WO2010021340A1 - 歯欠損部の修復方法及び修復材料の製造方法 - Google Patents
歯欠損部の修復方法及び修復材料の製造方法 Download PDFInfo
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- WO2010021340A1 WO2010021340A1 PCT/JP2009/064509 JP2009064509W WO2010021340A1 WO 2010021340 A1 WO2010021340 A1 WO 2010021340A1 JP 2009064509 W JP2009064509 W JP 2009064509W WO 2010021340 A1 WO2010021340 A1 WO 2010021340A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K6/00—Preparations for dentistry
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3813—Epithelial cells, e.g. keratinocytes, urothelial cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
- A61L27/3843—Connective tissue
- A61L27/3865—Dental/periodontal tissues
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3886—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells comprising two or more cell types
- A61L27/3891—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells comprising two or more cell types as distinct cell layers
Definitions
- the present invention relates to a method for repairing a tooth defect and a method for manufacturing a restoration material.
- Teeth are organs having a hard tissue of enamel in the outermost layer and dentin in the inner layer, further producing odontoblasts inside the dentin, and further having a pulp in the center. Teeth may be lost due to dental caries, periodontal disease, etc. The presence or absence of teeth greatly affects the appearance and taste of food, and tooth regeneration technology is used from the viewpoint of maintaining health and maintaining a high quality of life. Various developments have been made.
- J. Dent. Res., 2002, Vol. 81 (10), pp. 695-700 cells such as epithelial cells and mesenchymal cells isolated from tooth embryos are bioabsorbable. It has been disclosed that tooth-like tissue is regenerated by implantation into the abdominal cavity of a rat together with a carrier.
- JP-A-2004-331557 describes that tooth germ cells isolated from a living body are cultured in the presence of a physiologically active substance such as fibroblast growth factor. Has been.
- 2004-357567 proposes culturing at least one kind of tooth germ cells isolated from a living body and cells differentiable into these cells together with a carrier containing fibrin.
- the carrier containing fibrin forms a “tooth” having a specific shape using a target shape of a tooth germ.
- WO2003 / 101503 pamphlet a method of regenerating tooth germ by culturing tooth germ cells and these differentiable cells by mechanical stimulation, and a tooth germ regenerated thereby, Disclosed is a treatment method for implantation into the jawbone of a patient with a tooth germ defect or injury.
- Japanese Patent Laid-Open No. 2005-013261 discloses an artificial biomaterial in which colloidal silica particles are attached to the material surface as an implant material to be used embedded in a living body.
- an object of the present invention is to provide a method for producing a restoration material for repairing a tooth defect portion so as to have a hardness equivalent to that of a normal tooth, to be normally occluded, and to have a stimulus responsiveness equivalent to that of a normal tooth. And it is providing the repair method of a tooth
- the present invention provides a method for producing a restoration material used for repairing a tooth defect portion in the oral cavity and a method for repairing a tooth defect portion.
- a first aspect of the present invention is a method for producing a restoration material used for repairing a tooth defect in an oral cavity, and one of the mesenchymal cells and epithelial cells, one of which is derived from a tooth germ Either one of the first cell aggregate and the second cell aggregate, which are respectively configured, are brought into close contact with each other without being mixed and cultured inside the support carrier to form a reconstructed tooth germ or tooth.
- the tooth germ or tooth provides the manufacturing method, which is used as a restoration material for obtaining an equivalent of a missing tooth in the tooth defect portion.
- the second aspect of the present invention is a method for repairing a tooth defect in the oral cavity, wherein either one of the mesenchymal cells and epithelial cells each derived from a tooth germ is configured.
- the cell aggregate 1 and the second cell aggregate are brought into close contact with each other without being mixed and cultured inside the support carrier to form a reconstructed tooth germ or tooth.
- There is provided a method for repairing a tooth defect including embedding the reconstructed tooth germ or tooth.
- FIG. 1A is a photographic image confirmed in the tooth eruption period (left), the period from eruption to occlusion (center), and the period after occlusion (right) according to Example 2 of the present invention. It shows the appearance taken from 45 degrees oblique to the part.
- FIG. 1B is a photographic image confirmed in the tooth eruption period (left), the period from eruption to occlusion (middle), and the period after occlusion (right) according to Example 2 of the present invention. The appearance taken from the vertical direction of the part is shown.
- FIG. 1C is a photographic image of an occlusion diagram confirmed at the tooth eruption period (left), the period from eruption to occlusion (center), and the period after occlusion (right) according to Example 2 of the present invention. .
- FIG. 1A is a photographic image confirmed in the tooth eruption period (left), the period from eruption to occlusion (center), and the period after occlusion (right) according to Example 2 of the present invention. It shows the appearance taken from 45 degrees
- FIG. 1D is an appearance CT photograph image confirmed at the tooth eruption period (left), the period from eruption to occlusion (center), and the period after occlusion (right) according to Example 2 of the present invention.
- FIG. 1E is a cross-sectional CT photographic image (E) confirmed in the tooth eruption period (left), the period from eruption to occlusion (center), and the period after occlusion (right) according to Example 2 of the present invention. is there.
- FIG. 2A is a graph showing the Knoop hardness of the enamel of the erupted tooth according to Example 2 of the present invention.
- FIG. 2B is a graph showing the Knoop hardness of the dentin of the erupted tooth according to Example 2 of the present invention.
- FIG. 3 is a hematoxylin-eosin stained image of a regenerated tooth just before eruption according to Example 2 of the present invention (20 ⁇ ).
- FIG. 4A is a hematoxylin-eosin stained image (20 ⁇ ) for confirming the bone remodeling phenomenon in the periodontal ligament (B: compression side, C: traction side) bone when the correction force according to Example 2 of the invention is applied. ).
- FIG. 4B is an enlarged view (200 times) of the compression side (black frame B) in FIG. 4A.
- FIG. 4C is an enlarged view (200 times) of the pulling side (black frame C) in FIG. 4A.
- FIG. 5A is an immunostained image showing the expression of c-fos in uncorrected medullary medulla according to Example 2 of the present invention. 100 times.
- FIG. 5B is an immunostained image showing the expression of c-fos in the medulla oblongata 2 hours after the correction stimulus according to Example 2 of the present invention. 100 times.
- FIG. 5C is an immunostained image showing the expression of c-fos in the medulla at 48 hours after the correction stimulus. 100 times.
- FIG. 5D is an immunostained image showing the expression of c-fos in the medulla oblong after 2 hours after demyelination treatment. 100 times.
- the method for producing a restorative material of the present invention is a method for producing a restorative material used to repair a tooth defect part in the oral cavity, and one of them is a mesenchymal cell and an epithelial cell derived from a tooth germ.
- the first cell aggregate and the second cell aggregate each configured in any one of the above are closely mixed without being mixed, cultured inside the support carrier, and reconstructed tooth germ or tooth Confirming the directionality of the reconstructed tooth germ or tooth formed by the culture so that the tip of the tooth can be embedded as the inside of the oral cavity
- the tooth germ or tooth whose directionality has been confirmed (hereinafter referred to as a direction confirmation step) is used as a restoration material for obtaining an equivalent of the missing tooth in the tooth defect portion.
- the method for repairing a tooth defect part of the present invention is a method for repairing a tooth defect part in the oral cavity, and either one of mesenchymal cells and epithelial cells each derived from a tooth germ is used.
- the constructed first cell aggregate and second cell aggregate are brought into close contact with each other without being mixed and cultured inside the support carrier to form a reconstructed tooth germ or tooth (hereinafter, A reconstructed tooth germ forming step), and embedding the reconstructed tooth germ or tooth in the tooth defect portion (hereinafter referred to as an embedding step).
- the first cell mass and the second cell mass are brought into close contact with each other without being mixed in the reconstructed tooth germ formation step and disposed inside the support carrier.
