JP4991204B2 - Teeth manufacturing method - Google Patents

Description

  The present invention relates to a method for manufacturing a tooth, and particularly to a method for manufacturing a tooth using cells.

Teeth have a hard tissue called enamel in the outermost layer and dentin in the inner layer, and further have dentin blasts that produce dentin inside, and a pulp in the center, which is lost due to caries, periodontal disease, etc. It is an organ that can be broken. In general, it is considered that there is little concern about life with regard to tooth loss, and at present, it is often compensated mainly by dentures and implants. However, the presence or absence of teeth greatly affects the appearance and taste of food, and interest in the development of tooth regeneration technology has increased from the viewpoint of maintaining health and maintaining a high quality of life.
A tooth is a functional unit that is formed by induction of the developmental process of the fetal period and is constructed by a plurality of cell types, and is considered to be the same as an organ. Therefore, teeth are not generated by a stem cell system in which cell types are generated from stem cells such as hematopoietic stem cells and mesenchymal stem cells in adults, but only by stem cell transfer currently being promoted by regenerative medicine (stem cell transfer therapy) ) Cannot regenerate teeth. In addition, it is also considered to identify genes that are specifically expressed during tooth development and artificially induce tooth germs to regenerate teeth. I can't guide you.
Therefore, in recent years, studies have been conducted focusing on tooth regeneration by reconstituting a tooth germ using isolated tooth germ cells and transplanting the reconstructed tooth germ.

For example, Non-Patent Document 1 discloses that cells such as epithelial cells and mesenchymal dental follicle cells isolated from a tooth germ are transplanted into a rat abdominal cavity together with a bioabsorbable carrier. It is disclosed that the tissue is regenerated.
Non-Patent Document 2 describes that a system capable of realizing an epithelial-mesenchymal interaction by passaged cultured cells is effective and co-culture with collagen gel is effective.
As a method for regenerating tooth germ, for example, Patent Document 1 describes culturing tooth germ cells in the presence of a physiologically active substance such as fibroblast growth factor. Patent Document 2 proposes culturing at least one kind of tooth germ cells and cells differentiable into these cells together with a carrier containing fibrin, where the carrier containing fibrin is It is described that a tooth having a specific shape is formed using a tooth germ having a desired shape.

In Patent Documents 3 and 4, a cell mixture of a dental germ containing mesenchymal cells derived from dental pulp that forms dentin and epithelial cells that contribute to enamel formation from the mandible of 6-month-old pig, A method is disclosed in which a biodegradable polymer comprising a glycolic acid-polyacetic acid copolymer is seeded on a solidified carrier (Scaffold), transplanted into an animal body, and a tooth is formed. Here, it is described that a carrier having a target shape of a tooth germ is used to form a “tooth” having a specific shape.
On the other hand, Patent Document 5 discloses a tooth regeneration method for treating a patient having a bone defect or damage. According to this method, after seeding mesenchymal cells on a polyglycolic acid mesh carrier, epithelial cells are overlaid with collagen or wrapped with an epithelial cell sheet to form bone. In Patent Document 5, a carrier is used to construct a bone shape.
J. Dent. Res., 2002, Vol.81 (10), pp.695-700 "Regenerative medicine using cells derived from teeth and tooth germ and its potential", Regenerative medicine The Journal of Japanese Society for Regenerative Medicine, 2005, Vol.4 (1), pp.79-83 JP 2004-331557 A JP 2004-357567 A US Patent Application Publication No. 2002/0119180 US Patent Application Publication No. 2004/0219489 International Publication No. 2005/014070 Pamphlet

In any of the above techniques, the tooth germ is reconstructed using cells, cellular factors, etc., but it does not reproduce the characteristic cell arrangement and directionality that can express sufficient functions as a tooth. In addition, simply isolating and culturing a plurality of cells constituting a tissue makes it difficult to reconstruct a tissue having a specific cell arrangement.
The number of teeth required depends on the situation. Since it takes time to produce teeth by culturing, it is required from the viewpoint of efficiency to produce the target number from the beginning without excess or deficiency.

  An object of the present invention is to provide a method of manufacturing a tooth that can control the number of teeth to be manufactured and retain a specific cell arrangement, and an object thereof is to achieve the object.

Specific means for solving the above problems are as follows.
(1) A first preparation step for preparing a first cell aggregate substantially consisting of a mesenchymal cell derived from a tooth germ, and a second cell aggregate consisting essentially of an epithelial cell. a second preparation step, the inside of a support carrier gel, wherein the first cell mass, an arrangement step of arranging in contact with said second cell mass, the first and second Culturing a cell aggregate inside the support carrier to obtain a tooth as a dental aggregate composed of a plurality of teeth, and a cell aggregate consisting essentially of only the epithelial cells, This is a method for manufacturing a tooth, which includes cell regions constituting an enamel nodule in the same number as the target number of teeth.
(2) a pre-Symbol method for producing a tooth according to (1) at least one of the first cell mass and the second cell mass is characterized in that it is a single cell assemblage.
( 3 ) The method for producing teeth according to (1) or ( 2) , wherein the culturing step is performed in the presence of another animal cell.
( 4 ) The tooth manufacturing method according to any one of (1) to ( 3 ), wherein the culturing step is continued until periodontal tissue is formed.
( 5 ) The cell aggregate substantially consisting only of the epithelial cells further comprises a Shh expression region composed of cells expressing a sonic hedgehog gene (hereinafter abbreviated as Shh) (1 ) To ( 4 ).
( 6 ) The method for producing a tooth according to ( 5 ), wherein the number of the sonic hedgehog gene expression regions is determined according to a target tooth form.
( 7 ) The method for producing a tooth according to ( 5 ) or ( 6 ) above, wherein the sonic hedgehog gene-expressing cell is an epithelial cell.

