JP4153391B2 - Tissue formation promoting factor - Google Patents

Description

  The present invention relates to chondrocytes, progenitor cells, mesenchymal stem cells cultured in vitro, or these cells present in vivo in a three-dimensional environment containing fat-soluble vitamins, in vivo and in vitro. And an efficient method for producing cartilage tissue and a method for treating cartilage damage using the same.

  Cartilage diseases such as osteoarthritis tend to increase rapidly with an aging population, and the resulting social and economic costs are high, and an effective treatment that can restore joint function is strongly desired.

  Traditionally, based on the hypothesis that the damaged joint surface can be repaired by transplanting autologous cells that the damaged joint surface appears to reconstruct the appropriate extracellular matrix, The treatment to be introduced into the knee is considered promising, and this has been put into practical use and commercialized (Brittberg et al. (Non-patent Document 1, Non-patent Document 2). However, this treatment method targets small-scale cartilage damage such as sports disorders. It is impossible to apply to large-scale cartilage damage such as osteoarthritis with high severity and a large number of patients. The reality is that there are doubts about the therapeutic effect due to the decrease in cartilage tissue formation ability of the body, or the decrease in cartilage tissue formation ability caused by amplification outside the body regardless of age (non-patented) Document 3).

  Furthermore, in autotransplantation treatment using autologous tissue such as autologous joint-derived chondrocytes as a cell source, there is an invasive problem due to the need to collect the patient's own cells from the joint. Therefore, utilization of mesenchymal stem cells, which have a relatively low degree of invasiveness by collection, is expected, and this practical application is also being studied.

  Mesenchymal stem cells (MSCs) are found in bone marrow, blood, dermis, and periosteum that can differentiate into special types of mesenchymal or connective tissue including connective tissues such as fat, bone, cartilage, elasticity, muscle and fibers. Are pluripotent blast cells or embryonic cells. These cells are usually present at a very low frequency in the bone marrow. The process of separating, purifying, and amplifying these cell populations in tissue culture has already been reported by Friedenstein et al.

Many differentiations of MSCs into chondrocytes have already been reported (Non-patent document 5, Non-patent document 6, Non-patent document 7, Non-patent document 8). As for the creation of cartilage from MSCs in vivo, there is a report of Wakitani et al. Who conducted a cartilage defect treatment experiment using rabbits (Non-patent Document 9). In addition, there are reports on the creation of cartilage in vitro by Johnstone et al. Regarding the creation of cartilage using rabbit bone marrow-derived mesenchymal stem cells (Non-patent document 10, Non-patent document 11, Non-patent document 12).

Brittberg et al. The New England Journal of Medicine Vol. 331, no. 14, 889-895 (1994) Morohashi et al. (Example of commercialization of cultured autocartilage transplantation in the US) Tissue culture engineering, Vol. 23, 568-571 Breinan et al. Orthopedics Vol. 20: 525-538 (1997) Friedenstein et al. Cell Tissue Kinet. Vol. 20, 263-272 (1987) Grigoriadis et al. Journal of Cell Biology Vol. 106, 2139 (1988) Bab et al. Cell Sci. Vol. 84, 139-151 (1986) Friedenstein et al. Bone Miner Res. Vol. 7, 243-272 (1990) Caplan et al. Orthop. Res. Vol. 9, 641-650 (1991) Wakitani et al. Journalof Bone Joint Surgage Am. Vol. 76A, 579 (1994) Johnstone et al. Trans. Orthop. Res. Soc. Vol. 21, 65 (1996) Johnstone et al. Experimental Cell Research Vol. 238, 265-272 (1998) Mackay et al., Tissue Engineering Vol. 4, no. 4, 415-428 (1998)

  In the above situation, a problem that arises when using chondrocytes, progenitor cells or mesenchymal stem cells is how to maintain the ability of the cells to form cartilage tissue. In other words, chondrocytes suffer from a decrease or loss of chondrocyte function due to aging or amplification, and mesenchymal stem cells also suffer from a decrease or loss of differentiation ability into chondrocytes due to aging or amplification. Under such circumstances, it is desired to set an environment that leads to better cartilage formation while maintaining more stable chondrocyte function or ability to differentiate into chondrocytes.