- the reconstructed tooth germ or tooth obtained by culturing is used as a restoration material for embedding in a defect part.
- the equivalent of a tooth erupted from the embedded site indicates the length and hardness of the tooth that bites the opposing tooth, and Nerve invasion can also occur. For this reason, it has a hardness equivalent to that of normal teeth similar to the surrounding teeth, can be normally occluded, and can have stimulus responsiveness equivalent to that of normal teeth.
- the term “tooth” refers to a tissue having a dentin layer on the inside and an enamel layer on the outside in a continuous manner, and means a tissue having a direction having a crown and a root.
- the directionality of teeth can be specified by the arrangement of crowns and roots.
- the crown and root can be visually confirmed based on the shape and tissue staining.
- the crown refers to a portion having a layer structure of enamel and dentin, and there is no enamel layer in the tooth root.
- Dentin and enamel can be easily identified morphologically by those skilled in the art by tissue staining or the like. Enamel can be identified by the presence of enamel blasts, and the presence of enamel blasts can be confirmed by the presence or absence of 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. Confirmation of amelogenin and dentin sialoprotein can be easily carried out by methods well known in the art, and examples thereof include in situ hybridization and antibody staining. In addition, the directionality of teeth can be specified by the arrangement of crowns and roots. The crown and root can be visually confirmed based on the shape and tissue staining.
- teeth germ and “tooth germ” are expressions used when specifically referring to those distinguished based on the stage of development.
- the “tooth germ” in this case is an early embryo of a tooth that has been determined to become a future tooth, and is typically used in the tooth development stage from the Bud stage to the bell stage (Bell stage). stage), and is a tissue in which accumulation of dentin and enamel, which is a characteristic of the hard tissue of the tooth, is not recognized.
- the term “dental germ” refers to the development of the dentin and enamel that are characteristic of the hard tissue of the tooth at the stage transition of the “dental germ” used in the present invention.
- teeth refers to a tissue at a stage before the function as a tooth is expressed. Development from a tooth germ to a tooth is carried out through each stage of the rod-like, cap-like, early bell-like and late stages.
- the epithelial cells are invaginated so as to wrap the mesenchymal cells.
- the epithelial cell part becomes the outer enamel and the mesenchymal cell part forms dentin inside. Therefore, teeth are formed from tooth germs by cell-cell interaction between epithelial cells and mesenchymal cells.
- mesenchymal cell means a cell derived from mesenchymal tissue
- epithelial cell means a cell derived from epithelial tissue
- periodontal tissue refers to alveolar bone and periodontal ligament in which an outer layer of a tooth is mainly formed. The alveolar bone and periodontal ligament can be easily identified by those skilled in the art by histological staining or the like.
- a first cell aggregate and a second cell aggregate each configured with either one of mesenchymal cells and epithelial cells, one of which is derived from a tooth germ
- the body is placed in close contact without mixing and placed inside the support carrier and cultured to form a reconstructed tooth germ.
- the method described in International Publication No. 2006/129672 can be applied as it is.
- each of the first cell aggregate and the second cell aggregate is composed of only mesenchymal cells or epithelial cells, and each cell aggregate is substantially composed of any one of these cells. It is structured.
- Cell aggregate refers to a state where cells are densely packed, and may be a tissue state or a cell aggregate (cell aggregate) prepared from a single cell state. Further, “substantially” means containing as little as possible cells other than the target cells. The number of cells constituting this cell aggregate varies depending on the type of animal, the type of support carrier, hardness and size, but generally 10 1 to 10 8 cells, preferably 10 3 per cell aggregate. It can be ⁇ 10 8 pieces.
- At least one of the mesenchymal cells and epithelial cells used in this production method is a tooth in order to reproduce the cell arrangement in the living body and effectively form a tooth having a specific structure and direction. Any one may be used as long as it is derived from an embryo, but in order to reliably form a tooth, it is most preferable that both mesenchymal cells and epithelial cells are derived from a tooth germ.
- the tooth germ is preferably from the saddle stage to the cap stage from the viewpoint of juvenileity and homogeneity in the differentiation stage of the cells.
- 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 or mesenchymal stem cells that do not contain blood cells, more preferably oral cavity Examples include mesenchymal cells, bone marrow cells in the jawbone, mesenchymal cells derived from head neural crest cells, mesenchymal progenitor cells that can produce the mesenchymal cells, and stem cells thereof.
- the epithelial cells derived from other than the tooth germ are cells derived from other epithelial tissues in the living body, preferably the epithelial cells of the skin and oral mucosa and gingiva, more preferably the skin and Examples include immature epithelial progenitor cells, such as non-keratinized epithelial cells and stem cells thereof, which can produce differentiated, for example, keratinized or complexed epithelial cells such as mucous membranes.
- Tooth germs and other tissues include mammalian primates such as humans, monkeys, ungulates such as pigs, cows and horses, small mammal rodents such as mice, rats, rabbits, and other dogs, It can be collected from jawbones of various animals such as cats.
- To collect tooth germs and tissues the conditions usually used for the collection of tissues may be applied as they are, and they may be taken out in a sterile state and stored in an appropriate preservation solution.
- 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 the case of a mouse, it is preferable to use a tooth germ of 10 to 16 days of gestation.
- Preparation of mesenchymal cells and epithelial cells from the tooth germ is performed by first dividing the tooth germ isolated from the surrounding tissue into a tooth germ mesenchymal tissue and a tooth germ epithelial tissue according to the shape. At this time, an enzyme may be used for easy separation. Enzymes used for such applications include dispase, collagenase, trypsin and the like.
- 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.
- the mesenchymal cell and the epithelial cell may have undergone preliminary culture in order to obtain a sufficient number of cells.
- a medium used for culturing a medium generally used for culturing animal cells, such as Dulbecco's modified Eagle medium (DMEM), can be used, and serum for promoting cell growth is added to the serum.
- DMEM Dulbecco's modified Eagle medium
- cell growth factors such as FGF, EGF, and PDGF, and known serum components such as transferrin may be added.
- concentration in the case of adding serum can be suitably changed with the culture state at that time, it can usually be 10%.
- normal culture conditions for example, culture in an incubator with a temperature of 37 ° C. and a concentration of 5% CO 2 are applied.
- what added antibiotics, such as streptomycin, may be used suitably.
- the support carrier used in the present invention may be any support carrier capable of culturing cells therein, and is preferably a mixture with the above medium.
- support carriers include collagen, agarose gel, carboxymethyl cellulose, gelatin, agar, hydrogel, elastin, fibrin, fibronectin, laminin, extracellular matrix mixture, polyglycolic acid (PGA), polylactic acid (PLA), Lactic acid / glycolic acid copolymer (PLGA), cell matrix (trade name, manufactured by Nitta Gelatin Co., Ltd.), meviol gel (trade name, manufactured by Ikeda Rika Co., Ltd.), Matrigel (trade name, manufactured by Becton Dickinson Co., Ltd.), etc. Can be mentioned.
- These support carriers only need to have a hardness sufficient to maintain the position at which the cells are placed inside, and examples thereof include gels, fibers, and solids.
- collagen agarose gel, carboxymethylcellulose, gelatin, agar, hydrogel, cell matrix, meviol gel, matrigel, extracellular matrix mixture, elastin, fibrin, fibronectin, laminin from the viewpoint of providing appropriate hardness and holding power.
- the interaction between the cells of the mesenchymal cells and the epithelial cells constituting each cell aggregate and between the cell aggregates can be improved.
- the hardness that can maintain the position of the cells is usually a hardness that is applied as a three-dimensional culture, that is, a hardness that can maintain the arrangement of the cells and does not inhibit the enlargement due to proliferation. Can be determined.
- the support carrier only needs to have a thickness that allows the first and second cell aggregates to grow inside the carrier, and can be appropriately set depending on the size of the target tissue and the like. .
