Therapeutic components

Abstract

The present invention relates to compositions containing keratin, methods for producing such compositions, and various therapeutic applications thereof, particularly in dentistry and bone restoration. In particular, the present invention relates to keratin compositions produced by dissolving keratin in a solvent, casting the resulting solution, and drying it.

Description

[Technical Field]

[0001] The present invention relates to compositions containing keratin, methods for producing such compositions, and their use in various therapeutic applications, particularly in dentistry and bone restoration. [Background technology]

[0002] Dental caries is the most prevalent chronic disease in the world, affecting the hard tissues of teeth. It accounts for nearly half of all tooth extractions, and therefore remains a major public health problem. Caries affects both primary and permanent teeth. Caries ranges from mild white spot lesions (WSL) to severe tooth loss requiring extensive restorative treatment. 1 Amelogenin, an intrinsically disordered protein (IDP), is a major extracellular protein in developing enamel and is thought to be important for the formation of the hierarchical architecture of enamel. 2 The inventors used keratin as a model for IDP to develop a clinically manageable approach for treating WSL. In particular, they optimized keratin extraction from natural waste tissues, including wool and human hair, developed a keratin-specific crosslinking approach that optimizes the synergistic effect of disorder-order proteins, and further infiltrated WSL in situ, after which the keratin scaffold was calcified.

[0003] Biomineralization is the process by which living organisms produce minerals under the control of an organic matrix that plays a role in the nucleation and orientation of hierarchical growth and morphogenesis of calcified tissues. Research into biomineralization is not only important for better understanding how mineral-rich tissues are formed in living organisms, but also for gaining insights into advanced materials design. 3The biomimetic reconstruction of dental enamel could be an ideal method to regrow an organized enamel similar to apatite crystals with strong adhesion to the natural enamel surface as an alternative to conventional treatments. Such an approach would result in a strong tooth surface and solve the problem of secondary caries. Therefore, the biomimetic strategy for enamel restoration has been gaining interest in the fields of materials science and dentistry and is widely considered a promising approach for the prevention, repair, and treatment of defective enamel.

[0004] In the process of biomineralization of hard tissues, the microstructure of natural materials is precisely controlled and replicated. 2 Enamel formation, i.e., amelogenesis, is a highly controlled process involving protein-protein interactions, protein-mineral interactions, and interactions involving cell membranes in addition to strict genetic control. In the process of biomineralization of hard tissues, the microstructure of natural materials is accurately controlled and replicated. Amelogenesis, i.e., the formation of enamel, is a highly controlled process involving protein-protein, protein-mineral, and cell membrane interactions in addition to strict genetic control. 2 The inventors have found that the protein matrix can function as a template for guiding the nucleation of hydroxyapatite in calcified tissues and can induce hierarchical regeneration of enamel by mimicking the frontiers of biomineralization.

[0005] Despite numerous attempts to use various techniques for the remineralization of enamel and the restoration of enamel structure and properties, clinically useful restorations have not been found. This is at least partly due to the fact that the complex hierarchical structure of natural enamel cannot be reproduced on a large scale in the laboratory. A non-invasive remineralization strategy using a biomimetic approach for initial white spot lesions on tooth surfaces has been reported by Kirkham et al. 4has been attempted by (2007), and they considered Curodont™, a self - organizing peptide scaffold known to induce nucleation, which was shown to exhibit poorly crystalline hydroxyapatite. Chen et al. 5 (2006) also developed a wet - chemical method for synthesizing enamel - like prismatic and crystalline structures, but their dimensions were much larger than those of natural enamel and were only obtainable at high temperatures and pressures that are not clinically applicable. Yamagishi et al. 6 and Yin et al. 7 developed an inorganic - chemical method for growing enamel - like apatite nanocrystals oriented on tooth enamel, but were unable to form the important hierarchical architecture. Shao et al. 8 also succeeded in developing an inorganic - chemical method for growing oriented enamel - like apatite nanocrystals that can form a biomimetic crystalline - amorphous calcification frontier to induce epitaxial growth of enamel, but the thickness of the regenerated enamel was limited.

[0006] Also, in the study by Oldak et al. 9 using an amelogenin - containing chitosan hydrogel to reconstruct enamel and stabilize Ca - P clusters via amelogenin supramolecular organization, it was found that a high - density interface was formed between the newly grown layer. Natural enamel was formed, and bundles of organized crystals were observed within the repair layer. However, the formation of organized apatite nanocrystals with characteristic hierarchical order was not achieved. In recent years, Elsharkawy et al. 10 developed a material that mimics the structural hierarchy of tooth enamel. They reported a protein - mediated calcification process that utilizes the disorder - order interaction using elastin - like recombinamers. This technique was found to be able to form a calcification structure very similar to the hierarchical structure of enamel, presenting a potential strategy for material design that opens up the possibility of hard - tissue repair, but is not clinically applicable.

[0007] Protein-based biomaterials demonstrate great potential in various chemical and biological applications due to their ability to function as synthetic extracellular matrices that facilitate cell-cell and cell-matrix interactions. Protein polymers are particularly attractive for medical applications due to their extremely high biocompatibility and diverse physicochemical properties and biological activities. IDPs are known to play a fundamental role in calcification. IDPs contribute to intermolecular interactions at the protein-mineral interface. 11 Beniash et al. 12 They reported that amelogenin, a highly conserved IDP, undergoes a conformational transition from a disordered random coil structure to an ordered sheet structure when it interacts with the developing endocrine system. In the process of enamel formation, this conformational change is known to guide crystal growth. 13 Carneiro 14 However, we found that the conformational arrangement of amelogenin into amyloid-like nanoribbons may be necessary for the unique hierarchical structure of mature enamel.

[0008] In short, while many attempts have been made to synthesize enamel-like tissue, reconstructing enamel at all length scales remains a challenging task. This invention reproduces a highly ordered calcified structure using a keratin-based hydrogel. Keratin is the most abundant structural protein in epithelial cells and is considered one of the most important biopolymers in animals. 15Keratinous materials possess a high cysteine ​​content that distinguishes them from other proteins, and are typically considered to be highly durable, tough, inert to the natural environment, and provide mechanical support. Their inherent capabilities—spontaneous self-assembly, biocompatibility, biodegradability, mechanical longevity, and support for cell proliferation—give them great potential in the field of biomaterials research. These biomedical engineering functions stem from the highly ordered hierarchical structure of keratin fibers, ranging from nanoscale intermediate filaments to microscale cortical cells, and their easy self-assembly properties allow them to polymerize into porous scaffolds. 16,17

[0009] The inventors have created a composition that is useful for treating other pathological conditions, but is particularly useful for treating dental caries, and have developed a new manufacturing method for it. [Overview of the Initiative]

[0010] According to the present invention, a method is provided for producing a keratin composition, which is in particular a keratin film, and this method is (a) A step of providing a keratin solution containing keratin in a solvent, (b) A step of casting the solution onto the substrate, (c) A step of drying the solution, Includes.

[0011] The solution contains one, or preferably at least two, types of keratin. Keratin is a protein found in animal and human hair, body hair, and other tissues. Various types of keratin are well known in the art. For example, known human keratins include KRT23, KRT24, KRT25, KRT26, KRT27, KRT28, KRT31, KRT32, KRT33A, KRT33B, KRT34, KRT35, KRT36, KRT37, KRT38, KRT39, KRT40, KRT71, KRT72, KRT73, KRT74, KRT75, KRT76, KRT77, KRT78, KRT79, KRT8, KRT80, KRT81, KRT82, KRT83, KRT84, KRT85, and KRT86.

[0012] In certain embodiments, keratin or keratin group is wool keratin. Wool keratin includes keratin intermediate filaments (KIFs), S-carboxymethyl keratein A (SCMK-A) and S-carboxymethyl keratein B (SCMK-B), keratin-related proteins (KAPs) such as KAP1-1, KAP2, KAP3, KAP4, KAP5, KAP6, KAP7, KAP8, KAP11, KAP13, KAP24, and type I endoroot sheath keratins such as oIRSa1, oIRSa2, oIRSa3-1 and oIRSa3.2.

[0013] If the solution contains two or more types of keratin, it is preferable that these keratins differ from each other by having different molecular weights, being encoded by different keratin genes, or by modifications applied to them. If the solution contains one type of keratin, it is preferable that the keratin is not KR75 (or KRT75). KR75 (also known as K6hf) is known to form in the inner root sheath, nail bed, and lingual papilla of human hair follicles. KR75 is an isoform of keratin 6, with a molecular weight of 59.5 kDa and an isoelectric point pH of 7.9. In nature, keratin filaments containing KR75 are oriented perpendicular to the long axis of the hair, like a "barrel hoop".

[0014] When a solution contains multiple types of keratin, it is preferable to have a high level of diversity in the keratin within the solution or structure. For example, in one preferred embodiment, the lightest keratin present in the solution or structure has a molecular weight of less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, or less than 60% of the molecular weight of the heaviest keratin present in the solution or structure.

[0015] The solution may contain at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten types of keratin.

[0016] Optionally, one, both, more, or all of the keratin may be highly negatively charged. The term "highly negatively charged keratin" will be understood by those skilled in the art to mean keratin containing a large number of negatively charged functional groups such as COOH. Preferably, at least one of the keratin present in the keratin solution or keratin structure is highly negatively charged. "Highly negatively charged" refers to the pK of keratin. a This means that the value is less than 7.

[0017] As is known in this field, the zeta (ζ) potential is pK for complex molecules such as keratin. a It can be used as an indicator. In one embodiment, at least one of the keratins present in the keratin solution or keratin structure has a zeta potential of at least -2mV at physiological pH. In one embodiment, at least one of the keratins present in the keratin solution or keratin structure has a zeta potential of at most -40mV at physiological pH. Preferably, at least one of the keratins present in the keratin solution or keratin structure has a zeta potential of at least -4mV at physiological pH. More preferably, at least one of the keratins present in the keratin solution or keratin structure has a zeta potential of at least -6mV at physiological pH. More preferably, at least one of the keratins present in the keratin solution or keratin structure has a zeta potential of at least -8mV at physiological pH. More preferably, at least one of the keratins present in the keratin solution or keratin structure has a zeta potential of at least -10mV at physiological pH.

[0018] Optionally, at least one or both, multiple or all, keratin may be α-keratin. Optionally, one or more keratin may be β-keratin. α-keratin is found in all vertebrates and forms hair (including wool), the outer layer of skin, horns, nails, claws, hooves, and mammalian whiskers. β-keratin is found only in living reptiles and birds, and is particularly present in claws, scales, claws, shells, feathers, and beaks. Optionally, the keratin solutions and structures of the present invention may be derived from any such keratin source.

[0019] Optionally, at least one, both, more, or all of the keratins may be high-sulfur keratins. Optionally, at least one, both, more, or all of the keratins may be low-sulfur keratins. Preferably, the solution contains both high-sulfur and low-sulfur keratins. The terms "high-sulfur keratin" and "low-sulfur keratin" are well known in the art.

[0020] High-sulfur keratins can generally be divided into two groups: one group contains proteins with a cysteine ​​content (measured as S-carboxymethylcysteine ​​or SCMC) exceeding 30 moles and is known as "ultra-high-sulfur (UHS)" keratins, and the other group contains proteins with high glycine and / or tyrosine content (HGT).

[0021] Keratin, especially low-sulfur keratin, is known to contain substantial amounts of aspartic acid (ASP) and glutamic acid (Glu). While not bound by theory, the abundance of negatively charged functional groups such as COOH in aspartic acid (Asp) and glutamic acid (Glu) is thought to be due to calcium (Ca 2+ ) and (PO4 3- It is thought that its ability to attract ions promotes the precipitation of template-directed calcium and phosphate. Hydroxyapatite nucleation is involved in the regulatory process of bone mineralization, via charged amino acid (AA) domains and Ca 2+ and PO4 3-It is thought that the process is initiated by a group of negatively charged phosphorylated noncollagenous proteins that attract ions and increase local supersaturation to a level where critically sized nuclei can form, allowing these nuclei to grow into hydroxyapatite (HA) crystals. In addition to their role in calcification, charged AAs such as Asp and Glu bound to the HA surface have also been shown to promote protein uptake and osteoblast proliferation. (Song J.; Malathong V.; Bertozzi CR., 2005, "Calcification of Synthetic Polymer Scaffolds: A Bottom-Up Approach for Artificial Bone Development", J.Am.Chem.Soc., Vol. 127, pp. 3366-3372; George A.; Veis A., 2008, "Regulation of Nucleation, Crystal Growth, and Growth Inhibition of Phosphorylated Proteins and Apatite", Chem.Rev., Vol. 108, pp. 4670-4693; Tavafoghi, M.; Cerruti, M., 2016) "The Role of Amino Acids in the Calcification of Hydroxyapatite," Journal of The Royal Society Interface, Vol. 13 (123), 20160462).

[0022] Various solvents are useful in this invention. For example, the solvent used may be water, one or more polar aprotic solvents (e.g., dimethyl sulfoxide or dimethylformamide), one or more alcohols (e.g., ethanol), or a combination thereof. Optionally, the solvent may be ethanol, water, DMSO or DMF, or a combination of two or more of these.

[0023] For example, the solvent may include, or consist of, the following: water; ethanol; Water and ethanol; Water and DMF; DMSO; DMSO and DMF.

[0024] When two or more solvents are used, they may be used in equal proportions or in different proportions. For example, the solvents may be DMSO and DMF in a 50:50 ratio. Optionally, the solvent may contain 10, 20, 30, 40, 50, 60, 70, 80, or 90% water. Optionally, the solvent may contain 10, 20, 30, 40, 50, 60, 70, 80, or 90% DMSO. Optionally, the solvent may contain 10, 20, 30, 40, 50, 60, 70, 80, or 90% DMF.

[0025] The solvent may be deionized and / or may not contain any solutes other than keratin. Alternatively, salts such as sodium, sulfate, chloride, zinc, carbonate, phosphate, calcium, fluorine, iron, or potassium may be added to the solvent. Such salts can assist the calcification process, thereby aiding in the regeneration of bone or enamel.

[0026] The pH of the solvent may be 3-12, or 3.5-12, or 4-12, or 4.5-12, or 5-12, or 5.5-12, or 6-12, or 6.5-12, or 7-12, or 6.5-11.5, or 7-11.5, or 7-11, or 4-11, or 5-11. The pH of the solvent is preferably at least 4, or at least 4.5, or at least 5, or at least 5.5, 6, or at least 6.25, or at least 6.5, or at least 6.75, or at least 7. The pH of the solvent is preferably less than 12, or less than 11.75, or less than 11.5, or less than 11.25, or less than 11.

[0027] The keratin concentration in the solution is preferably 1-25% by weight, or 1-20% by weight, or 1-15% by weight, or 1-12.5% ​​by weight, or 1-10% by weight, or 2-25% by weight, or 2-20% by weight, or 2-15% by weight, or 2-12.5% ​​by weight, or 2-10% by weight, or 3-25% by weight, or 3-20% by weight, or 3-15% by weight, or 3-12.5% ​​by weight, or 3-10% by weight, or 4-25% by weight, or 4-20% by weight, or 4-15% by weight, or 4-12.5% ​​by weight, or 4-10% by weight, or 5-25% by weight, or 5-20% by weight, or 5-15% by weight, or 5-12.5% ​​by weight, or 5-10% by weight.

[0028] The keratin concentration is preferably at least 0.5% by weight, or at least 1% by weight, or at least 1.5% by weight, or at least 2% by weight, or at least 2.5% by weight, or at least 3% by weight, or at least 3.5% by weight, or at least 4% by weight, or at least 4.5% by weight, or at least 5% by weight.

[0029] The keratin concentration is preferably less than 25% by weight, or less than 20% by weight, or less than 15% by weight, or less than 14% by weight, or less than 13% by weight, or less than 12% by weight, or less than 11% by weight, or less than 10% by weight.

[0030] In one embodiment, the keratin solution has a neutral pH. In one embodiment, the pH of the keratin solution is 5.5-8.5, 6.0-8.0, 6.5-7.5, 6.6-7.4, 6.7-7.3, 6.8-7.2, 6.9-7.1, and preferably approximately 7 or 7.

[0031] Casting refers to applying a keratin solution onto a substrate. The solution may then be dried to form a structure such as a film. Any suitable substrate can be used in step (b). In some embodiments, the substrate is at least slightly hydrophobic so that the resulting film can be easily peeled off. Optionally, the substrate may be a high-molecular-weight organosilicon compound such as polymethylsiloxane or polydimethylsiloxane. In some embodiments, the substrate is a tooth or multiple teeth.