  It is known that culturing in a three-dimensional environment suppresses dedifferentiation and maintains the ability to differentiate into each tissue cell, and cartilage tissue is created to some extent by performing cartilage creation in vitro or in the body in a three-dimensional environment. There have been many reports that formation is maintained and enhanced. However, the actual clinical cartilage regeneration is still not enough, and there is a strong demand for the development of a further method for improving the decrease in differentiation ability.

  The simplest and most practical method for maintaining differentiation potential is the utilization of factors. Glucocorticoids such as dexamethasone, transforming growth factor-β family factor (TGF-β), such as bone morphology, which maintains the function as chondrocytes or has an effect of inducing differentiation from mesenchymal stem cells to chondrocytes Forming protein (preferably BMP-2 or BMP-4), basic fibroblast growth factor (bFGF), inhibin A or chondrogenic stimulating activity factor (CSA), type I collagen (especially in gel form), etc. Collagenous extracellular matrix, and vitamin A analogs such as retinoic acid, ascorbic acid and the like.

  In the more practical cartilage production research described in the “Background Art” section above, ascorbic acid is used as an effective factor in the creation of cartilage from chondrocytes and mesenchymal stem cells. Is not suppressed at a satisfactory level.

  As a result of searching and examining various factors to solve the above problems, one of the fat-soluble vitamins, reduced coenzyme Q10 and oxidized coenzyme Q10, is used in the regeneration of cartilage using chondrocytes or mesenchymal stem cells. The present invention provides a method for promoting cartilage tissue formation using this reduced coenzyme Q10 and oxidized coenzyme Q10, and further a method for treating cartilage damage using the same. The invention has been completed.

That is, the first of the present invention is that cells derived from cartilage tissue, progenitor cells or mesenchymal cells in a three-dimensional culture environment using a medium containing reduced coenzyme Q10, oxidized coenzyme Q10 or a mixture thereof in vitro. The present invention relates to a method for creating a cartilage tissue characterized by culturing stem cells.

  The present invention provides means for making the production of a three-dimensional cell conjugate for cartilage tissue regeneration in vivo and in vitro for the treatment of cartilage damage more practical on the premise of industrialization. .

  Glucocorticoids such as dexamethasone, transforming growth factor-β family factor (TGF-β), such as bone morphology, as a factor that maintains chondrocyte function or has an effect of inducing differentiation from mesenchymal stem cells to chondrocytes Forming protein (preferably BMP-2 or BMP-4), basic fibroblast growth factor (bFGF), inhibin A or chondrogenic stimulating activity factor (CSA), type I collagen (especially in gel form), etc. Collagenous extracellular matrix, vitamin A analogs such as retinoic acid, fat-soluble vitamins such as vitamin B12 and tocopherol, and water-soluble vitamins such as ascorbic acid. In particular, TGF-β is said to be essential in the creation of cartilage.

  In the search for non-protein cartilage differentiation factor of the present invention, the target is to promote the production of cartilage matrix of articular chondrocytes cultured in the presence of serum without coexisting with the protein factor having cartilage differentiation-inducing activity described above. Screening was performed. As a result, this strong activity was found in fat-soluble vitamins, particularly reduced coenzyme Q10 and oxidized coenzyme Q10.

  In the present invention, the concentration of reduced coenzyme Q10 and oxidized coenzyme Q10 in a three-dimensional culture environment is 1 ng / ml or more and 1000 μg / ml or less, preferably 100 ng / ml or more and 100 μg / ml or less. is there.

  The chondrocytes and progenitor cells can be used as a source of all living cartilage tissues, but are preferably chondrocytes derived from joints or ribs. Articular chondrocytes are the most preferred source. Chondrocytes can be isolated from these tissues by using collagenase. The isolated chondrocytes are cultured and amplified in a bovine or human serum environment or a synthetic serum-free environment, and the formation of cartilage tissue is a mixture with a support that provides these cells with a three-dimensional morphology, preferably a three-dimensional environment ( This was performed by culturing in a synthetic serum-free or serum environment as a three-dimensional cell conjugate). This three-dimensional cell conjugate can be applied to cartilage formation in vitro and in vivo.