- the “contact state” here is preferably a close (high density) state in order to ensure cell interaction in each cell aggregate or between cell aggregates.
- the state can be such that cells can be cultured with a holding force capable of maintaining a tightly attached state rather than simply touching.
- a final concentration of 2 to 3 mg / ml, that is, 120 g to 250 g is measured according to a method based on JIS-K6503-1996 (measured as a load required to push 4 mm with a 12.7 mm diameter plunger).
- the jelly strength is not limited, and other types of support carriers are preferably used as the support carrier of the present invention if they have the same strength by the same evaluation method. Moreover, you may obtain the support carrier of the hardness corresponding to the target jelly strength by mixing 1 type or multiple types of support carriers.
- the high density state means a density equivalent to the density at the time of composing the tissue.
- the density is preferably 1 ⁇ 10 8 to 1 ⁇ 10 9 cells / ml, most preferably 2 ⁇ 10 8 to 8 ⁇ 10 8 cells / ml to ensure cell interaction without impairing the activity.
- Such centrifugation may be performed for 3 to 10 minutes at a rotational speed corresponding to a centrifugal force of 300 to 1200 ⁇ g, preferably 500 to 1000 ⁇ g, which does not impair cell survival. Centrifugation lower than 300 ⁇ g may result in insufficient cell precipitation and lower cell density, while centrifugation higher than 1200 ⁇ g may result in cell damage, which is undesirable. .
- a single-cell suspension is usually prepared in a container such as a tube used for cell centrifugation, and then centrifuged to remove the cells as a precipitate. Leave as much of the supernatant as possible.
- the precipitate may be placed inside the support carrier as it is.
- components other than the target cells for example, a culture solution, a buffer solution, a support carrier, etc.
- components other than the target cells for example, a culture solution, a buffer solution, a support carrier, etc.
- components other than the target cells for example, a culture solution, a buffer solution, a support carrier, etc.
- cells are in close contact with each other, and cell-cell interaction is effectively exhibited.
- the cell aggregate further aggregates due to the solidification of the support carrier and becomes more tight.
- the contact between the first cell aggregate and the second cell aggregate is close, and it is particularly preferable that the second cell aggregate is pressed against the first cell aggregate.
- wrapping the surroundings of the first cell aggregate and the second cell aggregate with a culture medium and a solid material that does not inhibit oxygen permeation is also effective for bringing the cell aggregates into close contact with each other. It is also preferable to place a cell suspension having a high density in solutions having different viscosities and solidify the solution as it is because the contact of cells can be easily maintained.
- the enamel nodules of the tooth germ epithelium are Although it is preferable to arrange so as to contact one cell aggregate, the present invention is not limited to this.
- a solidification step for solidifying the support carrier after being placed inside the support carrier may be provided.
- solidification conditions for the support carrier may be applied as they are.
- a solidifiable compound such as collagen when used for the support carrier, it can be solidified by allowing it to stand for several minutes to several tens of minutes under the conditions usually applied, for example, at the culture temperature. As a result, it is possible to fix the bonds between the cells inside the support carrier and to make them strong.
- the culture period for forming the reconstructed tooth germ varies depending on the number of cells arranged in the support carrier and the state of the cell aggregate, the culture implementation conditions, and the animal species, but generally, it is at least a period of one day.
- the culture period is preferably 3 days or longer. If it is a reconstructed tooth germ or tooth obtained by culturing for this period, it can erupt as a functional tooth even if it is embedded in the defect. Further, by extending this culture period, the process of forming a reconstructed tooth germ proceeds, such as accumulation of dentin and enamel, crown formation, and root formation.
- the culture period for the formation of the reconstructed tooth germ can be appropriately adjusted according to the working conditions, the animal species, the state of the reconstructed tooth germ, etc.
- the process may proceed to the next embedding process in 6 days, or may be over 30 days depending on the condition of the reconstructed tooth germ and other culture methods, in some cases over 50 days, over 100 days, or over 300 days. Also good.
- the culture inside the support carrier may be a culture using the support carrier alone containing the first and second cell aggregates, or a culture in the presence of other animal cells.
- the culture inside the support carrier is a culture using only the support carrier, the culture can be performed under the normal conditions used for culturing animal cells.
- the culture conditions in animal cells may be applied as they are, and the conditions described above can be applied as they are.
- serum derived from mammals may be added to the culture, and various cellular factors known to be effective for the growth and differentiation of these cells may be added. Examples of such cellular factors include FGF and BMP.
- organ culture in general, a porous membrane is floated on a medium suitable for the growth of animal cells, and the first and second cell aggregates embedded with a support carrier are placed on the membrane and cultured.
- the porous membrane used here is preferably a membrane having a large number of pores of about 0.3 to 5 ⁇ m.
- a cell culture insert (trade name) and an isopore filter (trade name) are used. Can be mentioned.
- the culture period by organ culture may be about 1 to 7 days, and preferably 3 to 6 days.
- teeth having a specific cell arrangement can be formed at an early stage due to the action of various cytokines from the animal cells.
- Such culture in the presence of other animal cells may be performed by in vitro culture using isolated cells or cultured cells.
- the animals that can be used for this purpose are preferably mammals such as humans, pigs, mice, etc., and are preferably the same species as the tooth germ tissue or other species modified to immunodeficiency.
- a suitable living body part include subrenal capsule, mesentery, and subcutaneous transplantation in order to generate animal cell organs and tissues as normally as possible.
- the culture period in the presence of such animal cells can be appropriately adjusted depending on the origin of the cells, culture conditions, animal species for transplantation, etc., as in the case of organ culture and the like.
- a tooth germ that can have a specific cell arrangement (structure) as dentine inside and enamel outside can be obtained, or a tooth having these.
- the direction of the tip of the tooth (crown) and the root of the tooth is also provided.
- both the first cell aggregate and the second cell aggregate are formed as a single cell aggregate, it is preferable because a dental aggregate with a plurality of teeth having a tooth-specific cell arrangement is obtained. If this set of teeth is obtained, individual teeth can be used separately from the set.
- the direction confirmation step in the method for producing a restorative material of the present invention the direction of the tooth germ or tooth obtained by the reconstructed tooth germ formation step so that the tip of the tooth can be embedded in the tooth defect portion with the oral cavity inside. Confirm.
- the directionality of the tooth germ or tooth means the direction for embedding in the tooth defect part. If the formation of a crown is observed, the crown is indicated.
- the epithelial cell layer of the part or the epithelial cell layer of the reconstructed tooth germ is confirmed as the inside of the oral cavity.
- the open part of the epithelial / mesenchymal cell layer of the reconstructed tooth germ may be confirmed as the side opposite to the oral cavity.
- the tooth germ or tooth whose directionality has been confirmed in this directionality confirmation step is used as a restoration material for obtaining an equivalent of the missing tooth in the tooth defect portion. That is, the restoration material containing a tooth germ or a tooth after confirmation of directionality is used for embedding in a tooth defect portion.
- the tooth germ or tooth in the restorative material then grows in the defect over time and erupts as a tooth equivalent.
- the tip of the erupted tooth equivalent reaches almost the same position (occlusion plane) as the tip of the surrounding teeth, and beyond There is no extension.
- the burying period from eruption and occlusion varies depending on the animal species or the embedded tooth germ or the state of the teeth.
- the erupted tooth equivalent has the same hardness (hardness) as a natural tooth.
- Hardness is a measure by which dentin and enamel of tooth equivalents can withstand deformation due to press-fitting, scratching, etc., and can be confirmed by measurement using Knoop hardness.
- the adult mouse normal tooth enamel has a Knoop hardness of 300 to 600 KHN, preferably 300 to 500 KHN, and the normal dentin has a Knoop hardness of 60 to 120 KHN.
