Gel composition

Abstract

The present invention addresses the problem of providing a gel composition that can be molded and reshaped into an arbitrary three-dimensional structure. The problem is solved by a gel comprising three components: glycerin, collagen, and a polysaccharide.

Description

Gel composition

[0001] The present invention relates to a gel composition and the like.

[0002] Hydrogels are three-dimensional biomimetic materials having a structure and properties similar to those of the extracellular matrix, and are widely used as extracellular matrices during cell culture, cell adhesion scaffolds in the field of regenerative medicine, and sustained-release carriers in DDS. The cell adhesion scaffold in the field of regenerative medicine is essential for maintaining a space for cells to adhere and tissues to regenerate in vivo. For use in tissue regeneration in vivo, biodegradability, non-immunogenicity, the ability to maintain strength until tissue regeneration, cell affinity (cell adhesiveness, cell proliferation and / or differentiation induction ability), etc. are required, and the development of better cell adhesion scaffold materials having these characteristics has been actively carried out. Currently, materials such as collagen, gelatin, fibrin, polylactic acid, and polyglycol made into a predetermined shape such as sheet-like, disk-like, cylindrical, sponge-like, and fiber-like have been put into practical use as cell scaffold materials for regenerative medicine. However, cell scaffold materials that can freely and easily form a complex three-dimensional structure according to the shape of each defect site have not been put into practical use.

[0003] Non-Patent Document 1 discloses that a novel gel capable of freely forming and transforming into a three-dimensional structure was obtained by freeze-drying a polymer obtained by polymerizing acryloyl group-modified cholesterol-substituted pullulan. However, the preparation of the gel requires complicated processes such as polymer synthesis, self-assembly of the obtained polymer, cross-linking, freeze-thawing, freeze-drying, and hydration. Furthermore, since it is made of a synthetic polymer and requires a safety test, there is also a problem that it takes time to be put into practical use.

[0004] Sato et al., Sci. Rep., (2018) 8:15824

[0005] An object of the present invention is to provide a gel composition that can be formed and deformed into an arbitrary three-dimensional structure.

[0006] In view of the above problems, the inventors diligently conducted research and found that a gel consisting of three components—glycerin, collagen, and polysaccharides—can solve the above problems. Based on this finding, the inventors furthered their research and completed the present invention. That is, the present invention encompasses the following aspects.

[0007] Item 1. A gel composition comprising glycerin, collagen, and polysaccharides. Item 2. The gel composition according to Item 1, wherein the glycerin content is 75% or more, the collagen content is 15% or less, and the polysaccharide content is 20% or less. Item 3. The gel composition according to Item 1 or 2, wherein the total content of glycerin, collagen, and polysaccharides is 90% or more. Item 4. The gel composition according to any one of Items 1 to 3, comprising only glycerin, collagen, and polysaccharides. Item 5. The gel composition according to any one of Items 1 to 4, wherein the collagen is atelocollagen. Item 6. The gel composition according to any one of Items 1 to 5, wherein the viscosity at 37°C is 2000 mPa·s or more and 7500 mPa·s or less. Item 7. The gel composition according to any one of Items 1 to 6, wherein the average pore diameter in cross-section is 10 μm or more. Item 8. The gel composition according to any one of Items 1 to 7, further containing cells. Item 9. The gel composition according to claim 8, wherein the cells are bone marrow-derived mesenchymal cells. Claim 10. A pharmaceutical product comprising the gel composition according to any one of claims 1 to 9. Claim 11. The pharmaceutical product according to claim 10 for the regeneration of any one tissue selected from the group consisting of bone tissue, fibrous connective tissue, muscle tissue, adipose tissue, periodontal tissue, soft tissue, cartilage tissue, skin tissue, and nerve tissue. Claim 12. The pharmaceutical product according to claim 10 or 11, which is an injectable formulation. Claim 13. A method for tissue regeneration comprising administering the gel composition according to any one of claims 1 to 9 to a target. Claim 14. Use of a gel composition comprising glycerin, collagen, and polysaccharides for the manufacture of a pharmaceutical product for tissue regeneration. Claim 15. A sustained-release carrier for a bioactive substance comprising the gel composition according to any one of claims 1 to 9. Claim 16. The sustained-release carrier according to claim 15, wherein the bioactive substance is at least one selected from the group consisting of drugs, nucleic acids, extracellular vesicles, low molecular weight compounds, and proteins. Claim 17. A gel composition according to any one of claims 1 to 9, which is in a sol state and is used for the production of a gel.

[0008] According to the present invention, it is possible to provide a gel composition that can be molded and deformed into any three-dimensional structure.

