JP2005110542A - Cultured periosteum package, and method for preparing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cultured periosteum package which has an excellent carriable property and excellent preservability, and to provide a method for preparing the same. <P>SOLUTION: This method for preparing the cultured periosteum package comprises (a) a step for preparing the first container 1 having a cup-like portion and then putting a picked periosteum on the inner bottom side of the cup-like portion, (b) a step for continuously or periodically exchanging a culture medium without releasing the periosteum and simultaneously culturing the periosteum, and then (c) a step for sealing the first container 1 in a state filling a preserving liquid in the cup-like portion. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a cultured periosteal package containing a cultured periosteum, and a method for preparing and using the cultured periosteal package. The cultured periosteal package of the present invention is used, for example, to provide an implant for the treatment of periodontal tissue.

  In recent years, research on regenerative medicine that uses cells and tissues to treat defects and wounds in living tissues due to diseases and injuries has progressed, and has begun to move greatly toward industrialization. Particularly, tissues that are rapidly industrialized include skin, bone, and periodontium. Various techniques have been developed for the purpose of regeneration (reconstruction) of bone tissue or periodontal tissue, and one of them is an attempt to use the periosteum. The periosteum is a connective tissue covering the outer periphery of cortical bone, and it has been confirmed that stem cells having the ability to differentiate into bone cells, chondrocytes and the like are present therein (Non-Patent Documents 1 and 2).

In Vivo Differentiation of Bone and Hypertropic Cartilage from Periosteal-Derived Cells, Experimental Cell Research, 195, 492-503 (1991) In Vivo Osteochondrogenic Potential of Cultured Cells Derived From the Periosteum, Clinical Orthopedics and Related Research, 259, 223-232 (1990)

The present inventors have so far studied for the purpose of regeneration of bone tissue and periodontal tissue, and as a result, a membranous structure (cultured periosteum) is obtained by culturing periosteum under appropriate conditions. succeeded in. In addition, as a result of transplanting the obtained cultured periosteum to the alveolar bone defect, it was confirmed that a good regeneration effect was observed, and it was effective to apply the cultured periosteum to the regeneration of the alveolar bone (patent pending, special (Application 2002-64235).
At present, cultured periosteum is obtained by culturing the collected periosteum on a culture dish under predetermined conditions. At the research stage such as animal experiments, it is possible to prepare cultured periosteum in the vicinity of the site where transplantation is performed, and although it is general, the preparation of cultured periosteum has moved away from the treatment site in clinical applications. Rather, it is expected that it will be performed in the place. Therefore, in the practical application of cultured periosteum, it is required to provide the cultured periosteum in a form that can withstand transportation (or storage). Since the culture dish used in the current culture method is not suitable for transportation, it is necessary to peel the cultured periosteum from the culture dish and transfer it to a container suitable for transportation in order to meet the above requirements. . However, the thickness of cultured periosteum is generally very thin, about 50-100 μm, and the culture perioste once peeled from the culture dish tends to be deformed or folded during transportation. Has been pointed out. In other words, a complicated operation of expanding the cultured periosteum at the treatment site is required, and this complicates transplantation and increases the burden on doctors and patients. In addition, the periosteum is damaged by the operation, and there is a possibility that a sufficient therapeutic effect cannot be obtained. On the other hand, when the above transportation form is adopted, there is a possibility that the cultured periosteum is damaged by vibration / impact during transportation and the function cannot be sufficiently exhibited after transplantation. In addition, depending on the treatment target, transplanting either the front or back surface of the cultured periosteum as a contact surface to the affected area has a significant effect on the therapeutic effect. However, in the cultured periosteum provided to the medical site as described above, transplantation is performed. Since it is virtually impossible to distinguish the front and back and transplant at times (during use), there may be cases where a sufficient therapeutic effect cannot be expected.
On the other hand, the membranous composition immediately after culturing contains a periosteum piece that is the basis of the cultured periosteum. The periosteum piece after culturing contains cells that contribute to the therapeutic effect after transplantation, that is, highly active cells. It is thought that it no longer exists, and it is expected that transplanting it with cultured periosteum will not increase the therapeutic effect, although it may decrease the therapeutic effect. In consideration of this, it is preferable to remove the periosteum piece before the transplantation operation, but it is difficult to remove the periosteum piece from the thin graft material that is complicatedly folded during transportation as described above.
Furthermore, when transplanting the cultured periosteum, the cultured periosteum is usually peeled off from the culture dish using tweezers or the like. However, as described above, since the cultured periosteum is extremely thin, the tissue is easily damaged by this peeling operation. . In addition, the cultured periosteum is easily folded when peeled off, and an operation for expanding the cultured periosteum at the transplanted portion is required. Thus, even when transplanting cultured periosteum (during use), there were problems in both therapeutic effect and operability.

