mg) in collagen contained in the dentin-derived collagen-rich organic composition of 62 cases, consisting of 18 human cases, 36 bovine cases (including 19 bovine SRM cases and 17 Tokachi Young Beef (registered Japanese trademark)), and 8 domestic pig cases, was 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, which is the quantitative value of the amount of pyridinoline contained (ng / mg), for 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 distribution was determined by high-performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pair 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, from 18 human mammals, 36 cattle mammals and livestock animals (including 19 SRM cattle and 17 Tokachi Young Cattle (registered Japanese trademark)), and 8 domestic pigs (62 cases in total), 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), of soft tissue-derived Teruprag (n = 3, derived from bovine dermis), high-grade gelatin (n = 3), Tokachi Young Beef (registered trademark of Japan) gum (n = 3), and Tokachi Young Beef (registered trademark of Japan) muscle (n = 3) were all 50 or less.

[0116] From 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 cows, and 8 domestic pigs, which are mammals and livestock.

[0117] Furthermore, the distribution of PEN, which is the quantitative value of the pentosidine content (ng / mg), was measured by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion-pair reagents for collagen contained in the dentin-derived collagen-rich organic composition from a total of 62 cases, including 18 human cases, 36 bovine cases (19 of which were bovine SRM and 17 were Tokachi young cattle (registered Japanese trademark)), and 8 domestic pig cases. The distribution was shown in Figure 3.

[0118] As shown in Figure 3, the PEN, which is the quantitative value of the pentosidine content (ng / mg) of collagen contained in the organic composition with a high content of collagen derived from tooth dentin, which is an example of hard tissue, for 44 livestock animals (including 19 SRM cattle, 17 Tokachi young cattle (registered Japanese trademark), and 8 domestic pigs), was set to 0 < PEN < 0.4, based on the upper limit value for the 44 livestock animals.

[0119] Furthermore, collagen contained in the dentin-derived collagen-rich organic composition from a total of 62 cases, including 18 human cases, 36 bovine cases (19 of which were bovine SRM and 17 were Tokachi young cattle (registered Japanese trademark)), and 8 domestic pig cases, was quantified by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion pair reagents. Figure 4 shows the distribution of values ​​for 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 FIG. 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 were used as an indicator for PEN.

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

[0122] Collagen samples derived from teeth of six Tokachi young cattle (registered trademark of Japan) were prepared using the following method. (1) For collagen containing residual mineral components, a decalcifying solution containing 500 mM EDTA was used. The samples were shaken at 37°C for two days, while the decalcifying solution was replaced, to completely remove the mineral components, and then the EDTA was removed by washing with ultrapure water. (2) The resulting collagen was completely dried to obtain dried collagen. (3) The dried collagen was completely decomposed using Proteinase K (Takara Bio Inc.) at 56°C. (4) The Proteinase K was then inactivated by heating at 95°C for five minutes. (5) The samples were 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 samples of Tokachi Young Beef (registered trademark of Japan) were less than 0.125, the endotoxin levels of the gelatin from three samples of pigskin were around 1.25, and the endotoxin levels of the high-grade gelatin from three 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 were the endotoxin levels of the six samples of tooth-derived collagen from Tokachi Young Beef (registered trademark of Japan) and the three samples of high-grade gelatin extremely excellent, but the endotoxin levels of the three samples of pigskin-derived gelatin were 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, the collagen-rich organic composition being (i) or (i) and (ii) below; (i) PYD, which is a quantitative value of the pyridinoline amount (ng / mg) of 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) PpP (amount of pyridinoline contained (ng / mg) / amount of pentosidine contained (ng / mg)), which is a weight ratio of PYD, which is a quantitative value of the amount of pyridinoline contained (ng / mg) in collagen contained in the high-collagen-containing organic composition, to PEN, which is a quantitative value of the amount of pentosidine contained (ng / mg), as quantified by high performance liquid chromatography equipped 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 which contains endotoxin that has been substantially inactivated, or which contains endotoxin that 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, which uses 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 or bone extracted from a mammal to obtain a crushed material; A step of subjecting the obtained pulverized material to a strong alkali treatment; a step of deashing the strongly alkali-treated pulverized material under negative pressure conditions; A method for producing a collagen-rich organic composition according to any one of claims 1 to 3, comprising the steps of:

6. The method according to claim 5, wherein the step of subjecting the obtained pulverized material to a strong alkali treatment is a step of subjecting the obtained pulverized material to a strong alkali treatment 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 amount of pyridinoline contained (ng / mg) measured, and the PEN, which is the value of the amount of pentosidine contained (ng / mg) measured; selecting the tooth extracted from the mammal as a biomaterial, a biomaterial mainly composed of collagen, or a raw material for other products when the quantified pyridinoline content (ng / mg) PYD or the quantified pyridinoline content (ng / mg) PYD and the calculated weight ratio PpP (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) are predetermined values; 13. A method for identifying teeth extracted from a mammal as a source of biomaterial, collagen-based biomaterial 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 subjecting the obtained pulverized material to a strong alkali treatment; a step of demineralizing the pulverized material treated with strong alkali under negative pressure conditions to obtain an organic composition with a high collagen content; 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 amount of pyridinoline contained (ng / mg) measured, and the PEN, which is the value of the amount of pentosidine contained (ng / mg) measured; selecting the tooth or bone extracted from the mammal as a biomaterial, a biomaterial mainly composed of collagen, or a raw material for other products when the quantified pyridinoline content (ng / mg) PYD or the quantified pyridinoline content (ng / mg) PYD and the calculated weight ratio PpP (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) are predetermined values; 1. A method for identifying teeth or bones extracted from a mammal as a source of biomaterials, collagen-based biomaterials or other products, comprising:

9. The method according to claim 7 or 8, wherein the step of quantifying the pyridinoline content (ng / mg) of collagen contained in the obtained collagen-rich organic composition and the step of quantifying the pentosidine content (ng / mg) of collagen contained in the obtained collagen-rich organic composition 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 step of quantifying the pyridinoline content (ng / mg) of collagen contained in the obtained collagen-rich organic composition and the step of quantifying the pentosidine content (ng / mg) of collagen contained in the obtained collagen-rich organic composition, and the predetermined values ​​are PYD>100 or PYD>100 and PpP>740, respectively.

10. A method for evaluating a potential risk of a disease in a mammal and / or whether the mammal is potentially healthy, comprising using 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 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, A step of quantifying the amount of pyridinoline (ng / mg) contained in collagen contained in teeth of a mammal that is to be evaluated for potential risk of disease and / or potential health of the mammal to obtain a quantitative value 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 non-diseased mammals, diseased mammals, healthy mammals, and unhealthy mammals 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 non-diseased mammals, diseased mammals, healthy mammals, and unhealthy mammals to obtain a reference quantitative value R-PEN; calculating a weight ratio reference value 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 PppP and the R-PppP; The method includes:

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.

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 step of 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.