  ADVANTAGE OF THE INVENTION According to this invention, the number of the teeth to manufacture can be controlled and the manufacturing method of the tooth which hold | maintained specific cell arrangement | positioning can be provided.

The method for producing a tooth of the present invention comprises a first cell aggregate substantially consisting of only one of mesenchymal cells and epithelial cells, at least one of which is derived from a tooth germ, inside a support carrier. A placement step of placing the second cell aggregate substantially consisting of only the other in contact with each other, and a culture step of culturing the first and second cell aggregates inside the support carrier. In addition, the cell aggregate that is substantially composed only of the epithelial cells (hereinafter referred to as epithelial cell aggregates) includes cell regions constituting the enamel nodules in the same number as the target number of teeth.
The close contact state between the first and second cell aggregates can effectively reproduce cell-cell interactions, including dentin on the inside, enamel on the outside, crown, tooth root, pulp, periodontal tissue, etc. Teeth having a layer structure unique to the teeth can be produced.

  In the present invention, the “enamel nodule” means a cell population that is thickened from the peripheral portion in the central portion of the inner enamel epithelium at the interface with the mesenchymal tissue among the epithelial tissue constituting the tooth germ. The enamel nodule is a “signal center” that is a cell population that regulates the development and differentiation of organs by organizing itself and surrounding tissues, and expresses a plurality of genes such as Shh, p21, Wnt10b, and Edar. It is characterized by having.

Since the enamel nodule is a signal center for induction of tooth formation, a cell aggregate consisting essentially of epithelial cells containing the same number of teeth as the target cell region, and a mesenchyme A target is obtained by forming a reconstructed tooth germ by contacting and arranging a cell aggregate that is substantially composed of only a systemic cell (hereinafter referred to as a mesenchymal cell aggregate), and then culturing the reconstructed tooth germ. A number of teeth can be manufactured.
Since the number of teeth to be manufactured can be controlled, teeth can be manufactured efficiently. In addition, it is easy to select the size of the support carrier suitable for the manufacture of teeth, the number of mesenchymal cells to be used, or the number of cells that can differentiate into tooth germ mesenchymal tissue.

  Further, the epithelial cell aggregate may include a cell region constituting an enamel nodule and a Shh expression region composed of a sonic hedgehog gene (hereinafter, Shh) -expressing cell. Since the epithelial cell aggregate includes a cell region constituting an enamel nodule and a Shh expression region, the number of teeth to be prepared and the shape of the teeth can be arbitrarily controlled. The shape of the teeth is greatly different between incisors and molars, and in addition to having a characteristic cell arrangement as a tooth, it is significant that a tooth having the required shape can be produced.

The “tooth” in the present invention refers to a tissue having a dentin layer on the inner side and an enamel layer on the outer side, particularly a tissue having directionality 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. The number of crowns varies depending on the form of the teeth, and in molars, a plurality of crowns are fused to form a whole tooth.
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 expression of amelogenin or its gene. On the other hand, dentin can be identified by the presence of odontoblasts, and the presence of dentin blasts can be confirmed by the presence or absence of the expression of dentin sialoprotein or its gene. 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 the present invention, “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.
In the present invention, “mesenchymal cell” means a cell derived from mesenchymal tissue, and “epithelial cell” means a cell derived from epithelial tissue.

In the present invention, “tooth germ” and “tooth germ” are expressions used when particularly referring to those distinguished based on the developmental stage described later. The “tooth germ” in this case is an early embryo of a tooth 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), and is a tissue in which accumulation of dentin and enamel, which is a characteristic of the hard tissue of the tooth, is not observed. The term “dental bud” refers to the development of the stage of “dental germ” used in the present invention. It refers to the tissue at the stage before the function as a tooth is developed.
As shown in FIG. 1, the tooth germ is performed through each stage of the rod-like stage, the cap-like stage, the bell-like early stage, and the late stage in the process of ontogeny. Here, in the rod-like phase, epithelial cells invaginate so as to wrap mesenchymal cells (see FIGS. 1 (A) and (B)). Becomes the outer enamel, and the mesenchymal cell part forms dentin inside (see FIGS. 1C and 1D). Therefore, a tooth germ is formed by cell-cell interaction between epithelial cells and mesenchymal cells.
The mesenchymal cells and epithelial cells in the present invention may be those from the above rod-like stage to the bell-like stage (hereinafter simply referred to as “dental germ”) that form or possibly form tooth germs. From the viewpoint of juvenileity and homogeneity in the differentiation stage of the cell, those from the cocoon stage to the cap stage are preferable.

  The “cell aggregate” refers to a state where cells are densely packed, and may be a tissue state or a single cell state. Further, “substantially” means containing as little as possible cells other than the target cells. Since each cell aggregate can be a tissue itself or a part thereof, or an aggregate of a single cell, either one may be a cell aggregate composed of a single cell, both of which are simple. A cell aggregate composed of one cell may be used, but in order to efficiently achieve tissue reconstitution according to the present invention, both are preferably composed of a single cell.