In order to solve at least one of the above problems, the present invention provides a cultured periosteal package, a method for preparing a cultured periosteal package, and a method for using the cultured periosteal package having the following configurations.
[1] a container having a cup-shaped portion;
A culture periosteum housed in the container in a state of being adhered to the inner bottom surface of the cup-shaped portion,
A cultured periosteum package in which the container is sealed in a state in which the cup-shaped portion is filled with a storage solution.
[2] The cultured periosteum package according to [1], wherein the container has a portion that is continuous with an outer edge portion of the cup-shaped portion and can hold a liquid upward.
[3] Two grooves continuous in the vertical direction are formed on the surface of the side wall of the cup-shaped portion,
The cultured periosteum package according to [1] or [2], wherein a groove is formed on the bottom wall surface of the cup-shaped portion so as to connect the two grooves.
[4] The cultured periosteum package according to [3], wherein the two grooves are formed at positions that are substantially symmetrical with respect to a central axis of the cup-shaped portion.
[5] Any one of [1] to [4], wherein a mark indicating that the periosteum piece serving as a basis of the cultured periosteum is placed on the bottom wall surface of the cup-shaped portion is attached. The cultured periosteum package according to 1.
[6] The cultured periosteum package according to [5], wherein the mark is composed of grooves that are continuously or intermittently continuous.
[7] having a cup-shaped portion;
Two grooves continuous in the vertical direction are formed on the side wall surface of the cup-shaped portion,
A groove is formed on the bottom wall surface of the cup-shaped portion so as to connect the two grooves,
On the bottom wall surface of the cup-shaped portion, a container in which a groove continuously or intermittently connected to surround a region where a periosteum piece serving as a basis of the cultured periosteum is placed; and
A culture periosteum housed in the container in a state of being adhered to the inner bottom surface of the cup-shaped portion,
A cultured periosteum package in which the container is sealed in a state in which the cup-shaped portion is filled with a storage solution.
[8] The cultured periosteum package according to any one of [1] to [7], wherein the container includes a plurality of the cup-shaped portions.
[9] The cultured periosteum package according to [8], wherein a continuous or intermittent groove for separating adjacent cup-shaped portions is formed between the cup-shaped portions in the container.
[10] A container used for the preparation and transportation of cultured periosteum,
Having a cup-shaped part,
Two grooves continuous in the vertical direction are formed on the side wall surface of the cup-shaped portion,
A container in which a groove is formed on the surface of the bottom wall of the cup-shaped portion so as to connect the two grooves.
[11] A container used for the preparation and transportation of cultured periosteum,
A cup-shaped portion, and a portion that is continuous with the outer edge of the cup-shaped portion and can hold a liquid upward;
Two grooves continuous in the vertical direction are formed on the side wall surface of the cup-shaped portion,
A container in which a groove is formed on the surface of the bottom wall of the cup-shaped portion so as to connect the two grooves.
[12] The container according to [10] or [11], wherein the two grooves are formed at positions that are substantially symmetrical with respect to a central axis of the cup-shaped portion.
[13] Any one of [10] to [12], wherein a mark indicating that the periosteum piece serving as the basis of the cultured periosteum is placed on the bottom wall surface of the cup-shaped part is attached. Container as described in.
[14] The container according to [13], wherein the mark is composed of a continuous or intermittent groove.
[15] The container according to any one of [10] to [14], comprising a plurality of the cup-shaped parts.
[16] The container according to [15], wherein a continuous or intermittent groove for separating adjacent cup-shaped parts is formed between the cup-shaped parts in the container.
[17] A method for preparing a cultured periosteum package comprising the following steps:
a) preparing a first container having a cup-shaped portion and placing the collected periosteum piece on the inner bottom surface of the cup-shaped portion;
b) a step of culturing under continuous or periodic medium exchange under conditions in which cells contained in the periosteum pieces grow and so that the periosteum pieces do not exfoliate;
c) After culturing, the step of sealing the first container in a state where the cup-shaped portion is filled with a storage solution.
[18] The method for preparing a cultured periosteum package according to [17], wherein step b) includes the following steps:
b-1) preparing a box-shaped second container having a recess having a shape and a size capable of accommodating the cup-shaped part of the first container;
b-2) setting the cup-shaped portion of the first container in the recess of the second container;
b-3) a step of culturing under conditions where cells contained in the periosteum pieces grow and periodically changing the medium, and removing the medium in the second container from the region other than the recess Then, the medium exchange is performed by injecting a new medium from the region other than the recess into the second container.
[19] The method for preparing a cultured periosteum package according to [17], wherein the step b) includes the following steps:
b-1) preparing a box-shaped second container having a recess having a shape and a size capable of accommodating the cup-shaped part of the first container;
b-2) setting the cup-shaped portion of the first container in the recess of the second container;
b-4) a step of culturing under conditions in which cells contained in the periosteum pieces grow and continuously exchanging the medium, the medium in the second container from one region of the second container The medium is changed by continuously introducing a new medium into the second container from a region different from the one region.
[20] The culture according to [17], wherein the first container has a portion that is continuous with an outer edge portion of the cup-shaped portion and can hold a liquid upward, and the step b) includes the following steps: Preparation method of periosteum package:
b-5) a step of culturing under conditions where cells contained in the periosteum proliferate and periodically changing the medium, wherein the medium retained in the portion in the first container is removed Thereafter, the medium change is performed by injecting a new medium into the part.
[21] The method for preparing a cultured periosteum package according to any one of [17] to [20], wherein the second container includes a plurality of the recesses.
[22] Two longitudinally extending grooves are formed on the side wall surface of the cup-shaped portion, and a groove is formed on the bottom wall surface of the cup-shaped portion so as to connect the two grooves. The method for preparing a cultured periosteum package according to any one of [17] to [21].
[23] The method for preparing a cultured periosteal package according to [22], wherein the two grooves are formed at positions that are substantially symmetrical with respect to a central axis of the cup-shaped portion of the first container.
[24] The cultured periosteum package according to any one of [17] to [23], wherein a mark indicating that the periosteum piece is a region on which the periosteum piece is placed is attached to a bottom wall surface of the cup-shaped portion. Preparation method.
[25] The method for preparing a cultured periosteal package according to [24], wherein the mark is composed of a continuous or intermittent groove.
[26] The method for preparing a cultured periosteal package according to any one of [17] to [25], wherein the first container includes a plurality of the cup-shaped parts.
[27] The method for preparing a cultured periosteum package according to any one of [17] to [26], wherein the following steps are performed after step b):
b-6) A step of removing the periosteum piece from the membranous structure obtained in step b).
[28] A method for transplanting cultured periosteum, comprising the following steps:
The step of preparing the cultured periosteum package according to any one of [1] to [9];
Opening the cultured periosteum package and removing the stored cultured periosteum;
Transplanting the removed cultured periosteum to the affected area.
[29] A method for transplanting cultured periosteum, comprising the following steps:
Providing the cultured periosteum package according to [3] or [4];
Cleaving the container along the grooves formed in the side wall surface and the bottom wall surface of the container, and removing the cultured periosteum;
Transplanting the removed cultured periosteum to the affected area.
[30] A method for transplanting cultured periosteum, comprising the following steps:
Providing a cultured periosteum package according to [5];
Removing the periosteum piece from the marking on the bottom wall surface of the container;
Removing the cultured periosteum;
Transplanting the removed cultured periosteum to the affected area.
[31] A method for transplanting cultured periosteum, comprising the following steps:
Providing a cultured periosteum package according to [6];
Punching out a region surrounded by the groove using the groove formed on the bottom wall surface of the container to remove the periosteum piece;
Removing the cultured periosteum;
Transplanting the removed cultured periosteum to the affected area.
[32] A method for transplanting cultured periosteum, comprising the following steps:
Providing a cultured periosteum package according to [7];
Punching out a region surrounded by the groove using the groove formed on the bottom wall surface of the container to remove the periosteum piece;
Passing the crown through the hole formed in the container by the step and placing the container on the tooth;
Cleaving the container along the grooves formed in the side wall surface and the bottom wall surface of the container, and taking out the cultured periosteum so as to cover the periodontal tissue as an affected part.

  In the cultured periosteum package of the present invention, the cultured periosteum is stored in a state of being adhered to a part of the container, and since the container is sealed in a state filled with the preservation solution, the cultured periosteum is deformed during transportation, Folding is effectively prevented. Therefore, by using the cultured periosteum package of the present invention, a cultured periosteum that is easy to handle at the treatment site can be obtained. In addition, since tissue damage during transportation is also prevented, a cultured periosteum that can exhibit its original function and has an excellent therapeutic effect is provided. Furthermore, since the cultured periosteum package of the present invention does not obscure the front and back of the cultured periosteum during transportation (or during storage), the front and back of the cultured periosteum can be easily distinguished at the time of transplantation. Thereby, a good therapeutic effect can be expected.

