WO2012121485A2 - Method for inducing in vivo migration of stem cell - Google Patents

Abstract

The present invention relates to an implantable composition for treating a damaged tissue and a method for inducing an in vivo migration of a cell for treatment to a damaged tissue region. The present invention treats the damaged tissue by inducing/promoting homing of a cell for tissue generation by implanting a biodegradable scaffold reacted with chemotactic factors (for example, IL-8 or MIP-3α) to a damaged location (for example, joint cartilage or skin). Thus, the composition of the present invention can not only be applied to the treatment of a damaged bone tissue, a joint cartilage, or a skin tissue more conveniently and efficiently compared to the conventional technology, but can also be used as a useful treatment supplement agent in cell treatment using allogeneic cell by enabling efficient utilization of cell resources for treatment, the cell resources which are high in scarcity.

Description

 【Specification】

 [Name of invention]

 In vivo migration of stem cells

[Technical field]

 The present invention relates to an implant composition for treating damaged tissue and a method for in vivo migration of therapeutic cells to a damaged tissue site.

[Background technology]

Stem cells are cells that can differentiate the organisms that make up the tissue into a variety of cells. These stem cells collectively refer to the undifferentiated cells from the embryonic, fetal and adult tissues before they are differentiated. . Stem cells are differentiated into specific cells due to differentiation stimulation (environment), and unlike differentiated cells in which cell division is stopped, they can proliferate because they can produce the same cells as themselves by cell division. i ferat ion (expansion) is characterized in that it can be differentiated into other cells by different environment or differentiation stimulus, so that it has plasticity in differentiation. Articular cartilage is an avascular tissue consisting mainly of protein polysaccharide and type 2 collagen and contains about 5 »of chondrocytes in the tissue volume [1]. When articular cartilage is damaged, the movement of chondrocytes to defects is hardly induced and damaged articular cartilage has the disadvantage that spontaneous healing does not occur [2]. Autologous chondrocyte transplantation and bone marrow are the representative biological methods for treating damaged articular cartilage. Perforation, etc. Autologous chondrocyte transplantation is a method of culturing autologous chondrocytes and transplanting them into defective areas, but the disadvantages of repeated surgery and donor limitation are disadvantages. Bone marrow perforation is performed using the bone marrow-derived mesenchymal stem cells that have been perforated to the subchondral bone of the cartilage defect and introduced into the defect along with the bone marrow. How to treat cartilage. This method is relatively simple and economical, but the result is inconsistent, and tissue regeneration is not smooth when the number of incoming mesenchymal stem cells is insufficient [3-7].

Mesenchymal stem cells are able to differentiate into bone, cartilage and adipocytes, and because they proliferate faster than cartilage cells, they are important cell sources for articular cartilage regeneration. Attention. In order for damaged articular cartilage to be regenerated by mesenchymal stem cells, a large number of mesenchymal stem cells enter the cartilage defect area, and the collected mesenchymal stem cells attach to the articular cartilage matrix to proliferate and differentiate into cartilage cells. A substrate must be created. In the natural healing process, the mesenchymal stem cells move to the joint cartilage damage site and undergo differentiation into cartilage cells. For cartilage differentiation of mesenchymal stem cells, the pooling of collected mesenchymal stem cells and the interaction between cell-cell and cell-cartilage substrates are essential [ ₈ ]

 The pericellular microenvironment plays an important role in cartilage differentiation of mesenchymal stem cells.

 Interaction of mesenchymal stem cells with collagen adjacent to cells promotes cartilage differentiation of mesenchymal stem cells [9, 10].

 Sufficient influx of bone marrow-derived mesenchymal stem cells into tissue damage is critical for the treatment of various damaged tissues, including cartilage [11-13]. Homing of the mesenchymal stem cells circulating in the blood or bone marrow into tissue damage is called homing. Homing of mesenchymal stem cells is performed by cell adhesion proteins and chemokines in several stages [14-17]. ]. Chemokines are small cytokines of about 810 kDa. To date, about 50 chemokines have been known and function to induce chemotaxis of various cells [18-20]. Chemokines induce cell chemotaxis by attaching to chemokine receptors, a type of G-protein receptor. Currently, about 19 chemokine receptors have been reported, and tissue regeneration such as skin using chemokine has been reported in mesenchymal stem cells [21-23].

Currently used therapies for joint damage (eg, drug therapy, surgery, gene therapy, etc.) are normal at the site of articular cartilage injury. There is no recovery to articular cartilage. In addition, autologous chondrocyte transplantation is performed to complement the sufficient number of chondrocytes required for transplantation or regeneration of autologous or allogeneic bone-cartilage tissue in consideration of the physical characteristics of the implant. Due to the limitation of the donor or donor, there are many difficulties in the actual implementation, the patient has a high cost of treatment, has to undergo two or more surgeries, and the disadvantages of remarkably decreasing the function of transplanted cells are not overcome.

 Recently, in order to overcome these drawbacks, artificial substitutes composed of complexes coated with cells on biodegradable synthetic polymers have been actively studied and attempted for clinical application, but this method has also not overcome the existing problems. Various treatment methods such as grafting technology of cells and biomaterials have been developed for regeneration of damaged articular cartilage tissue, but the cell and biomaterial complex derivatives are limited and insufficient for clinical application.

