[DESCRIPTION]

[invention Title]

A COMPOSITION OF THE INJECTABLE AGENTS FOR TISSUES REPAIRE CONTAINING MESENCHYMAL STEM CELLS AND OPTIMIZED CULTURE MEDIA

[Technical Field]

The present invention relates to an injectable composition for tissue regeneration for correcting morphological or degenerative defects in the skin, soft tissue or bone of a recipient. More particularly, the present invention relates to an injectable composition for tissue regeneration, which contains a culture medium, obtained by culturing human mesenchymal stem cells in a serum-free medium, containing cell activating compounds, for at least one day, and to a method of regenerating tissue using the same.

[Background Art]

As used herein, the term "stem cells" refers to cells, which remain undifferentiated into particular cells, and, if necessary, has the potential to differentiate into all kinds of cells constituting the body, including nerve, blood and the like. Stem cells are classified into two categories: embryonic stem cells obtained from embryos developed from fertilized eggs; and adult stem cells potentially stored in each part of the adult human body.

Among them, the adult stem cells are isolated from adult autologous tissue, unlike the embryonic stem cells, and thus, they can avoid ethical problems and, in addition, have no risk of immune rejection. Also, the adult stem cells are distinguished from the embryonic stem cells, in that they can

be obtained in large amounts for treatment. In addition, the adult stem cells have advantages in that they do not have the potential to cause cancer when they are transplanted into the body for organ regeneration and in that they can undergo not only self- renewal to produce cells immediately after transplantation and create and store undifferentiated stem cells, if necessary, but also site-specific differentiation to differentiate according to the characteristics of the peripheral tissues. These various advantages of the adult stem cells suggest the medical safety and high utility of the adult stem cells. Thus, studies on the treatment of various diseases in vivo using such adult stem cells have been actively conducted.

The adult stem cells have an advantage in that they can be obtained from autologous tissue as described above, and indeed, the collection of the adult stem cells has been mainly conducted in bone marrow. However, there are various difficulties in obtaining the cells in an amount sufficient for treatment, because the bone marrow collection itself is a high-risk surgery and, in addition, the actual yield of the cells is low. Adipose tissue-derived stem cells, which are used in the present invention, have various advantages over prior bone marrow-derived stem cells. For past several years, there has been a considerable advance in the biomaterial field, but the production of adipose tissue or soft tissue, which can be transplanted into the human body, has not been successfully developed. Also, various studies on the use of human adipose tissue have not been conducted. However, since it was recently confirmed that adult stem cells are present in adipose tissue, various studies on the use of adipose stem

cells started to be conducted (Zuk et al., Molecular Biology of Cell, 13: 4279-4295 (2002); Rodriguez et al., Biochimie, 87: 125-128 (2005)). The adipose stem cells have advantages in that they have multipotent ability and proliferation ability, which are substantially similar or superior to bone marrow-derived stem cells, and, in view of actual clinical utility, a sufficient amount of the adipose stem cells can be isolated from a small amount of adipose tissue and cultured, and, in addition, a surgical process for collecting the adipose stem cells is significantly safe to bone marrow collection. Also, the clinical utility and superiority of the adipose stem cells can be maximized, because the collection of adipose tissue can be performed in local anesthesia to reduce the risk of general anesthesia and, in addition, the patient's wound site can be healed within one week (Rei Ogaw., Current Stem cell research & therapy, 1: 13- 20 (2006) ) .

Despite this superiority of the adipose stem cells, studies on the adipose stem cells are currently focused only on methods which use the pluripotent differentiation ability of the cells, and studies focused on maintaining and increasing the native potential of these cells were insufficient .

With respect to the clinical utilization of adult stem cells, a number of studies on tissue regeneration and therapy have been conducted (Cecilia Roh and Stephen lyle., Pediatr. Res., 59: 100-103 (2006); H. Nakagawa et al., British Journal of Dermatology, 153: 29-36 (2005); Rei Ogawa., Current Stem Cell Research & Therapy, 1: 13-20 (2006)), but there have been various problems in that, when the adult stem cells are

transplanted into a living body, the viability thereof is low and the transplanted cells do not show the sufficient effects thereof. Also, studies on the technology for increasing the viability of the adult stem cells upon transplantation and activating the adult stem cells to optimize the tissue regeneration ability of the adult stem cells are still insufficient.

With respect to studies in Korea, Korean Patent Laid- Open Publication No. 2004-0024900, entitled "Animal serum- free medium composition for culturing human stem cells and method for inducing differentiation into liver cells", discloses a method of differentiating human stem cells using an animal serum-free medium, and Korean Patent Laid-Open Publication 2003-0004565, entitled "Method for producing cells for transplantation" discloses the use of stem cells for cell transplantation. However, there have been no studies on the activation of mesenchymal stem cells.