The first aspect of the present invention relates to a cultured periosteum package and has the following configuration.
A container having a cup-shaped part, and a culture periosteum accommodated in the container in a state of being adhered to the inner bottom surface of the cup-shaped part, and the container is filled with a preservation solution in the cup-shaped part. A sealed periosteal package that is sealed.
In the above configuration, since the cultured periosteum is adhered to a part of the container, in principle, the cultured periosteum is not deformed or folded during storage (or transportation). On the other hand, the filling of the preservation solution minimizes the generation of water flow when vibration or impact is applied from the outside during transportation. Thereby, it is possible to prevent the cultured periosteum from being detached from the container during transportation, and at the same time, damage to the tissue is suppressed. As described above, by using the cultured periosteal package having the above-described configuration, it is possible to provide the cultured periosteum to the treatment site while maintaining the membranous tissue. Therefore, a high therapeutic effect can be expected by transplantation using the cultured periosteum. Moreover, handling of the cultured periosteum at the time of transplantation is facilitated, and transplantation is simplified. As a result, further improvement of the therapeutic effect can be expected.

  The container used for the cultured periosteum package of the present invention includes a cup-shaped portion. Here, the cup-shaped portion refers to a portion surrounded by a bottom wall and a side wall and opened upward (only a part of the upper portion may be opened). As long as these conditions are satisfied, the shape and size of the bottom wall and the side wall are not particularly limited. As a specific shape of the cup-shaped portion, a shape generally adopted as the shape of the culture dish, that is, a shape in which the shape of the bottom wall in plan view is circular and has a substantially cylindrical inner space can be exemplified. As another specific example, a shape in which the plan view shape of the bottom wall is also circular, and the cross-sectional area of the inner space gradually increases as the distance from the bottom wall (in this case, the side wall tapers outward toward the upper side). Can be mentioned. Note that the shape of the bottom wall in plan view is not limited to a circle, and may be, for example, an ellipse, a triangle, or a rectangle.

  In one embodiment of the present invention, a container having a portion (hereinafter, also referred to as “liquid holding portion”) that is continuous with the outer edge portion (upper end portion) of the cup-shaped portion and that can hold liquid upward is used. When such a container is used, the medium can be exchanged without using another container (second container) in the preparation stage of the cultured periosteum package, as will be described later.

Moreover, in another one aspect | mode of this invention, a container provided with the flange part which follows the outer edge part of a cup-shaped part is used. When a container having a liquid holding portion is used as described above, the flange portion is formed so as to be continuous with the cup-like portion via the liquid holding portion. That is, the liquid holding part is positioned between the upper part of the cup and the flange part.
The flange portion is formed continuously with the outer edge portion (upper end portion) of the cup-shaped portion. The flange portion is provided mainly for the purpose of facilitating sealing (sealing) described later. That is, if the flange portion is used as an adhesive surface, sealing can be easily performed.

  The material of the container is not particularly limited, and examples thereof include polystyrene, polycarbonate, silicone, and the like. A coating may be applied to the surface of the container. Such coating can improve the adhesion and / or proliferation of periosteum (periosteal cells). Specific examples of the coating material include collagen (such as type I) and polylysine.

The size of the cup-shaped portion can be determined in consideration of the size of the cultured periosteum housed therein, the purpose of use of the cultured periosteal package, and the like. For example, periodontal when tissues constituting the cultured periosteal package for use in the reconstruction of the area of the inner bottom surface of about 0.7 cm ² ~ about 100 cm ² about the cup-shaped portion, preferably from about 3.0 cm ² ~ about 40cm about ^2, more preferably it is set to about 12cm ² ~ about 20 cm ^2.

  The cultured periosteum housed in the container is a membranous structure obtained by culturing periosteum pieces under conditions that allow the cells contained therein to grow. In the present invention, such cultured periosteum is housed in the container in a state of being adhered to the inner bottom surface (surface of the bottom wall) of the cup-shaped portion of the container. The cultured periosteum in such a state can be obtained by culturing the collected periosteum pieces in a cup-shaped portion of a container used for the cultured periosteal package of the present invention. The cultured periosteum thus obtained is well adhered to the inner bottom surface of the cup-shaped part.

  In one aspect of the present invention, two grooves (hereinafter referred to as “first groove and second groove”) continuous in the vertical direction are formed on the surface of the side wall of the cup-shaped portion of the container. Similarly, a groove (hereinafter referred to as “third groove”) is formed on the bottom wall surface so as to connect the two grooves. In the cultured periosteum package of such an embodiment, the cultured periosteum can be easily removed by cleaving the container along these grooves at the time of use (transplantation). Therefore, since the operation of peeling the cultured periosteum by tweezers or the like is not necessary, damage to the cultured periosteum can be suppressed as much as possible. In addition, if the cultured periosteum is taken out immediately above the transplanted part, it is possible to directly cover the affected part with the cultured perioste in a state in which the membranous shape is maintained. The work of installing the cultured periosteum is not necessary.

It is preferable to form the first groove and the second groove on the outer surface side of the cup-shaped portion. Similarly, it is preferable to form a third groove on the outer bottom surface (back surface) side of the cup-shaped portion. Thus, if each groove | channel is formed in the outer surface side of a cup-shaped part, since the inner surface of a cup-shaped part can be made into a flat surface, the favorable adhesion | attachment of the culture periosteum with respect to a cup-shaped part wall surface is securable.
The first to third grooves are preferably formed in a mode (depth, width, etc.) that allows the container to be cleaved along the grooves without using a special tool or instrument. Specifically, for example, the unit length is about 1 mm to about 3 mm, the width is 0.5 mm to about 1 mm, the interval is about 0.5 mm to about 2 mm, and the groove can be a continuous groove on the dotted line. Alternatively, it can be a continuous groove with a width of about 0.5 mm to about 1 mm. In selecting the mode of each groove, it is preferable to consider the material of the container.
It is preferable that the first groove and the second groove are formed at positions that are substantially symmetrical with respect to the central axis of the cup-shaped portion of the container. By forming both grooves in this way, the container can be cleaved at the center of the cup-shaped part, so that workability is improved, as well as the external force applied to the cultured periosteum at the time of cleavage is balanced left and right Thus, the cultured periosteum can be reliably removed, and at the same time, deformation and damage of the cultured periosteum can be suppressed as much as possible.

  It is preferable that the mark which shows that it is the area | region where the periosteum piece used as the base of culture periosteum is mounted in the bottom wall surface of the cup-shaped part of a container is attached. By attaching such a mark, it becomes possible to grasp the position of the periosteum piece based on the mark. Here, it is considered that there are no more highly active cells that contribute to the regeneration effect in the periosteum piece after the formation of the cultured periosteum, and transplanting such periosteal alteration together with the cultured periosteum has a therapeutic effect. Although it can be lowered, it is not expected to increase the therapeutic effect. Therefore, it is preferable to remove the periosteum pieces before using the cultured periosteum. However, since the periosteum pieces can be easily removed by using the mark as described above, the cultured periosteal package is excellent in usability. Will be constructed. In addition, in order to abbreviate | omit the periosteum piece removal operation | work at the time of use, the culture | cultivation periosteum package of this invention may be comprised as the state from which the periosteum piece was removed previously.

The mark can be formed by, for example, a desired color ink, paint, tape, or the like. Alternatively, a continuous or intermittent groove can be formed on the surface of the bottom wall of the cup-shaped portion so as to surround a certain region, and this groove can be used as a mark here. If such a mark made of a groove is employed, the cup-shaped bottom wall and the periosteum piece in the region surrounded by the groove can be easily removed using a punching device (instrument).
The mark is preferably attached to the central region of the cup-shaped portion, but the position is not particularly limited.

