JP2014039517A - Method for controlling size and shape of crown, cusp, and root of tooth, regenerated tooth, and regenerated tooth germ using growth factors including igf - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a tooth regeneration method controlling the shape of the crown, cusp, and root such that the regenerated tooth occludes normally, exhibits hardness equivalent to that of a normal tooth, and functions equivalently to the normal tooth.SOLUTION: A culture method of a cell derived from a tooth or a tooth germ comprises the step of culturing the cell in the presence of growth factors including IGF. In the culture step, the cell derived from a tooth or a tooth germ is arranged in a support carrier, and in the support carrier, 0.5 to 8 μg/ml of IGF is blended.

Description

  The present invention is a method for regenerating organs, organs and tissues.

  A tooth is an organ having hard tissues of enamel and dentin and further having a pulp in the center. In addition, teeth are often lost due to caries, periodontal disease, etc., and the functionality of the maxillofacial region decreases, so tooth regeneration technology is expected from the viewpoint of maintaining health and maintaining a high quality of life. .

  For example, Non-Patent Document 1 and Patent Document 1 reported that regenerated teeth reconstructed by the organ primordium method erupt as functional teeth when transplanted into the adult jawbone. In addition, it was suggested that the regenerated tooth has a periodontal ligament derived from the transplanted reconstructed tooth germ and is connected to the surrounding alveolar bone by a sharpy fibrous tissue. Furthermore, the development of a technique in which the produced regenerated tooth germ is produced in a regenerative tooth unit with periodontal ligament and alveolar bone and transplanted into an adult mouse is described. However, there is no disclosure of a form control method for increasing the crown size, which is most important for the function of the regenerated tooth to chew. Also, the most important method for controlling the shape of the cusp for the teeth to chew is not disclosed.

  For example, Patent Document 2 discloses culturing tooth germ cells in the presence of fibroblast growth factor (FGF), transformed cell growth factor (TGF), and the like as physiologically active substances. However, a technique for controlling the form is not disclosed. Also, in the function of organs such as teeth, the normal form is extremely important, especially in the case of teeth, the form control that increases the size of the crown, and the masticatory function is missing if the form control of the cusp is not possible, It can be said that there is no clinical applicability and effectiveness. However, at present, in the process of tooth generation and regeneration, it has been impossible to control and regenerate the form.

  For example, Patent Document 3 discloses that a tooth germ is regenerated using a carrier to form a tooth having a specific shape, but a method using a growth factor is not disclosed. In addition, in the technique of shape production using a carrier, the reproduction of the entire histological structure of the tooth organ itself has not been reported.

  For example, Patent Document 5 describes a method of stimulating bone formation and bone remodeling with an IGF / IGFBP complex, but does not disclose a technique for controlling the morphology. The IGF stimulation technique in the present invention is a technique for teeth, and further realizes shape control of crowns and cusps and roots, which are extremely important for the functionality of the teeth, and is considered to be the most necessary technique for the dental regeneration technique.

Japanese Patent Application No. 2010-525698 JP 2004-331557 A JP 2004-357567 A Special table 10-512235

Proceeding of the National Academy of Sciences of the United States of America (PNAS), 106 (32): 13475-13480 2009

  A tooth can maintain a normal mastication function by having a tooth type specific form. Therefore, in the prosthetic treatment using artificial teeth, precise adjustment of the cusp shape such as occlusion adjustment is extremely important. In order to have the same function as a normal tooth, it is necessary to control the form. However, in the techniques described in Non-Patent Document 1 and Patent Document 1, the regenerated tooth has a reduced form, and the regenerated tooth and root cusp shape However, it is insufficient compared to normal teeth. It does not describe the form control for solving these problems. The technology of the present invention controls the shape of the crown and cusp as well as the root of the tooth, allowing the size to increase and decrease. Therefore, it solves the masticatory dysfunction caused by the unbalance of the size of the maxilla and mandible and the regenerative teeth, and is an indispensable technique for realizing dental regenerative medicine.

  More than 90% of the Japanese population has been crowded and has been reported to increase in recent years. Since crowding is caused by a mismatch between the size of the jawbone and the teeth, if the size of the teeth can be controlled, the occurrence of crowding can be suppressed. Furthermore, the occurrence of caries, periodontal disease, and temporomandibular disorders that are caused by crowding are also suppressed. These are solved by a form control for increasing the crown size and a form control for reducing the crown size.

  The morphogenesis of tissues, organs and organs is controlled by the external environment of the cells. Teeth are generated and formed by epithelial-mesenchymal interactions, but the mechanisms involved in tooth morphogenesis have hardly been reported. For this reason, it has been impossible to control the morphology of teeth and regenerated teeth, despite the fact that the teeth are organs that perform their functions due to the specific morphology of the crown. In the present invention, a method for controlling morphology by incorporating stimulation by growth factors including IGF into the generation mechanism of tooth epithelial-mesenchymal interaction has been developed. This technique enables morphological control not only in teeth but also in the formation of lungs, kidneys, liver, glandular tissues, limbs, fingers, etc., generated by epithelial-mesenchymal interactions.

  Appropriate size and form are indispensable for transplantation and regenerative medicine in organs, organs, and tissues to be functional. Therefore, even in the transplantation therapy and regenerative medicine that are activated, the functionality of the transplanted organ, the transplanted tissue, the regenerated organ, and the regenerated tissue becomes insufficient unless it is recommended in combination with an appropriate form control technique. However, in the developmental process and regeneration process, the mechanism of morphological control is hardly elucidated, and there are no paper reports. In addition, in order to improve the technological base of transplantation medicine and regenerative medicine, the development of morphological control technology using a mechanism that cooperates with the development process and regeneration process is also necessary to improve clinical results. Development is considered impossible. In the present invention, a tooth whose form is controlled is realized by combining stimulation with growth factors including IGF in the tooth generation process and regeneration process. Furthermore, since the present invention can control not only the crown size but also the cusp formation and root formation, it can be said that the technique can reproduce the functionality.

  In current dental treatment, functional replacement of an artificial product in bridge and implant treatment is being promoted, but the functionality is insufficient because the periodontal ligament function cannot be reproduced. It was also clarified that teeth were regenerated and erupted by reconstructing epithelial cells and mesenchymal cells derived from tooth germ. However, its size was small.

