Method for tooth creation

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine and concerns methods for creation a tooth of a required size, and repair of the lost teeth in the oral cavity by transplantation of the created tooth into the dental loss. Particularly, the method for the tooth creation of a required length in one direction involves the stages: placing the first cell aggregate and the second cell aggregate in a tight contact inside a supporting carrier, wherein the first cell aggregate and the second cell aggregate respectively consists of mesenchymal or epithelial cells; culturing the first and second cell aggregate inside the supporting carrier; the tooth size is corrected by a contact length of the first cell aggregate and the second cell aggregate in the same pre-set direction. A method for determining the contact length of the first and second cell aggregate for the tooth preparation of the required size involves preparing a number of structural types containing structures of various contact length of the first and second cell aggregate in the same pre-set direction; culturing each of a number of structural types inside the supporting carrier; measuring the tooth length prepared at the previous stage in one direction; correlating the given length and contact length providing the basis for calculating the required contact length of the first cell aggregate and the second cell aggregate.

EFFECT: group of inventions enables creating the tooth of the required size that enables preparing a single tooth that can be used as it is in the form of a transplant.

23 cl, 1 tbl, 6 ex, 13 dwg

 

The technical field to which the invention relates.

The present invention relates to a method of creating a tooth having a desired size.

Prior art

The tooth is a body with enamel as outer layer and a hard tissue called dentin, within a given layer, and odontoblast form dentin on the inside with the pulp in the center. Teeth can be lost due to caries and periodontal disease, and due to the fact that the presence of teeth has a great impact on a person's appearance and taste of food, there is growing concern about the methods of reproduction of the teeth. In addition, concerns about the methods of reproduction of the teeth is also growing for the sake of, for example, the health and maintain a high quality of life.

The tooth is a functional unit which is formed by the induction of the formation during the embryonic period and formed by many types of cells. The tooth is not formed by the system of stem cells in which types of cells are formed from stem cells such as hematopoietic stem cells and mesenchymal stem cells in adults, and at the present time, therefore, the teeth can not be regenerated only by stem cell transplant, carried out the field of regenerative medicine. Although there have been investigated for the STI regeneration of teeth by identifying genes involved in the formation of teeth and artificial induction of early tooth development, full induction of regeneration of teeth can be achieved only by the identification of genes.

Therefore, in recent years there has been studied a method of obtaining a regenerated tooth by reconstructing early tooth development using selected tissues and cells derived from a tooth germ, followed by transplantation of the reconstructed tooth germ.

The authors of the present invention have found that the placement of the first cell mass and the second cell mass in contact with each other inside the support carrier, made of collagen gel, where the first cell mass formed mainly of mesenchymal any of epithelial cells derived from a tooth germ, and the second cell mass formed mainly from the remaining cell type, and then by culturing the first and second cell masses inside the support carrier can efficiently induce differentiation of cells, and may receive the regenerated tooth germ and the regenerated tooth, with the specific arrangement of cells and the orientation (for example, see Document 1 from the list Patent literature).

Moreover, the authors of the present invention showed that the regenerated tooth germ and the regen is new tooth, with a certain arrangement of cells and the orientation, in a similar way can be obtained even with the epithelial cells of the oral cavity at the first stage of their cultivation as epithelial cells (for example, see Document 2 from the list Patent literature), or can be obtained by using cells derived from amnion as mesenchymal cells (for example, see Document 3 from the list Patent literature), or obtained in other ways using cells obtained by the induction of differentiation of totipotent stem cells as mesenchymal cells (for example, see Document 3 from the list Patent literature).

Also in the regenerated tooth germ and the regenerated tooth size, tooth differs depending on its position, and this size also varies among individuals. Therefore, it is important to control the size for the regeneration of the tooth, suitable for putting in place a lost tooth. However, in the above documents, the method of controlling the size of the regenerated tooth was not considered. Moreover, the above method can also be obtained from the regenerated set of teeth. In this case, each tooth separated from the set and used as a graft, but likely experience difficulty with control of the number of teeth and size of each of the second tooth in the set due to the lack of development of the technology of three-dimensional operations with cells and insufficient understanding of the control mechanism forms in developmental biology.

Also as a way of creating a regenerated tooth having desired size and shape, it was suggested that the method of inoculation of cell mixture early tooth development, including mesenchymal cells derived from dental pulp, which form the dental papilla and dentin, as well as including epithelial cells, which are involved in the formation of enamel, frame, obtained by hardening the biodegradable polymer made from a copolymer polyglycolic acid and polylactic acid, followed by transplantation into the body of the animal for the formation of the tooth. In this way control the shape of the tooth tried to do it using the frame of the desired shape. However, the regenerated tooth derived from a tooth germ, consisting of a layer of epithelial cells and a layer of mesenchymal cells, and dental primordium, as is well known, grows in a certain chronology through epithelial-mesenchymal interaction that occurs between cells. Thus, when using the framework does not provide enough interaction between cells. Consequently, the use of the framework may not be preferred (for example, see Document 1 from the list of non-Patent literature). Moreover, the rate of formation of the tooth is greater than the speed of destruction of the framework. Therefore, the tooth may be formed with a certain part of the frame, note Sanogo to him, and it is likely that the reproducibility of the location of the cells and the shape of the tooth will be low.

On the other hand, it was considered that, in General, the normal form of the crown of the tooth can not be obtained up until mesenchymal tissue in the reconstructed tooth germ will not be perfect. However, it was reported that even if instead of mesenchymal tissue to apply a mass of mesenchymal cells (mass, obtained by centrifugation after the separation of the tissue enzyme treatment), if the number of large cells in in vitro culture receive relatively large size of the tooth germ and the number of tubercles crown of the tooth will be increased (for example, see Document 1 from the list of non-Patent literature). However, even after transplantation of this tooth germ inside of a living organism form of dental crowns and number of tubercles dental crowns are changed compared to a normal tooth and the tooth with the correct form does not work. In accordance with the message, the tooth with the correct form is not obtained even when reverse engineering using a combination of epithelial and mesenchymal cell mass. In accordance with these data when reconstructing using epithelial tissue and mesenchymal cell mass the correct form can be obtained if you are using mesenchymal tissue, however, the limitation, the loser is by the use of homogeneous mesenchymal cells during reception of the teeth, indicates that a partial solution is achieved by increasing the number of cells.

Moreover, it was shown that upon receipt of the reconstructed tooth germ from epithelial tissue and mesenchymal cell mass, an increase in the number of teeth is achieved by increasing the number of mesenchymal cells, but the size of the teeth, this condition is not valid (Document 2 from the list of non-Patent literature). In accordance with these data, the final size of the tooth and tubercle of the crown of the tooth is determined by internal factors of mesenchymal or epithelial tissue, i.e. it is concluded that mesenchymal and epithelial cells have internal memory with respect to the final size of the tooth and tubercle dental crowns, respectively.

In addition, the authors of the present invention proposed to apply for the education unit of epithelial cells is the number of epithelial tissues in the configuration area of the enamel knot, what is required for a desired number of teeth upon receipt of the reconstructed tooth germ with the unit of epithelial cells and aggregation of mesenchymal cells, as a way of controlling the number and shape of the resulting teeth (Document 5 from the list Patent literature). In accordance with the method can be obtained Assembly of the teeth having a third what has been created the number of teeth. However, for the formation of the field configuration enamel knot must be received so many epithelial tissues as corresponds to the number of teeth required. In addition, the paper 5 from the list Patent literature does not address the control of the size of the teeth.

The level of technology

Patent literature

Document Patent literature 1 - WO 2006/129672

Document Patent literature 2 - pending patent application of Japan No. 2008-29756

Document Patent literature 3 - pending patent application of Japan No. 2008-206500

Document Patent literature 4 - pending patent application of Japan No. 2008-200033

Document Patent literature 5 - pending patent application of Japan No. 2008-29757

Non-patent literature

Document non-Patent literature 1 - No et al. Tissue Engineering Volume 12, Number 8,2006,2069-2075

Document non-Patent literature 2-3. Cai et al. Developmental Biology 304 (2007) 499-507

Summary of the invention

Problems solved by the present invention

The size and shape of teeth that are appropriate for their functions depend on the area where they grew up, and depending on the plot size and shape vary even for the same posterior tooth. Furthermore, the size of the tooth varies from individual to individual. Therefore, when creating a regenerated tooth germ and regenerierung what about the tooth for the treatment of missing teeth, it is very important to control the size, so that the tooth could properly function in the individual, in which he transplanted.

In addition, in accordance with the experience of the authors in the size of the regenerated tooth germ may vary due to different conditions, and because of the "memory"characteristic of the cells do not get the teeth with good reproducibility, and the same size.

Therefore, the present invention is to provide a method of creating a tooth having a desired size, in particular of the tooth, in which the width of the tooth crown has the desired length.

Money solutions

As a result of intensive studies to resolve the above problems, the authors of the present invention have found that the width of the crown of the regenerated tooth depends on the length of contact in a given direction unit mesenchymal cells and Assembly of epithelial cells in supporting the media and does not depend on the number of each type of cells, and therefore, the width of the crown of the regenerated tooth can be controlled by adjusting the length of the contact. In addition, it was found that when forming unit mesenchymal cells and Assembly of epithelial cells, respectively, in the form of an almost columnar structure, and subsequent control of the length of the contact in the axial voltage is the making of the columnar structure can be formed tooth, having a desired length in one direction; specifically, when establishing the length of the contact within the range of plus/minus 25% (±25%) of the desired length can be obtained regenerated tooth having a desired length; together with control of the size of the tooth you can also control the amount of bumps dental crowns; and when establishing the length below the specified numeric values can be obtained from a single tooth and, accordingly, this is the essence of the present invention.