[0009] This shows the viscosity of four types of the three-component gel compositions of the present invention at 37°C. The average pore size (top) and photographs of the surface structure taken with an electron scanning microscope (bottom) of the gel composition (Col / Gly / Pul gel) and collagen gel (Col gel) of the present invention are shown. The binding properties of the gel composition (Col / Gly / Pul gel), Pullulan, Atelocollagen, and Glycerin of the present invention, as determined by Fourier transform infrared spectroscopy, are shown. The dynamic viscoelasticity of the gel composition (Col / Gly / Pul gel) and collagen gel (Col gel) of the present invention in the angular velocity range of 1 to 10 rad / sec at 37°C are shown. The viability (initial adhesion amount) when cells were cultured in wells coated with either the gel composition (Col / Gly / Pul gel) or collagen gel (Col gel) of the present invention, and in uncoated wells (Control), is shown. Changes in cell morphology and cell number when cells were cultured for 24 hours in wells coated with either the gel composition (Col / Gly / Pul gel) or collagen gel (Col gel) of the present invention, and in uncoated wells (Control), are shown. The following shows the expression levels of bone formation-related genes (ALP, RunX2, and BMP-2) when cells were cultured in wells coated with either the gel composition of the present invention (Col / Gly / Pul gel) or collagen gel (Col gel), and in uncoated wells (Control). The following shows ALP activity (upper left panel), calcium precipitation (upper right panel), and alizarin red stained images (lower panel) when cells were cultured in wells coated with either the gel composition of the present invention (Col / Gly / Pul gel) or collagen gel (Col gel), and in uncoated wells (Control). The following shows CT images of rat skulls in which either the gel composition of the present invention (Col / Gly / Pul gel) or collagen gel (Col gel) was transferred to the bone defect site, or in an untreated (Control) state (upper panel). The area enclosed by the dashed line represents the bone defect site. The analysis results for the average bone mineral density (BMD), trabecular bone number (Tb.N), bone volume ratio (BV / TV), and bone surface area / bone volume ratio (BS / BV) at the bone defect site in each group are shown.The images show Sirius Red stained rat skulls in which either the gel composition of the present invention (Col / Gly / Pul gel) or collagen gel (Col gel) was transferred to the bone defect site, or untreated (Control) rat skulls. Photographs of the gel compositions of the present invention (Col / Gly / alginate gel, Col / Gly / hyaluronic acid gel, and Col / Gly / Pul gel) are shown. The viscosity at 37°C is shown when the gel composition of the present invention (Col / Gly / alginate gel and Col / Gly / Pul gel) is mixed with an equal amount of PBS. The amount of calcium precipitated when cells were cultured in wells coated with either the gel composition of the present invention (Col / Gly / alginate gel and Col / Gly / Pul gel) is shown. Photographs of FITC-ovalbumin release from the gel composition of the present invention (Col / Gly / Pul gel) on days 0, 1, and 7 are shown.

[0010] In this specification, the expressions “contains” and “includes” include the concepts of “contains,” “includes,” “substantially consists of,” and “consistes of only.”

[0011] 1. Gel Composition In one aspect, the present invention relates to a gel composition comprising glycerin, collagen, and polysaccharides (which may be referred to as "the gel composition of the present invention" in this specification). This will be described below.

[0012] In this specification, "gel composition" may be a gel state in which the dispersed phase cross-links to form a three-dimensional network structure throughout the dispersion, resulting in high viscosity and loss of fluidity (becoming a solid), or it may also include a sol state in which the dispersed phase is dispersed in a dispersion medium and maintains fluidity. The dispersion medium of the gel composition of the present invention contains glycerin. The "gel composition" in this specification also includes a gel composition in which the water content has been reduced or has become water-free by drying, and a gel composition obtained by swelling the dried gel composition with a liquid such as water, ethanol, culture medium, or pseudo-body fluid. In one embodiment, the gel composition of the present invention is a gel state composition, i.e., a gel. In another embodiment, the gel composition of the present invention is a sol state composition, i.e., a sol.

[0013] The gel composition of the present invention comprises three components: glycerin, collagen, and polysaccharide. The glycerin, collagen, and polysaccharide used as raw materials can be those that are commonly available and are not particularly limited. The polysaccharide is not limited as long as it is a water-soluble polysaccharide that gels without the need for a crosslinking agent. Examples include pullulan, hyaluronic acid, alginic acid, amylose, dextran, chitosan, cellulose, carrageenan, starch, agar, amylopectin, inulin, mannan, heparin, chondroitin sulfate, agarose, xanthan gum, and their derivatives (e.g., hydroxypropylcellulose, carboxymethylcellulose, etc.). Among these, from the viewpoint of high water solubility, for example, polysaccharides having α-links such as pullulan, polysaccharides having carboxyl groups such as hyaluronic acid and alginic acid, and water-soluble cellulose derivatives such as hydroxypropylcellulose and carboxymethylcellulose are preferred, with pullulan, alginic acid, and hyaluronic acid being more preferred, and pullulan being even more preferred. In one embodiment, the gel composition of the present invention may further contain components other than the three components described above. In one embodiment, it is preferable that the gel composition of the present invention does not contain any components other than the three components described above.

[0014] The content of the three components in the composition is not particularly limited as long as it can form a gel composition, but for example, the glycerin content is 71% or more, 72% or more, 73% or more, or 74% or more of 100% of the mass of the gel composition or 100% of the total mass of the three components in the gel composition, preferably the glycerin content is 75% or more, and more preferably the glycerin content is 80% or more, 82% or more, 85% or more, 88% or more, or 90% or more. The upper limit of the glycerin content is not particularly limited, but for example, it is 99% or less, 95% or less, 93% or less, 90% or less, 88% or less, 86% or less, 85% or less, 82% or less, or 80% or less. The collagen content in the composition is preferably 20% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, or 5% or less, relative to 100% of the mass of the gel composition or 100% of the total mass of the three components in the gel composition. In one embodiment, the collagen content in the composition is preferably 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, or 8% or less, relative to 100% of the mass of the gel composition or 100% of the total mass of the three components in the gel composition. In another embodiment, the collagen content in the composition is preferably 7% or less, 6.5% or less, 6% or less, 5.5% or less, or 5% or less, relative to 100% of the mass of the gel composition or 100% of the total mass of the three components in the gel composition. The lower limit of the collagen content is not particularly limited, but for example, it is 0.5% or more, 1% or more, 1.5% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, or 9% or more. The polysaccharide content in the composition is, for example, 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, or 11% or less, relative to 100% of the mass of the gel composition or 100% of the total mass of the three components in the gel composition, and preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, or 5% or less.In one embodiment, the polysaccharide content in the composition is preferably 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, or 8% or less, based on 100% of the mass of the gel composition or 100% of the total mass of the three components in the gel composition. In another embodiment, the polysaccharide content in the composition is preferably 7% or less, 6.5% or less, 6% or less, 5.5% or less, or 5% or less, based on 100% of the mass of the gel composition or 100% of the total mass of the three components in the gel composition. The lower limit of the polysaccharide content is not particularly limited, but for example, it may be 0.1% or more, 0.5% or more, 1% or more, 1.5% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, or 9% or more. By having the content of the three components in the composition within the above range, it is possible to obtain a gel composition that has viscosity sufficient to be molded and modified into any shape, stability of the three-dimensional network structure in liquid, and in which the shape of the gel does not change significantly after drying, and further possesses biodegradability and cell adhesion ability. In one embodiment, the total content of the three components in the composition is, for example, 70% or more, 80% or more, preferably 85% or more, more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, even more preferably 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% of 100% of the mass of the gel composition.