 Therefore, there is an urgent need for the development of therapeutic methods that use cells but maintain their properties to the maximum and overcome the existing shortcomings. Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

[Detailed Description of the Invention]

 [Technical problem]

 The present inventors endeavored to develop a method for effectively inducing cells necessary for tissue regeneration to damaged sites for the treatment of damaged tissue sites.

As a result, the present inventors have found that mesenchymal stem cells (preferably, bone marrow-derived mesenchymal stem cells) or keratinocytes are damaged at sites where the chemokines IL (inter leukin) -8 and MIPC Macrophage Inflammatory Protein (IIIa) are damaged. Promote influx with high efficiency and reduce the chemokine The present invention has been completed by discovering that biodegradable scaffolds, including the biodegradable scaffolds, can be easily and effectively treated by implanting them into a damaged area.

 Accordingly, an object of the present invention is to provide an implantable composition for treating damaged tissue.

 Another object of the present invention is to provide a method for in vivo migration of therapeutic cells to damaged tissue sites. Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

Technical Solution

 According to an aspect of the present invention, the present invention includes at least one chemotactic factor selected from the group consisting of IL (inter leukin) -8, Macrophage Inflammatory Protein (MIP) -3 α, derivatives thereof, and combinations thereof It provides an implantable composition for implanting damaged tissue (biodegradable scaffolds) comprising an active ingredient as an active ingredient. The present inventors endeavored to develop a method for effectively inducing cells necessary for tissue regeneration to damaged sites for the treatment of damaged tissue sites.

As a result, the present inventors have found that the induction of mesenchymal stem cells (preferably bone marrow-derived mesenchymal stem cells) or keratinocytes into the site where the chemokines IL (interleukin) -8 and MIPC Macrophage Inflammatory Protein) _3α are damaged. It has been found that the treatment can be carried out at high efficiency and the biodegradable scaffolds including the chemokine can be implanted in the damaged area for simple and effective treatment. We select chemokine receptors that express in human bone marrow-derived mesenchymal stem cells and whose expression increases under in vitro injury to cartilage or skin damage, and the biodegradable scaffolds containing their ligands are damaged for damaged bone tissue. Of mesenchymal stem cells by transplantation into joint or skin tissue It was confirmed that homing can be induced to effectively treat joint or skin damage.

 As used herein, the term “chemotaxis” refers to the positive (ie, direction toward the stimulus) or negative (ie, direction away from the stimulus) migration of mobile cells induced by chemical stimuli, wherein It is activated by chemotactic substances (chemokines), which are mediated by complimentary cell surface receptors (chemokine receptors).

 As used herein, the term “chemotactic factor” refers to extracellular matrix molecules and secreted proteins that diffuse from tissue to activate chemotaxis, for example, the TGF transforming growth factor family, the BMP bone morphogenetic protein family, CDMP (c art i lage ᅳ -derived morphogenetic proteins), f ibroblast growth factor (FGF) family, connect ive tissue growth factors (CTGF) family, platelet-derived growth factor (PDGF) family, VEGFCvascular endothelial growth factor (PDGF) family, extracellular Matrix molecules (eg, osteopontin, fibronectin, hyaluronic acid, heparin, thrombospondine, collagen, vitronectin, etc.) and chemokines.

 According to a preferred embodiment of the present invention, the chemotactic factor of the present invention includes chemokines.

 Chemokines are proteins (5-20 kDa) that play important physiological functions in a variety of processes, such as hematopoietic function of blood stem cells and chemotaxis of white blood cells.

 The amino acid sequence of all chemokines is similar and can be characterized by a contiguous arrangement of four cysteines. The chemokine family is divided into four subfamily: (a) CCL; (b) CXCL; (c) CX3CL; And (d) XCL (Murphy, et al., International union of pharmacology, XXII, Nomenclature of chemokine receptors, Pharmacol Rev, 52: 145 ᅳ 176 (2000)).

According to a preferred embodiment of the present invention, chemokines that can be used in the present invention are CCL20 (MIP-3 α), CCL 19, CCL21, CCL27, CCL28, CXCL8 IL-8), CXCL9, CXCL 10, CXCL11, CXCL12 ( SDF-1), CXCL 16, CXCL 13, CXCL5, CXCL6, CCL2 (MCP-1), CCL8, CCL 13, CCL25, CCL3, CCL4, CCL5, CCL7, CCL 14, CCL15, CCL 16, CCL23, CX3CL1, XCLl , XCL2, CCL1, CCL 17, CCL22, CCL11, CCL24, CCL26, CXCL1, CXCL2, CXCL3 and CXCL7, more preferably CCL20, CCL19, CCL21, CCL27, CCL28, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL16, CXCL13, CXCL5, CXCL6, CXCL6, 8 And CCL25, even more preferably CCL20, CXCL8, CCL2 and CXCL12, most preferably CCL20 and CXCL8.

 The amino acid sequences of CCL20 and CXCL8 are described in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

 According to a preferred embodiment of the present invention, the concentration of chemokine used in the present invention is 10-1,000 ng / ml per biodegradable support (1 g), more preferably 2 to 800 ng per biodegradable support (1 g). / ml, even more preferably 35 kPa 700 ng / ml per biodegradable support (1 g), most preferably 50-500 ng / ml per biodegradable support (1 g).