That is, the present invention relates to:

[1] the way to create a tooth having a desired length in one direction, including:

stage location of the first unit cell and the second unit cells in close contact inside a support carrier, where the first unit cell and the second unit cells respectively comprise either mesenchymal or from epithelial cells; and

the stage of culturing the first and second aggregates of cells inside the support carrier,

where the size of the tooth is adjusted by adjusting the length of contact of the first unit cell and the second unit cells in a predetermined one direction;

[2] the method of creating a tooth having a desired length in one direction, including:

a step for many types of structures in which the first unit of the glue is OK and second unit cells are arranged in close contact inside a support carrier, by changing the length of contact of the first unit cell and the second unit cells in one given direction, where the first unit cell and the second unit cells respectively comprise either mesenchymal or from epithelial cells;

the stage of culturing each of the many types of structures inside the support carrier;

stage length measurement of the tooth, obtained at the previous stage, in one direction, and determining the correlation between this length and the length of contact;

the stage is based on a correlation calculation of the length of contact of the first unit cell and the second unit cells, which are required for a tooth having a desired length in one direction.

stage location of the first unit cell and the second unit cells in close contact inside a support carrier so that they had calculated at the previous stage the length of the contact, where the first unit cell and the second unit cells respectively comprise either mesenchymal or from epithelial cells; and

the stage of culturing the first and second aggregates of cells inside the support carrier;

[3] the method of creating a tooth having a desired length in one direction, including:

a step for many types of structures in which the first unit tile is K and the second unit cell in the form of a nearly columnar structures are arranged in close contact inside a support carrier, so that the axial direction of each columnar structures are parallel, by changing the length of contact of the first unit cell and the second unit cell in the axial direction, where the first unit cell and the second unit cells respectively comprise either mesenchymal or from epithelial cells.

the stage of culturing each of the many types of structures inside the support carrier;

stage length measurement of the tooth, obtained at the previous stage, in one direction, and determining the correlation between this length and the length of contact;

the stage is based on a correlation calculation of the length of contact of the first unit cell and the second unit cells, which are required for a tooth having a desired length in one direction.

stage location of the first unit cell and the second unit cell in the form of a nearly columnar structure in close contact inside a support carrier, so that the contact length in the axial direction was the length calculated in the previous stage, and the axial direction of each of the columnar structures were parallel, where the first unit cell and the second unit cells respectively comprise either mesenchymal cells or from epithelial cells; and

the stage of culturing the first and second units to etoc inside the support carrier;

[4] the method of creation of a molar tooth having a desired length in mesio-distal and/or buccal-lingual direction, including:

a step for many types of structures, in which the first unit cell and the second unit cell in the form of a nearly columnar structures are arranged in close contact inside a support carrier so that the axial direction of each of the columnar structures were parallel, by changing the length of contact of the first unit cell and the second unit cell in the axial direction and/or length of contact in the direction perpendicular to the axis, where the first unit cell and the second unit cells respectively comprise either mesenchymal cells or from epithelial cells;

the stage of culturing each of the many types of structures inside the support carrier;

stage length measurement of a molar tooth, obtained at the previous stage, in mesio-distal direction and/or the buccal-lingual direction, with subsequent determination of the correlation between the contact length in the axial direction and length of a molar tooth in mesio-distal direction, and/or correlation between the contact length, perpendicular to the axis and length of a molar tooth in the buccal-lingual direction;

the stage is based on a correlation calculation of the length of contact of the first unit cells vtorogo unit cells in the axial direction and/or length of contact in the direction perpendicular to the axis, which is necessary to obtain the posterior tooth of the desired length in mesio-distal direction and/or in the buccal-lingual direction;

stage location of the first unit cell and the second unit cell in the form of a nearly columnar structure in close contact inside a support carrier, so that the contact length in the axial direction and/or the contact length in the direction perpendicular to the axis, was the length calculated in the previous stage and the axial direction of each of the columnar structures were parallel, where the first unit cell and the second unit cells respectively comprise either mesenchymal or from epithelial cells; and

the stage of culturing the first and second aggregates of cells inside the support carrier;

[5] the method of creating a tooth having a desired length in one direction, including:

stage location of the first unit cell and the second unit cell in the form of a nearly columnar structure in close contact inside a support carrier so that the axial direction of each of the columnar structures were parallel, whereby the contact length of the first unit cell and the second unit cell in the axial direction is within the range of plus/minus 25% of the required length, where the first EPR is gat cell and the second unit cells, respectively, consist of either mesenchymal, any of epithelial cells; and

the stage of culturing the first and second aggregates of cells inside the support carrier;

[6] the method according to the above [5], in which stage the location of the first and second cell aggregates inside the support carrier includes:

stage getting multiple structures, in which the first and second cell units are located inside the support carrier;

phase measurements of the length of contact of the first and second aggregates of cells in the axial direction and

stage selection structure in which the measured contact length is approximately within the range of plus/minus 25% of the required length.

[7] the method of creating a single tooth, including:

stage location of the first unit cell and the second unit cells in close contact inside a support carrier, where the first unit cell and the second unit cells respectively comprise either mesenchymal or from epithelial cells; and

the stage of culturing the first and second aggregates of cells inside the support carrier,

where the maximum contact length of the first unit cell and the second unit cell is equal to or less than the specified value.

[8] the method in accordance with any of the above [1]to[7], in which both unit cells are to locname masses;

[9] the method in accordance with any of the above [1]to[8], in which at least either mesenchymal cell or epithelial cell derived from a tooth germ;

[10] the method of restoring the missing parts of teeth in the oral cavity, including:

stage transplantation of tooth obtained by the method in accordance with any of the above [1]to[9], in the region of the loss of a tooth.

[11] the method according to above [10], in which the tooth obtained by the method in accordance with any of the above [1]to[9], transplanted as is in the area of the lost tooth without dividing the tooth into two or more parts;

[12] the method according to above [10] or [11], in which the mesenchymal cells and epithelial cell obtained from the individual, that part of the tooth was lost;

[13] the method in accordance with any of the above [10]-[12], in which the mouth is the mouth of memleketim;

[14] the design method of creating a tooth having a desired length in one direction, under specified conditions,

in which way design includes a method to determine the length of contact of both aggregates of cells in one given direction, which is required to obtain tooth that possesses the desired size, in conditions when the first choice of the unit cell and the second unit cells are arranged in close contact inside a support carrier and the first unit cell and the second unit cells respectively comprise either mesenchymal cells, any of epithelial cells, and

which method of determining the length of the contact further includes:

a step for many types of structures in which the first unit cell and the second unit cells are arranged in close contact inside a support carrier by changing the length of contact of the first unit cell and the second unit cells in one given direction, where the first unit cell and the second unit cells respectively comprise either mesenchymal or from epithelial cells;

the stage of culturing each of the many types of structures inside the support carrier;

stage length measurement of the tooth, obtained at the previous stage in one direction, and then determine the correlation between the contact length and the length of the tooth in one direction; and

the stage is based on a correlation calculation of the length of contact of the first unit cell and the second unit cells, which are required to produce tooth having a desired length in one direction.

[15] the design method of creating a single tooth under specified conditions

in which way design includes a method for determining the maximum length of contact of both cell aggregates, which are required for a single tooth, in conditions when the first unit cells of the second unit cells are arranged in close contact inside a support carrier and the first unit cell and the second unit cells respectively comprise either mesenchymal cells, any of epithelial cells, and

which method of determining the maximum length of the contact further includes:

a step for many types of structures in which the first unit cell and the second unit cells are arranged in close contact inside a support carrier by changing the maximum length of contact of the first and second aggregates of cells, where the first unit cell and the second unit cells respectively comprise either mesenchymal or from epithelial cells;

the stage of culturing each of the many types of structures inside the support carrier;

stage dimension number of teeth, obtained in the preceding stage, and determine the maximum length of contact of the first unit cell and the second unit cell, which is necessary to obtain a single tooth; and

[16] the method in accordance with any of the above [14] or [15], where at least either mesenchymal cell or epithelial cell derived from a tooth germ;

The implementation of the invention

In accordance with the present invention, if the aggregation of mesenchymal cells and epithelial unit cells are placed in close contact inside a support carrier, in this case, by adjusting the length of contact of mesenchymal cells and epithelial cells in the ass the direction it is possible to control the width of the tooth crown in the direction of the length of contact of the regenerated tooth germ, and in the resulting regenerated tooth.

Specifically, if the aggregation of mesenchymal cells and the Assembly of epithelial cells is formed in a nearly columnar structure, in this case, by controlling the length of the contact in the axial direction of the columnar structure can be formed in a tooth having a desired length in the axial direction.

Moreover, also on the basis of the present invention it is possible to design a method of creating a tooth, comprising determining the length of the contact, which allows you to get a tooth having a desired size.

Moreover, in accordance with the method of the present invention, regardless of the number of cells included in each cell mass, subject to receipt of a predetermined length of contact can be obtained a tooth having a desired length and, consequently, the desired size can be obtained with high efficiency by using fewer cells.

Moreover, in the present invention, by setting the length of contact between cell mass below a preset value, instead of the aggregate set of teeth can be obtained from a single tooth. Therefore, instead of going through the stages of separation, the tooth can be used as is in the form of graft.

Brief description of drawings

Figure 1 shows phase-contrast microscopy organ culture at day zero, the second day, the fifth day and the seventh on the HB, in those cases, when the length of the contact Assembly of epithelial cells and aggregation of mesenchymal cells during reproduction of the restored tooth germ with less than 450 μm, between 450 μm and 900 μm and 900 μm and 1500 μm. Arrow-pointers in the figure show both ends of the area of the tooth crown, which will form the future crown of the tooth.

Figure 2 is a schematic diagram showing the overall width of the tooth crown, which will form the future crown of the tooth, measured in the form of indices, showing the size of the regenerated tooth germ on the seventh day organ culture.

Figure 3 is a bar graph showing the ratio of the length of the contact Assembly of epithelial cells and aggregation of mesenchymal cells during reproduction of the reconstructed tooth germ and the size of the regenerated tooth germ on the seventh day organ culture.

Figure 4 is a chart showing the ratio of the length of the contact Assembly of epithelial cells and aggregation of mesenchymal cells during the production of the reconstructed tooth germ and the size of the regenerated tooth germ on the seventh day organ culture.

Figure 5 is a schematic illustration of the width of the tooth crown, measured in the form of indexes showing the size of regeneron the bathing of the tooth, formed by transplantation of a regenerated tooth germ under subrenal capsule.

Figure 6 shows stereomicroscopic image of the regenerated tooth on the 21-day analysis surrenales capsule, in the case when the length of the contact Assembly of epithelial cells and aggregation of mesenchymal cells during the process of obtaining a reconstructed tooth germ with less than 450 nm, between 450 nm and 900 nm and between 900 nm and 1500 nm. Arrow-pointers in the figure show the two ends of the dental crown.

7 is a bar graph showing the ratio of the length of the contact Assembly of epithelial cells and aggregation of mesenchymal cells during reproduction of the reconstructed tooth germ and the width of the crown of the regenerated tooth on the 21st day of analysis surrenales capsule.

Fig is a chart showing the ratio of the length of the contact Assembly of epithelial cells and aggregation of mesenchymal cells during reproduction of the reconstructed tooth germ and the width of the crown of the regenerated tooth on the 21st day of analysis surrenales capsule.

Figure 9 shows the CT image of the regenerated tooth on the 21-day analysis surrenales capsule, in cases where the length of the contact Assembly of epithelial cells and Assembly the mesenchymal cells during playback of the reconstructed tooth germ with less than 450 nm, between 450 nm and 900 nm and between 900 nm and 1500 nm.