[0015] Glycerin has two or more hydroxyl groups in its molecule, making it difficult to vaporize upon drying. By containing glycerin within the above-mentioned range in the gel composition, the glycerin is retained even after drying, allowing the gel composition to be molded and deformed into any desired shape.

[0016] In one embodiment, the collagen is preferably atelocollagen, in which the non-helical regions (telopeptide regions) at both ends of a type I collagen polypeptide chain are cleaved and removed. The form of use of the gel composition of the present invention is not particularly limited, but for example, when it is introduced into a living organism, using atelocollagen with low antigenicity can reduce the risk of rejection of the gel composition.

[0017] Polysaccharides have excellent properties as thickening and stabilizing agents, binding agents, and film-forming agents. Therefore, by including polysaccharides within the above-mentioned range, the viscosity of the gel composition increases, making it possible to mold and deform it into any desired shape. Polysaccharides may be labeled, and examples of such labels include fluorescent dyes such as FITC.

[0018] The average molecular weight of the polysaccharide is, for example, between 1,000 and 1,000,000, preferably between 2,000 and 500,000, and more preferably between 5,000 and 20,000. The average molecular weight can be measured by conventional methods, such as HPLC.

[0019] The viscosity of the gel composition at 37°C is preferably 2000 mPa·s or more, 2500 mPa·s or more, 3000 mPa·s or more, 3100 mPa·s or more, 3200 mPa·s or more, 3300 mPa·s or more, 3400 mPa·s or more, 3500 mPa·s or more, or 3700 mPa·s or more, and preferably 7500 mPa·s or less, 7000 mPa·s or less, 6500 mPa·s or less, 6000 mPa·s or less, 5500 mPa·s or less, 5000 mPa·s or less, 4500 mPa·s or less, or 4300 mPa·s or less. Having a viscosity within the above range makes it possible to mold and deform the gel composition into any three-dimensional structure. In this invention, the viscosity of the gel composition is the value measured using an E-type viscometer (Toki Sangyo) for the gel composition alone, with a cone rotor of 3°C × R14, 37°C, and a rotation speed of 10 rpm.

[0020] The method for molding the gel composition into any three-dimensional structure is not particularly limited, but examples include pouring it into a mold of any shape or molding it under pressure. From the viewpoint of being simple, allowing for shape adjustment as needed, and enabling aseptic operation, a preferred method is to mold it into any shape by aspirating and discharging it with a syringe at the in vitro and / or in vivo level. In one embodiment, the viscosity of the gel composition at 37°C is preferably 6000 mPa·s or less, more preferably 5500 mPa·s or less, and particularly preferably 5000 mPa·s or less, from the viewpoint of enabling aspiration and discharging of the gel composition with a syringe. Preferably, the gel composition of the present invention is a composition that can maintain its three-dimensional structure after being molded into one. The gel composition of the present invention can be deformed into any three-dimensional structure even after being molded into one.

[0021] In dynamic viscoelasticity measurements at 37°C, the gel composition of the present invention preferably has a storage modulus G' and loss modulus G'' in the angular frequency range of 1 to 10 rad / s, which are between 1,000 Pa and 30,000 Pa, or between 1,000 Pa and 20,000 Pa. Having the dynamic viscoelasticity within this range results in a composition with excellent strength and adhesion, enabling the molded three-dimensional structure to be maintained for a long period. Dynamic viscoelasticity can be measured, for example, using a rheometer such as Rheosol-G5000 (UBM).

[0022] The gel composition is preferably porous, from the viewpoint of increasing the surface area for cell adhesion and proliferation, and / or the volume that can hold physiologically active substances. The porosity by volume of the gel composition of the present invention is preferably 50 to 90%. The porosity can be measured, for example, by determining the pore area using image analysis software ImageJ. In one embodiment, porosity allows cells to easily penetrate not only the surface but also the interior of the gel composition when the gel is swollen with a solution containing cells, enabling three-dimensional culture.

[0023] The average pore diameter in the cross-section of the gel composition of the present invention is, for example, about 6 μm or more, preferably about 10 μm or more, about 20 μm or more, about 30 μm or more, about 40 μm or more, about 50 μm or more, about 60 μm or more, or about 70 μm or more, and for example, about 300 μm or less, about 200 μm or less, about 150 μm or less, or about 100 μm or less. The average pore diameter in the cross-section can be measured according to a conventional method, such as a method that combines observation with scanning electron microscopy with analysis using image analysis software. In one embodiment, having a pore diameter within the above range allows cells to easily enter the gel composition and works favorably for cell growth and adhesion, resulting in good cell adhesion.

[0024] The gel composition of the present invention has cell affinity and excellent cell adhesion properties, and moreover, it does not inhibit the proliferation and / or differentiation ability of adhered cells, and preferably promotes the proliferation and / or differentiation of adhered cells. Cell adhesion can be analyzed, for example, by culturing cells in a culture dish coated with the gel composition for a relatively short period of time, for example, 30 minutes to 12 hours, according to a conventional method, washing away unadhered cells, and then measuring the number of cells remaining in the culture dish by a known method. Cell proliferation and differentiation ability can be analyzed by known methods, for example, by measuring the number of cells, cell morphology, and differentiation markers after culturing cells for about 24 hours.

