WO2004052418A1

WO2004052418A1 - Graft for regenerating bone-cartilage tissue

Info

Publication number: WO2004052418A1
Application number: PCT/JP2003/015573
Authority: WO
Inventors: Guoping Chen; Hajime Ohgushi; Takashi Ushida; Tetsuya Tateishi; Yoshinori Takakura; Shigeyuki Wakitani
Original assignee: National Institute Of Advanced Industrial Science And Technology
Priority date: 2002-12-06
Filing date: 2003-12-05
Publication date: 2004-06-24
Also published as: JPWO2004052418A1; JP4923235B2; AU2003289197A1

Abstract

A bone-cartilage tissue graft having a structure in which a bone tissue is connected to a cartilage tissue; and a process for producing a bone-cartilage tissue graft comprising the step wherein at least one type of bone-forming cells selected from the group consisting of bone cells and stem cells capable of differentiating into bone cells and at least one type of cartilage-forming cells selected from the group consisting of cartilage cells and stem cells capable of differentiating into cartilage cells are sowed in a scaffold material capable of holding the cells in such a state that the bone-forming cells are substantially separated from the cartilage-forming cells.

Description

Specification

Osteochondral tissue regeneration implant

Technical field

The present invention relates to regeneration of bone and cartilage tissue used for repairing bone and cartilage diseases such as refractory osteoarthritis and cartilage wounds caused by accidents.

Background art

Osteoarthritis is a frequent disease in the field of orthopedics and often results in a high degree of dysfunction. Artificial joint surgery is the main body of surgical treatment, but current artificial joint parts made of metal or high molecular polymer have problems such as infection, wear, loosening, and breakage. In the case of tissue transplantation, in addition to the problem of a shortage of donors, if the donor is another person, there is also a problem of a giant response based on an immune response. Due to the existence of such various problems, it is considered that a regenerative medical engineering-based treatment is ideal at present, and research on regeneration of bone and cartilage tissue is being actively conducted. When transplanting cartilage tissue, fixation of cartilage tissue is important for wound healing, and it is most desirable to regenerate bone and cartilage tissue simultaneously.

Furthermore, in order to regenerate bone and cartilage tissue by regenerative medical engineering techniques, bone, cartilage cells or stem cells capable of differentiating into bone and cartilage cells are used as a scaffold for proliferation, and the living body that forms A three-dimensional porous carrier material is required as a tissue support. Such carrier materials are required to have conditions such as porosity, biocompatibility and bioabsorbability. Conventionally, polylactic acid (PLA) ゃ polyglycolic acid (PGA) or a copolymer of lactic acid and glycolic acid A three-dimensional porous carrier material prepared with a bioabsorbable synthetic polymer such as (PLGA) or a natural polymer such as collagen is often used (Japanese Patent Application Laid-Open Nos. 2002-146086 and 2002-2002). 146084; JP-A-2002-143291; JP-A-2002-143290).

However, those made of the above-mentioned bioabsorbable synthetic polymer have excellent mechanical strength, but are hydrophobic, and because of their large gaps and cracks, most cells pass through the gaps. Without it, it is extremely difficult to seed bone, chondrocytes or stem cells that can differentiate into bone and chondrocytes. For this reason, an effective cell seeding rate cannot be obtained, and a large amount of these cells cannot be accumulated on the support carrier. Therefore, there is a problem that the regeneration efficiency of bone and cartilage tissue is low. On the other hand, collagen sponge, which is a natural high molecular porous material derived from living organisms, is hydrophilic, has excellent interaction with cells, and is easy to seed cells. The problem was that it was difficult to handle clinically because it was low, and it was soft and easily twisted.

In addition, a porous carrier material for simultaneously regenerating a tissue having a hierarchical structure such as bone and cartilage tissue has not been developed yet.

An object of the present invention is to solve such problems of the related art. Specifically, it has good biocompatibility and is capable of differentiating into at least one type of osteogenic cell selected from the group consisting of osteocytes or stem cells capable of differentiating into osteocytes, or chondrocytes or chondrocytes It is easy to seed at least one type of chondrogenic cell selected from the group consisting of stem cells, and the seeding efficiency is good. Therefore, it is possible to accumulate a large amount of these cells on a support (scaffold). An object of the present invention is to provide a support for osteogenic cells or osteochondrogenic cells, which has good regeneration efficiency of tissues and cartilage tissues, has high mechanical strength, and is easy to handle even in clinical practice.

Further, the present invention provides a bone material having such a structure that bone-forming cells and cartilage-forming cells are laminated on such a support material in a state where they are substantially separated from each other, and the bone tissue and the cartilage tissue are connected to each other. An object of the present invention is to provide an in vivo implant for simultaneous regeneration of cartilage tissue. .

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Porous sheet made of a typical bioabsorbable synthetic polymer according to the present invention and another bioabsorbable material (bioabsorbable natural polymer, cell growth factor and cell differentiation control factor or their derivatives). 1 shows a schematic diagram of a quality scaffold material. In Figure 1:

(a) a porous body made of another bioabsorbable material (bioabsorbable natural polymer, cell growth factor, cell differentiation control factor or a derivative thereof);

(b) a sheet-like porous structure of a bioabsorbable synthetic polymer; and

(c) Composite porosity composed of a sheet-shaped bioabsorbable synthetic polymer and a porous body composed of another bioabsorbable material (bioabsorbable natural polymer, cell growth factor, cell differentiation control factor, or a derivative thereof) Quality structure. Figure 2: Representative sheet-shaped bioabsorbable synthetic polymer according to the present invention, another bioabsorbable material (bioabsorbable natural polymer, cell growth factor, cell differentiation control factor or their derivative) and inorganic material. The schematic diagram of the porous scaffolding material which consists of a dagger. In Figure 2:

(b) a sheet-like porous structure of a bioabsorbable synthetic polymer;

(c) A sheet-shaped bioabsorbable synthetic polymer and another bioabsorbable material (bioabsorbable natural macromolecule, cell growth factor, cell segment control factor or derivative thereof) A porous composite porous structure; and

(d) A sheet-shaped bioabsorbable synthetic polymer having a coating layer made of an inorganic compound on the outer surface of the porous body, and another bioabsorbable material (natural polymer, cell growth factor, cell differentiation inducer Or a derivative thereof) and a porous body having a layer of an inorganic compound.

Figure 3: Representative block-like bioabsorbable synthetic polymer according to the present invention and another bioabsorbable material (bioabsorbable natural polymer, cell growth factor, cell differentiation regulator or their derivatives). Schematic diagram of a porous scaffold material. In Figure 3:

(b) a block-like porous structure of a bioabsorbable synthetic polymer; and

(c) A porous composite of a block-shaped bioabsorbable synthetic polymer and a porous body made of another bioabsorbable material (natural bioabsorbable polymer, cell growth factor, cell differentiation control factor or their derivatives) Porous structure.

Figure 4: Typical block-like bioabsorbable synthetic polymer according to the present invention, another bioabsorbable material (bioabsorbable natural polymer, cell growth factor, cell differentiation control factor or derivative thereof) and FIG. 2 is a schematic view of a porous scaffold material made of an inorganic compound. In Figure 4:

(b) a block-shaped porous structure of a bioabsorbable synthetic polymer; (c) Porous composite with a porous body consisting of a block-shaped bioabsorbable synthetic polymer and another bioabsorbable material (bioabsorbable natural polymer, cell growth factor, cell differentiation control factor or their derivatives) A porous structure; and

(d) A block of a bioabsorbable synthetic polymer in block form and another bioabsorbable material (bioabsorbable natural polymer, cell growth factor, cell segment control factor or their derivatives) and an inorganic compound A composite porous structure of a porous body and a porous body having a layer.