Accordingly, the present inventors have developed an injectable agent for tissue regeneration, which contains components which optimize the viability and pharmacological action of mesenchymal stem cells in order to optimize the tissue regeneration effect of the mesenchymal stem cells. [Disclosure] [Technical Problem] It is an object to provide an injectable composition for optimizing the tissue regeneration ability of mesenchymal stem cells and to increase the viability of mesenchymal stem cells through the composition so as to maximize the activity of the stem cells, thus rapidly and effectively regenerating tissue.

[Technical Solution]

The injectable composition for tissue regeneration according to the present invention contains a culture medium and culture environment for optimizing the tissue regeneration ability of mesenchymal stem cells.

To achieve the above object, the present invention provides an injectable composition for tissue regeneration, which contains a culture medium, obtained by culturing mesenchymal stem cells in a serum-free medium, containing cell-activating compounds, for at least one day.

Herein, the injectable composition for tissue regeneration, which contains a culture medium, obtained by culturing mesenchymal stem cells in a serum-free medium, containing cell-activating compounds, for at least one day, is referred to as a mesenchymal stem cell-optimizing constituent, and the cell activating compounds include antioxidants, vitamins or analogues thereof, amino acids or analogues thereof, growth factors or analogues thereof, collagens or analogues thereof or inorganic substances. The antioxidants include one or more selected from the group consisting of L-lipoic acid, coenzyme Q-IO, C-MED 100, Trolox and GSH, the vitamins include one or more selected from the group consisting of vitamins A, B, C, D and E, the amino acids include one or more selected from the group consisting of L-alanine, L-aspartic acid, L-histidine, L- leucine and L-glutamine, and the growth factor is selected from the group consisting of vascular endothelial growth factor, hepatocyte growth factor, transforming growth factor- beta, platelet derived growth factor-A, keratin sulfate, basic fibroblast growth factor and cadherin (like epidermal

growth factor-2), and the collagens include one or more selected from the group consisting of collagens I-XIII and XVII and fibronectin.

In another aspect, the present invention provides a method of treating defects in skin tissue, soft tissue, tooth and bone by injecting into treatment sites the injectable composition for tissue regeneration together with mesenchymal stem cells. The injectable composition for tissue regeneration according to the present invention shows the effect of optimizing the tissue regeneration ability of mesenchymal stem cells by increasing the viability of the mesenchymal stem cells and increasing the proliferation ability of fibroblasts, when the injectable composition is transplanted. Hereinafter, the present invention will be described in detail .

The first aspect of the present invention relates to an injectable composition for tissue regeneration, which contains a culture medium, obtained by culturing mesenchymal stem cells in a serum-free medium, containing cell-activating compounds, for at least one day.

The injectable composition for tissue regeneration according to the present invention contains a mesenchymal stem cell-optimizing constituent, which refers to a culture medium, obtained by culturing mesenchymal stem cells in a serum-free medium, containing cell-activating compounds, for at least one day.

Herein, the mesenchymal stem cell-activating compounds include antioxidants, vitamins or analogues thereof, amino acids or analogues thereof, growth factors or analogues

thereof, collagens or analogues thereof and inorganic substances. Among them, the growth factors and the collagens may not be added to an initial culture medium, because they are secreted from mesenchymal stem cell, when the mesenchymal stem cells are cultured in the serum-free medium for at least one day.

The injectable composition for tissue regeneration according to the first aspect of the present invention contains the optimizing composition in order to maximize the viability of transplanted mesenchymal stem cells, when the composition is administered into treatment tissue.

The term "viability" of cells means the viability of subcutaneously injected mesenchymal stem cells up to the stage in which the injected mesenchymal stem cells migrate and adhere to skin tissue. Through the expression of various extracellular proteins secreted from the adhered stem cells, the proliferation and differentiation of fibroblasts are activated.

Specifically, the mesenchymal stem cells according to the present invention include mesenchymal stem cells isolated from mammalian adipose tissue, bone marrow, umbilical cord blood or placenta.

The stem cells according to the present invention are adult stem cells in adipose tissue and can be obtained from adipose tissue cells through a simple purification process. A process for the collection of adipose tissue is generally carried out using adipose tissue wasted in a general liposuction process and does not require additional invasive procedures, and thus the utility thereof is greatly increased. A process of isolating adult stem cells from mammalian

adipose tissue, bone marrow, cord blood or placenta and culturing the isolated cells is not limited only to the method of the present invention and can be carried out according to any conventional method known in the art. Adipose stem cells can be obtained by collecting human adipose tissue by liposuction under local anesthesia, treating the extracellular matrix of the collected adipose tissue with collagenase, centrifuging the treated tissue, and separating mononuclear cells, red blood cells and various cell fragments from the centrifuged tissue.

Specifically, adipose stem cells can be generally obtained by collecting adipose tissue, wasted in a liposuction process conducted in hospitals, in an aseptic state, and isolating only pure adipose stem cells from the collected liposuction material.

The isolated stem cells are cultured in a non-inducible medium of serum-containing Dulbecco ' s modified eagle's medium (DMEM) to remove non-adhesive cells.