- the tooth equivalent obtained by the present invention is: Knoop hardness is shown in this range. Thereby, the masticatory function equivalent to a normal tooth is exhibited.
- the erupted tooth equivalent has a pulp and periodontal ligament, and shows a tissue structure similar to that of a normal tooth. Since the tooth has a periodontal ligament, nerve invasion also occurs and the ability to respond to noxious stimuli such as compression is retained. Such a tissue structure can be confirmed by histological analysis, immunological staining, gene expression and the like according to a conventional method in addition to visual confirmation.
- the restoration material obtained by the method for producing a restoration material of the present invention can be used as a raw material for the equivalent of a tooth having a length and hardness of a tooth that can be engaged with a counter tooth.
- the tooth germ or tooth obtained by the reconstructed tooth germ forming process is embedded in the defect part as a restoration material.
- the direction of tooth germ or tooth embedding as in the direction confirmation step of the restoration material manufacturing method described above, if the formation of the crown is recognized, the crown is not formed, and the formation of the crown is not recognized.
- the epithelial cell layer of the coronal equivalent part or the epithelial cell layer of the reconstructed tooth germ is inside the oral cavity, or the open part of the epithelial / mesenchymal cell layer of the reconstructed tooth germ is defined as the oral cavity It is preferable to arrange so as to be on the opposite side. Thereby, the front-end
- deletion part should just be normally provided in gingiva by tooth extraction etc., and there is no restriction
- the tooth germ grows into a tooth inside the defect with time, and erupts as a tooth equivalent.
- the tip (cusp) of the erupted tooth equivalent is almost the same as the tip of the surrounding tooth, although it depends on the condition and size of the embedded tooth germ. It reaches the position (occlusal plane) and does not extend any further. Thereby, it can be set as the length of the tooth
- the burying period from eruption and occlusion varies depending on the animal species or the embedded tooth germ or the state of the teeth.
- the erupted tooth equivalent has the same hardness (hardness) as that of the natural tooth, similar to the matters described for the method of manufacturing the restoration material described above.
- Hardness is a measure by which dentin and enamel of tooth equivalents can withstand deformation due to press-fitting, scratching, etc., and can be confirmed by measurement using Knoop hardness.
- the adult mouse normal tooth enamel has a Knoop hardness of 300 to 600 KHN, preferably 300 to 500 KHN, and the normal dentin has a Knoop hardness of 60 to 120 KHN. Knoop hardness is shown in this range. Thereby, the masticatory function equivalent to a normal tooth is exhibited.
- the erupted tooth is provided with pulp and periodontal ligament in addition to enamel and dentin, similar to the matters described for the method of manufacturing the restoration material described above, and shows a tissue structure similar to that of a normal tooth. Is. Since the tooth has a periodontal ligament, nerve invasion also occurs and the ability to respond to noxious stimuli such as compression is retained. Such a tissue structure can be confirmed by histological analysis, immunological staining, gene expression and the like according to a conventional method in addition to visual confirmation.
- the method for repairing a tooth defect part according to the present invention has a hardness equivalent to that of a normal tooth and is normally occluded by repairing the tooth defect part using the tooth germ or tooth reconstructed as described above.
- the tooth defect portion can be repaired so as to have a stimulus responsiveness equivalent to that of a normal tooth.
- the tooth equivalent regenerated in the defect part acts as a functional tooth and can maintain the repaired state for a long period of time. For this reason, since the quality of life can be maintained, it is used favorably in the aesthetic field.
- the deterioration of health due to the presence of a tooth defect in the oral cavity can be avoided, the health of mammals other than humans such as domestic animals such as cattle, horses and pigs, and pets such as dogs and cats Can be maintained.
- Example 1 (1) Preparation of tooth germ epithelial cell and tooth germ mesenchymal cell To reconstruct the tooth, the tooth germ was reconstructed. A mouse was used as this experimental model. C57BL / 6N mice (purchased from CLEA Japan) embryonic day 14.5 days, mandibular molar tooth germ tissues were excised from the embryos by a conventional method under a microscope. Mandibular molar tooth germ tissue was washed with Ca 2+ , Mg 2+ -free phosphate buffer (PBS ( ⁇ )), and Dispase II (Roche, Mannheim, Germany) at a final concentration of 1.2 U / ml was added to PBS ( ⁇ ).
- PBS Mg 2+ -free phosphate buffer
- the treated enzyme solution was treated for 12.5 minutes at room temperature, and then washed three times with DMEM (Sigma, St. Louis, MO) supplemented with 10% FCS (JRH Biosciences, Lenexa, KS). Furthermore, DNase I solution (Takara, Siga, Japan) was added to a final concentration of 70 U / ml to disperse the tooth germ tissue, and surgically treated with a 25 G needle (Terumo, Tokyo, Japan) Tooth germ mesenchymal tissue was isolated.
- DMEM Sigma, St. Louis, MO
- FCS JRH Biosciences, Lenexa, KS
- tooth germ epithelial cells For tooth germ epithelial cells, the tooth germ epithelial tissue obtained above was washed three times with PBS ( ⁇ ), and collagenase I (Worthington, Lakewood, NJ) at a final concentration of 100 U / ml was dissolved in PBS ( ⁇ ). The treatment with the enzyme solution at 37 ° C. for 20 minutes was repeated twice. The cells collected by precipitation by centrifugation were further treated with 0.25% Trypsin (Sigma) -PBS ( ⁇ ) at 37 ° C. for 5 minutes. After washing the cells three times with DMEM supplemented with 10% FCS, a DNase I solution with a final concentration of 70 U / ml was added to the cells, and single tooth germ epithelial cells were obtained by pipetting.
- PBS collagenase I
- tooth germ mesenchymal tissue was washed three times with PBS ( ⁇ ), and PBS ( ⁇ ) containing 0.25% Trypsin (Sigma) and 50 U / ml Collagenase I (Worthington). Was processed. 70 U / ml DNase I (Takara) was added, and single tooth germ mesenchymal cells were obtained by pipetting.
- tooth germ reconstruction was performed using the tooth germ epithelial cells and tooth germ mesenchymal cells prepared above. Place tooth germ epithelial cells or tooth germ mesenchymal cells suspended in DMEM (Sigma) supplemented with 10% FCS (JRH) into a 1.5 mL microtube (Eppendorf, Hamburg, Germany) coated with silicone grease. Cells were collected as a precipitate by centrifugation. Remove the supernatant of the culture solution after centrifugation as much as possible, perform centrifugation again, and completely observe the culture solution remaining around the cell precipitate using a GELoader Tip 0.5-20 ⁇ L (eppendorf) while observing with a stereomicroscope. Removed. Thereafter, the cells were dispersed by tapping or pipetting to prepare cells used for preparation of reconstructed tooth germs.
- the reconstructed tooth germ prepared in the gel was allowed to stand in a CO 2 incubator for 10 minutes to solidify Cellmatrix type IA (Nitta Gelatin), and cell culture insert (pore size was added to 10% FCS (JRH) -added DMEM (Sigma).
- the cell mass is transferred together with the surrounding gel as a support carrier onto the membrane of the cell culture insert of a culture vessel set so that the 0.4 micron PET membrane (BD, Franklin Lakes, NJ) is in contact with the support carrier for 18 to 24 hours.
- Organ culture After culturing, organ culture on the cell culture insert was continued to analyze tooth development.
- Example 2 Preparation of individually separated tooth germ Organ culture was performed in the same manner as in Example 1 (1) to (2), and a reconstructed tooth germ in which a plurality of tooth germs occurred on the 2nd to 5th day of organ culture was obtained. Separated surgically using a needle and tweezers under a stereomicroscope. In the case of individual separated tooth germs, 30 ⁇ L of Cellmatrix type IA (Nitta gelatin, Osaka, Japan) was dropped on a Petri dish coated with silicon grease to prepare a collagen gel drop.