Figure 5: Typical sheet-shaped bioabsorbable synthetic polymer according to the present invention, another bioabsorbable 'ft material (bioabsorbable natural polymer, cell growth factor, cell differentiation regulator or their derivatives) FIG. 2 is a schematic diagram of a gel-like scaffold material. In Figure 5:

(a) a sheet-like porous structure of a bioabsorbable synthetic polymer;

(b) a gel made of another bioabsorbable material (bioabsorbable natural polymer, cell growth factor, cell differentiation controlling factor or a derivative thereof);

(c) Composite of a sheet-shaped bioabsorbable synthetic polymer and another bioabsorbable material (a bioabsorbable natural polymer, cell growth factor, cell differentiation control factor or their derivative) and a porous body consisting of a gel Structure.

Figure 6: Schematic representation of a bone-cartilage tissue regeneration implant. In Figure 6:

(a) cartilage tissue comprising a second scaffold material and chondrogenic cells; and

(b) Bone tissue composed of the first scaffold material and osteogenic cells.

Figure 7. An electron micrograph of a composite mesh of a bioabsorbable synthetic polymer and a bioabsorbable natural polymer.

Figure 8. An electron micrograph of a composite mesh of a bioabsorbable synthetic polymer, a bioabsorbable natural polymer, and an inorganic compound.

Fig. 9. Bone and cartilage tissue of the regenerated dog joint.

FIG. 10. A schematic longitudinal cross-sectional view of the laminated sheet-like scaffold material. In Figure 10: (a) Porous body or gel made of another bioabsorbable material (bioabsorbable natural polymer, cell growth factor, cell differentiation control factor or their derivatives);

(b) Sheet-like or block-like porous structure of bioabsorbable synthetic polymer Figure 11. (a) and (b) are schematic cross-sectional views of a typical sheet-like composite material according to the present invention. . In Figure 11: (a) a porous body or gel made of another bioabsorbable material (bioabsorbable natural polymer, cell growth factor, cell fraction control factor or derivative thereof); and

(b) Sheet-shaped or block-shaped porous structure of bioabsorbable synthetic polymer Figure 12: Photograph of bone and cartilage tissue of human ankle joint. In Figure 12:

(a) MR image of the ankle joint of the patient before the operation (osteochondral disorder inside the talus: arrow);

(b) CT image of the ankle of the patient before surgery (osteochondral disorder inside the talus: arrow);

(c) an external view of the gel of the stem cells; and

(d) Stem cell-bone structure (arrow) Radiograph after transplantation.

Disclosure of the invention

The present invention has been made to solve the above problems, and relates to the following implant and a method for producing the same.

Item 1. An osteo-cartilage tissue transplant having a structure in which bone tissue and cartilage tissue are connected. Item 2. The bone tissue comprises a scaffold material capable of retaining cells and at least one type of osteogenic cell selected from the group consisting of bone cells retained by the scaffold material or stem cells capable of differentiating into bone cells. The cartilage tissue according to claim 1, wherein the cartilage tissue comprises a scaffold material capable of retaining cells and at least one chondrogenic cell selected from the group consisting of chondrocytes retained in the scaffold material or stem cells capable of differentiating into chondrocytes. Transplant.

Item 3. The implant according to Item 1, wherein the scaffolding material is a sheet or a block.

Item 4. The implant according to Item 2, wherein the scaffold material further comprises a structure made of another compound formed in a mesh structure made of a bioabsorbable synthetic polymer or an internal structure matrix of a porous material.

Item 5. The implant according to Item 4, wherein the structure in the internal structure matrix is a porous structure or a gel.

Item 6. The structure in the internal structure matrix further includes one or more members selected from the group consisting of a natural polymer, a cell growth factor, a cell differentiation controlling factor and an inorganic compound, or a derivative thereof. Item 5. The transplant according to Item 4. Item 7. The implant according to Item 2, wherein the bone tissue scaffolding material is a porous ceramic. Item 8. The implant according to Item 2, wherein the cartilage tissue scaffolding material is a gel.

Item 9. The transplant according to Item 2, wherein the osteogenic cells and / or chondrogenic cells are bone marrow-derived stem cells.

Item 10. At least one type of osteogenic cell selected from the group consisting of bone cells or stem cells capable of differentiating into osteocysts and at least one type selected from the group consisting of chondrocytes or stem cells capable of differentiating into chondrocytes A method for producing an osteo-cartilage tissue transplant, comprising the step of seeding the chondrogenic cells of the present invention on a scaffold capable of retaining the cells in a state where the osteogenic cells and the chondrogenic cells are substantially separated.

Item 11. At least one type of bone selected from the group consisting of bone cells or stem cells capable of differentiating into bone cells as the first scaffolding material, wherein the scaffolding material is composed of the first and second scaffolding materials. Seeding morphogenic cells, seeding at least one chondrogenic cell selected from the group consisting of chondrocytes or stem cells capable of differentiating into chondrocytes on a second scaffold capable of retaining cells, osteogenesis Item 10. The method according to Item 10, comprising a step of laminating a first scaffold material holding osteoblasts and a second scaffold material holding chondrogenic cells.

Item 12. The method according to item 10, further comprising a step of culturing the osteogenic cells and the chondrogenic cells in separate media.

Item 13. The method according to Item 10, wherein the scaffold material is a sheet or a block.

Item 14. The item in which the scaffolding material is formed of a bioabsorbable synthetic polymer in a mesh body or a porous body having an internal structure matrix in which a structure made of another bioabsorbable material is formed. 10. The method according to 10.

Item 15. The bioabsorbable material constituting the structure includes at least one selected from the group consisting of a natural polymer, a cell growth factor, a cell differentiation control factor, and an inorganic compound or a derivative thereof. The method described in 14 above.

Item 16. The structure is produced by forming a structure of a natural polymer and then complexing it with at least one selected from the group consisting of a cell growth factor, a cell differentiation regulator and an inorganic compound. Item 15. The method according to Item 15. Item 17. The structure according to Item 15, wherein the structure is produced by forming a structure of a natural polymer and a cell growth factor and then complexing it with a cell differentiation control factor or an inorganic compound. Method.

Item 18. The structure according to Item 15, wherein the structure is produced by forming a structure of a natural macromolecule and a cell differentiation controlling factor and then complexing it with a cell growth factor or an inorganic compound. the method of.

Item 19. The structure according to Item 15, wherein the structure is produced by forming a structure of a natural polymer, a cell growth factor, and a cell differentiation controlling factor, and then complexing the structure with an inorganic compound. Method.

Item 20. The item described in 'Item 14, wherein the structure is produced by forming a structure of an inorganic compound and then complexing it with a natural polymer, a cell growth factor, or a cell differentiation controlling factor. the method of.

Item 21. A scaffold for bone tissue formation, in which a structure made of another bioabsorbable material is formed in a mesh structure or a porous body made of a bioabsorbable synthetic polymer.

Item 22. The material according to item 21, wherein the scaffolding material is a sheet or a block.

Item 23. The material according to item 21, which is a laminate of a sheet and / or a block.

Item 24. The structure according to Item 21, wherein the structure in the internal structure matrix includes one or more members selected from the group consisting of natural macromolecules, cell growth factors, cell differentiation control factors, and inorganic compounds. Material.

Item 25. A scaffold for bone and cartilage tissue formation, in which a structure made of another compound is formed in a mesh structure or a porous material made of a bioabsorbable synthetic polymer inside an internal structure matrix.

Item 26. The material according to item 25, having a bone tissue forming part and a cartilage tissue forming part, and having a portion that restricts cell migration between the bone tissue forming part and the cartilage tissue forming part.

Item 27. The material according to item 26, which is a laminate of a sheet-like material and a Z-like or block-like material. Hereinafter, the present invention will be described in more detail.

