WO2003075972A1

WO2003075972A1 - Tissue-forming composition

Info

Publication number: WO2003075972A1
Application number: PCT/JP2003/002661
Authority: WO
Inventors: Ken-Ichiro Hata; Hirokazu Mizuno; Keisuke Wada
Original assignee: Osteogenesis Co., Ltd.; Ueda, Minoru
Priority date: 2002-03-08
Filing date: 2003-03-06
Publication date: 2003-09-18
Also published as: AU2003211750A1

Abstract

It is intended to provide a composition which is usable in forming (regenerating) bone, periodontium or cartilage. Periost is collected and cultured under such conditions as enabling the proliferation of cells contained therein and the production of extracellular matrix to give a film-shaped construct. A composition containing this construct is used as a transplantation material.

Description

Description Tissue-forming composition Technical field

The present invention relates to a composition for tissue formation that can be used for repairing and regenerating bone tissue, periodontal tissue, or cartilage tissue. The present invention can be used, for example, for regeneration (reconstruction) of long stem bone, jaw bone, skull, periodontal tissue (specifically, alveolar bone and periodontal ligament) and articular cartilage. Background art

Various methods have been developed for the purpose of repairing or regenerating damage or defect of bone tissue or periodontal tissue. Known examples of clinical application include a guided tissue regeneration (GTR) method and a guided bone regeneration (GBR) method. In these methods, the tissue defect is covered with a film called a blocking film. The rate of regeneration of epithelial tissue and connective tissue is faster than the rate of regeneration of bone tissue.However, the use of such a blocking membrane can prevent epithelial tissue and connective tissue from penetrating into the tissue defect, and reduce bone tissue. A place for reproduction is secured. Thus, regeneration of bone tissue is promoted.

The GTR method or GBR method can be said to be technically established, but the clinical results vary greatly depending on the surgeon's technique, the state of the defect, or the difference in the material used, and a sufficient regenerative effect May not be obtained. In addition, it was pointed out that it takes a long time to obtain a sufficient regenerating effect, and that the amount of regenerated bone is relatively small.

Thus, an object of the present invention is to provide a novel method for regenerating bone or periodontal tissue instead of these methods. Disclosure of the invention

In view of the above problems, the present inventors have attempted to produce a graft material that can be used for regeneration of bone or periodontal tissue. First, we focused on periosteum, the connective tissue that covers the outer periphery of cortical bone. Periosteum has been conventionally used as a source of pluripotent cells or osteoblast-like cells contained therein (H. NAKAHARA et al., EXPERIMENTAL CELL RESEARCH 195, 492-503 (1991), .HJ KREDER et al., CRYOBIOLOGY 30, 107-115 (1993)). Since extracellular matrix produced by culturing periosteum makes it difficult to isolate these cells, cells are generally isolated and passaged before a thick layer of extracellular matrix is formed. The extracellular matrix has been removed by culturing or treating with trypsin-collagenase. On the other hand, osteoblast-like cells can be isolated from membrane-like structures obtained by culturing periosteum by enzymatic treatment, and bone is formed when these cells are transplanted with an appropriate artificial matrix. It is known. This means that the structure contains cells capable of forming bone. From this, the present inventors thought that the use of periosteum as a starting material could produce a graft material having a good bone regeneration effect. As a result of various studies, we succeeded in obtaining a membrane-like structure by culturing the collected periosteum under conditions that allow the cells contained therein to proliferate and produce extracellular matrix (matrix). did. Next, when this structure was transplanted into the alveolar bone defect, and its bone regeneration effect was examined, favorable regeneration of the alveolar bone was observed, and the structure is a useful material for the regeneration of bone or periodontal tissue. This was supported.

-On the other hand, the above-mentioned structure had an appropriate thickness and a certain strength, and could be directly sutured around a defect such as bone tissue at the time of transplantation. That is, the structure can be surely engrafted to the transplant site, and can be used for a long period of time. Therefore, conditions suitable for transplantation materials were provided, such that they could be held at the transplantation site. The handling was also easy.

From the above, the above-described structure obtained using periosteum as a starting material can provide an easy regeneration technique in addition to having a high bone regeneration effect, and is extremely useful in regeneration of bone or periodontal tissue. The finding that it is a useful transplant material was obtained.

Here, since the periosteum includes cells capable of differentiating into chondrocytes in addition to cells having the ability to differentiate into osteocytes, such cells will differentiate into chondrocytes. By culturing the periosteum under the conditions suitable for the above, it is considered that a transplant material containing cartilage cells or its precursor cells, which can be used for repair and regeneration of cartilage tissue can be obtained. The present invention is based on the above findings and provides the following configuration.

[1] A composition for tissue formation, comprising: a periosteum-derived cell; and an extracellular matrix of the cell.

[2] The composition for forming a tissue according to [1], which is a film-like structure.

[3] A tissue-forming composition comprising periosteum-derived cells, an extracellular matrix of the cells, and a bioabsorbable material.

[4] A film-like structure comprising: a first layer containing the cells and the extracellular matrix; and a second layer made of the bioabsorbable material and formed on the first layer. The composition for forming a tissue according to [3], which is a body.

[5] The tissue-forming composition according to [3] or [4], wherein a main component of the bioabsorbable material is polylactic acid, polydaricholic acid, or a lactate-glycolic acid copolymer.

[6] A membrane-like structure obtained by culturing periosteum under conditions in which cells contained therein can proliferate and produce extracellular matrix.

[7] The structure according to [6], wherein the condition is a condition in which the culture solution contains FBS.

[8] The above-mentioned condition is a condition in which the culture solution contains FBS and siascorbic acid. The structure according to [6].

[9] culturing the periosteum under conditions in which cells contained therein can proliferate and produce extracellular matrix to obtain a membrane-like structure;

A method for producing a composition for forming a tissue, comprising:

[10] On a support made of a bioabsorbable material, the periosteum is cultured under conditions in which cells contained therein can proliferate and produce extracellular matrix, and the support and the periosteum-derived cell layer Obtaining a membrane-like structure that is physically bonded,

A method for producing a tissue-forming composition, comprising:

[11] culturing the periosteum under conditions in which cells contained therein can proliferate and produce extracellular matrix; and

A step of forming a layer of a bioabsorbable material on the periosteum after culture to obtain a membrane-like structure,

A method for producing a composition for forming a tissue, comprising:

[1 2] a step of freezing or freeze-drying the film-like structure;

[9] The method for producing a tissue-forming composition according to any one of [9] to [11], further comprising:

[13] The production method according to any one of [9] to [12], wherein the condition is a condition that the culture solution contains FBS.

[14] The production method according to any one of [9] to [12], wherein the condition is a condition in which the culture solution contains FBS and siascorbic acid.