[0032] Casting may be performed in vivo, ex vivo, or in vitro.

[0033] This method may include a step of repeating step (b) and optionally step (c) in order to form structures of various shapes or sizes.

[0034] For example, to form a membrane structure in vitro, the casting process may include dropping and casting a solution onto an inert, hydrophobic surface as described above. Thicker structures may be fabricated by drying a layer and then casting another layer on top of it. This process may be repeated to obtain the desired thickness. This process can be automated using 3D printing with a keratin solution.

[0035] After casting, allow the solution to dry. Optionally, the keratin solution may be dried for at least 30, 40, 50, 60, 75, 90, 105, 120, or 150 seconds. Optionally, the solution may be dried at temperatures of 15°C to 50°C, or 15°C to 45°C, or 15°C to 40°C, or 15°C to 38°C. Optionally, the solution may be dried at temperatures of at least 10, 12, 15, 17, 18, 19, 20, 22, 25, 27, 30, 32, 35, 37, 40, 42, or 45°C. Optionally, the solution may be dried at temperatures below 55, 52, 50, 47, 45, 42, 40, 37, 35, 32, 30, 27, or 25°C.

[0036] Drying may be performed in vivo, ex vivo, or in vitro.

[0037] The solution may also contain crosslinking agents such as triethylene glycol dimethacrylate (TEGDMA), hexamethylene diisocyanate (HDI), and / or polyethylene glycol dimethacrylate (PEGDMA). Optionally, the crosslinking agent may also contain a photoinitiator such as lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), which can be used to shorten the drying time. Other photocrosslinking agents that may be used include trimethylbenzoyl diphenylphosphin oxide (TPO), benzoyl peroxide (BPO), 2,2-dimethoxy-2-phenylacetophenone (DMPA), camphorquinone (CQ), phenanthrenequinone (PQ), benzophenone (BP), and 1-phenyl-1,2-propanedione (PPD).

[0038] The crosslinking agent is a specific molar ratio relative to the average lysine residues and / or cysteine ​​residues in keratin. They may be used in ratios. For example, TEGDMA may be used in a ratio of approximately 1, 10, 20, 30, 40, or 50 per lysine residue and / or cysteine ​​residue. HDI may be used in a ratio of approximately 1, 2, 3, 5, 8, or 10 per lysine residue and / or cysteine ​​residue.

[0039] This method may include a step of irradiating the cast solution with light to initiate film formation using light.

[0040] The solution generally dries to form a film. The film generally contains spherulite, which is typically Maltese cross-shaped. Spherulite is generally considered to be a densely packed crystalline aggregate of chains that can impart great strength and rigidity to proteins. The diameter of the spherulite is generally 3 to 50 μm. Preferably, the diameter of the spherulite is 1 to 10 μm. Preferably, the diameter of the spherulite is at least 3, 4, 5, 6, 7, or 8 μm. The diameter of the spherulite is preferably less than 50, 45, 40, 35, and 30 μm. If the composition is in the form of a film, the film may optionally be 1 mm thick.2 Each may contain at least 10, 15, 20, 25, or 30 spherulites. If the composition is in the form of a film, the film may optionally be 1 mm thick. 2 The composition may contain less than 120, 115, 110, 105, or 100 spherulites per unit. If the composition is in the form of a film, the film may optionally be at least 10, 15, 20, 25, or 30 μm thick. If the composition is in the form of a film, the film thickness may optionally be less than 230, 225, 220, 215, 210, 205, or 200 μm thick.

[0041] Spherulites are known to those skilled in the art. Crystal lamellae self-assemble to form spherical superstructures, within which highly ordered lamellar sheets grow radially from the center. While we do not wish to be bound by theory, these spherulites are thought to act as nucleation sites that promote hierarchical mineral growth, thereby aiding in the regeneration of enamel or bone. Birefringence occurs due to the orientation of keratin within the lamellae, and when this structure is observed between orthogonal polarizers under an optical microscope, a "Maltese cross" pattern appears. Therefore, it is quite easy for those skilled in the art to determine whether a keratin structure contains spherulite structures.

[0042] In one embodiment, the film contains one or more large spherical bodies made up of two or more, three or more, four or more, five or more, ten or more, twenty or more, or fifty or more sferlites. In another embodiment, the film contains two or more, three or more, four or more, five or more, ten or more, twenty or more, or fifty or more sferlites, and these sferlites are linearly oriented.

[0043] In one embodiment, the keratinspherite includes a β-sheet and a random coil structure. In a preferred embodiment, the keratinspherite has a high proportion of β-sheets.

[0044] In one embodiment, the dried keratin film contains an organic network of nanospheres on its surface. In a preferred embodiment, the diameter of the nanospheres is 10-60 nm, 20-50 nm, and preferably 20-40 nm.

[0045] In one embodiment, the dried keratin film contains nano- and / or microfibril structures. In a particularly preferred embodiment, the fibrils are well organized and oriented substantially parallel to one another. In one embodiment, the dried keratin film contains first and second fibril populations. In one embodiment, the widths of the fibrils in the first population are 50-150 nm, 60-140 nm, 70-130 nm, 80-120 nm, 90-110 nm, preferably about 100 nm. In one embodiment, the widths of the fibrils in the second population are 400-600 nm, 450-550 nm, 470-530 nm, 480-520 nm, The wavelength is 490-510 nm, preferably around 500 nm.

[0046] The step of providing a keratin solution may include the step of dissolving keratin in a solvent. The keratin may be supplied in any form suitable for dissolving in a solvent. In particular, the keratin may be freeze-dried. The keratin may also be in the form of a dry powder.

[0047] This method may include a step of washing the composition, particularly in water, or a step of storing the composition, particularly in water.

[0048] This method, (d) A step of incubating the keratin composition in a calcification solution. It may include.

[0049] When the composition is incubated, a calcified keratin composition is generally obtained.

[0050] Calcification solutions generally contain calcium or sodium salts, or both. For example, a calcification solution may contain calcium phosphate. It may also contain sodium chloride or sodium fluoride. In one embodiment, the calcification solution contains approximately 0.1 mM to 1 M Ca 2+ and / or PO4 at approximately 0.1 mM to approximately 1 M 3- It contains F, optionally ranging from approximately 0.01 mM to approximately 1 M. - It may contain: Optionally, it may contain zinc, barium, strontium, lead, silver, potassium, carbonate, iron and / or magnesium.

[0051] Acids such as nitric acid, hydrochloric acid, or phosphoric acid may be added to the calcification solution to dissolve the calcium phosphate. Therefore, the calcification solution may contain acid. Optionally, the keratin composition may be incubated in a calcification solution with a pH of approximately 4, 5, 6, or 7.

[0052] Optionally, the keratin composition may be incubated in the calcification solution for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. Optionally, the keratin composition may be incubated in the calcification solution for less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days. Incubation may optionally be carried out at a temperature of 15 to 90°C, or at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60°C, or at a temperature of less than 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50°C, or at approximately 30, 35, 37, 40, 42, or 45°C. Preferably, incubation is carried out at 37°C.

[0053] After incubation, the method may include a step of removing the composition from the calcification solution. It may be washed.

[0054] The calcification solution may be a body fluid such as saliva, blood, interstitial fluid, serum, or plasma, or may contain these. Calcification may be carried out in vivo, ex vivo, or in vitro. For example, calcification may be carried out in the oral cavity using saliva as the calcification solution.

[0055] Alternatively, the calcification solution may be a hydroxyapatite solution. In a preferred embodiment, the hydroxyapatite solution is a hydroxyapatite solution containing a high concentration of fluorine, and most preferably a supersaturated hydroxyapatite solution containing a high concentration of fluorine.

[0056] When a calcification solution is added to the film, calcification occurs. The calcified film is spherulite. It contains a spherulite-like calcified structure. In one embodiment, the calcified film contains two or more, three or more, four or more, five or more, ten or more, twenty or more, or fifty or more spherulite-like calcified structures. In one embodiment, the calcified spherulite-like structure has a form in which aggregated mineral nanocrystals are helically oriented. In one embodiment, the nanocrystals have a needle-like shape. In a preferred embodiment, the nanocrystals contain oriented prism-like structures. In a preferred embodiment, the origin of the prism-like structure is the center of the spherulite, and the prism-like structure terminates at a single point on the periphery of the spherulite.

[0057] Preferably, the thickness of the prism-like structure is 0.5 to 2 μm, 1.0 to 1.7 μm, and most preferably 1.36 ± 0.33 μm. Preferably, the length of the prism-like structure is several tens of micrometers. Preferably, the diameter of the prism-like structure is 40 to 120 nm, 50 to 110 nm, 60 to 100 nm, and most preferably 83 ± 22.96 nm.

[0058] In one embodiment, the prism-like structure is an elongated apatite nanocrystal. In a preferred embodiment, the elongated apatite nanocrystals are oriented in a substantially parallel arrangement.

[0059] In a preferred embodiment, the sferrite-like calcified structure contains apatite crystals on both its surface and in its cross-section.

[0060] In one embodiment, the calcified film has increased hardness and / or Young's modulus compared to the film before the addition of the calcification solution. In one embodiment, the calcified film has a Young's modulus exceeding 5 GPa, 5.1 GPa, 5.5 GPa, 6 GPa, 6.5 GPa, 7 GPa, 7.5 GPa, or 8.0 GPa. In one embodiment, the calcified film has a hardness exceeding 0.3 GPa, 0.4 GPa, 0.5 GPa, or 0.6 GPa.

[0061] In one embodiment, the sferlite-like calcified structure includes a bottom layer, a top layer, and an interface layer, where the interface layer is located between the bottom layer and the top layer.

[0062] In one embodiment, the bottom layer does not contain apatite crystals. In a preferred embodiment, the bottom layer consists of a keratin organic matrix, or is substantially composed of a keratin organic matrix. In one embodiment, the bottom layer consists of carbon and sulfur, or is substantially composed of carbon and sulfur.

[0063] In one embodiment, the top layer includes a polycrystalline circular structure. In one embodiment, the top layer includes calcium, phosphorus, sulfur, carbon, and fluorine.

[0064] In one embodiment, the interface layer contains arched crystals. In one embodiment, the interface layer contains fluorine. In one embodiment, the interface layer has the highest fluorine content among the three layers.

[0065] In one embodiment, the interface layer and / or top layer comprises nanospheres, where the nanospheres comprise or substantially consist of a keratin organic matrix. In one embodiment, these nanospheres provide additional nucleation centers that promote the growth of further nanocrystals.

[0066] In one embodiment, the calcified spherulite contains rod-like (helical) structures of keratin.

[0067] A keratin solution containing keratin and a solvent is also provided. The keratin solution and its components may be the same as those described in connection with the method of the present invention.

[0068] Optionally, the keratin used in the method of the present invention may be obtained by extracting keratin from animal or human hair, body hair, wool, hooves, shells, horns, claws, nails, quills, or feathers. In particular, the keratin may be extracted from wool from sheep, goats, camelids, and rabbits, especially sheep.

[0069] Furthermore, a method for extracting keratin from animal or human tissues, particularly wool, such as hair, body hair, wool, hooves, shells, horns, claws, nails, quills, or feathers, is provided, and this method is... (a) A step of mixing the tissue with the extraction solution, (b) A step of heating the mixture, (c) A step of separating the supernatant containing keratin from the remainder of the mixture, Includes.

[0070] The extraction solution may optionally contain one or more of the following: urea, sodium dodecyl sulfate, 2-mercaptoethanol, thiourea, dithiothreitol (DTT), [3-(coramidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), L-cysteine, ampholite, glycerol, Triton X-100, sodium phosphate, sodium pyrosulfite, sodium hydroxide, peracetic acid, 1-butyl-3-methylimidazolium chloride (BMIM+Cl2), iodoacetic acid, zinc acetate, ethylenediaminetetraacetic acid (EDTA), trisaminomethane-hydrochloride (Tris-HCl) buffer, HEPES buffer, and other buffers.

[0071] The mixture may optionally be heated at 40-60°C, or 45-60°C, or 40-55°C, or 45-55°C, or approximately 50°C for approximately 8, 9, 10, 11, 12, 13, or 14 hours.

[0072] Optionally, the supernatant may be separated from the mixture by filtration, centrifugation, or a combination of both.

[0073] This method may also include a step of dialyzing the supernatant with water to obtain a keratin solution.

[0074] This method may also include a step of drying the keratin solution, particularly a step of freeze-drying.

[0075] Furthermore, keratin compositions, such as membranes containing keratin, are provided. In particular, the keratin composition contains keratinspherite. The composition may be calcified. Each aspect of the keratin composition may be the same as those described in relation to the method of the present invention. The keratin composition can be obtained or obtained using the method of the present invention.

[0076] Furthermore, pharmaceutical compositions containing a keratin composition or a calcified keratin composition according to the present invention are provided. The pharmaceutical composition may further contain a pharmaceutically acceptable carrier. Optionally, the pharmaceutical composition may be in the form of a hydrogel, solution, paint, varnish, coating, scaffold such as a 3D printed scaffold, film, toothpaste, strip, especially dental strip, dental restorative material, composite, gum, metal implant, cement, ceramic, paste, plastic putty, film, or dentin desensitizer, or may be added thereto.

[0077] Keratin solutions or compositions are also used in the preparation of devices and structures used in medical procedures. This is possible. Examples of medical structures or devices provided by the present invention include synthetic grafts, prostheses or orthoses, implants or tissue grafts, particularly dental or bone grafts, which include keratin solutions, keratin compositions, calcified keratin compositions or pharmaceutical compositions according to the present invention.

[0078] Furthermore, keratin solutions, keratin compositions, calcified keratin compositions, pharmaceutical compositions, structures, or devices according to the present invention are provided for use in treatment.

[0079] The solution, composition, structure, or device may be used in dental treatment, particularly in the treatment of WSL or caries. In particular, it may be used to treat or prevent tooth demineralization, dental erosion, tooth wear, alveolar bone resorption, periodontitis, peri-implantitis or pulp disease, tooth hypersensitivity, and soft tissue repair (e.g., grafting and augmentation). Alternatively, it may be used for bone repair, healing, or regeneration.

[0080] In one embodiment, the solution, composition, structure, or device is used to treat or restore defective enamel. In one embodiment, the solution, composition, structure, or device stimulates enamel reconstruction. In a preferred embodiment, the reconstructed enamel has an oriented prism structure. In a preferred embodiment, the reconstructed enamel has the same structure as healthy tooth enamel. In a preferred embodiment, the solution, composition, structure, or device treats or restores defective enamel over substantially the entire thickness of the lesion.

[0081] In one embodiment, the reconstructed enamel has a Knoop hardness exceeding 0.4 GPa, 0.5 GPa, 0.6 GPa, 0.7 GPa, 0.8 GPa, 0.9 GPa, 1.0 GPa, 1.1 GPa, 1.2 GPa, 1.3 GPa, 1.4 GPa, 1.5 GPa, 1.6 GPa, 1.7 GPa, 1.8 GPa, 1.9 GPa, or 2 GPa.

[0082] In one embodiment, the reconstructed enamel has a Young's modulus exceeding 10 GPa, 15 GPa, 20 GPa, 25 GPa, 30 GPa, 35 GPa, 40 GPa, 45 GPa, 46 GPa, 47 GPa, 48 GPa, 49 GPa, 50 GPa, 51 GPa, 52 GPa, or 53 GPa.

[0083] In one embodiment, the reconstructed enamel has a hardness exceeding 0.4 GPa, 0.5 GPa, 0.6 GPa, 0.7 GPa, 0.8 GPa, 0.9 GPa, 0.95 GPa, 1.0 GPa, or 1.05 GPa.

[0084] The solution, composition, structure, or device may be used for the treatment or prevention of bone loss, or for the repair of damaged bone.

[0085] Solutions, compositions, structures, or devices may be used to promote or induce bone healing, bone regeneration, and / or bone formation. In one embodiment, solutions, compositions, structures, or devices may be used to promote or induce bone healing, bone regeneration, and / or bone formation of the skull. In one embodiment, solutions, compositions, structures, or devices may be used to promote or induce bone healing, bone regeneration, and / or bone formation of the cranial vault.

[0086] Solutions, compositions, structures, or devices may be used to treat bone defects. In one embodiment, bone defects may be the result of trauma, infection, congenital abnormalities, pathological diseases, and / or premature tooth loss. That is the case.

