JP2011156329A - Bone/cartilage-like structure - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a bone/cartilage-like structure for transplantation wherein a cartilage-like structure prepared without using scaffold and an artificial bone material are firmly bonded, and a method for making the same. <P>SOLUTION: As a result of keen examinations to solve the problem, the invention is completed. That is, in the invention, a sheet-shaped animal cell aggregation obtained by carrying out centrifugal sedimentation is cultured in a container after inserting an animal cell suspension into the container including a flat inner bottom face orthogonal to the radial direction of the centrifugal rotation so as to carry out centrifugal separation, and after starting the culture, a bone-like solid is placed on the cell aggregation on a bottom surface of the culture container so as to make the bone/cartilage-like structure. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

Detailed Description of the Invention

Industrial application fields

The present invention relates to an osteochondral-like structure and its production.

In recent years, many basic findings regarding tissue regeneration using animal cells such as human cells have been discovered, and their clinical application is expected. It is necessary to create various shapes of tissues according to the types of tissues to be created. For cartilage, skin, and retina, it is necessary to create a sheet-like tissue. Until now, as a method of creating a sheet-like tissue, except for special cases such as skin cells, cells are adhered and cultured on the coating surface of a temperature-responsive polymer, the temperature is lowered after cell growth, and the polymer is degraded to decompose the cells. A method of collecting a sheet (WO01 / 068799) or a method of culturing a cell inside a scaffold such as a nonwoven fabric or a collagen gel (Journal of Bioscience and Bioengineering, 98 (6), 477-481) , 2004).
However, in these methods, there is a concern that a temperature-responsive polymer or a drug such as collagen is mixed in the prepared tissue and remains after the tissue is transplanted into the human body. In addition, the use of these drugs not only increases the manufacturing cost of the tissue, but also complicates the tissue creation process due to the operation of the drug, and hinders the practical application of transplantation treatment using these tissues. There are concerns.
Therefore, there is a demand for the development of a method for constructing a three-dimensional structure by a “scaffold-free” culture method that does not use a special drug or carrier. In contrast, the pellet culture method (cell culture technology, Tokyo Chemical Doujin, p.189, 1990), which is a classic culture method using chondrocytes, and the swollen aggregation culture method (regenerative medicine (Japanese Journal of Regenerative Medicine)) Vol.7 Suppl, p.124-125, 2008). However, cell agglomerates that can be prepared by the pellet culture method or the swirl agglutination culture method are not sheet-like but spherical, and the size and exact shape cannot be controlled.
An example of the cartilage tissue will be described in more detail. Cartilage is a part of connective tissue that forms a skeletal system together with bone, and is a very important tissue that participates in supporting the body and organs. Articular cartilage is composed of hyaline cartilage, and the characteristic physical characteristics of hyaline cartilage are defined by the biochemical characteristics of the extracellular matrix (ECM). The main components of ECM are type II collagen, aggrecan (a type of proteoglycan), hyaluronic acid, and other matrix proteins. Inside the cartilage, type II collagen fibrils form a network structure with IX and type XI collagen, and the gaps contain giant aggregates composed of highly hydrated aggrecan, hyaluronic acid, and link protein. Since such a three-dimensional structure is formed, hyaline cartilage has a high moisture content and viscoelasticity (anti-compression), and can withstand loads several times the weight. In addition, hyaluronic acid, which is a lubricant secreted from synovium and chondrocytes, has a low coefficient of friction on the surface of the cartilage, enabling smooth movement of the joint, and further suppressing wear of the cartilage itself. However, these abundant ECMs make it difficult for cells to infiltrate the surrounding tissues, and because they lack blood vessels, nerves, and lymph vessels and supply nutrients slowly, cartilage is a tissue with a very poor self-repairing ability. It is. For example, if the damage is shallow enough to remain in the articular cartilage layer, cells around the defect proliferate to promote ECM production, but do not excite a reaction that just fills the defect. Further, in the case of deep damage reaching the subchondral bone, bleeding from the bone marrow occurs, and cartilage progenitor cells and various cytokines are supplied, so that they are repaired in the cartilage-like tissue. However, since the repaired tissue is different from the original hyaline cartilage in terms of