- Cellmatrix type IA Nita gelatin, Osaka, Japan
- M1-deficient C57BL / 6 produced by the above method was sucked and anesthetized with diethyl ether, and physiological saline containing 5 mg / ml sodium pentobarbital was injected intraperitoneally at a ratio of 240 ⁇ l per 20 g body weight.
- a mouse with a numbness of pain was fixed to the dissecting table in the supine position, and the upper and lower jaws were fixed with rubber or thread in a state where the mouse was fully opened.
- the maxillary M1 part alveolar crest gingiva was incised with a scalpel and exfoliated with the periosteum to reveal the mandible.
- a pin vise (ultrafine drill) was used to make a hole with a diameter of 1 mm in the mandibular bone corresponding to the M1 portion while paying attention to perforation of the maxillary sinus, and individually separated tooth germs were transplanted. After transplantation, the wound was closed with a periosteal gingival flap and sutured using a needled thread. In order to adjust the orientation of the individual isolated tooth germ to be transplanted, the bite head of the individual isolated tooth germ was labeled with methylene blue, and the label was transplanted in accordance with the eruption direction. Eight-week-old C57BL / 6 transplanted intraorally was fed daily with food for breeding crushed into powder.
- the upper and lower jaws M3 of the head fixed with 4% paraformaldehyde-phosphate buffer are exposed, and the horizontal angle is based on the plane connecting the occlusal surfaces of the lower jaw M3 and the lower jaw M1.
- the angle was taken in accordance with the usual method of intraoral photography of the central occlusal position on the basis of the overlapping method when the mesial buccal cusp and mesial lingual cusp of the lower jaw M1 were viewed from the side.
- Example 2 (4) Knoop hardness Individual teeth generated in the oral cavity in Example 2 (1) were extracted, and the hardness of the enamel and dentin of the teeth was measured at three or more points with the same tooth. The measurement was performed using a microhardness tester (HM-102 manufactured by Mitutoyo Corporation) equipped with two counter-ridge angles 172 ° 30 'and a 130 ° Knoop hardness measurement square pyramid diamond indenter (19BAA061 manufactured by Mitutoyo Corp.). . The indenter was pressed against the regenerated tooth at a load of 10 g for 10 seconds, and the hardness was determined from the length of the long side of the 7.11: 1 diamond indentation produced thereby.
- HM-102 manufactured by Mitutoyo Corporation
- the regenerated teeth were fixed to a metal plate using a dental resin (Unifast III GC).
- the enamel was measured at a portion parallel to the ground, and the dentin was measured by cutting it horizontally with the dental engine (ULTIMATE 500 Nakanishi) and exposing the test surface.
- the results of Knoop hardness measurement are shown in FIG.
- the mean Knoop hardness of normal tooth enamel is 341.083 KHN at 3 weeks old, 457.5 KHN at 6 weeks old, 436 KHN at 9 weeks old, and the enamel of regenerated teeth is 469.
- the hardness was 81 KHN (see FIG. 2A).
- the average Knoop hardness of normal teeth was 66.87 KHN at 3 weeks, 76.53 KHN at 6 weeks, 88.58 KHN at 9 weeks, and the dentin of regenerated teeth was 81.83 KHN ( (See FIG. 2B).
- the regenerated teeth of both enamel and dentin are teeth having a hardness adapted to the normal masticatory function.
- FIG. 3 shows the regenerated tooth just before eruption (FIG. 3, white arrow).
- histological analysis revealed a tissue structure equivalent to that of a normal tooth having enamel, dentin, pulp, and periodontal ligament.
- enamel trabeculae radiated parallel to the enamel, and dentinal tubules were also observed in the dentin.
- the periodontal ligament has a sufficient thickness, and a structure capable of buffering mastication pressure was observed.
- the regenerated tooth in the course of eruption moves in the long axis direction while forming the root, erupts from the alveolar crest and reaches the occlusal plane, and in this process the periodontal ligament is retained and in harmony with the periodontal tissue It was suggested that
- the wire for buccal movement correction was molded into Saline containing 5 mg / ml pentobarbital sodium C57BL / 6 transplanted with individual tooth germs by the above method was injected intraperitoneally at a ratio of 240 ⁇ l per 20 g body weight.
- a mouse with a numbness of pain was fixed to the dissecting table in the supine position, and the upper and lower jaws were fixed with rubber or thread in a state where the mouse was fully opened.
- the length of the wire end on the opposite side of the loop was adjusted so as to reach the distal portion of the erupted tooth when attached.
- Fit the buccal movement correction wire loop to the buccal cervical part of the tooth that corrects the opposite wire end, and attach it to the maxillary bilateral incisor cervical part with Unifill Flow (Photopolymerization Resin, manufactured by GC Corporation) Glued and fixed.
- Unifill Flow Photopolymerization Resin, manufactured by GC Corporation
- the wire end that was fitted to the buccal tooth neck is moved to the lingual tooth neck, the correction force is activated, and then the bone remodeling phenomenon is analyzed.
- a histological analysis was performed 6 days after the correction, in which it became particularly prominent. The results are shown in FIGS. 4A to 4C.
- Pain due to tooth pressure is well known by correction, etc., and is especially mediated by nerves present in the periodontal ligament.
- -Fos protein production has been shown to be increased (Byers MR. Et al., Crit Rev Oral Biol Med 10, 4-39, 1999. Deguchi T. et al. J Dent Res 82: 677-681 , 2003., Fujiyoshi, Y. et al., Neuroscience Letters 283, 205-208, 2000.).
- Pain caused by exposure of the pulp is well known by processing such as demyelination, and in the same way as pain caused by correction, it is mediated by nerves present in the pulp, especially in nerve cells in the caudal nucleus of the medullary trigeminal spinal tract.
- C-Fos protein production has been shown to be increased (Byers MR. Et al., Crit Rev Oral Biol Med 10, 4-39, 1999. Deguchi T. et al.
- the extracted medulla was fixed with 4% paraformaldehyde-phosphate buffer for 16 hours, then embedded in OCT compound (Miles Inc., Naperville, IL) by a conventional method, and cryostat (Leica, Wetzlar, Germany). 50 ⁇ m sections were prepared. The prepared sections were c-fos (SANTA CRUZ BIOTECHNOLOGY, INC., Santa Cruz, California, USA) as the primary antibody, Goat IgG fraction to rabbit IgG (CAPPEL, Aurora, Ohio, USA) as the secondary antibody, Rabbit. Peroxidase anti-peroxidase (CAPPEL, Aurora, Ohio, USA) was immunostained using a tertiary antibody, and expression in the trigeminal tract nucleus of the medulla was compared.
- OCT compound Miles Inc., Naperville, IL
- cryostat Leica, Wetzlar, Germany
- FIG. 5A in the C57BL / 6 medullary trigeminal spinal cord caudal subnuclear region where the regenerated tooth was not corrected or exposed, c-Fos protein expression was not observed, but FIG. 5B And in the C57BL / 6 medullary trigeminal spinal cord caudal subnuclear region with regenerated teeth corrected as shown in FIG. 5C, high level expression of c-Fos protein was induced after 2 hours, as shown in FIG. 5C. The expression was maintained until 48 hours later. Further, as shown in FIG.