[0087] In a preferred embodiment, the solution, composition, structure, or device enables bone conduction and / or bone induction signals that promote bone formation and thereby induce biomineralization.

[0088] For example, a solution, composition, structure, or device may be applied and hardened during surgery to replace bone with an implant, or to aid in calcification and subsequent healing.

[0089] Alternatively, a keratin solution may be dried into a specific shape and then applied to fill cavities within the bone to aid in bone remineralization and healing. The shape of the bone cavities can be determined using scanning techniques known in this field, such as computed tomography or magnetic resonance imaging, or by surgical observation.

[0090] The solution, composition, structure, or device may be used for a single treatment or multiple treatments at the same site. In one embodiment, the solution, composition, structure, or device is intended for periodic use on a tooth to promote or maintain a healthy enamel layer. In one embodiment, the solution, composition, structure, or device is intended for periodic application to a healthy tooth to prevent enamel loss.

[0091] The solution or composition may be used on its own or in combination with one or more other active ingredients.

[0092] Furthermore, a method is provided for treating WSL or dental caries, or bone injury or bone disorder, the method comprising administering a keratin composition, calcified keratin composition or pharmaceutical composition according to the present invention, or utilizing an implant or tissue graft according to the present invention.

[0093] From another perspective, the present invention provides uses for the keratin solution or composition according to the present invention for the manufacture of pharmaceuticals used to treat any of the diseases described above.

[0094] In yet another aspect, the present invention provides a product or kit comprising (a) a keratin solution or composition according to the present invention and (b) another calcification solution and / or crosslinking agent. The product may be a combination formulation used separately, simultaneously, or sequentially in treatment.

[0095] The solutions, compositions, structures, or devices of the present invention may be formulated and / or packaged to suit their intended use. For example, in dental applications, the solution or composition may be applied to the teeth with a brush, like paint or varnish. Alternatively, the solution or composition may be applied to the teeth via a dental tray. Such trays are commonly used in the art, for example, to apply solutions for teeth whitening. In a preferred embodiment, in dental applications, the solution may be applied as a spray. Optionally, the spray may be applied after cleaning the teeth, for example, by brushing. With regard to bone defects, the solution or composition may be applied directly to the cavity so that the structure fills the cavity.

[0096] The present invention further provides a kit comprising a keratin solution, keratin composition, or pharmaceutical composition according to the present invention, and an applicator for applying the solution or composition to a site of interest. The applicator may be, for example, a brush, pad, spray, dropper, or dental tray. The kit may further include instructions on how to use the components.

[0097] The solution, composition, structure, or device of the present invention is for any animal having teeth and / or bones. It is intended to be used in relation to the following. In one embodiment, the animal is a mammal, preferably a human.

[0098] Throughout this specification and its claims, the words “comprise” and “contain,” and their variations, such as “comprising” and “comprises,” mean “including, but not limited to,” and do not exclude other components, numbers, or processes. Furthermore, unless the context requires otherwise, the singular form includes the plural form. In particular, where the indefinite article is used, this specification should be understood to assume both the singular and the plural, unless the context requires otherwise.

[0099] Preferred features of each aspect of the present invention may be the same as those described in relation to any other aspect. Within the scope of this application, the various aspects, embodiments, examples and alternatives described in the preceding paragraphs, claims and / or the following description and drawings, in particular their individual features, are expressly intended to be adopted independently or in any combination. That is, all embodiments and / or features of any embodiment can be combined in any way and / or combination unless such features are incompatible.

[0100] Hereinafter, one or more embodiments of the present invention will be described by illustration only, with reference to the attached drawings. [Brief explanation of the drawing]

[0101] [Figure 1] This diagram shows keratin extraction using a reduction process. The keratin source is obtained, for example, from wool (1). The keratin source is degreased by Soxhlet extraction (2). The keratin source is then filtered (3) and centrifuged (4). The supernatant is separated (5) and dialyzed (6) to obtain a keratin solution (7). [Figure 2a] This figure shows the in vitro process for preparing a keratin membrane. After keratin extraction, the keratin is dissolved in one of the following (1): Milli-Q water (pH 7-11), dimethyl sulfoxide (DMSO), or dimethylformamide (DMF). Subsequently, a crosslinking agent such as triethylene glycol dimethacrylate (TEGDMA), hexamethylene diisocyanate (HDI), or polyethylene glycol dimethacrylate (PEGDMA) may be optionally added (2). The solution is then dropped (3) onto a polydimethylsiloxane (PDMS) substrate (4) and left to dry. [Figure 2b] This figure shows images of keratin membranes formed in vitro using keratin solutions of various concentrations and triethylene glycol dimethacrylate (TEGDMA) crosslinking agents of various concentrations. "K5" represents a 5 wt% keratin solution, and "K10" represents a 10 wt% keratin solution. "TE10" indicates that the ratio of TEGDMA crosslinking agent to lysine and cysteine ​​amino acid residues in keratin is 1:10 (corresponding to a ratio of 100 μl keratin to 3.74 μl TEGDMA), and "TE20" indicates that the ratio of TEGDMA crosslinking agent to lysine and cysteine ​​amino acid residues in keratin is 1:20 (corresponding to a ratio of 100 μl keratin to 7.48 μl TEGDMA). If "TE" is not indicated (i.e., "K5" or "K10"), it indicates that no crosslinking agent was used. Handling score: 1 = difficult, 2 = moderate, 3 = easy. Likelihood of collapse: 2 = moderate, 3 = low. Fragility: 1 = High, 2 = Medium, 3 = Low. Opacity: 1 = Opaque, 2 = Medium opaque, 3 = Transparent. [Figure 3]This figure shows the SDS-PAGE of keratin aqueous solutions. Figure 3a shows the bands at various concentrations for two different batches. Bands corresponding to low-sulfur (LS) keratin (1), high-sulfur (HSPs) keratin (2), and high-glycine-tyrosine (HGT) keratin (3) are observed. Figure 3b shows that a solution of artificially expressed keratin 75 (KR75) yields a single band. [Figure 4] These are scanning electron microscope images of keratin fibers observed in a keratin film prepared with 5% by weight keratin (a) or 10% by weight keratin (b) using triethylene glycol dimethacrylate (TEGDMA) crosslinking agent. [Figure 5] This figure shows optical microscope images of keratin films. Figure 5a is a polarized light microscope image of a keratin film prepared by dissolving 5% by weight of wool-derived keratin in water only. The inset shows a high-magnification image of organic spherulite. Figure 5b is a white light microscope image of a keratin film prepared by dissolving 5% by weight of keratin 75 (KR75) in water only. [Figure 6] This figure shows scanning electron microscope images of biomimetic calcification. Figures 6a to 6f show the stages of membrane calcification over 14 days in a membrane formed from 10% by weight keratin and triethylene glycol dimethacrylate (TEGDMA) crosslinking agent (the ratio of crosslinking agent to lysine and cysteine ​​amino acid residues in keratin is 1:10 (corresponding to a ratio of 100 μl keratin to 3.74 μl TEGDMA)). Figures 6g and 6h show cross-sections of the membrane showing the calcified crystals. Furthermore, images of keratin films made from 5% by weight keratin (Figure 6i) or 10% by weight keratin (Figure 6j) prepared using a TEGDMA crosslinking agent (ratio of crosslinking agent to lysine and cysteine ​​amino acid residues in keratin: 1:10), 5% by weight keratin (Figure 6k) with polyethylene glycol dimethacrylate (PEGDMA) crosslinking agent and 3% by weight lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) photoinitiator added, and 5% by weight keratin (Figure 6l) without crosslinking agent are also shown. [Figure 7]This figure shows the hierarchical calcification of keratin-based films. Figure 7a is an image of a keratin film before calcification. Figure 7b is a polarized light microscope image of an organic keratin film showing organic sferrite on its surface. The inset shows a high-magnification image of keratin sferrite with a characteristic birefringent Maltese cross pattern. Figure 7c is a scanning electron microscope (SEM) image of organic sferrite before calcification. Figure 7d is an SEM image showing that keratin is self-organizing into densely packed nanofibrils. Figure 7e is an atomic force microscope (AFM) image showing the nanofibrils in three dimensions. Figure 7f is an SEM image of a calcified keratin film showing calcified sferrite growing on the surface. The inset shows inorganic sferrite observed under a polarizing filter, showing the size increase after calcification. High-resolution transmission electron microscope (HRTEM) images showing the growth direction of apatite crystals and the d-spacing of the crystal lattice are shown. Figures 7g to 7i show SEM images of calcified sferrite from the film surface at various length scales, illustrating the surface properties of the calcified sferrite. These images show nanocrystals growing spirally from the sferrite center toward the film surface. Figure 7j is a high-resolution transmission electron microscope (HR-TEM) image of the calcified structure obtained from focused ion beam (FIB) milling liftout, showing apatite crystals from the film surface. Figure 7k is a selected-region electron diffraction (SAED) image of the surface apatite crystals. Figures 7l to 7n show SEM images of the film cross-section from low to high magnification, illustrating the bulk properties of the calcified film, showing apatite nanocrystals oriented parallel to each other. Figure 7o is an HR-TEM image showing the growth direction of the apatite crystals and the d-spacing of the crystal lattice. Figure 7p is a SAED image of the bulk apatite crystals. Figure 7q shows the Fourier transform infrared (FTIR) spectra of the calcified film at three different time points (day 1, day 3, and day 7). Figure 7r shows the 19F solid-state MAS-NMR spectrum. Figure 7s shows the 31P MAS-NMR chemical shift. Figure 7t shows the Young's modulus and hardness measurements of the keratin film before and after calcification, with a statistically significant difference of p<0.05. [Figure 8]This figure shows the calcification pattern of keratin over time. Figure 8a is a polarized light microscope image of keratin calcified spherulite on day 1 of calcification. Figure 8b is an optical light microscope image of spherulite under polarized light on day 1. The inset shows an SEM image of a brushite-like crystal. Figure 8c shows keratin calcified spherulite on day 2 of calcification. Figure 8d is a polarized light microscope image of spherulite on day 2. Figure 8e shows the mineral coating on day 10 of calcification, showing a disordered, circular, calcified structure, which suggests that when keratin is inaccessible, calcification deposits on top without showing an ordered pattern. [Figure 9] This figure shows the crystal properties of a keratin-induced calcification structure. Figure 9a shows a high-resolution transmission electron microscope (HR-TEM) image of the calcification structure obtained from focused ion beam (FIB) milling liftout, showing three different zones: the upper calcification zone, the intermediate interface zone, and the lower organic zone. The inset shows a selected-region electron diffraction (SAED) image of the organic layer. Figures 9b and 9c show SAED images of the upper and intermediate zones, respectively. Figures 9d to 9h show HR-TEM images of different regions on the FIB lamellae corresponding to the numbers shown in Figure 9a, showing the d-spacing of the crystal lattice confirming the apatite phase. Figure 9i is an energy-dispersive X-ray analysis (EDX) image of the FIB cross section, showing the compound distribution in the three layers. [Figure 10] This figure shows the preparation and characterization of keratin films. Figures 10a and 10b show atomic force microscope (AFM) images of 5% and 10% by weight keratin solutions. Figure 10c shows a scanning electron microscope (SEM) image of a 3% keratin film. Figure 10d shows the quantitative evaluation of fibrils in the keratin film. Figure 10e shows 3% by weight keratin and triethylene glycol dimethacrylate (TEGDMA) crosslinking agent (ratio of crosslinking agent to lysine and cysteine ​​amino acid residues in keratin: 1:10). Figures 10f and 10g show polarized light microscope images of the spherulite arrangement pattern. Figure 10h is a Congo red stained image of the keratin film showing its organic structure. [Figure 11]This figure shows the characterization of keratin in solution. Figures 11a to 11d show the self-assembly of keratin films. Figure 11a shows 5 wt% keratin, Figure 11b shows 11 wt% keratin, Figure 11c shows 5 wt% keratin and triethylene glycol dimethacrylate (TEGDMA) crosslinking agent (ratio of crosslinking agent to lysine and cysteine ​​amino acid residues in keratin: 1:11), and Figure 11d shows 11 wt% keratin and TEGDMA crosslinking agent (same ratio of 1:11). Figure 11e shows the disorder-to-order restoration of keratin, showing the deconvolution of attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) spectra in the amide I region for two different films, a 3 wt% keratin film and an 11 wt% keratin film. It shows that as keratin concentration increases, the β-sheet structure increases and random coils decrease, and in the corresponding polarized light microscope images, a more ordered spherulite pattern is shown with increasing keratin concentration. Figure 11f shows Fourier transform infrared (FTIR) imaging of an organic film, indicating the regions where spherulites were imaged using an optical microscope. FTIR amide I (1600–1700 cm⁻¹) and amide III (1200–1350 cm⁻¹) spectra corresponding to four different regions of the film are outlined and measured on a chemical map, and the corresponding rates of secondary structure deconvolution are shown in the corresponding pie charts. [Figure 12] This figure shows the organic-inorganic mechanism and the possibility of controlling calcification. Figure 12a shows the attenuated total reflectance Fourier transform infrared (ATR-FTIR) deconvolution of the secondary structure in uncrosslinked (Figure 12a) or crosslinked (Figure 12b) organic keratin films. [Figure 13]This figure shows the characterization of inorganic sferrite. Figure 13a is a Fourier transform infrared (FTIR) spectroscopy imaging of a calcified film, where the heatmap on the left shows the distribution of the vibrational band region of phosphate groups (900-1100 cm-1), and the heatmap on the right shows the amide I band region of proteins (1600-1700 cm-1). Spectra were acquired in four different regions of the calcified keratin film depending on their proximity to proteins / minerals, and the corresponding secondary structure deconvolutions are shown in the corresponding pie charts. Figure 13b is a small-angle X-ray scattering (SAXS) scattering curve originating from an organic film, showing a peak indicating a coiled structure. Figure 13c is a SAXS scattering curve originating from a non-calcified film, showing a peak corresponding to a rod-like structure. Figure 13d is the SAXS diffraction pattern of calcified sferrite on a calcified film. Figure 13e shows the scattering curves for each corresponding color in Figure 13d. [Figure 14] This figure shows the cell viability and RNA expression of human bone marrow-derived stem cells cultured on membranes formed using one of the following: (i) 5% keratin solution (Group 1), (ii) 5% keratin solution crosslinked with triethylene glycol dimethacrylate (TEGDMA) (Group 2), (iii) 5% keratin solution crosslinked with TEGDMA followed by exposure to a calcification solution (Group 3), (iv) 5% keratin solution crosslinked with hexamethylene diisocyanate (HDI) (Group 4), or BioHorizons' Mem-Lok® absorbable collagen matrix. Cells were cultured for 7, 14, or 21 days before viability evaluation. Cells survived when cultured in mesenchymal stem cell growth medium (Figure 14a) or osteoblast growth medium (OGM) (Figure 14b). Furthermore, after 28 days of culture, the cells showed messenger RNA expression of runt-related transcription factor 2 (RUNX2) (Figure 14c), alkaline phosphatase (ALP) (Figure 14d), Osterix (OSX) (Figure 14e), bone morphogenetic protein 2 (BMP-2) (Figure 14f), osteocalcin (OCN) (Figure 14g), and osteopontin (OPN) (Figure 14h). *, **, ***, and **** indicate statistically significant differences of p<0.05, p<0.01, p<0.001, and p<0.0001, respectively. [Figure 15] This figure shows the improvement in Knoop hardness and structural integrity after drop-cast treatment of leukoplakia lesions (WSL) with either (i) a 5% keratin solution (K5), (ii) a 5% keratin solution crosslinked with triethylene glycol dimethacrylate (TEGDMA) (K5TE10), or (iii) ICON® resin infiltrate (positive control) (ICON). Knoop hardness was evaluated in deionized water (DW) (Figure 15a), artificial saliva (AS) (Figure 15b), or calcification solution (MS) (Figure 15c). The improvement in structural integrity can be visually confirmed by comparing the surface images of a healthy enamel surface in DW (Figure 15d), an untreated WSL in DW (Figure 15e), a WSL treated with a 5% keratin solution in DW (Figure 15f), a WSL treated with a 5% keratin solution in AS (Figure 15g), and a WSL treated with a 5% keratin solution in MS (Figure 15h) using scanning electron microscopy. The inset in (d) shows the boundary line separating the keratin-treated WSL from the untreated WSL. ns, **, and **** represent the differences when p>0.05, p<0.01, and p<0.0001, respectively. [Figure 16] This diagram schematically illustrates the artificial induction of vitiligo (WSL). (1) shows 0.1M lactate at pH 4.6, (2) shows filter paper, and (3) shows 8% methylcellulose gel. [Figure 17] This figure shows a WSL sample artificially induced in enamel. [Figure 18] This diagram schematically illustrates a pixel sampling method across the entire sample. (A) is the original sample image. (B) shows a dotted circle indicating the reference enamel control area of ​​the lesion. (C) shows a dotted circle indicating the intervention area of ​​the lesion. (D) shows a dotted circle indicating the WSL control area of ​​the lesion. [Figure 19] This figure shows the measurement protocol for optical coherence tomography. (A) shows the cross-sectional surface area of ​​the lesion WSL traced with a dotted line (top of the image), and the grayscale value of this region is recorded. (B) shows the lesion region from (A) copied onto the intervention lesion. (C) shows the surface area of ​​the lesion where the intervention was traced and recorded. [Figure 20]This figure shows images taken using white light microscopy (WLM) (left) and optical coherence tomography (right) before (lower part of WLM image) and after (upper part of WLM image) treatment with uncrosslinked 5% keratin. The sample was in deionized water (Figure 20a) or artificial saliva (Figure 20b). [Figure 21] This figure shows images taken using a white light microscope (WLM) (left) and optical coherence tomography (OMCT) (right) before (lower part of the WLM image) and after (upper part of the WLM image) treatment with 5% keratin crosslinked with triethylene glycol dimethacrylate (TEGDMA). The sample was present in deionized water (Figure 21a) or artificial saliva (Figure 21b). [Figure 22] This figure shows white light microscope (WLM) (left) and optical coherence tomography (OMCT) images (right) before (lower part of WLM image) and after (upper part of WLM image) treatment with ICON® resin infiltrate (positive control). The sample was in deionized water (Figure 22a) or artificial saliva (Figure 22b). [Figure 23] This figure shows the grayscale levels (calculated from optical coherence tomography) before and after treatment with ICON® resin infiltrate (positive control) in both deionized water (DW) and artificial saliva (AS). Higher grayscale values ​​indicate a higher severity of WSL. [Figure 24] This figure shows the grayscale levels (calculated from optical coherence tomography) before and after treatment with 5% keratin without crosslinking agents in both deionized water (DW) and artificial saliva (AS). Higher grayscale values ​​indicate a higher severity of WSL. [Figure 25] This figure shows the grayscale levels (calculated from optical coherence tomography) before and after treatment with 5% keratin using a TEGDMA crosslinking agent in both deionized water (DW) and artificial saliva (AS). Higher grayscale values ​​indicate a higher severity of WSL. [Figure 26] This figure compares the grayscale levels (calculated from optical coherence tomography) of a control and treatments with ICON® resin infiltrate, 5% keratin without a crosslinking agent, and 5% keratin with a TEGDMA crosslinking agent in deionized water. [Figure 27] This figure compares the grayscale levels (calculated from optical coherence tomography) of artificial saliva treated with a control, ICON® resin infiltrate, 5% keratin without a crosslinking agent, and 5% keratin with a TEGDMA crosslinking agent. [Figure 28]This figure shows data from preclinical studies of white spot lesions (WSL). Figures 28a and 28b show surface and cross-sectional images of natural polished enamel, respectively. Figures 28c and 28d show surface and cross-sectional images of induced untreated WSL, respectively. Figure 28e shows the interface between healthy and poor enamel, showing the lost prism / interprism continuum and pores formed between prisms. Figure 28f shows the treatment window on the buccal surface of a premolar, with WSL exhibiting the characteristic chalky white appearance on the right and a keratin-treated lesion with reduced opacity on the left, demonstrating the remineralization ability of keratin. Figure 28g shows the interface between WSL and a keratin-treated lesion, showing the boundary line between the untreated and treated lesions, and the presence of a repair layer. Figure 28h is a surface image of a keratin-treated lesion showing newly grown prism-like enamel that has filled the pores. Figure 28i is a cross-section of Figure 28g, showing newly formed enamel by keratin treatment on the left and the prism that disappeared in WSL on the right. Figure 28j shows the pores of the enamel formed by WSL induction, with dotted arrows indicating the relationship between the pore location and the c-axis. As a keratin-treated lesion incubated in a calcification solution, Figure 28k shows reconstructed enamel with pores filled by keratin. Figure 28l is a high-magnification SEM image showing keratin infiltration into the WSL in an ordered pattern, with some crystals having the same orientation as the prism along the c-axis and other crystals oriented perpendicular to it. Figure 28m shows the repaired enamel in a region approximately 50 μm away from the enamel surface, where the pores are repaired and some areas appear to be filled with organic matrix. Figure 28n shows a surface image of a keratin-treated lesion incubated in artificial saliva, illustrating the boundary between treated and untreated WSLs, with the inset showing nearly gapless keratin-repaired enamel. Figure 28o shows the boundary line separating the repaired enamel (upper part) from the lesion gap (lower part) at a depth of approximately 100 μm from the surface. Figure 28p is a high-magnification image of keratin-treated repaired enamel, showing elongated crystals filling the lesion gap, their orientations parallel to each other.Figures 28q and 28r are high-resolution transmission electron microscope (HR-TEM) images of FIB lamellae, showing continuous, non-porous enamel. Figure 28s is a selected-region electron diffraction (SAED) image of the repaired area. Figure 28t shows microhardness analysis. Figure 28u shows elastic modulus and hardness measured by nanoindentation, with a statistically significant difference of p<0.0001. P: prism, IP: between prisms, UPW: ultrapure water, MS: calcification solution, AS: artificial saliva. [Figure 29] This figure shows the surface characterization of treated enamel lesions. Figure 29a shows the integration of keratin and newly formed enamel-like crystals, indicating that they are attached to the organic matrix and that the matrix appears to be inducing crystal growth. Figure 29b shows the keratin coating in ultrapure water (UPW) as a keratin-treated WSL incubated in artificial saliva. Figure 29c shows the calcification pattern of an untreated WSL in a calcification solution. Figures 29d to 29f show resin-treated lesions incubated in a calcification solution, indicating the presence of calcified precipitates on top of the resin coating. [Figure 30] This figure shows focused ion beam (FIB) lift-out and mechanical characterization. Figures 30a and 30b are high-resolution transmission electron microscope (HR-TEM) images of keratin-treated WSLs. Figure 30c is the selected-area electron diffraction (SAED) pattern of the keratin-treated lesion at a distance of 50 μm from the surface. Figure 30d shows the bulk nanoindentation results of WSLs before and after treatment, with a statistically significant difference of p<0.0001. [Figure 31]This figure shows the regeneration of a rat cranial defect in vivo. Figure 31a is a schematic diagram showing the surgical procedure performed. Figure 31b shows the rat cranial defect before bone removal. Figure 31c shows the rat cranial defect after bone removal. Figure 31d shows the rat cranial defect after application of keratin film. Figures 31e to 31p show X-ray images (Figures 31e to 31j) and micro-CT three-dimensional reconstructions (Figures 31k to 31p) with a 6mm scale bar, showing empty defects (Figures 31e, 31k), Ker5 (Figures 31f, 31l), Ker5TE1 (Figures 31g, 31m), M-Ker5TE1 (Figures 31h, 31n), Ker5H3 (Figures 31i, 31o), and collagen membranes (Figures 31j, 31p). Figures 31q to 31u show the parameters measured by micro-CT, namely bone volume (BV) (mm³) (Figure 31q), the ratio of bone volume to trabecular volume (BV / TV) (%) (Figure 31r), trabecular thickness (Tb.Th) (mm) (Figure 31s), number of trabeculae (TN) (mm⁻¹) (Figure 31t), and trabecular spacing (mm) (Figure 31u). [Modes for carrying out the invention] [Examples]