  Therefore, an object of the present invention is to adjust the cusp shape to a cusp having a size equivalent to that of a normal tooth and capable of normal occlusion, and also to adjust sufficient root formation to support mastication, It is to provide a restoration method for repairing a tooth defect portion so as to have a function equivalent to that of the first embodiment.

  In current regenerative medicine, a method of regenerating a deficient tissue by transferring cells is being promoted. However, when the tissue to be regenerated is a tooth, there is a limit to the control of form and size.

  Accordingly, an object of the present invention is to provide a technique for regenerating an organ called a tooth in cell transfer therapy.

The present invention is as follows:
Item 1. A method for culturing cells derived from teeth or tooth germs, comprising culturing in the presence of a growth factor comprising insulin-like growth factor (IGF),
In the culturing step, cells derived from teeth or tooth embryos are arranged in a support carrier, and IGF is mixed in an amount of 0.5 to 8 μg / ml in the support carrier.

Item 2. A method for producing a cell having a tooth tissue regeneration ability, comprising a step of culturing a tooth or tooth germ-derived cell in the presence of a growth factor containing IGF,
In the culturing step, cells derived from teeth or tooth embryos are arranged in a support carrier, and IGF is mixed in an amount of 0.5 to 8 μg / ml in the support carrier.

Item 3. Regenerative tooth germ comprising a step of mixing a tooth or tooth germ-derived cell with a support carrier containing a growth factor containing IGF or constructing an aggregate of teeth or tooth germ-derived cells in the support A manufacturing method of
A method in which 0.5 to 8 μg / ml of IGF is blended in the support carrier.

Item 4. Mixing a cell derived from a tooth or tooth germ into a support carrier containing a growth factor containing IGF, or constructing an aggregate of cells derived from a tooth or tooth germ in the support, and the above step A method for producing a regenerated tooth germ or a regenerated tooth comprising a step of culturing a cell derived from a tooth germ mixed with a growth factor-containing support carrier or an aggregate in the presence of IGF,
A method in which 0.5 to 8 μg / ml of IGF is blended in the support carrier.

Item 5. Mixing a cell derived from a tooth or tooth germ into a support carrier containing a growth factor containing IGF, or constructing an aggregate of cells derived from a tooth or tooth germ in the support, and the above step Increasing the size of crowns, cusps and roots in regenerated teeth or regenerated tooth germs, including the step of culturing cells derived from tooth germs mixed with growth factor-containing support carriers or constructed aggregates in the presence of IGF And a method for controlling its form,
A method in which 0.5 to 8 μg / ml of IGF is blended in the support carrier.

Item 6. Increasing the size of the crown, cusp, and root of the regenerated tooth or regenerated tooth germ, including the steps of placing the regenerated tooth germ in a support carrier containing a growth factor containing IGF and culturing the regenerated tooth germ A method of controlling its form,
In the culturing step, IGF is mixed in the support carrier in an amount of 0.5 to 8 μg / ml.

  Item 7. Support carrier is collagen, fibrin, laminin, extracellular matrix mixture, polyglycolic acid (PGA), polylactic acid (PLA), lactic acid / glycolic acid copolymer (PLGA), hydrogel, proteoglycan, entactin, polysaccharide, hydroxy Item 7. The method according to any one of Items 1 to 6, comprising at least one selected from the group consisting of apatite, β-TCP, α-TCP, alumina ceramics, dry bone, dry teeth, dry roots, and polysaccharides.

  Item 8. A composition for tooth regeneration comprising a growth factor comprising IGF, the composition comprising IGF in an amount added to a support carrier or medium for tooth regeneration to a final concentration of 0.5 to 8 μg / ml .

  Item 9. Item 9. The tooth regeneration composition according to Item 8, further comprising mesenchymal cells and / or epithelial cells.

  Item 10. Support carrier is collagen, fibrin, laminin, extracellular matrix mixture, polyglycolic acid (PGA), polylactic acid (PLA), lactic acid / glycolic acid copolymer (PLGA), hydrogel, proteoglycan, entactin, polysaccharide, hydroxy Item 10. The composition according to Item 8 or 9, comprising at least one selected from the group consisting of apatite, β-TCP, α-TCP, alumina ceramics, dry bone, dry tooth, dry root, and polysaccharide.

Item 11.
A cell suspension of mesenchymal cells isolated from the mesenchymal tissue of the tooth or tooth germ, and a cell suspension of epithelial cells isolated from the epithelial tissue of the tooth or tooth germ have an IGF of 0.5 to 8 μg. A composition for regenerating tooth tissue having a compartment in which the mesenchymal cells are present and a compartment in which the epithelial cells are present in the collagen gel obtained by injecting into a collagen gel formulated with / ml.

  Item 12. Any one of Items 1 to 11, wherein the growth factor further comprises at least one selected from the group consisting of EGF, TGF, NGF, BDNF, VEGF, G-CSF, PDGF, EPO, TPO, FGF, HGF, and BMP. The method or composition according to Item.

  Item 13. Item 5. A method for repairing a tooth defect part, comprising a step of transplanting a cell, a regenerated tooth embryo or a regenerated tooth produced by the method according to any one of Items 1 to 4 to a tooth defect part.

  Item 14. Item 5. A method for treating a tooth having a defective part, comprising a step of transplanting a cell, a regenerated tooth embryo or a regenerated tooth produced by the method according to any one of Items 1 to 4 to a defective part of a patient's tooth.

  According to the present invention, by controlling the form of the crown, the cusp, and the root, the teeth are regenerated so that they normally occlude, have the same hardness as normal teeth, and have the same functionality as normal teeth. A method can be provided.