 As used herein, the term "biodegradable scaffolds" refers to a three-dimensional structure consisting of a biodegradable polymer containing the chemotactic factor, and is damaged to a target location (eg, damaged articular cartilage or skin tissue) through transplantation. It functions as a support for inducing the migration of cells required for tissue regeneration. Preferably, the biodegradable scaffold of the present invention is a biocompatible material, which generally forms a porous micro scaffold to provide a physical scaffold for the cells to migrate, and is provided for the influx of cells for treatment or regeneration to the implanted position. .

 The biodegradable scaffolds of the present invention are chemotactic and thereby promote homing of cells (preferably stem cells or keratinocytes) necessary for damaged tissue. Introduced cells exert regenerative efficacy at the damaged target site. According to the present invention, the implantation of the biodegradable scaffold of the present invention significantly increased the number of migrated cells (see FIGS. 2B and 4D), and also markedly increased the travel distance and travel speed (FIG. 2C). In addition, the transplant only increased cell migration but did not have any effect on cell differentiation (FIG. 3). This means that the biodegradable support of the present invention promotes only the influx of cells necessary for damaged tissue regeneration.

According to a preferred embodiment of the invention, the biodegradable support that can be used in the present invention is any size, under conditions that can be degraded in vivo, It can also be used in form or composition.

 According to a preferred embodiment of the present invention, the biodegradable support that can be used in the present invention is PLGA (poly (lactic-co-glycolic acid)), PLAC polylact ic acid (PGA), polyglycol ic acid (PGA), PCL (poly-ε) -capro lactone), poly (amino acid) (PAA), polyanhydrides, polyisoesters, derivatives thereof, copolymers, and combinations thereof; Collagen gels, polyvinyl alcohol sponges, polysaccharides (e.g. alginates), polyphosphazenes, polyacrylates and polyethylene oxide-polypropylene glycol blot copolymers, more preferably PLGA , PLA, PGA, PCL, PAA, polyanhydrides, polyisoesters and derivatives thereof, copolymers and combinations thereof, most preferably PLGA. According to a preferred embodiment of the present invention, the damaged tissue treated with the composition of the present invention is bone tissue, joint tissue or skin tissue.

 Joint or cartilage injury is rarely cured because the movement of chondrocytes rarely occurs to the site of injury. Treatments for articular cartilage damage include autologous chondrocyte transplantation and bone marrow perforation, but have the following disadvantages: (a) repeated surgery; (b) donor limitation; And (c) difficulty in introducing the stratified mesenchymal stem cells.

 Recently, in order to overcome the shortcomings of conventional articular cartilage damage treatment, mesenchymal stem cells have been noted as a cartilage damage treatment agent.

Mesenchymal stem cells (MSCs) are nonhematopoietic stromal cells that can differentiate and regenerate mesenchymal tissues such as bone, cartilage, muscle, ligaments, tendons and fat. Exists in For example, MSCs are known to be present in the bone marrow in small numbers (eg, about 1 per 10,000 monocytes). In addition, although not immortalized, MSCs may have the ability to multiply and multiply by multiples while maintaining growth and multisystem differentiation. In addition, although MSCs appear to migrate to damaged tissues, their trafficking and tissue homing are processes that are not accurately understood. For example, among chemokines and their receptors, CXCR4 is known to be important for hematopoietic stem cell and cancer cell metastasis (Zou YR, et al., Function of the chemokine receptor CXCR4 in haematopoiesi s and in cerebellar development, Nature, 393: 595-599 (1998)), the need for CXCR4 expression in MSC in vitro and MSC in vivo has not been evaluated. In addition, several reports have shown that MSCs carried to damaged tissues can induce recovery, but only limited MSCs have been differentiated into repaired tissues. Such discrepancies can be explained for the following reasons: (a) technical difficulties in the separation of MSCs in repaired tissues; (b) identifying the extent of MSC differentiation into cells involved in the healing phase; (c) the mechanism of MSC activity, likely to induce regeneration of MSC but not differentiation.

 As mentioned above, there have been numerous studies to apply MSCs clinically, but the development of effective methods for exerting in vivo therapeutic efficacy by survival of MSCs by recruiting to the site of injury is still insufficient.

 The compositions of the present invention can treat bone-related diseases by contributing to tissue regeneration by homing MSCs to common bone (bone tissue) injury sites other than joint tissue. Bone tissue to which the composition of the present invention can be implanted includes, but is not limited to, all skeletal parts of the body such as ribs, skull, iliac bone, humerus, vertebrae, pelvic neck, and shoulder blades.

 According to a preferred embodiment of the present invention, the joint site to which the composition of the present invention can be implanted includes a hip joint, a shoulder, a knee, an ankle, a wrist, an elbow, an ankle, an ulna, a vertebra, a wrist, an iliac and a jaw joint, It is not limited to this. Damaged articular cartilage areas of the present invention include hyaline cartilage (e.g., glass cartilage in the joints, nose, larynx or sternum), elastic cartilage (e.g., elastic cartilage in the ear) and fibrocartilage. For example, intervertebral discs, but is not limited thereto.