histology, biochemistry and biomechanics, it often degenerates and progresses to osteoarthritis in the long term. Therefore, surgical replacement using an artificial joint has been proposed and put to practical use as a treatment means for the damage or disease of the cartilage tissue as described above. However, due to the damage of the artificial joint due to repeated stress, etc., the useful life is limited and the artificial joint must be replaced by re-operation. However, because the burden on the patient is large, the application of the artificial joint is only for elderly people This is the current situation. There are also disadvantages such as the risk of infection and limited range of motion. (Biology of bone and cartilage from basic to clinical development, Kanehara Publishing Co., Ltd., 87-112 (2002), Regenerative Medicine From the basics of tissue engineering to the most advanced technology, NTS, 225-283, 691-701 (2002) , Joint Marker Molecular Biology Approach for Pathological Diagnosis, Medical Review, 96 (1997), Arthritis Rhem, 43 (9), 1916-1926 (2000), Biomaterials, 21, 431-440 (2002))
In order to solve these problems, it is considered effective to regenerate tissue using cells, and transplantation of articular cartilage tissue or isolated chondrocytes has been attempted. For example, mosaic transplantation (Mosaicplasty) is practiced in which a small amount of cartilage fragments are collected from a plurality of sites where the patient's weight is low and transplanted into a defect. However, there is a problem that the cartilage that can be transplanted cannot be applied to a large defect because it has a quantitative limit. Furthermore, although the function after transplantation is improved in the short term, there are problems such as fibrosis in the long term. Will arise. (Clin Orthorelate Res, 401, 170-184 (2002))
As another method, there is an autologous chondrocyte transplantation method (Carticel ^™ ) in which chondrocytes separated by enzymatic treatment from cartilage pieces are proliferated in monolayer culture and then transplanted. There are problems such as dedifferentiation like fibroblasts and adhesion, fibrosis, and hypertrophy after transplantation. (J Bone Joint Surg Am, 88, 503-507 (2006), J Bone Joint Surg Am, 86, 286-295 (2004)) Therefore, in order to successfully regenerate a defect by chondrocyte transplantation, defragmentation is necessary. It is necessary to develop a technology that can secure a large amount of normal chondrocytes that are not transformed.
By the way, in 1999, the presence of cells exhibiting pluripotency into mesenchymal tissues such as bone, cartilage, fat, and tendon was reported in the bone marrow, and it was named as mesenchymal stem cell (MSC: Mesenchymal Stem Cell). It was. (Sciene, 4, (276), 71-74 (1997)) MSC-specific surface antigens have not yet been fully identified, but CD90, CD105, CD166, CD29, CD44, etc. are positive, CD14, CD34, CD45 It is now common to call MSCs that are negative. (Biochemical and Biophysical Research Communication, 265, 134-139 (1999)) In addition, MSC can be expanded to 38 PDL (Population Doubling Level-Biol, Biol 64, Journal of Cellular Biochem, 2) while maintaining the differentiation ability to osteoblasts. 294 (1997)), it has been reported that the ability to differentiate into chondrocytes is up to 22 PDL. (Experimental Hematology, 28, 707-715 (2002)) That is, MSC can be expanded and then differentiated into chondrocytes to solve the quantitative problem described above and to reject the patient's own cells. Treatment that avoids this becomes possible. Other advantages of cartilage regeneration from MSCs include a light burden on the patient regarding bone marrow fluid collection, and ease of separation of MSCs from bone marrow fluid. Thus, although cartilage regeneration from MSC is very promising, this regeneration process requires the development of a series of steps of MSC separation, proliferation, differentiation, and three-dimensionalization.
Culture methods using various scaffold materials have been devised as methods for culturing cells three-dimensionally. (Tissue Eng, 13 (7), 1583-1592 (2007), J Biomed Master Res, 50, 138-143 (2000), J Biomed Master Res A (2005)) For example, natural polymers such as collagen and synthetic high A bioabsorbable polymer such as polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid-glycolic acid copolymer (PLGA), which is a molecule, as a main component. There is a method using a sponge or mesh material. Since cartilage requires anti-compressibility, sponges and meshes can be excellent scaffolding materials in terms of mechanical strength, but it is difficult to uniformly seed cells in a carrier and it is difficult to create a homogeneous tissue. Conceivable. Further, in PLA and PGA, there is a problem that degradation products (lactic acid in PLA and glycolic acid in PGA) cause an inflammatory reaction after transplantation. On the other hand, there is a gel-embedded culture method using a gel material such as collagen, fibrin, agarose, or alginic acid. Although this method has low mechanical strength, it has an advantage that cells can be uniformly seeded in a carrier. However, the three-dimensional culture using these carriers has a possibility of rejection by transplanting foreign substances into the living body.