It is a phase-contrast microscope image of the culture | cultivation 14th day of the control group (A) and IGF-1 addition group (B) of the reproduction | regeneration tooth germ concerning Example 1 of this invention. The size of the IGF-1 added group (B) was increased, indicating that the morphology was controlled. It is a graph which shows the size (A: long diameter, B: width diameter) of the reproduction | regeneration tooth germ concerning Example 1 of this invention. It is a phase-contrast microscope image on the 14th day of culture | cultivation of the control group (A) and IGF-1 addition group (B) of the developmental tooth germ concerning Example 1 of this invention. It was shown that the morphology of the IGF-1 added group (B) increased and the morphology was controlled. It is a graph which shows the size (A: major axis, B: width diameter, C: crown major axis) of the developmental tooth germ concerning Example 1 of this invention. It is a hematoxylin-eosin dyeing | staining image of the 14th day culture | cultivation of the IGF-1 addition group of the developmental tooth germ concerning Example 1 of this invention (10 time). It is a micro CT image 30 days after subrenal capsule transplantation of regenerated tooth germs of a control group (A) according to Example 2 of the present invention and an IGF-1 added group (B). The size of the IGF-1 added group (B) was increased, indicating that the morphology was controlled. It is a graph which shows the size (A: long diameter, B: width diameter) of the subrenal capsule transplanted regeneration tooth concerning Example 2 of this invention. It is a graph which shows the Knoop hardness of the subrenal graft transplanted regeneration tooth (A: enamel, B: dentin) concerning Example 2 of this invention. It is a micro CT image 90 days after the intra-mandibular transplantation of the regenerated tooth embryo of the IGF-1 addition group according to Example 3 of the present invention. It was shown that regenerated teeth with IGF-1 erupted with increasing size. Sagittal plane A and occlusal plane B: IGF- C: IGF + It is a graph which shows the size (A: major axis, B: width diameter) of the regenerated tooth in the jawbone concerning Example 3 of this invention. It was shown that the size of regenerated teeth with IGF-1 added increased. It is a graph which shows the Knoop hardness of the regenerated tooth (A: enamel, B: dentin) concerning the intra-mandibular bone concerning Example 3 of this invention. Regenerated teeth with IGF-1 added were shown to have the same hardness as the control group. It is a graph which shows the proliferation ability of the tooth germ mesenchymal cell concerning Example 4 of this invention. A schematic diagram of the method for producing the regenerated tooth germ is shown in FIG.

  The method for reconstructing a regenerated tooth according to the present invention is preferably an epithelial cell and a mesenchymal cell derived from a tooth germ. The first cell mass and the second cell mass are brought into close contact with each other without being mixed, placed inside the support carrier and cultured to obtain a reconstructed tooth germ or tooth.

  The present invention will be described below.

  In the present invention, the term “tooth” refers to a tissue having a directivity having a dentin on the inside and a layer of enamel on the outside as a hard tissue, a pulp disposed in the center, and a crown or root. means. The dental crown is the part of the layer structure of enamel and dentin, and there is no enamel layer in the root of the tooth, cementum is present, and it is connected to the alveolar bone by the periodontal ligament and supports the crown. .

  Dentin and enamel can be easily identified morphologically by immunostaining or the like. Enamel is also produced by enamel blasts. The presence of enamel blasts can be confirmed by the presence or absence of ameloblastin and amelogenin. On the other hand, dentin can be identified by the presence of odontoblasts, and the presence of odontoblasts can be confirmed by the presence or absence of dentin sialoprotein.

  In addition, the directionality of teeth can be specified by the arrangement of crowns and roots. The crown and root can be recognized based on the tissue structure and tissue staining.

  In the present invention, “dental germ” and “tooth” are expressions used when specifically referring to those distinguished based on the stage of development. The “tooth germ” in this case is a tooth primordium that has been determined to become a future tooth, and is generally used in the tooth development stage from the Bud stage to the Bell stage (Bell stage). stage). Tooth germs are initially grown from cells derived from the oral mucosal epithelium and mesenchymal tissue, and the tooth germs differentiate into enamel organs, tooth papilla, and dental follicles. The enamel is enamel, the tooth papilla is the pulp and dentin, the dental follicle is cementum, the periodontal ligament, and the alveolar bone, completing the tooth. In the present specification, “regenerated tooth” and “regenerated tooth germ” refer to a tooth and a tooth germ regenerated using a tooth or a tooth germ-derived cell, respectively. In the present specification, the term “regenerated tooth germ” refers to not only cells that have reached the stage from the rod-shaped stage to the bell-shaped stage as a result of cell culture, but also cells derived from teeth or tooth germs. Those mixed with a support carrier are also included.

  In the method of the present invention, “the tooth or tooth germ-derived cell” to be subjected to the culturing step includes not only the above-described “tooth” or “tooth germ” but also the cells appropriately enzyme-treated, etc. Those having the ability to regenerate teeth or tooth germs as well as these cells are also included. Examples of cells having the ability to regenerate teeth or tooth germs include stem cells and iPS cells having the ability to differentiate into teeth or tooth germs. Therefore, the method of the present invention also includes a method of regenerating teeth or tooth germs by culturing the stem cells and iPS cells. As conditions for regenerating a tooth or a tooth germ using stem cells and iPS cells, a method known per se can be appropriately used.

  In the present invention, “mesenchymal cell” means a cell derived from mesenchymal tissue, and “epithelial cell” means a cell derived from epithelial tissue.

  The present invention provides a method for culturing cells derived from teeth or tooth germs, which comprises culturing in the presence of a growth factor containing IGF. By this method, cells having the ability to regenerate dental tissues can be produced. Specific examples of the culture method include a method of reconstructing a regenerated tooth germ using a tooth or tooth germ-derived cell and organ-culturing the reconstructed tooth germ.

  Examples of IGF include IGF-1 and IGF-2, and preferably IGF-1 is used.

  As growth factors other than IGF, EGF, TGF, NGF, BDNF, VEGF, G-CSF, PDGF, EPO, TPO, FGF, HGF and BMP can be used.