[0025] The types of cells cultured using the gel composition of the present invention are not particularly limited, but examples include stem cells (ES cells, iPS cells, bone marrow-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, neural stem cells, hematopoietic stem cells, adipose-derived mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, skin stem cells, muscle stem cells, germline stem cells, etc.), osteoblasts, gingival cells (gingival fibroblasts, gingival epithelial cells), chondrocytes, osteoclasts, fibroblasts, keratinocytes, oral mucosal epithelial cells, airway mucosal epithelial cells, gastric mucosal epithelial cells, intestinal mucosal epithelial cells, vascular endothelial cells, conjunctival epithelial cells, smooth muscle cells, adipocytes, leukocytes, lymphocytes, muscle cells, etc. Preferably, the cells are mesenchymal stem cells, and particularly preferably bone marrow-derived mesenchymal stem cells. Furthermore, the organism from which the cells originate is not particularly limited, and examples include mammals such as humans, monkeys, mice (Musculus genus), rats, hamsters, guinea pigs, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, and deer.

[0026] In one embodiment, the gel composition of the present invention is used for three-dimensional culture and / or gel culture of cells. The culture method is not particularly limited, and examples include seeding cells on wells coated with the gel composition of the present invention, and swelling the gel composition of the present invention with a culture medium containing cells so that the cells are dispersed in the gel composition of the present invention. The culture temperature of cells using the gel composition of the present invention is about 37°C, and the culture period is about 1 to 6 weeks, preferably about 2 to 5 weeks, more preferably about 3 to 4 weeks. A solvent such as DMSO may be used in the culture medium.

[0027] The gel composition of the present invention is produced by drying a solution of glycerin, collagen, and polysaccharides. Drying methods include freeze-drying and drying using an evaporator, with freeze-drying being preferred, and the drying conditions are not particularly limited. Examples of pre-freezing temperatures include -10°C to -196°C, freezing temperatures include -40°C to -90°C, and pressures include 50 Pa or less. A gel composition is usually obtained in about 8 to 24 hours. The obtained gel composition is suitable for long-term storage because it contains no water or has a water content of 5% or less relative to 100% of the gel composition's mass. Such drying, preferably freeze-drying, allows the gel to dry while maintaining its three-dimensional network structure and shape. Furthermore, drying, preferably freeze-drying, can increase the number of pores and pore diameter of the gel composition.

[0028] In one embodiment, the gel composition of the present invention, which is in a sol state, can be used for the production of a gel.

[0029] 2. Pharmaceuticals: In one aspect, the present invention relates to a pharmaceutical (which may be referred to as "the pharmaceutical of the present invention" in this specification) comprising a gel composition containing glycerin, collagen, and polysaccharides. This will be described below.

[0030] The gel composition of the present invention exhibits high cell adhesion, does not inhibit cell proliferation or differentiation into tissue, and can be molded and deformed into any three-dimensional structure. Therefore, the composition of the present invention can be used as a tissue regeneration agent and / or cell adhesion scaffold material that can be molded and deformed as needed into a three-dimensional structure that matches the shape of individual defect sites. In one embodiment, the composition of the present invention may contain cells when implanted or injected into the body, or it can be used as a transplant material for cell therapy in which cells are cultured in vitro within the gel and then implanted directly into the body.

[0031] The pharmaceutical product of the present invention can take any form; for example, it can be used in gel form by molding and deforming it to match the shape of the implantation or injection site at the time of use, or it can be used after being pre-molded into a disc, sponge, sheet, block, or the like. The gel composition of the present invention has a viscosity within the aforementioned range when in gel form, so it can be used as an injectable tissue regeneration agent and / or an injectable cell adhesion scaffold material, which can be drawn up with a syringe or the like and directly injected into any three-dimensional structure that matches the shape of the defect site in the body.

[0032] The pharmaceutical product of the present invention can exert the above-mentioned effects on its own. Therefore, the pharmaceutical product of the present invention can exert its desired effects even without containing other components having these effects and / or actions, although it may contain other components having pharmacological effects. Other components are not particularly limited as long as they are pharmaceutically acceptable. Other components include, for example, cell adhesion promoting materials such as poly-L-lysine, poly-L-arginine, collagen, laminin, and fibronectin; pharmacologically active components such as vitamins such as ascorbic acid and nicotinamide; neurotrophic factors such as NGF and BDNF; bone morphogenetic factors such as BMP; epidermal growth factor, basic fibroblast growth factor, insulin-like growth factor; cytokines such as IL-2; and additives. Examples of additives include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, and chelating agents.

[0033] The cells that can be included in the pharmaceutical of the present invention are the same as those described in "1. Gel Composition". The pharmaceutical of the present invention can be suitably used for the regeneration of three-dimensional tissues, such as bone tissue, fibrous connective tissue, muscle tissue, adipose tissue, periodontal tissue, soft tissue, cartilage tissue, skin tissue, nerve tissue, etc. In one embodiment, the pharmaceutical of the present invention contains collagen, which is a main component of the extracellular matrix of bone tissue and is known to promote the ability to induce osteogenic differentiation. Therefore, it can be suitably used for bone tissue regeneration by enhancing the bone regeneration ability from bone marrow mesenchymal stem cells and / or osteoblasts.

[0034] The applications of the pharmaceutical product of the present invention are not particularly limited, but examples of mammals include humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, and deer. In one embodiment, the pharmaceutical product of the present invention can be suitably used in patients with tissue defects due to trauma or tumors, and particularly preferably bone defects or fractures.

[0035] The dosage of the drug of the present invention is appropriately determined considering the size of the tissue defect in the patient; for example, approximately 1 ml should be administered to a defect of about 1 cm in diameter.