The implant of the present invention has a structure in which the bone tissue and the cartilage tissue are connected, and the bone-cartilage tissue is integrated.In a preferred embodiment, the cartilage tissue is supported by the bone tissue Things. Transplantation is difficult with cartilage tissue alone, but by integrating it with bone tissue, transplantation can be performed more easily.

The bone tissue of the implant may be composed entirely of bone and may be bone, and a wide range of tissues that can become bone is included. For example, a tissue in which osteogenic cells are seeded on a scaffolding material capable of retaining cells, preferably a scaffolding material (scaffold) composed of a bioabsorbable material, or a scaffolding material retaining osteogenic cells in the presence of a culture solution. The tissue in which bone is partially formed by culturing is included in the bone tissue. As a scaffold material for bone tissue, a ceramic material such as hide-opening xiapatite can be used in addition to a bioabsorbable material.

Similarly, the cartilage tissue of the implant may be composed entirely of cartilage, and broadly encompasses tissue that can become cartilage. For example, a tissue in which chondrogenic cells are seeded on a scaffold material capable of retaining cells, preferably a scaffold material (scaffold) composed of a bioabsorbable material, and a scaffold material retaining chondrogenic cells are cultured in the presence of a culture solution. The tissue in which cartilage is partially formed is included in the cartilage tissue. Gels such as collagen gel can be used as a scaffold for cartilage tissue.

The structure in which the bone tissue and the cartilage tissue are connected means a structure in which the bone tissue is connected so as to support the cartilage tissue. For example, bone tissue and cartilage tissue are directly connected without any separation from each other, or when bone and cartilage tissue are connected via a bioabsorbable material, but when bone and cartilage are regenerated, The bioabsorbable material includes a structure that is absorbed by a living body.

Examples of the structure in which the bone tissue and the cartilage tissue are connected include, for example, a structure having a hierarchical structure in which the bone tissue and the cartilage tissue are connected in layers, and a structure in which one or more independent cartilage tissues exist in a mass on the bone tissue. Is mentioned. Further, a sponge-like or gel-like cartilage tissue may be placed on the bone tissue to obtain the implant of the present invention.

In one of the preferred embodiments of the present invention, the osteochondral tissue graft of the present invention comprises covering the osteochondral tissue with a periosteum obtained from another bone tissue such as a tibia, or a substitute thereof. Is preferred. In the present invention, the osteogenic cells include all cells capable of forming bone, for example, osteocytes, osteoblasts, bone marrow cells capable of inducing and inducing osteoblasts, mesenchymal stem cells, somatic stem cells Is exemplified. When an osteoclast system is obtained from living bone, it is preferable to use only osteoblasts. You may. It is most preferable that all cells are osteogenic cells as osteogenic cells, but as long as cells having osteogenic performance are present, cells with weak or no osteogenic performance They may be mixed. Osteoblasts are preferred as osteogenic cells. When stem cells such as bone marrow cells and mesenchymal stem cells are used as the osteogenic cells and seeded on the scaffold, it is preferable to induce the osteogenic cells to differentiate into osteoblasts in the bone tissue. As the differentiation-inducing medium, any medium suitable for differentiation induction can be used, and is not particularly limited, for example, 10% fetal serum or 10 to 15% human serum, 100 mg Og ZL glucose, 5 O DMEM medium supplemented with mg ZL-ascorbic acid, ΙΟΟηΜdexamethasone, and lOniM glycemic phosphate can be used. Stem cells capable of differentiating into bone cells are cultured using such a differentiation-inducing medium, and the cells can be separated.

In the present invention, the chondrogenic cells include all cells capable of forming cartilage, and include, for example, chondrocytes, bone marrow cells capable of differentiating and inducing chondrocytes, mesenchymal stem cells, and somatic stem cells.

As chondrogenic cells, chondrocytes are preferred. When seeded on a scaffold using stem cells such as bone marrow cells, mesenchymal stem cells, and somatic stem cells as chondrogenic cells, the chondrogenic cells are induced to differentiate into chondrocytes in cartilage tissue. As the differentiation-inducing medium, any medium suitable for inducing differentiation can be used, and is not particularly limited. For example, after the above-described scaffold material is impregnated with a cell solution for seeding stem cells capable of differentiating into chondrocytes, 45 DMEM containing 0 O mg / L glucose, 584 mg / L glutamine, 0.4 mM proline, 5 O mg / L ascorbic acid, transforming growth factor] 33 (TGF-] 33), ΙΟΟηΜdexamethasone A medium (differentiation medium) can be used. Stem cells capable of differentiating into chondrocytes are cultured using such a differentiation-inducing medium, and the cells can be separated.

Gaps between the mesh or porous body of the bioabsorbable synthetic polymer in the present invention, A porous structure or a gel is exemplified as a structure containing a bioabsorbable material such as a natural polymer such as collagen, a cell growth factor, a cell differentiation controlling factor, and an inorganic compound provided in the pore.

The bioabsorbable biosynthetic polymer mesh or porous body used in the present invention is mainly used to increase the mechanical strength of the scaffold material of the present invention, and the mesh body is woven, knitted, or woven cloth. Alternatively, it may be made of a nonwoven fabric or the like. Further, the porous body can be obtained by a well-known method such as a foam molding method using a foaming agent, or a porous agent removing method. In this foam molding method for a porous body, a foaming agent is added to a polymer compound, the foaming agent is foamed, and then the polymer is cured. A water-soluble sugar or salt may be added to the polymer solution, and after curing, the water-soluble substance may be washed and removed with water. As a scaffold for the osteogenic material, a porous ceramic material such as porous hydroxyapatite can be used.

As the mesh size of the mesh or the pore size of the porous body increases, the mechanical strength decreases, but the pore density of the natural polymer porous structure per mesh unit increases, Since the seeded cells are retained in these pores, the number of seeded cells in the complex can be increased, and the regeneration of bone tissue and cartilage tissue becomes efficient.

Therefore, the size of the stitch of the mesh or the size of the pores of the porous body depends on the location in the living body to be implanted, etc. It is appropriately determined in consideration of the speed and the like. The size of the mesh stitch is, for example, 0.1 mm to 5 cm.

Examples of bioabsorbable synthetic polymers that form a mesh or a porous body include polylactic acid, polydalicholic acid, a copolymer of lactic acid and glycolic acid, polymalic acid, polyester such as poly-ε-force prolactone, cellulose, or poly. Examples thereof include polysaccharides such as alginic acid. The bioabsorbable synthetic polymer preferably used in the present invention is polylactic acid, polyglycolic acid, or a copolymer of lactic acid and glycolic acid.

As a bioabsorbable material different from the bioabsorbable synthetic polymer constituting the structure of the present invention, a natural polymer, a cell growth factor, a cell differentiation controlling factor or an inorganic compound, or Are exemplified. Natural polymers can be used as long as they are naturally occurring or derived from living organisms and exhibit biocompatibility. Collagen, hyaluronic acid, chondroitin sulfate, gelatin, fipronectin, and 1One or more selected from laminin and the like, particularly collagen is preferably used. Collagen includes types I, II, III, IV, V, VI, VIII, IX, X, etc. In the present invention, any of these can be used, and derivatives thereof may be used.

Cell growth factor and cell differentiation control factor can be used as long as they can control cell growth and differentiation. Epidermal growth factor (EGF), insulin, platelet-derived growth factor (PDGF), fibroblast proliferation One or more selected from factor (FGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), transforming growth factor j3 (TGF-j8), bone morphogenetic factor (BMP), dexamethasone, etc. And derivatives thereof, and any of them can be used in the present invention.

Any inorganic compound can be used as long as it promotes the adhesion, growth, and differentiation induction of bone cells or stem cells that can be differentiated into bone cells, and any of them can be used.One or more selected from hydroxyapatite, calcium triphosphate, and the like Alternatively, there are derivatives thereof, and any of these can be used in the present invention.