As the preservation solution to be filled in the container, one that can maintain the properties of the cultured periosteum until the time when the cultured periosteal package of the present invention is used is employed. Typically, a medium used for preparation (culture) of cultured periosteum can be used as a preservation solution.
For example, when it is expected that the time until use is relatively short (that is, when it is considered that active growth or proliferation action is not necessary for the preservation solution), phosphate buffer solution or physiological saline Etc. can also be used as a preservation solution.
A preservative solution to which an anti-freezing agent (for example, glycerol or DMSO) or a preservative (for example, sodium azide (NaN ₃ ) or antibiotics) is added may be used. If such a preservation solution is used, a cultured periosteum package that can be stored or transported in a frozen environment is constructed.
In the present invention, the container is sealed in a state where the cup-shaped portion is filled with the preservation solution, but gas unavoidable in the sealing operation may be mixed into the cup-shaped portion.

In the cultured periosteum package of the present invention, the container is sealed. For example, the container is sealed by sealing the opening of the container. In this case, it is sufficient that the cup-shaped portion is sealed so that at least the cup-shaped portion is sealed. Therefore, the sealing may be performed at the opening of the cup-shaped portion. For sealing, a material used for sealing in the medical field or food field such as a resin sheet (film) or a sheet (film) obtained by metal coating the surface of the resin sheet can be used.
The container may be sealed with a lid (lid body). For example, lids of various mechanisms such as a screw type, a screw type, and a push type can be used. The lid may be configured as a part of the container or may be configured as a separate body from the container.

  In one embodiment of the present invention, the container includes a plurality of cup-shaped portions, and the cultured periosteum is stored in each cup-shaped portion. In such an aspect, it is preferable that a continuous or intermittent groove for separating adjacent cup-shaped portions is formed between the cup-shaped portions. By forming such a groove, it becomes possible to separate each cup-shaped part at the time of use, and it becomes easy to use. An example of such a container is shown in FIG. In the illustrated container 80, a total of six cup-shaped portions 81 are provided, and a groove (cut) 82 on the dotted line is formed between each cup-shaped portion 81.

Next, a method for preparing a cultured periosteal package, which is a second aspect of the present invention, will be described. The preparation method disclosed here can be suitably used for preparing the above-described cultured periosteum package. However, it is not intended that the above-described cultured periosteal package be limited to those obtained by the preparation method described below.
A second aspect of the present invention relates to a method for preparing a cultured periosteal package, and one embodiment thereof is characterized by including the following steps. That is,
a) preparing a first container having a cup-shaped portion and placing the collected periosteum piece on the inner bottom surface of the cup-shaped portion;
b) a step of culturing under continuous or periodic medium exchange under conditions in which cells contained in the periosteum pieces grow and so that the periosteum pieces do not exfoliate;
c) After culturing, the first container is sealed in a state where the cup-shaped portion is filled with a storage solution.
In step a), the collected periosteum piece is placed in a predetermined position in the first container, that is, on the inner bottom surface of the cup-shaped portion. Periosteum is collected from a suitable donor, but considering the problem of immune rejection, it is preferable to collect periosteum from the subject (patient) himself who uses the cultured periosteum package. Here, “periosteum” is a connective tissue covering the outer periphery of the cortical bone, and its configuration is largely divided into an inner layer and an outer layer. The inner layer includes a cell layer composed of multipotent cells, and the outer layer includes collagen fibers and fibroblasts.
The periosteum can be collected from the long trunk bone, the jawbone, the skull, etc., for example, openly under local anesthesia. The collected periosteum is cut into an appropriate size as necessary, and then placed at a predetermined position in the first container. It is preferable to place the periosteum so that the surface that was on the bone side is down (that is, the adhesive surface). That is, it is preferable that the periosteum is placed so that the inner layer side of the periosteum adheres to the inner bottom surface of the cup-shaped portion, and is used for culture. This is because better proliferation of periosteum cells can be expected. The collected periosteum may be treated with an antibiotic before culturing.

After step a), a medium is added to the first container, and the medium is changed continuously or periodically under the condition that the cells contained in the periosteum pieces grow and the periosteum pieces do not peel off. Culture is performed (step b)). Before adding the culture medium for the first time, it is preferable to confirm the state in which the periosteum piece is adhered to the inner bottom surface of the cup-shaped part. The culture conditions are not particularly limited as long as the cells (periosteal cells) contained in the periosteum pieces can grow. By culturing under such conditions, periosteal cells proliferate and, accordingly, an extracellular matrix (extracellular matrix) is produced. As a result, a membranous structure (cultured periosteum) having a strength required for transplantation, particularly a strength capable of withstanding suturing, is formed. In addition, as a culture medium which can be used, what added serum to commercially available Medium 199 (made by Gibco) can be illustrated. As the serum, fetal bovine serum (FBS) or human serum can be used. The former is marketed by many manufacturers and can be easily obtained. On the other hand, in the case of human serum, those collected from the subject who uses the cultured periosteum package of the present invention (that is, the recipient of the cultured periosteum) itself can be preferably used.
Culture conditions that can proliferate cells having multipotency, which are one of the periosteal cells, and promote differentiation into bone cells can also be employed. Such a culture condition may be adopted in a part of the culture period (for example, the late stage of the culture). Examples of such culture conditions include conditions using a medium containing serum and L-ascorbic acid.

  Although the culturing time varies depending on the condition of the periosteum piece to be used, for example, by culturing for about 1 week to about 2 months, a structure (cultured periosteum) in which the extracellular matrix has a layered structure is obtained. If the obtained membranous structure is too thin, sufficient strength cannot be obtained. Therefore, the culture is preferably continued for a predetermined time so that a thickness of 0.3 mm or more (for example, 0.3 mm to 1 mm) is obtained. preferable.

During the culture, the medium is changed continuously or periodically. This medium exchange is performed under mild conditions so that the periosteum pieces (periosteum pieces and cultured periosteum after formation of the cultured periosteum) do not peel off. If the culture medium is directly removed from the cup-shaped part in which the periosteum pieces are stored, and a new culture medium is directly injected into the cup-shaped part in the same manner, the periosteum pieces may be detached due to the water flow accompanying the medium exchange. Therefore, in the present invention, for example, medium replacement is performed by the following procedure. First, prepare a box-shaped second container (typically open at the top) having a recess having a shape and size that can accommodate the cup-shaped part of the first container (step b-1)), The cup-shaped part of the first container is set in the concave part of the second container (step b-2). The shape and size of the cup-shaped part of the first container and the concave part of the second container so that the cup-shaped part is completely accommodated in the concave part of the second container when the cup-shaped part of the first container is set. Etc. are preferably designed. In addition, when the cup-shaped portion of the first container is set, the cup-shaped portion of the first container and the second container are arranged so that there is no substantial gap between the cup-shaped portion and the concave portion of the second container. It is preferable to design the shape, size, etc. of the recess. By satisfying the above conditions, it is possible to more efficiently perform the medium exchange described later, and it is possible to prevent the first container from vibrating or dropping from the recess of the second container during the medium exchange.
It is preferable to prepare a container having a plurality of recesses as the second container, and set the first container in each recess (in this case, the same number of first containers as the number of recesses of the second container are prepared). ). In this way, a large number of periosteum pieces can be cultured simultaneously. That is, the culture efficiency is improved.

