Method for dental restoration and method for making restoration material

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine, namely to dentistry, and may be used for making a restoration material used for partial dental restoration in the oral cavity. That is ensured by placing the first cell mass formed by mesenchymal or epithelial cells/cell and a second cell mass formed by other mesenchymal or epithelial cells/cell on a carrier. One of the mesenchymal or epithelial cells is made from a dental germ, and the above cell masses are placed in tight contact to each other, but not mixed. The above cell masses are grown to form the whole restored tooth or its germ. That is followed by localising the whole restored tooth or its germ grown that enables implanting the whole restored tooth or is germ within the lost tooth so that a dental crown is directed inside the oral cavity with the dental germ or the tooth being used as the restoration material for preparing an equivalent of the lost tooth within the lost tooth area.

EFFECT: group of inventions enables performing partial dental restoration within the lost tooth area by implanting the restored dental germ or the whole restored tooth, as prepared by the above method.

10 cl, 5 dwg, 2 ex

The technical FIELD

[0001] the Present invention relates to a method of restoring the area of the lost tooth and methods for producing a restorative material.

PRIOR art

[0002] the Tooth is an organ consisting of: enamel at the outer layer and dentin in the inner layer, which refers to solid tissues; odontoblasts forming dentin and the inside layer of dentin; the pulp of the tooth, filling the Central space. Teeth can be lost due to dental caries, periodontal disease and similar diseases. Given the large value of the presence or absence of teeth for appearance and for the perception of taste, and in order to preserve the health and quality of life have been developed various methods of tooth restoration.

[0003] Thus, in a publication in J. Dent. Res. (2002, Vol.81 (10), pp.695-700) described regeneration subatomnoy tissue in the intraperitoneal transplantation of mouse cells with a biodegradable carrier, in particular epithelial or mesenchymal cells isolated from early tooth development.

Another example: in Japanese published patent application (JP-A) No. 2004-331557 proposed method for regenerating tooth germ, in which the cells of the dental germ isolated from a living organism, grown in the presence of physiologically active substances, e.g. the p fibroblast growth factor. In another patent document, JP 2004-357567 And proposed a method comprising cells of at least one type selected from cells of early tooth development in vivo, and cells capable of differentiation with the formation of this type are grown together on the media containing fibrin. This media allows us to give the formed dental germ of a desired shape, thus forming a "tooth" with a unique morphology.

[0004] In WO 2006/129672 described method, according to which epithelial cells and mesenchymal cells derived from a tooth germ, is converted into cell mass, which is then placed in a collagen gel, bringing them into close contact with each other, and grow in these conditions, thus creating a tooth, with a unique arrangement of cells.

[0005] on the other hand, in document WO 2003/101503 described the way in which for the regeneration of tooth germ cells of the dental germ cells capable of differentiation with the formation of the appropriate type, grown with mechanical stimulation, as well as the modality in which the regenerated thus dental embryo transplanted into the jaw bone of the patient with a lost or damaged tooth Bud.

In addition, the document JP 2005-013261 As described artificial biomaterial particles of colloidal oxide cream is Oia, attached to its surface, is used as an implant material that is implanted into a living organism.

[0006] it is noted, however, that the introduction of the screw-shaped implant materials such as titanium, modern implant therapy suppresses the growth of the jaw bone during growth of the jaw and prevents movement of the tooth, which is undesirable. In addition, although the regenerated tooth to provide the same function as that of the regular tooth, requires the same normal occlusion, in the above-mentioned methods using cells isolated from a living organism, the pursuit of such occlusion has not been sufficiently confirmed. It should also be noted that the nerves of the periodontal ligament have sensitivity to damaging stimuli in the mouth (such as compression)that biologically important from the point of view of perception, for example, sensations from food. To the regenerated tooth had those same functions as regular tooth, you must restore the area of the missing tooth to such an extent that the regenerated tooth provided normal occlusion hardness, equal to the hardness of a normal tooth, and included the nerves, providing a normal sensitivity to stimuli acting on the tooth.

Summary of the INVENTION the MEANS to solve the aforementioned PROBLEMS

0007] Thus, the purpose of the present invention is to provide a method of manufacturing a restorative material to restore the area of the lost tooth, which will provide a regenerated tooth hardness, equal to the normal hardness of the tooth, which will allow to achieve normal occlusion, and sensitivity to stimuli, equal to the normal sensitivity of the tooth; and the way to restore the area of the lost tooth.

[0008] the Present invention provides a method of manufacturing a restorative material used to restore the area of the lost tooth in the mouth, and the way to restore the area of the lost tooth.

The first aspect of the present invention provides a method of manufacturing a restorative material used to restore the area of the lost tooth in the oral cavity, comprising the steps:

placed on the carrier of the first cell mass formed by the cells or by cells of mesenchymal or epithelial cells and a second cell mass formed another cell or other cells of mesenchymal or epithelial cells, one of mesenchymal or epithelial cells derived from a tooth germ and the first and second cell masses come in close contact with each other without mixing;

grown first and second is th cell mass with the formation of the rudiment of the restored tooth or the whole of the restored tooth and determine the orientation of the embryo of the restored tooth or the whole of the restored tooth, formed in cultivation that allows you to embed a root of the restored tooth or a restored tooth so that the crown part of the tooth was directed inside the mouth and

use the tooth germ or tooth, the orientation of which was defined as a restorative material to obtain the equivalent of a lost tooth in the area of the lost tooth.

[0009] the Second aspect of the present invention provides a method of restoring the area of the lost tooth in the oral cavity, comprising the steps:

placed on the carrier of the first cell mass formed by the cells or by cells of mesenchymal or epithelial cells and a second cell mass formed another cell or other cells of mesenchymal or epithelial cells, one of mesenchymal or epithelial cells derived from a tooth germ, and the first and second cell masses come in close contact with each other, without mixing;

grow first and the second cell mass with the formation of the rudiment of the restored tooth or the whole of the restored tooth and implement the principle of the restored tooth or the whole of the restored tooth in the area of the lost tooth.

BRIEF DESCRIPTION of DRAWINGS

[0010] On Figa presents pictures of the appearance of the tooth prima is and 2 of the present invention during extrusion (left), in the period from extrusion to occlusion (center) and in the period after occlusion (right), which were removed at an angle of 45° to transplantation in the upper jaw.

On FIGU presents pictures of the appearance of the tooth of example 2 of the present invention during extrusion (left), during the period from extrusion to occlusion (center) and in the period after occlusion (right), which were removed in the vertical direction relative to the location of the transplant in the upper jaw.

On Figs presents photographs of the occlusion of the teeth of example 2 of the present invention, taken during extrusion (left), during the period from extrusion to occlusion (center) and in the period after occlusion (right).

On Fig.1D presents the CT image the appearance of a tooth of example 2 of the present invention, taken during extrusion (left), during the period from extrusion to occlusion (center) and in the period after occlusion (right).

On Five presents the CT slice images (E) of the tooth of example 2 of the present invention, taken during extrusion (left), during the period from extrusion to occlusion (center) and in the period after occlusion (right).

On Figa presents a graph of Knoop hardness for tooth enamel after extrusion in example 2 of the present invention.

On FIGU presents a graph of the Knoop hardness of dentin after extrusion in example 2 of the present invention.

Figure 3 presents stained with hematoxylin and eosin image of the regenerated tooth of example 2 of the present invention immediately prior to extrusion (magnification ×20).

On Figa presents stained with hematoxylin and eosin image, confirming the phenomenon of bone reconstruction in bone in periodontal ligament (In: side compression With: side stretch), which occurred in the application of orthodontic force in example 2 of the present invention (magnification ×20).

On FIGU presents an enlarged view of the side of the compression zone In the black frame) on Figa (magnification ×200).

On Figs presents an enlarged view of the side of the stretch (area With the black frame) on Figa (magnification ×200). On Figa presents immunoalkaline image expression of the protein c-Fos in the medulla observed in the absence of orthodontic impact in example 2 of the present invention (magnification ×100).