The pores of a structure made of another bioabsorbable material, formed in the internal structure matrix of a mesh body or a porous body made of a bioabsorbable synthetic polymer, allow the growth of seeded cells and tissue regeneration And the pores are preferably continuous. Its size is l to 300 m, preferably 20 to: about 100 im.

Also, in the present invention, the thickness of the mesh-like (including sheet-like) scaffold material may be determined as appropriate depending on the use mode of the biocomposite material, and is usually 0.1 to 5 mm, preferably 0.1 to 1 mm. ~ L mm. Its porosity is usually above 50%. The thickness of the block-like scaffolding material is usually 3 to 50 mm, preferably 5 to 20 mm. Its porosity is usually above 50%.

The structure in one preferable internal structure matrix of the present invention is a porous structure, and is a mesh structure of a bioabsorbable synthetic polymer or an internal structure matrix of a porous material, that is, a stitch or a porous structure of a mesh body. Bioabsorbable natural high in body pores A porous body containing a molecule or a derivative thereof and further containing at least one selected from the group consisting of a cell growth factor, a cell differentiation controlling factor, and an inorganic compound or a derivative thereof (FIGS. 1 and 2). . The scaffold material having a porous structure of the present invention can be produced by various methods. For example, a mesh or porous body of the bioabsorbable synthetic polymer and a bioabsorbable natural polymer, cell It can be obtained by crosslinking or adsorbing a porous structure made of a bioabsorbable material such as a growth factor, a cell differentiation controlling factor, an inorganic compound or a derivative thereof. In the following method, the porous structure contains at least one kind of bioabsorbable material. The material contains a bioabsorbable natural polymer or a derivative thereof as an essential component, and further contains a cell growth factor. And at least one selected from the group consisting of a cell differentiation controlling factor and an inorganic compound or a derivative thereof.

For example, the structures in the internal structure matrix

After forming the structure of the natural polymer, it may be complexed with at least one selected from the group consisting of cell growth factors, cell differentiation regulators and inorganic compounds by impregnation, immersion, coating, etc. After forming a structure of a natural polymer and a cell growth factor, a complex may be formed by impregnation, immersion, coating, or the like with a cell differentiation control factor and Z or an inorganic compound.

After forming a structure of a natural macromolecule and a cell differentiation controlling factor, a complex may be formed by impregnation, immersion, coating or the like with a cell growth factor and a metal or inorganic compound.

After forming a structure of a natural polymer, a cell growth factor, and a cell fraction control factor, a complex with an inorganic compound may be formed by impregnation, dipping, or coating. The structure of the natural polymer and the cell growth factor can be produced in the same manner by using a solution of the natural polymer and the cell growth factor instead of the natural polymer solution. The same applies to a structure of a natural polymer and a cell differentiation controlling factor, and a structure of a natural polymer, a cell growth factor and a cell differentiation controlling factor. Further, the structure may be formed after the internal structure matrix of the mesh body or the porous body is composited with an inorganic compound by impregnation, dipping, coating, or the like in advance.

One of the preferable methods of this production method is as follows: (1) a bioabsorbable natural polymer which forms a secondary structure in a mesh or porous body of a bioabsorbable synthetic polymer as a primary structure; Cell growth factor, cell differentiation control factor or their derivatives 1 After adhering and impregnating more than one kind of solution, subsequently, (2) freeze-drying, and preferably crosslinking by treating with a gaseous or liquid crosslinking agent.

In the above step (1), at least one kind of bioabsorbable natural polymer, cell growth factor, cell differentiation controlling factor or a derivative thereof is mixed and then impregnating force or at least one kind of each is mixed. Done.

In the above step (1), the bioabsorbable synthetic polymer mesh (porous structure), which is the primary structure, is replaced with the bioabsorbable natural polymer, cell growth factor, cell differentiation controlling factor, or any of these. Treat with an aqueous solution of the derivative. Although there are various treatment methods, a dipping method and a coating method are preferably employed.

The immersion method is effective when the concentration or viscosity of an aqueous solution of a bioabsorbable natural polymer, a cell growth factor, a cell differentiation controlling factor, or a derivative thereof is low. Specifically, the bioabsorbable natural polymer is used. It is carried out by immersing the bioabsorbable synthetic polymer mesh in a low-concentration aqueous solution of a cell growth factor, a cell differentiation controlling factor or a derivative thereof. The application method is effective when the concentration and viscosity of the aqueous solution of the bioabsorbable natural polymer, cell growth factor, and cell differentiation control factor are high and the immersion method cannot be applied. This is performed by applying a high-concentration aqueous solution of a molecule, a cell growth factor, and a cell differentiation regulator to a bioabsorbable synthetic polymer mesh.

As the crosslinking agent used in the present invention, any conventionally known crosslinking agents can be used. Crosslinking agents which are preferably used are aldehydes such as daltaraldehyde, formaldehyde, paraformaldehyde, in particular glutaraldehyde.

As described above, in the crosslinking of the present invention, it is preferable to use the above-mentioned crosslinking agent in a gaseous state.

Specifically, when the above-mentioned natural polymer porous body is crosslinked, crosslinking is carried out at a constant temperature under a constant concentration of a crosslinking agent or in an atmosphere of a crosslinking agent vapor saturated with an aqueous solution thereof for a predetermined time. The crosslinking temperature may be selected within a range in which the bioabsorbable synthetic polymer mesh does not dissolve and vapor of the crosslinking agent can be formed, and is usually set at 20 ° C to 50 ° C.

The cross-linking time depends on the type of the cross-linking agent and the cross-linking temperature. Is desirably set in a range in which is performed. If the cross-linking time is short, the cross-linking fixation becomes insufficient, and the natural polymer porous body may be dissolved in a short time after transplantation in the living body, and the longer the cross-linking time, the more the cross-linking proceeds. If the time is too long, the hydrophilicity decreases, the seeding density of osteogenic cells and chondrogenic cells on the scaffold decreases, and cell donation and tissue regeneration are not performed efficiently. It is not preferable because it causes problems such as a decrease in bioabsorbability. The preferred crosslinking time is about 10 minutes to 12 hours.

Other production methods include (1) a bioabsorbable synthetic polymer as a primary structure, a mesh or a bioabsorbable natural polymer that forms a secondary structure in a porous body, or one of its derivatives. After adhering and impregnating the above solution, (2) freeze-drying, and preferably crosslinking by treating with a gaseous or liquid crosslinking agent, and then (3) a water-soluble crosslinking accelerator such as lipopositimide And a crosslinking reaction with a cell growth factor and / or a cell division controlling factor or a derivative thereof.

In the above step (1), one or more bioabsorbable natural polymers or derivatives thereof are mixed and then impregnated. The impregnation method is the same as the method described above.

In the above step (3), the bioabsorbable natural polymer of the secondary structure or the porous body of the derivative thereof is treated in advance with a water-soluble cross-linking accelerator such as carbodiimide, and then the cell growth factor and the cell differentiation control factor are treated. It is preferable to carry out the treatment by impregnating each with one or more mixed aqueous solutions.

The second scaffold material of the present invention is a gel-like structure, and is in a mesh structure of a bioabsorbable synthetic polymer or in an internal structure matrix of a porous body, that is, in a mesh of a mesh body or in a pore of a porous body. In addition, a gel structure comprising a bioabsorbable natural polymer, a cell growth factor, a cell differentiation controlling factor and a derivative thereof is further formed. The method of preparation is as follows: (1) A mesh structure or a porous material of a bioabsorbable synthetic polymer of the primary structure is used to prepare one or more solutions of bioabsorbable natural polymer, cell growth factor, cell differentiation control factor, etc. After adhering and impregnating, subsequently, (2) it is crosslinked by gelling.