Figure 10 is a chart showing the ratio of the length of the contact Assembly of epithelial cells and aggregation of mesenchymal cells during reproduction of the reconstructed tooth germ and the number of tubercles crowns regenerated tooth on the 21st day of analysis surrenales capsule.

Figure 11 presents the results of measuring the width of the area of the tooth crown of the regenerated tooth germ derived in the case when the length of the contact Assembly cylindrical epithelial cells and aggregation of mesenchymal cells in the reconstructed tooth germ mounted in a fixed range, and the number of cells included in each unit, changed.

On Fig presents the results of measuring the width of the tooth crown of the regenerated tooth obtained in the case when the length of the contact Assembly cylindrical epithelial cells and aggregation of mesenchymal cells in the reconstructed tooth germ mounted in a fixed range, and the number of cells included in each unit, changed.

On Fig presents the results of measuring the amount of bumps regenerated tooth obtained in the case when the length of the contact Assembly cylindrical epithelial cells and aggregation of mesenchymal cells in reconstru the new dental source mounted in a fixed range, and the number of cells included in each unit, changed.

The description of the various embodiments of the invention

The way to create the desired tooth in accordance with the present invention includes: a stage the location of the first unit cell and the second unit cell consisting either of mesenchymal cells or from epithelial cells in close contact inside a support carrier; and the stage of culturing the first and second aggregates of cells inside the support carrier, where the size of the tooth is adjusted by adjusting the length of contact of the first and second aggregates of cells in one direction.

In the present invention under the term "tooth" refers to a fabric containing a layer of dentin on the inside and a layer of enamel on the outside and having an orientation that includes the tooth crown and tooth root. The orientation of the tooth can be identified by the location of the tooth crown and tooth root. The crown of the tooth and the root of the tooth can be measured visually, based on the shape and histological staining. The crown of the tooth has a layered structure containing enamel and dentin and the tooth root does not have a layer of enamel.

Dentin and enamel can be easily identified morphologically by an expert in the field using histological staining and p Moreover, the enamel can be identified by the presence of enamel blast cells (precursor cells), and the presence of enamel blast cells can be confirmed by the presence 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 of sialoprotein dentin. Proof of amelogenin and sialoprotein dentin can be easily implemented well known in the field of ways, for example you can apply in situ hybridization and staining antibodies.

In the present invention "tooth germ" and "tooth Bud" are expressions used to denote the various stages of the formation of teeth. In this case, the tooth germ refers to the original root of the tooth, which will be the tooth in the future, and reflects the period of development from the stage of the kidney to the stage of "bell", which is usually used to denote the stage of the formation of the tooth, and is the cloth in which the accumulation of dentin and enamel, which are referred to as hard tissues of the tooth, not yet determined. On the other hand, the term "tooth Bud"used in the present invention, refers to the fabric after the stage of "early tooth development" and is a fabric, which varies from stage, when it begins to form estimates is giving dentin and enamel, referred to as hard tissues of the tooth, until the stage of eruption of the tooth from the gums and in General, since the functioning of the tooth. The formation of the tooth of early tooth development proceeds through a stage of kidney, the stage of "cap"early stage "bell" and late stage "bell". At the stage of kidney epithelial cells embedded mesenchymal cells, which leads to thickening, and in the stage of "cap" penetrate epithelial cells surrounded by mesenchymal cells. Then comes the turn early and late stages of "bell", with some epithelial cells becomes the outer enamel, and some mesenchymal cells forms the dentin on the inside. Education early tooth development is controlled by intercellular communication between epithelial cells and mesenchymal cells through cytokine that leads to the formation of the tooth.

In the present invention "mesenchymal cells" refers to cells derived from the mesenchymal tissue, and cells obtained by culturing such cells, and "epithelial cells" refers to cells derived from epithelial tissues, and cells obtained by culturing such cells.

In addition, in the present invention, the periodontal tissue" refers to the alveolar bone and periodontal membrane, educated, main the way on the outer layer of the tooth. The person skilled in the art will easily be able to identify the shape of the alveolar bone and the root shell using histological staining.

In the present invention "stage occupancy of the first unit cell and the second unit cell consisting either of mesenchymal cells or from epithelial cells in close contact inside a support carrier (hereinafter referred to as "the stage of allocation") is described, for example, in documents 1-5 Patent literature, and the full contents of these documents are incorporated herein for reference purposes.

Above the first unit cell and the second unit cells formed essentially only of mesenchymal or epithelial cells, respectively. "Mainly comprised of mesenchymal cells implies that in the present invention one unit cell performs the same functions as if it consisted only of mesenchymal cells, and does not include, to the extent possible, cells other than mesenchymal cells. The same applies to "mainly comprised of epithelial cells.

In this document the unit cell is in close contact with the cells, which can be cell mass prepared from dispersed cells, and in some cases even cloth. PR is the application of tissue has the advantage of which makes it easy to get your teeth with a regular arrangement of cells and form, but the quantity that can be obtained is limited. As the cell mass can be used cultured cells that are relatively easy to obtain, and therefore they are preferable. In accordance with the method of the present invention the regenerated tooth with the correct location of the cells and form can be obtained even when using cell mass.

Mesenchymal and epithelial cells, forming the unit cells can be obtained from any tissue of a living organism, provided that the regenerated tooth can be formed from regenerated tooth germ, educated in the use of these cells. From the point of view of effective education of a tooth having a specific structure and orientation, by reproducing cells located within a living organism, preferably, if at least one of these units of cells was derived from a tooth germ. More preferably, if and mesenchymal, and epithelial cells derived from a tooth germ. From the point of view of juveniles and homogeneity at the stage of cell differentiation is desirable, if the tooth germ is in the stage between the stage of the kidney and the stage of "cap"./p>

Examples of mesenchymal cells, is not derived from a tooth germ, include cells derived from other mesenchymal tissues of a living organism. Preferably, if they are bone marrow cells and mesenchymal cells do not contain blood cells, more preferably, if they are mesenchymal cells of the oral cavity, bone marrow cells inside the jaw bone, mesenchymal cells derived from the cells of the cranial nerve cushion mesenchymal cells-precursors that can differentiate into mesenchymal cells and stem cells. Because they are treated mesenchymal cells, an example of use derived from amnion cells is described in document 3 from the list Patent literature, and an example of using cells obtained by the induction of differentiation of totipotent stem cells as mesenchymal cells, described in document 4 from the list Patent literature, and the full contents of this document are incorporated herein for reference purposes.

Epithelial cells can also occur from early tooth development, examples of such cells are cells derived from other epithelial tissues of a living organism. Preferred examples of epithelial cells include epithelial cells of the skin, mucosa and des the s in the mouth, as more preferred examples of epithelial cells include immature epithelial precursor cells that can form differentiated, for example keratinized or parakeratinized epithelial cells, such as cells of the skin or mucosa. Examples of such immature epithelial precursor cells include nitratenitrogen epithelial cells and their stem cells. Example of the use of epithelial cells from the mouth and cells obtained in the first stage of cultivation of epithelial cells, are described in the document 2 from the list Patent literature, and the full contents of this document are incorporated herein by reference for reference purposes.

Tooth primordium and other fabrics can be selected from the jaw bone or the like of various animals-mammals, such as dogs and cats, and in addition, primates, such as humans and monkeys, ungulates, such as pigs, cows and horses, and small mammals such as rodents, such as mice, rats and rabbits. To collect early tooth development and tissue, as a rule, can be applied unmodified conditions for collection of tissue and the tooth germ and the fabric can be collected under sterile conditions and stored in the appropriate preservative solution. Examples of human tooth germ include dental conceived is to the third of a molar tooth, also called the wisdom tooth, as well as embryonic tooth germ, and from the point of view of the use of autogenous tissues using germ of wisdom teeth is preferred. In the case of mice, you can apply tooth mouse embryo at embryonic age 10-16 days.

During the preparation of mesenchymal and epithelial cells of early tooth development, tooth germ isolated from the surrounding tissue, initially divided into mesenchymal tissue of the tooth Bud and epithelial tissue early tooth development, based on their form. To facilitate selection, this point can be applied enzymes. Examples of enzymes include dispute, collagenase and trypsin.

Cell mass according to the present invention means a mass of cells derived from mesenchymal or epithelial tissue, and can be obtained by the aggregation of cells, obtained by dispersion of mesenchymal or epithelial tissue, or aggregation of cells obtained from the first stage or passage culture of these cells.

For dispersion of the cells can be applied enzymes, such as dispute, collagenase and trypsin. To obtain sufficient number of cells from the environment, typically used for culture of animal cells, such as the Modified Dulbecco Wednesday Needle (eng. Dulbecco''s Modified Eagle Medium, DMEM), can be used as a medium for cultiv the simulation of the first stage or passage culture of dispersed cells before preparation of cell mass. To stimulate growth of cells can be added serum, or alternatively, the serum can be added to cell growth factor such as FGF, EGF or PDGF, or a component of serum, such as transferrin. In cases when you add the whey, its concentration may be changed appropriately depending on the stage of cultivation and may typically be about 10%. For cell culture can be applied to normal culture conditions, such as culturing in an incubator at 37°C With 5% CO2. If necessary, can be added to an antibiotic, such as streptomycin.

For the aggregation of cells in the cell suspension centrifuged. When forming close contact mesenchymal and epithelial cell mass for interoperability cells need to maintain their high density. The condition of high density means that the density is almost equivalent density, which creates a fabric, for example a high density is in the range from 5×107cells/ml to 1×109cells/ml, preferably from 1×108cells/ml to 1×109cells/ml, and more preferably from 2×10 cells /ml to 8×10 cells/ml. a method of obtaining a cell mass with such a high cell density is not restricted, for example, cells could the t to be aggregated and precipitated by centrifugation. Centrifugation is particularly preferred because it is convenient to achieve high density without impairing cellular activity. This centrifugation can be performed at speeds equivalent to centrifugal force in the amount of 300×g to 1200×g, and preferably from 500×g to 1000×g for three to ten minutes. Centrifugation at speeds below 300×g may be unable to sufficiently increase the density of the cells, whereas centrifugation at speeds higher than 1200×g may cause damage to the cells.

In the case where high-density cell mass obtained by centrifugation, centrifugation, as a rule, is carried out after obtaining a cell suspension in a container such as a test tube for centrifugation of the cells and the supernatant removed to the extent possible, leaving the cells in the form of precipitation. In this document the amount of components other than interest of cells (for example, the volume of culture medium or buffer solution), preferably not more cell volume, and most preferably, if the components other than interest cells are absent. If such high-density aggregates of cells are in close contact with each other inside the support carrier in accordance with the method described below, you get what I tightly Packed cells and cells communicate effectively manifested.