After the first container is set in the recess of the second container as described above, the culture is performed while periodically or continuously changing the medium under conditions where cells contained in the periosteum pieces grow. In the case of periodic medium exchange, first, the medium in the second container is removed from the area other than the recesses (that is, other than the area where the first container is installed) in the second container. By this operation, in principle, all the medium existing in the region in the second container except in the first container is removed. Of course, the condition may be such that not only the first container but also a part of the medium existing in the other region remains, but in order to enhance the effect of the medium exchange, as much medium as possible is replaced. Thus, it is preferable that the amount of the remaining medium is as small as possible.
Following the medium removal operation, a new medium is injected into the second container. In addition, before completely removing the culture medium in the second container, injection of a new culture medium can be started. As in the case of the above removal operation, the medium injection operation is also performed in a region other than the concave portion of the second container. As described above, by performing the operation of removing and injecting the medium in the region other than the concave portion of the second container, it is possible to perform the medium exchange without causing a large water flow in the first container. Accordingly, the influence of the culture medium exchange on the cultured periosteum (and the periosteum piece) in the first container is extremely small, and the culture periosteum is effectively prevented from being detached or damaged by the culture medium exchange.
Examples of the medium exchange frequency include once a day, once every two days, or once every three days, but are not limited thereto.
In addition, removal and injection | pouring of a culture medium are performed using a pipette and a tube, for example.

When continuously exchanging the medium, the medium is continuously discharged from one area of the second container, and a new medium is continuously introduced into the second container from an area different from the area. Typically, the medium in the container is discharged from one end side of the second container, and the medium is introduced into the container from the other end side of the second container. By providing a partition in the second container, a water flow along a predetermined route can be generated along with the medium exchange.
The medium exchange rate (frequency) for continuous medium exchange is 1/1 to 1/600, 1/5 to 1/300, or 1/10 to 1/1 / of the volume of the second container per minute. The rate at which the medium corresponding to 120 is introduced and discharged can be exemplified, but is not limited thereto.
Typically, the medium change is continuously performed throughout the culture step, but there may be periods during which no medium change is performed during the culture step. Such a case is, for example, a case where continuous medium exchange is performed again after a predetermined time interval after continuous medium exchange.

  By adopting a first container that has a part (liquid holding part) that can be held at the upper part of the cup-shaped part and is continuous with the outer edge part, the medium can be changed without using the second container. You can also. When such a container is used, the medium is exchanged by removing the medium held in the liquid holding part of the first container and then injecting a new medium into the liquid holding part (step b-5)). . Such medium exchange is performed periodically. Examples of the medium exchange frequency include once a day, once every two days, or once every three days, but are not limited thereto. For example, the container of the form shown in FIG. 8 can be used as the first container here. In the illustrated container 90, a step 93 corresponding to the liquid holding portion is provided between the cup-shaped portion 91 and the flange portion 92, and the medium is exchanged using the step 93 portion. This prevents the culture medium from directly flowing into (and outflowing) into the cup-shaped portion 91 and effectively preventing the cultured periosteum cultured in the cup-shaped portion 91 from being peeled off or damaged. .

  It is not necessary to use a medium of the same composition throughout the culture step (including both by regular medium exchange and by continuous medium exchange). For example, media having different compositions may be used in the early and late stages of the culture step.

  The culture step may be performed while applying continuous, intermittent, periodic or intermittent vibration.

After the culturing step, a step (step c)) of sealing the first container in a state where the cup-shaped portion is filled with the storage solution is performed. Specifically, for example, the first container after the culture is taken out from the second container, and then the first container is filled with the preservation solution, and then sealed. As the preservation solution, the medium used in the culture step, phosphate buffer solution, physiological saline, or the like can be used. As the preservation solution, a frost damage prevention agent (for example, glycerol or DMSO) or a preservation agent (for example, sodium azide (NaN ₃ ) or antibiotics) may be used. If such a preservation solution is used, a cultured periosteum package that can be stored or transported in a frozen environment is prepared. When a culture medium is used as the preservation solution, typically, the first container is filled with the medium (preservation solution) so that the first container after the culture is supplemented, and then sealed.
The sealing operation is performed by sealing the opening of the container (or the opening of the cup-shaped portion) using a resin sheet (film) or a sheet (film) obtained by metal-coating the surface of the resin sheet. it can. Sealing can be performed by a conventional method. For example, various known sealing methods can be employed in the fields of medicine and food.
Alternatively, the container may be sealed using a suitable lid such as a screw type, a screw type, or a push type. The lid may be configured as a part of the container or may be configured as a separate body from the container.
In addition, it is preferable to perform the above sealing operation aseptically.

  Prior to step c), a step (step b-6)) of removing the periosteum pieces used for the culture from the membranous structure obtained by the culture step can also be performed. By carrying out this step, a cultured periosteal package containing a cultured periosteum that does not contain a periosteum piece is prepared. In such a cultured periosteum package, an operation for removing the periosteum piece is not necessary when the package is used. Thus, if the said step is performed, the culture | cultivation periosteum package by which a user's operation burden is eased can be provided.

  Each of the above operations (only some operations or all operations may be performed) can be automated.

  Although the second aspect of the present invention has been described above, the description in the first aspect is incorporated for matters not particularly mentioned. Therefore, for example, a mark indicating that the first container in which two grooves continuous in the vertical direction are formed on the side wall surface of the cup-shaped portion can be used, or that the periosteum piece is placed is the first. The fact that it may be attached to the bottom wall surface of the cup-shaped portion of the container is naturally taken into consideration in the second aspect.