  Mesenchymal stem cells (MSCs) from bone marrow, blood (including peripheral blood), periosteum and dermis, and other tissues with mesodermal origin, etc. Density gradient fraction in bovine or human serum environment or synthetic serum-free environment Can be separated. The MSCs contained in these tissues decrease significantly with age, but can be isolated from the bone marrow, among other things, and the isolated fractional preparation contains at least about 90% of the cells, and preferably at least about 95% of the cells. Is likely to contain cells of human mesenchymal stem cells.

  Further, the same fractionally prepared cells are cultured and amplified in an environment containing bovine or human serum medium or an environment containing serum-free synthetic medium, and the formation of cartilage tissue causes these cells to have a three-dimensional morphology, preferably a three-dimensional environment. It was carried out by culturing in an environment containing a serum-free synthetic medium as a mixture (referred to as a three-dimensional cell conjugate) with a support to which the above is applied. This three-dimensional cell conjugate can be applied to cartilage formation in vitro and in vivo.

  The following can be used as the three-dimensional culture environment. By processing the chondrocytes, progenitor cells, and mesenchymal stem cells such as centrifugation, a simple three-dimensional culture environment in a cell mass state can be constructed. The simple cell mass referred to here is a cell mass without a special support that provides a three-dimensional culture environment. In addition, a support produced using a biocompatible material derived from a natural product or a non-natural product can be used as a three-dimensional culture environment.

  As biocompatible materials derived from natural products, collagen, gelatin, chitin, chitosan, agar, basement membrane components, fibronectin, laminin, glycosaminoglycan, hyaluronic acid and mixtures thereof can be used.

  Non-naturally occurring biocompatible materials include polylactic acid, polyglycolic acid, lactic acid / glycolic acid copolymer, polyε caprolactone, lactic acid / ε caprolactone co-polymer, which are hydrolyzable polymers of α and β-hydroxycarboxylic acids. A polymer or the like can be used.

  In the present invention, the “support for tissue regeneration” means a support for regeneration of various tissues constituting a living body. Examples of the structure of the tissue regeneration support of the present invention include gels, nonwoven fabrics, foams, sponges, and fabric structures.

  In addition, the tissue regeneration support used in the present invention can be appropriately sized to compensate for the affected area by appropriately setting the thickness. The thickness of the support of the present invention is preferably 50 μm to 1 cm. When used for treatment of human knees or hip joints, a thickness of 1 to 4 mm is practically preferable.

  Moreover, there is no limitation in particular about the shape and area of the said support body, The thing of sufficient size in order to fill an affected part can be produced. For example, when used for treatment of a human knee or hip joint, it is preferably a cylindrical one having a diameter of 10 to 20 mm.

  Next, a method for producing a three-dimensional cell conjugate for cartilage tissue regeneration according to the present invention will be described. In the production method described above, a three-dimensional support used in the present invention is seeded with chondrocytes, the same progenitor cells or mesenchymal stem cells, and a synthetic medium is used in an artificial environment and / or in a living body in a three-dimensional culture environment. It is characterized by culturing in.

  As used herein, the term “synthetic medium” means that the composition of the present invention is capable of undergoing in vitro chondrogenesis according to the method of the present invention, and that the minimum essential medium, ascorbate or analog thereof, iron source Means a maintenance, growth or culture medium containing equimolar metals and insulin or insulin-like growth factor. In addition, it is preferable to use bovine albumin that has a proven record in humans as another protein factor. In this experiment, DMEM, a trace amount of iron source, insulin, and bovine albumin were additionally used as minimum essential media.

In the above production method, seeding of mesenchymal stem cells or progenitor cells can be performed by a known method, but seeding of cells or progenitor cells so that the density is 10 ⁶ to 10 ⁸ per 1 cm ^{3 of} the support. preferable.

  In the above method, “culturing in an artificial environment” means culturing in vitro such as a test tube or an incubator. Batch culture by static or swirling or circulating continuous culture can be performed, preferably swiveling batch culture or circulating continuous culture.