Support media used in the present invention may be a medium in which cells can be grown, and preferably is a mixture with the above-described environment. Material supporting carrier nothing specific is not limited to, for example, can be used collagen, agarose gel, carboxymethyl cellulose, gelatin, agar, hydrogel, "Cellmatrix" (trade name), "Mebiol Gel" (trade name), "Matrigel" (trade name), elastin, fibrin, laminin, a mixture of extracellular matrix, polyglycolic acid (eng. polyglycolic acid, PGA), polylactic acid (eng. polylactic acid, PLA) and copolymers of lactic acid/glycolic acid (eng. lactic acid/glycolic acid copolymer, PLGA). These supporting media may have a density, through which cells can in fact be maintained in places, in which were placed the aggregates of cells, and examples of these support media include those that are in the form of a gel, fibers and solids. Among them, most preferred are materials having the appropriate density and fixation, such as collagen, agarose gel, carboxymethyl cellulose, gelatin, agar, hydrogel, "Cellmatrix", "Mebiol Gel", "Matrigel", a mixture of extracellular matrix, elastin, fibrin, and laminin. In this case, the density through which cells can in fact p derivatise in their places, can be density, which is applicable to three-dimensional culture, i.e. density, which can be stored position of the cells, while cell hypertrophy resulting from their growth is not inhibited, and this density can be easily determined by the expert in this field.

Moreover, supporting the carrier used in the present invention, may have fixation, by which cells can maintain close contact cell aggregates without going in dispersed state. "Close contact" means that the above-mentioned high-density aggregation of mesenchymal cells and the Assembly of epithelial cells retain the same level of density even in the immediate vicinity of the contact surface of mesenchymal cells and epithelial cells. If supporting media, which can maintain a close contact is collagen, for example, its use at a final concentration of 2 mg/ml to 3 mg/ml, that is, when the strength of the jelly from 120 g to 250 g according to the method corresponding to JIS-K6503-1996 (measured as the load required to penetrate to 4 ml of the piston diameter 12.7 mm), provides the appropriate density. Other types of supporting media can be used in the present invention upon receipt of such a strength similar to the way assessment is key. Moreover, the supporting carrier with a density equivalent to the required strength of the jelly can also be obtained by combining together one or more types of supporting media.

The location of the first unit cell and the second unit cells inside the support carrier nothing specific is not limited, but if aggregates of cells are the cell mass, for example the precipitate obtained by centrifugation as above, you can enter them inside the support carrier and place it, for example, using microspace. If the cell Assembly is cloth, it can be located in any position within the supporting carrier using the tip of the needle of the syringe.

In the present invention the methods of the location of the first unit cell and the second unit cells in supporting the carrier in close contact with each other does not specifically limited, for example, after the location of one cell unit in supporting the media another cell Assembly can be positioned so that it will put pressure on the first unit cell, and thus, both units can be installed in close contact with each other. More specifically, by appropriately changing the position of the tip above the needle of the syringe into the supporting medium is one of agrega the s cells can exert pressure on the other unit cells. When used as the unit cells of the epithelial tissue or mesenchymal tissue surface of the tissue, which was in contact with mesenchymal tissue or epithelial tissue in the original tooth germ may be positioned so that she was in contact with other unit cells.

In addition, after the location is also preferable to arrange the stage of solidification supporting media. This will allow the cells to further aggregate that will result in the condition of high density. For example, if you use a collagen gel, the solidification can be achieved by leaving the gel at a temperature of cultivation for a period of from several minutes to several tens minutes. Stage time is reduced to a minimum number of non-cell components inside the unit and achieved the status of high density.

In the present invention "stage of culturing the first and second aggregates of cells inside a support carrier (hereinafter referred to as "stage cultivation") is described in documents from 1-5 list Patent literature, the full contents of which are incorporated herein for reference purposes.

The cultivation period varies depending on the number of cells placed in a supporting medium, and from the state of cell mass, as well as about the conditions, which is the stage of cultivation, and on the type of animal, and the person skilled in the art can appropriately select a time period. In the case of transplantation inside the oral cavity in order to cut out the functional tooth requires at least a one-day cultivation period, and more preferably a three-day or longer period.

By increasing the length of the cultivation period can be made great progress in the formation of the reconstructed tooth germ, including the formation of accumulations of dentin and enamel, the formation of the tooth crown and tooth root formation. To achieve the desired state of the cultivation is carried out, for example, for 6 days or more 30 days or more, 50 days, 100 days or more, or 300 days or more, and during cultivation can be changed environment and the cultivation conditions.

Stage cultivation inside the support carrier may be carried out either in the presence of supporting media, which includes first and second units of the cell or the cultivation can be carried out in the presence of other animal cells.

In cases when the cultivation is carried out only in the presence of a supporting medium, culturing can be carried out in the normal conditions, used for cultivation of animal cells. In this case, the culture can be added to serum obtained from a mammal, and various cellular factors that are known to be effective in the growth and differentiation of such cells. Examples of such cellular factors include FGF and BMP.

From the point of view of gas exchange and supply nutrients aggregates of cells, as well as from the point of view of the implementation of full stages in vitro without any contact or mixing with other animal cells, preferably the use of organ culture to cultivate inside the support carrier. In organ culture, as a rule, the cultivation carried out by means of a porous membrane, floating in a medium suitable for growth of animal cells, and by placing a supporting carrier with the first and second aggregates of cells on the membrane. The porous membrane used herein is preferably a membrane with many pores with a diameter from 0.3 to 5 μm, and specific examples include Cell Culture Insert" (trade name) and "Isopore Filter" (trade name).

On the other hand, the implementation of cultivation within the supporting medium in the presence of other animal cells makes possible the early education of the tooth with a certain arrangement of cells in response the actions of various cytokines, etc. animal cells. Such cultivation in the presence of other animal cells can be carried out by culturing ex vivo using the selected cells or cultured cells, and in addition the support carrier with the first and second aggregates of cells can be transplanted into a living body for carrying out cultivation in vivo.

Such transplantation and cultivation in vivo are particularly preferred because of the tooth and/or tissue of the periodontium can be formed at an early stage. Preferred examples of animals that can be used as a living organism, include mammals, preferably mammalian, non-human, such as pigs and mice, and the animal is preferably from the same species as the fabric of early tooth development. In cases when the cultivation is carried transplantation in an animal that does not belong to the same species as the fabric of early tooth development, it is preferable to use an animal in which the change was made, which makes it immunodeficient. Examples of suitable for growth in vivo site in a living organism, in which the organ or tissue of animal cells develop, to the extent possible, normal, preferably include the area under surrenales capsule, mesentery (omental) and subcutaneous site.

Period Kul is iferouane after transplantation varies depending on the size of the tooth at the time of transplantation and size of developed tooth and can be typically, from 3 to 400 days. For example, the period of time of transplantation under surrenales capsule is preferably from 7 to 60 days, although it varies depending on the size of the dental embryo transplants and size of the regenerated tooth.

Preliminary ex vivo cultivation can be carried out before transplantation in a living organism. Preliminary cultivation strengthens the bonds between cells and the communication between the first and second aggregates of cells, making intercellular interaction stronger. In the intercellular interaction can be amplified and total growth period may be shortened.

Period of pre-culturing nothing specific is not limited. Preferably the period is for three days or longer, even more preferably seven days or longer, because the tooth Bud can develop from early tooth development during this period and, therefore, the cultivation period after transplantation can be reduced. For example, in the case of transplantation and cultivation under surrenales capsule organ culture as a pre-culture time is preferably from 1 to 7 days.

Tooth formed in accordance with the above-mentioned stage location and stage of cultivation, has a specific tooth arrangement of the notches (structure), has the dentin inside and enamel on the outside and preferably has the focus, then there is a vertex (crown of the tooth and the root of the tooth in the correct position, allowing him sufficiently to function as a tooth. Thus formed, the tooth can be widely used as an alternative tooth. Moreover, it can be used in the study to clarify the process of formation of teeth.

In addition, by extending the cultivation period in addition to the tooth can be formed tissues of the periodontium, such as alveolar bone and periodontal membrane, which support and stabilize the teeth in the jaw bone. As a result, the practicality of the tooth after transplantation can be further improved. In addition, only the tissue of the periodontium can be selected and used.

The present invention is characterized by the fact that the length of the thus obtained tooth in one direction is corrected by a correction of the length of contact of the first unit cell and the second unit cells in a predetermined one direction in the above-mentioned stage location.

The contact length can be adjusted depending on the size, shape and position of the unit cells, which should be located inside the support carrier. For example, at the location of the cell mass within the TF is rivalshigh media using microspace, the size, shape and position of the unit cell can be changed accordingly by changing the diameter of the needle of the syringe and the movement of the needle tip inside the support carrier during extrusion of cellular mass and length of contact between two aggregates of cells in any direction can be adjusted. If the unit cell is used mesenchymal tissue and epithelial tissue, the shape and size of the fabric can be adjusted to the location of the tissue inside the support carrier, and by correcting the position of their location inside the support carrier can be adjusted the length of contact between two aggregates of cells.

In addition, by obtaining many types of structures in which the first and second units of cells were located in close contact with each other inside the support carrier, and then measuring the length of contact of both aggregates of cells and selection of patterns, in which the measured contact length is the desired length can be obtained reconstructed tooth germ with the required contact length, and this stage is also included in the "correction of the length of contact" in the present invention. The length measurement of the contact can be performed, for example, by monitoring the phase-contrast microscope.

In this case, the length of the tooth in one direction against the seeking of the width of the tooth crown in either direction, for example, the width in the buccal-lingual direction (the direction perpendicular to the tooth row) and the width in mesio-distal direction (the direction parallel to the tooth row) are ideal, but the direction is not limited to them. Measure the width of a tooth crown can be performed properly by a specialist in this field.

It should be noted that if the regenerated tooth germ is formed by correcting the length of contact of the first unit cell and the second unit cells in one given direction, then, as a rule, the crown is formed of a tooth is adjusted length in the same direction as the length of the contact.

In accordance with the present invention one aspect of the method of producing a tooth having a desired length in one direction, includes: a step for many types of structures in which the first unit cell and the second unit cells are arranged in close contact inside a support carrier by changing the length of contact of the first unit cell and the second unit cells in one given direction; the stage of culturing each of the many types of structures inside the support carrier; stage length measurements obtained at the previous stage of the tooth in one direction, in order to determine the correlation between the contact length and the length of the tooth in one direction the attachment; and the stage of calculating, based on the correlation, the length of contact of the first unit cell and the second unit cells required to obtain a tooth having a desired length in one direction.