In order to solve the problems of these carriers, it is considered that a culture method that does not use carriers is useful. A typical example is pellet culture of chondrocytes. (Cell Processing Engineering-From Antibody Drugs to Regenerative Medicine-Corona, 111-112 (2007)) This culture method involves creating and culturing spherical cell aggregates by centrifuging the cell suspension. is there. It has been confirmed that MSCs can also be differentiated into chondrocytes by pellet culture (Tissue Engineering, 4 (4), 415-428 (1998)), and the cell density is high in the pellet, and intercellular interaction including paracrine and juxtacrine. Because of its strong action, it is considered that MSC can be efficiently differentiated into chondrocytes. Therefore, it was considered that a three-dimensional cartilage tissue can be prepared from MSC without using a carrier, but in this method, only one pellet can be prepared with one centrifuge tube, and the diameter is about 0.03 mm, which is used for clinical research. Has the disadvantage of being too small. (Tissue Engineering, 14 (12), 2041-2049 (2008))
In contrast, we studied a method of creating a sheet-like tissue that is scaffold-free and can be controlled in shape and size, and put the animal cell suspension in a container that has a flat inner surface perpendicular to the radial direction of centrifugal rotation. Invented is a sheet-like animal cell mass culture composition obtained by centrifuging and starting culture in a corresponding container while forming a sheet-like animal cell mass. (Japanese Patent Application No. 2008-244903) Further, even if an animal cell suspension is placed in a container including a bottom surface of a flat inner surface in a horizontal direction, and the cells that have naturally settled are cultured, the sheet-shaped animal has a biased shape. It has also been found that cell clusters can be created. (Scheduled to be presented at the Society of Regenerative Medicine. March 18 and 19, 2010, Hiroshima International Congress Center)
By the way, when transplanting the created tissue for transplantation into the affected area, the created tissue structure for transplantation should not move until the created tissue structure for transplantation is bonded to the original tissue and fixedly engrafted. It is necessary to fix to the affected part. In the case of a tissue structure created using the above-mentioned nonwoven fabric scaffold, the created tissue structure for transplantation can be sutured to the affected area and fixedly engrafted. However, in the case of the tissue structure for transplant made by scaffold free, it is inferior in mechanical strength and cannot be sutured. In addition, since the main component of the transplant structure is an extracellular matrix produced by chondrocytes or differentiated MSCs, binding between the mother cartilage tissue is inhibited by the newly produced extracellular matrix when transplanted. It is difficult to engraft. (Osteoartritis Cartage, 9 (SupplA), S6-15 (2001), Spo Sport Journal, Vol. 27 (2), 215-221)
Articular cartilage diseases include a partial defect in which only the surface layer of the cartilage layer is shallow and partially defective, and a deep full-layer defect that reaches the bone (lower bone) in the subchondral layer. In the latter case, it is not sufficient to produce a cartilage tissue structure and transplant it, and the regeneration of the lower bone is not observed. On the other hand, the bond engraftment between the prepared and transplanted cartilage structure and the surrounding normal cartilage tissue is difficult as described above, whereas the transplanted bone-like structure such as an artificial bone and the surrounding normal bone are It is also known that the connection with the tissue is easily and firmly performed. Here, as the artificial bone material for transplantation, hydroxyapatite, calcium triphosphate, and the like formed into various shapes such as a columnar shape and a rod shape are used.
Therefore, if an osteochondral-like structure in which the scaffold-free cartilage structure and the artificial bone material are firmly bonded is created and this osteochondral-like structure is transplanted into the lower bone defect, It was considered that the cartilage structure fixed to the upper part of the structure was also well fixed to the affected part, the cartilage tissue was regenerated well, and the lower bone was also regenerated. In this regard, there has been a report of trying to create an osteochondral structure by seeding chondrocytes on hydroxyapatite and culturing for 3 weeks, but the bond strength between the cartilage-like structure and hydroxyapatite is weak and not fixed firmly. . (The Open Biomedical Engineering Journal, 2, 64-70 (2008))
Therefore, it was necessary to develop an osteochondral-like structure for transplantation in which a scaffold-free cartilage-like structure and an artificial bone material were firmly bound.