Next, the method of using the cultured periosteal package of the present invention will be described taking the case of transplantation for the purpose of reconstruction of periodontal tissue as an example.
First, the cultured periosteum package is opened and the preservation solution is removed. Instead of removing all the preservation solution, only a part of the preservation solution may be removed (for example, a part of the preservation solution is left for the purpose of preventing drying at the time of transplantation or immediately after transplantation). After removing the preservation solution, the cultured periosteum may be washed. Such washing is preferably performed under conditions where the cultured periosteum is not damaged and the cultured periosteum does not peel from the container. For example, the cleaning liquid is gently injected into the container, and then the injected cleaning liquid is gently removed. The above cleaning may be performed a plurality of times. As the washing solution, a phosphate buffer solution, physiological saline, or the like can be used.
Subsequently, the periosteum piece is removed. However, this operation is omitted when the periosteum pieces have been removed in the state of the cultured periosteum package. The present invention is not limited to this case, and the following operation can also be performed without the removal operation. When a mark indicating the position of the periosteum piece is attached to the bottom wall of the container, the periosteum piece can be removed using the mark. Preferably, a part of the bottom wall of the container cup-shaped part is punched out simultaneously with the removal of the periosteum piece. If the mark is formed by a groove as described above, this punching operation can be easily performed. In addition, when it is possible to use an instrument having sufficient shearing force to punch the bottom wall of the cup-shaped part, the periosteum piece can be removed and the cup-shaped part bottom wall can be removed without using the groove as described above. Punching can also be performed.
Next, the cultured periosteum is removed from the container. In the case where grooves are formed in the side wall and the bottom wall of the container cup-shaped portion, the container can be cleaved along the groove and taken out. According to such a method, cultured periosteum can be taken out without using other tools such as tweezers, and workability is improved. Further, it is possible to effectively prevent the cultured periosteum from being damaged or deformed, which becomes a problem when it is removed by tweezers. In addition, it becomes possible to take out the cultured periosteum directly above the affected area (transplanted area), and directly transplant the cultured perioste in a state of holding the membranous form to the affected area. The work which installs becomes unnecessary.
The removed cultured periosteum is placed so as to cover the affected area (on the periodontal tissue). When the periosteum fragment is removed to form a cultured periosteum having a hole in part, the crown is passed through the hole (through the hole), and the cultured periosteum is placed over the affected area (on the periodontal tissue). It is preferable to place it. This is because the cultured periosteum can be easily and reliably transplanted to the affected area.
When a hole is formed in the bottom wall of the container cup-shaped part when removing the periosteum piece, the cultured periosteum can be removed and transplanted to the affected area of the removed cultured periosteum at the same time. That is, if the container is opened with the container placed on the tooth by passing the crown through the hole formed in the bottom wall of the container cup-shaped part, and the cultured periosteum is removed by this, the removed cultured periosteum Is placed on the affected area (on the periodontal tissue) without any substantial spacing. According to such transplantation, workability is further improved, and more reliable transplantation is possible.

[Preparation of cultured periosteal package]
A container (hereinafter also referred to as “first container”) 1 used for the cultured periosteum package is shown in FIG. This container is made of polystyrene, and its inner surface is uniformly coated with collagen I. As illustrated, the container 1 includes a cup-shaped portion 11 and a flange portion 12 that is continuous with an outer edge portion (upper end portion) thereof. The cup-shaped portion 11 has a circular shape with a bottom wall 11a having a diameter of about 4 cm, and the cross-sectional area of the inner space gradually increases as the distance from the bottom wall 11a increases. Two grooves (hereinafter referred to as “a first groove for cleavage and a second groove”) 21 and 22 are formed on the outer surface of the cup-shaped side wall 11b. These two grooves 21 and 22 are formed at positions that are symmetrical with respect to the central axis of the cup-shaped portion 11 of the first container 1. On the other hand, a straight groove (hereinafter referred to as “third groove for cleavage”) 23 is formed on the outer surface (back surface) of the cup-shaped portion bottom wall 11a so as to connect the first groove for cleavage and the second groove. Yes. A continuous groove (hereinafter referred to as “periosteal position display groove”) 24 is formed on the outer surface of the cup-shaped bottom wall 11a so as to surround the central region in a circular shape.

The periosteum is cultured using the first container 1 having the above configuration. First, after local anesthesia of the patient, the gingiva is incised using a scalpel, and about 10 cm ² of periosteum is collected from the mandible. The collected periosteum is cut into an appropriate size (for example, about 0.1 cm ² to about 1 cm ² ), and then transferred to the first container 1. At this time, the periosteum is placed in a region surrounded by the periosteum position display groove 24 formed on the bottom wall of the first container 1. In addition, the periosteum is placed so that the inner layer side (bone side) faces down.
In the present embodiment, a total of six first containers 1 on which periosteum pieces collected from the same patient are placed by the above operation are prepared.

  Next, each 1st container 1 is set to the 2nd container 2 (refer FIG. 2). The 2nd container 2 has the six recessed parts 30 formed at substantially equal intervals. Each of these recesses 30 is formed in a shape and size (depth) in which the first container 1 can be fitted without substantial gaps. In FIG. 3, sectional drawing of the state which set each 1st container 1 to each corresponding recessed part 30 is shown. Reference numeral 5 in the figure indicates a periosteum piece. As shown in FIG. 3, in this embodiment, the cup-shaped portion 11 of the first container 1 is completely accommodated in the corresponding recess 30, while the edge 12 of the first container 1 is the edge of the corresponding recess 30. Engage with. By setting the first container 1 in such a manner, it is possible to prevent the position of the first container 1 from being shifted or the first container 1 from being removed from the recess 30 when exchanging the medium described later. In addition, if the edge part 12 is utilized, the 1st container 1 can be set and taken out easily.

After the first container 1 is set, the second container 2 is moved into a CO ₂ incubator kept at about 37 ° C. and left to stand for a predetermined time (for example, about 15 to 45 minutes). Thereafter, the second container 2 is taken out from the CO ₂ incubator, and after confirming that the periosteum piece 5 adheres to the bottom wall surface of the first container cup-shaped part 11, the area where the recess 30 is not formed in the second container 2. From (for example, the region indicated by arrow A in FIG. 3), the medium is slowly poured into the second container 2 using a dropper. An amount of the medium corresponding to, for example, about 70% to about 90% of the volume of the second container 2 is used. However, the amount of the medium is not limited to the range shown here, and may be a smaller amount of medium as long as at least all the first containers 1 are filled with the medium. A larger amount of the medium may be used as long as the medium replacement operation is not hindered.
As the medium, commercially available Medium 199 (manufactured by Gibco) supplemented with serum, L-ascorbic acid, and Antibiotic-Antimycotic (manufactured by Invitrogen) (for example, 10%, 25 μg / ml, and 1% in order) / v) can be used. As the serum, fetal bovine serum (FBS) or human serum can be used.

An adhesive material can be used to promote adhesion of the periosteum to the first container surface. For example, fibrin glue can be used as the adhesive material. In the case of using an adhesive material, (1) a method of placing the periosteum after dropping or applying the adhesive material in advance to the region where the periosteum is placed in the first container, or (2) the first After the periosteum is placed at a predetermined position of the container, the periosteum is adhered to the first container surface by, for example, a method of coating the periosteal surface with an adhesive material by dropping or applying an adhesive material on the periosteum. Will be strengthened. Instead of using an adhesive material, or in addition to this, a force in the direction of the first container is applied to the periosteum by placing a cover glass on the placed periosteum, and thereby, between the periosteum and the first container. It is also possible to strengthen the adhesive force. When a cover glass or the like is used, after the periosteum adheres well to the surface of the first container (for example, after about 1 to 2 weeks have passed since the start of the culture, depending on the culture conditions) Recovery can be performed.
In the case where the adhesion between the periosteum and the first container is reinforced by the above-described means, the operation of leaving for a predetermined time before injecting the medium as described above may be omitted.