Stage getting multiple types of structures in which the first unit cell and the second unit cells are arranged in close contact inside a support carrier by changing the length of contact of the first unit cell and the second unit cell in one direction and the next stage of culturing each of the many types of structures inside the support carrier can be performed under the above explanation stage, location, stage of cultivation and method of adjusting the length of the contact.

The correlation between the contact length and the length of the tooth in one direction can be determined according to well known method or a similar method. For example, can be created in various charts, expressing the ratio of the length of contact and the length of the tooth width of the tooth crown), or formula expressing the ratio of the length of contact and the length of the tooth. In addition, can be analyzed the length distribution of contact, providing the length of one tooth, and can be defined length range of contacts for a given size of a tooth.

The stage of calculating the length of contact of the first unit cleto and the second unit cell, required to obtain a tooth having a desired length in one direction based on the obtained correlation and can be made by substituting the values of the requested size of a tooth in the above formula and chart.

Thus, after determining the required length of contact can be obtained tooth with a desired size by the location of the first unit cell and the second unit cells with the required contact length in close contact with each other inside the support carrier in almost the same conditions as in the above stage, the location of many types of structures, with subsequent implementation of cultivation in almost the same conditions as the above-mentioned conditions for the cultivation of many types of structures. In this document, "almost the same conditions" refer to conditions in which the tooth, have the same length in the same direction, can be obtained with good reproducibility, if the contact length is set the same. Under location and stage of cultivation to determine the length of contact and the stage location and stage of cultivation to obtain tooth having a desired length in one direction, it is desirable, for example, to culturing conditions, such as the type of support media, temperature, medium composition and location of the cult of the market (or organ culture, or in vivo culture) were the same.

In addition, when the location of the first and second aggregates of cells inside the support carrier, it is desirable that the length of the contact part expected forming position, and must have a specified length in the tooth, which is formed in the future, has the length calculated above. The person skilled in the art can appropriately determine the direction of the contact surface of the first and second aggregates of cells will be directions in the tooth, which is formed in the future. For example, upon receipt of a molar tooth, in which the length And mesio-distal direction longer than the length In the buccal-lingual direction, the contact surface of the first and second aggregates of cells in General should be rectangular and longer side should form the contact length, which provides the length And mesio-distal direction.

Another aspect of the method of producing a tooth having a desired length in one direction in accordance with the present invention, includes: a step for many types of structures in which the first unit cell and the second unit cell in the form of a nearly columnar structure, consisting either of the mesenchymal cells or from epithelial cells are in close contact inside a support carry the El so to the axial direction of each of the columnar structures were parallel, by changing the length of contact of the first unit cell and the second unit cell in the axial direction; the stage of culturing each of the many types of structures inside the support carrier; a stage of measuring the length of the tooth in one direction, obtained in the preceding stage, in order to determine the correlation between the contact length and length; and the stage is based on a correlation calculation of the length of contact of the first unit cell and the second unit cell, which is necessary to obtain a tooth having a desired length in one direction.

Stage getting multiple types of structures with different lengths of contact and subsequent implementation stage of the cultivation can be carried out in accordance with the above explanation stage, location, stage of cultivation and method for adjusting the length of the contact. As described above, the correlation between the contact length and the length of the tooth in one direction can be expressed by the formula or chart can also be determined from the length of contact, providing a predetermined length of the tooth. After that, on the basis of these correlations can be determined contact length of the first and second aggregates of cells required to obtain tooth, with t Buemi length in one direction.

In the present invention form an almost columnar structure" refers to an elongated shape that extends in one direction, such as almost cylindrical shape and almost prismatic shape. If the unit cell is fabric, the fabric may be formed in the columnar structure, and then located inside the support carrier. Moreover, if the unit cell is the cell mass, for example, the tip of the needle microspace can be placed inside the support carrier and cell mass can be located in almost cylindrical shape inside a support carrier by squeezing the cells during the movement of the needle tip.

Thus, after determining the necessary length of contact of a tooth having a desired length in one direction, can be obtained by the arrangement of the first unit cell and the second unit cell in the form of a nearly columnar structure when the contact length in close contact with each other inside the support carrier in almost the same conditions as in the above stage, the location of many types of structures, with subsequent implementation of cultivation in almost the same conditions as the above-mentioned conditions for the cultivation of many types of structures.

Another aspect of the method of producing a tooth in accordance with this the current invention is a method of obtaining a posterior tooth, with the required length in mesio-distal direction and/or in the buccal-lingual direction, and includes: a step for many types of structures in which the first unit cell and the second unit cell in the form of a nearly columnar structure, consisting either of the mesenchymal cells or from epithelial cells are in close contact inside a support carrier so that the axial direction of each of the columnar structures were parallel, by changing the length of contact of the first unit cell and the second unit cell in the axial direction and/or length of contact in the direction perpendicular to the axis; a stage of cultivation of each of many types of structures inside the support carrier; a stage of measuring the length of a molar tooth, obtained in the preceding stage in mesio-distal direction and/or in the buccal-lingual direction in order to determine the correlation between the contact length in the axial direction and length of a molar tooth in mesio-distal direction and/or the correlation between the contact length, perpendicular to the axis, and a length of a molar tooth in the buccal-lingual direction.

As described above, as a rule, due to the fact that the width of a molar tooth in the buccal-lingual direction is longer than the width in mesio-distal direction, if the unit cells strmilov the n in the form of a columnar structure, the width of the tooth crown in mesio-distal direction can be controlled by controlling the length of the contact in the axial direction, and the width of the tooth crown in the buccal-lingual direction can be controlled by controlling the length of contact perpendicular to the axis.

The contact length in the axial direction and the contact length, perpendicular to the axis can be changed using any method, for example, you can change the length of the unit cell in the form of a columnar structure, the diameter, the distance between the axes of both aggregates of cells. If the unit cell is a tissue, forming it to the desired diameter and length before you location inside the support carrier, and then adjusting its location inside the support carrier, you can change the contact length in the axial direction and the contact length, perpendicular to the axis. In addition, if the unit cell is the cell mass, for example, when placing it inside a support carrier by using microspace diameter of the unit cell can be changed by changing the diameter of the needle, and by changing the distance by which the needle tip is moved inside the support carrier, you can change the length of the unit cell in the axial direction. In addition, after placing one of the unit cells by adjusting the position in which RAS is alagaesia other unit cells, you can change the distance between the axes of both aggregates of cells. By reducing the distance between the two axes, so that they put pressure on each other, the contact surface of the unit cell becomes more General, and, thus, it is possible to change the contact length in the axial direction and the contact length in the direction perpendicular to the axis.

In addition, stage location, the stage of cultivation stage and determine the correlation between different lengths can be implemented in accordance with the previously mentioned methods.

Thus, after determining the length of the contact in the axial direction and/or length of contact in the direction perpendicular to the axis, molar tooth having a desired length in mesio-distal direction and/or in the buccal-lingual direction, can be obtained by the arrangement of the first unit cell and the second unit cell in the form of a nearly columnar structure at a given length of contact in close contact with each other inside the support carrier in almost the same conditions as in the above stage, the location of many types of structures, with subsequent implementation of cultivation in almost the same conditions as above conditions for the cultivation of many types of structures.

In addition, another aspect of the method of producing a tooth having demand is th length in one direction in accordance with the present invention, involves the step of positioning the first and second aggregates of cells in the form of a nearly columnar structure in close contact under location, so that the axial direction of each of the columnar structures were parallel, and the contact length in the axial direction of the first and second units of cells was within the range of plus/minus 25%, preferably ±10% of the above-mentioned desired length.

As described below, the inventors found that if the mass of mesenchymal cells and a mass of epithelial cells in the form similar to cylindrical, come in close contact inside a support carrier so that the axial direction of each of the columnar structures were parallel, the length of the thus obtained of the tooth depends on the length of contact in the axial direction of the columnar structures. In addition, it was found that by setting the length of the contact is approximately within the range of plus/minus 25%, preferably ±10% of the required length can be obtained from the tooth, in which the width of the tooth crown in mesio-distal direction is the desired length. Therefore, for example, if you receive the tooth, in which the width of the tooth crown in mesio-distal direction is approximately equal to X microns, then the first unit cell and the second unit cells can be formed in the columnar structure is ture, and the contact length in the axial direction can be set between 0,H μm and 1,25X μm, preferably between 0,9ץ μm and 1.1 μm.

The method of controlling the length of contact of the first and second aggregates of cells in the columnar structure can be carried out according to the already explained method.

In addition, instead of receiving the unit cells having a desired length in the axial direction, you can get many types of structures in which the first unit cell and the second unit cell in the form of a nearly columnar structures are arranged in close contact inside a support carrier, a contact length of both aggregates of cells in the axial direction can be measured, the structure in which the measured contact length is the desired length, you can select and run through a stage of cultivation. The length measurement of the contact can be accomplished, for example, the observation of phase-contrast microscope.

A method of obtaining a single tooth in accordance with the present invention includes: a stage the location of the first unit cell and the second unit cell consisting either of mesenchymal cells or from epithelial cells in close contact inside a support carrier; and the stage of culturing the first and second aggregates of cells inside the support carrier, where the maximum is Naya contact length of the first unit cell and the second unit cell is equal to or less than the specified value.

When in accordance with the method of the document 1 from the list Patent literature was received unexpected Assembly of the teeth, the authors realized that by controlling the length of contact of the first and second aggregates of cells and by controlling the size of the tooth, you can get a single tooth with good reproducibility. Probably, this is due to the fact that the first enamel knot, which determines the number of teeth formed from the dental Bud, not produced in quantities of more than one within a given distance. If you can get a single tooth, it is not necessary to carry out the selection before transplantation acquired tooth.

It should be noted that in the method of obtaining a single tooth in accordance with the present invention, the maximum length of contact" refers to the length of the longest straight line among straight lines included in the contact surface of the first unit cell and the second unit cell.

In addition, if the method of obtaining a single tooth in accordance with the present invention is applied in mice, the contact length of the first and second aggregates of cells preferably equal to or less than 3000 μm, and more preferably equal to or less than 1500 nm. It should be noted that the contact length is preferably equal to or more than 100 μm, and more preferably is avna or more than 200 microns. The contact length can be controlled according to the above mentioned description.

In the present invention a single tooth refers to the structure of the tooth, which can be transplanted into a living body and which is characterized by the presence of a tooth crown, tooth root, dental pulp and dentin formed continuous with the periodontal bone and alveolar bone, which are formed around each tooth. The person skilled in the art can easily determine the number of teeth.