Bone disease or articular cartilage disease that can be treated by the composition of the present invention includes arthritis, osteoporosis, osteochondrosis, osteochondritis, incomplete osteoplasia, osteomyelitis, osteoprogenitors, cartilage dysplasia, including osteoarthritis and rheumatoid arthritis, Cartilage, chondroma, chondrosarcoma, intervertebral herniation, Klipel-pile syndrome, deformative osteoarthritis, cystic fibrosis, and articular cartilage disease associated with tissue damage from accidents, fractures, wounds, joint damage, autoimmune diseases, diabetes and cancer Including It is not limited to this.

 The composition of the present invention can also be usefully used for the regeneration and treatment of damaged skin tissue. According to the present invention, the chemotactic factor selected in the present invention

IL-8 and MIP-3C1 can greatly increase the influx of human keratinocytes into damaged skin tissues and promote regeneration of the skin epidermis, dermis and subcutaneous tissue.

 As used herein, the term "keratinocyte" is also referred to in the art as "skin cells", "keratin cells" or "keratin forming cells," and is present in the basal layer of the epidermis and thus, "basal cell" or "basal." It is also called "basal keratinocyte", it is meant to include all of them.

 Skin regeneration or wound healing takes place through a variety of processes, including immunoreaction, re-epithelialization, granulocyte formation, fibroplasia, and contraction of the wound tissue. The movement, proliferation and differentiation of keratinocytes play an important role in a series of processes. Keratin cells make up 95% of the epidermis, which constitutes the outermost part of the skin, and its primary function is to protect against external damage such as pathogens (bacteria, parasites, viruses, etc.), heat, ultraviolet rays and water loss. To form. Keratin cells are produced in the basal layer and directed towards the granular layer of the surface, where the resulting keratin replaces existing cellular composition and skin tissue and ultimately aids skin regeneration. Therefore, the composition of the present invention which induces the efficient movement of keratinocytes to the damaged area of skin tissue can be used as an effective composition for treating skin diseases.

 Skin diseases that can be treated by the compositions of the present invention include burns, frostbite, wounds, keloids, chemical destruction of tissues, abrasions, bone fractures, lacerations, avulsions, penetrating wounds ( penetrated wounds, cuts, contusion or bruise, skin ulcers, keratosis, bedsores, ulcers and acne.

According to a preferred embodiment of the present invention, the skin tissue to which the composition of the present invention can be implanted includes, but is not limited to, epidermis, dermis and subcutaneous tissue. According to a preferred embodiment of the present invention, the induction-induced cells in the present invention are stem cells or keratinocytes. The stem cells include autologous stem cells or non-auto logous stem cells, preferably autologous stem cells, more preferably autologous mesenchymal stem cells or autologous ectoderm stem cells, most Preferably bone marrow-derived mesenchymal stem cells or keratinocyte stem cells.

 The composition of the present invention comprises (a) a pharmaceutically effective amount of the biodegradable support described above; And (b) a pharmaceutically acceptable carrier.

 As used herein, the term “pharmaceutically effective amount” means an amount sufficient to achieve the therapeutic or regenerative activity of the damaged tissue described above.

 When the composition of the present invention is made into a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation of lactose, dextrose, sucrose, sorbetle, manny, starch, acacia rubber, calcium phosphate, alginate, Gelatin, silicate, microcrystalline cellulose, polyvinylpyridone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil Including, but not limited to. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

 The pharmaceutical compositions of the present invention can be administered directly to the injured site, for example by subcutaneous injection, intramuscular injection, transdermal administration, intraarticular injection, or the like.

Suitable dosages of the pharmaceutical compositions of the present invention may be prescribed in various ways depending on factors such as formulation method, mode of administration, age of patient, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and reaction response. Can be. The pharmaceutical composition of the present invention can be easily carried out by those skilled in the art according to the present invention, Formulated with pharmaceutically acceptable carriers and / or excipients may be prepared in unit dose form or may be prepared by incorporation into a multi-dose container. The formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or may be in the form of axes, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers. According to the invention, the compositions of the invention can be implanted in living mammals, more preferably in damaged joint sites (eg articular cartilage) or skin tissue.

 According to a preferred embodiment of the present invention, the mammal is not particularly limited, and preferably includes humans, mice, rats, rabbits, monkeys, pigs, horses, cows, sheep, antelopes, dogs and cats. Preferably humans and mice are included. According to another aspect of the present invention, the present invention provides a method for in vivo migration of therapeutic cells to a damaged tissue site comprising the following steps:

 (a) Biodegradable in a solution comprising at least one chemoactic factor selected from the group consisting of IL (inter leukin) -8, Macrophage Inflammatory Protein (MIP) 一 3a, derivatives thereof, and combinations thereof. Dipping the biodegradable scaffolds; And

 (b) implanting the biodegradable scaffold into the damaged joint site. Since the chemotactic factor and biodegradable support used in the method of the present invention have been described above, the description thereof is omitted in order to avoid excessive duplication. According to another aspect of the invention, the invention provides a method of treating damaged tissue comprising the following steps:

(a) biodegradable support in a solution comprising one or more chemoactic factors selected from the group consisting of IL (inter leukin) -8, MIPC Macrophage Inflammatory Protein (3 α), derivatives thereof, and combinations thereof dipping (biodegradable scaffolds); And (b) implanting the biodegradable support into a damaged tissue site. Since the chemotactic factor and biodegradable support used in the method of the present invention have already been described above, the description thereof is omitted to avoid excessive duplication.