On FIGU presents immunoalkaline image expression of the protein c-Fos in the medulla, the observed 2 hours after the application of orthodontic impact in example 2 of the present invention (magnification ×100).

On Figs presents immunoalkaline image expression of the protein c-Fos in the medulla observed after 48 hours after application of orthodontics the impact (magnification ×100).

On Fig.5D presents immunoalkaline image expression of the protein c-Fos in the medulla, the observed 2 hours after opening the pulp of the tooth (magnification ×100).

The PREFERRED IMPLEMENTATION of the INVENTION

[0011] under the present invention a method of manufacturing a restorative material relates to methods of manufacture of the material used to restore the area of the lost tooth in the mouth, and includes the steps:

placed on the carrier of the first cell mass formed by the cells or by cells of mesenchymal or epithelial cells and a second cell mass formed another cell or other cells of mesenchymal or epithelial cells, one of mesenchymal or epithelial cells derived from a tooth germ and the first and second cell masses come in close contact with each other without mixing;

grow first and the second cell mass with the formation of the rudiment of the restored tooth or the whole of the restored tooth (this step is referred to below as "the stage of formation of the rudiment of the restored tooth") and

determine the orientation of the embryo of the restored tooth or the whole of the restored tooth is formed by growing that allows you to embed the principle of the restored tooth or a whole in ostanavlivaemsya tooth so to the crown part of the tooth was directed inside the mouth (this step is referred to below as "the stage of orientation");

use the tooth germ or tooth, the orientation of which was defined as a restorative material to obtain the equivalent of a lost tooth in the area of the lost tooth.

[0012] also provided by the present invention a method of restoring the area of the lost tooth relates to a method of restoring the area of the lost tooth in the mouth and includes the steps:

placed on the carrier of the first cell mass formed by the cells or by cells of mesenchymal or epithelial cells and a second cell mass formed another cell or other cells of mesenchymal or epithelial cells, one of mesenchymal or epithelial cells derived from a tooth germ and the first and second cell masses come in close contact with each other without mixing;

grow first and the second cell mass with the formation of the rudiment of the restored tooth or the whole of the restored tooth (this step is referred to below as "the stage of formation of the rudiment of the restored tooth"); and

introduce the principle of the restored tooth or the whole of the restored tooth in the area of the lost tooth (this step is additional what is below as the "implementation phase").

[0013] According to the present invention a method of manufacturing the restorative material and the method of restoring the area of the lost tooth include the use as a restorative material, introduced into the area of the lost tooth, a germ of a restored tooth or a restored tooth that will be obtained at the stage of forming the rudiment of the restored tooth location of the first cell mass and the second cell mass on the carrier, and the first and second cell masses are in close contact with each other without mixing. Using the fact that in the area of the missing tooth as a restorative material will be introduced the germ of the restored tooth or a restored tooth, from implementing extruded equivalent of the tooth, acquired such length and hardness, which provide occlusion and tooth-antagonist, and the equivalent of the tooth can grow nerves. As a result, the equivalent of the tooth may have a hardness equivalent hardness regular tooth, that is the same as the surrounding teeth can provide normal occlusion sensitivity to incentives, equal to the sensitivity of the normal tooth.

[0014] In the present invention, the term "equivalent tooth" and the like denote the tooth having such length and hardness,which make possible occlusion and tooth-antagonist after extrusion from the area of the lost tooth, in which was embedded the specified value.

In the present invention, the expression "stage of the formation of the rudiment of the restored tooth" refers to the method of manufacturing the restorative material and the method of restoring the area of the lost tooth.

Further, the word "phase"used herein means not only a separate stage, but other stages, which cannot be clearly separated from him, and serve to achieve the desired effect of the considered phase.

In addition, ranges, marked with prepositions "from" and "to"include also the boundary numeric values are specified directly after these prepositions.

The following is a description.

[0015] In the present invention, the term "tooth" is denoted by a continuous fabric comprising an inner layer of dentin and the outer layer of enamel and with orientation, through the allocation of crown and root. The orientation of the tooth may be determined by the mutual location of the crown and root. The distinction between crown and root can be confirmed visually on the basis of their form, histological staining, etc. the Crown is the part in which there are layers of enamel and dentin, whereas in the root layer of the enamel is missing.

[0016] the Professionals in this field can easily recognize the dentin and enamel morfologicheskie histological staining or similar methods. Enamel can also be determined by the presence of ameloblasts, which can be confirmed by the presence/absence amelogenin. On the other hand, dentin can be defined by the presence of odontoblasts, which can be confirmed by the presence/absence of sialoprotein dentin. The presence amelogenin and sialoprotein dentin can be easily confirmed known in the field manner, for example by in situ hybridization and immunoablative antibodies, etc. And, finally, the orientation of the tooth can be determined by the location of the crown and root. The distinction between crown and root can be confirmed visually on the basis of their form, histological staining, etc.

[0017] In the present invention, the terms "tooth germ" and "dental flask" is used for precise designations corresponding to certain stages of development. In this case, the "tooth germ" refers to education at an early stage of tooth development, which in the future will turn into a tooth and development which includes a step flask and the stage of "the bells" in accordance with the usual periods of tooth development and, in particular, implies the presence of tissue in the condition in which no accumulation is observed in dentin and enamel, which is the characteristic components of hard tissues of the tooth. On the other hand, a dental flask" implies the presence of a certain tissue at p is ragogna stage from "tooth germ" in the sense of in which the term is used in the present invention, that is, indicates the stage at which begins the accumulation of dentin and enamel, characteristic of the hard tissues of the tooth, and prior to germination of the tooth from the gums, which are prerequisites to perform the typical functions of a tooth. Evolving from early tooth development, tooth passes the stage bulb, the stage of "the Cup"early stage "bells" and late stage "bells". At the stage bulb epithelial cells spaceways, forming a shell around the mesenchymal cells and at early and late stages of the "bell" of the epithelial of the turns in the outer enamel and of the mesenchymal part begins to form inner dentin. So in the intercellular interaction of early tooth development between epithelial and mesenchymal cells is formed a tooth.

[0018] In the present invention, the term "mesenchymal cell" refers to a cell derived from mesenchymal tissue, and epithelial cell" means a cell originating from epithelial tissue. Below in the present invention, the term "periodontal" means the alveolar bone and periodontal ligament, formed mainly in the outer layer of the tooth. Specialists in this field can easily recognize the alveolar bone and periodontal ligament morphologically by the histologist is ical staining or similar methods.

[0019] In the step of forming the rudiment of the restored tooth according to the present invention is placed on the carrier of the first cell mass formed by the cells or by cells of mesenchymal or epithelial cells and a second cell mass formed another cell or other cells of mesenchymal cells, one of mesenchymal or epithelial cells derived from a tooth germ, and the first and second cell masses are in close contact with each other without mixing; then the first and the second cell mass grown as a cell culture with the formation of the rudiment of the restored tooth.

With regard to the stage of forming the rudiment of the restored tooth, the corresponding method described in document WO 2006/129672, can be used in its original form without modification.

[0020] As the first cell mass and the second cell mass form or exclusively from mesenchymal cells, either exclusively of epithelial cells, and each of these cell masses essentially consists of cells of one of these types. The term "cell mass" refers to a dense formation, consisting of cells, and the cells can be either in a state of tissue, either in a state of mass (of aggregate)prepared from single cells. The expression "consists essentially of" the hereafter is denotes the minimum possible inclusion of substances, non-interest cells. The number of cells that make up every cell mass, varies depending on animal species, as well as the type, hardness and size of the carrier and may vary in General from 10¹up to 10⁸cells, preferably from 10³up to 10⁸cells per cell mass.

[0021] as for the mesenchymal cells and epithelial cells used in the present invention, at least one of the cells of these two types can be derived from a tooth germ, so they are able to reproduce the location of cells in a living organism and effectively form a tooth having a specific structure and orientation. For greater reliability in the formation of the tooth as mesenchymal and epithelial cells should preferably be derived from a tooth germ (or tooth germs). Tooth primordium take preferably at the stage of the flask or on the stage "bowl", so that the cells were immature and homogeneous with respect to their differentiation.