[15] A composition for forming bone or periodontal tissue, comprising a cell derived from periosteum, and an extracellular matrix of the cell.

[16] The composition according to [15], wherein the composition comprises a film-like structure.

[17] The composition according to [16], wherein the membrane-like structure comprises a single layer in which cells and extracellular matrix are mixed. [18] A sheet-like structure obtained by culturing periosteum under conditions in which cells contained therein can proliferate and produce extracellular matrix.

[19] The structure according to [18], wherein the condition is a condition in which the culture solution contains FBS.

[20] The structure according to [18], wherein the condition is a condition in which the culture solution contains FBS and L-ascorbic acid.

[21] A method for producing a composition for forming bone or periodontal tissue, comprising a step of culturing periosteum under conditions under which cells contained therein can proliferate and produce extracellular matrix. .

[22] a step of culturing the periosteum under conditions in which cells contained therein can proliferate and produce an extracellular matrix to obtain a membrane-like structure;

Freezing the film-like structure;

A method for producing a composition for forming bone or periodontal tissue, comprising:

[23] The production method according to [21] or [22], wherein the condition is a condition in which the culture solution contains FBS.

[24] The production method according to [21] or [22], wherein the condition is a condition in which the culture solution contains FBS and L-ascorbic acid.

[25] A composition for forming bone or periodontal tissue, comprising a periosteum-derived cell, an extracellular matrix of the cell, and a matrix material.

[26] The matrix material is one or more materials selected from the group consisting of collagen, hyaluronic acid, polyglycolic acid (PGA), polylactic acid (PLA), and inorganic bioabsorbable materials. [25] The composition according to [25].

[27] The matrix material is selected from the group consisting of / 3-tricalcium phosphate, α-triphosphate calcium, tetracalcium phosphate, octacalcium phosphate, and amorphous calcium phosphate. One or two or more materials [2 5. The composition according to [5].

[28] The composition according to [27], wherein the matrix material has an average particle diameter of 0.5 im to 50 im.

[29] The composition according to any one of [25] to [28], wherein the matrix material is contained in an amount of 50% by weight to 75% by weight.

[30] The composition according to any one of [25] to [29], further comprising a cell having an osteogenic ability.

[3 1] further comprising an extracellular matrix of cells having osteogenic ability

The composition according to [30].

[3 2] It further includes PRP (Platelet-rich Plasma). [25 :! -The composition according to any one of [31].

[33] The composition according to any one of [25] to [32], further comprising freeze-dried allogeneic bone.

[3 4] [1] ~ [: 5], [15] ~ [: 17] and [25 :! Or any one of [33] or [6 :! A method for regenerating bone tissue, comprising implanting the structure of any one of [8] and [18]-(: 20) into a bone defect.

[3 5] [1] ~ [: 5], [15] ~ [17] and [25] ~! : 3 3] or [6 ;! A method for regenerating periodontal tissue, comprising: transplanting the structure of any one of [8] and [18] to [20] into a periodontal tissue defect.

[3 6] [1] ~ [: 5], [1 5 :! To [17] and [25] to [: 33], or [6 :! A method for regenerating bone tissue, comprising implanting the structure of any one of [1] to [8] and [18] to [20] so as to block the bone defect from surrounding tissue.

[3 7] [1] ~! : 5], the composition of any one of [15] to [17] and [25] to [33], or [6 :! ~ [8] and [18] ~! : 20. A method for regenerating periodontal tissue, comprising implanting any one of the structures of [20] so as to block the periodontal tissue defect and the surrounding tissue. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph (temporal change in the area of the cultured periosteum (CP)) showing the measurement results in Example 2.

FIG. 2 is a graph showing the measurement results of alkaline phosphatase (ALP) activity in Example 2 (time-dependent change of alkaline phosphatase activity of cultured periosteum in vitro).

FIG. 3 shows the results of ALP activity staining in Example 2.

FIG. 4 shows the results of HE staining (HE-stained images of cultured periosteal sections) in Example 2.

FIG. 5 shows the results of immunostaining in Example 2.

FIG. 6 is a diagram showing the results of Western plotting in Example 2. FIG. 7 is a diagram showing the result of Alcian blue staining (Alcian blue stained image of cultured periosteum) in Example 2.

FIG. 8 is a diagram showing the state of the transplanted part in Example 3 three months after transplantation. Arrow a indicates the transplant group (the site where the transplantation material was transplanted), and arrow b indicates the control group (the defect was left alone).

FIG. 9 is a tissue section image (Truidine blue staining, 4 times) of the transplant group (A) and the control group (B) 3 months after transplantation in Example 3. In the transplantation group (A), the transplantation site (a) is filled with new bone having a lamellar structure. New cementum is also found on the root surface (b), which contacts the new bone and the root via fibrous tissue perpendicular to the root. On the other hand, in the control group (B), a small amount of fibrous tissue is seen at the bifurcation (c), but no bone is observed. Also, no regeneration of cementum on the root surface (d) was observed.

FIG. 10 shows the state of periodontal tissue defect (Class II) formed in Example 5. FIG.

FIG. 11 is a diagram showing a state immediately after transplantation of a periodontal tissue defect site (A: transplanted PRP gel and cultured periosteum, B: transplanted PRP gel and bioabsorbable membrane, C: Transplant only PRP gel (control)).

Fig. 12 is a diagram showing the state of the transplanted part at the time point three months after transplantation (A: transplanted PRP gel and cultured periosteum, B: transplanted PRP gel and bioabsorbable membrane, C: PRP gel only Transplant (control)).

Fig. 13 shows the soft X-ray images of the transplanted part (in order from the left, the group in which PRP gel and cultured periosteum were transplanted, the group in which PRP gel and bioabsorbable membrane were transplanted, and the group in which only PRP gel was transplanted. Group).

FIG. 14 is a diagram showing a micro CT tomogram of a CP (cultured periosteum) transplant specimen. a: periodontal tissue defect (parallel to the mandible), b: root (perpendicular to the mandible), C: partially enlarged view of b.

FIG. 15 is a view showing a non-decalcified tissue section image of a CP (cultured periosteum) transplant specimen. a: Root partially polished specimen, b: Regenerated periodontal tissue (left) and healthy dog periodontal tissue (right). FIG. 16 is a diagram showing a state of the culture dish before culturing the periosteum in Example 6, where a state in which a rectangular bioabsorbable membrane is placed in the culture dish is observed.