In the present invention, the number of cells constituting the first and second cell aggregates varies depending on the type of animal, the type of support carrier, hardness, size, and the number and form of teeth to be produced. aggregates per generally 10 ¹ to 10 ^8, preferably to ¹⁰ 3 to 10 ^8.

In the arrangement step in the production method of the present invention, the first cell aggregate and the second cell aggregate are placed in contact with each other inside the support carrier.
In the arrangement step, the first and second cell aggregates are arranged inside a support carrier capable of maintaining the contact state of the cells, so that the cells constituting each cell aggregate are another cell aggregate. It does not mix with the cells that make up. Thus, in the arrangement step, the cell aggregates are arranged without being mixed, so that a boundary line is formed between the cell aggregates. Such an arrangement is appropriately expressed as “partitioning” in this specification.

Here, the epithelial cell assembly and the mesenchymal cell assembly are separated from each other in a separate preparation step (the first cell preparation step and the second cell preparation step) so as to be substantially composed of mesenchymal cells and epithelial cells. The cell preparation step) is preferably prepared.
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 can 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 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.
In addition, 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.

  The tooth germ and other tissues used in the present invention include mammalian primates such as humans and monkeys, ungulates such as pigs, cows and horses, and small mammal rodents such as mice, rats and rabbits. From various animal jaw bones. To collect tooth germs and other tissues, the conditions used for the collection of the tissues may be applied as they are. They may be taken out aseptically and stored in an appropriate preservation solution or cultured. Examples of human tooth germs include third molars, so-called wisdom tooth germs, deciduous teeth, and fetal tooth germs. From the viewpoint of use of autologous tissues, it is preferable to use wisdom tooth germs. . Other tissues can be collected from, for example, the mucous membrane and tongue in the oral cavity, gums, bone marrow, skin, placenta, amniotic membrane, fetal tissue, and the like.

The preparation of mesenchymal and epithelial cells from this tooth germ or other tissue involves first dividing the tooth germ or other tissue isolated from the surrounding tissue into mesenchymal tissue and epithelial tissue according to shape. Done.
At this time, since the tooth germ tissue can be structurally distinguished under a microscope, it can be easily separated by cutting or peeling off with scissors for anatomy or tweezers. Further, the separation of the tooth germ mesenchymal tissue and the tooth germ epithelial tissue from the tooth germ tissue can be easily performed by cutting or peeling with an injection needle, a tungsten needle, tweezers or the like according to the shape. It can carry out similarly to a tooth germ tissue also in tissues other than a tooth germ.

In addition, the cell region constituting the enamel nodule can be easily separated from the tooth germ epithelial tissue by cutting or peeling it with an injection needle, tungsten needle, tweezers or the like according to its shape.
Preferably, enzymes may be used to easily separate tooth germ cells from surrounding tissues and / or to separate epithelial and mesenchymal tissues from tooth germ tissues. Enzymes used for such applications include dispase, collagenase, trypsin and the like.

  Mesenchymal cells and epithelial cells may be prepared from mesenchymal tissue and epithelial tissue to a single cell state, respectively. In the preparation process, an enzyme may be used so that it can be easily dispersed in a single cell. Examples of such enzymes include dispase, collagenase, trypsin and the like. For separation of mesenchymal cells from mesenchymal tissue, it is preferable to treat with collagenase and trypsin at the same time, and finally treat with DNase. At this time, DNase treatment is carried out because some cells are damaged by the enzyme treatment, and the cells are aggregated by the DNA released into the solution when the cell membrane is dissolved, resulting in a decrease in the amount of collected cells. This is to prevent it.

  The epithelial cell aggregate preparation method according to the present invention includes (1) a method in which a tooth germ epithelial tissue including a cell region constituting an enamel nodule is used as it is, and (2) a tooth germ epithelial tissue that constitutes an enamel nodule. (3) a tooth germ epithelial tissue not containing an enamel nodule or a cell derived from the tooth germ epithelial tissue and a cell region constituting the enamel nodule or the cell region A method of coexisting with a cell aggregate composed of cells derived therefrom; (4) comprising an epithelial cell other than a tooth germ that can differentiate into a tooth germ, and a cell region constituting an enamel nodule or a cell derived from the cell region Examples thereof include a method of allowing a cell aggregate to coexist. Here, in the methods (3) and (4), cell aggregates composed of cell regions constituting enamel nodules or cells derived from the cell regions are allowed to coexist in a number corresponding to the number of target teeth. You can also.

  In particular, from the viewpoint of surely controlling the number of teeth to be produced, the methods (1), (3) and (4) are preferable. In the methods (3) and (4), the cells in the enamel nodule are formed. A method using a cell aggregate composed of cells derived therefrom is more preferable.

  In the present invention, the cells constituting the cell aggregate may have undergone preliminary culture prior to the placement step in order to obtain a sufficient number of cells. For the culture of mesenchymal cells and epithelial cells, conditions such as temperature generally used for culturing animal cells can be used as they are.

As 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. As an alternative, for example, cell growth factors such as FGF, EGF, and PDGF, and known serum components such as transferrin may be added. In addition, although the density | concentration in the case of adding serum can be suitably changed with the culture state at that time, it can usually be 10%. For cell culture, normal culture conditions, for example, culture in an incubator with a temperature of 37 ° C. and a concentration of 5% CO ₂ are applied. Moreover, what added antibiotics, such as streptomycin, may be used suitably.