Then, the adipose stem cells are cultured in a serum- free medium, containing cell activating compounds, for at least one day, thus obtaining a mesenchymal stem cell- optimizing constituent asccording to the present invention.

In order to increase the efficiency of the injectable agent for tissue regeneration, the optimizing constituent of the present invention also contains cell-activating compounds, which are effective for the viability of mesenchymal stem cells, the proliferation ability of fibroblasts and the prevention of cell aging. The cell-activating compounds include antioxidants, vitamins or analogue thereof, amino acids or analogues thereof, amino acids or analogues thereof,

growth factors or analogues thereof, collagens or analogues thereof and inorganic substances.

The antioxidants include one or more selected from the group consisting of L-lipoic acid, coenzyme Q-IO, C-MED 100, retinoic acid, vinpocetine, picamilon, quinic acid, quinate, adenine dinucleotide, acetyl-L-carnitine, dimethylamino ethanol and L-hydroxyl acid. The antioxidants include cell aging-related retinoic acid, vinpocetine as a precursor thereof, picamillon serving as an assistant in this cycle, and quinic acid and quinate, which serve as protein kinase. In addition, carbohydrate synthesis factors, such as adenine dinucleotide and acetyl-L-carnitine, which are involved in metabolism, perform an important role in cell nutrition, and such antioxidants include other additives, such as dimethylaminoethanol, which acts to stop apoptosis, L- lipoic acid and L-hydroxy acid, which are involved in cell proliferation, and coenzyme Q-IO, which is involved in amino acid production.

The vitamins include one or more selected from the group consisting of vitamins A, B, C, D and E. Vitamin A is known to be involved in primary immune reactions, cell development processes related thereto, and a series of apoptosis reactions, and then a typical example thereof is retinoic acid, which binds to a retinoic acid receptor (RAR) to regulate and activate metabolic processes associated therewith. Among them, complexes bound to RAR-alpha, RXR- alpha and RXR-beta were reported to promote or inhibit the expression of about 128 genes, which are involved in the development of T lymphocytes as major immune cells. In particular, it was reported that bcl2 family genes known as

typical anti-apoptotic proteins are definitely increased in apoptosis mechanisms (Rasooly, R. et al., J. Immunol., 175: 7916 - 7929 (2005); Spilianakis, CG. et al., Eur. J. Immunol., 35(12): 3400-4 (2005); Evans T, Exp. Hematol., 33(9): 1055-61 (2005)). This suggests that vitamin A can show the effect of inhibiting apoptosis reactions.

Vitamin B is generally known as riboflavin and performs an important role in maintaining human health. One research team in Sweden reported that treatment with this substance shows an effect on neutrophil migration, thus causing an increased primary immune response (Verdrengh, M. and Tarkowski, A., Inflamm. Res., 54(9): 390-3 (2005)). It is expected that this substance can increase initial immune responses, caused by the migration of primary immune cells. Vitamin C has important intracellular functions of promoting the synthesis of collagen and fibroblasts, and typical examples thereof include ascorbic acid. When it is used in combination with other cytokines TGF- and IFN- Y, the effect thereof is further increased (Chung, J. H. et al., J. Dermatol. Sci . , 15(3): 188-200 (1997)). Vitamin D3 has been frequently used, because it is known to influence cell development and differentiation, unlike other vitamins. Vitamin D3 mediates an important signaling system in cell growth and development processes, particularly formation processes of epidermal keratinocytes and osteogenic cells such as osteoblasts and osteoclasts. Also, it has an inhibitory effect against various cytokines, such as IL-I α, IL-6 and IL-8d, which are involved in inflammatory reactions (Alper, G. et al., Endocr. Rev., 23: 763 (2002)) The amino acids include one or more selected from the

group consisting of L-alanine, L-aspartic acid, L-histidine, L-leucine and L-glutamine . Such substances assist in oxidation nutrients and various metabolisms (Kaori Shigemitsu. et al., J. Biol. Chem. , 274: 1058 (1999)). The growth factors include one or more selected from vascular endothelial cell growth factor, hepatocyte growth factor, transforming growth factor-beta, platelet derived growth factor, keratin sulfate, basic fibroblast growth factor and cadherin (like epidermal growth factor-2). Such growth factors are proteins binding to receptors on the cell surface and act to activate the proliferation and differentiation of cells (Connolly, D. T. J. Cell. Biochem, 47(3): 219-23 (1991); Schott, R.J. and Morrow, L.A. Cardiovasc, Res., 27(7): 1155-61 (1993); Neufeld, G. et al., Prog. Growth Factor Res., 5(1): 89-97 (1994); Senger, D. R. et al., Cancer and Metastasis Reviews, 12(3-4): 303-24 (1993)). When the growth factor binds to a specific receptor, a signaling chain is activated, so that a signal is transferred from the outside of cells to the cellular nuclei. When the signaling is continued, the growth factor acts with a large amount of other messengers to finally activate new proteins. According to this method, the growth factor forms an important signaling system in the transfer, function and control of intracellular interaction (Lawrence, D.A. Eur. Cytokine Netw, 7(3): 363-74 (1996); Cox, D. A. and Maurer, T., Clin. Immunol. Immunopathol, 83(1): 25-30 (1997); Alevizopoulos, A. and Mermod, N., Bioessays. 19(7): 581-91 (1997)) .