Problems to be solved by the invention

An object of the present invention is to provide a bone-cartilage-like structure for transplantation in which a cartilage-like structure to be created in a scaffold-free manner and an artificial bone material are firmly bonded, and a method for producing the same.

Means for solving the problem

As a result of intensive studies to solve the above-mentioned problems, the present invention has been completed. That is, the present invention at least puts an animal cell suspension into a container including a bottom surface of a flat inner surface perpendicular to the centrifugal rotation radius direction, and centrifuges the sheet-shaped animal cell mass formed by centrifugal sedimentation. The present invention relates to an osteochondral-like structure characterized in that a bone-like solid is placed on the cell mass on the bottom surface of the culture container after culturing in the cultivation of a sheet-like animal cell mass cultured in (1).
Examples of the shape of the bottom surface of the flat inner surface perpendicular to the centrifugal rotation radius direction used in the present invention include a circle, an ellipse, a triangle, a square, a rectangle, a regular pentagon, and a donut.
The area of the bottom surface of the vertical flat inner surface in the centrifugal radial direction to be used in the present invention, may be any of 0.15~1000cm ^2, 0.2~10cm ² is preferred.
The depth of the container used in the present invention may be 3 to 100 mm, but is preferably 5 to 50 mm.
The material of the container used in the present invention should be one in which the components are eluted in the culture solution and adversely affect the cells, and examples thereof include polystyrene, polypropylene, stainless steel, glass and the like.
In particular, materials suitable for the adhesion of animal cells are materials that have been hydrophilized so that animal cells can easily adhere to them, or materials that have been coated with adhesion proteins, and have been subjected to plasma discharge treatment for adhesion culture. As an example, a polystyrene incubator marketed as “for cell culture” can be mentioned.
The centrifuge used for the centrifuge used in the present invention may be any centrifuge as long as it can centrifuge a container including a flat inner surface perpendicular to the radial direction of centrifugal rotation, but a swing type is desirable.
The centrifugal speed may be 50 to 15000 rpm, but is preferably 100 to 3000 rpm.
The animal cell used in the present invention is an animal-derived cell, and examples of the animal include birds, reptiles, amphibians, fish, mammals and the like. Examples of mammals include humans, monkeys, cows, pigs, sheep, horses, mice, and the like. In addition, both primary cells that can divide and proliferate only a limited number of times, typically up to about 50 times after collection from animals, and cell lines that can divide and proliferate more than 50 times after collection from animals. Can be used. Examples of primary cells include human articular chondrocytes, rat primary hepatocytes, mouse primary bone marrow cells, porcine primary hepatocytes, human primary umbilical cord blood cells, and the like. Examples of cell lines include Chinese hamster ovary cell line CHO cell, human uterine cancer cell line HeLa, African green monkey kidney cell line Vero cell, human hepatoma cell line Huh7 cell and the like. In addition, cells obtained by performing genetic manipulation on the above-described cells by means such as introduction of a plasmid or viral infection can also be used in the present invention.
Chondrocytes used in the present invention include chondrocytes collected from articular cartilage tissue, mesenchymal stem-like cells contained in bone marrow fluid and umbilical cord blood, chondrocytes obtained by differentiating them in vitro, and fertilized eggs at a certain period. Examples include embryonic stem cells (ES cells) to be separated and chondrocytes obtained by differentiation from them in vitro. In addition, cells obtained by performing genetic manipulation on the above-described cells by means such as introduction of a plasmid or viral infection can also be used in the present invention.
The area of the sheet-like animal cell mass prepared in the present invention is not more than the area of the flat inner surface perpendicular to the centrifugal rotation radius direction of the container used for centrifugation.
The thickness of the sheet-like animal cell mass prepared in the present invention may be 0.1 to 100 mm, but is preferably 0.5 to 15 mm.
The thickness of the sheet-like animal cell mass prepared in the present invention may vary depending on the location even in the same cell mass, but the deviation may be within a range of ± 50%, but is preferably within ± 20%.
The culture medium used in the present invention includes a standard medium containing growth factors and nutrients used to support cell growth and maintenance, and a medium obtained by adding various additives such as animal serum to a standard medium. As an example. The standard medium to be used varies depending on the cell type desired to be cultured, and media such as Iskov medium, RPMI medium, Dulbecco MEM medium that are usually used for culturing animal cells can be used, but it is effective for cell growth and maintenance according to known literatures. Non-serum factors known to be present, such as serum albumin, transferrin, lipid and fatty acid sources, cholesterol, pyruvate, glucocorticoids, nucleosides for DNA and RNA synthesis, growth factors (eg epidermal growth factor, fibroblasts) Growth factors, platelet-derived growth factors and insulin), and extracellular matrix cells (eg, collagen, fibronectin and laminin) may be added.
Although the culture | cultivation period performed by this invention may be any of 1-2000h, 24-504h is preferable.
The bone-like solid material used in the present invention should have a mechanical strength of a certain level or more and does not have harmful effects on animal cells, such as eluting components harmful to animal cells during culture. Examples thereof include calcium triphosphate (such as βTCP) and hydroxyapatite.
Examples of the shape of the bone-like solid used in the present invention include a cylinder, a triangular prism, a cube, a rectangular parallelepiped, and the like.