FIG. 17 is a diagram showing a state at the time when about one month has elapsed after the culturing in Example 6, and it was observed that a film derived from periosteum was formed in a state of being in close contact with the surface of the bioabsorbable film. Is done. BEST MODE FOR CARRYING OUT THE INVENTION

The composition for tissue formation of the present invention contains periosteum-derived cells and extracellular matrix of the cells. The composition of the present invention can be obtained by culturing periosteum under conditions in which cells contained therein (also referred to herein as “periosteal cells”) can proliferate and produce extracellular matrix. Prepared from the resulting film-like structure. Here, “periosteum” is a connective tissue that covers the outer periphery of cortical bone, and its structure is largely divided into an inner layer and an outer layer. The inner layer has a cell layer composed of pluripotent cells, and the outer layer is composed of collagen fibers and fibroblasts. On the other hand, the membrane-like structure obtained by culturing periosteum in the present invention is not clearly divided into two layers unlike periosteum, that is, it is composed of a single layer in which cells and extracellular matrix are mixed. It is characterized by the following. A single layer here means that there is no clear histological boundary within the structure. Therefore, the cell density need not be uniform throughout the structure.

On the other hand, in the present invention, “periosteum-derived cells” refer to cells that are produced by growing, differentiating, etc., the cells that originally existed as a result of culturing the collected periosteum. The composition for tissue formation of the present invention may contain a bioabsorbable material in addition to the periosteum-derived cells and the extracellular matrix of the cells. Such a composition for tissue formation is obtained by first culturing a periosteum under the above-mentioned conditions to obtain a membrane-like structure. Then, the surface of the membrane-like structure (the surface on the culture vessel side or the opposite side thereof) A layer (membrane) made of a bioabsorbable material is formed on the surface, and the structure can be prepared from the resulting structure. Further, the periosteum is cultured on a layer (membrane) made of a bioabsorbable material, and a structure obtained by this, that is, a layer derived from the periosteum is integrally formed on the layer made of the bioabsorbable material. It can also be prepared from a bonded structure. The structure obtained by these methods includes a first layer containing periosteum-derived cells and an extracellular matrix of the cells, and a second layer made of a bioabsorbable material and formed on the first layer. It will be. Here, the second layer made of the bioabsorbable material may have a multilayer structure. The multilayer in this case may include layers having different compositions. Bioabsorbable materials include collagen, gelatin, hyaluronic acid, fibrin, Polyglycolic acid (PGA), polylactic acid (PLA: poly-L-lactic acid or poly-D, lactic acid), lactic acid-glycolic acid copolymer (L-lactic acid-glycolic acid polymer, or D, L-lactic acid-glycolic acid Unified), glycolic acid-ε-force prolactone copolymer, L-lactate-ε-force prolactone copolymer, poly-ε-force prolactone, and / 3-tricalcium phosphate (hereinafter “/ that 3 _ TCPJ), α- tricalcium phosphate (hereinafter, gamma _alpha - TCP "hereinafter), tetracalcium phosphate, octacalcium phosphate, and inorganic bioresorbable material such as amorphous-phosphate calcium Examples can be given. These materials may be used alone or in any combination of two or more. Preferably, a bioabsorbable material containing polyglycolic acid, polylactic acid, or a lactic acid-glycolic acid copolymer as a main component is used. If such a bioabsorbable material is used, lactic acid or dalicholate, which is a decomposition product thereof, is a substance existing in the living body, so that high safety can be ensured. As a material containing a lactic acid-glycolic acid copolymer as a main component, for example, a commercially available GS membrane (trade name, GS Corporation, Tokyo) can be used. The bioabsorbable material can function as a support in the tissue forming composition. This function of the bioabsorbable material not only facilitates storage and transportation of the composition of the present invention, but also prevents deterioration in quality during storage and transportation, and furthermore, at the time of use (ie, at the time of transplantation). Also becomes easy. In addition, when the composition of the present invention is transplanted, good stability at the transplanted part can be obtained. In addition, the use of a bioabsorbable material makes it possible to impart appropriate strength to the composition of the present invention, so that the invasion of epithelial tissue and connective tissue into defects such as bone tissue can be prevented. When the composition of the present invention is used for the purpose of promoting tissue regeneration while preventing it, that is, when the composition of the present invention is used in expectation of a function as a barrier film, an excellent regenerative effect is expected. it can. The periosteum can be collected from the long bone, jaw, skull, etc. under local anesthesia. The collected periosteum is cut to an appropriate size if necessary, then transferred to an appropriate culture vessel (for example, a commercially available culture dish), and used for culture. The collected periosteum may be treated with an antibiotic before culture. When culturing the periosteum, it is preferable that the surface on the bone side of the periosteum be the surface side of the culture vessel. That is, it is preferable to perform culture in a state where the inner layer side of the periosteum adheres to the surface of the culture vessel. This is because good proliferation of periosteal cells is performed. When transferring the periosteum to the culture vessel, it is preferable to perform culture by adding a medium after confirming that the periosteum has adhered to the surface of the culture vessel. The culture conditions are not particularly limited as long as cells contained in the periosteum (periosteal cells) can proliferate. By culturing under these conditions, the periosteal cells proliferate, and the extracellular matrix (extracellular matrix) is produced accordingly. As a result, the thickness and strength of the explant increases, and a membrane-like structure having the strength required for transplantation, particularly the strength enough to withstand suturing, is formed. Examples of a medium that can be used include a medium containing FBS (ゥ fetal serum). Culture conditions that can proliferate multipotent cells, which are one of the periosteal cells, and promote differentiation into bone cells can be employed. Such culturing conditions may be employed in a part of the culturing period (for example, at the latter stage of culturing). Examples of such culture conditions include, for example, conditions using a medium containing FBS and L-ascorbic acid. On the other hand, it is possible to employ culture conditions in which multipotential cells contained in periosteal cells can proliferate and differentiation into cartilage cells is promoted. By adopting such culture conditions, a membrane-like structure containing cartilage cells can be obtained, and a composition suitable for regeneration of cartilage tissue can be prepared from the structure. Examples of such culture conditions include, for example, conditions using a medium containing bFGF (basic fibroblast growth factor) and / or TGF / 3. Of course, such culture conditions that promote the differentiation into cartilage cells may be employed in a part of the culture period (for example, late in the culture). The culturing time varies depending on the condition of the periosteum to be used. For example, culturing for 1 week to 2 months gives a structure in which the extracellular matrix has a membrane shape. If the obtained film-like structure is too thin, sufficient strength cannot be obtained. It is preferable to continue the culture for a time. Incidentally, a part of the tissue may be cut out during the culturing, transferred to another culturing vessel and used for subculture. The membrane-like structure obtained by culturing the periosteum is taken out of the culture vessel using tweezers or a scraper. In addition to these physical means, or instead of these physical means, chemical means such as enzyme treatment can be used. For example, trypsin is used to weaken the adhesiveness between the structure and the culture vessel, and then the structure can be peeled off from the culture vessel surface by tweezers or the like. Such a chemical treatment is used as long as the function of the structure as a transplant material is not impaired.