In the present invention, it is preferable that the epithelial cell aggregate further includes a Shh expression region composed of Shh expressing cells.
In the present invention, it is preferable that the number of the Shh expression regions is determined according to the target tooth form.

  The epithelial cell aggregate further includes a Shh expression region in addition to the cell region constituting the enamel nodule, thereby inducing the formation of a new crown by the Shh expression region on the side having the enamel and the dentin. Can do. Furthermore, when the epithelial cell aggregate includes two or more Shh expression regions, formation of two or more crowns can be induced according to the number of Shh expression regions. That is, by setting the number of Shh expression regions included in the epithelial cell aggregate to an arbitrary number, teeth having an arbitrary number of dental crowns can be formed in the same number as the number of cell regions constituting the enamel nodule. Although the tooth form differs greatly between incisors and molars, it is possible to arbitrarily control the form of the teeth to be prepared by controlling the formation of the crown. Here, the Shh expression region includes a cell region constituting an enamel nodule expressing Shh.

  Furthermore, in the present invention, the Shh-expressing cell is preferably an epithelial cell. The epithelial cells are more preferably tooth germ-derived epithelial cells or epithelial cells that can differentiate into tooth germ. By using epithelial cells as Shh-expressing cells, the Shh-expressing cells themselves can differentiate into cells that form tooth germs, in addition to having the activity of controlling the number of dental crowns.

  Shh-produced protein is the most important morphogen in development and regulates the morphogenesis of most organ systems generated by limbs, cerebrospinal midline structure and epithelial-mesenchymal interactions, and epithelial cells in the early stage of organogenesis It is known to be expressed in The gene sequence of Shh in the mouse is available as Genbank NM009170.

  The Shh-expressing cells in the present invention can be prepared by methods known to those skilled in the art. For example, it can be prepared by incorporating a DNA fragment containing the full length of Shh into an appropriate viral vector and introducing it into a wide variety of cell types including epithelial cells. Since general cells do not express the Shh gene, an expression level that can be induced by introducing a Shh expression vector is sufficient as a Shh expression cell. In the Shh-expressing cell, when a gene in which the Shh gene and the green fluorescence protein (GFP) gene are fused is expressed in the cell and analyzed by a flow cytometer, compared to a cell not introduced with the Shh expression vector, The fluorescence intensity is preferably 100 times or more.

  Moreover, the expression level of Shh in a cell can be confirmed by calculating the expression level of m-RNA using a Shh specific primer pair. A publicly known thing can be used as a Shh specific primer pair.

In the present invention, the Shh expression region is more preferably contained in the epithelial cell aggregate as a cell aggregate composed of Shh expressing cells. Thereby, the form control of the tooth | gear formed can be performed more reliably. Cell aggregates of Shh-expressing cells can be prepared by aggregating Shh-expressing cells by centrifugation. Alternatively, a suspension of Shh-expressing cells can be dropped on the surface of the culture plate, and the culture plate can be turned upside down. It can also be prepared as a cell mass by the hanging drop method of culturing. It can also be produced by injecting a cell suspension into a solidifiable carrier. Since it is preferable to control the cell aggregates of Shh-expressing cells at a high density and an arbitrary size to be used for reconstructing a tooth germ, it is preferable to prepare by a method in which carrier components and the like do not adhere to the outside of the cell aggregates. It is preferable to prepare a cell aggregate by the hanging drop method.
In the present invention, the number of cells constituting the cell aggregate of Shh-expressing cells varies depending on the size and the number and shape of teeth to be prepared, but generally 1 to 10 ⁸ cells, preferably 1 cell aggregate. The number can be 10 ¹ to 10 ⁵ .

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. As such a support carrier, collagen, fibrin, laminin, extracellular matrix mixture, polyglycolic acid (PGA), polylactic acid (PLA), lactic acid / glycolic acid copolymer (PLGA), cell matrix (trade name), Examples include meviol gel (trade name) and matrigel (trade name). 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. Here, 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. For example, in the case of collagen, use at a final concentration of 2.4 mg / ml provides adequate hardness.
Here, 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. .

In addition, the support carrier may be any cell as long as it can maintain the contact state of the cells. The term “contact state” as used herein refers to a cell interaction in each cell assembly and between cell assemblies. A high density state is preferable.
The high density state means that the density is equivalent to the density at the time of composing the tissue. For example, in the case of a cell aggregate, 5 × 10 ⁷ to 1 × 10 ⁹ cells / ml at the time of cell placement, In order to ensure cell interaction without impairing the activity, the density is preferably 1 × 10 ⁸ to 1 × 10 ⁹ cells / ml, most preferably 2 × 10 ⁸ to 8 × 10 ⁸ cells / ml. In order to prepare a cell aggregate at such a cell density, it is preferable to aggregate and precipitate cells by centrifugation because the density can be easily increased without impairing the cell 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. .

  When preparing high-density cells by centrifugation, 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 container such as a tube used at this time is preferably silicone-coated from the viewpoint of completely removing the supernatant.