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Abstract
Description
歯胚の再生方法としては、例えば、特開2004-331557号公報には、生体から単離された歯胚細胞を、線維芽細胞増殖因子等の生理活性物質の存在下で培養することが記載されている。また、特開2004-357567号公報には、生体から単離された歯胚細胞及びこれらの細胞に分化可能な細胞のうち少なくとも1種類を、フィブリンを含む担体と一緒に培養することが提案されており、ここでフィブリンを含む担体は、歯胚の目的形状のものを使用して、特有の形態を有する「歯」を形成すると記載されている。
また特開2005-013261号公報では、生体内に埋め込まれた使用されるインプラント材料として、コロイダルシリカ粒子を材料表面に付着させた人工生体材料を開示している。
本発明の第一の態様は、口腔内の歯欠損部を修復するために用いられる修復材料の製造方法であって、いずれか一方が歯胚由来である間葉系細胞と上皮系細胞とのいずれか一方でそれぞれ構成された第1の細胞集合体及び第2の細胞集合体を、混合することなく密着させて支持担体内部に配置して培養し、再構成された歯胚又は歯を形成させること、歯の先端部を口腔内側として埋設し得るように、前記培養によって形成された前記再構成された歯胚又は歯の方向性を確認すること、を含み、方向性が確認された前記歯胚又は歯は、前記歯欠損部において、欠損した歯の等価物を得るための修復材料として用いられるものである当該製造方法を提供する。
なお本発明における「再構成歯胚形成工程」は、修復材料の製造方法及び歯欠損部の修復方法の双方で適用される。
また、本明細書において「工程」との語は、独立した工程だけでなく、他の工程と明確に区別できない場合であっても本工程の所期の作用が達成されれば、本用語に含まれる。
また本明細書における数値範囲を示す表現は、その前後の数値を含むものとする。
以下、本発明について説明する。
また、歯の方向性は、歯冠や歯根の配置によって特定することができる。歯冠や歯根は、形状や組織染色などに基づいて目視にて確認することができる。
また本発明において「歯周組織」とは、歯の主として外層の形成された歯槽骨及び歯根膜をいう。歯槽骨及び歯根膜は、当業者には、組織染色などによって形態的に容易に特定することができる。
この再構成歯胚形成工程としては、国際公開第2006/129672号に記載されている方法をそのまま適用することができる。
また、歯胚以外に由来する上皮系細胞としては、生体内の他の上皮系組織に由来する細胞であり、好ましくは、皮膚や口腔内の粘膜や歯肉の上皮系細胞、更に好ましくは皮膚や粘膜などの分化した、例えば角化した、あるいは錯角化した上皮系細胞を生み出しうる未熟な上皮系前駆細胞、たとえば非角化上皮系細胞やその幹細胞等を挙げることができる。
培養に用いられる培地としては、一般に動物細胞の培養に用いられる培地、例えばダルベッコ改変イーグル培地(DMEM)等を用いることができ、細胞の増殖を促進するための血清を添加するか、あるいは血清に代替するものとして、例えばFGF、EGF、PDGF等の細胞増殖因子やトランスフェリン等の既知血清成分を添加してもよい。なお、血清を添加する場合の濃度は、そのときの培養状態によって適宜変更することができるが、通常10%とすることができる。細胞の培養には、通常の培養条件、例えば37℃の温度で5%CO2濃度のインキュベーター内での培養が適用される。また、適宜、ストレプトマイシン等の抗生物質を添加したものであってもよい。
なお、ここで支持担体は、第1及び第2の細胞集合体が担体内部で成育することができる程度の厚みを有すればよく、目的とする組織の大きさ等によって適宜設定することができる。
遠心分離による沈殿物とした場合には、沈殿物をそのまま支持担体の内部に配置すればよい。このとき、目的とする細胞以外の成分(例えば、培養液、緩衝液、支持担体等)は、細胞の容量と等量以下であることが好ましく、目的とする細胞以外の成分を含まないことを最も好ましい。このような高密度の細胞集合体では、細胞が緊密に接触しており、細胞間相互作用が効果的に発揮される。特に目的とする細胞以外の成分が極端に少ない細胞集合体を支持担体の内部に配置すると、支持担体の固化等によって更に凝集し、より一層緊密状態となる。
また、この培養期間を延長させることによって、象牙質及びエナメル質の蓄積、歯冠形成、及び歯根の形成のように、再構成歯胚の形成過程が進行する。このため、この再構成歯胚形成のための培養期間は、実施条件、動物種、再構成歯胚の状態等に応じて適宜調整可能であり、例えば、後述する器官培養の場合には7日、場合によって6日で次の埋設工程に移行してもよく、或いは、再構成歯胚の状態や他の培養法によっては30日超、場合によって50日超、100日超又は300日超としてもよい。
支持担体内部における培養を支持担体のみによる培養とした場合には、動物細胞の培養に用いられる通常の条件下での培養とすることができる。ここでの培養は、一般に動物細胞での培養条件をそのまま適用すればよく、前述した条件をそのまま適用することができる。また培養には、哺乳動物由来の血清を添加してもよく、またこれらの細胞の増殖や分化に有効であることが既知の各種細胞因子を添加してもよい。このような細胞因子としては、FGF、BMP等を挙げることができる。
器官培養による培養期間としては、1日間から7日間程度とすればよく、好ましくは3~6日とすることができる。
また、第1の細胞集合体と第2の細胞集合体を共に単一細胞の集合体とした場合には、歯特有の細胞配置を有する複数の歯による歯の集合体が得られるため好ましい。この歯の集合体が得られた場合には、個々の歯を集合体から分離して用い得る。
即ち、方向性確認後の歯胚又は歯を含む修復材料は、歯欠損部への埋設に用いられる。修復材料中の歯胚又は歯は、その後、時間の経過と共に欠損部内で成長し、歯の等価物として萌出する。修復材料中の歯胚又は歯の状態や大きさ等によって異なるが、萌出した歯等価物の先端部(咬頭)は周囲の歯の先端部とほぼ同じ位置(咬合平面)に達し、それ以上、延長することはない。萌出、咬合までの埋設期間は、動物種又は埋設された歯胚若しくは歯の状態によって異なる。
この際、歯胚又は歯の埋設方向としては、上述した修復材料の製造方法の方向確認工程と同様に、歯冠の形成が認められれば歯冠を、歯冠の形成が認められない場合には、歯冠相当部の上皮細胞層又は再構成歯胚の上皮細胞層を口腔内側となるように配置すること、或いは再構成歯胚の上皮・間葉細胞層の開放部分を口腔内とは反対側となるように配置することが好ましい。これにより、歯等価物の先端部(歯冠)が口腔内側となって、周囲の歯と同様の向きに揃えることができる。
このような欠損部は、通常、顎骨、口腔の歯槽骨などに位置する。また歯が喪失することによって歯槽骨量が低下している場合には、この欠損部位に対して、GTR法(guided tissue regeneration:組織再生誘導法)など、インプラントの埋設のために臨床で用いられている公知の方法により骨の再生を行って骨量を増加させてもよい。歯胚又は歯を孔部へ配置した後は、通常の処理に従って縫合等を行うことが好ましい。
このため、生活の質を維持することができるので、審美分野において良好に用いられる。また口腔内の歯欠損部の存在に起因した健康状態の悪化を避けることができるので、ウシ、ウマ、ブタなどの家畜や、イヌ、ネコなどのペットなど、ヒト以外の哺乳動物の健康状態を維持することができる。
(1)歯胚上皮系細胞と歯胚間葉系細胞の調製
歯の形成を行うために歯胚の再構築を行った。この実験モデルとしてマウスを用いた。
C57BL/6Nマウス(日本クレアから購入)の胎齢14.5日、胚仔から下顎臼歯歯胚組織を顕微鏡下で常法により摘出した。下顎臼歯歯胚組織をCa2+,Mg2+不含リン酸緩衝液(PBS(-))で洗浄し、PBS(-)に最終濃度1.2U/mlのDispase II (Roche, Mannheim, Germany)を添加した酵素液で室温にて12.5分間処理した後、10%FCS(JRH Biosciences, Lenexa, KS)を添加したDMEM(Sigma, St. Louis, MO)で3回洗浄した。さらにDNase I溶液 (Takara, Siga, Japan)を最終濃度70U/mlになるよう添加し歯胚組織を分散させ、25G注射針 (Terumo, Tokyo, Japan)を用いて外科的に歯胚上皮組織と歯胚間葉組織を分離した。