  In the method for producing a three-dimensional cell conjugate used in the present invention, the culture in the artificial environment is performed under the condition that the culture solution is moved relative to the support at a speed of 0.1 cm / second to 50 cm / second, This is preferable from the viewpoint of supplying a fresh medium to the three-dimensional cell conjugate and from the viewpoint of removing waste products from the three-dimensional cell conjugate.

  In the above method, “culturing in vivo” means culturing in a living tissue, for example, by culturing cells by placing a support or a mixture of a support and cells in the living tissue.

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

(Example 1) Creation of cartilage tissue Cartilage tissue was collected from a Japanese Saanen goat knee joint approximately one year after birth, and chondrocytes were separated by collagenase. The cells were cultured using Ham's F-12 medium containing 10% fetal bovine serum and ascorbic acid at a final concentration of 50 μg / ml, and chondrocytes passaged twice were used for the production of cultured cartilage.

1 ml of 1 × 10 ⁷ cells / 5 ml cell suspension (Ham's F-12 medium containing 10% fetal bovine serum and final concentration of 50 μg / ml ascorbic acid) was placed in a 15 ml polypropylene centrifuge tube, By centrifuging at 1200 rpm for 5 minutes, a three-dimensional culture environment consisting of a supernatant medium and a simple cell mass at the bottom was prepared in a centrifuge tube. Oxidized coenzyme Q10 and reduced coenzyme Q10 were added to the medium to give 0.175 μg / ml and 35 μg / ml, respectively, and cultured at 5% CO ₂ and 37 ° C. for 14 days. Medium change was performed every 3 days. As a comparison, culture without addition of oxidized coenzyme Q10 or reduced coenzyme Q10 was performed in the same manner. The cell mass after culture was evaluated histologically and biochemically. As a histological evaluation, Alcian blue staining was used, and as a biochemical evaluation, glycosaminoglycan production was determined by a dye method. As a result, the tissue staining of the cell cluster showed remarkable Alcian blue positivity as shown in FIG. When this cultured cartilage was solubilized with 4M guanidine hydrochloride solution and glycosaminoglycan, one of the matrix characteristic of cartilage, was quantified by a dye method called DMMB (Dimethyl Methylene Blue) method, reduced coenzyme was determined. Significant production enhancement was observed by the addition of Q10 or oxidized coenzyme Q10 (FIG. 2). In FIG. 2, DNA measurement is performed as an internal standard for offsetting variations due to differences in extraction efficiency. Therefore, the amount of glycosaminoglycan is indicated by a number per unit DNA amount (glycosaminoglycan production ratio). GAG / DNA refers to glycosaminoglycan / DNA.

The results of tissue staining (alcian blue staining) of goat chondrocyte mass cultured for 14 days are shown. This is a quantification of glycosaminoglycan in a goat chondrocyte mass cultured for 14 days.

Explanation of symbols

1 QX; oxidized coenzyme Q10
2 QH; Reduced Coenzyme Q10
3 GAG; glycosaminoglycan

Claims (7)

Characterized by culturing reduced coenzyme Q10 or oxidized coenzyme Q10 or cartilage-derived cells under three-dimensional culture environment using a medium containing the mixture, the progenitor cells or mesenchymal stem cells in vitro A method of creating cartilage tissue. The method for creating a cartilage tissue according to claim 1, wherein the medium used in the three-dimensional culture environment is a medium containing ascorbic acid. The method for creating a cartilage tissue according to claim 1, wherein the cells in the three-dimensional culture environment are simple cell clusters. The method for creating a cartilage tissue according to claim 1, wherein the cells in the three-dimensional culture environment are three-dimensional cell conjugates in which the cells are seeded on a tissue regeneration support prepared from a biocompatible material derived from a natural product. The method for creating a cartilage tissue according to claim 1, wherein the cells in the three-dimensional culture environment are three-dimensional cell conjugates in which the cells are seeded on a tissue regeneration support prepared from a biocompatible material derived from a non-natural product. Claim 1 cartilage creation method according cells in three-dimensional culture environment is a three-dimensional cell combination of cell mass or claim 4 or 5 of Claims 3, wherein transplanted into cartilage lesion. The method for creating a cartilage tissue according to claim 1 , wherein the cartilage tissue is an articular cartilage tissue.

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