[0022] Mesenchymal cells released from a source other than early tooth development, can be taken from other mesenchymal tissues of a living organism and represent, for example, preferably, the bone marrow cells that do not contain blood cells, and mesenchymal stem cells, more PR is doctitle - mesenchymal cells from the oral cavity, bone marrow cells from the bone and mesenchymal cells are selected from cells of the cranial neural crest-derived mesenchymal precursor cells, which can be produced by mesenchymal cells and stem cells.

Epithelial cells released from a source other than early tooth development, can be taken from other epithelial tissues of a living organism and represent, for example, preferably epithelial cells of the skin, the mucous membranes of the mouth and gums, more preferably immature epithelial precursor cells, which upon differentiation can for example be creationsim or parakeratosis changes, forming the epithelial cells of the skin, mucous membranes, and the like. Examples of such immature epithelial precursor cells are nitratenitrogen epithelial cells and their stem cells.

[0023] the Tooth germ and other tissues can be distinguished in the jaw bone and similar sources a variety of different animals, such as primates (humans, apes), ungulates (pigs, cows, horses), that is, in mammals; rodents (mice, rats, rabbits), i.e. small mammals; dogs, cats, etc. For selection of early tooth development and tissue can be no change to the conditions applied, is commonly used for selection of fabrics; tooth Bud or cloth should be allocated under sterile conditions and stored in a suitable place. As early tooth development may be used, for example, the tooth germ of the third molars, also called the wisdom tooth or tooth Bud of the embryo, but from the point of view of the use of autogenous tissues using early tooth development tooth preferable. If you use a tooth Bud mouse, take it preferably 10-16 day development of the embryo.

[0024] the Preparation of mesenchymal and epithelial cells from this early tooth development perform, separated tooth germ isolated from the surrounding tissue, mesenchymal tissue and epithelial tissue on the basis of their form. In this procedure, to facilitate the selection of fabrics can be used enzymes. Examples of enzymes used for this purpose are dispute, collagenase and trypsin.

[0025] the Mesenchymal cells and epithelial cells can be prepared in the form of single cells from mesenchymal tissue and epithelial tissue. In order to facilitate the dispersion of the tissue to single cells, can be used such enzymes as dispose, collagenase and trypsin.

[0026] as the medium for cultivation can be used in the environment, typically used for cultivation of animal cells, for example the environment of Iglau modification of Dulbecco SIMD (DMEM), which to enhance cell proliferation can be added serum, or, in the alternative, the cell growth factor, for example fibroblastic FFR (FGF), EGF epidermal (EGF) or platelet-derived TGF (PDGF), or known components of the serum, such as transferrin. In that case, if added to the serum concentration may be appropriately changed depending on growing conditions and can usually be 10% by volume. When growing cells can be created usual in these cases, conditions such as those used for growing cells in incubators at 37°C and 5% CO₂. If necessary, you can also add antibiotics, such as streptomycin.

[0027] According to the present invention can be used medium suitable for the cultivation of cells and preferably represents a mixture with the above environment. Examples of such media are collagen, agarose gel, carboxymethyl cellulose, gelatin, agar, hydrogel, elastin, fibrin, fibronectin, laminin, extracellular matrices mixed polyglycolic acid ISC (PGA), polylactic acid PLA (PLA), copolymers of lactic and glycolic acids SMGC (PLGA), "Cellmatrix" (trade name, manufacturer Nitta Gelatin Inc.), "Mebiol Gel" (trade name, manufacturer Ikeda Scientific Co., Ltd.) and "Matrigel" (tor the TV title, manufacturer, Beckton, Dickinson and Company), which can also be used in combination. These carriers can have sufficient rigidity to cells effectively kept in those places where they were placed on a carrier, which may be in the form of a gel, fibers or solids. From the standpoint of ease provide the required rigidity, and the holding capacity of these carriers are preferred collagen, agarose gel, carboxymethyl cellulose, gelatin, agar, hydrogel, "Cellmatrix", "Mebiol Gel", "Matrigel", the extracellular matrices of mixed composition, elastin, fibrin, fibronectin and laminin, and combinations thereof; most preferred of these collagen, fibrin, fibronectin, laminin, extracellular matrices of mixed composition, "Cellmatrix", "Mebiol Gel", "Matrigel" and their combinations. These carriers provide good intercellular interaction between mesenchymal and epithelial cells within the corresponding cell mass and good interaction between cell mass. Given this level of rigidity, which helps to keep the cells in their places may be taken stiffness, which is usually used in three-dimensional cultures, i.e. rigidity, ensuring the preservation state of the cells and not hindering at the same time their hypertrophy due to growth. This size is dosti can easily be determined.

This media may have a thickness of not preventing growth within the first and second cell masses, and this thickness can be set in accordance with the size of this fabric and similar parameters.

[0028] in Addition, the media can have such holding capacity, which allows you to keep the contact state of the cells, preventing their dispersion. In this document, the term "contact state" refers to a dense (high density) state that provides intercellular interaction within each cell mass and between cell mass, and due to the condition of high density, in which the unit cells, it is possible to grow cells with such holding capacity, which, for example, provides contact state more powerful than the simple touch of cells. For example, when using collagen appropriate rigidity is provided at a final concentration of 2 to 3 mg/ml, i.e. at a concentration, which has a jelly strength from 120 to 250 g, measured according to JIS-K-1996 (as the load required to compress by 4 mm by using a rod with a diameter of 12.7 mm). This value is the strength of the jelly is not restrictive, and as the carrier of the present invention can preferably use Atisa and other types of media, if they have the same strength, determined by the same method of measurement. In addition, the media stiffness corresponding to the desired strength of the jelly can be obtained by mixing one or more different media.

[0029] the status of the high density means a density, which is almost equivalent to the density of the forming fabric, for example in the case of cell mass, it ranges from 5×10⁷up to 1×10⁹cells/ml at the location of cells on the carrier, preferably from 1×10⁸up to 1×10⁹cells/ml, which is required for intercellular interaction without negative effects on the activity of cells, and most preferably from 2×10⁸up to 8×10⁸cells/ml to prepare cell mass with a density of cells, it is preferable to condense and precipitate the cells by centrifugation, since it is thus possible to achieve a high density without negatively affecting the activity of the cells. Centrifugation can be performed with a speed of rotation which corresponds to the centrifugal force from 300 g to 1200 g, not adversely affecting the survival of cells, preferably from 500 g to 1000 g, for 3 to 10 minutes. Centrifugation with the power below 300 g can create a lack of precipitation cells, and the density of the cells would then be too low, whereas zentrifugenbau with power above 1200 g can cause damage to cells, therefore, these use cases are not classified as preferred.

[0030] In cases where a high density of cells is ensured by centrifugation, this process is usually performed by preparing a suspension of single cells in the container, for example a test tube, used for centrifugation of the cells, and the supernatant is removed carefully, leaving a residue composed of cells.

In those cases, when the precipitate is prepared by centrifugation, it can be placed directly inside the media. The volume of the other components, excellent prepared from cells (e.g., environment, culture, buffer solution, the media, and the like), preferably should not exceed the volume of the cells and most preferably, other components, other than the prepared cells were absent. In the cell mass of such high density cells are in close contact with each other, which enables efficient intercellular interaction. In those cases, when the cell mass with a very low content of components other than the prepared cells are placed inside carrier, cells are particularly highly compacted due to the curing media and similar processes, creating a condition in which the cells are very tightly.

[0031] the closer the contact between the first cell is the mass and a second cell mass, the better; especially preferably, the second cell mass rested in the first cell mass. In addition, to provide more intimate contact between cell mass, effective coverage of the first cell mass and the second cell mass of a solid membrane, permeable to the environment and culture of oxygen. Preferably also, when placed in suspension cells of high density in the solution is to use a solution that is different from the suspension viscosity, which subsequently can harden not undergoing changes that will allow you to save the contact to the cells. In those cases, when the first cell mass represents the mass of mesenchymal cells single tooth primordia, and the second cell mass represents the epithelial tissue of the dental germ, it is preferable to place the enamel knot epithelial tissue of a tooth Bud so that it is in contact with the first cell mass, but the present invention is not limited to this approach.