The natural polymers that make up the gel include gelatin, collagen, starch, pectin, hyaluronic acid, chitin, chitosan or alginic acid and the attraction of these materials. Conductors.

Specific examples of the preparation of a gel-like structure include a biostructure that absorbs at low temperature and one or more types of raw natural macromolecules, cell growth factors, and cell differentiation control factors, or a mixed solution of one or more types of each. A method of gelling by increasing the temperature to 37 ° C after introduction into the porous body of the body, or a method using a bioabsorbable natural polymer, cell growth factor, and cell division control factor at a low temperature. There is a method in which a mixed solution of more than one kind is further mixed with a crosslinking agent, introduced into the porous material of the primary structure, and heated to 37 ° C to gel. Examples of the cross-linking agent include an epoxy compound, carbodiimide, carbonyldiimidazole, glutaraldehyde, hexamethylene diisocyanate, and the like.

By using the block-like porous structure shown in FIG. 3 instead of the mesh-like porous structure shown in FIG. 1 above, the corresponding block-like scaffold material can be obtained in the same manner as above. It is possible to obtain

The third scaffold material of the present invention is a porous structure, and is provided in a mesh body of a bioabsorbable synthetic polymer or an internal structure matrix of a porous body, that is, in a mesh of a mesh body or in a pore of a porous body. It is further formed of a porous body composed of an inorganic compound and at least one selected from the group consisting of a bioabsorbable natural polymer, a cell growth factor and a cell differentiation control factor (Fig. 2).

This production method is performed by depositing or coating fine particles of an inorganic compound on the surface of the porous structure described above. A method for depositing or coating fine particles of an inorganic compound is described in Japanese Patent Application Laid-Open No. 2002-143921.

By using the block-like porous structure shown in FIG. 4 instead of the mesh-like porous structure shown in FIG. 2 above, the corresponding block-like scaffolding material is obtained in the same manner as above. It is possible to obtain

The desirable shape of the composite material of the present invention is a sheet-like shape, and the inside structure matrix of a mesh body or a porous body of a bioabsorbable synthetic polymer having such a shape, that is, a stitch or a porous body of the mesh body A porous structure of a natural polymer is formed in the pores. The overall thickness of the sheet is usually 0.1 to 5 mm, preferably 0.1 to 1 mm, and the thickness of the natural polymer porous structure. Can be appropriately prepared, but is preferably formed to have substantially the same thickness as the mesh or porous body of the bioabsorbable synthetic polymer. The porosity of the porous structure is usually 80% or more. In this specification, the term “sheet-like material” includes a mesh-like material, a film-like material, and a film-like material (FIGS. 1, 2, and 5).

In the present invention, the porous structure of a natural polymer such as a collagen sponge formed in a sheet-like scaffold is preferably thin. In the case of a thin porous structure, the cell solution for seeding can be impregnated into the pores of the porous body without leaking, and as a result, the density of cells retained in the scaffold material increases, and the regeneration of bone and cartilage tissue can be performed quickly and It will be done efficiently.

When a scaffold material seeded with osteogenic or chondrogenic cells is used on a single sheet, thin bone-cartilage tissue can be regenerated, but as shown in Fig. 10, a scaffold seeded with these cells can be used. Materials can be stacked and used. In this case, the thickness of the regenerated bone and cartilage can be adjusted by the number of sheets used for the bone tissue and the cartilage tissue. In the case of Fig. 10, cells were seeded on each of the stacked sheet-like scaffolds, so the density of the seeded cells was as high as that of one sheet-like scaffold. When transplanted into a living body as a body, osteo-cartilage tissue can be regenerated satisfactorily.

When laminating bone and cartilage, the thickness of the bone and cartilage may be the same or different (Figure 6). The thickness of cartilage tissue is usually about 0.4 to 7.0 mm, and the thickness of bone tissue is about 0.4 to 30 mm.

The transplant of the present invention may be used without culturing after seeding osteogenic cells or chondrogenic cells, but is preferably formed into a transplant after culturing for about 2 weeks to 2 months. .

For example, in order to produce the sheet-like scaffolding material of the present invention as shown in Fig. 11 (1), a sheet-like mesh of a bioabsorbable synthetic polymer or a porous body is made of a natural material derived from a body. Place in the aqueous solution of the polymer, freeze, and freeze-dry. As a result, a sheet-like scaffold material in which the mesh or porous body of the bioabsorbable synthetic polymer is sandwiched in the natural polymer porous structure is formed.

Also, as shown in Fig. 11 (2), a sheet of bioabsorbable synthetic polymer The solid or porous body is frozen on the upper or lower surface of an aqueous solution of a natural polymer derived from a living body, and freeze-dried. One side is a bioabsorbable synthetic polymer mesh or porous body and the other side is a natural high polymer. A sheet-like scaffold material that is a molecular porous structure is formed. FIGS. 11 (1) and (2) are schematic diagrams showing that a natural high molecular porous structure is formed on the surface of a mesh or porous body of a bioabsorbable synthetic polymer. However, as is apparent from the schematic diagram of FIG. 1, in practice, the natural polymer porous structure is a mesh or porous material of a bioabsorbable synthetic polymer mesh. It is also formed in body pores.

In another preferred embodiment of the scaffold material for retaining chondrogenic cells and osteoblasts according to the present invention, collagen or the like is contained in a mesh or a porous body of a bioabsorbable synthetic polymer and pores. Natural macromolecules, epidermal growth factor (EGF), insulin, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), liver cell growth factor (HGF), vascular endothelial growth factor (VEGF), trans Further formed a structure consisting of cell growth factors such as forming growth factor j3 (TGF-J3), bone morphogenetic factor (BMP), and dexamethasone, and inorganic compounds such as cell differentiation regulators, hydroxyapatite, and calcium triphosphate. It is composed of scaffolding materials.

The chondrocytes used in the present invention and stem cells capable of differentiating into chondrocytes are prepared from living tissues by a conventional method.

For chondrocytes, living cartilage tissue is treated with enzymes such as collagenase, trypsin, liberase, and proteinase to degrade extracellular matrix, and then a serum medium is added, followed by centrifugation to isolate chondrocytes. The isolated chondrocytes were plated on a culture brassco, and 10% fetal calf serum, 450 mg / L glucose, 584 mg / X glutamine, 0.4 mM proline and 5 O mg ZL ascorbic acid Culture in DMEM medium containing DMEM (DMEM serum medium). Subculture 2 or 3 times until the number of cells becomes sufficient. Collect the subcultured cells by trypsinization, and use them as a cell fluid for seeding. ,

Stem cells capable of differentiating into chondrocytes are isolated by direct centrifugation, or isolated by centrifugation of a bone marrow extract by density gradient centrifugation using a density gradient medium composed of Percoll (perco 11). Seed these cells into a culture flask and add DMEM T / JP2003 / 015573

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Subculture 2-3 times in serum medium until sufficient cells are obtained. The subcultured cells are collected by trypsinization and used as a cell solution for seeding.

In order to seed the scaffold material of the present invention with chondrocytes or stem cells capable of differentiating into chondrocytes, the scaffold material is wetted with a culture solution, and the scaffold material is impregnated with the cell solution for seeding, or This is carried out by directly impregnating the above scaffold material with a cell solution for seeding. Alternatively, the chondrogenic cells are mixed with at least one solution selected from the group consisting of a bioabsorbable natural polymer, a cell growth factor and a cell differentiation controlling factor at a low temperature, and then the porous material of the primary structure is mixed. Or at least one solution selected from the group consisting of bioabsorbable natural polymers and cell growth factors and cell differentiation regulators at low temperature. Further, a method is exemplified in which a chondrogenic cell is mixed with a crosslinking agent, and then the mixture is introduced into a porous material having a primary structure, and the temperature is increased to 37 ° C. to cause gelation.