Advantageous Effects

 The features and advantages of the present invention are summarized as follows:

 (a) The present invention provides an implant composition for treating damaged tissue and a method for in vivo migration of therapeutic cells to a damaged tissue site.

 (b) Tissue regeneration by implanting a biodegradable scaffold with the chemotactic factor (e.g. IL-8 or MIP-3a) of the present invention at an injured location (e.g. bone tissue, articular cartilage or skin). Damaged tissues can be treated by inducing / promoting homing of cells.

 (c) Therefore, the composition of the present invention can be applied to the treatment of damaged bone tissue, articular cartilage or skin tissue more simply and efficiently compared to the prior art itself, as well as in the treatment of cells using taga cells. It can be used as a useful therapeutic aid by making efficient use of high therapeutic plastic resources.

[Brief Description of Drawings]

1 shows the results of altered gene expression of chemokine receptors in human bone marrow-derived mesenchymal stem cells stimulated by proinflammatory cytokines. A: RT-PCR; Β: reverse dot-blot. Control, normal mesenchymal stem cell group; IL-4h, IL-Ιβ-stimulated group for 4 hours; IL-24h, IL-Ιβ-stimulated group for 24 hours; IL-48h, IL-Ιβ-stimulated group for 48 hours; TNF-4h, TNF-α-stimulated group for 4 hours; TNF-24h, TNF-a-stimulated group for 24 hours; And TNF-48h, TNF-a—stimulated group for 48 hours. 2 shows the effect of chemokines on cell proliferation and migration. It is a result. Figure 2a is a result showing the cell proliferation (cell proliferation), Figure 2b is a result showing the wound healing effect, Figure 2c is a result showing the migratory capacity (migratory capacity). 3 is a result showing the effect of chemokines on cell differentiation. Degree

The upper panel of 3 _a shows the results of the Masson trichrome staining, and the lower panel shows the expression of cartilage marker genes. 3B shows the results of Von kossa staining. Abbreviation: NC, negative control; And PC, positive control. 4 shows the results of in vitro chemotaxis of bMSCs and keratinocytes, respectively. 5 shows the in vivo chemotaxis of bMSCs. The upper panel shows the results of animal experiments (W, Note) and the lower panel shows the PLGA scaffold transplantation diagram containing chemokines. Figure 6 is a result showing the visual observation (A) and pathological findings (B) after in vivo chemotactic observation of bMSCs.

[Form for implementation of invention]

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples according to the gist of the present invention to those skilled in the art to which the present invention pertains. Will be self explanatory. Example Targets and Methods Cell culture

Human bone marrow-derived mesenchymal stem cells undergo IRB deliberation and aspirate bone marrow in the posterior iliac crest after centrifugation to precipitate pellets in the bone marrow. After removing the solution, 10% (v / v) fetal bovine serum (FBS, fetal bovine serum; GIBCO BRL, Grand) in DMEM-LG (Dulbecco's modified eagle's medium-low glucose; GIBCO-BRL, Grand Island, NY, USA) Island, NY, USA), 100 units / ml penicillin G (GIBCO) and 0.1 mg / ml streptomycin (GIBCO) were added to wash the basal medium and T-75 cm ² cell culture flask (Falcon, Germany) Aliquots were incubated for 4 days. Subsequently, the cells not attached to the bottom of the flask were removed, and the mesenchymal stem cells attached to the bottom were cultured at intervals of three days, and then passaged to about 9OT in the flask. The isolated bone marrow-derived mesenchymal stem cells were grown to cells up to 2 or 3 passages, and all experiments used cells of 2 or 3 passages. In vitro cartilage damage condition formation

 In vitro cartilage damage conditions were established by treatment of the proinflammatory cytokine IL-1 | 3 (R & D Systems Inc., Minneapolis, MN, USA) or TNF-α (R & D Systems Inc.). After mesenchymal stem cells were cultured in DMEM-LG medium without fetal bovine serum for 12 hours, IL-1β or TNF-α was added to the medium at a concentration of 10 ng / ml for 4, 24, 48 hours. The cells were cultured and used for the experiment. Reverse Transcritase-Polymerase Chain Reaction

To select chemokine receptors expressed in normal mesenchymal enjoyment cells, total RNA of mesenchymal stem cells was extracted using RNeasy kit (Qiagen, Valencia, CA, USA), and chemokine receptors were subjected to reverse transcriptase-polymerase chain reaction. Gene expression was analyzed. The chemokine receptors analyzed were CCR1-10, CXCR1-7, CX3CR, and XCR1 in total (Table 1). Amplified Chemokine DNA of the receptor was confirmed by the DNA sequence analysis method using 3100 Genetic Analyser (Applied Biosystemslnc., Foster City, CA, USA) there is no overlapping sequence with each other. Table 1

 Primer sequence used for RT-PCR.