[0032] If it is a carrier in the form of a gel, solution or similar form, the location of the cells on the carrier may provide for the subsequent stage of the curing medium. For curing medium may apply conditions used for curing media in General, without any changes. So, if you wear the El is used capable of curing the composition, for example, collagen, curing can be achieved with commonly used terms, such as during curing at a temperature of cultivation for from several minutes to several tens minutes. This will set between the cells in medium dense and stable adhesion.

[0033] the Time required for the formation of the rudiment of the restored tooth in the growth process, depending on the number of cells placed on a carrier, from the state of the cell mass, the growing conditions and the species of animal, and may be preferably at least one day, more preferably not less than 3 days. The rudiment of the restored tooth or restored tooth obtained by cultivation during this time, you may ekstrudirovaniya in the form of a functional tooth immediately after its introduction in the area of the lost tooth.

In addition, if you extend the time of growing, it will continue the process of forming the rudiment of the restored tooth, i.e. the accumulation of dentin and enamel, the formation of crown and root development. Thus, the cultivation in the formation of the rudiment of the restored tooth can be adjusted depending on conditions of cultivation, the species status of the embryo of the restored tooth and the like, for Example, when growing organ cult of the market, below, you can proceed to the implementation phase after 7 days or in some cases after 6 days of cultivation. In other cases, depending on the status of the embryo of the restored tooth or by using another method of growing the cultivation period may last more than 30 days or in some cases more than 50 days, 100 days, or more than 300 days.

[0034] the Growing media can be carried out either only with the carrier containing the first and the second cell mass, or in the presence of other animal cells.

If growing on the media is the only media that may apply conditions commonly used for the cultivation of animal cells. In this case, as the General conditions for the cultivation of animal cells, and the above-mentioned conditions can be applied without any changes. However, in addition to the culture can be added serum mammals and various factors contributing to the growth and differentiation of these cells. Such factors may constitute, for example, (FGF) and bone morphogenetic protein (BMP).

[0035] in Addition, from the point of view of gas exchange and supply of tissues and cell mass nutrients and taking into account the fact that the whole process of preparation can be carried out in vitro without contact with other animals cells, versiani the media preferably runs as a growing organ culture. Usually in organ culture, the cultivation is carried out by overlaying the porous membrane on the environment, culture, suitable for cultivation of animal cells, and areas on the membrane of the first and second cell masses embedded in the media. The porous membrane preferably is a membrane with a large number of pores with a diameter of from 0.3 to 5 μm. Examples of such membranes are Cell Culture Insert" (trade name) and "Isopore Filter" (trade name). The cultivation of organ culture may be approximately from 1 to 7 days, preferably from 3 to 6 days.

[0036] If the growing medium is performed in the presence of other animal cells, the tooth with the specific arrangement of cells may be formed at an early stage under the influence of various cytokines and other similar factors from animal cells. Cultivation in the presence of other animal cells can be performed in vivo using dedicated or cultured cells.

[0037] as animals in this process, preferably mammals such as humans, pigs, mice, and it is preferable to use the same view, from which was obtained a fabric early tooth development, or other type whose status was changed and given to the immunodeficiency. If you are using a living organism, to p is impactfully species transplantation in the body are transplantation in potochechnoi capsule, in mesenteric and subcutaneous tissue transplantation, which allow the organ or tissue, consisting of animal cells, to develop more normally. The cultivation in the presence of animal cells can be adjusted accordingly depending on the origin of cells, growing conditions, species of animal, which was performed transplantation, and the like, as in the case of organ culture as described above.

[0038] Due to such a stage of the formation of the rudiment of the restored tooth can be obtained tooth germ or tooth arrangement (structure) cells, specific for a tooth, that is, having the dentin inside and enamel outside. In addition, the tooth germ or tooth will also be preferable to have a desired orientation, i.e. it will include the crown and the tooth root.

As the first cell mass and the second cell mass preferably represent a mass of single cells, as it allows you to get a unit consisting of several teeth, with specific teeth arrangement of cells. When such a unit is received, before using him separate individual teeth.

[0039] In the step of determining the orientation in the method of manufacturing the restorative material of the present invention is the determination of the orientation of the tooth C is Chatka or tooth, obtained in the above step of forming the rudiment of the restored tooth that allows you to embed the tooth germ or tooth in the area of the missing tooth so that the crown part of the tooth was directed inside the mouth.

[0040] Under the orientation of the tooth germ or tooth refers to the orientation of the attached dental germ or tooth at the introduction into the area of the lost tooth. If it is detected the formation of crowns, ensure that the crown was directed inside the mouth. If the formation of the crown is not found, ensure that the inside of the mouth was referred that part of the layer of epithelial cells, which corresponds to the crown, or the layer of epithelial cells in the embryo of the restored tooth. Alternatively, you can ensure that the open part of the layer of epithelial/mesenchymal cells of the embryo of the restored tooth was directed in the direction opposite to the oral cavity. If you put a tooth germ or tooth in the area of the lost tooth under his orientation is determined, as described above, the tooth will be directed inside the oral cavity, allowing the orientation of this tooth will coincide with the orientation of the surrounding teeth.

[0041] the Tooth germ or tooth, the orientation of which was determined during orientation, is used as vos is canonicalname material to obtain the equivalent of a lost tooth in the area of the lost tooth.

To this end restorative material containing tooth germ or tooth, after determining the orientation of the implement in the area of the lost tooth. After that, the tooth germ or tooth restoration material begins to grow in the area of the lost tooth, and after a while extruded as the equivalent of the tooth. After extrusion tip (tubercle) equivalent of the tooth reaches almost the same position, which is occupied by the surrounding teeth (line occlusion), and does not grow beyond this position, although the situation may be different depending on condition, size, etc. of a tooth germ or tooth restorative material. The time during which the tooth germ or tooth remains embedded before extrusion and occlusion, different animal species and depends on the conditions in which is embedded the tooth germ or tooth.

[0042] After the extrusion, the equivalent of the tooth has a hardness close to the hardness of the natural tooth. Hardness is a measure that indicates the degree of deformation of the dentin and enamel of the equivalent of the tooth under compression, abrasion and/or similar processes, and can be determined by measurement of Knoop hardness. The normal enamel of the tooth adult mouse Knoop hardness ranges from 300 to 600, preferably from 300 to 500 units, and normal dentin hardness is about Hardness is 60 to 120 units. The equivalent of the tooth is obtained according to the present invention has a Knoop hardness in the same range. This ensures his chewing function similar to the functions of a normal tooth.

[0043] After the extrusion, the equivalent of the tooth has, in addition to enamel and dentin, dental pulp and periodontal ligament, i.e. has a fabric composition similar to the composition of the normal tooth. Because the tooth has periodontal ligament, it can grow nerves, which provides sensitivity to damaging stimuli in the teeth, such as compression. Such tissue composition can be confirmed visually or by histological analysis, immunostaining, analysis of gene expression or similar known methods.

[0044] Thus, the restorative material obtained by the method of manufacturing the restorative material of the present invention, can be used as source material equivalent of the tooth, the length and the hardness of which provide occlusion and tooth-antagonist.

[0045] At the implementation phase in the method of restoring the area of the lost tooth according to the present invention the tooth germ or tooth obtained at the stage of forming the rudiment of the restored tooth, described above, are implemented in the area of the missing tooth as a restorative material.

As for the direction the program implementation, the tooth germ or tooth is preferably placed as well as at the stage of determining the orientation of the above method of manufacturing a restorative material, that is, the crown sent inside the mouth, if formed crown is detected, or part of a layer of epithelial cells, corresponding to the crown, or the layer of epithelial cells in the embryo of the restored tooth is directed to the inside of the mouth, if the formation of the crown is not detected. In an alternative embodiment, the open portion of the layer of epithelial/mesenchymal cells of the embryo of the restored tooth is directed in the direction opposite to the oral cavity. Due to this, the crown equivalent of the tooth is oriented inside the oral cavity, i.e. the orientation of the equivalent of the tooth will coincide with the orientation of the surrounding teeth.