Subsequently, the second container 2 is again transferred into the CO ₂ incubator and cultured under conditions of about 37 ° C. and about 5% CO ₂ . Periodically change medium during culture. Specifically, at the initial stage of culture, the medium is changed once every three days. When the cells can be visually confirmed to grow, the medium is changed once a day. In the medium exchange, first, similarly to the operation of pouring the medium, the medium in the second container 2 is slowly sucked from the region where the recess 30 is not formed in the second container 2, thereby Remove the media present in the area. Subsequently, the medium kept at 37 ° C. is slowly poured into the second container 2 by the same operation as described above. After exchanging the medium consisting of this series of operations, the culture is continued again in a CO ₂ incubator under conditions of about 37 ° C. and about 5% CO ₂ .

  By culturing as described above, a membranous structure (cultured periosteum) is gradually formed around the periosteum piece 5. For example, the culture is continued until a culture periosteum having a size enough to completely cover the bottom wall surface of the first container 1 is formed. Although it naturally depends on the condition of the periosteum piece used and the composition of the culture medium, it is expected that a period of, for example, about 1 week to about 2 months will be required before the culture periosteum of such a size is formed.

  After completion of the culture, each first container 1 is taken out from the second container 2. Then, the first container 1 is filled with a medium (preservation solution) having the same composition as the medium used for culture, and the cup-shaped portion 11 of the first container 1 is hermetically packaged (sealed) so as not to contain air (see FIG. 4). By the above operation, a cultured periosteal package 60 that encloses the cultured periosteum 6 while being adhered to the inner wall and is filled with the culture medium is obtained. The cultured periosteum package 60 is stored under predetermined conditions until use (including transportation). Preferably, it is stored under light-shielding and low-temperature conditions. Cryopreservation is possible by adding a freezing protection agent to the medium (preservation solution). According to cryopreservation, it is possible to preserve for a long time while maintaining quality.

[Transplantation using cultured periosteum package]
The cultured periosteum package 60 obtained in Example 1 is opened, and the first container 1 is slowly tilted to discard the preservation solution in the container. Next, as shown in FIG. 5, the periosteum piece 5 portion is removed using a perforating apparatus 40 that can be punched out along the groove 24 formed in the cup-shaped bottom wall 11 a of the first container. At the same time, a part of the cup-shaped bottom wall 11a is punched out. After such an operation, as shown in FIG. 6A, the first container 1 is cleaved immediately above the affected part (periodontal tissue). The first container 1 is cleaved using the grooves 21, 22, and 23 formed on the side wall and the bottom wall of the cup-shaped portion 11. Specifically, the first container is held so as to be sandwiched from above and below at the two points farthest from the two grooves 21 and 22 formed on the side surface of the cup-shaped portion 11, and the cup-shaped portion 11 is moved downward from above. Apply force to cleave left and right. By cleaving the first container 1 as described above, the cultured periosteum 6 peels from the surface of the cup-shaped bottom wall 11a of the first container 1 (FIG. 6 (a)). At this time, the position of the cultured periosteum 6 is adjusted so that the crown 71 passes through the hole 6 a formed in the cultured periosteum 6. In this way, the peeled cultured periosteum 6 covers the periodontal tissue and is simultaneously held on the tooth 70 by the hole 6a formed in the central region thereof (FIG. 6 (b)). For example, prior to cleaving the first container 1, the dental crown 71 is passed through a hole formed in the bottom wall of the first container 1 (the first container 1 is temporarily held by the tooth 70). In this state, the above cleavage operation is performed. According to such a procedure, the cultured periosteum 6 can be transplanted so as to be surely held in the tooth 70 as described above.
Finally, in order to allow engraftment to the transplanted part and long-term retention at the transplanted part, the peripheral part of the established cultured periosteum 6 is sutured to the tissue around the defect part. In addition, a collagen membrane, a Gore-tex membrane, or the like used in the conventional GTR method or the like may be placed so as to cover the cultured periosteum 6. In this way, it is possible to more reliably prevent the epithelial tissue and the like from entering the tissue defect portion, and a higher periodontal tissue regeneration effect can be expected.

  The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.

  According to the present invention, a cultured periosteal package that can be used for the reconstruction of periodontal tissue and bone tissue is provided. The cultured periosteal package of the present invention can be applied to various fields where periodontal tissue, bone tissue, or cartilage tissue needs to be repaired and regenerated. For example, the present invention can be applied to regeneration of alveolar bone and periodontal tissue in an alveolar bone defect due to periodontal disease or the like. In addition, the present invention can be applied to bone augmentation when artificial roots are planted on a ridge with a high degree of bone resorption. Furthermore, the present invention can be applied to bone tissue regeneration in bone defects caused by trauma, various bone diseases, bone reinforcement or supplementation. In the cultured periosteum package of the present invention, the cultured periosteum housed therein is in a state where its shape and quality can be maintained at a high level even during storage or transportation, handling is easy at the treatment site, and high therapeutic effect is also achieved. An expected cultured periosteum can be provided. Moreover, according to one aspect of the present invention, there is provided a cultured periosteal package that can realize a usage pattern particularly suitable for the reconstruction of periodontal tissue. By using such a cultured periosteum package, it is possible to realize transplantation with extremely excellent workability.

FIG. 1 is a perspective view showing a container (first container 1) used in one embodiment of the present invention. FIG. 2 is a perspective view showing a state in which the first container 1 is set in the second container 2. FIG. 3 is a cross-sectional view showing a state in which the first container 1 is set in the second container 2. FIG. 4 is a cross-sectional view showing a cultured periosteum package 60 according to an embodiment of the present invention. FIG. 5 is a diagram showing an operation for removing the periosteum piece 5 when the cultured periosteum package 60 is used. FIG. 6 is a diagram showing the transplantation operation of the cultured periosteum 6, (a) shows a state immediately after the cultured periosteum 6 is taken out from the first container 1, and (b) shows the removed cultured periosteum 6 on the periodontal tissue 75. Each transplanted state is shown in. FIG. 7 is a diagram showing an example of a container that can be used for the cultured periosteum package of the present invention. FIG. 8 is a view showing an example of a container that can be used for the cultured periosteum package of the present invention.

Explanation of symbols

1: first container, 2: second container, 5: periosteum piece, 6: cultured periosteum, 11: first container cup-shaped part, 21: first groove for cleavage, 22: second groove for cleavage, 23: cleavage Third groove for use, 24: Periosteal position display groove, 30: Second container recess, 60: Cultured periosteum package

Claims (32)