CXCR2 520 정방향 GTTCCTCCCTTCTCTTCACA

CXCR2 520 Forward GTTCCTCCCTTCTCTTCACA

 Reverse CTGAGACAGAGTCTCACTGT

 CXCR3 528 Forward CCAGACTTCATCTTCCTGTC

 Reverse AGGTCTCAGACCAGGATGAA

 CXCR4 517 Forward ACACAGTCAACCTCTACAGC

 Reverse CTCGGTGATGGAAATCCACT

 CXCR5 490 Forward GAAACGCATGCCTGGTTCAC

 Reverse ACCTAGAACGTGGTGAGAGA

 CXCR6 518 Forward TTCTTCTTGCCACTGCTCAC

 Reverse AAACAAAGCCTGCCTCACCA

 CXCR7 516 Forward AACTTCTCGGACATCAGCTG

 Reverse GGTCTCAGGTAGTAGGTGT

 CX3CR 539 Forward GATCTGCTGTTTGTAGCCAC

 Reverse CATAGAGCTTAAGCGTCTCC

 XCR 550 Forward GAGTCCCTCACCMCATCTT

 Reverse ACAGGGTGAAGTTGTAGGGA

 GAPDH 444 Forward ATCACTGCCACCCAGAAGAC

 Reverse ATGAGTCCACCACCCTGTT Reverse Dot -Blot Hybridization

In order to select chemokine receptors with increased expression by IL-Ιβ or TNF-α, DNAs of 19 chemokine receptors were synthesized using reverse transcriptase-polymerase chain reaction and used as sample DNA for 4, 24 or 48 hours. CDNAs of mesenchymal enjoyment cells stimulated with IL-Ιβ or TNF-α were synthesized using Omniscript RT Kit (Qiagen) and used as probe cDNAs. The DNA of the chemokine receptor was immobilized on the nylon membrane (Amersham, GE healthcare, UK) and irradiated with ultraviolet (1200X100 jules) to fix the cDNA of mesenchymal stem cells stimulated with proinflammatory cytokines. prime labeling system (GE Healthcare UK Limited.Amersham Place, Little Chalfont, Buckinghamshire, UK) and ³² P-dCTP (NEN Life Sciences, Boston, USA) were used for labeling. Membrane-immobilized chemokine receptor DNA and labeled mesenchymal stem cells were shaken at 42 ^° C for 15 hours. After washing the membrane, the signal was detected by a fluorescence image analyzer (FLA-7000, Fujifilm, Japan) and quantified using a density analyzer (TINA pixel analyzer version 2.10, Ray test I sot open megerate GmbH, Straubenhardt, Germany). Quantified results were verified for statistical significance using paired t-tests (P-value <0.05). Effect on Cell Proliferation

 In order to confirm the effect on cell proliferation, 50-500 ng / ml of chemokine was treated in MSC and cultured for 1 to 4 days. After chemokine stimulation, the cell proliferation rate was measured by ΜΊΤ method on each day. Effect on Cell Differentiation

 10 ng / ml TGF-P3 (R & D system Inc, Minneapolis, MN, USA) and 30 // g / ml ascorbic acid (Sigma Co., St. Louis, M0, USA) to induce differentiation into chondrocytes ) Induced cartilage differentiation for 14 days in basal culture [DMEM—LG (GIBCO) containing 10% fetal bovine serum (GIBC0), 1% antibiotic (ant ibioti cant Mycotic) solution (GIBCO), TGF Cartilage differentiation was examined in comparison with mesenchymal stem cells not treated with -P3. Osteoblast differentiation culture [10 mM β -glycerophosphate (Sigma), 100 μΜ dexamethasone (Si ma), 50 μ g / ml ascorbic ac i d-2-phosphate (S i gma) to induce differentiation into osteoblasts 7} added basal culture] was compared with mesenchymal stem cells cultured in basal culture after induction of differentiation for 14 days. Wound Healing and Real-time Cell Migration Rates

MSC was incubated using a silicon culture insert (IBIDI, Germany) to form wounds, and 500 ng / mL chemokine stimulation was applied to the cells to count the number of cells that shifted the extent of wound healing. Tracking the cells in real time to measure the distance and speed. Cytochemistry

In vitro cell chemotaxis was assessed by culturing cells using 8 / i-hole sized transwell inserts (Falcon, Germany) and counting cells that migrated through the micropores toward chemokines. In vitro cell chemotaxis was performed by PLGA scaffolds containing chemokines (IL-8, MIP-3a) implanted subcutaneously into the tail blood vessels by injecting fluorescently labeled 1.5X10 ⁶ MSCs (Seohyun Kim, Korea Research Institute of Science and Technology) Movement toward the doctor was observed with a real-time imaging device (Optix, ART, USA).

Chemokine Receptor Expressed in Mesenchymal Stem Cells

 The primer sequences for human chemokine receptors prepared for reverse transcription-polymerase chain reaction are as follows (Table 1). DNA sequencing of the amplified by the primers of Table 1 was confirmed by DNA sequencing does not overlap with the sequence of the other chemokine receptor (not shown). Expression of 19 chemokine receptors in human bone marrow-derived normal mesenchymal stem cells was analyzed by RT—PCR (FIG. 1). Analysis of chemokine receptor expression in mesenchymal stem cells from three or more donors revealed that CCR2, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR5 and CXCR7 are common in three or more mesenchymal stem cells. It was expressed as. Increased expression in bone marrow-derived mesenchymal stem cells during articular cartilage injury