[0036] The gel composition contained in the pharmaceutical of the present invention is bioabsorbable. Because it is bioabsorbable, it serves as a scaffold for cells in the early stages of tissue regeneration and secures space for tissue regeneration, while not remaining in the body once tissue regeneration has progressed sufficiently and not hindering tissue regeneration. Therefore, it can be suitably used for the regeneration of three-dimensional tissues. The gel composition of the present invention is usually absorbed within 10 to 30 days after introduction into the body.

[0037] Although cell adhesion scaffolds for regenerative medicine are being actively researched, commercially available collagen gels and Matrigels have low viscosity and cannot maintain a three-dimensional structure, and pre-processed materials in sponge-like or sheet form cannot be deformed. Therefore, it has not been possible to provide materials with arbitrary three-dimensional structures that can be shaped to suit individual defect sites. The gel composition disclosed in Non-Patent Literature 1 can be molded into any three-dimensional structure, but the manufacturing process requires complex steps such as the synthesis of complex polymers, self-assembly of polymers, crosslinking, freeze-thaw cycles, freeze-drying, and hydration. Furthermore, there is a hurdle in that safety testing of synthetic polymers is required for practical use. With the present invention, it is possible to obtain a pharmaceutical suitable for tissue regeneration that contains a gel composition that can be molded and deformed into any three-dimensional structure, can be easily obtained by simply mixing three components and freeze-drying, and does not use synthetic polymers, etc., thus offering high safety and practicality.

[0038] 3. Sustained-Release Carrier The present invention relates, in one aspect, to a sustained-release carrier for physiologically active substances (which may also be referred to as "the sustained-release carrier of the present invention" in this specification), comprising a gel composition containing glycerin, collagen, and polysaccharides. This will be described below.

[0039] Since the gel composition of the present invention is porous with a large pore size and the solvent has been removed while maintaining its original shape, when it is swollen with a liquid containing a physiologically active substance, the physiologically active substance can easily penetrate into the gel interior. Furthermore, since the gel composition of the present invention consists only of natural components, it has high safety and is further biodegradable. Therefore, the gel composition of the present invention can be suitably used as a carrier for introducing physiologically active substances such as nucleic acids, proteins, and drugs into the living body. In a preferred embodiment, the sustained-release carrier of the present invention can be used as a sustained-release carrier that gradually releases the carried physiologically active substance.

[0040] The physiologically active substance carried by the sustained-release carrier of the present invention is not particularly limited, and examples include nucleic acids such as DNA, RNA, chimeric nucleic acids of DNA and RNA, and hybrids of DNA / RNA, proteins such as enzymes, receptors, antibodies, antigens, cytokines such as interferon and interleukin, extracellular vesicles, low molecular weight compounds, or drugs. These physiologically active substances such as nucleic acids, proteins, low molecular weight compounds, and drugs may be used alone or in combination of two or more.

[0041] The subjects into which the sustained-release carrier of the present invention is introduced include mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, cows, pigs, sheep, and horses, birds such as chickens, and non-human animals such as fish.

[0042] The sustained-release agent of the present invention is preferably used in dosage forms such as injections, eye drops, nasal drops, inhalants, and suppositories. In this case, it is suitably provided as a pharmaceutical composition, and an introduction agent for various dosage forms can be obtained using appropriate carriers commonly used.

[0043] 4. Other uses The gel composition of the present invention has high cell adhesiveness, does not inhibit cell proliferation and differentiation into tissues, can be molded and deformed into an arbitrary three-dimensional structure, can carry a physiologically active substance, and is biodegradable. Therefore, the composition of the present invention can be used alone or mixed with tissue stem cells or a physiologically active substance as other uses where the above properties are useful, such as a cosmetic composition for local injection.

[0044] Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited by these examples.

[0045] Example 1. Preparation and property evaluation of gel composition 1.1 Preparation of Col gel The collagen gel (Col gel) was prepared according to the manufacturer's (Koken CO., LTD) instructions. 1 mL of 10×PBS (Nacalai Tesque, INC.), 0.1 mL of 1M HEPES pH7.4 (Nacalai Tesque, INC.), 0.1 mL of 1M NaHCO3, 0.8 mL of distilled water, and 8 mL of IPC-50 atelocollagen (Koken CO., LTD) were mixed in order to prepare a sterile precursor solution. 300 μL of this solution was added to each well of a 24-well plate and left to gel in an incubator at 37°C for 2 hours.

[0046] 1.2 Preparation of Col / Gly / Pul gel 510 mg of glycerin (Nacalai Tesque Co., Ltd.), 30 mg of pullulan (Tokyo Chemical Industry Co., Ltd.), 12 mL of IPC-50 atelocollagen (5 mg / ml), and 24 mL of 1 mM NaOH were stirred with a magnetic stirrer for 5 hours. After stirring, it was frozen at -80°C for 30 minutes and dried sufficiently in a freeze dryer (EYELA FDU-2110; Tokyo Rika Kikai Co., Ltd.) for about 12 hours. Then, ethanol was added for sterilization and dried again by the freeze dryer. By changing the blending ratios of glycerin, pullulan, and atelocollagen, a total of 5 kinds of gels were prepared (Table 1). The prepared gels could be aspirated with a syringe and molded into any shape, and could be freely deformed even after being molded once. Also, they could maintain their shape without dissolving in water for more than one week.