Method for Reconstructing Morphologically Controlled Regenerated Tooth Germ Using IGF-1 In the method of the present invention, first, tooth germ-derived cells are mixed with a support carrier containing a growth factor containing IGF. Thereby, the regenerated tooth germ is reconstructed. For example, the following method is mentioned. Single cells collected from tooth germ epithelial tissue and tooth germ mesenchymal tissue are placed in a silicon-coated 1.5 ml microcentrifuge tube (Watson, Parsippany, NJ, USA) and centrifuged at 2500 rpm for 3 minutes. Is completely removed with GELoader Tip 0.5-20 μl (Eppendorf, Hamburg, Germany) to prepare a high-density cell suspension of epithelial and mesenchymal cells. Next, 30 μl of type I collagen gel is formed in a drop shape on a silicon-coated 35 mm Petri dish, and IGF-1 is mixed into the collagen gel. Furthermore, 0.05 μl of a high-density cell suspension of mesenchymal cells is injected into the gel using a Hamilton syringe (Osaka chemical, Osaka, Japan). Subsequently, 0.05 μl of an epithelial cell suspension is injected so as to be joined to an aggregate of mesenchymal cells to produce a regenerated tooth germ in which epithelial cells and mesenchymal cells are compartmentalized at high density. As described above, in the method of the present invention, a method of mixing a tooth or tooth germ-derived cell with a support carrier containing a growth factor containing IGF includes, for example, a tooth or tooth germ-derived cell on the support carrier. Injecting methods are also encompassed.

Here, the number of cells per cell aggregate can be generally 10 ¹ to 10 ⁸ , preferably 10 ³ to 10 ⁸ .

  In the present invention, when the growth factor containing IGF is added to the support carrier, for example, the concentration is 0.5 to 25 μg / ml. From the viewpoint of morphological control to increase the size of the crown, cusp and root, IGF is 0.5 to 8 μg / ml, and more preferably 0.5 to 4 μg / ml. However, the concentration varies depending on the organs, tissues, and cells to be regenerated. The concentration is also changed depending on the type of support carrier to be added.

  The mesenchymal cells derived from other than the tooth germ are cells derived from other mesenchymal tissues in the living body. Preferably, bone marrow cells and mesenchymal stem cells, more preferably oral mesenchymal cells such as dental pulp cells and periodontal ligament cells, bone marrow cells in the jawbone, mesenchymal progenitor cells and stem cells thereof, etc. However, the present invention is not limited to this.

  In addition, epithelial cells derived from other than tooth germs are cells derived from other epithelial tissues in the living body. Preferably, epithelial cells of skin and oral mucosa and gingiva, enamel blasts, more preferably differentiated, eg keratinized or keratinized epithelial cells and their epithelial progenitor cells such as skin and mucous membranes, Examples include non-keratinized epithelial cells and stem cells thereof, but are not limited thereto.

  Tooth germs and other tissues include mammalian primates such as humans, monkeys, marmosets, ungulates such as pigs, cows, horses, carnivorous dogs, small mammalian rodents such as mice, It can be collected from jawbones of various animals such as rats and rabbits. Examples of human tooth germs include fetal tooth germs in addition to third molars, so-called wisdom tooth germs, but it is preferable to use wisdom tooth germs from the viewpoint of use of autologous tissue. In addition, it is also preferable to use the dental pulp tissue and periodontal ligament tissue of extracted permanent teeth and deciduous teeth. In the case of a mouse, it is preferable to use a tooth germ of 10 to 16 days of gestation.

  Preparation of mesenchymal and epithelial cells from the tooth germ is performed by first surgically dividing the tooth germ isolated from the surrounding tissue into tooth germ mesenchymal tissue and tooth germ epithelial tissue according to the shape. Is called. At this time, an enzyme may be used for easy separation. Examples of the enzyme used for such applications include dispase, collagenase, trypsin, hyaluronidase, and elastase.

  Mesenchymal cells and epithelial cells may be prepared in a single cell state from mesenchymal tissue and epithelial tissue, respectively. Enzymes such as dispase, collagenase, and trypsin may be used so that they can be easily dispersed in a single cell.

  In addition, the mesenchymal cell and the epithelial cell may have undergone preliminary culture in order to obtain a sufficient number of cells. Desirably, those having 2 to 4 passages are desirable. Further, it may be frozen and thawed.

  The support used in the present invention for reconstituting organs and organs is preferably a mixture with the above medium. Such support carriers include collagen, agarose gel, carboxymethylcellulose, gelatin, agar, hydrogel, proteoglycan, entactin, elastin, fibrin, laminin, extracellular matrix mixture, polyglycolic acid (PGA), polylactic acid (PLA) , Lactic acid / glycolic acid copolymer (PLGA), polysaccharides, and the like. Usually, the hardness applied as three-dimensional culture is preferable.

  Supporting carriers used for tissue regeneration are collagen, fibrin, laminin, extracellular matrix mixture, polyglycolic acid (PGA), polylactic acid (PLA), lactic acid / glycolic acid copolymer (PLGA), hydrogel It is preferably at least one selected from the group consisting of proteoglycan, entactin, hydroxyapatite, β-TCP, α-TCP, alumina ceramics, dry bone, dry teeth, dry roots and polysaccharides. Commercially available support carriers for organ and organ reconstruction or tissue regeneration include cell matrix (collagen), meviol gel ((thermoreversible hydrogel that responds to temperature using polymer material), matrigel (main component) : Laminin, collagen IV, heparan sulfate proteoglycan, and a mixture of entactin / nidogen).

Culturing method As a culturing method, a method known per se as an organ culturing method can be appropriately used. For example, the regenerated tooth germ reconstructed above is cultured on a cell culture plate using an appropriate medium.

  Examples of the culture medium include Dulbecco's modified Eagle (DMEM) medium, αMEM medium, DMEM / F12 medium, RPMI medium, and the like. Further, Dulbecco's modified Eagle medium can be used as long as the component system is the same. IGF may be further added to the culture medium. In this case, the blending amount can be appropriately set, for example, in the range of 50 to 200 ng / ml, preferably 100 ng / ml, as the final concentration in the medium. Moreover, you may add growth factors other than IGF mentioned above to the said culture medium. In that case, the compounding quantity of growth factors other than IGF can be suitably set in the range of 50 to 200 ng / ml, preferably 100 ng / ml, for example, as the final concentration in the medium. The culture temperature is not particularly limited, but is appropriately set at 37 ° C., for example. In addition, the culture time is not particularly limited, but can be appropriately set, for example, in the range of 14 days, more preferably in the range of 10 to 14 days. When transplanted to animals, a culture period of 7 days or less is preferred.