一方、歯胚間葉系細胞は、歯胚間葉組織をPBS(-)で3回洗浄し、0.25%Trypsin (Sigma)、50U/mlのCollagenase I (Worthington)を含むPBS(-)で処理した。70U/mlのDNase I (Takara)を添加して、ピペッティングにより単一の歯胚間葉系細胞を得た。
次に、上記で調製された歯胚上皮系細胞及び歯胚間葉系細胞を用いて、歯胚再構築を行った。シリコングリースを塗布した1.5mLマイクロチューブ (Eppendorf, Hamburg, Germany)に、10%FCS(JRH) 添加DMEM(Sigma)で懸濁した歯胚上皮系細胞、あるいは歯胚間葉系細胞を入れ、遠心分離により細胞を沈殿として回収した。遠心後の培養液の上清をできる限り除去し、再度遠心操作を行い、実体顕微鏡で観察しながら細胞の沈殿周囲に残存する培養液を GELoader Tip 0.5-20μL (eppendorf) を用いて完全に除去した。その後、タッピングあるいはピペッティングにより細胞を分散させて、再構成歯胚作製に用いる細胞を準備した。
器官培養2~5日目において歯胚が複数発生している再構成歯胚を、実体顕微鏡下で注射針とピンセットを用いて外科的に個別の歯胚に分離した。シリコングリースを塗布したペトリディッシュにCellmatrix type I-A (Nitta gelatin, Osaka, Japan) を30μL滴下してコラーゲンゲルドロップを作製した。この溶液に、上記個別分離歯胚を入れ、CO2インキュベーターに10分間静置してCellmatrix type I-A (Nitta Gelatin)を固形化し、10%FCS(JRH) 添加DMEM(Sigma)にセルカルチャーインサート(ポアサイズが0.4ミクロンのPETメンブレン;BD, Franklin Lakes, NJ)が接するようにセットした培養容器のセルカルチャーインサートの膜上に、個別分離歯胚を支持担体である周囲のゲルと共に移して、18~24時間、器官培養した。
上記に従って作製した個別分離歯胚から周囲のゲルを注射針とピンセットで外科的に除去し、個別分離歯胚を、腎皮膜下へ移植せずにそのまま7~10週齢C57BL/6の上顎第一臼歯(M1)部歯槽骨に移植した。本発明により口腔内において個別の歯を萌出させ、咀嚼などの口腔機能を実現させた。
(1)個別分離歯胚の作製
実施例1(1)~(2)と同様にして器官培養を行い、器官培養2~5日目において歯胚が複数発生している再構成歯胚を、実体顕微鏡下で注射針とピンセットを用いて外科的に個別に分離した。個別分離歯胚の場合、シリコングリースを塗布したペトリディッシュにCellmatrix type I-A (Nitta gelatin, Osaka, Japan) を30μL滴下してコラーゲンゲルドロップを作製した。この溶液に、上記個別分離歯胚を入れ、CO2インキュベーターに10分間静置してCellmatrix type I-A (Nitta Gelatin)を固形化し、10%FCS(JRH) 添加DMEM(Sigma)にセルカルチャーインサート(ポアサイズが0.4ミクロンのPETメンブレン;BD, Franklin Lakes, NJ)が接するようにセットした培養容器のセルカルチャーインサートの膜上に、個別分離歯胚を支持担体である周囲のゲルと共に移して、5日間、器官培養し、複数の再構成歯胚が形成された場合には、外科的に単一の再生歯胚に分離した。培養後、周囲のゲルを注射針とピンセットで外科的に除去し、8週齢C57BL/6の上顎M1部歯槽骨に移植した。
口腔内移植の3日前にジエチルエーテルで吸引麻酔した8週齢C57BL/6に、5mg/mlのペントバルビタールナトリウムを含む生理食塩水を、体重20gに対して240μlの割合で腹腔注射した。痛覚の麻痺したマウスの上顎M1を、ピンセットを用いて上顎からM1を抜歯し、残根のないことを確認し、噴出してくる血液を脱脂綿で拭き取り止血した。食物摂取について、粉末状に砕いた飼育用の餌を毎日与えた。3週間以上、飼育して抜歯窩の治癒をはかり、M1欠損のC57BL/6を作製した。
これらの結果から、再生歯は正常歯と同様に安定した咬合関係を形成し咀嚼機能を有することが明らかになった。
個別分離歯胚を移植した上顎骨を含んだ頭部を摘出し、頭部全体を4%パラホルムアルデヒド-リン酸緩衝液で12時間固定した後、inspeXio SMX-90CT(島津製作所 社製)でCT撮影を行った。CT撮影時の設定は、ビュー数を600、アベレージを10、スキャンを1、画像を512×512、スケーリングを50、スライス厚を1、BHCをなしに設定し、撮影を行った。CT撮影して得たデータは、解析ソフトImaris(Zeiss社製)を用いて3次元立体再構築し、マイクロCTの断面画像の作成を行った。
実施例2(1)において口腔内に発生させた個別の歯を摘出し、その歯のエナメル質と象牙質の硬度を同一歯で3点以上測定した。
測定は微小硬さ試験機(HM-102 ミツトヨ社製)に二つの対稜角172°30′と130°のヌープ硬さ測定用四角錐ダイヤモンド圧子(19BAA061 ミツトヨ社製)を装着したもので行った。再生歯へ圧子を負荷10g、10秒間圧接して、それにより生じた7.11:1の菱形圧痕の長辺の長さから硬度を求めた。再生歯は金属板に歯科用レジン(ユニファストIII ジーシー社製)を用いて固定した。エナメル質は地面と水平な部分において測定を行い、象牙質は歯科技工用エンジン(ULTIMATE 500 ナカニシ社製)にて地面と水平に切削し、被検面を露出させて測定を行った。ヌープ硬度測定の結果を図2に示す。
移植後約20日から50日経過すると、M1歯肉部より再生歯の咬頭が認められ、萌出が開始した。萌出状況を観察して、萌出前、萌出直前、萌出直後、萌出中、咬合面到達状態の上顎骨を摘出した。4%パラホルムアルデヒド-リン酸緩衝液で16時間固定した後、22.5%のギ酸で72時間脱灰し、常法によりパラフィン包埋して、10μmの切片を作製した。脱灰液は上顎骨2つにつき50mlとし、脱灰48時間目に全量を交換した。組織学的解析のためには常法に従い、ヘマトキシリン-エオジン染色を行った。
歯は歯根膜を介して周囲の歯槽骨と結合しているため、歯に矯正力を与えると、歯根膜によってメカニカルストレスが周囲環境に伝達される。矯正力によって圧迫された歯根膜部位では歯槽骨の破骨細胞による吸収が、牽引された歯根膜部位では骨芽細胞による骨形成が行われ、歯と歯槽骨の間の空間的距離を一定に保つようになっている。このような歯根膜の機能を評価するために、再生歯に矯正力を加え、正常な歯の移動で見られる圧迫側での破骨細胞の出現、牽引側での骨芽細胞の出現といった骨のリモデリングを確認するために、以下の解析を行った。
その結果、頬側において、歯根膜腔が狭窄して歯根膜線維が圧縮する歯根膜の圧迫が認められた(図4Aの黒枠Bと図4B参照)。一方、逆側の舌側では、歯根膜腔が拡大して歯根膜線維が緊張する歯根膜の牽引が認められた(図4Aの黒枠Cと図4C参照)。また、歯根膜の牽引側の歯槽骨壁に添って一層の細胞の配列が認められたため、骨を形成する骨芽細胞(図4C矢印頭 参照)の出現が示唆された。さらに、牽引側の反対側の圧迫側では、細胞体に多数の核を含む多核巨細胞が歯槽骨に認められ、窩状な骨の吸収像が認められたので、破骨細胞(図4B矢印 参照)の発現と骨の吸収が示唆された。
これらの結果から、再生歯が正常歯と同様に、矯正力などのメカニカルストレスによって歯根膜が応答し、骨のリモデリング現象が誘起されることが示唆された。
歯の圧迫による痛みは矯正などによってよく知られており、特に歯根膜に存在する神経が媒介し、延髄三叉神経脊髄路核尾側亜核領域の神経細胞でのc-Fosタンパク質の産生が上昇することが明らかにされている(Byers MR. et al., Crit Rev Oral Biol Med 10, 4-39, 1999. Deguchi T. et al. J Dent Res 82:677-681, 2003.、Fujiyoshi, Y. et al., Neuroscience Letters 283, 205-208, 2000.)。また歯髄の露出による痛みは露髄処理などによってよく知られており、矯正による痛みと同様に、特に歯髄に存在する神経が媒介し、延髄三叉神経脊髄路核尾側亜核領域の神経細胞でのc-Fosタンパク質の産生が上昇することが明らかにされている(Byers MR. et al., Crit Rev Oral Biol Med 10, 4-39, 1999. Deguchi T. et al. J Dent Res 82:677-681, 2003.)そこで再生歯の神経機能を解析する目的で、マウスの再生歯に矯正と同様の圧迫刺激、および象牙質に歯科用ドリルで穴をあける露髄処理を行い、延髄三叉神経脊髄路核尾側亜核領域においてc-Fosタンパク質の発現が上昇するかどうかを解析した。