[0046] the size and depth of the area of the missing tooth are usually not restricted, insofar as this area is formed in the gums during tooth extraction or similar procedure. On the shape of the area of the lost tooth is also not limited in any way. As soon as the it will be possible to implement the regenerated tooth germ in this area, all these parameters can be appropriately adjusted depending on the nature of the area, the type of animal, type of the restored tooth, etc.</>

The area of the lost tooth is usually located in the jaw bone, the alveolar ridge of the mouth, etc. If as a result of tooth loss weight alveolar ridge has decreased, it is possible to increase the bone regeneration with the use of known methods used clinically for introduction of the implant, for example directed tissue regeneration STD (GTR). The premise of the tooth germ or tooth in the area of the holes is preferably accompanied by suturing or similar techniques according to usual methods.

[0047] subsequently implemented in the area of the lost tooth tooth primordium grows and turns into a tooth, passing the stage of extrusion as the equivalent of the tooth. Just as it was described above for the method of manufacturing a restorative material, the edge (tubercle) equivalent of the tooth after the extrusion reaches almost the same position, which is occupied by the surrounding teeth (line occlusion), and does not grow beyond this position, although the situation may be different depending on condition, size, etc. introduced early tooth development. This makes it possible to increase the length of the tooth to a value, which will be provided by the occlusion and tooth-antagonist. The time during which the tooth germ or tooth remains embedded before extrusion and occlusion, different types of LM is now and depends on the conditions in which is embedded the tooth germ or tooth.

[0048] Similar to as described above for the method of manufacturing a restorative material, the equivalent of the tooth after the extrusion has a hardness close to the hardness of the natural tooth. Hardness is a measure that indicates the degree of deformation of the dentin and enamel of the equivalent of the tooth under compression, abrasion and/or similar processes, and can be determined by measurement of Knoop hardness. The normal enamel of the tooth adult mouse Knoop hardness ranges from 300 to 600, preferably from 300 to 500 units, and normal dentin Knoop hardness is 60 to 120 units. The equivalent of the tooth is obtained according to the present invention has a Knoop hardness in the same range. This ensures his chewing function similar to the functions of a normal tooth.

[0049] Further, similar to the way it was described above for the method of manufacturing a restorative material, the equivalent of the tooth after the extrusion has in addition to enamel and dentin to the pulp of the tooth and periodontal ligament, i.e. has a fabric composition similar to the composition of the normal tooth. Because the tooth has periodontal ligament, it can grow nerves, which provides sensitivity to damaging stimuli in the teeth, such as compression. This tissue is left can be confirmed visually or by histological analysis, immunostaining analysis of gene expression or similar known methods.

[0050] the Method of the present invention to restore the area of the lost tooth involves the use of a tooth germ or tooth restored, as described above, therefore, in the recovery area of the lost tooth can be achieved with normal occlusion and sensitivity to damaging stimuli, equivalent to the same characteristics of normal teeth. Due to this the equivalent of the tooth, regenerated in the area of the lost tooth, performs the function of the tooth and the restored state can be maintained for a long time.

This allows you to maintain the quality of life, and this method can be successfully used to meet the aesthetic needs. In addition, as it is possible to prevent the deterioration of health associated with the presence in the mouth area of the lost tooth, it is possible to maintain a healthy condition of animals-mammals, such as livestock, including cows, horses and pigs, and animals, home maintenance, including dogs and cats.

EXAMPLES

[0051] Next will be described examples of the present invention, however, the scope of the present invention is not restricted by them. Percentages in the examples are listed by weight (mass), if n is specified otherwise.

[0052] Example 1

(1) Preparation of epithelial and mesenchymal cells of the dental germ

With the aim of forming a tooth recovered early tooth development. This experiment was conducted on a mouse.

Fabric tooth germ of a lower molar in a known manner isolated from mouse embryos of C57BL/6N (purchased from CLEA Japan, Inc.) with the period of embryonic development of 14.5 days under the microscope. Fabric tooth germ of the lower molar teeth were washed in phosphate buffer solution (PBS(-)) without CA²⁺/MD²⁺and within 12.5 minutes was treated at room temperature the enzyme solution, prepared by adding dispute II (Roche, Mannheim, Germany) to a solution of PBS(-) to a final concentration of 1.2 units/ml Then the fabric of the dental germ washed three times in medium SIMD (DMEM) (Sigma, St. Louis, Missouri, USA) supplemented with 10% serum of newborn calves (FCS) (JRH Biosciences, Lenexa, Kansas, USA). After this dispersion tissue of a tooth germ was added to the solution Gnkazy I (Takara, Shiga, Japan) to a final concentration of 70 units/ml, after which the epithelial and mesenchymal tissue of the tooth germ has separated from each other by using the injection needle 25 gauge (Terumo, Tokyo, Japan).

[0053] To obtain the epithelial cells of the epithelial tissue of the tooth germ, selected as described above, washed three times with a solution of PBS(-) and twice for 2 minutes, treated at 37°C. the enzyme solution, prepared with addition of collagenase 1 (Worthington, Lakewood, NJ, USA) in PBS(-) to a final concentration of 100 units/ml of Cells was collected by sedimentation using a centrifuge and within 5 minutes was treated with 0.25% trypsin solution (Sigma) in PBS(-) at 37°C. the cells washed three times in medium SIMD (DMEM) supplemented with 10% fetal calf serum, and then thereto was added a solution Gnkazy I to a final concentration of 70 units/ml by pipetting took epithelial cells of one tooth Bud.

To obtain mesenchymal cells mesenchymal tissue of the dental germ washed three times with a solution of PBS(-) and treated with a solution of PBS(-)containing 0.25% trypsin (Sigma) and 50 units/ml collagenase I (Worthington). To the resulting added Tnkase I (Takara) to a concentration of 70 units/ml, after which the pipetting took mesenchymal cells of one tooth Bud.

[0054] (2) preparation of the rudiment of the restored tooth

Following this, using epithelial and mesenchymal cells of the dental germ, prepared as described above was carried out restoration of the tooth Bud. Epithelial cells of the dental germ or mesenchymal cells of the dental germ, suspended in the environment SIMD (DMEM) (Sigma), was placed in microprobing 1.5 ml (Eppendorf, Hamburg, Germany) coated with silicone grease, which was added 10% fetal those who Yak serum FCS (JRH). After that, cells were recovered by precipitation with the aid of a centrifuge. After centrifugation the supernatant liquid medium culture was removed to the maximum extent possible, and then centrifugation was performed again and completely deleted the environment, culture, remaining at the cellular precipitate, under a stereoscopic microscope using microdontia GELoader of 0.5-20 µl (Eppendorf). After that, the cells were dispersively light shaking or pipetting to obtain cell - material for the preparation of a germ of a restored tooth.

[0055] In a Petri dish coated with silicone grease dropwise added 30 μl of "Cellmatrix" type I-A (Nitta gelatin, Osaka, Japan)to form a drop of the collagen gel. In this solution introduced from 0.2 to 0.3 μl of the above-mentioned epithelial or mesenchymal cells of the dental germ using microdontia of 0.1-10 (Quality Scientific plastics)to prepare the unit cells (5×10⁴cells/aggregate). To form the rudiment of the restored tooth, in the same way introduced and epithelial cells, resulting in preparation of unit cells they were in contact with the unit mesenchymal cells of the dental germ, prepared as described above. This helped to prepare the embryo, in which both types of cells were in close contact with each other.