The cell concentration of the cell solution for seeding is preferably ⁵ × 10 ⁵ cells / m 1 to 5 × 10 ⁷ cells / m 1, and it is preferable to seed a cell solution having a volume equal to or larger than the volume of the scaffold material. When chondrocytes are used to regenerate the cartilage tissue of the present invention, the scaffold material is impregnated with the cell solution for seeding, and then a culture solution is further added. A cartilage tissue transplant is obtained by culturing and growing the medium in an incubator at 37 ° C. in an atmosphere of 5% CO ₂ .

In the case of stem cells, a further step of differentiation into chondrocytes is required.After impregnating the scaffold material with a cell solution for seeding stem cells capable of differentiating into chondrocytes, 4500 mg / L glucose, 584 mg / L glutamine, The cells were cultured in a DMEM medium (differentiation medium) containing 0.4 mM proline, 50 mg ZL ascorbic acid, lOng / mL transforming growth factor] 33 (TGF-β3), and ΙΟΟηΜdexamethasone. obtain.

Stem cells capable of differentiating into bone cells used in the present invention are prepared from living tissues by a conventional method. Stem cells capable of differentiating into osteocytes are isolated by direct centrifugation or by centrifuging the bone marrow extract by density gradient centrifugation using a density gradient medium consisting of Percoll (perco 11). Inoculate these cells into a culture flask and subculture 2-3 times in DMEM serum medium until the number of cells is sufficient. Subcultured cells 15573

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Collect by trypsinization and use as cell solution for seeding.

Bone cells can be obtained by subjecting living bone tissue to a treatment with an enzyme such as collagenase to degrade extracellular matrices and releasing the cells. In addition, the crushed bone fragments are cultured on a culture vessel, and isolated from cells that have migrated from around the bone fragments. Culture the isolated bone cells in DMEM medium supplemented with 10% fetal calf serum, 100 Omg ZL glucose, and 50 mg / L ascorbic acid.

In the present invention, in order to regenerate the bone tissue, either a bone cell or a stem cell capable of being divided into a bone cell may be used, but it is desirable to use a stem cell capable of differentiating into a bone cell. No.

In order to inoculate the scaffold material of the present invention with osteogenic cells, the scaffold material is wetted with a culture solution, and the scaffold material is impregnated with the cell solution for seeding, or the scaffold material is directly seeded on the scaffold material. It is performed by impregnating the cell solution for use.

The cell concentration of the cell solution for seeding is preferably ⁵ × 10 5 cells / m 1 to 5 × 10 ⁷ cells / m 1, and it is preferable to seed a cell solution having a volume equal to or larger than the volume of the scaffold material. Stem cells that can be differentiated into osteocytes are cultured in DMEM medium supplemented with 10% fetal calf serum or 10-15% human serum, 100 mg / L glucose, 5 mg / L ascorbic acid, ΙΟΟηΜdexamethasone, and 10 mM glycemic phosphate. Then, it can be differentiated to obtain a transplant of bone tissue.

Further, the cartilage tissue portion and the bone tissue portion are laminated, and the cartilage tissue culture medium and the bone tissue culture medium are cultured using a bioreactor that passes through the cartilage, the tissue portion, and the bone tissue portion, respectively, to regenerate the bone-cartilage tissue.

Seeding the above chondrocytes or stem cells capable of differentiating into chondrocytes, seeding the laminated cartilage tissue portion with the above stem cells capable of differentiating into the osteocytes or osteocytes, and further stacking the stacked bone tissue portion to obtain a normal The cartilage and bone tissue parts are brought into contact by sewing with a bioabsorbable suture system or sandwiched between plastic clips such as Teflon. Put this in a normal bioreactor, 10% fetal serum or 10-15% human serum, antibiotics, 100 Omg / L glucose, 584 mg / L glutamine, 0.4 mM proline, 5 Omg ZL ascorbic acid , Iotaomikuron'omikuron'itamyu dexamethasone in DMEM medium supplemented with 10mM glycerin port phosphate, 37 ° C, 5% C_〇 ₂ atmosphere 2003/015573

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Culture in an incubator. Alternatively, the cartilage tissue portion and the bone tissue portion are superimposed and cultured in a bioreactor that provides the cartilage tissue portion and the bone tissue portion with a cartilage tissue culture medium and a bone tissue culture medium, respectively. The culture medium for cartilage tissue culture was 450 Omg ZL gnorecose, 584 mg Lg / retamine, 0.4 mM proline, 50 mg 1 ascorbic acid, transforming growth factor) 83 (TGF-j33) and ΙΟΟη Consist of DMEM medium containing dexamethasone. Bone tissue culture medium is DMEM medium supplemented with 10% fetal fetal serum or 10 to 15% human serum, 100 mg Og ZL glucose, 50 mg L ascorbic acid, 、 ηΜdexamethasone, and 10 mM glycerol phosphate. Constitute. The flow rate of each medium can be adjusted.

In the implant of the present invention, the bone tissue and the cartilage tissue are connected, and the osteogenic cells and the chondrogenic cells are preferably present in the scaffold material in a substantially separated state. The “substantially separated state” means that near the interface between bone tissue and cartilage tissue, there may be a portion where osteogenic cells and chondrogenic cells overlap, but the overlapping portion is as small as possible. The smaller the better, the overlapping of osteogenic cells and chondrogenic 14 cells has little or no effect on the osteo-cartilage junction, as long as the cartilage is supported by bone. For example, osteogenic cells and chondrogenic cells are seeded on a first scaffold material and a second scaffold material, respectively, and then the obtained first scaffold material (bone tissue) and second scaffold material (cartilage tissue) are laminated, Cultures of this, if necessary, fall under the “substantially separated state”. Alternatively, in one scaffold material, the bone tissue forming part and the cartilage group. The tissue forming part forms a part where the movement of the bone forming cells and the chondrogenic cells is restricted, such as the bioabsorbable sheet shown in FIG. However, those in which osteogenic cells and chondrogenic cells are seeded on the scaffold and cultured as necessary also fall under the “substantially separated state”. Since the scaffold structure is gradually degraded or resorbed during the culture, the osteogenic and chondrogenic cells can mix near the interface, but this is within the range of a substantially separate state. In addition, chondrogenic cells may be mixed in a small proportion with osteogenic cells, and chondrogenic cells may be mixed with a small proportion of osteogenic cells. However, as long as the bone and cartilage are formed so as to be suitable for transplantation, they fall under the “substantially separated state”.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1

A copolymer of lactic acid and dalicholate (PLGA), a bioabsorbable polymer with high mechanical strength, is immersed in 0.3 wt% of a pig tendon-derived pepsin-solubilized type I collagen acidic aqueous solution, -80 ° C for 12 hours. Next, this frozen product was freeze-dried under vacuum reduced pressure (0.2 Torr) for 24 hours to produce a sheet-like uncrosslinked scaffold material of a PLGA mesh body and a collagen sponge.

The obtained uncrosslinked composite biomaterial was crosslinked at 37 ° C. for 4 hours with glutaraldehyde vapor saturated with a 25 wt% aqueous solution of glutaraldehyde, and then washed 10 times with a phosphate buffer. Furthermore, it was immersed in a 0.1 M glycine aqueous solution for 4 hours, washed 10 times with a phosphate buffer solution, washed 3 times with distilled water, and frozen at -80 ° C for 12 hours. This is freeze-dried under vacuum reduced pressure (0.2 Torr) for 24 hours, and seeded with chondrocytes or stem cells capable of being divided into chondrocytes or osteocytes or stem cells capable of differentiating into osteocytes according to the present invention. We obtained PLGA-collagen composite mesh, a composite porous body of bioabsorbable synthetic polymer and bioabsorbable natural polymer, which is one of the scaffold materials to be nourished.