[0102] [Example 1] Keratin extraction, production of keratin 75, and in vitro membrane formation [Keratin Extraction] The extraction process is shown in Figure 1. The wool was thoroughly washed with deionized water and dried. Since the wool (and other keratin sources such as hair) is covered with a thin covalent lipid layer, it was degreased by Soxhlet extraction for 6 hours using reflux with hexane and dichloromethane in a 1:1 v / v ratio. This improves the extractability of keratin. The washed wool (10 g) was mixed in a 300 mL round-bottom flask with 7 M urea (180 mL), sodium dodecyl sulfate (SDS) (0.021 mol, 6 g), and 2-mercaptoethanol (0.21 mol, 15 mL). The mixture was heated at 60 °C for 24 hours with continuous stirring, maintaining a neutral pH range. 2-mercaptoethanol helps solubilize keratin by cleaving disulfide bonds between cysteine ​​residues in the amino acid chain. By increasing the temperature, protons are removed from the -NH3 group, and hydrogen bonds are cleaved. Urea was used at high concentrations to weaken the hydrophobic interactions between keratin fibers, causing them to swell and thus facilitating the reduction of disulfide bonds by 2-mercaptoethanol. This increased the rate at which the reduction buffer diffused into the fibers and the rate at which the protein diffused out of the fibers. SDS was used to cleave strong intermolecular interactions and increase the extraction rate, and also contributed to the stability of the aqueous solution.

[0103] The resulting mixture was filtered through a 120-mesh stainless steel sieve and then centrifuged at 6,000 rpm for 30 minutes. The supernatant was then dialyzed against deionized water (3.5 L) for 3 days (5 kDa cutoff), with the extra solution being changed periodically (2-3 times) until a colorless, clear solution was obtained. Aliquots of the reduced keratin solution were stored in a freezer at -80°C for 4 hours, then freeze-dried (VirTis SP Scientific Sentry 2.0, Ipswich, UK) until a fine freeze-dried white interpenetrating fibrous powder remained.

[0104] [Expression and purification of keratin 75 in Escherichia coli] Keratin 75 (KR75) was produced in Rosetta BL21 Escherichia coli cells (Novagen) using a plasmid pET-15b(+) expression system (Synbio Technologies, LLC) into which an HRV 3C protease site was introduced, and constructed with the addition of six histidine tags. The inoculum contained 100 mg / ml of the selected transformant. -1 The culture medium was prepared by culturing in ampicillin-containing LB broth, and then incubated at 37°C for 4 hours until an OD of 600 nm ≈ 0.6 was reached. Recombinant protein expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) overnight at 18°C ​​under shaking culture. Cells were harvested by centrifugation at 6,000 g for 30 minutes, resuspended in 0.05 M Tris buffer (pH 6.0), and then lysed by sonication. The sonicated mixture was centrifuged at 7,000 rpm for 30 minutes. The cell pellet containing expressed KRT75 was washed four times with a pH 7.5 wash buffer containing 0.05 M Tris-Cl, 5 mM EDTA, and 0.5% Triton-X100 to remove cell debris.

[0105] Subsequently, the cell pellet was solubilized in 0.05 M Tris buffer (pH 12) containing 1 M urea and 2 mM DTT. The resulting mixture was dialyzed against 0.05 M Tris buffer (pH 7.5) using a dialysis bag (12-14 kDa cutoff). The isolated proteins were purified using a HisPur® Ni-NTA spin column (Thermo Scientific®). Subsequently, to remove imidazole, the column was dialyzed against Milli-Q water (pH 7.0) and then freeze-dried at -60°C (VirTis, SP Scientific Sentry 2.0) to obtain KRT75 powder.

[0106] [In vitro membrane formation] After keratin extraction or production, the lyophilized keratin powder was dissolved in Milli-Q water (pH 7-11), dimethyl sulfoxide (DMSO), or dimethylformamide (DMF) at a concentration of 2-10% by weight. The resulting solution was either a non-crosslinked solution without a crosslinking agent, or it was crosslinked with triethylene glycol dimethacrylate (TEGDMA), hexamethylene diisocyanate (HDI), or polyethylene glycol dimethacrylate (PEGDMA) (with the addition of 3% by weight of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) photoinitiator). The solution was then dropwise cast onto a polydimethylsiloxane (PDMS) substrate and left to dry at room temperature, 37°C, or 45°C to induce self-assembly (Figure 2a).

[0107] Figure 2b shows various films that can be formed when parameters such as the keratin concentration of the starting keratin solution, the presence or absence of crosslinking agent use, and the crosslinking agent concentration are varied. Films formed with a crosslinking agent required 10-15 minutes to dry, while films formed without a crosslinking agent required 15-20 minutes to dry.

[0108] [Example 2] Analysis of keratin diversity 〔method〕 To determine the molecular weight of the keratin structure after self-assembly, the samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Each aliquot of LDS sample buffer (10 μL) was mixed with 20 μL of all soluble fractions and 1 mg / mL powder diluent, and then heated at 70°C for 10 minutes. Next, 20 μL of each solution was loaded into 10 lanes of a 4-12% Bis-Tris NuPAGE® precast polyacrylamide gel (Thermo Fisher Scientific, UK) using ethanesulfonic acid (MES buffer). Electrophoresis was performed for 40 minutes under conditions of 200 V voltage and 125 mA current. The gel was then sieved. The samples were stained with a mixture of 2 volumes of Lilian Blue and 3 volumes of 10% acetic acid for 25 minutes, followed by treatment overnight with 10% acetic acid and deionized water.

[0109] Further confirmatory analyses were performed to validate the results of the previous SDS-PAGE. The SDS-PAGE bands of the purified protein were subjected to MS analysis to confirm KRT75 protein expression. Gel bands were prepared for enzymatic digestion. Cys residues were reduced with DTT and derivatized to specific carbamide methyl derivatives via iodoacetamide. Trypsin digestion was performed overnight at room temperature after a 2-hour preliminary incubation at 37°C. Raw mass spectrometer data were imported into a peak list folder using Proteome Discoverer (Thermo Scientific; v2.5). The data files were processed and searched against the Uniprot Human Taxonomy database (50,590 entries) using the Sequest (Eng et al.; PMID 24226387) search algorithm. Database searches were performed with a strictness of FDR 1%, including decoy searches. The database output files were uploaded to Scaffold software (version 5.1.2; www.proteomesoftware.com) for visualization and manual validation.

[0110] 〔result〕 Figure 3a shows that keratin obtained from wool by the method described in Example 1 yields a wide variety of keratin with different molecular weights. The figure shows distinct bands of 45-50 kDa corresponding to type I keratin and distinct bands of 55-60 kDa corresponding to type II keratin. These low-sulfur (LS) keratins form intermediate filaments in the wool fiber cortex. A series of high-sulfur (HSPs) keratins (12-28 kDa) and high-glycine-tyrosine (HGT) keratins (less than 10 kDa) are also observed, and these keratins originate from the wool matrix.

[0111] In contrast, artificially expressed keratin 75 yielded a single molecular weight keratin, as expected (Figure 3b).

[0112] [Example 3] Imaging of keratin structure and calcification 〔method〕 To induce protein-mediated calcification in the membrane, a supersaturated calcium-phosphate calcification solution was prepared by adding 2 mM hydroxyapatite (HAP) powder and 2 mM sodium fluoride to 100 mL of deionized water under continuous stirring. To completely dissolve the powder, 69% nitric acid was gradually added dropwise to the solution until the powder was completely dissolved. Then, 30% ammonium hydroxide solution was added dropwise until the pH reached 6. The crosslinked keratin membrane was incubated in 50 mL of HAP solution at 37°C for 3–5 days. After calcification, the membrane was removed from the solution, thoroughly washed with deionized water, and stored at room temperature until evaluation.

[0113] The morphology of self-assembled keratin fibrils was analyzed by scanning electron microscopy (SEM). The keratin film was fixed onto an aluminum stub using double-sided carbon tape. The sample was sputtered with an 8 nm conductive gold coating (Leica EM ACE600 sputter coater, Milton Keynes, UK). SEM images were acquired using a Schottky field emission JSM-7610F SEM (JEOL Ltd., Hertfordshire, UK), and surface morphology was observed using a secondary electron detector.

[0114] The film was observed using a cross-polarized light microscope (GXM-XPLPOLTEC-5, UK) with 4x and 10x air objectives, under conditions where the polarizer and analyzer were orthogonal to each other.