上記の方法にて矯正力活性後2時間と48時間、および露髄処理を行った2時間後で、C57BL/6を4%パラホルムアルデヒド-リン酸緩衝液で灌流固定した。摘出した延髄を4%パラホルムアルデヒド-リン酸緩衝液で16時間固定した後、常法によりOCT compound(Miles Inc., Naperville, IL)に包埋して、クリオスタット (Leica, Wetzlar, Germany)で50μmの切片を作製した。作製した切片は、c-fos(SANTA CRUZ BIOTECHNOLOGY, INC., Santa Cruz, Calfornia, USA)を1次抗体として、Goat IgG fraction to rabbit IgG (CAPPEL, Aurora, Ohio, USA)を2次抗体、Rabbit peroxidase anti-Peroxidase を(CAPPEL, Aurora, Ohio, USA)3次抗体として用いて免疫染色し、延髄の三叉神経路核における発現を比較した。
本明細書に記載された全ての文献、特許出願、および技術規格は、個々の文献、特許出願、および技術規格が参照により取り込まれることが具体的かつ個々に記された場合と同程度に、本明細書中に参照により取り込まれる。
Claims (10)
- 口腔内の歯欠損部を修復するために用いられる修復材料の製造方法であって、
いずれか一方が歯胚由来である間葉系細胞と上皮系細胞とのいずれか一方でそれぞれ構成された第1の細胞集合体及び第2の細胞集合体を、混合することなく密着させて支持担体内部に配置して培養し、再構成された歯胚又は歯を形成させること、
歯の先端部を口腔内側として前記歯欠損部に埋設し得るように、前記培養によって形成された前記再構成された歯胚又は歯の方向性を確認すること、
を含み、方向性が確認された前記歯胚又は歯は、前記歯欠損部において、欠損した歯の等価物を得るための修復材料として用いられるものである当該製造方法。 - 前記欠損した歯の等価物が、300~600KHNのエナメル質のヌープ硬度及び60~120KHNの象牙質のヌープ硬度を備えたものである請求項1記載の修復材料の製造方法。
- 前記培養が器官培養であり、前記修復材料が、器官培養後の再構成された歯胚又は歯と支持担体とを含む請求項1又は請求項2記載の修復材料の製造方法。
- 前記間葉系細胞及び上皮系細胞が共に歯胚由来である請求項1~請求項3のいずれかに記載の修復材料の製造方法。
- 前記第1の細胞集合体及び第2の細胞集合体が共に単一細胞集合体である請求項1~請求項4のいずれかに記載の修復材料の製造方法。
- 前記支持担体が、コラーゲン、アガロースゲル、カルボキシメチルセルロース、ゼラチン、寒天、ハイドロゲル、エラスチン、フィブリン、ファイブロネクチン、ラミニン、細胞外マトリクス混合物、ポリグリコール酸(PGA)、ポリ乳酸(PLA)、乳酸/グリコール酸共重合体(PLGA)、セルマトリクス(商品名)、メビオールゲル(商品名)及びマトリゲル(商品名)からなる群より選択された少なくとも1種である請求項1~請求項5のいずれかに記載の修復材料の製造方法。
- 口腔内の歯欠損部の修復方法であって、
いずれか一方が歯胚由来である間葉系細胞と上皮系細胞とのいずれか一方でそれぞれ構成された第1の細胞集合体及び第2の細胞集合体を、混合することなく密着させて支持担体内部に配置して培養し、再構成された歯胚又は歯を形成させること、
前記歯欠損部に、前記再構成された歯胚又は歯を埋設すること、
を含む歯欠損部の修復方法。 - 前記間葉系細胞及び上皮系細胞が共に歯胚由来である請求項7に記載の歯欠損部の修復方法。
- 前記第1の細胞集合体及び第2の細胞集合体が共に単一細胞集合体である請求項7又は請求項8に記載の歯欠損部の修復方法。
- 前記支持担体が、コラーゲン、アガロースゲル、カルボキシメチルセルロース、ゼラチン、寒天、ハイドロゲル、エラスチン、フィブリン、ファイブロネクチン、ラミニン、細胞外マトリクス混合物、ポリグリコール酸(PGA)、ポリ乳酸(PLA)、乳酸/グリコール酸共重合体(PLGA)、セルマトリクス(商品名)、メビオールゲル(商品名)及びマトリゲル(商品名)からなる群より選択された少なくとも1種である請求項7~請求項9のいずれかに記載の歯欠損部の修復方法。
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- 2009-08-19 RU RU2011109664/15A patent/RU2521195C2/ru not_active IP Right Cessation
- 2009-08-19 JP JP2010525698A patent/JP5514110B2/ja active Active
- 2009-08-19 KR KR1020117006131A patent/KR20110045067A/ko not_active Application Discontinuation
- 2009-08-19 AU AU2009283516A patent/AU2009283516B2/en not_active Ceased
- 2009-08-19 US US13/059,847 patent/US20110212414A1/en not_active Abandoned
- 2009-08-19 WO PCT/JP2009/064509 patent/WO2010021340A1/ja active Application Filing
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- 2009-08-19 CN CN2009801326316A patent/CN102131489A/zh active Pending
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WO2011064553A2 (en) | 2009-11-26 | 2011-06-03 | Blade Dynamics Limited | An aerodynamic fairing for a wind turbine and a method of connecting adjacent parts of such a fairing |
WO2011125425A1 (ja) * | 2010-04-07 | 2011-10-13 | 株式会社オーガンテクノロジーズ | 再生歯ユニットの製造方法 |
Also Published As
Publication number | Publication date |
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JPWO2010021340A1 (ja) | 2012-01-26 |
RU2011109664A (ru) | 2012-09-27 |
AU2009283516B2 (en) | 2014-02-13 |
CN102131489A (zh) | 2011-07-20 |
KR20110045067A (ko) | 2011-05-03 |
EP2322133A1 (en) | 2011-05-18 |
TW201014912A (en) | 2010-04-16 |
CA2734706A1 (en) | 2010-02-25 |
EP2322133A4 (en) | 2012-03-14 |
RU2521195C2 (ru) | 2014-06-27 |
JP5514110B2 (ja) | 2014-06-04 |
US20110212414A1 (en) | 2011-09-01 |
AU2009283516A1 (en) | 2010-02-25 |
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