[0056] the Embryo vosstanavlivat the constituent of the tooth, the prepared gel was left in CO₂-the incubator for 10 minutes for curing "Cellmatrix I-A (Nitta Gelatin). Was prepared vessel with the culture in which the environment SIMD (DMEM) (Sigma) supplemented with 10% fetal calf serum FCS (JRH) was in contact with the membranes of Cell Culture Insert (polyethylene terephthalate membrane with pore size of 0.4 μm; BD, Franklin lake, NJ, USA). The unit cell together with the surrounding gel as the carrier moved in the vessel with the culture on the membrane of Cell Culture Insert" and grew up there organ culture for 18-24 hours. At the end of this period the growing organ culture continued in "Cell Culture Insert"to analyze the development of the tooth.

[0057] (3) separation Technique to separate the beginnings

From the second to the fifth day of cultivation organ culture the embryo of the restored tooth, which has developed several dental germs, subjected to surgical division into separate beginnings under a stereoscopic microscope using a needle and tweezers. In a Petri dish coated with silicone grease dropwise added 30 μl of "Cellmatrix" type I-A (Nitta gelatin, Osaka, Japan)to form a drop of the collagen gel. Each selected separately tooth germ was placed in this solution and left in the CO₂-the incubator for 10 minutes for curing "Cellmatrix I-A (Nitta Gelatin). Was th who sponsored the vessel with the culture, in which environment SIMD (DMEM) (Sigma) supplemented with 10% fetal calf serum (JRH) was in contact with the membranes of Cell Culture Insert (polyethylene terephthalate membrane with pore size of 0.4 μm; BD, Franklin lake, NJ, USA). Allocated separately tooth primordium together with the surrounding gel as the carrier moved in the vessel with the culture on the membrane of Cell Culture Insert" and grew up there organ culture for 18-24 hours.

[0058] (4) Transplantation in the mouth

With allocated separately early tooth development, prepared as described above, surgically removed the surrounding gel using a needle and tweezers. Allocated separately dental embryo transplanted into the alveolar bone in the area of the first molar (M1) in the upper jaw mouse C57BL/6 aged 7 to 10 weeks without transplantation in potochechnoi capsule. In accordance with the present invention separate the tooth in the oral cavity was brought to extrusion, to ensure that functions such as chewing.

[0059] Example 2

(1) Preparation allocated separately early tooth development

Organ culture was grown in the same manner as in Example 1, steps (1) and (2), and from the second to the fifth day of cultivation, the rudiment of the restored tooth, which has developed several dental primordia, were subjected to the surgical division at otdelnosti under a stereoscopic microscope using a needle and tweezers. To get the selected separately dental embryo in a Petri dish coated with silicone grease dropwise added 30 μl of "Cellmatrix" type I-A (Nitta gelatin, Osaka, Japan)to form a drop of the collagen gel. Each selected separately tooth germ was placed in this solution and left in the CO₂-the incubator for 10 minutes for curing "Cellmatrix I-A (Nitta Gelatin). In prepared container of culture medium SIMD (DMEM) (Sigma) supplemented with 10% fetal calf serum FCS (JRH) was in contact with the membranes of Cell Culture Insert (polyethylene terephthalate membrane with pore size of 0.4 μm; BD, Franklin lake, NJ, USA). Allocated separately tooth primordium together with the surrounding gel as the carrier moved in the vessel with the culture on the membrane "Celt Culture Insert" and grew up there organ culture for 5 days. When he emerged a few rudiments of the restored teeth, they were surgically separated at the beginnings of the teeth. After growing embryo surrounding the gel was removed surgically using a needle and tweezers and transplanted tooth root in the alveolar bone in the area of the first molar (M1) in the upper jaw mouse C57BL/6 to 8 weeks of age.

[0060] (2) the Technique of transplantation in the mouth

Three days before transplantation 8-week mouse 57BL/6 after inhalation anesthesia diethyl ether was injected intraperitoneally with saline at a dose of 240 μl/20 g body weight, containing 5 mg/ml pentobarbital sodium. From the upper jaw of the mouse under anesthesia with pliers pulled the top of the M1, was convinced that remained unremoved root, and stopped the bleeding absorbent cotton. To provide food, mice were daily fed powdered food. To prepare the mouse C57BL/6 without M1, it was observed not less than 3 weeks to heal the wounds of the extracted tooth.

[0061] the Mouse C57BL/6 without M1, prepared as described above was subjected to inhalation anesthesia with diethyl ether and introduced her intraperitoneally with saline at a dose of 240 μl/20 g body weight, containing 5 mg/ml pentobarbital sodium. The mouse under anesthesia recorded on the back of the breakout table, while the upper and lower jaw fixed with rubber bands or threads, so his mouth was wide open. In the gum at the alveolar ridge in the area of the upper M1 made an incision with a scalpel and turned the gums together with the periosteum to expose the lower jaw. Using direct drill and ultrafine drills prepared in the lower jaw, corresponding to M1, the hole diameter of 1 mm, not touching while the maxillary cavity, and the aperture is selected individually transplanted tooth primordium. After transplantation, the wound was closed peristyle-gingival flap and sewed with a thread attached is her needle. To control the orientation of the selected separately early tooth development, prepared for transplantation, bump this early tooth development was marked with methylene blue and transplant the embryo has established this point in the direction of extrusion. After transplantation 8-week-old mice of the C57BL/6 daily fed powdered food.

[0062] the Mouse C57BL/6, which as described above was transplanted individual tooth primordium, introduced intraperitoneally with saline at a dose of 240 μl/20 g body weight, containing 5 mg/ml pentobarbital sodium. The mouse under anesthesia recorded on the back of the breakout table, while the upper and lower jaw fixed with rubber bands, so his mouth was wide open. The regenerated tooth, the distal region M2 and M3 and the adjacent gingiva was observed before extrusion, immediately prior to extrusion, directly after extrusion, during extrusion, and when the tooth has reached the surface of the occlusion. The results of observations are presented in figure 1. On Figa-1E left image shows the state during extrusion from the period before extrusion until directly after extrusion); average image shows the state during the period occurring after extrusion to the occlusion; and the right image shows the state after occlusion. On each image the clusters Figa-1E regenerated tooth presented in the right part of the image.

[0063] Approximately the 20th to the 50th day after transplantation could be observed tubercle of the regenerated tooth in the gum area corresponding to M1, which indicates the start of the extrusion process. Over time the height of the crown of the regenerated tooth is increased, and this tooth has reached the line of occlusion. Reaching the line of occlusion, the regenerated tooth was in the position of bugorkova-fissure contact with the tooth-antagonist (see Figa-1C).

These results indicate that the regenerated tooth is in a state of stable occlusion is similar to a normal tooth and capable of chewing function.

[0064] (3) Analysis using computed microtomography

Head with the upper jaw, which was transplanted allocated separately tooth germ was removed and the whole was placed in 4%paraformaldehyde in phosphate buffer for 12 hours, then made visualization using computer tomography device inspeXio SMX-90 CT (production Shimadzu Corporation). Visualization was performed with the following parameters: number of slices - 600; average - 10; scans - 1; size of 512×512; scale:1 : 50; slice thickness - 1; without correction for increasing the rigidity of radiation. Data obtained through computed tomography, were treated by three-dimensional reconstruction using the software Imaris (the production Zeiss) to prepare microtomographic images in the transverse projections.

[0065] as a result microtopographical analysis was set as shown in Fig.1D and 1E, in the extrusion process the regenerated tooth during root development is moved in the longitudinal direction and extruded from the alveolar crest, and then reaches the line of occlusion. Between the root section and the alveolar bone was observed cavity corresponding to the periodontal ligament and the transverse projection reveals the presence of the pulp chamber and apical opening holes in the top of the root, like a normal tooth. It was found that the regenerated tooth has reached bugorkova-fissure contact with the tooth-antagonist in the state of occlusion. These results show that in the extrusion process the regenerated tooth moves along the alveolar ridge in the longitudinal direction, while maintaining periodontal ligament and pulp chamber and reaches the line of occlusion, as with conventional tooth.