When culturing the periosteum on the bioabsorbable material layer (membrane), the structure consisting of the bioabsorbable material layer and the periosteal-derived layer (membrane) formed thereon is removed from the culture vessel. Taken out. Here, an example of a culturing method when a bioabsorbable material is used during culturing will be described below.

First, an appropriately sized membrane-shaped (sheet-shaped) bioabsorbable material is placed in a culture vessel. At this time, a part of the bioabsorbable material may be adhered and fixed to the culture vessel surface using an adhesive. Subsequently, after pouring the culture solution into the culture vessel, the collected periosteum is placed on a bioabsorbable material. Thereafter, culture is performed under the same conditions as described above. The collected periosteum may be treated with an antibiotic before culturing, and culturing is preferably performed with the periosteal bone side facing the bioabsorbable material.

In such a culture method using a bioabsorbable material, the periosteum placed on the bioabsorbable material can be rapidly and favorably adhered to the living body due to the high adhesiveness of the bioabsorbable material to the periosteum. Adhered and fixed to absorbent material surface. Therefore, the operation of confirming the attachment of the periosteum to the surface of the culture vessel, which was required when a bioabsorbable material is not used, is not required, and the culturing operation is simplified, and more reliable culturing is performed. It is possible to do. In order to remove unnecessary components, the structure taken out of the culture vessel may be washed with PBS, physiological saline, or the like.

When a layer made of a bioabsorbable material is formed after culturing the periosteum, for example, the layer is prepared in advance on the surface of the membrane-like structure taken out of the culture vessel and adhered to the culture vessel. Place the bioabsorbable material membrane and adhere. Such an operation can be performed in a container filled with physiological saline, an appropriate buffer, a culture medium, and the like. Membranous structure obtained by the above operation (with a layer of bioabsorbable material) Can be used as it is or after being cut to an appropriate size, as a composition (graft material) for transplantation into a defective portion of bone, periodontal tissue, or cartilage tissue. In addition, it may be frozen or freeze-dried once, thawed at an appropriate time before use, and then used. Freezing allows long-term storage, and also facilitates handling before use. Further, effects such as a decrease in antigenicity can be expected. In the case of freezing or the like, it is preferable to add an antifreeze agent (eg, glycerin, DMS0) in advance. On the other hand, a composition for transplantation (transplant material) is prepared by mixing the material obtained by the above operation into finely cut membrane-like structures (including those that have been subjected to treatment such as freezing) and other materials. You can also. The composition may be frozen or lyophilized once, thawed at an appropriate time before use, and then used. Other materials here include, for example, collagen, gelatin, hyaluronic acid, fibrin, polyglycolic acid (PGA), polylactic acid (PLA: poly-L-lactic acid, or poly-D, L-lactic acid), Lactic acid-glycolic acid copolymer (lactic acid-glycolic acid polymer or D, lactic acid-dalicolic acid polymer), glycolic acid-ε-force prolactone copolymer, lactate-ε-force prolactone copolymer , Poly-ε-force prolacton, matrix materials such as inorganic bioabsorbable materials such as i8_TC C, cells having bone forming ability, extracellular matrix of cells having bone forming ability (extracellular matrix), BMP ( Growth factors such as bone morphogenetic factors, and PRP (Platelet-rich Plasma). Lyophilized allogeneic bone and the like can be used. These materials may be used alone or in combination of two or more. If a matrix material such as collagen is used, it becomes a scaffold for bone cells (eg, osteoblasts) or cartilage cells at the transplantation site, and promotes the proliferation of the cells. Playback is performed. It is preferable to select an appropriate matrix material according to the tissue to be regenerated. For example, when the purpose is to regenerate bone tissue or periodontal tissue, In this case, it is preferable to use an inorganic bioabsorbable material such as / 3-TCP. When the purpose is to regenerate cartilage tissue, it is preferable to use, for example, type II collagen specific to cartilage tissue.

Similarly, when an extracellular matrix of cells having bone forming ability is used, a high regenerative effect can be obtained, but in this case, an effect of promoting bone formation by a growth factor such as BMP contained therein can be expected.

On the other hand, if bone-forming cells are used, direct regeneration of bone tissue by itself can be expected. In addition, if a growth factor such as BMP is used, it acts on cells present in the transplant site or cells in the composition of the present invention, and can promote bone formation.

Similarly, since PRP contains abundant growth factors that have the effect of promoting the proliferation and differentiation of osteogenic cells, effective regeneration of bone tissue and the like at the transplantation site can be expected by using this.

When the membrane-like structure is used as it is as a composition for transplantation (including a case where it is cut into a suitable size and then transplanted), the transplantation method is to cover a defect such as bone tissue, for example. Placement of the structure. It is preferable that the periphery of the structure be sutured to the tissue around the defect in order to allow the graft to survive and remain in the transplant for a long period of time. In addition, a membrane-like structure that is folded regularly or irregularly can be used for transplantation. For example, after the above-mentioned membrane-shaped structure is folded into a spherical shape or a polyhedral shape according to the shape of the defective portion, it can be transplanted so as to fill the tissue defective portion. At this time, other materials (eg, collagen, gelatin, hyaluronic acid, fibrin, polyglycolic acid (PGA), polylactic acid (PLA: poly-lactic acid or poly-D, L-lactic acid), lactic acid-glycolic acid copolymer -Lactic acid-dalico-co-lactic acid polymer or D, l_-lactic acid-glycolic acid polymer), Daricholic acid- ε-force prolactone copolymer, L-lactic acid-ε-force prolactone copolymer, poly -ε-force prolactone ;; 3 – matrix material such as TCΡ and other inorganic bioabsorbable materials; Cells, extracellular matrix (extracellular matrix) of cells with osteogenic potential, growth factors such as BMP (bone morphogenetic factor), and PRP (Platelet-rich Plasma). It may be transplanted to. Specifically, for example, after PRP is gelled by the action of tribohydrate, applied or filled in the defect, and then transplanted a membrane-like structure obtained by culturing the periosteum so as to cover the defect can do. In the above, the type of the inorganic bioabsorbable material is not particularly limited, and is selected from the group consisting of: 3-TCP, α-TCP, tetracalcium phosphate, octacalcium phosphate, and non-crystalline phosphate. Can be used. These materials can be used alone, or two or more arbitrarily selected materials can be used in combination. Preferably,) any of 8-TCP or a-TCP, or a combination thereof at an arbitrary ratio is used. More preferably, 3-TCP is used as the inorganic bioabsorbable material.