  In the case of a precipitate by centrifugation, the precipitate may be placed inside the support carrier as it is. At this time, it is preferable that components other than the target cells (for example, a culture solution, a buffer solution, a support carrier, etc.) are equal to or less than the volume of the cells and do not contain components other than the target cells. Most preferred. In such a high-density cell assembly, cells are in close contact with each other, and cell-cell interaction is effectively exhibited.

  When used in a tissue state, it is preferable to remove the connective tissue other than the target cells by performing an enzyme treatment or the like. When there are many components other than the target cell, for example, when the amount is equal to or greater than the volume of the cell, cell-cell interaction is not sufficiently exhibited, which is not preferable.

  Further, it is preferable that 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. In addition, wrapping the surroundings of the first cell aggregate and the second cell aggregate with a culture medium or a solid material that does not inhibit oxygen permeation is also effective for bringing the cell aggregates into close contact with each other, and viscosity It is also preferable to place a high-density cell suspension in different solutions and solidify the solution as it is because the contact of cells can be easily maintained.

  When the support carrier is in the form of a gel or a solution, a solidification step for solidifying the support carrier may be provided after the arrangement step. By the solidification step, the cells arranged inside the support carrier are fixed inside the support carrier. For solidification of the support carrier, generally used solidification conditions for the support carrier may be applied as they are. For example, when a solidifiable compound such as collagen is 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.

In the culturing step in the production method of the present invention, the first cell aggregate and the second cell aggregate are cultured inside the support carrier. In this culturing step, cell-cell interaction is effectively performed by the first cell aggregate and the second cell aggregate in close contact with each other, and the tissue, that is, the tooth is reconstructed.
The culturing step may be performed by maintaining the contact state between the first cell aggregate and the second cell aggregate by the support carrier, and the culture step is performed by culturing with the support carrier alone having the first and second cell aggregates. Alternatively, it may be cultured in the presence of other animal cells.
The culture period varies depending on the number of cells arranged inside the support carrier and the state of the cell aggregate, and further the conditions of the culture process, but generally has an enamel on the outside for 1 to 300 days. In order to form the teeth on the inside, it is preferably 1 to 120 days, and preferably 1 to 60 days from the viewpoint of enabling rapid provision. Furthermore, in order to obtain a tooth having a periodontal tissue, it is generally 1 to 300 days, preferably 1 to 60 days.

When the culture is carried out using only the support carrier, the culture can be carried out under the usual conditions used for culturing animal cells. For the culture here, generally, the culture conditions in animal cells may be applied as they are, and the conditions described above can be applied as they are. Moreover, 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.
Moreover, it is preferable to use organ culture from the viewpoint of gas exchange and nutrient supply of tissues and cell aggregates. In organ culture, generally, a porous membrane is floated on a medium suitable for the growth of animal cells, and a cell aggregate embedded with a support carrier is placed on the membrane to perform culture. The porous membrane used here is preferably a membrane having a large number of pores of about 0.3 to 5 μm, and specifically includes cell culture inserts (trade names) and isopore filters (trade names). be able to.

  In the case of culturing in the presence of other animal cells, teeth having a specific cell arrangement can be formed at an early stage under the action of various cytokines from animal cells. Such culture in the presence of other animal cells may be performed by in vitro culture using isolated cells or cultured cells.

In addition, it is particularly preferable that the support carrier having the first and second cell aggregates is transplanted into a living body and cultured in the living body because teeth and / or periodontal tissue can be formed at an early stage. . In this case, the first and second cell aggregates are transplanted into the living body together with the support carrier.
Preferred examples of animals that can be used for this purpose include mammals such as humans, pigs, mice, and the like, and it is more preferable that they are derived from the same species as the tooth germ tissue. When transplanting human tooth germ tissue, it is preferable to use humans or mammals other than humans modified to immunodeficiency. In order to generate animal cell organs and tissues as normally as possible, such biological sites suitable for in vivo growth are preferably under the kidney capsule, mesentery, subcutaneous transplantation, and the like.
The growth period by transplantation varies depending on the size at the time of transplantation and the size of the teeth to be generated, but can generally be 3 to 400 days. For example, the transplantation period under the kidney capsule varies depending on the size of the culture to be transplanted and the size of the tooth to be prepared, but is 7 to 60 days from the viewpoint of the preparation of the tooth and the size of the tooth generated at the transplant destination. It is preferable.

Prior to transplantation into a living body, in vitro culture (pre-culture) may be performed. This pre-culture is preferable because the binding between cells and the binding between the first and second cell aggregates can be strengthened and the interaction between cells can be further strengthened. As a result, the overall growth period can be shortened.
The preculture period may be short or long. When the period is long, for example, 3 days or more, preferably 7 days or more, it is possible to shift from the tooth germ to the tooth bud, and as a result, the period until the teeth are formed after transplantation can be shortened. preferable. The pre-culture period is preferably 1 to 7 days, for example, for organ culture when transplanting under the kidney capsule, in order to efficiently regenerate teeth.

  The tooth manufactured by the manufacturing method of the present invention has a specific cell arrangement (structure) as a dentine inside and an enamel outside, and preferably further, the tip of the tooth (crown) It also has the direction of tooth root. By having directionality in addition to at least such a specific cell arrangement, preferably cell arrangement, it can also function as a tooth. For this reason, it can be widely used as a substitute for teeth. In particular, when mesenchymal cells and epithelial cells derived from autologous tooth germs and tissues are used, they can be used while avoiding problems due to rejection. Moreover, it is possible to avoid the problem due to rejection even when using cells derived from another person's tooth germ, which are generally compatible with the transplantation antigen.