[0115] 〔result〕 For some of the membranes, the resulting solutions were crosslinked with triethylene glycol dimethacrylate (TEGDMA) with varying molar ratios of cysteine ​​and lysine residues in keratin to control membrane rigidity and co-self-assembly. These crosslinked membranes could self-assemble into nanofibers with diameters of 100-500 nm. Increasing the keratin concentration from 5% by weight (Figure 4a) to 10% by weight (Figure 4b) while maintaining other conditions similarly enabled more orderly self-assembly. With increasing keratin concentration, it was observed that the more orderly self-assembled fibers aggregated into bundles.

[0116] Polarized light microscopy revealed the formation of spherulite structures (appearing as a Maltese cross shape) from wool-derived keratin (Figure 5). Spherulite is an anisotropic polycrystalline structure formed by the self-organization of crystalline lamellae to form a spherical superstructure, within which lamellar sheets grow radially from the center. Spherulite was observed in both 5% and 10% keratin, and with and without the crosslinking agent. In contrast, spherulite was not observed when an equivalent film was prepared from keratin 75.

[0117] Figures 6a to 6h illustrate how calcification progresses and how sferlites can function as nucleation sites mediating ordered calcification patterns.

[0118] In vitro calcification generated nanocrystals, and it was shown that keratin nanofibers functioned as nucleation sites for biomimetic calcification under keratin concentrations of 5% (Figure 6i) and 10% (Figure 6j) in the presence of triethylene glycol dimethacrylate (TEGDMA) crosslinking agent.

[0119] Figure 6k shows the calcification of keratin crosslinked with polyethylene glycol dimethacrylate (PEGDMA) after the addition of 3 wt% lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) photoinitiator. Figure 6l shows the calcification of a keratin film formed without the use of a crosslinking agent. Spherulite is thought to act as nucleation sites.

[0120] [Example 4] Mineral nucleation and crystal growth in keratin film Natural keratin was extracted, and the resulting powder was solubilized in ultrapure water (UPW) at concentrations of 3%, 5%, and 10% w / v. Subsequently, the film was air-dried to promote self-crosslinking of cysteine ​​residues and reconstruct the disulfide bonds, thereby making the film insoluble.

[0121] By introducing functional groups into the protein backbone, reactivity and supramolecular properties can be improved. Therefore, in addition to the group consisting solely of keratin, films were also prepared by mixing solubilized keratin with triethylene glycol dimethacrylate (TEGDMA) and treating the mixture.

[0122] Organic matrices for keratin-based films were investigated.

[0123] Figure 7a shows the keratin film before calcification.

[0124] Figures 7b and 7c show the network of birefringent organic spherulites found in all keratin-based films, and Figures 7d and 7e show the microfibril structure within their matrix. Spherulites are densely packed crystalline aggregates of chains and are thought to contribute to the strength and rigidity of proteins. These spherulites have a diameter in the range of 1 to 10 μm and are characterized by exhibiting a Maltese cross pattern when observed between polarizing filters.

[0125] Next, these films were incubated at 37°C in a supersaturated hydroxyapatite solution containing a high concentration of fluorine, and their calcification ability was evaluated. The calcification pattern was tracked over time using scanning electron microscopy (SEM).

[0126] The samples were fixed onto aluminum stubs via carbon adhesive tape and coated with a 10 nm thick gold coating using an automated sputter coating system (Leica EM ACE600, Milton Keynes, UK). SEM images were acquired using a Schottky field emission JSM-7610F SEM (JEOL Ltd., Hertfordshire, UK) operated at 10 kV, and surface morphology was observed using a secondary electron detector. The images were then imported into ImageJ (NIH, USA) for quantitative analysis. Induced enamel lesions were prepared by etching with 35% phosphoric acid for 20 seconds and washing for 20 seconds to remove the smear layer.

[0127] Figure 7f shows the sferlite-like calcification structure detected under SEM.

[0128] Figures 8a and 8b show that on the first day of calcification, the number of spherulites is small, and several platy crystals are present.

[0129] Figures 8c and 8d show that on the second day, some apatite crystals begin to grow, and as the number of sferlite increases, the platy crystals begin to disappear.

[0130] Figures 7g to 7i show that on day 3, the calcified spherulite structure exhibits a helical orientation morphology composed of needle-shaped mineral nanocrystals, appearing to be composed of oriented prism-like structures with a thickness of 1.36 ± 0.33 μm and a length of several tens of micrometers. These prisms are composed of elongated nanocrystals, with an average diameter of 83 ± 22.96 nm, starting from the center of the spherulite and ending at a single point on the periphery of the spherulite.

[0131] Figures 7l to 7n show that within the bulk of the calcified structure, the prisms are composed of elongated apatite nanocrystals and are oriented in a more ordered parallel arrangement.

[0132] High-resolution transmission electron microscopy was performed. Lamellars prepared by FIB were Ultra-X The samples were evaluated using a Thermo Fisher 60-300kV Spectra Ultra TEM equipped with an EDS detector and a Cs aberration corrector. Images were acquired using a 4k×4k Ceta-S detector. Data were post-processed using Velox® software (version 3.8). The obtained images were analyzed using Gatan Microscopy Suite® (GMS3) software. To analyze the crystalline phases present in the samples, the d values ​​obtained from the SAED patterns were compared with the PDF2 database (ICDD, USA, 2009 edition).

[0133] Figures 7j and 7o show the apatite phase confirmed by high-resolution transmission electron microscopy (HR-TEM) and selected-region electron diffraction (SAED). Further analysis of crystallite d-spacing data confirmed the presence of apatite crystals on both the surface and cross-section of the calcified sferrite.

[0134] Figure 7k shows the SAED data analysis illustrating the polycrystalline pattern corresponding to the apatite on the film surface.

[0135] Figure 7p shows a crystal pattern exhibiting an arch shape at 002 in the bulk. This arch shape is thought to be due to the crystal structure and preferred orientation within the keratin bulk.

[0136] [Example 5] Evaluation of the chemical and mechanical properties of the calcified film To investigate the mineral compounds in the film, Fourier transform infrared spectroscopy (FTIR) analysis was performed at three different time points: day 1, day 3, and day 7 of calcification.

[0137] FTIR analysis was performed using Spectrum One, which is equipped with a white light source and an InGaAs detector. Spectroscopy was performed on keratin solutions and films using an FTIR spectrometer (PerkinElmer®, Buckinghamshire, UK) in ATR mode. Spectra were obtained by averaging 32 scans for each sample, with a resolution of 2 cm. -1 And the wave number is 4000-700cm -1 The spectrum was obtained within the following range: Amide I spectral region (1700-1600 cm⁻¹). -1 The keratin secondary structure composition was analyzed using OriginPro 8.5 software (Microcal Inc.) to compare the results. The spectral band assignments were as follows: 1610–1627 cm⁻¹. -1 : Intermolecular β-sheet, 1628~1642 cm -1 :β-sheet, 1643~1650cm -1 , 1643~1650cm -1 Random coil, 1650~1659cm -1 : α-helix, 1643~1650cm -1 , 1660~1699cm -1 :β-sheet / β-turn.

[0138] Magic angle rotation nuclear magnetic resonance (MAS-NMR) spectroscopy was also performed. Solid-state 19F MAS-NMR analysis was performed using a 14.1 Tesla spectrometer (600 MHz, Bruker, Coventry, UK) with a 2.5 mm rotor, 22 kHz spinning conditions, and a Larmor frequency of 564.5 MHz to investigate fluorine and phosphorus compounds in calcified films. All samples were pulverized into fine powder using a Gyro mill (Glen Cresto, London, UK) before analysis. Spectra were obtained from single-pulse experiments with a 60-second recycle time. The 19F chemical shift scale was calibrated using the -120 p.pm peak of 1 M NaF solution and trichlorofluoromethane (CFCl3) as a second reference. H3PO4 was used as the reference material for the 31P chemical shift. Spectra were acquired over 4 hours by integrating 240 scans.

[0139] Figure 7q shows the phosphate (PO4) corresponding to a typical apatite structure. 3- This indicates that the band was detected at all three time points. In infrared (IR) analysis, the band was detected at 1000-1100 cm. -1 PO4 in the phosphate band 3- Strong peaks were detected attributable to the ν3' and ν3''' antisymmetric stretching vibration modes of PO4. 3- The ν1 symmetrical stretching vibration mode is also 960-965 cm -1 Observed between these two points.

[0140] Figures 7r and 7s show the crystalline phase verified by magic-angle rotation nuclear magnetic resonance (MAS-NMR). The 1F MAS-NMR spectrum of the film on day 1 showed only a sharp peak at approximately -103 ppm corresponding to fluoroapatite (FAp). On day 3, in addition to FAp, a peak of calcium fluoride (CaF2) at -108 ppm was observed, and this became more pronounced on day 7 (Figure 7r). The keratin film at different time points... 31 In the P MAS-NMR spectrum (Figure 7s), a sharp peak of approximately 3 ppm, which is attributed to crystalline apatite, was observed.

[0141] Young's modulus and hardness measurements were performed. Keratin film and enamel samples were bonded to aluminum holders. Nanoindentation tests were performed using a Nanoindenter (iNano) manufactured by Nanomechanics, Inc. (maximum indentation load 50 mN). The mechanical properties of the calcified film were recorded at an indentation depth of 30 nm on the film to reduce the influence of the underlying organic structure. Young's modulus and hardness of the enamel samples were evaluated according to the previously reported method 45, with a maximum indentation depth of 1000 nm and 50 to 100 indentations per sample.

[0142] Figure 7t shows the Young's modulus and hardness of 5% keratin tested before and after calcification. Before calcification, sample 5 exhibited a Young's modulus (5.11 ± 3.0 GPa) and hardness (0.3 ± 0.2 GPa). After calcification, both the rigidity and hardness of the film increased, showing an elastic modulus of 8.1 ± 3.94 GPa and a hardness of 0.64 ± 0.35 GPa.

[0143] [Example 6] Crystallographic characterization of the calcification structure To further investigate the preferential growth and crystallographic diffraction of nanocrystals, a series of ultrathin sections of calcified sferlite were milled using focused ion beam (FIB), and the lamellae prepared by FIB were evaluated using high-resolution transmission electron microscopy (HR-TEM) and selected-region electron diffraction (SAED).

[0144] Focused ion beam-SEM was performed as follows: FIB milling and deposition were performed using a Thermo Scientific® Helios® 5 UC DualBeam or Zeiss Crossbeam 350 FIB-SEM under Ga ion beam conditions of 30kV, 1nA, with only the final low-voltage cleaning step performed with a 2kV beam to reduce Ga implantation and amorphous damage while allowing for additional gentle thinning. The region of interest (ROI) (approximately 20 × 20 μm² wide) was coated with a thin carbon layer and a thick tungsten layer of approximately 2 μm by electron beam and ion beam deposition. The sample was removed with a micromanipulator and mounted on a Cu grid. The pores of the sample were filled with tungsten deposition to improve structural integrity during thinning. Lamellars were thinned with 3 or 4 windows approximately 3 μm wide, depending on the size of the selected mineral sample. Electron beam deposition was performed with a stage tilt of 0°, ion beam deposition and trench milling were performed at 52° (with a small over-tilt angle of approximately 0.7° added to make the front and back parallel for thin-film formation), and lift-out and mounting were performed at 0°.

[0145] High-resolution transmission electron microscopy was performed as follows: Lamellars prepared by FIB were scanned using a Thermo Fisher microscope equipped with an Ultra-X EDS detector and a Cs aberration corrector. Evaluation was performed using a 60-300kV Spectra Ultra TEM. Images were acquired using a 4k×4k Ceta-S detector. Data were post-processed using Velox® software (version 3.8). The obtained images were analyzed using Gatan Microscopy Suite® (GMS3) software. To analyze the crystalline phases present in the sample, the d-values ​​obtained from the SAED patterns were compared with the PDF2 database (ICDD, USA, 2009 edition).

[0146] Figures 9a to 9h show that the calcified sferrite is composed of three distinct regions. Specifically, a bottom layer composed entirely of a keratin organic matrix, where no crystal diffraction pattern is observed; multiple circular structures with an average diameter of 22.55 ± 4.36 nm are shown on TEM (Figures 9a, 9d); these structures decrease in number and size from top to bottom, forming a top layer (Figure 9b) that shows a polycrystalline pattern on SAED; and an interface layer located between the other two layers, where the circular structures almost disappear, and its SAED image shows an arched crystal ring (Figures 9e to 9h). Interval analysis confirmed that these diffraction patterns match those of hydroxyapatite and fluoroapatite crystals.

[0147] Figure 9i shows energy-dispersive X-ray analysis (EDX), indicating that the top layer is composed of large amounts of calcium and phosphorus, and small amounts of sulfur, carbon, and fluorine, while the interface layer is mainly composed of fluorine, and the bottom layer is composed entirely of carbon and sulfur.

[0148] [Example 7] Organic matrix surface morphology, self-assembly, and variability of keratin structure In the calcification process mechanism, the association and conformation of the keratin organic matrix To investigate the role of ions, the keratin secondary structure in aqueous solutions at various pH levels was analyzed using circular dichroism (CD) and dynamic light scattering (DLS).

[0149] The secondary structure of keratin in aqueous solutions of various pH levels was observed using a CD spectrometer (Chirascan® CD Spectrometer, Applied) equipped with a temperature controller. The analysis was performed using Photophysic Limited (UK), and the final pH of both solutions remained stable over time. The keratin solution (0.2 mg / mL) was prepared in Milli-Q water. In addition, to investigate the effect of calcium ions on keratin secondary structure, lyophilized keratin (0.2 mg / mL) was dissolved in 5 mM calcium chloride (CaCl2). A quartz cell with a path length of 0.5 mm was used for the measurement, and the CD spectrum was acquired by scanning the wavelength range of 190–260 nm three times at a sweep rate of 50 nm / min and a bandwidth of 1 nm, and integrating the signals. The spectrum was acquired at 25°C. The solution was equilibrated for 5 minutes before scanning. Subsequently, the CD dataset was deconvoluted using the Dichroweb web server to calculate the protein secondary structure.

[0150] To optimize keratin film formation, DLS measurements were performed to compare and measure changes in keratin particle size and charge in solution. A Zetasizer (Nano-ZS ZEN 3600, Malvern Instruments, UK) was used to measure both zeta potential and Z-average. Keratin solutions (0.2 mg / mL) were prepared at 25°C in Milli-Q water of different pH levels. In addition, to investigate the interaction between keratin and calcium salts, lyophilized keratin (0.2 mg / mL) was dissolved in 5 mM CaCl2. Each sample was equilibrated for 5 minutes before measurement.

[0151] Atomic force microscopy (AFM) was also performed. Regardless of the presence or absence of calcification, keratin films were imaged in tapping mode at room temperature and pressure using a MultiMode® AFM (with Nanoscope III controller, Digital Instruments, Santa Barbara) and an OTESPA-R3® cantilever (Bruker, California). Image analysis was performed using NanoScope® analysis (Bruker, California), and the images were flattened to remove curvature and slope.

[0152] Figures 10a and 10b show that when keratin is solubilized in water, numerous nanospheres with a diameter of 20–50 nm and an average diameter of 33.02 ± 6.87 nm are observed in the solution using AFM.

[0153] Figure 10c shows an organic network of nanospheres packed onto the surface of a dried 3 wt% keratin solution.

[0154] Figures 11a and 11b show the organic networks of nanospheres packed onto the surface of dried 5% by weight and 10% keratin solutions.

[0155] These nanospheres are in the range of 20–40 nm, with average diameters of 23.1±6.64 nm, 27.97±4.75 nm, and 30.72±6.78 nm, respectively.

[0156] Figure 10d shows the nanofibril and microfibril structures on the film. Different fibril populations are observed, and the average fibril diameter increases proportionally with keratin concentration.

[0157] Figure 10e shows fibrils observed on a 3 wt% keratin film crosslinked with TEGDMA. These fibrils are cross-oriented and consist of two distinct groups: one approximately 100 nm wide and the other approximately 500 nm wide.

[0158] Figure 11c shows fibrils observed on a 5 wt% keratin film crosslinked with TEGDMA. These fibrils are also cross-oriented and consist of two distinct groups, one with a width of approximately 100 nm and the other with a width of approximately 500 nm.

[0159] Figure 11d shows fibrils observed on a 10 wt% keratin film crosslinked with TEGDMA. These fibrils are well organized and oriented nearly parallel to one another.

[0160] Figures 10f and 10g show that numerous spherulite structures are observed on a 10 wt% keratin film, forming large spherical bodies composed of multiple spherulites. Some of the spherulites are arranged in long linear patterns.