The collagens include one or more selected from the group consisting of collagen types I to XIII and XVII and

fibronectin. The collagen is a protein most abundantly contained in the animal body and represents about 30% of the total protein of the human body. Although intracellular proteins or blood proteins remain dissolved in water, collagen is present as a structure as fiber or membrane in the human body (Bell, E. et al., Proc. Natl. Acad. Sci. U. S. A. 76: 1274-1278 (1979)). Thus, the first function of collagen is to make and support organs in the body to make the backbone of the body. The second role of collagen is to fill between the cell and cell of the body, and the collagen acts as a cell adhesive to increase the adhesion of adipose stem cells. Although the degradation and synthesis of collagen in the body are constantly repeated, the synthesis becomes slower than the degradation with the progression of aging, the balance in the body collapses, leading to a decrease in the amount of collagen in the body. For example, the dermal collagen in the skin forms a fundamental structure, thus making an entangled network structure. The network structure determines the elasticity and flexibility of the skin and is deformed when force is applied thereto. When the force is removed, the network structure is restored to the original shape. However, as aging progresses, the amount of collagen is reduced to weaken the network structure, and thus when collagen is thinned or destroyed, the elasticity or flexibility of the skin is reduced, so that the skin droops or creases into wrinkles (Leung, D. Y. et al., Science., 191: 475-477 (1976); Stopak, D., and Harris, A. K., Dev. Biol. 90: 383-398 (1982)). Thus, the cell-activating compounds will assist in the prevention of aging. As described above, the mesenchymal stem cell-

activating compounds include linoleic acid, quinic acid, quinate, coenzyme Q-IO, L-lipoic acid, L-hydroxyl acid, niacin, dimethylamino ethanol (DMAE) , α-tocopherol, ascorbyl palmitate, cartenoid, glycolic acid, dehydroepiandrosterone (DHEA), C-MED 100 (Optigene-X LLC, Sherwsbury, NJ), carboxyalkyl ester, estriol, acetyl-L-carnitine, lycopene, nicotinamide adenine dinucleotide (NADH) , picamilon, vinpocetine, retinoic acid, anti-neoplaston, procystein, vitamin A, B, C and D series, growth factors (vascular endothelial growth factor (VEGF) , insulin-like growth factor- I (IGF-I), transforming growth factor-β (TGF-β) , fibroblast growth factor (FGF), human growth hormones, etc.) and collagen.

Useful reinforcing compounds, which can cause a stronger effect than that of the activating compounds, include transcriptional factors, histone acetyl transferase, methyl-binding proteins, membrane protective factors, differentiation factors, acetylcholine and precursors, estriol, acetylcholinesterase, aminocaproic acid and the like. In addition, they may include cartenoid and carboxyalkyl ester, which influence DNA recovery, and calcium triphosphate and calcium monophosphate, which can induce the development of osteocytes, and these substances fall in the scope of the mesenchymal stem cell-activating compounds. In the present invention, a serum-free medium containing a Ham's F-12 nutrient mixture, which comprises amino acids or analogues thereof and vitamins or analogues thereof, is preferably used as a medium for mesenchymal stem cells. The Ham's F-12 nutrient mixture-containing serum-free medium according to the present invention is based on DMEM

not containing a pH indicator such as phenol red, and the Ham's F-12 nutrient mixture is added thereto at a ratio of approximately 1: 0.5-2. Herein, it is possible to add oxidation nutrients such as L-glutamine, energy metabolites such as sodium pyruvate, and carbon sources such as sodium bicarbonate.

Also, antioxidants, growth factors or analogues thereof, and collagens or analogues thereof may further be added to the serum-free medium, and among them, the growth factors or analogues thereof and collagens or analogues thereof may not be added to the initial culture medium, because they are secreted from mesenchymal stem cells, when the mesenchymal stem cells are cultured in the serum-free medium for at least one day. Then, the mesenchymal stem cells are subcultured in serum-free medium containing the cell activating compounds, and the culture medium is then collected. The culture medium was named "mesenchymal stem cell-optimizing constituent", because it contains useful growth factors and collagen, which are secreted from the mesenchymal stem cells.

The final concentrations of the cell-activating compounds in the mesenchymal stem cell-optimizing constituent according to the present invention are preferably 1-20000 uM for the antioxidant, 0.5-100 uM for vitamins or analogues thereof, 10-1000 uM for amino acids or analogues thereof, 10 pg/ml-100 ng/ml for growth factors or analogues thereof, 2.5- 100 ug/ml for collagens or analogues thereof and 0.001 uM-150 mM for inorganic substances.