  Furthermore, in the present invention, by extending the culture period, in addition to the teeth themselves, periodontal tissues such as alveolar bone and periodontal ligament can be formed to support and fix the teeth on the jawbone. As a result, it is possible to provide a usable tooth after transplantation.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. Moreover,% in an Example is a mass reference | standard unless there is particular notice.

[Reference example]
(Preparation of tooth germ mesenchymal cells and tooth germ epithelial cells)
To perform tooth formation, tooth germs were reconstructed. A mouse was used as this experimental model.
C57BL / 6N mice (purchased from Clea Japan) were born at 14.5 days, and the mandibular incisor tooth tissue was removed from the embryo by a conventional method under a microscope. Mandibular incisor 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 in PBS (−) After treatment with an enzyme solution supplemented with 12.5 minutes at room temperature, the plate was 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 the tooth germ epithelial tissue was surgically treated with a 25 G injection needle (Terumo, Tokyo, Japan). Tooth germ mesenchymal tissue was isolated.

  Tooth germ mesenchymal cells were obtained by washing the tooth germ mesenchymal tissue obtained above three times with PBS (−) and containing 0.25% trypsin (Sigma) and 50 U / ml collagenase I (Worthington). Treated with PBS (−) at 37 ° C. for 10 minutes. After washing the cells three times with DMEM supplemented with 10% FCS, DNase I (Takara) at a final concentration of 70 U / ml was added to the cells, and single tooth germ mesenchymal cells were obtained by pipetting.

  On the other hand, for tooth germ epithelial cells, the tooth germ epithelial tissue obtained as described above was washed three times with PBS (−), and collagenase I (Worthington, Lakewood, NJ) at a final concentration of 100 U / ml in PBS (−). The treatment for 20 minutes at 37 ° C. with the enzyme solution in which was dissolved 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 having a final concentration of 70 U / ml was added to the cells, and single tooth germ epithelial cells were obtained by pipetting.

(Preparation of reconstructed tooth germ)
Place tooth germ epithelial cells suspended in DMEM (Sigma) supplemented with 10% FCS (JRH Biosciences) into a 1.5 mL microtube (Eppendorf, Hamburg, Germany) coated with silicone grease, and centrifuge (580 × g) To collect the cells as a precipitate. 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 GELoader Tip 0.5-20 μL (eppendorf) while observing with a stereomicroscope. The cells used for the preparation of reconstructed tooth germs were prepared.

(Preparation of reconstructed tooth germ)
Next, 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 Biosciences) in a 1.5 mL microtube (Eppendorf, Hamburg, Germany) coated with silicone grease. The cells were collected as a precipitate by centrifugation (580 × g). Remove the supernatant of the culture solution after centrifugation as much as possible, perform the centrifugation again, and completely observe the culture solution remaining around the cell precipitate using GELoader Tip 0.5-20 μL (eppendorf) while observing with a stereomicroscope. The cells used for the preparation of reconstructed tooth germs were prepared.
30 μL of Cellmatrix type IA (Nitta gelatin, Osaka, Japan) prepared in the above culture solution at a concentration of 2.4 mg / ml was dropped onto a Petri dish coated with silicon grease to produce a collagen gel solution drop (gel drop). did. To this solution, 0.2 to 0.3 μL of the precipitate after centrifugation of the tooth germ mesenchymal cells was applied using a 0.1 to 10 μL pipette tip (Quality Scientific plastics) to obtain a cell aggregate. Cell aggregates were made. Next, the tooth germ epithelial cells are applied by the same method so as to be in contact with the previously produced tooth germ mesenchymal cell clumps, and the cell clumps are made in close contact with each other. A component tooth germ was prepared.

This will be described with reference to FIG.
The cell aggregate 12 previously arranged in the gel drop 10 with the pipette tip 16 constitutes a sphere in the gel drop 10 (see FIG. 2B). By pushing the other cell aggregate 14 after this, the spherical cell aggregate 12 is often crushed and wraps around the other cell aggregate 14 (see FIG. 2C). Thereafter, the gel drop 10 is solidified, thereby strengthening the bond between cells (see FIG. 2D).

(Culture of reconstructed tooth germ)
The reconstructed tooth germ prepared in the gel drop 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) in DMEM (Sigma) supplemented with 10% FCS (JRH). Is transferred onto the membrane of the cell culture insert of the culture vessel set so that the 0.4 μm PET membrane (BD, Franklin Lakes, NJ) is in contact with the surrounding gel as a support carrier. Organ culture was performed for 24 hours. After culturing, the surrounding gel was transplanted under an 8 week-old C57BL / 6 renal capsule to develop ectopic teeth, and teeth were prepared.

(Histological analysis)
On day 10 after transplantation, the reconstructed tooth germ was removed together with the surrounding kidney tissue, fixed with 4% paraformaldehyde-phosphate buffer for 6 hours, and then removed with 4.5% EDTA (pH 7.2) for 24 hours. Ashed, embedded in paraffin by a conventional method, and sectioned at a thickness of 10 μm. Each section was stained with hematoxylin-eosin according to a conventional method for histological analysis. The results are shown in FIG.