[0161] Figure 10h shows a Congo red stained image of the keratin structure in a 10 wt% keratin film. Under a white light microscope, dendritic structures of similar size to organic spherulites were observed.

[0162] To investigate keratin secondary structure conformation as a model for intrinsically disordered proteins (IDPs), deconvolution of the amide I spectral region obtained by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was performed. The secondary structure was quantified before and after self-assembly induction, and the effect on the arrangement of calcification motifs in various keratin films was examined.

[0163] FTIR analysis was performed using Spectrum One, which is equipped with a white light source and an InGaAs detector. Spectroscopy was performed on keratin solutions and films using an FTIR spectrometer (PerkinElmer®, Buckinghamshire, UK) in ATR mode. Spectra were obtained by averaging 32 scans for each sample, with a resolution of 2 cm. -1 And the wave number is 4000-700cm -1 The spectrum was obtained within the following range: Amide I spectral region (1700-1600 cm⁻¹). -1The keratin secondary structure composition was analyzed using OriginPro 8.5 software (Microcal Inc.) to compare the results. The spectral band assignments were as follows: 1610–1627 cm⁻¹. -1 : Intermolecular β-sheet, 1628~1642 cm -1 :β-sheet, 1643~1650cm -1 , 1643~1650cm -1 Random coil, 1650~1659cm -1 : α-helix, 1643~1650cm -1 , 1660~1699cm -1 :β-sheet / β-turn.

[0164] By increasing the keratin concentration and maintaining a neutral pH (pH 7), we were able to regulate the pattern and number of organic spherulites.

[0165] Figures 12a and 12b show that, with or without crosslinking, the highest concentration of keratin increases the number of β-sheets at the expense of a decrease in random coil conformation in the dried film.

[0166] Figure 12m shows that in dry films with a high keratin concentration, the number of organic spherulites increases, and the Maltese cross pattern becomes more clearly visible.

[0167] To understand the contribution of protein secondary structure to organic keratin spherulite formation, Fourier transform infrared spectroscopy (FTIR) imaging techniques were used. Deconvolution data of the amide I band for organic spherulite and its surroundings were also analyzed.

[0168] Figure 12n shows that keratin spherulites primarily adopt a β-sheet structure, in addition to also possessing a random coil structure. As we move away from the spherulite, the amount of β-sheets decreases (45.75%, 30.83%, 25.27%, 13.95%), the region with the number of random coils increases (31.98%, 52.35%, 59.0%, 61.17%), and α-helices are observed only in the region surrounding the spherulite (13.72%).

[0169] Therefore, by adjusting the keratin concentration during water evaporation, it became possible to control the disorder-order ratio of keratin in the resulting film, as well as control the formation of keratin spherulite.

[0170] [Example 8] Variability of keratin structure and its effect on calcification To investigate changes in secondary structure conformation, Fourier transform infrared (FTIR) imaging was performed after calcification. To do this, spectra were acquired from four different regions selected based on their proximity to the protein and calcified regions on the calcified keratin film. Deconvolution analysis of the amide I band was then performed.

[0171] Figure 13a shows that β-sheets / turns are dominant in regions close to the protein, while α-helix structures consistently increase as we move away from the organic region towards the mineral side, and random coils also increase slightly.

[0172] These results suggest that during the calcification process, proteins undergo structural changes, with a decrease in β-sheets and an increase in α-helices and random coils.

[0173] Small-angle X-ray scattering (SAXS) analysis was performed to evaluate the anisotropy of keratin films before and after calcification. SAXS scattering experiments were conducted at the cSAXS beamline of the Swiss Light Source (SLS) located within the Paul Scherrer Institute (PSI) in Switzerland. A microfocused X-ray beam was used for these experiments, and the photon energy was set to 12.4 keV using a Si(111) dicrystalline monochromator. Measurements were performed on both non-calcified and calcified keratin films using a beam size of 20 μm within a 2 × 2 mm probe area. A vacuum flight tube was placed between the sample and the detector to minimize air scattering and absorption. The keratin film samples were mounted on a motorized stage capable of two-axis movement in a plane perpendicular to the incident beam (xy plane), enabling raster scanning. During the experiment, fly scanning was performed in the vertical direction (y) with a step size of 20 μm and an exposure time of 0.05 seconds. A two-dimensional SAXS pattern was acquired at each scan point using a Pilatus 2 M detector positioned at a sample-detector distance of 2 m. This detector configuration covered a q range of approximately 0.03 to 5 nm-1, where q is the scattering vector, defined as q = 4π λ sin(θ) (where λ is the X-ray wavelength and θ is the half-angle of the scattering angle). A 1.5 mm steel beam stop was installed inside the flight tube to shield the detector from direct beam and protect it from damage.

[0174] Figure 13b shows that the scattering curve originating from the non-calcified keratin film exhibits a prominent SAXS peak within the q range of 0.1–0.25 Å⁻¹, suggesting a coiled structure.

[0175] Figure 13c shows that additional peaks in the 0.01–0.1 Å-1 range are observed in the calcified film and are associated with a rod-like (helical) structure.

[0176] Figures 13d and 13e show scattering curves obtained for each pixel from calcified sferlite, demonstrating a transition from a rod-like structure to a more coil-like structure as the distance from the mineral increases.

[0177] [Example 9] Biocompatibility analysis of keratin 〔method〕 Mesenchymal stem cell proliferation medium: Low-glucose Dulbecco modified Eagle medium (DMEM) (Sigma), 10% fetal bovine serum (FBS) (ThermoFisher Scientific, Ireland), 1% penicillin / streptomycin, 1% non-essential amino acids, and 1% sodium pyruvate.

[0178] Osteoblast differentiation medium (OGM): High glucose DMEM (4500 mg / L) D5671 (Sigma-Aldrich, Ireland), 10% FBS, 100 nM dexamethasone, 100 μM ascorbic acid-2-phosphate, 10 mM β-glycerophosphate, 1% penicillin / streptomycin, 1% non-essential amino acids, and 1% sodium pyruvate.

[0179] Film pretreatment: This study used four different experimental groups and a positive control group (n=12): (i) 5% keratin solution (Group 1); (ii) 5% keratin solution crosslinked with triethylene glycol dimethacrylate (TEGDMA) (ratio of crosslinking agent to lysine and cysteine ​​amino acid residues in keratin: 1:10 (corresponding to a ratio of 100 μL keratin: 3.74 μL TEGDMA)) (Group 2); (iii) 5% keratin solution crosslinked with TEGDMA (same ratio of 1:10) followed by exposure to a calcification solution (Group 3); (iv) 5% keratin solution crosslinked with hexamethylene diisocyanate (HDI) (Group 4) and BioHorizons Mem-Lok® absorbable collagen matrix. Before culturing, the membranes were immersed in 80% (v / v) ethanol at room temperature for 30 minutes. The ethanol was removed and the membranes were washed three times with sterile phosphate-buffered saline (PBS). The PBS was removed, and the membrane was immersed in sterile DMEM until use.

[0180] Quantitative reverse transcription polymerase chain reaction (qRT-PCR): To investigate osteoblast differentiation, human bone marrow-derived stem cells (HMSCs) (P4; RoosterBio, USA) were seeded at 10,000 cells / cm2 on membranes and cultured at 37°C under 5% (v / v) CO2 conditions. Cells were allowed to adhere to the membranes overnight in growth medium. Subsequently, the medium was removed, and the MSCs on each membrane were cultured in either growth medium or osteoblast differentiation medium. Cells were cultured for 28 days, with the medium changed every two days, and osteoblast-related gene expression was evaluated. In summary, after removing the medium and washing the cells with ice-cold PBS, Trizol reagent was added to each membrane. The membranes were incubated in Trizol for 10 minutes at room temperature with vortex stirring. Then, chloroform (100 μL per 1 mL of Trizol) was added to the sample, and the tubes were vortexed. The tubes were centrifuged at 13,000 g at 4°C for 20 minutes. After centrifugation, the aqueous phase was collected and transferred to a new microcentrifuge tube. Equal volumes of 70% ethanol were added to these tubes and mixed 20 times by inversion. This solution was loaded onto a Qiagen RNeasy column (mini kit), and RNA extraction was performed according to the manufacturer's instructions. The RNA was eluted in nuclease-free water and quantified using NanoDrop. cDNA was synthesized using the Quantitect Reverse Transcription Kit (Qiagen) and normalized to 5 ng / μL. qRT-PCR was used to evaluate the expression of two early osteoblast markers (runt-related transcription factor 2 (RUNX2) and alkaline phosphatase (ALP)), two mid-stage markers (Osterix (OSX®) and bone morphogenetic protein 2 (BMP-2)), and two late-stage osteoblast markers (osteopontin (OPN) and osteocalcin (OCN)). The tests were performed on an Eppendorf Realplex 4 Mastercycler using Qiagen Quantitect validated primers and the Sybr Green method (Sensimix SYBR Lo-ROX mix, Meridian Bioscience). Gene expression was normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the change in expression multipliers was calculated compared to MSCs cultured on standard tissue culture plastic under growth medium conditions.

[0181] AlamarBlue assay - metabolic viability: At predetermined points during culture, the cell culture medium was removed and the cells were washed with pre-warmed sterile PBS. AlamarBlue resazurin (10%, v / v; Bio-Rad) was diluted in phenol red-free medium (D5030, Sigma-Aldrich) and added to each culture well. The cells were incubated in AlamarBlue working solution for 4 hours at 37°C under 5% (v / v) CO2 conditions. After incubation, the supernatant was transferred to a 96-well plate, and AlamarBlue metabolism was evaluated by measuring absorbance at 570 nm and 600 nm. The reduction rate of AlamarBlue was calculated using the following formula. AlamarBlue reduction rate (%) = ((O2 × A1) - (O1 × A2) / (R1 × N2) - (R2 × N1)) × 100 Here, O1 and O2 are the molar extinction coefficients of oxidized AlamarBlue at wavelengths of 570 nm and 600 nm, respectively. R1 and R2 are the molar extinction coefficients of reduced AlamarBlue at wavelengths of 570 nm and 600 nm, respectively. A1 and A2 are the measured absorbances of the test wells at wavelengths of 570 nm and 600 nm, respectively. N1 and N2 are the measured absorbances of the negative control wells at wavelengths of 570 nm and 600 nm, respectively.

[0182] 〔result〕 Mem-Lok® was used as a positive control for the gold standard because it is known to support bone regeneration in vivo. Regardless of the type of culture medium used, all test membranes showed at least a 50% higher survival rate compared to the gold standard after 7 days of culture (Figures 7a and 7b). Regardless of the type of culture medium, an improvement in survival rate was observed in all groups when comparing 7 days and 21 days. After 21 days in mesenchymal stem cell proliferation medium, the calcified membrane significantly outperformed the gold standard in terms of survival rate.

[0183] For all osteoblast markers analyzed (RUNX2, ALP, OSX, BMP-2, OPN, and OCN), favorable levels of mRNA expression were observed in all test membranes compared to the gold standard.

[0184] [Example 10] Mechanical analysis of calcified keratin film 〔method〕 Preparation of enamel sections: Eighteen caries-free molars and premolars were collected, and sections were prepared using a water-cooled rotary diamond saw (XL 12205, Benetec Ltd., UK). The slabs were then washed with deionized water, wiped with tissue paper, and visually confirmed to be free of caries and carious foci. Next, the samples were placed buccal-side down in rectangular silicone molds and embedded in transparent acrylic resin. The resin was prepared by mixing acrylic polymer beads and monomer (Oracryl®, Bracon, UK) in a 2:1 (volume ratio). The acrylic resin was then poured in until the sections were completely covered, and cured for 30 minutes before polishing.

[0185] Polishing the enamel surface: To obtain a flat, high-gloss surface for morphological analysis, the sample was polished using a polishing machine (MetaServ 3000, Buehler, USA). Silicone waterproof abrasive paper with different grit sizes was used for polishing. The polishing protocol was as follows: 5 seconds at P500, then at P1200. 10 seconds at P2500, 10 seconds at P4000, and 2 minutes at each step. After each step, the polishing direction was changed by 90 degrees and ultrasonic cleaning was performed. After polishing, all samples were stored in distilled water until the next processing step.

[0186] Randomization of samples: Polished enamel sections were randomly assigned to three different groups (n=6): (i) 5% keratin solution (K5); (ii) 5% keratin solution crosslinked with triethylene glycol dimethacrylate (TEGDMA) (the ratio of crosslinking agent to lysine and cysteine ​​amino acid residues in keratin was 1:10 (corresponding to a ratio of 100 μL keratin to 3.74 μL TEGDMA)) (K5TE10 (iii) ICON® resin infiltrate (positive control) (ICON). Each group was then divided into three subgroups based on the incubation medium (deionized water (DW), artificial saliva (AS), or calcification solution (MS)) used to incubate the sample, according to the assigned group.

[0187] Induction of white spot lesions (WSL): Demineralization of polished enamel sections was performed using the two-layer acidic gel method described by Zhang et al. (2018) (Zhang, J., Lynch, RJM, Watson, TF, Banerjee, A. (2018) "Remineralization of enamel white spot lesions pre-treated with chitosan in the presence of salivary pellicle", J Dent, 72, pp.21-28). The polished enamel surface was then covered with adhesive tape, leaving a 1 mm × 3 mm window to induce WSL. The demineralization gel was prepared by dissolving 8 wt% methylcellulose powder (Sigma-Aldrich, USA) in 100°C deionized water in a 500 mL glass beaker with continuous stirring until it reached room temperature. Subsequently, five enamel sections were immersed in the gel with their surfaces facing upward and allowed to solidify overnight at 4°C. The following day, filter paper was placed on top of the gel, and 100 mL of 0.1 M lactic acid (Anala R, UK) at pH 4.6 was added on top of the paper. The gel was incubated at 37°C for 7 days. After 7 days, the samples were removed, and the WSL was washed with deionized water for 1 minute to remove any remaining gel.

[0188] Preparation of storage media: Nothing was added to the deionized water (DW) used as the DW storage medium before use.

[0189] The previously reported protocol was used to prepare 1 L of experimental artificial saliva. 0.1029 g of calcium chloride dihydrate, 0.01904 g of magnesium chloride, 0.544 g of potassium dihydrogen phosphate, 5.206 g of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and 2.2365 g of potassium chloride were added to 1 L of deionized water and thoroughly mixed using an orbital shaker. This solution was titrated to pH 7.0 (±0.05) using 0.1 M sodium hydroxide and measured with a pH meter (Mettler Toledo). The solution was sealed with Bemis Parafilm M and stored at room temperature.

[0190] The calcification solution was prepared according to the following protocol: 600 mL of calcification solution was prepared using 0.60276 g of hydroxyapatite powder, 0.0084 g of sodium fluoride, and 1.2554 g of bis-trismethane. Under fume hood conditions, bis-trismethane was dissolved in 35 mL of deionized water to adjust the pH to 6.0 (±0.05). Next, sodium fluoride and hydroxyapatite powder were added to 500 mL of deionized water. 69% nitric acid was added dropwise using a micropipette to completely dissolve the hydroxyapatite and sodium fluoride powder at pH 2.4. The solution was then titrated with bis-tris buffer to a pH of 6.0 (±0.05). The solution was then covered with Parafilm (Bemis It was sealed with Parafilm M and stored at room temperature.

[0191] Application of measures to WSL: Keratin was extracted from wool as described in Example 1. The K5 sample was prepared by adding 5% by weight of keratin to deionized water. K5TE 10 The samples were prepared by adding 5% by weight of keratin to deionized water, followed by the addition of TEGDMA as a crosslinking agent to bind to the lysine and cysteine ​​of the keratin. All mixtures were dropwise cast onto the WSL surface according to pre-assigned groups, dried at 37°C for 5 minutes, and then placed in their assigned storage media.

[0192] Leaf lesion sections to be subjected to ICON resin infiltration were treated with 99% ethanol (ICON dry DMG) and held on the lesion surface for 30 seconds. The surface was then cleaned with oil-free compressed air. Next, a TEGDMA-based resin matrix (ICON®-DMG infillrant) was applied to the lesion surface and held for 3 minutes. Additional infillrant was applied to the surface as needed to maintain moisture. The infillrant was then dispersed with oil-free compressed air and photopolymerized with ultraviolet light at a wavelength of 200-400 nm for 40 seconds. Further infillrant was applied to the surface for 1 minute, dispersed again with oil-free compressed air, and then photopolymerized for an additional 40 seconds.