The scaffolds were coated with gold and their structure was observed with a scanning electron microscope (SEM). Fig. 7 shows a photograph enlarged 100 times.

Example 2

The PLGA-collagen composite mesh obtained in Example 1 was immersed in a 100 mM aqueous solution of calcium chloride (20 mL) buffered with 50 raM Tris buffer (pH 7.4), and incubated for 3 hours in a thermostat at 37 ° C. did. The PLGA-collagen composite mesh was taken out of the Shiidai calcium aqueous solution and centrifuged at a speed of 600 rpm. The centrifuged PLGA-collagen composite mesh was immersed in lOOmM aqueous sodium hydrogen phosphate solution (20 mL) buffered with 50 mM Tris buffer, and incubated in a thermostat at 37 ° C for 3 hours. The PLGA-collagen composite mesh was removed from the aqueous solution of sodium hydrogen phosphate and centrifuged at a speed of 600 rpm. The centrifuged PLGA-collagen composite mesh was alternately immersed in the above-described calcium chloride aqueous solution and sodium hydrogen phosphate aqueous solution, and this alternate immersion was repeated up to six times. The obtained PLGA-collagen-hydroxyapatite porous composite structure was made of gold, and the structure was observed with a scanning electron microscope (SEM). Fig. 8 shows a photo magnified 200 times.

According to this SEM photograph, a state in which hydroxyapatite fine particles are deposited on the surface of the pores of the PLGA-collagen composite mesh is observed. In addition, it was found that the amount of deposited hydroxyapatite fine particles and the particle size became larger as the number of times of repeated alternate soaking increased.

The sheet-like porous structure of the PLGA mesh body and the collagen sponge prepared in Example 1 was sterilized with ethylene oxide gas.

Test example 1

(1) Formation of cartilage tissue

A thin piece of cartilage was shaved off from the cartilage of the dog elbow joint with a scalpel and minced, and then incubated in DMEM medium containing 0.2% (w / v) collagenase at 37 ° C for 12 hours. Then, the supernatant filtered through a nylon filter having a pore size of 70 μπι is centrifuged at 1500 rpm for 5 minutes, and 10% fetal bovine serum, antibiotics, 45 Omg / L gnorecose, 584 mg / L gnorecamine, 0.4 mM proline After washing twice with DMEM medium containing 5 and 5 Omg / 1 ascorbic acid, canine elbow chondrocytes were obtained. The resulting chondrocytes were cultured in DMEM medium containing 10% fetal serum, antibiotics, 4500 mg ZL gnorecose, 58 ArngZL gnoretamine, 0.4 mM proline and 50 mg / L ascorbic acid at 37 ° C and 5% C0. Culture was performed under _two atmospheres. Chondrocytes subcultured twice were detached and collected with 0.025% trypsin / 0.01% EDTA / PBS (-) to prepare 5 × 10 ⁶ cells / ml cell solution.

Next, the PLGA-collagen mesh sterilized with ethylene oxide gas was wetted with a DMEM serum medium, and the edge of the complex (membrane) was surrounded by a rubber ring, and 0.5 ml / cm ^{2 of the} cell solution was dropped. The cells were cultured in an incubator at 37 ° C. in a 5% CO ₂ atmosphere for 4 hours. Thereafter, the rubber ring was removed, a large amount of culture solution was added, and the culture was continued. After 24 hours, the cells PLGA were seeded - fall collagen composite mesh, the edges of the composite (film) enclosed with a rubber ring, PLGA - 0. 5 ml / cm 2 cell fluid to the lower surface of the collagen composite mesh Was dropped, and the chondrocytes were seeded. In the incubator The culture was allowed to stand for 4 hours at 37 ° (5% CO ₂ atmosphere. The rubber ring was removed, a large amount of culture solution was added, and the culture was continued. The PLGA-collagen composite mesh seeded twice was laminated, and a cartilage tissue layer was obtained by sandwiching the periphery of the laminate with a Teflon tulip.

(2) Bone tissue formation

Cells were isolated by centrifugation of bone marrow collected from dog femurs at 1500 rpm in 10% fetal serum, antibiotics, 100 Omg / L glucose, 5 OmgZL in Falcon T-75 culture flasks. The cells were cultured in a DMEM medium supplemented with ascorbic acid at 37 ° C. in a 5% CO ₂ atmosphere. After 24 hours, change the medium, discard the non-adhered cells, and add 10% fetal serum, antibiotics, 100 Omg / L glucose, and 5 Omg ZL ascorbic acid to the bone marrow cells adhered to the surface of the flask. Added new DMEM medium, 37. C, 5% C_〇 the culture was continued under ₂ atmosphere. The medium was changed every two days.

Bone marrow cells subcultured twice were detached and collected with 0.025% trypsin / 0.01% EDTA / PBS (-) to prepare a 5 × 10 ⁶ cell _S / ml bone marrow cell solution. Next, the PLGA-collagen-hydroxypatite porous composite structure of Example 2 sterilized with ethylene oxide gas was wetted with a DMEM serum medium, and the edge of the composite (membrane) was surrounded by a rubber ring. , 0.5 ml / cm ² cell solution was added dropwise. The cells were cultured in an incubator at 37 ° C. in a 5% CO ₂ atmosphere for 4 hours. Thereafter, the rubber ring was removed, a large amount of culture solution was added, and the culture was continued. After 24 hours, PLGA cells were seeded - collagen composite mesh fall, and the edges of the composite (film) enclosed with a rubber ring, PLGA-collagen composite to the lower surface of the mesh 0. 5m 1 / cm ² The cell solution was dropped, and bone marrow cells were seeded. In an incubator, 37 ° (:., Under 5% C0 ₂ atmosphere, and cultured by standing for 4 hours then remove the rubber ring, 10% © shea calf serum or 1% human serum, antibiotics, l OO OmgZL A large amount of DMEM medium supplemented with glucose, 5 Omg / L ascorbic acid, ΙΟΟηΜdexamethasone, and lOmM glycerol phosphate was added, and the culture was continued 24 hours later, PLGA-collagen-hydroxylate seeded twice with cells The sheapatite porous composite structure was laminated and sandwiched around the laminate with a Teflon clip to obtain a bone tissue layer. (3) Bone-cartilage tissue transplantation

The above cartilage and bone tissue layers are sandwiched between Teflon clips, and 10% fetal serum, antibiotics, 100 Omg 1 glucose, 584 mg / L glutamine, 0.4 mM proline, 50 mg / L ascorbic acid, ΙΟΟηΜ The cells were cultured in a DMEM medium supplemented with dexamethasone and 10 mM glycerol phosphate in an incubator at 37 ° C under a 5% CO ₂ atmosphere. After two weeks of culture, the complex was implanted subcutaneously on the back of nude mice. Specimens were collected 8 weeks after transplantation. As shown in FIG. 9, after 8 weeks, the surface of the cartilage layer was glossy and the color was observed to be milky white. The specimens were subjected to HE staining and safranin-0 staining. As a result, small round cells in the pits and extracellular matrix with Safranin-0 staining were observed in the cartilage layer. In addition, m-RNA expressing type II collagen and aggrecan was detected and identified in the m-RNA sample extracted from the sample. M-RNA expressing osteocalcin was detected and identified in the m-RNA sample extracted from the bone layer. From these, it was confirmed that the regenerated tissue was bone'cartilage tissue.