The utility of the mesenchymal stem cell-activating compound-containing serum-free medium, established in the

present invention, can be seen in the Ham's F-12 nutrient mixture. In this mixture, various inorganic substances and amino acids, which help to maintain the growth and homeostasis of the cells and are involved in increasing the safety and maintenance of the cells in the late-stage culture following the initial-stage culture of the adipose-derived stem cells, vitamin nutrients that can stimulate the higher production of growth factors selected from the adipose- derived stem cells, and other factors, are mixed with each other at a given ratio. The serum-free medium containing the Ham's F-12 mixture, established in the present invention, do not show any reduction or negative effect on the culture of the adipose-derived stem cells and the production of growth factor compared to the conditions of general serum media containing animal serum. Also, some of growth factors show a higher productivity in the serum-free medium, all the components and contents of the culture medium can be determined, unlike serum media having unknown components caused by serum. This suggests that various variables, which can result from animal serum contained in serum media, can be minimized, and the effects of the present invention can be achieved at a low cost of about 50% compared to the prior art.

When the mesenchymal stem cell-optimizing constituent according to the present invention is injected or transplanted in a mixture with subcultured or non-subcultured mesenchymal stem cells, it can activate the mesenchymal stem cells to have a good effect on the surrounding environment, making it possible to rapidly correct morphological or degenerative defects in the skin, soft tissue or bone of a recipient .

The second aspect of the present invention relates to a method of treating defects of skin, soft tissue, tooth or bone, in which an injectable composition for tissue regeneration containing a culture medium, obtained by culturing mesenchymal stem cells in a serum-free medium containing cell-activating compounds, for at least one day, is injected into treatment sites together with the mesenchymal stem cells.

Also, when the serum-free medium is a medium containing 1-20000 uM antioxidant, 0.5-100 uM vitamins or analogues thereof, 10-1000 uM amino acids or analogues thereof, 10 pg/ml-100 ng/ml growth factors or analogues thereof, 2.5 ug/ml-100 ug/ml collagens or analogues thereof and 0.001 uM- 150 mM inorganic substances, it can be used as an injectable composition without undergoing a step of culturing mesenchymal stem cells.

Defects of skin, bone, soft tissue or tooth tissue can occur due to wounds, diseases, aging or the like. Thus, there is a need to develop a method of recovering and enhancing the growth of tissue, having defects therein, while avoiding scars or wounds.

The method of the present invention comprises injecting or transplanting mesenchymal stem cells and the inventive injectable composition for tissue regeneration into either sites adjacent to defects or tissue having defect sites. Defects, which can be corrected using this method, include wrinkles, chapped skins, pit scars, nontraumatic skin depressions, acne scars, lip hypoplasia, periodontal defects, soft tissue defects, bone tissues, skin ulcers and the like. Because the cells, which are injected as suggested in

the present invention, are autologous tissues, the cells have tissue compatibility with the recipient and can be subcultured in a cell culture system.

Also, the mesenchymal stem cells and the injectable composition for tissue regeneration according to the present invention can be administered together with a biodegradable cell-free matrix. The cell-free matrix playing a structural role provides a tissue-like environment to the administered stem cells even in vitro, and thus it increases the adhesion of the administered stem cells to the surrounding tissue and provides stability to the stem cells so as to allow the stem cells to function as normal cells, when the stem cells are injected or transplanted.

The biodegradable cell-free matrices are divided, according to the intended use of the cell scaffolds, into gels, beads, sponges, sheets and frame types. In the present invention, commercially available matrices, such as collagen sponges, chitosan alginate sponges, chitosan beads and hyaluronic acid sponges, may be used. [Advantageous Effects]

As described above, the mesenchymal stem cell- optimizing constituent according to the present invention contains mesenchymal stem cell-act Lvating compounds, such as various growth factors and collagen types, and thus it increases the in vivo adhesion of mesenchymal stem cells and optimizes the tissue regeneration ability of the stem cells. It is useful as a composition for tissue regeneration, which contains mesenchymal stem cells for correcting morphological or degenerative defects in the skin, soft tissue and tooth tissue of a recipient.

[Description of Drawings]

FIG. 1 is a cell culture micrograph showing the characteristics of adipose stem cells isolated from adipose tissue; FIG. 2 is a photograph showing the results of flow cytometry of adipose stem cells isolated from adipose tissue;

FIG. 3 is a photograph showing the tissue-specific multipotency of adipose stem cells isolated from adipose tissue; FIG. 4 is a micrograph of adipose stem cells anchored on a plate, which shows the effect of a mesenchymal stem cell-optimizing constituent on the increase in the viability of adipose stem cells;

FIG. 5 is a graphic diagram showing quantitative results for the number of adipose, bone marrow and umbilical core stem cells anchored through the mesenchymal stem cell- optimizing constituent;

FIG. 6 is a photograph showing the effects of the adipose stem cell-optimizing constituent on the activation of fibroblasts and the promotion of fibroblast migration;

FIG. 7 is a graphic diagram quantitatively showing the results of FIG. 6;

FIG. 8 is a photograph showing that dermis is increased, when stem cells isolated from adipose tissue are injected into a porcine animal model in a mixture with the adipose stem cell-optimizing constituent;

FIG. 9 is a photograph showing the change in dermis upon injection or transplantation of adipose-derived stem cells in a mixture with the adipose stem cell-optimizing constituent, observed with the Dermascan;

FIGS. 10 to 13 are photographs showing that the reduction of wrinkles is activated when an injectable agent for tissue regeneration with the autologous adipose stem cells is injected or transplanted into either a site adjacent to defects or a defect site in order to correct morphological defects or degenerative defects in the skin, soft tissue and bone of a recipient. [Mode for Invention]

Hereinafter, the present invention will be described in further detail with reference to examples, but the scope of the present invention is not limited to these examples.