[0193] Microhardness measurement: Surface microhardness was measured for all samples using a microhardness tester (Duramin, Struer, Denmark) equipped with a Knoop diamond indenter. For each sample, five indentations were measured at intervals of at least 100 μm. A load of 0.2 kgf and a holding time of 10 seconds were used for measuring untreated polished enamel and untreated WSL. For the intervention group, the samples were imaged with a white light microscope (Keyence, America) after indentation, and then HK = 14.299 × F / D 2 The microhardness was calculated using the following formula (where F is the applied load (N) and D is the indentation length (mm)).

[0194] Scanning electron microscopy (SEM): After measuring the microhardness, cross-sections were prepared for each group, etched with 35% phosphoric acid for 20 seconds, and then washed for 20 seconds to remove the smear layer, preparing the samples for SEM imaging. The samples were fixed onto aluminum stubs with double-sided carbon tape and then sputtered with an 8 nm conductive gold coating (Leica EM ACE600 sputter coater, Milton Keynes, UK). SEM images were acquired using a Schottky field emission type JSM-7610F SEM (JEOL Ltd., Hertfordshire, UK), and the surface morphology was observed using a secondary electron detector to evaluate the changes in sample morphology before and after treatment.

[0195] Statistical analysis: The results were statistically analyzed using Graphpad Prism software. The Shapiro-Wilk test showed that the data followed a normal distribution, and one-way analysis of variance (ANOVA) was used to compare microhardness across all groups. Tukey's HSD post-hoc test was used to assess statistical significance between groups at p<0.001.

[0196] 〔result〕 Microhardness measurement: As shown in Figure 15a, the average values ​​for the DW storage medium were 333.80 ± 24.65 kg / mm³ for polished enamel and WSL, respectively. 2 and 24.94±6.16 kg / mm 2 The WSL processed by ICON was 33.3±21.09 kg / mm². 2 The Knoop microhardness values ​​were as follows: K5 and K5TE10 were 136.9 ± 24.65 kg / mm², respectively. 2 and 158.48±45 kg / mm 2 This shows the average microhardness.

[0197] As shown in Figure 15b, the average values ​​for the AS storage medium were 340.8 ± 19.7 kg / mm³ for polished enamel and WSL, respectively. 2 and 27.36±6.05 kg / mm 2 The WSL processed by ICON was 44.68±11kg / mm². 2 The Knoop microhardness values ​​were as follows: K5 and K5TE10 were 222.48 ± 48.5 kg / mm², respectively. 2 and 136.4±26.62 kg / mm 2 This shows the average microhardness.

[0198] As shown in Figure 15c, the average values ​​for polished enamel and WSL for MS storage media are 350.4 ± 53 kg / mm², respectively. 2 and 11.2±3.3 kg / mm 2 The WSL processed with ICON was 13.2±1.17 kg / mm². 2The Knoop microhardness values ​​were as follows: K5 and K5TE10 were 173.8 ± 39.6 kg / mm², respectively. 2 and 144.24±50.419 kg / mm 2 This shows the average microhardness.

[0199] In summary, polished enamel surfaces showed a significant difference compared to all other samples. In all storage media, ICON-treated WSL did not show a significant difference compared to untreated WSL, but both keratin-treated lesions showed a significant difference compared to the WSL and ICON-treated groups, with improvement in hardness observed in the keratin group.

[0200] SEM analysis: Figure 15d shows an SEM image of untreated polished enamel, and Figure 15e shows an SEM image of WSL. The microstructure of sound enamel consists of prisms, interprism continuum, and densely packed hydroxyapatite crystals. However, in WSL, the prism core is significantly affected, and the hydroxyapatite crystallites and their arrangement are lost.

[0201] K5-treated WSLs incubated in various media for 7 days were observed by SEM (Figures 8f, 8g, and 8h). In DW, keratin infiltrated the WSLs, forming a capsule covering the lesions. Keratin incubated in AS formed a uniform new mineral layer after remineralization, with nanorods oriented parallel to each other. Keratin-treated WSLs incubated in MS also had a uniform reparative calcification layer consisting of enamel-like oriented apatite crystals approximately 50-70 nm wide, some of which existed as dense bundles with specific orientations.

[0202] [Example 11] Vitiligo Lesion Test 〔the purpose〕 To investigate the remineralization ability of enamel-like crystals in infiltrative carious leukoplakia lesions using a novel, clinically manageable keratin-based membrane.

[0203] 〔method〕 Enamel sections (n=18) obtained from caries-free molars and premolars were embedded in acrylic resin, polished, and then randomly assigned to three groups. These were immersed in a demineralization solution for 7 days to induce whitish leukoplakia (WSL) lesions (Figures 9 and 10). Subsequently, all samples were etched with 15% hydrochloric acid for 2 minutes to enhance the penetration of the treatment agent, washed with deionized water for 30 seconds, and dried with oil-free air using an air compressor. Six samples were assigned to each group, which consisted of a positive control (ICON® resin infiltrate) and two different test groups. ICON® is a WSL treatment agent provided by DMG Chemisch-Pharmazeutische Fabrik GmbH, containing camphorquinone and triethylene glycol dimethacrylate (TEGDMA) as photoinitiators. In Group 1, 50 μL of 5% keratin dissolved in Milli-Q water was pipetteed onto the WSL samples for treatment. On the other hand, the samples from the other test group were treated by adding 5% by weight keratin, which had been dissolved in Milli-Q water and crosslinked with TEGDMA, dropwise using a pipette (ratio of crosslinking agent to lysine and cysteine ​​amino acid residues in keratin: 1:10 (100 μL of keratin:T This corresponds to a ratio of 3.74 μL of EGDMA). All samples were incubated at 37°C for 2 minutes to allow the keratin solution to form a hydrogel within the lesion, and then immersed at 37°C in the assigned storage medium (deionized water (DW) or artificial saliva (AS)).

[0204] WSL was analyzed using a visual evaluation based on the International Caries Detection and Assessment System (ICDAS) criteria, and the change in grayscale values ​​before and after intervention was measured using a white light microscope (WLM) and optical coherence tomography.

[0205] WLM was used to obtain surface characterization before, during, and after each procedure, as well as in cross-sections, and to assist in the evaluation of the ICDAS scoring method. Images were acquired at 20x magnification using an E20 lens. ImageJ (NIH, USA) was used for grayscale analysis of the data. After comparing all images, it was determined that a 400 x 300 pixel elliptical selection region was sufficient for acquiring grayscale data, as it could obtain sufficient pixel information without being affected by lesion boundaries or transition surfaces. The selection region was dragged onto each region of interest, and the grayscale value of each pixel was recorded (Figure 18).

[0206] Optical coherence tomography (OCT) (VivoSight, Kent, UK) was also used to measure the WSL lesion depth and surface area density in baseline samples, as well as before and after treatment. The scan area was 6 mm wide to include the entire cross-section of the lesion. Multislice mode was used, with a slice interval of 0.001 mm. For each sample, two-dimensional cross-sectional images of the control region and the intervention region were acquired. Subsequently, the images were imported into ImageJ (NIH, USA) and grayscale analysis was performed. The image analysis procedure is shown in Figure 19.

[0207] 〔result〕 The changes in grayscale values ​​of OCT and WLM after image analysis were plotted as shown in Figures 20-22. OCT and Keyence analysis revealed a consistent difference in grayscale values ​​between the control and intervention areas of each sample (Figures 23-27). Both wool-derived keratin test groups showed a decrease in lesion depth and density, and in both the deionized water and artificial saliva cases, ImageJ analysis after application of both keratin solutions detected a decrease in grayscale values ​​comparable to that of the positive control (ICON®).

[0208] These results indicate that the film is as effective in treating vitiligo lesions as the gold standard ICON® resin infiltrate. Furthermore, since the keratin film promotes tooth remineralization (unlike current treatments such as ICON®), it is thought that it may provide long-term benefits in addition to short-term effects.

[0209] [Example 12] Evaluation of surface characteristics of induced enamel lesions To investigate the possibility of keratin treating artificially induced enamel pores and restoring their mechanical properties, we conducted an in vitro preclinical trial using an induced enamel WSL model.

[0210] Extracted human non-cariogenic molars (HRA approved by the NHS Research Ethics Committee, approval number: 16 / SW / 0220) were observed under a white light microscope (GXM-XPLPOLTEC-5, UK) to confirm the absence of caries or caries fossae. Next, the samples were placed in a silicone mold with the facial side facing downwards and embedded in transparent acrylic resin (Oracryl®, Bracon, UK). Enamel sections were prepared using a water-cooled rotary diamond saw (XL 12205, Benetec Ltd., UK). The sections were carefully polished using a polishing device (MetaServ 3000, Buehler, USA) and silicon carbide polishing paper (Struers®, UK) in the order of P500, P1200, P2500, and P4000, from coarse to fine. After each step, the polishing direction was changed by 90 degrees and ultrasonic cleaning was performed. After polishing, all samples were stored in distilled water until the next processing step.

[0211] The polished enamel surface was covered with tape, leaving a window approximately 1 mm wide and 2 mm long on the facial side of each molar. Using a known protocol, WSL was guided onto this window. For comparison, all WSL windows were symmetrically divided into two parts: the lower half was protected with adhesive tape to serve as a negative control, and the upper half was used for restorative treatment. The restorative treatment side was pre-etched with 15% hydrochloric acid for 2 minutes to open the enamel pores and ensure penetration of the treatment agent, then washed with deionized water, sonicated in a water bath for 2 minutes to remove residual contaminants, air-dried, and stored at 4°C until use.

[0212] WSL blocks were treated with either Ker5 or resin infiltrate (ICON®, DMG America). Each treatment group was incubated in three different storage media: ultrapure water (UPW), artificial saliva, or calcification solution. For keratin treatment, the same preparation procedure as described above was followed, with 20 μL pipetted onto the exposed area to infiltrate the vitiligo lesions, followed by drying and storage at 37°C for 7 days. The treated enamel blocks were then washed with UPW, sonicated for 2 minutes to remove debris, and air-dried.

[0213] Figures 28a to 28e show scanning electron microscope images confirming WSL induction.

[0214] Figures 28a and 28b show that the microstructure of sound polished enamel consists of prisms, interprism continuum, and densely packed hydroxyapatite crystals.

[0215] Figures 28c and 28d show that the prism core is significantly affected, resulting in the loss of hydroxyapatite crystallites, creating gaps between the prisms, and disrupting the crystal arrangement.

[0216] Figures 28f to 28i show the improvement in the color and appearance of lesions after WSL treatment with keratin.

[0217] Figures 28g to 28m show films observed from the treated lesion surface incubated in a calcification solution, indicating signs of enamel reconstruction. As a result of inducing WSL in the enamel, the gaps and voids formed both within and between the prisms (Figure 28j) are filled with novel nanocrystals throughout almost the entire layer of the treated WSL (Figures 28k to 28m).

[0218] Figure 30a shows the integration of HA crystals and keratin on the surface, and it appears that keratin is infiltrating between the mineral crystals and inducing their growth.

[0219] Figures 28n to 28p show that similar results are obtained even when teeth are incubated in artificial saliva, and that the lesion gap is repaired by enamel-like nanocrystals continuously growing from the lesion crystals, and the pores are filled up to the deep part.

[0220] Figure 30b shows that when keratin-treated WSL is incubated in UPW, only a film is formed on the surface, and no evidence of mineral repair is observed.

[0221] In addition, WSL that had not been subjected to any treatment after being remineralized in a calcification solution was also examined.

[0222] Figure 30c shows that when no treatment is applied after remineralization in a calcification solution, mineral precipitates with no specific orientation are deposited on the surface, not following the enamel prism orientation.

[0223] Figure 30d is a SEM image showing that a resin infiltrant incubated in a calcification solution fills the gaps in porous enamel and forms a resin layer on the WSL.

[0224] Figures 30e and 30f show that the calcification detected within the resin-infiltrated enamel prisms exists as a massive structure showing no distinct crystal form on the resin surface.

[0225] Furthermore, focused ion beam (FIB) lamellas of keratin-treated lesions were milled and the crystal phase was investigated. High-resolution transmission electron microscope (HR-TEM) images of keratin-treated lesions were obtained from the region near the enamel surface and the bulk region approximately 50 μm away from the surface.

[0226] Figures 28q to 28s and 30a to 30c are TEM images showing bundles of nanocrystals, and newly formed nanocrystals are detected between existing crystals oriented parallel to the growth direction along the c-axis at any position, forming a continuous pattern between the prisms. Selected area electron diffraction (SAED) also confirmed the apatite phase.

[0227] These results demonstrate that keratin has the ability to repair damaged enamel across almost the entire thickness of the lesion.

[0228] Microhardness analysis was performed to evaluate the mechanical properties of the reconstructed enamel.

[0229] Microhardness analysis was performed as follows. Surface microhardness was measured for all samples using a microhardness tester (Duramin-20, Struers Ltd, Rotherham, UK) equipped with a Knoop diamond indenter. Microhardness of sound enamel was obtained from polished enamel around the WSL window in each experimental group. For each sample, five indentations were measured at intervals of at least 100 μm. A load of 0.2 kgf and a holding time of 10 seconds were used for the measurement of untreated polished enamel and untreated WSL. For the intervention group, the samples were imaged with a white light microscope (VHX-7000 series, Keyence, America) after indentation, and then the microhardness was calculated using the following formula: HK = 14.299 × F / D²; F = applied load (N), D = length of the diagonal of the indentation (mm).

[0230] Figure 28t shows the Knoop hardness of healthy enamel and untreated WSL, measured in various preservation media. In ultrapure water (UPW), keratin-treated lesions showed a microhardness of 1.23 ± 0.22 GPa, compared to 3 ± 0.19 GPa for enamel, 0.07 ± 0.02 GPa for WSL, and 0.3 ± 0.05 GPa for resin-infiltrated lesions. In the calcification solution, the microhardness of 5% keratin after calcification increased to 1.65 ± 0.3 GPa, and the hardness of enamel and WSL was estimated to be 2.89 ± 0.28 GPa and 0.1 ± 0.1 GPa, respectively. Furthermore, 5% keratin significantly improved the microhardness of WSL in artificial saliva from 0.1 ± 0.02 GPa to 2.1 ± 0.35 GPa. On the other hand, no significant improvement was observed with resin infiltrants. The hardness of lesions incubated in calcification solution was estimated at 0.34 ± 0.03 GPa, while the hardness of lesions incubated in artificial saliva was estimated at 0.31 ± 0.05 GPa.

[0231] Young's modulus and hardness measurements were also performed. Keratin film and enamel samples were bonded to aluminum holders. Nanoindentation tests were performed using a Nanoindenter (iNano) manufactured by Nanomechanics, Inc. (maximum indentation load 50 mN). The mechanical properties of the calcified film were recorded at an indentation depth of 30 nm on the film to reduce the influence of the underlying organic structure. Young's modulus and hardness of the enamel samples were determined according to the previously reported method 45, with a maximum indentation depth of 1000 nm and 50 to 100 indentations per sample. I went and evaluated it.

[0232] Figure 28u shows the results of nanoindentation measurements evaluating the improvement in enamel stiffness and elastic modulus before and after keratin repair in artificial saliva and calcification solution. The average stiffness and hardness of enamel before WSL induction were estimated to be 86.42±8.67 GPa and 2.62±0.67 GPa, respectively. In lesions after WSL induction, the Young's modulus was 4.97±3.45 GPa and the hardness was 0.11±0.12 GPa. After keratin treatment within the defective enamel, the Young's modulus and hardness significantly improved to 53.27±19.78 GPa and 1.07±0.78 GPa, respectively. Similar results were obtained in lesions treated with keratin in artificial saliva, where the repaired enamel showed a stiffness of 48.14±21.16 GPa and a hardness of 0.94±0.68 GPa. Resin-treated lesions exhibited a Young's modulus of 8.23 ​​± 2.95 GPa and a hardness of 0.31 ± 0.2 GPa, which is consistent with previous studies.

[0233] Figure 30d shows that nanoindentation measurements obtained from the bulk sample confirmed an improvement in mechanical properties.

[0234] These results complement SEM and HR-TEM findings that indicated keratin has the ability to treat and infiltrate nearly the entire thickness of induced enamel lesions.

[0235] [Example 13] Animal study to evaluate the potential of keratin for bone healing The inventors further investigated the possibility that keratin membranes induce bone healing in in vivo studies.