The inorganic bioabsorbable material can be obtained by a known method. In addition, commercially available inorganic bioabsorbable materials can also be used. 3) As TCP, for example, those manufactured by Olympus Optical Industries, Ltd. can be used. The inorganic bioabsorbable material is preferably in a powder form. In this way, the material to be implanted together with the composition of the present invention (or the composition itself when the composition of the present invention is composed of an inorganic bioabsorbable material) is formed into a slurry or paste. It can be prepared in a state with high fluidity. As a result, transplantation becomes easy, and engraftment and retention at the transplanted part are improved.

The powdered inorganic bioabsorbable material can be prepared by crushing and pulverizing an inorganic bioabsorbable material processed to an appropriate size to a desired particle size. Wear. The inorganic bioabsorbable material preferably has an average particle size of 0.5 m to 50 m. More preferably, an inorganic bioabsorbable material having an average particle size of 0.5 μηι to 1 μμηι is used. Still more preferably, an inorganic bioabsorbable material having an average particle size of 1 tm to 5 μηι is used. It is also possible to use a combination of a plurality of types of inorganic bioabsorbable materials having different particle diameters.

On the other hand, the cells having osteogenic ability refer to cells capable of forming bone tissue, and include osteoblasts, preosteoblasts, mesenchymal stem cells that have acquired the ability to differentiate into osteogenic cells, and the like. These cells may be used in any combination. Preferably, a mesenchymal stem cell that has acquired the ability to differentiate into bone cells is used as the cell having bone formation ability. Here, “acquired the ability to differentiate into bone cells” refers to a state in which an undifferentiated state has been oriented to differentiate into bone cells.

As the mesenchymal stem cells that have acquired the ability to differentiate into bone cells, not only autologous cells but also allogeneic allogeneic cells can be used. Mesenchymal stem cells that have acquired the ability to differentiate into bone cells (hereinafter referred to as bone differentiation-acquiring cells J) can convert undifferentiated mesenchymal stem cells (MSCs) into bone cells. It can be prepared by culturing under conditions that induce differentiation, for example, 3) -glycosephosphate (8-glycerophosphate), dexamethasone (Dexamethason), and L-ascorbic acid (L-ascorbic acid). By culturing undifferentiated mesenchymal cells in a medium containing the cells, differentiation into bone cells can be induced The culture conditions are not limited to these, and the differentiation into bone cells can be induced Known sources can be used as the conditions Sources of undifferentiated mesenchymal stem cells include bone marrow, periosteum, adipose tissue, and peripheral blood Can be. After collecting them according to a conventional method, undifferentiated mesenchymal cells are selected based on the presence or absence of adhesiveness. That is, undifferentiated mesenchymal stem cells can be obtained by selecting cells having adhesive properties from cells contained in bone marrow and the like. The extracellular matrix of cells having osteogenic ability is a matrix (matrix) existing around cells having osteogenic ability. Not only the extracellular matrix of the autologous cells but also the extracellular matrix of the same kind of allogeneic cells can be used. This is because BMPs and the like contained in such extracellular matrix are of the same species, so that similar effects can be expected even when allogeneic cells are used. Thus, the fact that not only the extracellular matrix of autologous cells but also the extracellular matrix of allogeneic cells can be used facilitates the preparation of the extracellular matrix of cells with the ability to acquire bone differentiation potential. PRP refers to platelet-rich PI asma, that is, plasma that is rich in platelets. In other words, it refers to blood whose platelets have been contracted. PRP is performed by subjecting blood collected according to the method of Whman et al. (Dean H. Whitman et al .: J Oral Max I lofac Surg, 55, 1294-1299 (1997)) to centrifugation. It can be manufactured in different ways. PRP is known to be rich in growth factors such as platelet-derived growth factor (PDGF), transforming growth factor β1 (TGF-β1), and transforming growth factor β2 (ΊGF-) 32). (Jarry J. Peterson: Oral surg Oral Med Oral Pathol Oral Radiol Endod, 85, 638-646 (1998)).

Here, an example of a method for preparing PRP is as follows. First, an anticoagulant such as sodium citrate is added to the collected blood and left at room temperature for a predetermined time. Thereafter, centrifugation is performed under the conditions where blood cells and buffy coat separate (for example, about 5,400 rpm). This separates into two layers (the upper layer is called platelet poor plasma; the lower layer contains blood cells and buffy coat). After removing the upper layer, the conditions under which red blood cells are separated For example, centrifuge at about 2,400 rpm). The resulting fraction substantially free of red blood cells (Platelet-rich Plasma: PRP) is collected. The method for preparing PRP is not limited to this method, and can be prepared by a method modified as necessary.

There is no general definition of the amount of platelets contained in PRP, but plasma containing about 5 to about 20 times platelets compared to the collected blood can be used as PRP in the present invention. . In addition, as long as the preparation is possible and there is no unreasonable burden in the preparation, it is preferable to use a platelet having as large a content of platelets as possible.

Preferably, autologous PRP is used. This eliminates the risk of toxicity or immune rejection.

PRP is rich in platelets, growth factors such as TGF-β1, TGF-β2, and fibrinogen. Therefore, platelets, growth factors such as TGF-β1, and fibrinogen are prepared, and a mixture of these can be used as {R} in the present invention. Platelets, various growth factors, and fibrinogen can be prepared by known methods or commercially available.

Platelets, fibrinogen, or platelets and fipurinogen may be used instead of PRII. It is also possible to use fibrin instead of fibrinogen here. The composition of the present invention obtained from the above membrane-like structure has bone cells or cells capable of differentiating into bone cells, or cartilage cells or cartilage cells. Since it contains cells, a positive bone formation effect can be obtained at the transplant site when transplanting You.

On the other hand, if the defect is covered with the film-like structure as described above, epithelial tissue and connective tissue can be prevented from invading the defect, and a place for regeneration of bone tissue and the like can be secured. Become. That is, the same operation and effect as the barrier film in the conventional GTR method or the like can be obtained. Here, a collagen film, a goatex film and the like used in the conventional GTR method and the like are further placed so as to cover the transplanted composition of the present invention so as to cover the defect such as bone tissue. You can also. In this way, invasion of epithelial tissue and the like to the defect can be more reliably prevented, and a higher bone and other regeneration effect can be expected.

Hereinafter, the present invention will be described in more detail with reference to Examples.

[Example 1] Collection of periosteum and preparation of cultured periosteum

After general anesthesia of a dog (hybrid, 1.5 years old, about 15 kg), the mandibular gingiva was incised using a scalpel for surgery, and about 1 cm ² of periosteum was collected from the mandible. The collected periosteum was subjected to explant culture (explant culture: explant culture). First, the collected periosteum was transferred to a culture dish (Falcon) having a diameter of 10 cm. At this time, the periosteum was placed with the inner layer side (bone side) facing down. Transferred culture dishes under this condition 3 7 ° kept the C0 ₂ incubator in beta and C, and allowed to stand for 30 minutes. C0 ₂ incubator removed from the culture dish, after the explant (periosteum) was confirmed to have adhered to the surface of the petri dish, poured media. The medium was commercially available Medium 199 (manufactured by Gibco) and FBS (manufactured by Gibco), L-ascorbic acid, and Antibiotic-Antimoicic (manufactured by Invitrogen) were respectively 10%, 25Mg / mk and 1% (v / v) was used.