Chemokine receptor

When articular cartilage is damaged, proinflammatory cytokines (IL-Ιβ or TNF-α) are secreted and the secretion of various chemotactic factors is increased by stimulating chondrocytes [23]. Therefore, the mesenchymal obtained from three donors using inverse spot-taking matching method to determine which receptors are increased among chemokine receptors expressed in mesenchymal stem cells in articular cartilage injury model. Increased chemokine receptors were stimulated by stimulation of IL-Ιβ or TNF-α at 4, 24 and 48 hours in stem cells. In this paper, only one representative donor was shown, but repetitive experiments confirmed that the expression patterns of chemokine receptors were similar in mesenchymal stem cells derived from three donors (FIG. 2). The results of the inverse spot-matching method were quantified using mesenchymal stem cells of each donor using densitometry (TINA Pixel Analyzer version 2.10 (Raytest Isotopenmegerate GmbH, Straubenhardt, Germany). change> 2) As a result of arranging the increased receptors according to the stimulation time, the chemokine receptors, which are more than doubled by proinflammatory cytokines, are common in mesenchymal stem cells of each donor and mesenchymal stem cells of three donors. The expression of CCR2, CCR4, CCR6, CXCR1, CXCR2, and CXCR3 were increased in mesenchymal stem cells of three donors.

 Taken together, the results of CCR2, CCR4, CCR6 and CXCR1 round receptors are significantly expressed in normal mesenchymal stem cells, and their expression is significantly increased in mesenchymal stem cells of three donors by pro-inflammatory cytokines. (More than 2 times). Effect on cell migration

Wound healing techniques were performed to evaluate the effect of selected candidates on cell migration. Previously reported, SDF1 is known to induce chemotaxis of mesenchymal stem cells through CXCR4 and CXCR7 [17]. Therefore, we compared SDF1 with candidate factors selected in this study. First, the effect on cell proliferation was examined to confirm that the wound healing effect was not cell proliferation. There was no effect on cytotoxicity and cell proliferation at concentrations of 50, 100 or 500 ng / ml (FIG. 2A). . Among the four candidates, IL-8 and ΜΙΡ-3α showed the fastest wound healing results, and among the various concentrations, the best result was confirmed at the concentration of 500 ng / ml (FIG. 2B). Next, moving cells were tracked in real time to measure the moving distance and speed of the cells. MIP-3a is the fastest Many distances were identified as moving cells (Figure 2c). Effect on Cell Differentiation

 As a result of examining the influence of candidate factors during bone and cartilage differentiation induction of mesenchymal stem cells for 14 days, it was confirmed that there was no effect on differentiation (FIG. 3). Therefore, the chemokines found in this study promote the influx of autologous mesenchymal stem cells necessary for damaged articular cartilage regeneration. Chemotaxis Assessment

 The chemotaxis of mesenchymal stem cells of candidates selected in vitro and in vivo was evaluated. First, cytochemistry towards TNFa was observed in vitro using a transwell insert (FIG. 4A). Meanwhile, among the four candidates selected, IL-8 and MIP-3a induced cytochemistry most frequently in vitro (FIG. 4B), and chemotaxis increased more significantly when these two materials were used in combination. It was confirmed (FIG. 4C). In addition, IL-8 and MIP-3a confirmed that the keratin cells essential for skin tissue regeneration can be introduced experimentally. As a result, IL-8 and MIP-3a induced cytochemistry of keratinocytes (FIG. 4D). ). Based on these results, we tried to confirm the cytochemistry induced by IL-8 and MIP-3 a in vivo. The PLGA scaffold was soaked in PBS or IL-8 and MIP_3a for 24 hours, and then subcutaneously implanted in nude mice, and then identified using fluorescence labeled on the transfer ol cells of mesenchymal stem cells injected through the tail blood vessel. . Injected mesenchymal stem cells at about 2 weeks were identified in IL-8 and MIP-3a and cell migration increased by 6 weeks (FIG. 5). Visual observation and pathological findings

In order to examine the influx of inflammatory cells by the chemotactic factor transplanted with the support and to confirm the inflammatory response, the transplanted support was recovered at 6 weeks, and visual observation and H & E staining were performed. As a result of visual observation, scaffolds of IL8 and ΜΙΡ3α groups are more fused to surrounding tissues than the PBS group. There was a lot of angiogenesis (FIG. 6A). As a result of H & E staining, the PBS group showed partial angiogenesis only at the edge of the implanted support, followed the pores and permeated the cells. The mesenchymal stem cells of the injected human cells penetrated to the center of the support and formed extracellular matrix. In PBS, IL-8, ΜΙΡ-3α group, some inflammatory cells including macrophages were observed around the vessels, but there was no difference between the groups and all three groups could not confirm the inflammatory reaction (FIG. 6b).

 The specific parts of the present invention have been described in detail, and for those skilled in the art, these specific technologies are merely preferred embodiments, and it is obvious that the scope of the present invention is not limited thereto. It is intended that the substantial scope of the invention be defined by the claims appended hereto and their equivalents.

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SDF-1 and its receptor CXCR4 in human stem eel 1 homing and repopulat ion of trans lanted immune-deficient N0D / SCID and N0D / SCID / B2m (null) mice. Leukemia 2002; 16: 1992-2003.