[0236] A total of 42 healthy male rats, 6-8 weeks old and weighing approximately 250-350g, were used. Surgery was performed to create a cranial vault defect in each rat. A 2cm longitudinal incision was made along the cranial midline to create the defect. The periosteum was carefully incised, elevated, and pulled bilaterally to fully expose the cranial vault. A 6.0mm diameter treffin bur was used to perforate a circular, segmented cranial vault defect in the parietal bone of each rat, and care was taken to avoid damaging the dura mater when removing the entire defect layer. During perforation, the site was continuously irrigated with sterile saline solution, and then the cranial vault bone fragments were carefully removed with forceps.

[0237] Next, the defects were randomly assigned to six different groups. Each rat provided one cranial defect, resulting in seven defects in each group.

[0238] Four different keratin membrane experimental groups were examined. 5% by weight keratin film (Ker5), Keratin (Ker5TE1) crosslinked with triethylene glycol dimethacrylate, Calcified keratin-TEGDMA (M-Ker5TE1), Keratin-HDI (Ker5H3).

[0239] To evaluate spontaneous healing without treatment, positive controls (collagen membrane) and negative controls (void defects) were also established.

[0240] Prior to surgical intervention, the membrane was sterilized under ultraviolet light in a sterile hood for 20 minutes. The hydrated membrane was transplanted to cover the defect, and the ends of the membrane were fixed subperiosteally. The skin was then closed with layered sutures using 4-0 Vicryl sutures. The animals were observed daily until euthanasia to check for complications or abnormal behavior.

[0241] Figures 31a to 31c show circular segmental cranial vault defects in rats.

[0242] FIG. 31d shows a hydrated keratin film transplanted to cover the defect.

[0243] The rats were euthanized by an overdose of carbon dioxide (CO 2 ) after 8 weeks. After death, all animals were decapitated, and the skulls were immersed in 10% neutral buffered formaldehyde. The biocompatibility and bone healing ability of these films were evaluated by radiography and micro-computed tomography (micro-CT).

[0244] To obtain X-ray images of the skulls, a 2D intraoral X-ray device was used, and the samples were irradiated at 60 kVp, 7 mA, and 6 seconds.

[0245] FIGS. 31e to 31j show the X-ray images of each experimental group.

[0246] In all experimental groups, effective bone healing was observed compared to the negative control. To perform micro-CT, three-dimensional images of the scanned samples were obtained using a high-resolution micro-CT system. The X-ray generator was operated at an accelerating voltage of 81 kV and a current of 123 μA. The image resolution was 9 μm. The parameters evaluated were as follows: bone volume [BV (mm 3 )], ratio of bone volume to trabecular volume [BV / TV (%)], trabecular thickness Tb.Th (mm), trabecular number T.N (mm-1), and trabecular separation T.Sp (mm).

[0247] FIGS. 31j to 31u show the micro-CT images and analysis results. In all experimental groups, effective bone healing was observed compared to the negative control.

[0248] References 1. Palmer, L.C.; Newcomb, C.J.; Kaltz, S.R.; Spoerke, E.D.; Stupp, S.I. "A Biomimetic System for Hydroxyapatite Mineralization Inspired by Bone and Enamel" Chem. Rev., Vol. 108, pp. 4754 - 4783. 2. Marie, B.; Joubert, C.; Tayale, A.; Zanella-Cleon, I.; Belliard, C.; Piquemal, D.; Cochennec-Laureau, N.; Marin, F.; Gueguen, Y.; Montagnani, C. "Different secretory repertoires control the biomineralization process of the prism layer and nacre deposition in pearl oyster shells." Proc. Natl. Acad. Sci. USA, Vol. 109, pp. 20986-20991 (2012). 3. Addadi, L.; Weiner, S. "Control and design principles in biocalcification." Angew. Chem. Int. Ed. Engl., Vol. 31, pp. 153-169 (1992). 4. Kirkham, J.; Firth, A.; Vernals, D.; Boden, N.; Robinson, C.; Shore, RC et al. "Self-assembling peptide scaffolds promote enamel remineralization." J Dent Res., Vol. 86, No. 5, pp. 426-430 (2007). 5. Chen, H.; Tang, Z.; Liu, J.; Sun, K.; Chang, SR; Peters, MC et al. "Cell-free synthesis of human enamel-like ultrastructure." Advanced Materials, Vol. 18, No. 14, pp. 1846-1851 (2006). 6. Yamagishi, K et al. "Synthetic enamel for rapid tooth restoration." Nature, Vol. 433, p. 819 (2005). 7. Yin, Y.; Yun, S.; Fang, J.; Chen, H. "Chemical regeneration of human tooth enamel under conditions close to physiological conditions." Chem. Commun., pp. 5892-5894 (2009). 8. Shao, C.; Jin, B.; Mu, Z.; Lu, H.; Zhao, Y.; Wu Z.; Yan, L.; Zhang, Z.; Zhou, Y.; Pan, H.; Liu, Z.; Tang, R. "Restoration of tooth enamel by a biomimetic calcification frontier that guarantees epitaxial growth." Science Advances, Vol. 5, Eaaw9569 (2019). 9. Ruan, Q.; Zhang, Y.; Yang, X.; Nutt, S.; Moradian-Oldak, J. "Amelogenin-chitosan matrix promotes the construction of enamel-like layers with high-density interfaces." Acta Biomater., Vol. 9, pp. 7289-7297 (2013). 10. Elsharkawy, S.; Al-Jawad, M.; Pantano, MF; Tejeda-Montes, E.; Mehta, K.; Jamal, H.; Agarwal, S.; Shuturminska, K.; Rice, A.; Tarakina, NV; Wilson, RM; Bushby, AJ; Alonso, M.; Rodriguez-Cabello, JC; Barbieri, E.; Hernandez, AdR; Stevens, MM; Pugno, NM; Anderson, P.; Alvaro Mata, "Disorder-order interactions of proteins that regulate the growth of hierarchical calcification structures," Nature Communications, Vol. 9, 2145 (2018). 11. Boskey, AL; Villarreal-Ramirez, E. "Intrinsically disordered proteins and biomineralization." Matrix biology:j.matbio., vols. 52-54, pp. 43-59 (2016). 12. Beniash, E.; Simmer, JP; Margolis, HC. "Structural changes of amelogenin associated with self-assembly and interaction with minerals." J. Dent. Res., Vol. 91, pp. 967-972 (2012). 13. Fincham, AG et al. "Evidence for amelogenin 'nanospheres' as functional components of the secretory enamel matrix." J. Struct. Biol., Vol. 115, pp. 50-59 (1995). 14. Carneiro, KMM et al. "Amyloid-like ribbon structure of amelogenin in enamel calcification." Sci. Rep., Vol. 6, 23105 (2016). 15. Busch, S.; Schwarz, U.; Kniep, R. "Morphogenesis and structure of human teeth in relation to biomimetically grown fluoroapatite-gelatin composites." Chem. Mater., Vol. 13, pp. 3260-3271 (2001). 16. Delak, K et al. "Porcine amelogenin, a tooth enamel protein, is an intrinsically disordered protein that adopts an elongated molecular structure in monomeric form." Biochemistry, Vol. 48, pp. 2272-2281 (2009). 17. Coulombe, PA; Omary, MB. "The 'hard' and 'soft' principles governing the structure, function, and control of keratin intermediate filaments." Curr. Opin. Cell. Biol., Vol. 14, pp. 110-122 (2002).

Claims

1. A method for producing a keratin composition, particularly a keratin film, (a) A step of providing a keratin solution containing keratin in a solvent, (b) A step of casting the solution onto a substrate, (c) A step of drying the solution, A method that includes this.

2. A keratin solution containing at least one type of keratin in a solvent.

3. A keratin composition containing keratin, in the form of a film or similar.

4. The keratin composition according to claim 3, comprising keratinspherite.

5. A calcified keratin composition containing the composition described in claim 3 or 4.

6. The method or solution according to claim 1 or 2, wherein the solution contains at least two types of keratin.

7. The method, solution, or composition according to any one of claims 1 to 6, wherein the keratin or multiple types of keratin are wool keratin.

8. The method, solution, or composition according to any one of claims 1 to 7, wherein, when the composition contains a plurality of types of keratin, the molecular weight of the lightest keratin present in the solution or composition is less than 60% of the molecular weight of the heaviest keratin present in the solution or structure.

9. The method, solution, or composition according to any one of claims 1 to 8, wherein the keratin is highly negatively charged.

10. The method, solution, or composition according to any one of claims 1 to 9, wherein at least one, both, more than or all of the keratins is α-keratin.

11. The method, solution, or composition according to any one of claims 1 to 10, wherein at least one, both, more than or all of the keratins are high-sulfur keratins.

12. The method, solution, or composition according to any one of claims 1 to 10, wherein at least one, both, more than or all of the keratins are low-sulfur keratins.

13. The method, solution, or composition according to any one of claims 1 to 10, wherein the solution or composition contains both high-sulfur keratin and low-sulfur keratin.

14. The method, solution, or composition according to any one of claims 1 to 5, wherein the solution or composition contains one type of keratin, and the keratin is not KRT75.

15. The method or solution according to any one of claims 1, 2, or 6 to 14, wherein the solvent used is water, one or more polar aprotic solvents, one or more alcohols, or a combination thereof.

16. Claims 1, 2, wherein the solvent comprises one or more of the following: water, ethanol, water and ethanol, water and DMF, DMSO, and DMSO and DMF, or is selected from among them. Or the method or solution described in any one of items 6 to 15.

17. The method or solution according to any one of claims 1, 2, or 6 to 16, wherein the solvent is deionized and / or essentially does not contain any solute other than keratin.

18. The method or solution according to any one of claims 1, 2, or 6 to 17, wherein the solution contains salts such as sodium, sulfate, chloride, zinc, carbonate, phosphate, calcium, fluorine, iron, or potassium, and these salts may be added to the solvent.

19. The method or solution according to any one of claims 1, 2, or 6 to 18, wherein the pH of the solvent is 3 to 12.

20. The method or solution according to any one of claims 1, 2, or 6 to 19, wherein the concentration of keratin in the solution is 1 to 25% by weight.

21. The method according to any one of claims 1 and 6 to 20, wherein the substrate is at least slightly hydrophobic.

22. The method according to any one of claims 1 and 6 to 21, wherein the base material is a high-molecular-weight organosilicon compound such as polymethylsiloxane or polydimethylsiloxane.

23. The method according to any one of claims 1 and 6 to 21, wherein the substrate is a tooth or a plurality of teeth, or bone.

24. The method according to any one of claims 1 and 6 to 23, wherein the keratin solution is dried for at least 30 seconds.

25. The method according to any one of claims 1 and 6 to 24, wherein the keratin solution is dried at a temperature of 15°C to 50°C.

26. The method or composition according to any one of claims 1 to 25, wherein the solution contains a crosslinking agent such as triethylene glycol dimethacrylate (TEGDMA), hexamethylene diisocyanate (HDI), and / or polyethylene glycol dimethacrylate (PEGDMA).

27. The method or composition according to claim 26, wherein the crosslinking agent contains a photoinitiator.

28. The method according to claim 27, further comprising the step of irradiating the solution with light after casting the solution to initiate film formation by photocatalysis.

29. The keratin composition according to any one of claims 4 to 20, 26, or 27, wherein the diameter of the spherulite is approximately 3 to 50 μm.

30. The keratin composition according to any one of claims 4 to 20, 26, 27, or 29, wherein the composition is in the form of a film.

31. The keratin composition according to claim 30, wherein the thickness of the film is at least 10, 15, 20, 25, or 30 μm.

32. The thickness of the aforementioned film is less than 230, 225, 220, 215, 210, 205, or 200 μm. The keratin composition according to claim 30 or 31.

33. The method according to any one of claims 1 or 6 to 28, wherein the step of providing the keratin solution includes the step of dissolving the keratin in the solvent.

34. The method according to any one of claims 1, 6 to 28 or 33, further comprising the steps of washing the composition in water and / or storing the composition in water.

35. The method according to any one of claims 1, 6 to 28, 33, or 34, further comprising the step of incubating the keratin composition in a calcifying solution.

36. The method according to claim 35, wherein the calcification solution contains calcium or a sodium salt, or both.

37. The method according to claim 35 or 36, further comprising the step of adding an acid such as nitric acid, hydrochloric acid, or phosphoric acid to the calcification solution.

38. The method according to any one of claims 35, 36, or 37, wherein the calcification solution is a body fluid such as saliva, blood, interstitial fluid, serum, or plasma, or contains the same.

39. The method or composition according to any one of claims 1 to 38, wherein the keratin is obtained or can be obtained by extracting keratin from animal or human hair, body hair, wool, hooves, shells, horns, claws, nails, quills, or feathers.

40. A method for extracting keratin from animal or human tissue, particularly wool, such as hair, body hair, wool, hooves, shells, horns, claws, nails, quills, or feathers, (a) A step of mixing the tissue with the extraction solution, (b) A step of heating the mixture, (c) A step of separating the supernatant containing keratin from the remainder of the mixture, A method that includes this.

41. The aforementioned extract solution contains urea, sodium dodecyl sulfate, 2-mercaptoethanol, thiourea, dithiothreitol (DTT), [3-(coramidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), L-cysteine, ampholite, glycerol, Triton X-100, sodium phosphate, sodium pyrosulfite, sodium hydroxide, peracetic acid, 1-butyl-3-methylimidazolium chloride (BMIM+Cl). ２ The method according to claim 40, comprising one or more of the following: iodoacetic acid, zinc acetate, ethylenediaminetetraacetic acid (EDTA), Tris-HCl buffer, HEPES buffer, and other buffers.

42. The method according to claim 40 or 41, wherein the mixture is heated at 40 to 60°C for approximately 8 hours.

43. The method according to any one of claims 40 to 42, further comprising the step of drying the keratin solution.

44. A keratin solution, keratin composition, or calcified composition obtained or obtainable by the method described in any one of claims 1, 6 to 28, or 33 to 39.

45. A pharmaceutical composition containing a keratin solution, keratin composition, or calcified keratin composition according to any one of claims 2 to 20, 26, 27, 29 to 32, 39, or 44.

46. The pharmaceutical composition according to claim 45, wherein the composition is in the form of a hydrogel, a solution, a paint, a varnish, a coating, a scaffold such as a 3D printed scaffold, a film, a toothpaste, a strip, especially a dental strip, a dental restorative material, a composite, a gum, a metal implant, a cement, a ceramic, a paste, a plastic putty, a film, or a dentin hypersensitivity inhibitor, or is added thereto.

47. A medical structure or device, such as a synthetic graft, prosthesis or orthotic device, implant or tissue graft, particularly a dental or bone graft, comprising a keratin solution, keratin composition, calcified keratin composition or pharmaceutical composition according to any one of claims 2 to 20, 26, 27, 29 to 32, 39 or 44 to 46.

48. A keratin solution, keratin composition, calcified keratin composition, pharmaceutical composition, structure, or device for use in treatment, according to any one of claims 2 to 20, 26, 27, 29 to 32, 39, or 44 to 47.

49. A solution, composition, structure, or device according to any one of claims 2 to 20, 26, 27, 29 to 32, 39, or 44 to 47, for use in dental treatment, particularly in the treatment of leukoplakia (WSL) lesions or dental caries.

50. A solution, composition, structure, or device according to any one of claims 2 to 20, 26, 27, 29 to 32, 39, or 44 to 47, for use in the treatment or prevention of tooth demineralization, dental erosion, tooth wear, alveolar bone resorption, periodontitis, peri-implantitis or pulp disease, tooth hypersensitivity, and soft tissue repair, such as transplantation and augmentation.

51. A solution, composition, structure, or device according to any one of claims 2 to 20, 26, 27, 29 to 32, 39, or 44 to 47, for use in bone repair, healing, or regeneration.

52. A method for treating WSL or dental caries, or bone injury or bone disorder, comprising administering a keratin composition, calcified keratin composition, or pharmaceutical composition according to any one of claims 2 to 20, 26, 27, 29 to 32, 39, or 44 to 46, or using a device according to claim 47.

53. A product or kit comprising (a) a keratin solution or keratin composition, and (b) another calcifying solution and / or crosslinking agent.

54. A kit comprising a keratin solution, keratin composition, or pharmaceutical composition according to any one of claims 2 to 20, 26, 27, 29 to 32, 39, or 44 to 46, and an applicator for applying the solution or composition to a site of interest.

Images