A container having a cup-shaped portion;
A culture periosteum housed in the container in a state of being adhered to the inner bottom surface of the cup-shaped portion,
A cultured periosteum package in which the container is sealed in a state in which the cup-shaped portion is filled with a storage solution.   The cultured periosteum package according to claim 1, wherein the container has a portion that is continuous with an outer edge portion of the cup-shaped portion and can hold a liquid upward. Two grooves continuous in the vertical direction are formed on the side wall surface of the cup-shaped portion,
The cultured periosteum package according to claim 1 or 2, wherein a groove is formed on a bottom wall surface of the cup-shaped portion so as to connect the two grooves.   The cultured periosteum package according to claim 3, wherein the two grooves are formed at positions that are substantially symmetrical with respect to a central axis of the cup-shaped portion.   The cultured periosteum according to any one of claims 1 to 4, wherein a mark indicating that the periosteum piece serving as a basis of the cultured periosteum is placed on the bottom wall surface of the cup-shaped portion is placed. package.   The cultured periosteum package according to claim 5, wherein the mark is formed of a continuous or intermittent groove. Having a cup-shaped part,
Two grooves continuous in the vertical direction are formed on the side wall surface of the cup-shaped portion,
A groove is formed on the bottom wall surface of the cup-shaped portion so as to connect the two grooves,
On the bottom wall surface of the cup-shaped portion, a container in which a groove continuously or intermittently connected to surround a region where a periosteum piece serving as a basis of the cultured periosteum is placed; and
A culture periosteum housed in the container in a state of being adhered to the inner bottom surface of the cup-shaped portion,
A cultured periosteum package in which the container is sealed in a state in which the cup-shaped portion is filled with a storage solution.   The cultured periosteum package according to any one of claims 1 to 7, wherein the container includes a plurality of the cup-shaped portions.   The cultured periosteum package according to claim 8, wherein a continuous or intermittent groove for separating adjacent cup-shaped parts is formed between the cup-shaped parts in the container. A container used for the preparation and transport of cultured periosteum,
Having a cup-shaped part,
Two grooves continuous in the vertical direction are formed on the side wall surface of the cup-shaped portion,
A container in which a groove is formed on the surface of the bottom wall of the cup-shaped portion so as to connect the two grooves. A container used for the preparation and transport of cultured periosteum,
A cup-shaped portion, and a portion that is continuous with the outer edge of the cup-shaped portion and can hold a liquid upward;
Two grooves continuous in the vertical direction are formed on the side wall surface of the cup-shaped portion,
A container in which a groove is formed on the surface of the bottom wall of the cup-shaped portion so as to connect the two grooves.   The container according to claim 10 or 11, wherein the two grooves are formed at positions that are substantially symmetrical with respect to a central axis of the cup-shaped portion.   The container in any one of Claims 10-12 to which the mark which shows that it is the area | region where the periosteum piece used as the base of the said cultured periosteum is mounted is attached to the bottom wall surface of the said cup-shaped part.   14. A container according to claim 13, wherein the indicia consists of continuously or intermittently continuous grooves.   The container in any one of Claims 10-14 provided with the said some cup-shaped part.   The container according to claim 15, wherein a continuous or intermittent groove for separating adjacent cup-shaped parts is formed between the cup-shaped parts in the container. A method for preparing a cultured periosteal package comprising the following steps:
a) preparing a first container having a cup-shaped portion and placing the collected periosteum piece on the inner bottom surface of the cup-shaped portion;
b) a step of culturing under continuous or periodic medium exchange under conditions in which cells contained in the periosteum pieces grow and so that the periosteum pieces do not exfoliate;
c) After culturing, the step of sealing the first container in a state where the cup-shaped portion is filled with a storage solution. 18. The method for preparing a cultured periosteal package according to claim 17, wherein said step b) comprises the following steps:
b-1) preparing a box-shaped second container having a recess having a shape and a size capable of accommodating the cup-shaped part of the first container;
b-2) setting the cup-shaped portion of the first container in the recess of the second container;
b-3) a step of culturing under conditions where cells contained in the periosteum pieces grow and periodically changing the medium, and removing the medium in the second container from the region other than the recess Then, the medium exchange is performed by injecting a new medium from the region other than the recess into the second container. 18. The method for preparing a cultured periosteal package according to claim 17, wherein said step b) comprises the following steps:
b-1) preparing a box-shaped second container having a recess having a shape and a size capable of accommodating the cup-shaped part of the first container;
b-2) setting the cup-shaped portion of the first container in the recess of the second container;
b-4) a step of culturing under conditions in which cells contained in the periosteum pieces grow and continuously exchanging the medium, the medium in the second container from one region of the second container The medium is changed by continuously introducing a new medium into the second container from a region different from the one region. The cultured periosteal package according to claim 17, wherein the first container has a portion that is continuous with an outer edge portion of the cup-shaped portion and can hold a liquid upward, and the step b) includes the following steps. Preparation method:
b-5) a step of culturing under conditions where cells contained in the periosteum proliferate and periodically changing the medium, wherein the medium retained in the portion in the first container is removed Thereafter, the medium change is performed by injecting a new medium into the part.   The method for preparing a cultured periosteal package according to any one of claims 17 to 20, wherein the second container includes a plurality of the recesses. Two grooves continuous in the vertical direction are formed on the side wall surface of the cup-shaped part, and a groove is formed on the bottom wall surface of the cup-shaped part so as to connect the two grooves. Item 22. A method for preparing a cultured periosteum package according to any one of Items 17-21.   23. The method for preparing a cultured periosteal package according to claim 22, wherein the two grooves are formed at positions that are substantially symmetrical with respect to a central axis of the cup-shaped portion of the first container.   The method for preparing a cultured periosteum package according to any one of claims 17 to 23, wherein a mark indicating that the periosteum piece is a region on which the periosteum piece is placed is attached to a bottom wall surface of the cup-shaped portion.   25. The method for preparing a cultured periosteal package according to claim 24, wherein the mark comprises a continuous or intermittent groove.   The method for preparing a cultured periosteal package according to any one of claims 17 to 25, wherein the first container includes a plurality of the cup-shaped portions. The method for preparing a cultured periosteal package according to any of claims 17 to 26, wherein the following steps are performed after step b):
b-6) A step of removing the periosteum piece from the membranous structure obtained in step b). A method for transplanting cultured periosteum comprising the following steps:
Preparing a cultured periosteum package according to any one of claims 1 to 9;
Opening the cultured periosteum package and removing the stored cultured periosteum;
Transplanting the removed cultured periosteum to the affected area. A method for transplanting cultured periosteum comprising the following steps:
Preparing a cultured periosteal package according to claim 3 or 4;
Cleaving the container along the grooves formed in the side wall surface and the bottom wall surface of the container, and removing the cultured periosteum;
Transplanting the removed cultured periosteum to the affected area. A method for transplanting cultured periosteum comprising the following steps:
Preparing a cultured periosteum package according to claim 5;
Removing the periosteum piece from the marking on the bottom wall surface of the container;
Removing the cultured periosteum;
Transplanting the removed cultured periosteum to the affected area. A method for transplanting cultured periosteum comprising the following steps:
Preparing a cultured periosteal package according to claim 6;
Punching out a region surrounded by the groove using the groove formed on the bottom wall surface of the container to remove the periosteum piece;
Removing the cultured periosteum;
Transplanting the removed cultured periosteum to the affected area. A method for transplanting cultured periosteum comprising the following steps:
Preparing a cultured periosteal package according to claim 7;
Punching out a region surrounded by the groove using the groove formed on the bottom wall surface of the container to remove the periosteum piece;
Passing the crown through the hole formed in the container by the step and placing the container on the tooth;
Cleaving the container along the grooves formed in the side wall surface and the bottom wall surface of the container, and taking out the cultured periosteum so as to cover the periodontal tissue as an affected part.

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