The way to restore the missing parts of teeth in the oral cavity in accordance with the present invention includes a step transplantation into the loss of the tooth obtained by the method for obtaining a tooth in accordance with the present invention. In accordance with this method the tooth, suitable in size to the area of loss can be obtained and transplanted.

The way to restore the missing parts of teeth in the oral cavity in accordance with the present invention there is the possibility of transplantation of tooth germ or tooth at any stage, obtained in the method of creating a tooth according to the present invention. If the formation of the tooth crown can be seen, it is preferable to place the tooth in the inner part of the mouth. If the formation of dental crowns cannot be seen, predpochtitelnei layer of epithelial cells, the corresponding part of the dental crown, or a layer of epithelial cells reconstructed tooth germ toward the inside of the mouth. In addition, it is preferable to have the open part of the layer of epithelial-mesenchymal cells reconstructed tooth germ on the side opposite the inner side of the mouth. Thus, the tip of the tooth (crown) will be directed to the inside of the mouth and will have the same orientation as the surrounding teeth.

The loss of the tooth means located in the gum part of the lost teeth, and its form is nothing specific is not limited. Since the regenerated tooth germ or tooth can be built, there are no specific limitations as to the area of loss of the tooth and the type of tooth.

The area loss of the tooth, usually located in the jaw bone or alveolar bone inside the mouth. In addition, together with the loss of teeth if the alveolar bone mass also damaged, to increase the missing part of the bone is possible to apply well-known clinical method to restore the bone to facilitate embedding an implant, such as the GTR (guided tissue regeneration; eng. Guided Tissue Regeneration). After placing the tooth germ or tooth cavity suppose the equipment put on the plot seam according to normal process.

The way to restore the missing parts of teeth in the oral cavity in accordance with the present invention the animal, which will be implemented transplantation, preferably should be of the same type as that from which the extracted tooth root used to retrieve the tooth, and more preferably should be the same individual from which the extracted tooth primordium. As animals can be used in mammals, such as humans, cows, horses, pigs, dogs, cats and mice. Namichaetsa can also be used.

In addition, the present invention also provides a method of designing a method of producing a tooth having a desired length in one direction at a given condition. "Specified conditions" mean the conditions which define the supporting media, environment and way of cultivation. When running under a given condition of the production method, designed according to the method for designing a method of producing a tooth having a desired length in one direction at a given condition, you can get a tooth having a desired length in one direction.

The above design method in accordance with the present invention includes a method for determining, when the first unit cell and the second unit cleto is composed either of mesenchymal cells, any of epithelial cells are in close contact inside a support carrier, the length of contact of both cell aggregates, which are required for a tooth having a desired length in one direction.

In this case, the method of determining the length of the contact includes: a step for many types of structures in which the first unit cell and the second unit cell, consisting either of the mesenchymal cells or from epithelial cells are in close contact inside a support carrier by changing the length of contact of the first unit cell and the second unit cells in a predetermined one direction; the stage of culturing each of the many types of structures inside the support carrier; a stage of measuring the length of the tooth, obtained at the previous stage, in one direction, in order to determine the correlation between the contact length and the length of the tooth in one direction; and the stage is based on a correlation calculation of the length of contact of the first unit cell and the second unit cells necessary to obtain a tooth having a desired length in one direction.

In addition, the present invention also provides a method of designing a method of producing a single tooth in specified conditions.

The above design method in accordance with the laws the AI with the present invention includes a method for determining, when the first unit cell and the second unit cell, consisting either of the mesenchymal cells or from epithelial cells are in close contact inside a support carrier, the length of contact of both cell aggregates, which are necessary to obtain a single tooth.

Furthermore, the method of determining the maximum length of a contact includes: a step for many types of structures in which the first unit cell and the second unit cell, consisting either of the mesenchymal cells or from epithelial cells are in close contact inside a support carrier, to change the maximum length of contact of the first unit cell and the second unit cell; the stage of culturing each of the many types of structures inside the support carrier and the stage of measuring the number of teeth obtained at the previous stage, in order to determine the maximum contact length of the first unit cell and the second unit cells necessary to obtain a single tooth.

It should be noted that the terms used in this document, are used to explain specific embodiments and are not intended to limit the invention.

Moreover, as used herein, the term "include", is designed to oboznacheny the presence of the described entity (member, stage, element, number, etc) except when in the light of the context of possible different interpretation, and does not exclude the presence of an entity other than the above entity (member, stage, element, number, etc).

If no other definitions used in this document, the terms (including technical terms and scientific terms) are the same value, which is widely known to experts in the field, belongs to the present invention. Used in this document, the terms shall be construed as bearing integral with respect to the present invention and a related technical field value, unless a different definition is expressly provided, and thus, they should neither idealize nor be interpreted in an overly superficial value.

The case where the embodiment of the present invention is explained with reference to the schematic drawing and schematic diagram for a detailed explanation can be entered extended explanation.

The terms "first", "second", etc. are used for the expression of different elements, and it is clear that these elements should not be limited by these terms. These terms are used only to distinguish one element from another element. For example, it is possible to describe the first element as the second element, and so is E. the second element as the first element, without departing from the scope of the claims of the present invention.

Even in the above-mentioned design of mesenchymal cells and epithelial cells can originate from any tissue of a living organism, provided that the regenerated tooth can be obtained from the reconstructed tooth germ generated by these cells. Preferably, if at least one type of these cells derived from a tooth germ, and more preferably, if both types of cells derived from a tooth germ.

Hereinafter the present invention will be described in more detail with reference to examples. However, the present invention can be embodied in various ways, and the present invention should not be construed as limited to the examples described in this document.

Examples

(1) Obtaining epithelial cells early tooth development and mesenchymal cells of the dental germ

Dental reconstructed embryo to form the tooth. As the experimental model used mice.

From the embryo (the gestational age of 14.5 days) of mice C57BL/6N (purchased in Japan SLC, Inc.), under the microscope was removed in the usual way the fabric of the dental germ of a lower molar. Fabric tooth germ of the lower molar teeth were washed in phosphate buffer, not containing Ca2+, Mg2+(PBS(-)and treated at room is the temperature within two minutes with a solution of enzyme, which is a (PBS(-)), with the addition of 40 U/ml (final concentration) Dispose (produced by "BD", mA, USA). After that, three times washed with DMEM (cut Needle in the modification of Dulbecco) (produced by Sigma, St. Louis, Montana) supplemented with 10% FBS (fetal calf serum) (production Invitrogen, carlsbad, CA). The next step was added a solution Gnkazy I (production Takara, Shiga, Japan) to a final concentration of 70 Units/ml for dispersion tissue of a tooth germ, and surgically separated epithelial and mesenchymal tissue of a tooth Bud using 25 G injection needle (production Terumo, Tokyo, Japan).

Epithelial tissue of a tooth germ was washed three times with PBS(-), treated at 37°C for 30 minutes with a solution of the enzyme, which is a PBS(-) dissolved in 100 Units./ml (final concentration) Collagenase I (production, "Worthington", Lakewood, NJ) and this process was repeated twice. Cells from the sediment obtained by centrifugation, and treated at 37°C for 10 minutes with a solution of the enzyme, which is a PBS(-) dissolved therein 0.25% trypsin (final concentration) (produced by Sigma). After that, three times washed cells DMEM (manufactured by Sigma) supplemented with 10% FBS (manufactured "Invitrogen"). The next step was added to the cell solution of DNOC is s I (production of "Takara") to a final concentration of 70 Units/ml and obtained a suspension of epithelial cells of the dental germ, separated by pipetting.

At the same time mesenchymal tissue of the tooth germ was washed three times with PBS(-) and treated at 37°C for 20 minutes with a solution of the enzyme, which is a PBS(-) dissolved in 100 Units./ml (final concentration) Collagenase I (production of "Worthington"). Then further processed PBS(-) supplemented with 0.25% Trypsin (manufactured by Sigma) for ten minutes. The next step was added 70 Units/ml Gnkazy I (produced by Takara) to obtain a suspension of mesenchymal cells of the dental germ, separated by pipetting.

(2) the Manufacture of the reconstructed tooth germ

Next carried out the reconstruction of early tooth development, prepared using epithelial and mesenchymal cells of the dental germ. 1.5 ml of microprobing (production Eppendorf, Hamburg, Germany) coated with silicone grease was added to the epithelial or mesenchymal cells of the dental germ, suspended in DMEM (manufactured by Sigma) supplemented with 10% FBS (manufactured "Invitrogen") and collect the cells by centrifugation (within three minutes at 600×g) in the form of precipitation. After centrifugation removed so far as possible the supernatant of culture medium and again were centrifuged for three minutes at 600×g, followed by complete removal of culture medium, OST is usasa around precipitation cells, under a stereoscopic microscope using "GELoader Tip 0.5-20 u.1" production "Eppendorf").

To the Petri dish, which was applied silicone grease, added Pocatello 30 ál "Cellmatrix type I-A" (produced by "Nina gelatin, Osaka, Japan) in order to obtain a drop of collagen gel as a supporting medium. This solution was placed quantitatively by using a Hamilton syringe (CN RT-3 production "HAMILTON", Reno, Nevada) obtained after centrifugation mesenchymal cells of the dental germ of sediment, for the preparation of cell mass in the shape of the unit cell of cylindrical shape. The next step epithelial cells early tooth development in a number equal to the number of mesenchymal cells of the dental germ, had the same way with the formation of cell mass, so that the corresponding sides of the two cell masses were in contact with each other, and the axial direction was parallel cell mass of cylindrical form, prepared previously from mesenchymal cells of the dental germ, and received the reconstructed tooth germ.

Then a drop of the collagen gel was hardened for 20 minutes at 37°C, the connection between the two cell masses became strong. Prepared culture vessel, so that DMEM (manufactured by Sigma) supplemented with 10% FBS (production is a "Invitrogen") were in contact with inserts "Cell Culture Inserts (PET membrane, having a pore size of 0.4 μm; production "BD"). Hardened reconstructed tooth germ transfer on membrane inserts "Cell Culture Inserts" in the culture vessel for holding organ culture on the insert Cell Culture Insert" in the traditional way at 37°C, 95% RH and 5% CO2.

(3) analysis of the amount of the regenerated tooth germ derived from organ culture

Aggregates of cells cylindrical epithelial and mesenchymal cells were placed in contact with each other when the contact length is less than 450 μm, between 450 μm and 900 μm and 900 μm and 1500 μm with the formation of three groups reconstructed tooth germs, and reconstructed tooth germ was created using organ culture (figure 1). The length of contact between the two cell mass was measured using phase-contrast microscopy.