[0047]

[0048] 1.3 Characterization of Materials The viscosity of four types of Col / Gly / Pul gels at 37°C was measured using an E-type viscometer (Toki Sangyo) with a 3°×R14 cone rotor at a rotation speed of 10 rpm (Figure 1). The internal structure of the Col gel and Col / Gly / Pul gel was observed using a scanning electron microscope (SEM; S-4800; Hitachi), and the average pore diameter in the cross-section was calculated using ImageJ software (Figure 2). A porous network structure was observed in all gels. The pores of the Col / Gly / Pul gel were neatly shaped, mostly circular or elliptical. On the other hand, the Col gel had finer fibers and disordered bonding. Furthermore, while the average pore diameter of the Col gel was 5.44±0.35 μm, the average pore diameter of the Col / Gly / Pul gel was 78.06±2.76 μm, showing a significant increase in pore diameter. The binding properties of the Col / Gly / Pul gel were analyzed using Fourier transform infrared (FT-IR) spectroscopy (IRAffinity-1S; Shimadzu Corporation) (Figure 3). As a result, the Col / Gly / Pul gel contained an amide I (C=O stretching vibration) band (1659 cm²) similar to that of collagen. -1 ), amide II (N-H bending vibration) band (1557 cm) -1 ), and the amide III (C-N stretching vibration) band (1238 cm) -1 This revealed that collagen was well maintained. The dynamic viscoelasticity of the Col gel and Col / Gly / Pul gel at 37°C was observed using a rheometer (Rheosol-G5000; UBM) (Figure 4). The Col / Gly / Pul gel showed significantly higher values ​​than the Col gel in both G' (an indicator of elasticity) and G'' (an indicator of viscosity), indicating stronger strength and surface adhesion.

[0049] Example 2. In vitro experiment 2.1 Cell culture and seeding. Mesenchymal stem cells (rBMSCs) derived from rat bone marrow were obtained from the femurs of 8-week-old Sprague-Dawley rats (Shimizu Experimental Materials Co., Ltd.) and cultured under normal conditions in Eagle's minimum essential medium (E-MEM) containing 10% fetal bovine serum (FBS) and 1% antibiotic-anti-mycoplasma solution (both from Nacalai Tesque Co., Ltd.). Subsequently, the cells were cultured in α-MEM (containing 10% FBS, antibiotic-anti-mycoplasma solution, 10 mM β-glycerophosphate bone formation supplement, and 10 nM dexamethasone) as a differentiation induction medium, and the expression of bone formation-related genes, ALP activity, and calcium precipitation were examined. Third-generation cells were digested with 0.5 g / L trypsin and 0.53 mmol / L EDTA, centrifuged, resuspended, and 6 × 10⁶ cells were collected. 4 Cells were added to three types of 24-well plates at a density of cells / well: (i) without gel coating, (ii) coated with 300 μl of Col gel, and (iii) coated with 100 μl of Col / Gly / Pul gel. The cell culture medium was changed every three days. This study was conducted in accordance with the Osaka Dental University Animal Experiment Guidelines (Approval Number 23-01009).

[0050] 2.2 Cell Adhesion Cell adhesion was evaluated at 1, 3, and 6 hours using CellTiter-Blue® Cell Viability Assay (Promega Corporation). After incubation for 1, 3, and 6 hours, the samples were washed twice with PBS, and 300 μL of diluted CellTiter-Blue® reagent (50 μL of CellTiter-Blue® reagent diluted in 250 μL of PBS) was added. After incubation at 5% CO2 and 37°C for 2 hours, 100 μL of the reagent was added to each well in a 96-well plate. The fluorescence of the solution was analyzed at 560 / 590 nm using a microplate reader (SpectraMax M5; Molecular Devices). As shown in Figure 5, at all time points, the Col / Gly / Pul gel showed significantly higher cell adhesion than the wells without gel coating and the Col gel. (Results are shown based on the mean ± standard deviation. ns represents P > 0.05, *** represents P < 0.001, and **** represents P < 0.0001.)

[0051] 2.3 Cell morphology. Cell morphology of all samples after 24 hours of culture was evaluated using fluorescent staining. After washing the 24-hour cultured samples three times with PBS, 1 mL of 4% paraformaldehyde (PFA) solution was added for fixation, and the samples were cultured at room temperature (23–25°C) for 20 minutes. Subsequently, the samples were washed three times with PBS. 0.2% (v / v) Triton X-100 was added to the samples to make the cells permeable. After shaking for 30 seconds and culturing for 30 minutes, the samples were treated with Blocking One reagent (Nacalai Tesque Co., Ltd.) at room temperature (23–25°C) for 30 minutes, and stained with Alexa Fluor 488 phalloidin and 40,6-diamidino-2-phenylindole (DAPI) at 37°C in the dark for 1 hour. F-actin and cell nuclei were observed using a confocal laser scanning microscope (LSM700; Carl Zeiss AG). As shown in Figure 6, cells cultured in wells without gel coating were flattened and had a small cell count. Cells cultured on Col gel and Col / Gly / Pul gel were three-dimensional, and communication between cells via pseudopods was observed. Furthermore, in wells cultured on Col / Gly / Pul gel, the cell count was large, and the cells were not confined to a part of the gel but dispersed throughout.

[0052] 2.4 Expression of Osteogenesis-Related Genes The expression levels of osteoogenesis-related genes (ALP, RunX2, BMP-2) were evaluated using the TaqMan real-time PCR assay. After culturing on Col gel and Col / Gly / Pul gel for 7 and 14 days, cells were treated with collagenase type 2 for 30 minutes, centrifuged to remove the hydrogel, and harvested. Total RNA of rBMSCs was recovered using the RNeasy Mini Kit (Qiagen). Each RNA sample was reverse transcribed to cDNA in 10 μL using the Prime Script RT Kit. The expression levels of ALP and Runx2 after 7 days of culture and the expression level of BMP-2 after 14 days of culture were accurately measured using the PCRmax ECO48 Real-time qPCR system. The relative expression levels of the reactive genes in each group were standardized against the expression level of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and determined using the ΔΔCt method. As shown in Figure 7, cells cultured on Col / Gly / Pul gel showed significantly higher gene expression levels compared to cells cultured on Col gel and in wells without gel coating. (Results are shown as mean ± SD. * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and **** indicates P<0.0001.)