Tooth regeneration composition The present invention provides a tooth regeneration composition containing a growth factor comprising IGF. As the active ingredient IGF and other growth factors, those described above can be used.

  Moreover, a carrier and an additive can be mix | blended with the composition for tooth | gear reproduction | regeneration of this invention. Carriers and additives include solvents, excipients, coating agents, bases, binders, lubricants, disintegrants, solubilizers, suspending agents, thickeners, emulsifiers, stabilizers, buffers, Examples include isotonic agents, preservatives, and coloring agents.

  Further, in the present invention, a cell suspension of mesenchymal cells separated from the mesenchymal tissue of the tooth or tooth germ, and a cell suspension of epithelial cells separated from the epithelial tissue of the tooth or tooth germ, Dental tissue having a section where the mesenchymal cells are present and a section where the epithelial cells are present in the collagen gel obtained by injecting into a collagen gel containing 0.5-8 μg / ml of IGF A regenerating composition is provided.

Here, the cell density of the cell suspension is not particularly limited, but can be appropriately set within a range of 1 × 10 ⁸ to 1 × 10 ⁹ cells / ml, for example.

  The tooth regeneration composition of the present invention can be used in the above-described method for producing a regenerated tooth embryo and regenerated tooth, and a method for forming a form thereof.

The present invention relates to a method for repairing a tooth defect, comprising the step of transplanting a cell, a regenerated tooth germ or a regenerated tooth produced by the above method into a tooth defect, and There is provided a method for treating a tooth having a defect, which comprises the step of transplanting a cell, a regenerated tooth embryo or a regenerated tooth produced by the above-described method into a tooth defect of a patient.

  The method of transplanting cells, regenerated tooth embryos or regenerated teeth can be performed using a method known per se or in accordance with this method.

  In the present invention, the control of the morphology of the dental crown and cusp of a regenerated tooth produced by mesenchymal cells and epithelial cells of a mouse tooth germ has been clarified by Examples. Mice have genes, organs, and tissues that are homologous to humans. In addition, since a mouse can be deficient in a specific gene at the individual level, the mouse is regarded as the best model animal for elucidating the role of human genes in tissue formation at the individual level. In the dental field, analysis of stem cells derived from teeth is progressing in both humans and mice. For example, in the document PLoS ONE. 2006; 1 (1): e79. And the document Lancet. 2004 Jul 10-16; 364 (9429): 149-55, dental pulp stem cells have been reported in humans. It has also been reported in mice. Furthermore, stem cells derived from teeth are described in literature PLoS ONE. 2006; 1 (1): e79., Literature Lancet. 2004 Jul 10-16; 364 (9429): 149-55, literature Proceeding of the National Academy of Sciences of the. Culture methods as reported in United States of America (PNAS) 2003 May 13; 100 (10): 5807-12.Epub 2003 Apr 25, J J Dent Res. 2003 Dec; 82 (12): 976-81 Therefore, the technique of the present invention is expected to be applied to humans.

  In the present invention, a technique capable of controlling cusp formation by adding IGF-1 is disclosed. During tooth development, a signal center called an enamel knot is generated, and cusp formation is regulated (Mech Dev. 1996 Jan; 54 (1): 39-43.). Further, in the document J Cell Physio. 2012 April 227 (4): 1455-1464, it is reported that Sonic Hedgehog interacts with IGF-1 and affects myocyte proliferation and differentiation. It is surmised that IGF-1 cooperates with Sonic Headgehog by the same mechanism and influences the shape control of the tooth cusp in the present invention.

  Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to these Examples.

Example 1
[1-1] Preparation of single cells of tooth germ-derived epithelial cells and mesenchymal cells C57BL / 6J mice were treated with 50 U / ml Dispase (BD, Franklin Lakes, NJ) , USA) and 35 U / ml Deoxyribonuclease I (DNase I, Sigma, St. Louis, MO, USA) with phosphate buffer saline (PBS) for 4 minutes at room temperature. Separated using a 25G needle.
Tooth germ epithelial tissue was subjected to enzyme treatment twice at 37 ° C. for 15 minutes using PBS containing 100 U / ml Collagenase I (Worthington, Lakewood, NJ, USA) and 35 U / ml DNase I, and then 0.25% Trypsin ( (Sigma) and PBS containing 35 U / ml DNase I were subjected to enzyme treatment at 37 ° C. for 5 minutes to form single cells. Tooth germ mesenchymal cells were treated with PBS containing 0.25% Trypsin, 50 U / ml Collagenase I and 35 U / ml DNase I at 37 ° C. for 15 minutes to form single cells.

[1-2] Preparation of regenerated tooth germ and IGF-1-added tooth germ epithelial tissue and singulated cells collected from tooth germ mesenchymal tissue were each coated with a silicon-coated 1.5 ml microcentrifuge tube (Watson, Parsippany, NJ, USA) ), Centrifuge at 2500 rpm for 3 minutes, and completely remove the supernatant with GELoader Tip 0.5-20 μl (Eppendorf, Hamburg, Germany) to prepare high-density cell suspensions of epithelial cells and mesenchymal cells. (The cell densities of the epithelial cell suspension and the mesenchymal cell suspension were 5 × 10 ⁸ cells / ml, respectively). Next, 30 μl of type I collagen gel is formed in a drop shape on a silicon-coated 35 mm Petri dish, and 0.05 μl of high-density cell suspension of mesenchymal cells using a Hamilton syringe (Osaka chemical, Osaka, Japan) Was injected into the gel to produce mesenchymal cell aggregates. Subsequently, 0.05 μl of an epithelial cell suspension was injected so as to be joined to an aggregate of mesenchymal cells, and a regenerated tooth germ in which epithelial cells and mesenchymal cells were partitioned at high density was produced. In the IGF-1 addition group, regenerated tooth germs were prepared using a gel to which IGF-1 was added so as to be 4 μg / ml (Example 1-1). A regenerated tooth germ was prepared by performing the same operation as described above except that a gel to which IGF-1 was not added was used (Comparative Example 1-1). A schematic diagram of the method for producing the regenerated tooth germ is shown in FIG.