Example 1: Isolation and identification of mesenchymal stem cells

Human mesenchymal stem cells could be obtained from bone marrow, umbilical cord blood or placenta, but in this

Example, the isolation of mesenchymal stem cells from adipose tissue, from which the stem cells could be obtained in a relatively large amount, was performed.

Human adipose tissue was obtained through liposuction under local anesthesia, and the liposuction material was treated to obtain a, adipose cell population. Generally, adipose tissue wasted in a liposuction process carried out in hospitals can be collected in an aseptic state.

Human liposuction fat obtained in a hospital (Leaders Clinic, Seoul, Korea) was washed with the same volume of phosphate buffered saline (PBS), and only adipose tissue was isolated therefrom.

The extracellular matrix of the adipose tissue was enzymatically treated with 0.075-0.125% collagenase in a 2-5% CO ₂ incubator at 30-37 ^° C for 45 minutes, and the

enzymatically treated adipose tissue was centrifuged at 1200- 150Og for 5 minutes to obtain a stromal vascular fraction containing a high density of stem cells. Then, the fraction was washed with PBS and filtered through a 70-μm nylon ell filter to remove other tissues. Then, cell fragments, including red blood cells, and mononuclear cells, were separated from each other by centrifugation with Histopaque- 1077.

The isolated mononuclear cells were cultured in a non- inducible DMEM medium (containing 5-10% fetal bovine serum (FBS), 1-2% penicillin/streptomycin and 0.17-0.34% sodium bicarbonate) in a 2-5% CO ₂ incubator at 30-37 ^° C for 24-48 hours, and then non-adhesive cells were removed therefrom, thus isolating stem cells (see FIG. 1) . For the experiment of not only adipose-derived stem cells, but also bone marrow- and umbilical cord-derived stem cells, umbilical cord blood-derived mesenchymal stem cells (SciencellTM Research Laboratories, San Diego) were cultured in DMEM medium (containing 5-10% FBS, 1-2% penicillin/streptomycin and 0.17-0.34% sodium bicarbonate) in a 2-5% CO ₂ incubator at 30-37 ^° C .

As the cells cultured in the flask in the above- described medium composition and culture environment reached a confluence of 80%, the cells were subcultured. In the subculture process, the cells, from which the medium has been removed, was washed with PBS and suspended in 0.25-0.5% trypsin-EDTA. The cell suspension was centrifuged, and the cell pellets were suspended in 3 ml of non-inducible medium. After the number and viability of the cells in the cell suspension were measured, 1 ml of the cell suspension was

cultured in each of three T75 flasks containing 9 ml of non- inducible medium.

Specifically, as the non-inducible medium, DMEM containing the Ham's F-12 nutrient mixture shown in Table 1 below was used. [Table l]

Composition and concentration of Ham's F-12 nutrient mixture

The measurement of cell number and the examination of cell viability were performed by mixing 0.1 ml of the cell suspension with the same amount of 0.2% trypan blue, counting the stained cells with a hemocytometer in a microscope field and calculating the percentage of the dyed cells in the total cells .

The subculture was carried out one time to several times, such that adipose stem cells reached a number sufficient for obtaining an adipose stem cell-optimizing constituent .

Also, in the present invention, flow cytometry for the

identification of adipose stem cells was carried out. Stem cells express a number of adhesion and surface proteins. These proteins, SH-2, SH-3 and SH-4 monoclonal antibodies recognize the cell surface epitope of human adipose stem cells. As a results of the analysis of base sequence and absorbance of the peptides, SH-3 and SH-4 are identified by CD73 (ecto-5 ' -nucleotidase) , and SH-2 is identified by CD105 (endoglin) . These cell surface markers are shared by adipose stem cells (Barry F et al., Biochem Biophys Res Commun, 289(2) : 519-24 (2001) ) .

The subcultured adipose stem cells were analyzed using a Fluorescence Activated Cell Sorter (Beckman Coulter) . Specifically, the cells were collected with 0.25% trypsin- EDTA, washed with PBS, and then collected at a concentration of 10 ⁵ cells/ml. The collected cells were allowed with react with the mesenchymal stem cell-specific markers CD73-PE and CD105-FITC antibodies and analyzed with an argon laser at a wavelength of 488 nm.