For analysis of the width of the area of the tooth crown of the regenerated tooth germ on the seventh day organ culture in the regenerated tooth germ using phase-contrast microscopy was measured as indicated by the arrows-pointers in figure 2 the width of the tooth crown, which in the future will become the crown of the tooth. The area measurement is also shown by the arrows on the pictures of the seventh day in figure 1.

The results of the measurements are presented in figure 3. For the length of contact is less than 450 μm, the width of the area of the tooth to the Ronchi was 366±103,1 μm, for the length of contact between 450 μm and 900 μm width region of the tooth crown was 584,0±103,3 μm, and the length of contact between 900 μm and 1500 μm width region of the tooth crown was 934.9±239,8 μm. This indicates that the longer the contact Assembly of epithelial cells and aggregation of mesenchymal cells during formation of the reconstructed tooth germ, the wider is the area of the dental crown on the regenerated tooth germ.

In addition, figure 4 is the diagram of dispersion of the measured values of the length of the contact and the width of the region of the crown, and a linear approximation to a straight line is carried out on the figure by the method of least squares. The formula for a straight line is y=0,7114x+133,95.

(4) a Study of the size of the regenerated tooth with an analysis on surrenales capsule

On the eight-week C57BL/6 mice under anesthesia on the back was shaved off my hair located above the kidneys, the skin and the peritoneum was opened about 1 cm, and using ring forceps (production Natsume, Tokyo, Japan) were removed kidney. In surrenales capsule made an incision 2-3 mm with blades (produced by "Feather", Tokyo, Japan). In the space between the kidney and surrenales capsule, inserted three groups reconstructed tooth germs with different lengths of contact with collagen gel, as shown in example (2), the kidney was placed back and muscle pokr is involved and the skin sutured.

The regenerated tooth was extracted on day 21 after the start of the analysis on surrenales capsule. The regenerated tooth that was extracted, shown in Fig.6. The part marked with arrows-pointers on 6, was measured using a stereoscopic microscope as the width of the tooth crown.

The results of the measurements are presented in table 1.

Table 1
AndInD
The contact length in the reconstructed tooth germ (µm)The actual measured value of the width of the crown of the regenerated tooth (µm)The discrepancy in the size of the crown of the tooth; (B-A) (µm)The percentage of discrepancies and |B-A|/A×100(%)
437,91482,2944,3810,13
410,77529,76118,9928,97
318,38317,34(1,04)0,33
336,7786,35 49,5814,72
460,00491,9231,926,94
619,87762,57142,7023,02
549.87578,2828,415,17
295,25387,8892,6331,37
446,70459,6112,912,89
458,72490,3731,656,90
511,48402,97(108,51)21,22
384,75502,64117,8930,64
426,23468,4042,179,89
924,661017,4792,81 10,04
1020,241240,64220,4021,60
996,84979,63(17,21)1,73
1000,001083,8183,81scored 8.38
1223,271293,1769,905,71
915,86989,7673,908,07

The average percentage value, denoted as D in the table, was 13,03, and the standard deviation was equal 10,00.

In addition, the width of the tooth crown, obtained by dividing the above-mentioned measurement results on three based on the length of the contact group less than 450 μm, between 450 μm and 900 μm and 900 μm and 1500 μm, presented on Fig.7. For the length of contact is less than 450 μm width region of the dental crown was 497±118,0 μm, the length of contact between 450 μm and 900 μm width region of the tooth crown was 727,0±271,4 μm, and the length of contact between 900 μm and 1500 μm width region of the tooth crown was 1073,9±of 186.0 μm. This indicates that the longer the contact unit is epithelially cells and aggregation of mesenchymal cells during formation of the reconstructed tooth germ, the wider the crown of the tooth on the regenerated tooth.

In addition, Fig is the dispersion of the measured values of the length of the contact and the width of the crown, and a linear approximation to a straight line is carried out on the figure by the method of least squares. A straight line is expressed by a formula y=0,7257x+272,15.

(5) analysis of the number of tubercles on the regenerated tooth using micro CT

Using 3D microroentgens CT for experimental animals (produced by "RIGAKU, Tokyo, Japan) regenerated tooth obtained by the method presented in (4), photographed at a voltage 90,0 kV, electric current And 150,0 with 10 µm/pixel. The results are shown in Fig.9.

Further, the image was analyzed using "i-View" (produced by "RIGAKU, Tokyo, Japan), received 3D image of the regenerated tooth, and counted the number of tubercles on the regenerated tooth. If you put on a graph the length of the contact cell masses of epithelial cells and mesenchymal cells during the process of obtaining a reconstructed tooth germ and the number of tubercles regenerated tooth extracted under surrenales capsule, and calculate the correlation coefficient, we can see a strong correlation existing between the contact length during the reconstruction, and the number of tubercles regenerated tooth (R2=0,658) (figure 10). This indicates that the o the larger the contact of epithelial and mesenchymal cells during the reconstruction, the more bumps formed in the regenerated tooth.

(6) a Study of the reconstructed tooth germ by changing the number of cells and the length of contact of the unit cells in a fixed range.

The contact length of the cell mass was set in the range from 300 to 500 μm. By making cell mass using a cell suspension volume of approximately 0.05 μl and Hamilton syringe having an inner diameter 0,330 mm (KN RT-3 production "HAMILTON", Reno, Nevada)used in the example (2), and preparation of cell mass using a cell suspension volume of approximately 0.02 ál and Hamilton syringe having an inner diameter 0,203 mm (KN RT-3 production "HAMILTON"), received dental primordium in which the number of cells used in cell mass was changed. The shape of the regenerated tooth germ and the regenerated tooth formed from this reconstructed tooth germ, were analyzed by the methods provided in examples (3), (4) and (5).

The width of the area of the dental crown of the regenerated tooth germ is presented figure 11, the width of the crown of the regenerated tooth presents on Fig, and the number of tubercles in the regenerated tooth presents on Fig. Even if the number of cells is smenyaetsya while maintaining the length of the contact within a fixed range, did not observe any significant changes in the form of a regenerated tooth germ and the regenerated tooth.

1. A method of creating a tooth having a desired length in one direction, including:
stage location of the first unit cell and the second unit cells in close contact inside a support carrier, where the first unit cell and the second unit cells respectively comprise either mesenchymal or from epithelial cells; and
the stage of culturing the first and second aggregates of cells inside the support carrier,
where the size of the tooth is adjusted by adjustment of the length of contact of the first unit cell and the second unit cells in one given direction;

2. The method according to claim 1, where the two unit cells are cell mass.

3. A method of creating a tooth having a desired length in one direction, including:
a step for many types of structures containing the first unit cell and the second unit cells, which are arranged in close contact inside a support carrier, where many types of structures include structures with different contact length of the first and second unit cells, by changing the length of contact of the first unit cell and the second unit cells in one given direction, where the first unit cell and the second EPR is gat cells respectively comprise either mesenchymal cells, any of epithelial cells;
the stage of culturing each of the many types of structures inside the support carrier;
stage length measurement of the tooth, obtained at the previous stage, in one direction, and determining the correlation between this length and the length of the contact; and
the stage is based on a correlation calculation of the length of contact of the first unit cell and the second unit cell, which is necessary to obtain a tooth having a desired length in one direction;
stage location of the first unit cell and the second unit cells in close contact so that they had calculated at the previous stage the length of the contact inside the support carrier, where the first unit cell and the second unit cells respectively comprise either mesenchymal or from epithelial cells; and
the stage of culturing the first and second aggregates of cells inside the support carrier.

4. The method according to claim 3, where both unit cells are cell mass.

5. A method of creating a tooth having a desired length in one direction, including:
a step for many types of structures containing the first unit cell and the second unit cell in the form of a nearly columnar structures, which are arranged in close contact inside a support carrier, so cebysev direction of each of the aggregates of cells in the form of a nearly columnar structures were parallel, where many types of structures include structures with different contact length of the first and second unit cells, by changing the length of contact of the first unit cell and the second unit cell in the axial direction, where the first unit cell and the second unit cells respectively comprise either mesenchymal cells or from epithelial cells;
the stage of culturing each of the many types of structures inside the support carrier;
stage length measurement of the tooth, obtained at the previous stage, in one direction, and determination of correlation between its length and the length of the contact; and
the stage is based on a correlation calculation of the length of contact of the first unit cell and the second unit cell, which is necessary to obtain a tooth having a desired length in one direction;
stage location of the first unit cell and the second unit cell in the form of a nearly columnar structure in close contact inside a support carrier, so that the contact length in the axial direction was the length calculated in the previous stage, and the axial direction of each of the aggregates of cells in the form of a nearly columnar structures were parallel, where the first unit cell and the second unit cells respectively comprise either mesenchymal cells or from epithelial CL is current; and
the stage of culturing the first and second aggregates of cells inside the support carrier.

6. The method according to claim 5, where both unit cells are cell mass.

7. The method of creation of a molar tooth having a desired length in mesio-distal and/or buccal-lingual direction, including:
a step for many types of structures containing the first unit cell and the second unit cell in the form of a nearly columnar structures, which are arranged in close contact inside a support carrier, so that the axial direction of each of the aggregates of cells in the form of a nearly columnar structures were parallel, where many types of structures include structures with different contact length of the first and second unit cells, by changing the length of contact of the first unit cell and the second unit cell in the axial direction and/or length of contact in the direction perpendicular to the axis, where the first and second unit cells respectively comprise either mesenchymal or from epithelial cells;
the stage of culturing each of the many types of structures inside the support carrier;
stage length measurement of a molar tooth, obtained at the previous stage, in mesio-distal and/or in the buccal-lingual direction and defining a correlation between the length of the con is the act of a molar tooth in the axial direction and a length in mesio-distal direction and/or correlation between the contact length in the direction perpendicular to the axis, and a length of a molar tooth in the buccal-lingual direction;
the stage is based on a correlation calculation of the length of contact of the first unit cell and the second unit cell in the axial direction and/or length of contact in the direction perpendicular to the axis, which is necessary to obtain the posterior tooth of the desired length in mesiodistal and/or in the buccal-lingual direction;
stage location of the first unit cell and the second unit cell in the form of a nearly columnar structure in close contact inside a support carrier, so that the contact length in the axial direction and/or the contact length in the direction perpendicular to the axis, was the length calculated in the previous stage, and the axial direction of each of the aggregates of cells in the form of a nearly columnar structures were parallel, where the first unit cell and the second unit cells respectively comprise either mesenchymal cells or from epithelial cells; and
the stage of culturing the first and second aggregates of cells inside the support carrier.