[1-3] Preparation of regenerated tooth germ with increased morphology by addition of IGF 0.4 μm pore diameter cell culture in which the regenerated tooth germ obtained in [1-2] above was placed in 24-well cell culture plates (BD) on inserts (BD) using 10% FBS (MP Biomedicals, Solon, OH, USA), 100 μg / ml ascorbic acid (Sigma), 2 mM L-glutamine (Sigma), Dulbecco's modified eagle medium (DMEM, Sigma) Organ culture was performed. Furthermore, in the IGF-1 addition group, it culture | cultivated using the said culture medium which added IGF-1 so that it might become 100 ng / ml.
On the first day of culture, a translucent zone, an index of epithelial-mesenchymal interaction, was formed at the interface between epithelial cells and mesenchymal cells, and on day 14 of culture, it developed into a regenerated tooth germ corresponding to the bell-shaped phase . In addition, as shown in FIG. 1B, an increase in size was observed in the IGF-1 added group (Example 1-1) in the phase contrast microscope image.

[1-4] Comparison of Regenerated Tooth Germ Increased in Morphology with Addition of IGF and Regenerated Tooth Germ without Addition of IGF Organ culture of [1-3] above is performed, and the regenerative tooth germ's cervical loop is observed with a phase contrast microscope The maximum diameter of the tooth germ perpendicular to the line connecting the two was taken as the long diameter, the maximum diameter of the crown part perpendicular to the long diameter was taken as the width diameter, and the long diameter and the width diameter at the 14th day were measured. As shown in FIG. 2 (A), the major axis was 944 μm in the IGF-1 added group and 857 μm in the control group on the 14th day of culture. Further, as shown in FIG. 2 (B), the width was 969 μm in the IGF-1 added group and 814 μm in the control group on the 14th day of culture. From these results, it was suggested that the addition of IGF-1 during the production process of the regenerated tooth germ increases the long diameter and width diameter of the regenerated tooth germ.

[1-5] Preparation of histological specimens of regenerative tooth germs whose morphology was increased by addition of IGF and regenerative tooth germs without addition of IGF 4% paraform of tooth germ on the 14th day of culture in [1-3] above After fixation with aldehyde (PFA), decalcification with 10% ethylenediaminetetraacetic acid (EDTA) and embedding in paraffin, 8 μm-thick serial sections were prepared and histologically stained with hematoxylin and eosin (HE) Evaluation. In the HE staining, formation of enamel and dentin was observed in both the control group and the IGF-1 addition group, and it was observed that they occurred until the late bell stage.

[1-6] Preparation of developmental tooth germ with increased morphology by addition of IGF Fetal mandibular molar tooth embryos of C57BL / 6J mice at 14.5 gestational age were surgically removed. A 30 μl type I collagen gel was formed in a drop shape on a silicon-coated 35 mm Petri dish, and a tooth germ was positioned therein. In the IGF-1 added group, regenerated tooth germs were prepared using a gel added with 4 μg / ml IGF-1 (Example 1-2). 10% FBS (MP Biomedicals, Solon, OH, USA), 100 μg / ml ascorbic acid (Sigma) on 0.4 μm pore diameter cell culture inserts (BD) placed on 24-well cell culture plates (BD). ), 2 mM L-glutamine (Sigma) and Dulbecco's modified eagle medium (DMEM, Sigma). Furthermore, 100ng / ml IGF-1 was added to the IGF-1 addition group and cultured. In the phase contrast microscope image, as shown in FIG. 3 (B), on the 14th day, an increase in size was observed in the IGF-1 added group.

[1-7] Comparison of developmental tooth germ with increased morphology by addition of IGF and developmental tooth germ without addition of IGF Organ culture of the above [1-6] was performed, and the dental loop of the tooth germ was observed with a phase-contrast microscope image. The maximum diameter of the tooth germ orthogonal to the connected line is the long diameter, the maximum diameter of the crown part orthogonal to the long diameter is the width diameter, and the maximum diameter of the crown part orthogonal to the width diameter is the crown long diameter. The long diameter, the width diameter, and the crown long diameter were measured. As shown in FIG. 4 (A), the major axis was 937 μm in the IGF-1 added group and 857 μm in the control group on the 14th day of culture. As shown in FIG. 4B, the width was 1084 μm in the IGF-1 added group and 947 μm in the control group on the 14th day of culture. Further, as shown in FIG. 4C, the crown diameter was 588 μm in the IGF-1 added group and 459 μm in the control group on the 14th day of the culture. From these results, it was suggested that the long diameter, the width diameter, and the crown long diameter of the developing tooth germ increase by adding IGF-1.

[1-8] Preparation of Histological Specimens of Developmental Tooth Germ Increased in Morphology by Addition of IGF and Developmental Tooth Germ without Addition of IGF On the 14th day of culture in [1-6] After fixation with aldehyde (PFA), decalcification with 10% ethylenediaminetetraacetic acid (EDTA) and embedding in paraffin, 8 μm-thick serial sections were prepared and histologically stained with hematoxylin and eosin (HE) Evaluation. As shown in FIG. 5, in the HE staining, enamel and dentin formation was observed in both the control group and the IGF-1 addition group, and it was observed that they occurred until the late bell-like period.
[2-1] Regenerated tooth germ transplanted under renal capsule with increased morphology by addition of IGF For the purpose of avoiding the influence of the pressure of renal capsule, transplantation was performed using a spacer for regenerated tooth germ transplantation. OneTouch tips 200 (Sorenson BioScience, Salt Lake City, UT, USA) was cut into a ring shape so as to have an inner diameter of 1.3 mm and a height of 2.3 mm to prepare a spacer, and the collagen gel was filled inside and used. Collagen gels used in the IGF-1 added group (Example 2-1) and the control group (Comparative Example 2-1) were the same as those used in the production of regenerated tooth germs. The regenerated tooth germ obtained in the above [1-2] was arranged so that the crown side was brought close to the wall surface of the spacer in order to promote extension in the root direction. Under general anesthesia injected intraperitoneally with 5 mg / ml pentobarbital, a spacer containing regenerated tooth germ was implanted under the bilateral renal capsule of 7-week-old C57BL / 6 mice.