As a result, the PLA cells isolated from the adipose tissue showed a homology of 5.27% with stem cells, and after the PLA cells were subcultured twice, they showed a homology of 92.32% with stem cells for CD73-PE and a homology of 90.67% with stem cells for CD105-FITC (see FIG. 2).

Also, in order to observe the tissue-specific differentiation ability of the isolated adipose stem cells, the following test was performed.

The subcultured cells were cultured in a 6-well plate to a confluence of about 90%, and then allowed to differentiate into osteoblasts and adipose cells. For differentiation into osteoblasts, the subcultured cells were

allowed to differentiate in a differentiation medium

(containing 10 mM β-glycerophosphate, 50 ug/ml L-ascorbic acid and 100 nM dexamethason) for 10 days, while the medium was replaced with fresh medium at 3-day intervals. Then, the differentiated cells were stained and observed with alizarin red S. Meanwhile, for differentiation into adipose cells, the subcultured cells were cultured in a differentiation medium (containing 0.5 mM isobutyl-l-methyl-3-xanthine, 100 uM indomethasin, 1 uM dexamethason and 10 ug/ml insulin) for 3 days, and then in 10 ug/ml insulin for 1 day. The culture process was repeated three times, and then the cells were stained and observed with oil red O.

As a result, as shown in FIG. 3, the photographs on the right side of FIG. 3 showed that the cells differentiated into adipose cells, unlike the photographs on the left side.

Example 2: Measurement of amount of activating proteins in composition capable of optimizing adipose stem cells

The amount of various cytokines in the adipose stem cell-optimizing constituent obtained in Example 1 was measured using an ELISA kit (R&D) , and the measurement results are shown in Table 2 below. [Table 2]

Contents of adipose cell-activating proteins in adipose cell-optimizing constituent

Name of protein Concentration (pg/ml)

PDGF AA (Platelet-Derived Growth 44. 407 (±2 563 ) Factor A-chain)

PIGF (placenta growth factor) 37 873 (±1 695 )

KGF (Keratan sulfate proteoglycan 86. 28 (+20 330 )

In Table 2 above, the contents of collagen type I and fibronectin are given in ng/ml.

Example 3: Examination of increase in viability of adipose stem cells Parts (lxl0 ⁵ -lxl0 ⁷ cells) of the adipose stem cells isolated in Example 1 were stored in a serum-free cell freezing solution, and the remaining cells (lxl0 ⁵ -lxl0 ⁷ cells) were cultured in a serum-free medium containing cell activating compounds, thus obtaining an adipose stem cell- optimizing constituent, comprising various growth factors and a large amount of collagen types.

Also, the uncultured adipose stem cells (IxIO ⁵ to IxIO ⁷ cells) stored in the cell freezing solution were thawed in a water bath at 37 ^° C for 10-30 seconds and suspended in 1-10 ml of serum-free medium. The cell suspension was centrifuged, and the resulting cell pellets were cultured in a more than 100-fold volume of the adipose stem cell-optimizing constituent on a plate. For use as a control group, adipose stem cells were cultured in the same ratio of serum-free

medium as that of the test group on the same plate. The viability of each of the adipose stem cells was measured.

In the measurement, the anchorage of the cells was observed through real-time photographing (see FIG. 4), and after 7 days of cell culture, the number of cells adhered to the plate was measured using an MTT assay. As a result, it could be observed that the number of the anchored and survived cells in the test group was about 1.5-fold larger than that in the control group (see FIG. 5) . According to the same method as described above, the viability of the bone marrow- and umbilical cord blood- derived mesenchymal stem cells, obtained in Example 1, was measured. As a result, the cells were anchored in a number about 1.5-fold larger than that in the control group, in a manner similar to the case of the adipose stem cells (see FIG. 5) .

Example 4; Migration of fibroblasts by composition capable of optimizing adipose stem cells

In order to make artificial wounds, 5X10 ⁵ fibroblasts were cultured in DMEM medium (containing 5-10% fetal bovine serum, 1-10% penicillin/streptomycin and 0.17-0.34% sodium bicarbonate) in a 24-well plate in a 2-5% CO2 incubator at a humidity of 95% and a temperature of 30-37 ^° C to a confluence of 100%. Then, the cells were wounded with a plastic micropipette, and the former medium and the fibroblast debris were removed using 0.5-2 ml of PBS. To a portion under destroyed dermis, 0.5-2 ml of a mixture of the adipose stem cell-optimizing constituent, containing the adipose stem cells obtained in Example 1, with fibroblasts, was added as a test group. As a control group, only fibroblasts were added.

The cells were photographed at 24-hr intervals (see FIG. 6) to measure the migration distance of the cells, and the measurement results were expressed as numerical values (see FIG. 7) . As a result, when only the fibroblasts were added, they had indirect effects on the proliferation and migration of keratinocytes above the dermis. When the mixture of the fibroblasts with the adipose stem cells was added, the migration of the fibroblasts inserted into the dermis occurred due to the various growth factors and collagens secreted from the adipose stem cells, and the secreted growth factors and collagens had direct effects on the proliferation and migration of keratinocytes.