Then dishes were transferred to C0 ₂ incubator, lines cultured in 37 ° C, of 5% C0 ₂ Conditions I got it. At the beginning of the culture, the medium was changed once every three days. The medium was replaced once a day when the cells could be visually observed to grow. The medium was exchanged using a dropper. First, a small amount of the medium was removed by suction, and then the medium kept at 37 ° C was poured slowly and slowly until the explants were sufficiently immersed.

When cultured under the above conditions, the extracellular matrix (matrix) accumulates and the size and thickness of the explants increase from around the 10th day after the start of culture. Was done. By continuing the culture for about one month, a membranous explant (cultured periosteum) of about 50 cm ² and a thickness of about 0.3 to 0.5 mm was obtained. The explant (cultured periosteum) was peeled off from the surface of the culture dish using tweezers, and used as a material for transplantation.

[Example 2] Characterization of cultured periosteum

(2-1) Growth curve

The periosteum was collected from the mouse and the human, and subjected to an explant culture in the same manner as in Example 1. The resulting translucent circular film (CP: cultured per iosteum) was visually observed and its diameter was measured temporarily. The measurement was also performed on the dog CP obtained in Example 1 in parallel. The diameter (r) of the membrane on days 7, 10, 14, 21 and 28 after the start of the culture was measured on a lOOirnn petri dish, and the area was calculated. Assuming that the shape of the CP is circular, the area was determined by the following equation.

Area = 7t (1/2 r) ²

Figure 1 shows a graph summarizing the measurement results. As shown in this graph, good CP was obtained in all animal species under similar culture conditions. In particular, the growth rate of human CP was comparable to that of other animal species, and clinical application was fully expected. (2-2) ALP activity measurement

The dog periosteum was cultured on a 100-well Petri dish in the same manner as in Example 1, and the resulting CP was taken out over time and extracted on ice (0.05 M Tris-HCI, pH 9.0, 0.075% This was homogenized in Triton X-100) and stirred to prepare a sample solution for ALP activity measurement.

P-Nitrophenylphosphate was used as a substrate for ALP activity measurement, and the measurement was performed by a conventional method. To measure the amount of DNA, CP was homogenized in a 500 / zL extract (0.05 M Tris-HCI, pH 7.4) on ice to prepare a sample solution. For the measurement, IOO L of sample solution, 700 μL of 0.05- Tris-HCI solution (pH 7,4), 965 L of 0.05Μ phosphate buffer (containing 4.0Μ Na CI, pH 7.4), 165 μΙ_ of H20, A 70 / tiL staining solution (10 μg / mL bisbenzimi amidazole in 0.05 M Phosphate buffer, pH 7.4, 2. OM NaCI) was mixed, and the fluorescence at 458 nm was measured (excitation light: 365 nm). High molecular weight DNA derived from thymus was used as a standard for measuring the amount of DNA.

Fig. 2 shows a graph summarizing the measurement results. In the cultured periosteum (CP), ALP activity reached a maximum 4 weeks after culture. This suggests that cultured periosteum after 4 weeks of culture is suitable as a transplant material for bone regeneration.

(2 — 3) ALP activity staining

After 4 weeks of culturing on a 100 mm petri dish, CP was stained with an al-riphosphatase activity staining kit (LEUKOCYTE ALKALINE PHOSPHATASE SIGMA) and observed with a microscope (Fig. 3). The stained (red) cells can be observed throughout the CP, and it can be confirmed that the ALP is positive in the CP after 4 weeks of culture.

(2-4) HE (Hematoxy)

After 4 weeks of culture on a 100 mm Petri dish, CP was fixed with 10% phosphate buffered formalin and embedded in paraffin. After making a 3μηι thick paraffin section with a microtome, Was stained with HE and observed with an optical microscope (Fig. 4). It can be confirmed that the thickness of CP increased with the cultivation period, and reached a thickness of at least 100 to 200 m 4 weeks after the start of culture. In addition, it can be confirmed that ECM (extracellular matrix: extracellular matrix) exists around the cells existing in the CP. Considering that the accumulation of ECM and the increase in the thickness of the CP are necessary for the CP to have sufficient physical strength to withstand suturing, it is appropriate to use CP after a certain culture period as a transplant material. It is considered to be.

(2 — 5) Immunostaining

Block the paraffin section prepared in (2-4) with a blocking agent (Block Ace ™, Dainippon Pharmaceutical), wash with phosphate buffered saline, and wash the primary antibody (anti-osteocalcin antibody, anti-type I collagen antibody) , And anti-steanectin antibody and anti-osteobontin antibody). After washing with phosphate buffered saline, the cells were reacted with a secondary antibody (ant mouse, rabbit IG Hist fine simple stin MAX-PO (MULT I) Nichirei) for 1 hour, sealed, and observed under a microscope. (Figure 5).

CP was positive by analysis with anti-type I collagen antibody, anti-old steocalcin antibody, anti-osteobontin antibody, and anti-osteonectin antibody. Confirmation of the presence of these proteins, which are osteoblast differentiation markers, strongly suggested that cells capable of forming bone were present in CP.

(2 — 6) Western Blotting

After 4 weeks of culture on a 100-well petri dish, the CP was finely divided. After freezing CP with liquid nitrogen, it was ground in a mortar, and a protein extract (Cy to Buster Protein Extraction Reagent (Novagen)) was added. The supernatant obtained by centrifugation at 15000 rpm for 5 minutes is subjected to gel electrophoresis, transferred to a PVDF membrane, and the antibody (anti-steocalcin antibody, anti-type I The antibody was reacted with a gen antibody, an anti-steronectin antibody, and an anti-steobontin antibody) and then chemiluminescent (Fig. 6). Analysis with anti-type I collagen antibody, anti-steobontin antibody and anti-osteonectin antibody confirmed that various osteoblast differentiation markers were present in CP.

(2 — 7) Alcian blue staining

Alcian blue staining was performed using the paraffin section prepared in (2-4) in a conventional manner (Fig. 7). As a result, good staining properties were confirmed, and it was confirmed that glycosaminodalican was present in CP in a large amount. In other words, it was confirmed that CP also contained a glycoprotein characteristic of cartilage. On the other hand, immunostaining also confirmed the presence of type II collagen (data not shown), suggesting that CP also has the characteristics of cartilage.