  As shown in FIG. 3, the mesenchymal cell aggregate and the epithelial cell aggregate were partitioned and brought into close contact with each other to have a specific cell arrangement having dentin on the inside and enamel on the outside. It can be seen that four teeth are formed.

[Example 1]
(Preparation of reconstructed tooth germ)
In the same manner as in the Reference Example, using the prepared tooth germ epithelial tissue and tooth germ mesenchymal cells, the epithelial tissue separated from one tooth germ (including one enamel nodule) and the other tooth germ Using the separated mesenchymal tissue-derived cell aggregates, tooth germ reconstruction was performed in the same manner as in the reference example. In particular, the tissue interface side in the tooth germ of the epithelial tissue was brought into close contact with the cell aggregate using a tungsten needle.
The reconstructed tooth germ was cultured in the same manner as in the reference example, and histological analysis was performed. In the histological analysis, the entire region of all section samples was photographed, and the tissue structure was reconstructed three-dimensionally and observed using image analysis software (imaris, manufactured by Zeiss). The results are shown in FIG.

  As shown in FIG. 4, by providing a close contact between the mesenchymal cell aggregate and the epithelial tissue including enamel nodules, a tooth having a specific cell arrangement having dentin on the inside and enamel on the outside is formed. I understand that. At this time, since the epithelial tissue contains one cell region constituting the enamel nodule, one tooth could be produced.

[Example 2]
In preparation of reconstructed tooth germ, teeth were prepared in the same manner as in Example 1 except that two epithelial tissues containing enamel nodules were used and each epithelial tissue was closely attached to one mesenchymal cell aggregate. And histological analysis was performed. Each epithelial tissue was separated from a separate tooth germ. The results are shown in FIG.

  As shown in FIG. 5, it can be seen that two teeth can be produced by placing and culturing two epithelial tissues including enamel nodules in contact with a cell assembly composed of mesenchymal cells derived from tooth germ. That is, it can be seen that the number of teeth corresponding to the number of epithelial tissues including enamel nodules can be produced.

[Example 3]
In the production of the reconstructed tooth germ, the same procedure as in Example 1 was used, except that two partial tissues obtained by removing peripheral tissues other than the enamel nodules from the tooth germ epithelial tissue were used instead of the epithelial tissue separated from the tooth germ. Teeth were manufactured and histologically analyzed. The two partial tissues were prepared by surgically removing regions other than the thickened enamel nodules from the tooth germ epithelial tissues including enamel nodules separated from separate tooth germ tissues. The results are shown in FIG.

  As shown in FIG. 6, by arranging and culturing two partial tissues obtained by removing peripheral tissues other than enamel nodules from tooth germ epithelial tissue in a cell aggregate consisting of mesenchymal cells derived from tooth germ, It can be seen that teeth having a specific cell arrangement with dentin on the inside and enamel on the outside are formed. Moreover, it turns out that two teeth can be produced according to the number of enamel nodules arranged in contact.

[Example 4]
(Preparation of Shh-expressing cell mass)
By a conventional method, Mouse Shh cDNA clone (purchased from Mouse IMAGE cDNA Clones, EMM1002, Open Biosystems) was treated with restriction enzymes (EcoRI and NotI) to obtain a DNA fragment (SEQ ID NO: 1) containing the full length of Shh. This was subcloned into pMXs-IG vector (Reference: Exp. Hematol., 2003, Vol.31, pp.1007-1014) treated with the same restriction enzyme. Using FuGENE (trade name, manufactured by Roche), the resulting vector was transferred to PLAT-E cells (reference document; Exp. Hematol., 2003, Vol.31, pp.1007-1014) using the method specified by the manufacturer. And introduced. PLAT-E was cultured in a medium in which 1 μg / mL Puromycin (Sigma) and 10 μg / mL Blastcidine (Invitrogen) were added to DMEM supplemented with 10% FCS. Using the culture supernatant of PLAT-E, the vector was introduced into a cell line established from embryonic molar molar epithelial cells of C57BL / 6N mice (purchased from Japan SLC) at the embryonic age of 18.5 days did. Shh-expressing cells were obtained from the cell line into which the vector was introduced using a cell sorter EPICS ALTRA (Beckman, Fullerton, CA, USA).
About the obtained Shh expression cell, it confirmed that Shh was expressed using the Shh specific primer pair (Sense primer: Sequence number 2, Antisense primer: Sequence number 3) shown in following Table 1.

  Next, a cell suspension of Shh-expressing cells was prepared in a medium in which 10 μg / mL insulin and 10 μg / mL transferrin (Sigma) were added to DMEM / F12 (Sigma) supplemented with 10% FCS, and a concentration of 300 cells / 20 μL was prepared. 20 μL of the cell suspension was dropped onto the bottom of a 24-well culture plate bordered with silicon grease. The plate was turned upside down and cultured for 2 days (hanging drop method) to prepare a Shh-expressing cell mass composed of 300 cells.

(Preparation of reconstructed tooth germ)
The Shh-expressing cell mass was pressed onto a cell aggregate (gelatin-derived mesenchymal cell aggregate) in a gel drop prepared in the same manner as in Example 1. Furthermore, after transferring only one epithelial tissue containing the enamel nodule into the gel drop so that the Shh-expressing cell mass and the enamel nodule do not overlap therewith, the tissue interface side in the tooth germ of the tissue is used with a tungsten needle. Thus, a reconstructed tooth germ was prepared by closely adhering to the cell aggregate.