[2-2] Micro-CT analysis of regenerative tooth germ with increased morphology due to addition of IGF and regenerative tooth germ without addition of IGF in subrenal capsule transplantation The transplant sample transplanted in [2-1] above is transplanted 30 It was extracted at the time of the day and photographed with a micro CT (In vivo Micro X-ray CT System; R_mCT, Rigaku, Tokyo, Japan). For the image data, integrated image processing software (i-VIEW-3DX, Morita, Osaka, Japan) was used.
As shown in FIG. 6, the transplanted regenerated tooth germ was calcified, an alveolar bone was formed, and a periodontal ligament cavity was also observed. As shown in FIG. 7, the regenerative tooth germ added with IGF-1 showed an increase in size (B) by subrenal transplantation compared to the regenerated tooth germ without addition.

[2-3] Comparison of size by micro-CT analysis between regenerative teeth with increased morphology due to IGF addition and non-IGF-added regenerative teeth in subrenal capsule transplantation The longest diameter and the maximum diameter of the orthogonal crown part were taken as the width diameter, and both were measured. As shown in FIG. 7A, the major axis was 1.37 mm in the IGF-1 added group and 0.95 mm in the control group. As shown in FIG. 7B, the width was 0.49 mm in the IGF-1 added group and 0.41 mm in the control group. It was suggested that regenerated tooth germs cultured with IGF-1 increased in length and width after subrenal transplantation.

[2-4] Regenerative tooth germ with increased morphology by IGF addition, regenerative tooth germ without addition of IGF, and Knoop hardness of normal teeth in subrenal transplantation Diamond indenter (19BAA061; Mitutoyo, Tokyo, Japan) and microhardness The Knoop hardness of the enamel and dentin of the regenerated teeth 30 days after the transplantation of the above [2-2] was measured with a length testing machine (HM-102; Mitutoyo, Tokyo, Japan). The measurement was performed on the teeth of each of the control teeth of the upper first molar (natural tooth), regenerated teeth 30 days after subrenal transplantation, and the IGF-1 added group of 3-week-old mice. The measurement conditions were a 25 g pressure for 10 seconds for enamel and a 10 g pressure for 10 seconds for dentin. As shown in FIG. 8, both the enamel (A) and dentin (B) Knoop hardness were found to be equivalent in the IGF-1 added group and the control group.

Example 3
[3-1] Preparation of animal model for intraoral transplantation
The maxillary first molar was extracted from 3-week-old C57BL / 6 mice, and a 3-week healing period was provided. Thereafter, gingival incision and detachment of the same part were performed, and the alveolar bone was cut using a dental micromotor (PAL; Morita) to form a graft fovea having a near-centrifuge diameter of 0.8 mm and a buccal tongue diameter of 0.8 mm. The regenerated tooth germ obtained in [1-2] above, which had been cultured for 7 days, was embedded in the jaw bone graft fossa and gingival sutured with 8-0 nylon suture thread (BEAR Medic, Chiba, Japan).

  [3-2] Micro-CT analysis of regenerated tooth germs with increased morphology due to the addition of IGF and regenerated tooth germs without addition of IGF in intraoral transplantation.

The transplanted sample of the regenerated tooth transplanted in [3-1] above was photographed with a micro CT (In vivo Micro X-ray CT System; R_mCT, Rigaku, Tokyo, Japan) at the 90th day of transplantation. For the image data, integrated image processing software (i-VIEW-3DX, Morita, Osaka, Japan) was used.
As shown in FIG. 9 (A), it was suggested that regenerated tooth embryos cultured with the addition of IGF-1 erupt and reach the occlusal plane after intramaxillary transplantation.

  Furthermore, the maximum diameter of the regenerative tooth was taken as the long diameter, and the maximum diameter of the crown portion perpendicular to the long diameter was taken as the width diameter, and both were measured. As shown in FIG. 10 (A), the major axis was 1.49 mm in the IGF-1 added group and 1.21 mm in the control group. As shown in FIG. 10 (B), the width was 0.64 mm in the IGF-1 addition group and 0.48 mm in the control group. It was suggested that regenerated tooth germs cultured with IGF-1 increased in width. Regenerated teeth with IGF-I added had clear cusp morphology and increased in size.

[3-3] Regenerative tooth germ with increased morphology due to addition of IGF, regenerative tooth germ without addition of IGF, and Knoop hardness of normal teeth in oral transplantation Diamond indenter (19BAA061; Mitutoyo, Tokyo, Japan) and microhardness The Knoop hardness of enamel and dentin was measured with a testing machine (HM-102; Mitutoyo, Tokyo, Japan). Measurements were made on the teeth of each of the upper 1st molar (natural teeth) of 3-week-old mice, the regenerated tooth control group 90 days after intra-maxillary transplantation, and the IGF-1 added group. The measurement conditions were a 25 g pressure for 10 seconds for enamel and a 10 g pressure for 10 seconds for dentin. As shown in FIG. 11, the Knoop hardness of enamel and dentin were not significantly different in the IGF-1 added group and the control group compared to natural teeth, but showed a tendency to be slightly higher.

Example 4
[4-1] Tooth germ-derived mesenchymal cells whose proliferation ability was increased by the addition of IGF From the mesenchymal tissue of the mandibular molar tooth embryo of a 14.5-day-old mouse embryo, 0.25% Trypsin, 50 U / ml Collagenase I and 35 U / ml DNase I was used for 15 minutes of enzyme treatment, tooth germ mesenchymal cells were isolated and cultured in DMEM medium supplemented with 10% FBS, and the proliferative capacity of the first passage cells was measured . The measurement was performed on the IGF-1 added group, the control group, and the group treated with LY294002 (Promega, Madison, WI, USA) and added with IGF-1 (inhibitor-treated / IGF added group). Inhibitor treatment was performed by adding 10 μM LY294002 to the cell suspension and allowing to stand at 37 ° C. for 30 minutes. Molar embryonic mesenchymal cells were seeded at 1000 cells / well on a 96 well plate (BD) and cultured using DMEM supplemented with 10% FBS. Furthermore, 100ng / ml IGF-1 was added to the IGF-1 addition group and cultured (Example 4-1). The subject group was cultured in the same manner as described above except that IFG-1 was not added (Comparative Example 4-1). Proliferation ability was measured using Cell counting kit-8 (WST8; DOJINDO, Kumamoto, Japan). After adding 10 μl / well of WST8, the mixture was allowed to stand at 37 ° C. for 3 hours, and absorbance was measured at a measurement wavelength of 450 nm and a reference wavelength of 620 nm using LS-PLATE manager (Wako, Osaka, Japan).