[0053] 2.5 ALP Activity After culturing for 7 or 14 days, cells were harvested as in 2.4 and lysed with 300 μL of 0.2% Triton X-100. The lysate was transferred to a microcentrifuge tube. ALP activity was measured using the ALP pNPP Liquid Substrate enzyme-linked immunosorbent assay kit (Sigma-Aldrich). p-Nitrophenol production was determined by detecting the optical density of the solution at 405 nm using a 96-well microplate reader. DNA content was measured using the PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. ALP content was normalized by the DNA content of the corresponding cell lysate. As shown in the upper left panel of Figure 8, cells cultured on Col / Gly / Pul gels had significantly higher ALP activity compared to cells cultured on wells without gel coating and on Col gels. (Results are shown based on the mean ± standard deviation. ns represents P > 0.05, and **** represents P < 0.0001.)

[0054] 2.6 After culturing in calcium deposition differentiation induction medium for 21 days, the calcium deposited in the sample was dissolved with 10% formic acid and collected. The amount of calcium in these deposits was quantified using the Calcium E-Test Kit (Fujifilm Wako Pure Chemical Industries, Ltd.). 1 mL of calcium release test reagent and 2 mL of kit buffer were accurately added to 50 μL of collected medium. The reaction product was detected at 610 nm. Calcium concentration was calculated from the absorbance of the relative standard curve. As shown in the upper right panel of Figure 8, cells cultured on Col / Gly / Pul gel showed significantly higher calcium precipitation compared to cells cultured on wells without gel coating and on Col gel. (Results are shown as mean ± SD. *** represents P<0.001, and **** represents P<0.0001.)

[0055] 2.7 After culturing for 21 days in Alizarin Red-stained differentiation-inducing medium, the culture medium was removed, washed three times with 1 mL of PBS, 500 μL of methanol was added, and the cells were fixed at 4°C for 20 minutes. The methanol was removed, washed three times with 1 mL of purified water, 400 μL of staining solution was added, and the cells were allowed to stand for 5 minutes. The cells were observed using a BZ-X800 fluorescence microscope (Keyence Corporation). As shown in the lower panel of Figure 8, cells cultured on Col / Gly / Pul gel showed significantly more calcium deposition stained red compared to cells cultured on wells without gel coating and on Col gel. From these results, it was shown that cells cultured on Col / Gly / Pul gel underwent advanced osteogenic differentiation, indicating that the Col / Gly / Pul three-component gel has high osteogenic differentiation-inducing ability.

[0056] Example 3. In vivo experiment 3.1. Animal model and surgical procedure Twelve 8-week-old male Sprague-Dawley rats weighing 180-200g were randomly divided into three groups. After general anesthesia and surgical irrigation, a 20mm longitudinal incision was made along the center of the rat's head to separate the muscle and periosteum and expose the parietal bone. A 5mm diameter defect was created on both sides of the sagittal suture using trefin bales and washed with sterile saline. In the Col gel group, a Col gel with a diameter of 5mm and a thickness of 1mm was placed. In the Col / Gly / Pul gel group, 0.2ml of Col / Gly / Pul gel was placed. The periosteum and muscle were sutured in layers. To prevent postoperative infection and reduce postoperative pain, gentamicin (1mg / kg) and buprenorphine (0.05mg / kg) were injected daily for 3 days postoperatively.

[0057] 3.2. Micro-CT Analysis Immediately after dissection, skulls were placed in saline solution and scanned using a micro-CT system (SkyScan1275, Bruker) at 110kV, 55μA, and without filters. The images are shown in the upper panel of Figure 9. In the Col / Gly / Pul gel group, new bone formation was clearly observed in the defect area within the dotted line, indicating that the defect was closed. Bone regeneration was evaluated by quantifying the mean bone density (BMD), trabecular number (Tb.N), bone volume ratio (BV / TV), and bone surface area / bone volume ratio (BS / BV) of the region of interest, defined as having a diameter of 5.0 mm and a height of 0.505 mm around the defect, using morphometric software (CTAn; Bruker). The results are shown in the lower panel of Figure 9. In the Col / Gly / Pul gel group, the mean bone density and bone formation rate of the newly formed bone were significantly higher than in the other groups, and the bone surface area / bone volume ratio was lower, indicating that more bone was generated per unit area. (Results shown are mean ± +SD. ns represents P>0.05, * represents P<0.05, ** represents P<0.01, *** represents P<0.001, and **** represents P<0.0001.)

[0058] 3.3. Sirius Red Staining After micro-CT scanning, specimens were fixed in a 70% ethanol solution for 3 days and stained with Sirius Red to evaluate bone formation. Histological characteristics of sections were analyzed using a BZ-9000 fluorescence microscope. The results are shown in Figure 10. In the control group, the area of ​​newly formed bone was small, and the boundary between the newly formed bone and the original bone tissue was clearly visible (green arrow in Figure A), suggesting that the connection between the old and new bone was not strong. The deposition of fibrous tissue inside the newly formed bone was irregular (Figure B). In the Col gel group, the newly formed bone was incompletely covered, and more internal cavities were observed. The boundary between the old and new bone was still visible. The yellow areas (white arrow in Figure D) indicate more immature collagen fibers, suggesting that the newly formed bone was still in the process of formation and mineralization. In the Col / Gly / Pul gel group, bone production was most significant, and the entire defect was almost completely filled with newly formed bone. Furthermore, there was no clear boundary between the old and new bone, indicating the highest degree of fusion between the newly formed bone and the original bone tissue (green arrow in Figure E). The fibrous tissue of the newly formed bone had a large amount of regularly matured fibrous tissue deposited, indicating a high degree of mineralization in the newly formed bone (Figure F). These results demonstrate that the Col / Gly / Pul three-component gel exhibits high bone tissue differentiation induction ability as a cell adhesion scaffold in vivo, and can improve new bone formation.