Through the above experiments, it could be observed that the various growth factors and collagens promoted the migration of fibroblasts and, furthermore, had effects on the proliferation and migration of keratinocytrs in the dermis.

That is, when the composition for tissue regeneration containing adipose stem cells is injected or transplanted into either a site adjacent to defects or a effect site in a recipient, the viability of the adipose stem cells will be increased (see FIGS. 4 and 5), and the substances secreted from the stem cells will bind to the various growth factors and collagens of the tissue regenerative composition to promote the migration of keratinocytes and the proliferation and migration of keratinocytes in the recipient (see FIGS. 6 and 7). Thus, the composition according to the present invention will be effectively used for tissue regeneration.

Example 5: Animal test through injection or transplantation of adipose stem cells

Male micropigs (9.8 kg) were systemically anesthetized, and then the back thereof was shaved with an electric hair remover and sectioned in the form of checks, each having an area of 2 cm x 2 cm. A control group, containing only adipose stem cells, and a test group, containing the adipose stem cell-optimizing constituent and adipose stem cells, were administered into each section through a 1-cc insulin administration syringe at a concentration of 1 x 10 ⁶ cells per section. The depth of injection targeted the layer between the dermis and the epidermis, and the experiment was performed by dermatologists.

At 4 weeks and 8 weeks after the cell injection, the cells were injected into the same sites, and in order to confirm errors, the experiment was performed on a total of two micropigs. After 12 weeks, the micropigs were systemically anesthetized, and then slaughtered, and the subcutaneous skin tissue was extracted from the sectioned areas of the slaughtered animals. After the skin at the injection sites was collected, 10~jC/m frozen tissue sections were constructed from the collected skin according to a conventional method. The constructed tissue sections were fixed in 4% paraformaldehyde for at least 24 hours, stained according to the hematoxylin-eosin staining method and the Masson' s trichrome staining method, and then observed with an optical microscope.

As a result, as shown in FIG. 8, it could be seen in the hematoxyline-eosin staining that, when the adipose stem cell-optimizing constituent was added, the thickness of the dermal layer was increased by about 10% compared to the case of the control group. Also, the accumulation of collagen in

the upper dermal layer was increased, and fibrils adhered in parallel, making the Grenz zone thick. Also, collagen fibers distributed in the dermal layer were deeply stained, while the thickness thereof was increased, indicating that the collagen fibers were newly produced and remodeled. In the Masson's trichrome staining, it could be seen that, when the adipose stem cell-optimizing constituent was added, collagen fibers and ground substances were abundantly present in the dermis compared to the case of the control group. Example 6: Skin improvement effects through injection or transplantation of autologous adipose stem cells

The use of ultrasonic imaging technology enables the effect on the skin to be qualitatively and quantitatively analyzed. This technology can measure the change in the skin thickness and assists in evaluating the effect of laser treatment or other skin treatments. Ultrasonic waves use high-frequency sound waves and can be imaged. When a high- frequency signal transmitted from a signal emitting source onto the skin is in contact with a site having different tissues, the site will emit an echo signal, and the layer of each tissue will emit an echo signal. According to the magnitude, amplitude and return time of such eco signals, two-dimensional skin images are indicated.

When such two-dimensional skin images are indicated, the structure of the skin can be understood, while the thicknesses of the epidermal, dermal and subcutaneous fat layers can be measured. In measuring the skin thickness, the distance between two points can be calculated using a computer, and the distance (thickness) can be measured with an accuracy of 0.01 mm.

The autologous adipose stem cells, isolated in Example 1, were injected into wound sites together with the stem cell-optimizing constituent in order to treat the wound sites. The skin thickness was measured with a skin thickness measuring instrument and, as a result, the thickness was increased from 2.054 mm to 2.317 mm, and the effects of the stem cell-optimizing constituent on ten persons were similar with each other (see FIG. 9) .

Example 7: Wrinkle reduction effect through injection or transplantation of autologous adipose stem cells

In order to prove the wrinkle reduction effect of autologous adult stem cells on 30-50-year-old women, 50 persons per test group were selected and subjected to the following test. Autologous stem cells together with the stem cell- optimizing constituent were injected around one eye of the subjects twice a day for 8 weeks, and a portion around the other eye was used as a comparative example. After 4 weeks and 8 weeks, the wrinkle reduction effect of the stem cells was visually observed (see FIGS. 10 to 13), and the observation results are summarized in Table 3 below. [Table 3]

[industrial Applicability]

As described above, the injectable composition for tissue regeneration according to the present invention increases the in vivo adhesion of mesenchymal stem cells and optimizes the tissue regeneration ability of the stem cells.

That is, the injectable composition for tissue regeneration, containing various growth factors and mesenchymal stem cells having tissue-specific differentiation ability, will be useful in drugs, quasi drugs and cosmetics for wrinkle reduction and treatment, wound healing and the improvement and treatment of burns, scars and skin.