8. The method according to claim 7, where both unit cells are cell mass.

9. A method of creating a tooth having a desired length in one direction, including:
stage location of the first unit cell and the second unit cell in the form of almost starchitecture in close contact inside a support carrier, so that the axial direction of each of the aggregates of cells in the form of a nearly columnar structures were parallel, and that the contact length of the first unit cell and the second unit cell in the axial direction was within the range of plus/minus 25% of the required length, where the first unit cell and the second unit cells respectively comprise either mesenchymal cells or from epithelial cells; and
the stage of culturing the first and second aggregates of cells inside the support carrier.

10. The method according to claim 9, where both unit cells are cell mass.

11. The method according to claim 9, in which stage the location of the first and second aggregates of cells inside the support carrier includes:
stage getting multiple structures containing the first and second aggregates of cells that are located inside the support carrier;
phase measurements of the length of contact of the first and second aggregates of cells in the axial direction; and
stage selection patterns, the measured contact length which is about within the range of plus/minus 25% of the required length.

12. The method according to claim 11, where both unit cells are cell mass.

13. The way to create a single tooth, including:
a step for many types of structures containing the first unit cell and the second unit cells is to, which are arranged in close contact inside a support carrier, where many types of structures include structures with different contact length of the first and second unit cells, by changing the length of contact of the first unit cell and the second unit cells in one given direction, where the first unit cell and the second unit cells respectively comprise either mesenchymal cells or from epithelial cells;
the stage of culturing each of the many types of structures inside the support carrier;
stage length measurement of the tooth, obtained at the previous stage, in one direction, and determining the correlation between this length and the length of the contact; and
the stage is based on a correlation calculation of the length of contact of the first unit cell and the second unit cell, which is necessary to obtain a tooth having a desired length in one direction;
stage location of the first unit cell and the second unit cells in close contact inside a support carrier; and
the stage of culturing the first and second unit cells inside the support carrier, where the first unit cell and the second unit cells respectively comprise either mesenchymal cells or from epithelial cells;
where the maximum contact length of the first unit cell and the whose unit cell is equal to or less than a previously defined value.

14. The way po, where both unit cells are cell mass.

15. The method according to any one of claims 1 to 14, where at least either mesenchymal cell or epithelial cell derived from a tooth germ;

16. The way to restore the missing parts of teeth in the oral cavity, including:
stage transplantation of tooth obtained by the method according to any one of claims 1 to 15, in the region of the tooth is lost.

17. The method according to clause 16, in which the mesenchymal cells and epithelial cell obtained from the individual, that part of the tooth was lost.

18. The method according to clause 16, in which the tooth obtained by the method according to any one of claims 1 to 15, transplanted into the region of a tooth is lost, without dividing the tooth into two or more parts.

19. The method according to p, in which mesenchymal cell and epithelial cell obtained from the individual, that part of the tooth was lost.

20. The method according to any of PP-19, in which the mouth is the mouth of memleketim.

21. The way to determine the length of the contact in order to get a tooth having a desired size,
in which the method includes determining the length of contact of both aggregates of cells in one given direction, which is required to obtain tooth that possesses the desired size, in conditions when the first unit cell and the second unit cells are in close contact wew and supporting media and the first unit cell and the second unit cells respectively comprise either mesenchymal cells or from epithelial cells, and
which method of determining the length of the contact further includes:
a step for many types of structures containing the first unit cell and the second unit cells, which are arranged in close contact inside a support carrier, where many types of structures include structures with different contact length of the first and second unit cells, by changing the length of contact of the first unit cell and the second unit cells in one given direction, where the first unit cell and the second unit cells respectively comprise either mesenchymal cells or from epithelial cells;
the stage of culturing each of the many types of structures inside the support carrier;
stage length measurement of the tooth in one direction, obtained at the previous stage, and determining the correlation between the contact length and the length of the tooth in one direction; and
the stage of calculating, based on the correlation length of contact of the first unit cell and the second unit cells, which are required to produce tooth having a desired length in one direction.

22. The method of determining the maximum length of contact between two aggregates of cells, which required the attendance for receiving a single tooth,
in which the method includes determining the maximum length of contact of both aggregates of cells, which is required to obtain a single tooth, in conditions when the first unit cell and the second unit cells are arranged in close contact inside a support carrier and the first unit cell and the second unit cells respectively comprise either mesenchymal cells or from epithelial cells, and
which method of determining the maximum length of the contact further includes:
a step for many types of structures containing the first unit cell and the second unit cells, which are arranged in close contact inside a support carrier, where many types of structures include structures with different contact length of the first and second unit cells, by changing the maximum length of contact of the first unit cell and the second unit cell, where the first unit cell and the second unit cells respectively comprise either mesenchymal cells or from epithelial cells;
the stage of culturing each of the many types of structures inside the support carrier; and
stage dimension number of teeth, obtained in the preceding stage, and determine the maximum length of contact of the first unit cell and the second unit cell, which is necessary for the floor the treatment of a single tooth.

23. The method according to any of PP or 22, where at least either mesenchymal cell or epithelial cell derived from a tooth germ.



 

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16 cl, 2 dwg, 1 ex

FIELD: biotechnologies.

SUBSTANCE: method is proposed to extract stem cells, including whirling of heparinised bone marrow with hydroxyethyl starch at the ratio of source ingredients of 1:2 with speed of 700g for 15 min. in the closed system of three haematological containers connected to each other with tubes with subsequent removal of fat admixtures and plasma into the container No.1, transfer of the mononuclear fraction of bone marrow, a part of supernatant and erythrocytes adjoining the interface of two media into the container No.2. Sludged erythrocytes and bone fragments remain in the main container, whirling of the produced sample with the speed of 900g for 15 min. in the container No.2 to produce cell material for intravascular introduction, at the same time after the specified whirling a part of supernatant is removed into the container No.1 without unsealing of the system.

EFFECT: production of paracrine effect of bone marrow mononuclear cells and provision of safety.

1 tbl, 2 ex

FIELD: biotechnologies.

SUBSTANCE: method is proposed to produce epithelioid cells of buffalo cow light foetus by means of long-term no-reseeding cultivation having high sensitivity to the virus of infectious rhinotracheitis of cattle, parainfluenza-3, viral diarrhea-disease of mucous membranes and adenoviral infection.

EFFECT: possibility to use in diagnostics of virus infections.

4 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and veterinary science; it is used in transplantology, traumatology, surgery and oncology, and may be used to fill bone defects. What is described is a bioimplant which represents a donor bone deimmunised with chlorine-containing oxidants, a surface of which is covered with a multifunctional bioactive nanostructured coating (MBNC) of M-Ca-P-C-O-N or M-Ca-CON, wherein M is a metal specified in a group consisting of Si, Ti, Zr, Hf, Nb, Ta, and colonised by recipient's mesenchymal stem cells (MSC).

EFFECT: MBNC-coated bioimplant is in line with the anatomical and morphological characteristics of the replaced bone, provides cell adhesion, no transplant rejection, accelerated formation of connective tissue and callus.

8 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and veterinary science, namely to reconstructive surgery it aims at the applications in transplantation, traumatology, surgery, and oncology. What is described is a method for producing bioengineered constructs for bone defect replacement, which is based on a bone anatomically compatible with the replaced one which is deimmunised in 5-10% solution prepared of a dry mixture of sodium chlorite, sodium perchlorate, sodium chloride in a ratio of 7:2:1 and distilled water; it is coated with heterogeneous implantable gel and colonised with multipotent mesenchymal stromal cells recovered from the recipient's bone marrow using immunomagnetic separation technique.

EFFECT: method provides sizeable bone defect replacement, high strength, fast fixation and repair of the construct in the implantation region, causes no reject phenomena.

1 tbl, 4 dwg

FIELD: medicine.

SUBSTANCE: what is described is a composition for fistula treatment in an individual, including stromal stem cells of fatty tissue where at least approximately 50 % of stromal stem cells of fatty tissue making a composition, express CD9, CD10, CD13, CD29, CD44, CD49A, CD51, CD54, CD55, CD58, CD59, CD90 and CD105 markers and where the contents of the stromal stem cells of fatty tissue in the composition makes at least approximately 3×106 cells/ml.

EFFECT: invention extends the range of products for fistula treatment.

8 cl, 7 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to chemistry of high-molecular compounds, namely to preparing film and spongy chitosan and collagen materials effective in human skin cell culture and wound grafting. A method for preparing the composite chitosan and collagen resorbable matrixes for human skin cell culture involves preparation of solutions polysaccharide chitosan and protein collagen, mixing them in preset proportions and formation of film and spongy matrixes of mixed polymer solutions. For this purpose, chitosan and collagen solutions in the concentration 1.0-4.0 wt % in a general solvent (aqueous 2 % acetic acid) are prepared, mixed in preset proportions, and the film and spongy matrix materials are formed from the prepared solutions. An amount of collagen in polymer mixtures makes 2.5-10 wt % (of chitosan). Further, films and sponges are heated to temperatures within 50-100°C for 1.0-5.0 h in an atmospheric environment.

EFFECT: use of the declared method allows preparing the film and spongy resorbable composite materials of natural polymers effective in human skin cell culture.

2 cl, 6 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to cardiovascular surgery. A biological material for cardiovascular surgery is made of a preserved carp swim bladder.

EFFECT: reduced calcification with maintained physical-mechanical properties.

1 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to cardiovascular surgery. Described is biological material for prostheses, in which Glisson's capsule of horse is used. As base, Glisson's capsule of horse is used.

EFFECT: increase of elastic-strength characteristics of prostheses.

1 ex

FIELD: medicine.

SUBSTANCE: invention relates to dentistry and is intended for manufacturing a 3D surgical pattern in order to carry out transgingival dental implantation. A device for manufacturing the 3D surgical pattern in planning transgingival dental implantation contains a bearing part and means of fixation in the device of an X-ray pattern. The bearing part is made in the form of a base with a hole, in which vertically installed is a tube with a possibility of deviation from its vertical axis, and with protrusions on both sides from the hole, in which installed are screws with a possibility of interaction with the tube, as well as with two horizontal shoulders. Means of the X-ray pattern fixation are made in the form of plates with fastening elements for the X-ray pattern, installed on both the said horizontal shoulders. The device is additionally provided with an arrow, installed in the said tube with a possibility of extraction from it, and a scale plate, installed on one of the horizontal shoulders and in this position being in interaction with the said arrow in the tube. For more accurate installation the base and the horizontal shoulders are provided with a measuring scale.

EFFECT: invention provides opportunity of operative conversion of the X-ray pattern into the 3D surgical template in a patient's presence, reduction of planning duration and cost of transgingival implantation.

6 dwg

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