Example 3

With informed consent, the first bone marrow lOmL was collected from the iliac bone of patients with osteochondral disorders in the talus. The collected bone marrow Ri by the centrifugation at 1500 rpm, cells were isolated and 15% autologous serum in Falcon T-75 culture flask, antibiotic substance, in αΜΕΜ medium, 37 ° C, 5% C0 2 atmosphere And cultured. After 2 days, replace the media, it continued adhered have not cells cultured in adherent bone marrow cells 37 ° C, 5% C0 ₂ atmosphere on the surface of the flask discarded. The medium was changed every two days. As a result, an adherent cell population containing a large amount of stem cells capable of differentiating into chondrocytes or bone cells is expanded. The above cultured cells were subcultured twice and detached and collected with 0.025% trypsin / 0.01% EDTA / PBS (-) to prepare a cell solution of ⁵ × 10 ⁵ cells / mL. After immersing porous hydroxyapatite ceramic in this cell solution, add 82 μg / ral ascorbic acid, 10 mM glycerol phosphate, and 100 nM dexamethasone to 15% autologous serum, antibiotics, and αΜΕΜ medium for an additional 2 minutes. After culturing for a week, osteoblasts and a bone matrix, i.e., osteogenesis, were generated in the pores on the ceramic surface. Two weeks after the first bone marrow collection, a second bone marrow collection was performed in the same manner as the first bone marrow to grow adherent cells. The cultured cells passaged twice to 0.025% trypsin / 0 01% EDTA / PBS. (-) peeling-harvested 1 X 1 0 ⁷ cells / mL of cell solution was prepared by. This cell solution was mixed with a collagen aqueous solution, and cultured at 37 ° C. in a 5% CO ₂ atmosphere for 2 days to gel the collagen and the cell solution.

From the bone marrow collected twice, a ceramic with osteogenesis could be produced, and stem cells capable of differentiating into cartilage or bone cells could be maintained in a gel state. Therefore, surgery was performed on the patient. The patient's ankle joint was deployed to expose the talus with osteochondral disorders. About 1 X 2 cm of cartilage degeneration was found inside the surface of the talus. This part was extracted with the cartilage and the underlying bone as one.

The stem cells which can be divided into the above-mentioned collagen gel-based chondrocytes are placed on a ceramic (porous hydroxyapatite) -based bone tissue layer, and an osteo-cartilage tissue transplant is prepared. The extracted part was filled. In addition, sutures were placed over the periosteum collected from the tibia.

The tissue formation after transplantation was observed by radiograph. Postoperative radiographic observation showed that the ceramic and the surrounding bone tissue were integrated. In addition, pain in the ankle joint was reduced, and stability of the ankle joint was obtained. From the above, it could be inferred that bone and cartilage tissue were formed.

Thus, by creating a stem cell-bone structure, the stem cells performed bone formation on the ceramic base, and a stable bond was formed with the ceramic surface. Furthermore, cartilage formation occurred at the part in contact with the joint surface, indicating that simultaneous regeneration of osteochondral was possible.

According to the present invention, the cartilage tissue can be supported by the bone tissue, so that the cartilage tissue can be efficiently regenerated.

Claims

The scope of the claims

I. An osteo-cartilage tissue transplant having a structure in which bone tissue and cartilage tissue are connected.

2. The bone tissue comprises a scaffold material capable of retaining cells and at least one type of osteogenic cell selected from the group consisting of bone cells retained by the scaffold material or stem cells capable of differentiating into bone cells. The cartilage tissue according to claim 1, wherein the cartilage tissue comprises a scaffold material capable of retaining cells and at least one type of chondrogenic cell selected from the group consisting of chondrocytes retained in the scaffold material or stem cells capable of differentiating into chondrocytes. The implant of claim.

3. The implant according to claim 1, wherein the scaffolding material is a sheet or a block.

4. The structure according to claim ² , wherein the scaffold material has a structure made of a further compound formed in a mesh structure or a porous structure body made of a bioabsorbable synthetic polymer. Transplant.

5. The implant according to claim 4, wherein the structure in the internal structure matrix is a porous structure or a gel.

6. The structure in the internal structure matrix further comprises one or more members selected from the group consisting of natural macromolecules, cell growth factors, cell differentiation regulators and inorganic compounds, or derivatives thereof. Item 5. The transplant according to Item 4.

7. The implant according to claim 2, wherein the scaffold material of the bone tissue is a porous ceramic. .

8. The implant of claim 2, wherein the scaffold of cartilage tissue is a gel.

9. The transplant of claim 2, wherein the osteogenic cells and Z or chondrogenic cells are bone marrow-derived stem cells.

10. At least one selected from the group consisting of bone cells or stem cells capable of differentiating into osteocytes and at least one selected from the group consisting of chondrocytes or stem cells capable of differentiating into chondrocytes A method for producing an osteo-cartilage tissue transplant, comprising the step of seeding chondrogenic cells of a species on a scaffold capable of retaining cells in a state where the osteogenic cells and chondrogenic cells are substantially separated.

II. The scaffold material is composed of a first scaffold material and a second scaffold material, and the first scaffold material capable of retaining cells is selected from the group consisting of bone cells or stem cells capable of differentiating into bone cells. Seeding at least one type of osteogenic cell, wherein at least one type of chondrogenic cell selected from the group consisting of chondrocytes or stem cells capable of differentiating into chondrocytes is used as the second scaffold material capable of retaining cells 10. The method according to claim 10, comprising a step of seeding, and a step of laminating a first scaffold material holding osteogenic cells and a second scaffold material holding chondrogenic cells.

12. The method according to claim 10, further comprising the step of culturing the osteogenic cells and the chondrogenic cells in separate media.

13. The method according to claim 10, wherein the scaffolding material is a sheet or a block.

14. The scaffold material according to claim 10, wherein a structure made of another bioabsorbable material is formed in a mesh body made of a bioabsorbable synthetic polymer or an internal structure matrix of a porous body. The described method.

15. The bioabsorbable material constituting the structure includes at least one selected from the group consisting of a natural polymer, a cell growth factor, a cell differentiation control factor, and an inorganic compound or a derivative thereof. The method described in 4.

16. After the structure forms a structure of a natural polymer, the structure is combined with at least one selected from the group consisting of a cell growth factor, a cell differentiation regulator and an inorganic compound. 16. The method of claim 15, wherein the method is manufactured.

17. The claim according to claim 15, wherein the structure is produced by forming a structure of a natural polymer and a cell growth factor and then complexing it with a cell differentiation controlling factor or an inorganic compound. the method of.

18. The method according to claim 15, wherein the structure is produced by forming a structure of a natural macromolecule and a cell differentiation controlling factor and then complexing it with a cell growth factor or an inorganic compound. The described method.

19. The method according to claim 15, wherein the structure is formed by forming a structure of a natural polymer, a cell growth factor, and a cell differentiation control factor, and then complexing the structure with an inorganic compound. .

20. The structure is manufactured by complexing with a natural polymer, a cell growth factor, or a cell differentiation controlling factor after the formation of the inorganic compound structure. The method of claim 14.

2 1. A scaffold for bone tissue formation, in which a structure made of another bioabsorbable material is formed in a mesh structure made of a bioabsorbable synthetic polymer or an internal structure matrix of a porous body.

22. The material according to claim 21, wherein the scaffolding material is a sheet or a block.

23. The material according to claim 21, which is a laminate of a sheet and / or a block.

24. The structure according to claim 21, wherein the structure in the internal structure matrix contains one or more members selected from the group consisting of a natural polymer, a cell growth factor, a cell differentiation control factor, and an inorganic compound. The described material.

25. A scaffold for bone-cartilage tissue formation in which a structure made of another compound is formed in a mesh structure made of a bioabsorbable synthetic polymer or an internal structure matrix of a porous material.

26. The material according to claim 25, comprising a bone tissue forming part and a cartilage tissue forming part, wherein the material has a portion for restricting cell migration between the bone tissue forming part and the cartilage tissue forming part.

27. The material according to claim 26, which is a laminate of a sheet and / or a block.