[4-2] Analysis of proliferative ability of tooth germ-derived mesenchymal cells whose proliferation ability was increased by addition of IGF by WST8 As shown in FIG. 12, the IGF-1 addition group had a proliferation ability compared to the control group. Was found to be rising. This suggests that the addition of IGF-1 promotes the proliferation of tooth germ mesenchymal cells. Furthermore, in the LY294002-treated / IGF-1 addition group, the promotion of the proliferation of tooth germ mesenchymal cells observed in the IGF-1 addition group was not observed, and no significant difference was found between the control group and the control group. This suggests that inhibition of the PI3 kinase pathway inhibits the growth promoting effect of IGF-1 in tooth germ mesenchymal cells.

Example 5
The method according to [1-1] to [1 to 3] above, except that the concentration of IGF in the gel is 0.5 μg / ml, 1 μg / ml, 2 μg / ml, 4 μg / ml and 8 μg / ml, respectively. According to the procedure, preparation of a single cell of tooth germ-derived epithelial cells and mesenchymal cells, preparation of regenerated tooth germ, and organ culture were performed. The increase in size was observed at 0.5 μg / ml, 1 μg / ml, 2 μg / ml, 4 μg / ml, and 8 μg / ml, and the morphology of the crown and cusp could be controlled. Among these concentrations, regenerative tooth germs prepared using gels having a concentration of 0.5 to 4 μg / ml were particularly remarkably increased in size.

Claims (12)

A method for culturing cells derived from teeth or tooth germs, comprising culturing in the presence of a growth factor comprising insulin-like growth factor (IGF),
In the culturing step, cells derived from teeth or tooth embryos are arranged in a support carrier, and IGF is mixed in an amount of 0.5 to 8 μg / ml in the support carrier. A method for producing a cell having a tooth tissue regeneration ability, comprising a step of culturing a tooth or tooth germ-derived cell in the presence of a growth factor containing IGF,
In the culturing step, cells derived from teeth or tooth embryos are arranged in a support carrier, and IGF is mixed in an amount of 0.5 to 8 μg / ml in the support carrier. Regenerative tooth germ comprising a step of mixing a tooth or tooth germ-derived cell with a support carrier containing a growth factor containing IGF or constructing an aggregate of teeth or tooth germ-derived cells in the support A manufacturing method of
A method in which 0.5 to 8 μg / ml of IGF is blended in the support carrier. Mixing a cell derived from a tooth or tooth germ into a support carrier containing a growth factor containing IGF, or constructing an aggregate of cells derived from a tooth or tooth germ in the support, and the above step A method for producing a regenerated tooth germ or a regenerated tooth comprising a step of culturing a cell derived from a tooth germ mixed with a growth factor-containing support carrier or an aggregate in the presence of IGF,
A method in which 0.5 to 8 μg / ml of IGF is blended in the support carrier. Mixing a cell derived from a tooth or tooth germ into a support carrier containing a growth factor containing IGF, or constructing an aggregate of cells derived from a tooth or tooth germ in the support, and the above step Increasing the size of crowns, cusps and roots in regenerated teeth or regenerated tooth germs, including the step of culturing cells derived from tooth germs mixed with growth factor-containing support carriers or constructed aggregates in the presence of IGF And a method for controlling its form,
A method in which 0.5 to 8 μg / ml of IGF is blended in the support carrier. Increasing the size of the crown, cusp, and root of the regenerated tooth or regenerated tooth germ, including the steps of placing the regenerated tooth germ in a support carrier containing a growth factor containing IGF and culturing the regenerated tooth germ A method of controlling its form,
In the culturing step, IGF is mixed in the support carrier in an amount of 0.5 to 8 μg / ml.   Support carrier is collagen, fibrin, laminin, extracellular matrix mixture, polyglycolic acid (PGA), polylactic acid (PLA), lactic acid / glycolic acid copolymer (PLGA), hydrogel, proteoglycan, entactin, polysaccharide, hydroxy The method according to any one of claims 1 to 6, comprising at least one selected from the group consisting of apatite, β-TCP, α-TCP, alumina ceramics, dry bone, dry teeth, dry roots and polysaccharides. . A composition for tooth regeneration comprising a growth factor comprising IGF, the composition comprising IGF in an amount added to a support carrier or medium for tooth regeneration to a final concentration of 0.5 to 8 μg / ml .   The composition for tooth regeneration according to claim 8, further comprising mesenchymal cells and / or epithelial cells.   Support carrier is collagen, fibrin, laminin, extracellular matrix mixture, polyglycolic acid (PGA), polylactic acid (PLA), lactic acid / glycolic acid copolymer (PLGA), hydrogel, proteoglycan, entactin, polysaccharide, hydroxy The composition according to claim 8 or 9, comprising at least one selected from the group consisting of apatite, β-TCP, α-TCP, alumina ceramics, dry bone, dry teeth, dry tooth roots and polysaccharides.   A cell suspension of mesenchymal cells isolated from the mesenchymal tissue of the tooth or tooth germ, and a cell suspension of epithelial cells isolated from the epithelial tissue of the tooth or tooth germ have an IGF of 0.5 to 8 μg. A composition for regenerating tooth tissue having a compartment in which the mesenchymal cells are present and a compartment in which the epithelial cells are present in the collagen gel obtained by injecting into a collagen gel formulated with / ml. The growth factor according to any one of claims 1 to 11, further comprising at least one selected from the group consisting of EGF, TGF, NGF, BDNF, VEGF, G-CSF, PDGF, EPO, TPO, FGF, HGF, and BMP. The method or composition of claim 1.