[0059] Example 4. Preparation and Characterization of Gel Compositions (2) 4.1 Preparation and Characterization of Alginate Gel and Hyaluronic Acid Gel Glycerin, IPC-50 atelocollagen (Koken CO., LTD, prepared to be 2% of 100% of the mass of the gel composition after freeze-drying), and sodium alginate (Fujifilm Wako Pure Chemical Industries, Ltd., prepared to be 1-5% of 100% of the mass of the gel composition after freeze-drying), sodium hyaluronate (Fujifilm Wako Pure Chemical Industries, Ltd., prepared to be 1% of 100% of the mass of the gel composition after freeze-drying), or pullulan (Tokyo Chemical Industries, Ltd., prepared to be 5-20% of 100% of the mass of the gel composition after freeze-drying) as a comparison was mixed in water, the pH was adjusted to 6-6.5 with an aqueous NaOH solution, and then freeze-dried in the same manner as in Example 1.2 to create gels with varying concentrations of glycerin and polysaccharides (Figure 11). Gels containing 2% collagen, 97-78% glycerin, 1-5% alginate, 1% hyaluronic acid, or 5-20% pullulan could all be inhaled with a syringe, molded into any shape, and could be freely deformed even after being molded.

[0060] To confirm the stability in solution, the viscosity of 200 mg of prepared alginate gel or pullulan gel, when 200 mg of PBS was added, was measured using an E-type viscometer (Toki Sangyo) with a cone rotor at 3°C ​​× R14, 37°C, and a rotation speed of 100 rpm. The measurement results are shown in Figure 12. In the pullulan gel, the viscosity increased as the pullulan concentration increased from 5% to 20%. On the other hand, the alginate gel showed a viscosity similar to that of the pullulan gel in the above range from 1% to 5%. These results clearly show that by using alginate as a polysaccharide, it is possible to obtain a gel that exhibits equivalent viscosity at a lower concentration than pullulan. The alginate gel is expected to have pharmaceutical applications in situations where greater strength is required, such as in biomedical implants for fracture treatment.

[0061] 4.2 Evaluation of the osteogenic differentiation-inducing ability of alginate gel Similar to Example 4.1, atelocollagen was fixed at 2% relative to 100% mass of the lyophilized gel composition, and gels were prepared by varying the ratio of glycerin (90 or 97%) and polysaccharide (8% pullulan, or 1% sodium alginate). Mouse bone marrow-derived mesenchymal stem cells (Cyagen Biosciences) were cultured under normal conditions using the MesenCult Expansion Kit (Mouse, Veritas). 100 mg of the pullulan gel or alginate gel was attached to each well using the cap of a microtube (Watson 131-7155C), and bone marrow-derived mesenchymal stem cells were cultured. 5 100 μL of cells / mL was added, and after 4 hours, 2 mL of MesenCult Osteo Stimulatory Kit (Mouse, Veritas) was added to induce differentiation into osteoblasts. After one week of culture, the amount of calcium deposited on the gel was quantified using the Calcium E-Test Kit (Fujifilm Wako Pure Chemical Industries, Ltd.) to evaluate bone formation. The results are shown in Figure 13. Calcium precipitation was confirmed in both pullulan gel and alginate gel, confirming their ability to induce differentiation of stem cells into osteoblasts (n=3).

[0062] Example 5. Evaluation of sustained-release properties of gel composition Atelocollagen, glycerin, and pullulan (prepared to have concentrations of 2%, 78%, and 20% of the mass of the gel composition after lyophilization) were mixed in water, the pH was adjusted to 6-6.5 with an aqueous NaOH solution, and then a protein labeled with the fluorescent dye FITC (FITC-ovalbumin (45kDa)) was mixed in. The mixture was lyophilized in the same manner as in Example 1.2 to prepare a gel. 50 mg of the obtained gel was added to 0.50 mL of a PBS solution containing 2% bovine serum albumin, and incubated at 37°C for up to 7 days to confirm the release of protein from the gel. The results are shown in Figure 14. After 1 day, it was confirmed that ovalbumin was retained in the gel. After 7 days, almost all of the ovalbumin had been released, confirming that this gel has sustained-release properties for proteins.

Claims

1. A gel composition containing glycerin, collagen, and polysaccharides.

2. The gel composition according to claim 1, wherein the glycerin content is 75% or more, the collagen content is 15% or less, and the polysaccharide content is 20% or less.

3. The gel composition according to claim 1, wherein the total content of glycerin, collagen, and polysaccharides is 90% or more.

4. The gel composition according to claim 1, comprising only glycerin, collagen, and polysaccharides.

5. The gel composition according to claim 1, wherein the collagen is atelocollagen.

6. The gel composition according to claim 1, wherein the viscosity at 37°C is 2000 mPa·s or more and 7500 mPa·s or less.

7. The gel composition according to claim 1, wherein the average pore diameter in the cross-section is 10 μm or more.

8. The gel composition according to claim 1, further containing cells.

9. The gel composition according to claim 8, wherein the cells are bone marrow-derived mesenchymal cells.

10. A pharmaceutical product comprising the gel composition described in any one of claims 1 to 9.

11. The pharmaceutical agent according to claim 10 for the regeneration of any one tissue selected from the group consisting of bone tissue, fibrous connective tissue, muscle tissue, adipose tissue, periodontal tissue, soft tissue, cartilage tissue, skin tissue, and nerve tissue.

12. The pharmaceutical preparation according to claim 10, which is an injectable preparation.

13. A carrier for sustained release of a physiologically active substance, comprising the gel composition according to any one of claims 1 to 9.

14. The sustained-release carrier according to claim 13, wherein the physiologically active substance is at least one selected from the group consisting of drugs, nucleic acids, extracellular vesicles, low molecular weight compounds, and proteins.

15. A gel composition according to any one of claims 1 to 9, which is in a sol state and is used for the production of a gel.

Images