[0066] Similarly, in the head, placed in 4%paraformaldehyde in phosphate buffer, opened the molars M3 upper and lower jaw and made the inside of the oral cavity in a known manner snapshots in Central occlusion. The horizontal angle was determined for plane between the surfaces of the occlusion M3 lower and lower M1 and the vertical angle to overlap the anterior buccal bug the RCA and anterior lingual tubercle of lower M1 when viewed from the side.

[0067] as a result, it was confirmed that the regenerated tooth was in a state of occlusion with distal tubercle of lower M1, creating a flat surface occlusion with the upper M2 and M3. This allowed to conclude that the regenerated tooth after extrusion had a stable line of occlusion, necessary to establish the desired position of the lower jaw.

[0068] (4) Knoop Hardness

After the restoration of individual teeth, the development of which in the mouth proceeded as described in Example 2 (1), was performed measuring the hardness of his enamel and dentin compared with the same tooth is not less than three positions.

The measurement was performed by using a microhardness tester (NM-102 production Mitutoyo Corporation)equipped with a diamond indenter to measure Knoop hardness in the form of a quadrangular pyramid with vertical angles between the opposite faces of 172°30' and 130° (WAA production Mitutoyo Corporation). The indenter was pressed to the regenerated tooth with a load of 10 g for 10 seconds to determine the hardness along the length of the longest side of the diamond-shaped imprint 7,11:1. The regenerated tooth was fixed on a metal plate using denture curing resin (Unifast III production GC CORPORATION). Measurement of the hardness of the enamel was performed for the site, parallel to the earth's surface; measuring the hardness of dentin done is whether after as the test surface exposed by the cutting tooth in the horizontal direction using the drill (ULTIMATE 500 production NAKANISHI INC.). The results of measurement of Knoop hardness is shown in Figure 2.

[0069] As shown in figure 2, the normal enamel of the tooth Knoop hardness averages 341,083 units at the age of 3 weeks, the rate of 457.5 units at the age of 6 weeks and 436 units at the age of 9 weeks, and the enamel of the regenerated tooth Knoop hardness averaged 469,81 units (see Figa). In normal dentin of the tooth Knoop hardness averages 66,87 units at the age of 3 weeks, 76,53 units at the age of 6 weeks, and 88,58 units at the age of 9 weeks, whereas the regenerated dentin of the tooth Knoop hardness averaged 81,83 units (see Figv). Thus, it was recognized that the regenerated tooth has the required hardness to perform normal chewing function.

[0070] (5) Histological analysis

From approximately the 20th to the 50th day after transplantation could be observed tubercle of the regenerated tooth in the gum area corresponding to M1, which was indicated at the beginning of the extrusion process. To monitor the extrusion removed the upper jaw before extrusion, the upper jaw immediately prior to extrusion, the upper jaw directly after extrusion, the upper jaw in the extrusion process and Verhny the jaw where the tooth line of occlusion. The upper jaw was placed in 4%paraformaldehyde in phosphate buffer for 16 hours and decalzinirute 22.5%formic acid for 72 hours, then poured into paraffin in a known manner and has prepared a slice thickness of 10 μm. For every two of the upper jaw was spent 50 ml decalcifying fluid, and its complete replacement by decalcomania performed every 48 hours. Histological analysis was performed by staining with hematoxylin and eosin in a known manner.

[0071] figure 3 shows a regenerated tooth immediately before extrusion (white arrow). Figure 3 you can see the fabric structure equivalent to the structure of normal tissue of the tooth and including enamel, dentin, pulp, and periodontal ligament. In the relevant tissues enamel includes located close to each other enamel prisms, and dentin includes dentinal tubules. The periodontal ligament has sufficient thickness and has a structure that allows you to amortize the masticatory load. During extrusion, it was noted that the regenerated tooth during root development moved in the longitudinal direction and abstrogirovalsya of the alveolar ridge, subsequently reaching the line of occlusion. This process has not damaged periodontal ligament, which indicates that the regenerated tooth harmoniously combined with periodontal.

Because the tooth is connected with the surrounding alveolar bone by the periodontal ligament, the impact on the tooth orthodontic forces will lead to the transmission of mechanical stresses to the environment through the periodontal ligament. On the site of the periodontal ligament, compressible orthodontic force is demineralization of bone by osteoclasts, and on the site of the periodontal ligament, undergoes stretching occurs osteogenesis through osteoblast, which allows to keep the distance between the tooth and the alveolar process of the constant. To evaluate these functions of the periodontal ligament, the regenerated tooth was applied orthodontic force and subsequent analysis confirmed the rebuilding of bone, which included the appearance of osteoclasts on the compression side and the appearance of osteoblasts on the side of the tension caused by the normal movement of the tooth.

[0073] From the Nickel-titanium wire with diameter of a 0.012 inch (orthodontic wire VIM-NT, round, OralCare) prepared orthodontic wire for buccal movement, and one end of each wire curved in the form of a loop that you can hook over the neck of any incisor in the upper jaw. Mouse C57BL/6, which as described above was transplanted individual tooth primordium, was administered intraperitoneally the physiological rastvorov dose of 240 μl/20 g body weight, containing 5 mg/ml pentobarbital sodium. The mouse under anesthesia recorded on the back of the breakout table, while the upper and lower jaw fixed with rubber bands or threads, so his mouth was wide open. The end of the wire opposite the loop, made long enough so that this end can reach the distal section extruded tooth. Loop orthodontic wire for buccal move hooked on the neck of the randomly selected incisors in the upper jaw, securing the other end of the wire with the buccal side of the neck of the tooth intended for orthodontic therapy. Then loop stuck with UNIFIL FLOW (light-cured resin production GC CORPORATION). The end of the wire attached to the buccal side of the neck of the tooth, moved on the lingual side of the neck of the tooth to create orthodontic force. Following this, for the study of the realignment of the bones caused by periodontal ligament was performed histological analysis conducted on day 6 after orthodontic therapy, when rebuilding the bones of the most notable. The results are presented on Figa-4C.

[0074] to evaluate the sensitivity of the periodontal ligament of the regenerated tooth to mechanical stress, this tooth has applied orthodontic force that causes displacement in the buccal direction, and studies which followed fabric painting rebuilding of bone by staining with hematoxylin and eosin.

With buccal side was marked compression of the periodontal ligament, in which the fibers of the periodontal ligament were closed due to the narrowing of the periodontal space (see the area In a black frame on Figa and Figv). And, on the contrary, the opposite, i.e. lingual, side periodontal space has expanded, causing stretching of the periodontal ligament, in which the fibers of the periodontal ligament stretched (see the area in a black frame on Figa and Figs). In addition, as was discovered single layer of cells aligned along the walls of the alveolar ridge by stretching the periodontal ligament, the conclusion was made about the appearance of osteoblasts, which form bone (see the arrows on Figs). By compression, the opposite side of the tension in the alveolar ridge were found multinucleated giant cells with multiple nuclei in one cell body and a cavity formed by resorption of bone tissue, from which it was concluded that the formation of osteoclasts (see the arrows on Figv) and bone resorption.

Based on this, it was recognized that the regenerated tooth mechanical stress, such as orthodontic force to cause a response of the periodontal ligament in the form of a realignment of the bone, as in a normal tooth.

[0075] (8) Analysis of pain irritation

Well Izv the STN, what in orthodontic therapy and similar measures pain due to compression of the teeth, and found that the pain passed mainly nerves periodontal ligament and that this increases the production of the protein C-Fos in the nerve cells of the caudal padyatra spinal nucleus of the trigeminal nerve (dorsal horns of the spinal cord) (MR Byers. et al. // Crit Rev Oral Biol Med. 10, 4-39, 1999; Deguchi T. et al. // J Dent Res. 82:677-681, 2003; Fujiyoshi, Y. et al. // Neuroscience Letters. 283, 205-208, 2000). In addition, it is well known that therapy with the opening of the pulp of a tooth causes pain due to exposure of the dental pulp, and found that, as in the case of pain during orthodontic therapy, pain transmit primarily the nerves of the tooth pulp, and that this increases the production of the protein c-Fos in the nerve cells of the caudal padyatra spinal nucleus of the trigeminal nerve (dorsal horns of the spinal cord) (MR Byers. et al. // Crit Rev Oral Biol Med. 10, 4-39, 1999; Deguchi T. et al. // J Dent Res. 82:677-681, 2003). Thus, for analysis of functions of the regenerated nerves in the tooth restored tooth mouse was subjected to compressive impact, similar to that used in orthodontic therapy, and was completed with the opening of the pulp of the tooth by drilling holes in the dentin using a dental drill. When this was investigated, cause these impacts increase in expression of the protein c-Fos in audlem padyatra spinal nucleus of the trigeminal nerve (dorsal horns of the spinal cord).