  In the same manner as in Example 1, the reconstructed tooth germ was cultured, teeth were prepared, and histological analysis was performed. The results are shown in FIG.

As shown in FIG. 7, in addition to the cell assembly consisting of enamel nodules in the mesenchymal cell aggregate, in addition to the contact portion of the enamel nodule by culturing the Shh expressing cell aggregate in contact, It can be seen that crown formation is also induced at the contact portion of the Shh-expressing cell aggregate. That is, in the present invention, it can be seen that the shape of the tooth to be manufactured can be controlled. Also in this case, it can be seen that the number of teeth corresponding to the number of enamel nodules is formed.

[Comparative Example 1]
In the preparation of the reconstructed tooth germ in Example 1, two partial tissues composed of tooth germ-derived epithelial cells from which enamel nodules were surgically removed were used instead of the partial tissues composed of epithelial cells including enamel nodules. Then, teeth were prepared and analyzed histologically in the same manner as in Example 1 except that each partial tissue was brought into close contact with the mesenchymal cell aggregate so as not to contact each other. The results are shown in FIG.

  As shown in FIG. 8, even when cells composed of tooth germ-derived epithelial cells from which enamel nodules have been removed are placed in contact with cell aggregates composed of mesenchymal cells derived from tooth germs, induction of tooth formation occurs. I understand that there is no.

A conceptual diagram (FIG. 9) schematically showing the above results will be described.
One tooth is formed from one epithelial tissue including a cell region constituting an enamel nodule and a mesenchymal cell aggregate derived from a tooth germ (FIG. 9A, Example 1). Further, two teeth are formed by using two epithelial tissues including cell regions constituting enamel nodules (FIG. 9B, Example 2). Further, when enamel nodules are removed from the epithelial tissue derived from tooth germ, teeth are not formed (FIG. 9C, Comparative Example 1). Also, when only two partial tissues obtained by removing peripheral tissues other than enamel nodules from the tooth germ epithelial tissue are used, two teeth are formed (FIG. 9D, Example 3). Furthermore, in addition to one tooth germ-derived epithelial tissue including a cell region constituting an enamel nodule, an Shh-expressing cell aggregate is used to form one tooth in which two crowns are induced ( FIG. 9E, Example 4).

  Based on the above, cell aggregates consisting of cell regions constituting enamel nodules are essential for induction of tooth formation, and the number of cell aggregates consisting of cell regions constituting enamel nodules placed in contact with mesenchymal cell aggregates It can be seen that the number of teeth corresponding to the number can be produced.

It is the conceptual diagram which represented formation of the tooth germ typically. (A)-(D) are the figures which showed notionally the procedure of tooth germ reconstruction using the tooth germ origin mesenchymal cell and epithelial cell concerning the reference example of this invention. It is a cross-section dyeing | staining image which concerns on the reference example of this invention. It is the cross-section dyeing | staining image which image | photographed the tooth | gear obtained by Example 1 of this invention in three different cross sections. It is a cross-section dyeing | staining image which image | photographed the tooth | gear obtained by Example 2 of this invention in three different cross sections. It is the cross-section dyeing | staining image which image | photographed the tooth | gear obtained by Example 3 of this invention in three different cross sections. It is the cross-section dyeing | staining image which image | photographed the tooth | gear obtained by Example 4 of this invention in two different cross sections. It is a cross-section dyeing | staining image of the reconstructed tooth germ after the culture | cultivation obtained by the comparative example 1 of this invention. (A)-(D) are the conceptual diagrams which show the presence or absence of the cell area | region which comprises an enamel nodule, and a Shh expression area | region and tooth | gear formation, respectively.

Explanation of symbols

10 Gel pack (support carrier)
12 Cell aggregate (first cell aggregate)
14 Cell aggregate (second cell aggregate)
16 Pipette tips

Claims (7)

A first preparation step of preparing a first cell aggregate substantially consisting of mesenchymal cells derived from a tooth germ, and a second preparing a second cell aggregate substantially consisting of epithelial cells A preparation process;
Inside of a support carrier gel, and disposing step of placing the a first cell mass, contacting the second cell mass,
Culturing the first and second cell aggregates inside the support carrier to obtain teeth as a tooth aggregate composed of a plurality of teeth ,
A method for producing a tooth, wherein the cell aggregate substantially consisting only of the epithelial cells includes a cell region constituting an enamel nodule in the same number as a plurality of intended teeth. Method for producing a tooth according to claim 1, wherein at least one of the first cell mass and the second cell mass is a single cell assemblage. The method for producing teeth according to claim 1 or 2, wherein the culturing step is performed in the presence of another animal cell. The said culturing process is continued until periodontal tissue is formed, The manufacturing method of the tooth of any one of Claims 1-3 characterized by the above-mentioned. Cell aggregates consist essentially the epithelial cells, one of the claims 1 to 4, further comprising a sonic hedgehog gene expression area composed of sonic hedgehog gene expressing cells 1 The manufacturing method of the tooth according to item. 6. The method for manufacturing a tooth according to claim 5 , wherein the number of the sonic hedgehog gene expression regions is determined according to a target tooth shape. The method for producing a tooth according to claim 5 or 6, wherein the sonic hedgehog gene-expressing cell is an epithelial cell.