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

Porcine femoral knee articular cartilage was collected with a scalpel, suspended in 0.25% collagenase solution (dissolved in DMEM + 10% FCS), and incubated at 37 ° C. for 4 hours to obtain a chondrocyte suspension.
18.6 × 10 ⁶ cells / ml cell suspension (DMEM + 10% FCS + 50 mg / L ascorbic acid phosphate ester) 0.3 ml was put into 96-well multiwell (SUMILON MS-8096F) and centrifuged at 1200 rpm for 5 minutes. Thereafter, static culture was performed at 37 ° C. under 5% CO ₂ . At any time on the first, second, third, fourth, fifth, sixth, and seventh days after the start of culture, a βTCP cylinder (diameter 5 mm, height 3 mm) is gently placed on the cell mass for a total of 3 weeks. Cultured. In all cases, the medium was changed every two days.
After completion of the culture, osteochondral-like structures were fixed (formaldehyde), embedded in paraffin, sections (thickness 3 μm) were prepared, stained with Alcian blue, and the thickness of the chondroid cell sheet was set on top of TCP and TCP It measured about inside. (Figure 1)
As a result, it was found that a part of the cartilage-like cell sheet was in the TCP. The total sheet thickness on and in the TCP was almost constant regardless of the incubation time until the TCP was placed. On the other hand, it was recognized that the sheet thickness in TCP tends to be thicker when the incubation time until TCP is placed is shorter. That is, it was suggested that the sheet and TCP can be sufficiently bonded when TCP is placed in the early stage of culture.