[Example 3] Transplantation of explants and evaluation of bone formation ability (1)

Using a dental drill on the alveolar bone of the premolar part of the dog from which the periosteum was collected, use the dental drill to relocate to the alveolar bifurcation. Defects). The material for transplantation (cultured periosteum) obtained in Example 1 was wrapped around the root of the tooth so as to cover the defect, and was further fixed to the cervical region with a suture in a collar-wrap shape. A similar defect was formed in the adjacent premolar alveolar bone, which was left untreated and used as a control.

Three months after the transplantation, the condition of the transplanted part was visually observed, and a tissue section image of the transplanted part was also prepared and examined from a histological viewpoint to evaluate the osteogenic ability of the transplantable material. . Fig. 8 shows a photographic image of the transplanted part 3 months after transplantation. In the non-transplanted site (arrow b), which was a control group, no tissue filling was observed at the defect site. On the other hand, it can be seen that the transplant site (arrow a) has been replaced by some tissue. FIG. 9 is a tissue section image of the transplanted group (A) and the control group (B) 3 months after the transplantation. Toluidine blue was used for staining.

In the transplant group (A), the transplant site (a) is filled with new bone having a lamellar structure. The root surface (b) also contains new cementum, which is in contact with the new bone and the root via fibrous tissue perpendicular to the root. On the other hand, in the control group (B), a small amount of fibrous tissue is seen in the surgically cut branch (c), but no bone is observed. There is no regeneration of cementum on the root surface (d).

[Example 4] Preparation of PRP

(4-1) Blood sampling

About 20 mL of blood was collected from the hind limb and saphena vein of the dog from which the periosteum was collected using a vacuum blood collection tube (TERUM0, Venoject II vacuum blood collection tube, 3.13 sodium citrate 0.5 mL). (4-2) Separation of PRP

PRP was prepared from the collected blood by centrifugation. Centrifugation was performed according to the Japanese Red Cross separation method. The blood in the vacuum blood collection tube was transferred to a 50 mL centrifuge tube (manufactured by Greiner), and centrifuged at room temperature (22 ° C) and 230G for 5 minutes (HITACHI HimacCT6D rotor RT 5S5 50 mL tube I100rpm). This centrifugation splits the blood into two layers with a buffy coat (membrane) in between. The upper layer is rich in platelets, while the lower layer is rich in red blood cells. Therefore, the upper layer was aspirated with a pipette so as not to inhale the red blood cell components in the lower layer, and transferred to a 15 mL centrifuge tube (Greiner). The transferred blood was subjected to centrifugation at 2,500 rpm for 5 minutes using the same centrifuge. After centrifugation, gently aspirate the liquid from the liquid surface so that about 1/10 of the blood volume at the start of processing remains (leave 2 niL of liquid if the processing blood is 20 mL). Transferred to container. In this 15 mL centrifuge tube The remaining liquid was used as PRP.

[Example 5] Explant transplantation and evaluation of bone formation ability (2)

By completely penetrating the dental drill into the alveolar bone of the premolar region of the dog from which the periosteum was collected (ie, without leaving the lingual cortical bone), the periodontal tissue defect including the interdental bifurcation (Clas si II Model). The size of the defect was 3 mni wide and 4 heights (Fig. 10). When making the defect, the bone tissue covering the root was also removed, and the other periodontal tissues (periodontal membrane, cementum) of the root and the root bifurcation were carefully removed with a dental scaler. .

Separately, 1.2 ml of PRP obtained in Example 4 and 0.2 ml of a dicitrombin solution (10000 units, in 10% calcium chloride solution) were mixed in a syringe to obtain a PRP gel. After filling the thus obtained PRP gel into the periodontal tissue defect, the material for transplantation (cultured periosteum) obtained in Example 1 was wrapped around the root to cover the defect, and The collar was fixed to the cervical region using a suture (PRP + CP transplantation). A similar defect formed in the adjacent premolar alveolar bone was filled with PRP gel and covered with a bioabsorbable membrane (GC membrane (trade name, GC Corporation, Tokyo)). The control group was a suture-fixed group (PRP + GTR membrane), and a group in which the defect was filled with PRP gel only (PRP). FIG. 11 shows the state of the defect after each transplantation operation.

(5-1) Appearance observation

Three months after the transplantation, the gingiva of the transplanted part was opened and the appearance was observed (Fig. 12). No recovery of periodontal tissue was observed in the group transplanted with PRP gel alone (C: PRP). In the group using PRP gel and bioabsorbable membrane (B: PRP + GTR membrane), some recovery of bone tissue was observed, but it was far from complete recovery of the defect. On the other hand, the group using PRP gel and cultured periosteum (A: (PRP + CP transplantation), the defect was almost completely restored, and more surprisingly, the periodontal tissue was restored to the root.

(5-2) Soft X-ray analysis

Next, soft X-ray analysis was performed according to the following procedure. After the dog was sacrificed, the mandible was removed, fixed with 10% phosphate buffered formalin solution, and photographed with a S0FTEX closed radiograph (Fig. 13). The imaging was performed under the conditions of 70 kV, 3 mA, and X-ray irradiation time of 180 seconds.

As a result of soft X-ray analysis, the alveolar bone at the defect was hardly recovered in the group to which only the PRP gel was transplanted (PRP), whereas the root bifurcation in the group to which cultured periosteum was combined (PRP + CP transplantation) The alveolar bone was found to be almost completely recovered. In the group combined with bioabsorbable membrane (PRP + GTR membrane), the results were intermediate between these two.

(5 — 3) Micro CT (Computed Tomography) Analysis

X-ray micrographs were taken by X-ray micro-CT of a CP transplant specimen fixed with 10% phosphate buffered formalin solution (PRP gel and cultured periosteum transplanted group) (Fig. 14). The radiographs were taken of the root bifurcation defect preparation site (parallel to the mandibular surface) and the tooth root (vertical surface to the mandible).

From Fig. 14, it can be confirmed that the root bifurcation is filled with dense bone tissue as confirmed by the soft X-ray photograph. In addition, there is no direct contact between the tooth and the alveolar bone, and a shadow is observed (Fig. 14a). This suggests that the tooth and the regenerated alveolar bone are connected via soft tissues such as periodontal ligament. In addition, periodontal tissue is clearly regenerated on the root surface (Fig. 14b).

(Fig. 14c), it can be clearly confirmed that the teeth and the alveolar bone are not in direct contact. (5-4) Confirmation of regenerated periodontal tissue by photograph of non-decalcified tissue section

Non-decalcified section specimens were prepared for CP transplant specimens fixed with 10% phosphate buffered formalin solution (group to which PRP gel and cultured periosteum were transplanted). Polishing specimens were prepared using polymetylmethacrylate as the embedding agent. Staining was performed by a conventional method using toluidine blue.