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Claims

[Patent Claims] [Claim 11  Biodegradable scaffolds comprising one or more chemotactic factors selected from the group consisting of IL (interleukin) -8, Macrophage Inflammatory Protein (MIP) 一 3α, derivatives thereof, and combinations thereof as an active ingredient Implantable composit ion for treating a damaged tissue. [Claim 2]  The composition of claim 1, wherein the chemotactic factors are IL (inter leukin) -8 and MIP (Macrophage Inflammatory Protein) -3a. [Claim 3]  According to claim 1, wherein the biodegradable support is PLGA (poly (lacdc-co- glycolic acid)), PLA (polylact ic acid), PGA (polyglycol ic acid), PCLC poly-ε-caprolactone (PAG), poly ( amino acid)), polyanhydrides, polyisoesters, collagen gels, hydrogels, polyvinyl alcohol sponges, gelatin, polysaccharides, polyphosphazenes, polyacrylates, polyethylene oxide-polypropylene glycol blot copolymers, and Compositions selected from the group consisting of these mixtures. [Claim 4]  The composition of claim 1, wherein the damaged tissue is bone tissue, joint tissue, or skin tissue. [Claim 5] The composition of claim 4, wherein the joint tissue is a hip, shoulder, knee, ankle, wrist, elbow, ankle, ulna, vertebra, carpal, iliac or jaw joint. [Claim 6]  6. The composition of claim 5 wherein the skin tissue is epidermis, dermis or subcutaneous tissue. [Claim 7]  The composition of claim 1, wherein the composition enhances cell migration of stem cells or keratinocytes to damaged tissue sites. [Claim 8]  8. The composition of claim 7, wherein the stem cells are autologous stem cells. [Claim 9]  8. The composition of claim 7, wherein the stem cells are mesenchymal stem cells or ectoderm stem cells. [Claim 10]  The composition of claim 1, wherein the composition can be applied to a mammal. [Claim 111]  A method of in vivo migration of therapeutic cells to a damaged tissue site comprising the following steps:  (a) 1b (inter leukin) 8, M IP (Macrophage Inflammatory Protein) -3a, a solution comprising one or more chemoactic factors selected from the group consisting of derivatives and combinations thereof Dipping biodegradable scaffolds; And (b) implanting the biodegradable support into a damaged tissue site. [Claim 12]  The method of claim 11, wherein the chemotactic factors are inter leukin (IL-8) and macrophage inflammatory protein (MIP) -3 α. [Claim 13]  The biodegradable support according to claim 11, wherein the biodegradable support is polylactic acid (PLGA), polylactic acid (PLA), polyglycol ic acid (PGA), poly-ε-capro lac tone (PCL), Poly (amino acid), polyanhydride, polyoester, collagen gel, hydrogel, polyvinyl alcohol sponge, gelatin, polysaccharide, polyphosphazene, polyacrylate, polyethylene oxide-polypropylene glycol And blot copolymers and combinations thereof. [Claim 14]  The method of claim 11, wherein the damaged tissue is bone tissue, joint tissue, or skin tissue. [Claim 15]  15. The method of claim 14, wherein the joint tissue is hip, shoulder, knee, ankle, wrist, elbow, ankle, ulna, vertebra, carpal, iliac or jaw joint. [Claim 16]  15. The method of claim 14, wherein said skin tissue is epidermis, dermis or subcutaneous tissue. [Claim 17] 12. The method of claim 11, wherein said therapeutic cell is a stem cell or keratinocyte. [Claim 18]  18. The method of claim 17, wherein the stem cells are autologous stem cells. [Claim 19]  18. The method of claim 17, wherein the stem cells are mesenchymal stem cells or ectoderm stem cells. [Claim 20]  12. The method of claim 11, wherein the method is applicable to mammals. [Claim 21]  Method of treating damaged tissue comprising the following steps:  (a) Biodegradable in a solution comprising at least one chemoactic factor selected from the group consisting of IL (interleukin) -8, Macrophage Inflammatory Protein (MIP) -3, derivatives thereof, and combinations thereof. Dipping the biodegradable scaffolds; And  (b) implanting the biodegradable support into a damaged tissue site. [Claim 22]  22. The method of claim 21, wherein the chemotactic factors are inter leukin (IL-8) -8 and macrophage inflammatory protein (MIP) -3a. [Claim 23] The method of claim 21, wherein the biodegradable support is PLGA (poly (lactic-cx- glycolic acid)), PLA (polylact ic acid), PGA (polyglycol ic acid), PCL (poly-ε-caprolactone), PAA ( poly (amino acid)), polyanhydrides, polyisoesters, collagen gels, hydrogels, polyvinyl alcohol sponges, gelatin, polysaccharides, polyphosphazenes, polyacrylates, polyethylene oxide-polypropylene glycol blots Consisting of coalescing compounds and combinations thereof Characterized in that it is selected from the group [Claim 24]  The method of claim 21, wherein the damaged tissue is bone tissue, joint tissue, or skin tissue. [Claim 25]  25. The method of claim 24, wherein the joint tissue is a hip, shoulder, knee, ankle, wrist, elbow, ankle, ulna, vertebra, carpal, iliac or jaw joint. [Claim 26]  The method of claim 24, wherein the skin tissue is epidermis, dermis or subcutaneous tissue. [Claim 27]  The method of claim 21, wherein the therapeutic cell is a stem cell or keratinocyte. [Claim 28]  28. The method of claim 27, wherein said stem cells are autologous stem cells. [Claim 29]  28. The method of claim 27, wherein said stem cells are mesenchymal stem cells or ectoderm stem cells. [Claim 30]  The method of claim 21, wherein the method is applicable to mammals.