Porcine femoral knee articular cartilage was collected with a scalpel, suspended in 0.25% collagenase solution (dissolved in DMEM + 10% FCS), and incubated at 37 ° C. for 4 hours to obtain a chondrocyte suspension.
18.6 × 10 ⁶ cells / ml cell suspension (DMEM + 10% FCS + 50 mg / L ascorbic acid phosphate ester) 0.3 ml was put into 96-well multiwell (SUMILON MS-8096F) and centrifuged at 1200 rpm for 5 minutes. Thereafter, static culture was performed at 37 ° C. under 5% CO ₂ . At any time on the 5th, 7th, or 10th day after the start of the culture, βTCP cylinders (diameter 5 mm, height 3 mm) were gently placed on the cell mass and cultured for a total of 3 weeks. In all cases, the medium was changed every two days.
After culturing, an attempt was made to take out the structure in which TCP was bound on the sheet from the 96-well by pinching the TCP with tweezers and gently pulling it up.
As a result, the samples in which βTCP was placed 5 days after the start of the culture were able to take out the sheet-like cell mass from the well by holding the TCP with tweezers without detaching the TCP from the cell sheet for all 12 cultured cells. (Fig. 2) On the other hand, those in which βTCP was placed 7 days after the start of culturing, 7 out of 11 cultivated cells had TCP in tweezers without holding TCP away from the cell sheet. Although the cell clumps could be removed from the wells, in 4 cases, the TCP was detached from the sheet and the sheet could not be removed from the well. Furthermore, in those in which βTCP was placed 10 days after the start of culture, TCP was detached from the sheet in all of the 6 cultured cells, and the sheet could not be removed from the well. That is, in the case where βTCP was placed 5 days after the start of culture, the TCP and the cell sheet were strongly bound, but in the case where βTCP was placed 7 days after the start of culture, βTCP was slightly weaker, and after 10 days after the start of culture, βTCP was added. What was put on was clearly found to be weakly bonded.
This was considered to be a result consistent with the result of Example 1. That is, it is considered that the TCP is submerged deeply in the sheet when the cell sheet is still soft and the TCP is submerged deeply, and the sheet part in the TCP becomes thick, thereby strengthening the bond between the sheet and the TCP.

The invention's effect

As described above, according to the present invention, at least a sheet shape formed by centrifugal sedimentation after putting animal cell suspension into a container including a bottom surface of a flat inner surface perpendicular to the radial direction of centrifugal rotation. A sheet-like animal cell formed by natural sedimentation after putting an animal cell suspension into a container including a bottom surface of an inner surface flat in the horizontal direction, or culturing an animal cell mass in a corresponding container. In culturing a sheet-like animal cell mass in which the mass is cultured in the corresponding container, the cell mass (sheet) and the bone-like solid are obtained by placing a bone-like solid on the cell mass on the bottom surface of the culture container after the start of culture. It is possible to provide an osteochondral-like structure in which is firmly bound.

Relationship between the thickness of the cartilage-like cell sheet on and in TCP and the incubation time until TCP is placed Relationship between culture time until TCP is placed and sheet / TCP bond strength

Claims (1)

1. After the animal cell suspension is placed in a container including a bottom surface of a flat inner surface perpendicular to the centrifugal rotation radius direction and centrifuged, the sheet-like animal cell mass formed by centrifugal sedimentation is cultured in the container. An osteochondral-like structure produced by placing a bone-like solid on the cell mass on the bottom surface of the culture vessel after culturing, in culturing the mass.
2. Incubation of sheet-shaped animal cell mass in which the animal cell suspension is placed in a container containing the bottom surface of the flat inner surface in the horizontal direction and then the sheet-shaped animal cell mass formed by natural sedimentation is cultured in the corresponding container. An osteochondral-like structure produced by placing a bone-like solid on the cell mass on the bottom surface of the culture container.
3. The osteochondral-like structure according to claim 1 or 2, wherein the animal cell used is a chondrocyte.
4). The osteochondral-like structure according to claim 1 or 2, wherein the animal cells used are mesenchymal stem cells.
5. The osteochondral-like structure according to any one of claims 1 to 4, wherein the area of the flat inner surface is 0.3 cm ² or more.
6). The osteochondral-like structure according to any one of claims 1 to 5, wherein the flat inner surface is made of a material suitable for adhesion of animal cells.
7). The osteochondral structure according to any one of claims 1 to 6, wherein the bone-like solid is ceramics.
8). The osteochondral structure according to any one of claims 1 to 6, wherein the bone-like solid is βTCP.
9. The osteochondral structure according to any one of claims 1 to 6, wherein the bone-like solid is hydroxyapatite.
10. The osteochondral-like structure according to any one of claims 1 to 9, wherein the osteochondral-like structure is produced by placing a bone-like solid within 168 hours after the centrifugation operation.

Images