The alveolar bone, periodontal ligament, tooth cementum, and tooth dentin were confirmed from the reconstructed slice images of the periodontal tissue (Fig. 15a). In addition, it was confirmed that the periodontal tissue was regenerated to such an extent that it was almost indistinguishable from the comparison with the normal periodontal tissue image (Fig. 15b). From the results of each analysis described above, it was confirmed that the cultured periosteum of the present invention had extremely excellent periodontal tissue regeneration ability. In addition, it has been shown that the use of cultured periosteum also regenerates periodontal tissue other than bone tissue such as cementum and periodontal ligament to the same level as healthy ones. It was confirmed that the material had very excellent properties. Therefore, it can be said that the use of the cultured periosteum of the present invention enables regeneration of bone or periodontal tissue, which is clearly superior to existing methods such as GTR.

[Example 6] Preparation of implant material using bioabsorbable material

First, a bioabsorbable membrane made of a copolymer of lactic acid and glycolic acid (Gee Membrane (trade name, Gishi Ichi, Tokyo) was used in this example) was placed in a culture dish (10 cm diameter, manufactured by Falcon). After cutting to the appropriate length, the cells were transferred into a culture dish filled with the medium. After confirming that the bioabsorbable membrane had completely settled to the bottom of the culture dish, the periosteum (about 1 _cm square) collected by the method of Example 1 was placed on the bioabsorbable membrane. The medium was the same as that in Example 1 (commercially available Medium 199 (manufactured by Gibco), FBS (manufactured by Gibco), L-ascorbic acid, and Ant biotic-Ant imyotic (I πν rogen) Medium added at 10%, 25 / ιηΚ and 1% (v / v). Then dishes were transferred to C0 ₂ incubator, lines cultured in 37 ° C, of 5% C0 ₂ conditions ivy. The subsequent culture conditions and culture method were the same as in Example 1.

After culturing under the above conditions, as in Example 1, the cultured periosteum formed on the periphery of the explant began to be observed macroscopically from around day 10 after the start of culturing. Was. Then, by continuing the culture for about one month, a substantially circular cultured periosteum of about 10 cm ² as a whole was formed on the bioabsorbable membrane. As a result, a membrane-like structure in which the bioabsorbable membrane and the cultured periosteum derived from the periosteum were physically bonded was obtained. FIG. 16 is a diagram showing a state before periosteum culture. It is observed that the bioabsorbable membrane formed into a rectangular shape is placed in the culture dish. FIG. 17 is a diagram showing a state at the time when about one month has elapsed after the culture. It can be seen that a substantially circular cultured periosteum derived from the periosteum was formed in close contact with the surface of the bioabsorbable membrane. When the bioabsorbable membrane was removed from the culture dish and the cultured periosteum formed thereon was observed, the periosteum subjected to the culture remained at the center of the bioabsorbable membrane. The membrane was formed, and the periphery where the cultured periosteum was present was milky white. In addition, it was confirmed that the cultured periosteum was formed on the bioabsorbable membrane in a tightly contacted state. The present invention is not limited to the description of the embodiment and the example of the above invention. Various modifications are included in the present invention without departing from the scope of the claims and within the scope of those skilled in the art. Industrial applicability

According to the present invention, there is provided a composition for tissue formation using a membranous structure obtained by culturing periosteum. The composition of the present invention can be applied to various fields requiring repair and regeneration of bone tissue, periodontal tissue or cartilage tissue. For example, periodontal disease It can be applied to regeneration of alveolar bone and periodontal tissue in a defective part of alveolar bone due to the above. It can also be applied to bone augmentation when artificial roots are implanted in the ridge where high bone resorption is observed. Further, the present invention can be applied to regeneration of bone tissue at a bone defect caused by trauma or various bone diseases, and reinforcement or supplementation of bone. According to one aspect of the present invention, since cells having osteogenic ability are contained, bones can be actively regenerated by proliferating at the transplant site. As a result, bone regeneration can be performed in a relatively short time, and regeneration with sufficient bone mass can be expected.

On the other hand, since the film-like structure constituting the composition of the present invention has strength enough to withstand suturing, it can be surely engrafted to the transplanted part and can be held in the transplanted part for a long period of time. Is possible. Therefore, it can be used as a barrier membrane for preventing invasion of epithelial tissue and connective tissue into a defect such as a bone tissue, and a place for bone regeneration at the defect can be secured. In particular, when the composition of the present invention is constituted by using a bioabsorbable material, the bioabsorbable material imparts more strength and more effectively functions as a barrier film.

Claims

The scope of the claims

1. A tissue-forming composition comprising periosteum-derived cells and an extracellular matrix of the cells.

2. The tissue-forming composition according to claim 1, which is a film-like structure.

3. A tissue-forming composition comprising periosteum-derived cells, an extracellular matrix of the cells, and a bioabsorbable material.

4. A film-like structure comprising: a first layer containing the cells and the extracellular matrix; and a second layer made of the bioabsorbable material and formed on the first layer. 4. The tissue-forming composition according to claim 3, which is a body.

5. The tissue-forming composition according to claim 3, wherein the main component of the bioabsorbable material is polylactic acid, polydaricholic acid, or a lactic acid-glycolic acid copolymer.

6. A membranous structure obtained by culturing periosteum under conditions in which cells contained therein can proliferate and produce extracellular matrix.

7. The structure according to claim 6, wherein the condition is a condition that the culture solution contains FBS.

8. The structure according to claim 6, wherein the condition is a condition in which the culture solution contains FBS and sescorbic acid.

9. culturing the periosteum under conditions in which cells contained therein can proliferate and produce extracellular matrix to obtain a membranous structure;

A method for producing a composition for forming a tissue, comprising:

10. On the support made of a bioabsorbable material, the periosteum is cultured under conditions in which the cells contained therein can proliferate and produce extracellular matrix, and the support and the cell layer derived from the periosteum are cultured. Obtaining a physically bonded membrane-like structure;

A method for producing a tissue-forming composition, comprising:

11. culturing the periosteum under conditions in which the cells contained therein can proliferate and produce extracellular matrix; and

A step of forming a layer made of a bioabsorbable material on the periosteum after culture to obtain a membrane-like structure,

A method for producing a composition for forming a tissue, comprising:

1 2. Freezing or freeze-drying the membrane-like structure,

10. The method for producing a tissue-forming composition according to claim 9, further comprising:

13. The production method according to claim 9, wherein the condition is a condition that the culture solution contains FBS.

14. The production method according to claim 9, wherein the condition is a condition in which the culture solution contains FBS and L-ascorbic acid.