[0076] the Mouse C57BL/6, which as described above was transplanted individual tooth primordium, introduced intraperitoneally with saline at a dose of 200 μl/20 g body weight, containing 5 mg/ml pentobarbital sodium. The mouse under anesthesia recorded on the back of the breakout table.

In two hours and 48 hours after activation of the orthodontic force as described above, as well as two hours after opening the pulp of the tooth, C57BL/6 were subjected to perfusion fixation with 4%paraformaldehyde in phosphate buffer. Selected the medulla was placed in 4%paraformaldehyde in phosphate buffer for 16 hours and signed in the casting Wednesday OCT (Miles Inc., NAPERVILLE, Illinois, USA) in a known manner, after which the prepared slices with a thickness of 50 μm using a cryostat (Leica, Wetzlar, Germany). The prepared sections were subjected to immunoablative using C-Fos (SANTA CRU Z BIOTECHNOLOGY, INC., Santa Cruz, CA, USA) as primary antibodies, rabbit immunoglobulin G mixed with goat immunoglobulin G (CAPPEL, Aurora, Ohio, USA) as secondary antibody and rabbit peroxidase-antiperoxidase (CAPPEL, Aurora, Ohio, USA) as a tertiary antibody, and then were compared to the expression in the spinal nucleus of the trigeminal nerve.

[0077] After removal of the medium OST to block endogenous Fe is tentatively activity of the sample for 1 hour kept in 0.3%hydrogen peroxide solution in 80% methanol at room temperature. After this was done blocking blocking solution (3%solution of goat serum in Tris-buffered diluent) at room temperature for 1 hour and spent the reaction of the primary antibody with the sample at 4°C for 72 hours. After rinsing, samples were performed blocking blocking solution at room temperature for 1 hour and spent the reaction of the secondary antibody with the sample at room temperature for 1 hour. After rinsing, samples were performed blocking blocking solution at room temperature for 1 hour and spent the reaction of the tertiary antibody with the sample at room temperature for 1 hour. After sufficient washing to sample for coloring was added dropwise a solution of substrate - diaminobenzidine/ammonium sulphate, Nickel (from 0.08 to 0.1% ammonium sulphate, Nickel and 0.04% of diaminobenzidine - of 0.003% hydrogen peroxide in Tris buffer diluent). After staining the sections sufficiently washed and made into glycerol. Observation of the sample were in direct microscope (Axioimager production Zeiss). The results are presented in figure 5.

[0078] as a result, as shown in Figa, caudal padyatra spinal nucleus of the trigeminal nerve (dorsal horns of the spinal cord) mouse C57BL/6, which is not subject to orthodontic therapy regenerative the aqueous tooth, no therapy with the opening of the pulp of the tooth, the expression of the protein c-Fos were observed. At the same time, as shown in Figv and Figs, caudal padyatra spinal nucleus of the trigeminal nerve (dorsal horns of the spinal cord) mouse C57BL/6, which was subjected to orthodontic therapy, after 2 hours was found to have a high level of expression of the protein c-Fos, and this expression was maintained after 48 hours of stimulation, as shown in Figs. Next, as shown Fig.5D, caudal padyatra spinal nucleus of the trigeminal nerve (dorsal horns of the spinal cord) mouse C57BL/6, which opened the regenerated pulp of the tooth, after 2 hours was found to have a high level of expression of the protein c-Fos. These results indicate that nerve fibers are present in the area of periodontal ligament, perceive the compressive effects caused by the use of orthodontic therapy to the regenerated tooth, and pain, and that these impacts are transmitted to the Central nervous system, as normal tooth.

[0079] Thus, it was shown that through the application of the recovery method of the present invention the area of the lost tooth can be restored so that the restored tooth will have a hardness equivalent hardness usual tooth will be able to provide normal occlusion and possess sensitivity is lnasty to incentives, equivalent normal sensitivity. [0080] the Japanese patent application of Japan No. 2008-211870 fully incorporated herein by reference.

All references, patent applications and technical standards described in this document is included by reference to the same extent as in cases where there is a clear and specific indication that a particular source literature, individual application for an invention or a specific technical standard described for inclusion by reference.

1. A method of manufacturing a restorative material used to restore the area of the lost tooth in the oral cavity, comprising the steps:
placed on the carrier of the first cell mass formed by the cells or by cells of mesenchymal or epithelial cells and a second cell mass formed another cell or other cells of mesenchymal or epithelial cells and the epithelial cells of the first and second cell masses derived from a tooth germ, and the first and second cell masses come in close contact with each other without mixing;
grow first and the second cell mass with the formation of the rudiment of the restored tooth or the whole of the restored tooth; and
determine the orientation of the embryo recovered memory is and or the whole of the restored tooth, formed in cultivation that allows you to embed a root of the restored tooth or a restored tooth in the area of the missing tooth so that the crown part of the tooth was directed inside the mouth,
use the tooth germ or tooth, the orientation of which was defined as a restorative material with obtaining the equivalent of a lost tooth in the area of the lost tooth, and the equivalent of a lost tooth has a length, which is designed to occlusion and tooth-antagonist.

2. A method of manufacturing a restorative material according to claim 1, characterized in that the enamel equivalent of a lost tooth Knoop hardness ranges from 300 to 600 units, and dentin Knoop hardness is 60 to 120 units.

3. A method of manufacturing a restorative material according to claim 1 or 2, wherein the cultivation is carried out in organ culture and restorative material includes a rudiment of the restored tooth or a restored tooth formed in organ culture, and media.

4. A method of manufacturing a restorative material according to claim 1 or 2, wherein the mesenchymal cells derived from a tooth germ.

5. A method of manufacturing a restorative material according to claim 1 or 2, characterized in that as the first cell mass and the second is I cell mass consists of a single cell.

6. A method of manufacturing a restorative material according to claim 1 or 2, characterized in that the media represents at least one of the following groups of substances: collagen, agarose gel, carboxymethyl cellulose, gelatin, agar, hydrogel, elastin, fibrin, fibronectin, laminin, extracellular matrix mixed polyglycolic acid, polylactic acid, copolymer of lactic and glycolic acids.

7. The way to restore the area of the lost tooth in the oral cavity, comprising the steps:
placed on the carrier of the first cell mass formed by the cells or by cells of mesenchymal or epithelial cells and a second cell mass formed another cell or other cells of mesenchymal or epithelial cells and the epithelial cells of the first and second cell masses derived from a tooth germ, and the first and second cell masses come in close contact with each other without mixing;
grow first and the second cell mass with the formation of the rudiment of the restored tooth or the whole of the restored tooth; and
introduce the principle of the restored tooth or the whole of the restored tooth in the area of the lost tooth, receiving the equivalent of a lost tooth having a length for its occlusion and tooth-antagonist.

8. Method of recovery of the region of the missing tooth according to claim 7, wherein the mesenchymal cells derived from a tooth germ.

9. The way to restore the area of the lost tooth according to claim 7 or 8, characterized in that as the first cell mass and the second cell mass consists of a single cell.

10. The way to restore the area of the lost tooth according to claim 7 or 8, characterized in that the carrier is at least one of the following groups of substances: collagen, agarose gel, carboxymethyl cellulose, gelatin, agar, hydrogel, elastin, fibrin, fibronectin, laminin, extracellular matrix mixed polyglycolic acid, polylactic acid, copolymer of lactic and glycolic acids.