JP5843189B2 - Cartilage tissue regeneration composition containing SDF-1 inhibitor - Google Patents

Description

  The present invention relates to a composition (composition for cartilage tissue regeneration) used for regeneration / reconstruction of cartilage tissue. Specifically, the present invention relates to a composition containing an SDF-1 (stromal cell-derived factor-1) inhibitor and its use.

  Cell transplantation has been performed for the purpose of repairing and regenerating various tissues or treating diseases. Cell transplantation involves the administration of cells that make up the tissue itself or cells that assist in the construction of the tissue to the living body, so it can be expected to have a direct therapeutic effect. On the other hand, invasion associated with surgery, transplantation safety (infection and carcinogenesis) ), There are various problems such as the stability of cell quality and the time and cost required for cell culture.

  Cartilage tissue is one of the tissues expected to be regenerated by regenerative medicine. So far, regeneration methods targeting cartilage tissue have been proposed, such as a method using cell-derived components (see, for example, Patent Document 1) and a method using bone marrow cells (see, for example, Patent Document 2). Yes. However, since there are many problems to be overcome in the conventional regeneration method, the creation of a technology that has a high regeneration effect and can be applied clinically is eagerly desired.

Special table 2010-504093 gazette JP 2004-49626 A

  Under the above background, an object of the present invention is to provide a regenerative medical system targeting cartilage tissue that can be expected to have a high regenerative effect.

  In view of the above problems, the inventors of the present application have advanced research aiming at development of a new tissue regeneration therapy for controlling an in vivo stem cell accumulation system, and have focused on bone extension. Bone extension is a surgical operation that regenerates large tissues by making the best use of self-renewal ability. Accumulation and differentiation of in vivo stem cells are thought to play a major role, but the actual status and mechanism remain largely unknown. The inventors of the present application clarified the accumulation dynamics of various stem cells in the extended healing process and examined the relationship with tissue regeneration. The role of chemokine SDF-1, which is known to function in cell accumulation, in stem cell accumulation and healing processes of bone elongation was examined. SDF-1 is one of the chemokine ligands and has been reported to contribute to accumulation of vascular endothelial progenitor cells at the ischemic site, differentiation from hematopoietic stem cells to vascular endothelial progenitor cells, construction of vascular networks, and the like. However, there are many reports of only cell migration experiments in vitro, and many in vivo functions are unknown. In particular, its function in the tissue regeneration process is unclear. Therefore, we investigated the role of SDF-1 in bone elongation and its potential for therapeutic application. The specific examination method was as follows. Specifically, an 8-week-old female ICR mouse was used to produce a tibial bone extension model, tissue was collected over time, and the accumulation of stem cells in vivo was analyzed. In addition, the mouse bone marrow mononuclear cell fraction cells were labeled with a near-infrared fluorescent dye and transplanted into the tibia mesial bone marrow at the time of bone extension surgery. On the other hand, fluorescence signals were observed before and at the end of extension using an in vivo imager. In addition, the expression of SDF-1 and its receptor was analyzed. Furthermore, the effect of SDF-1 inhibition was examined from various angles. We examined how SDF-1 inhibition affects bone differentiation in vitro. In addition, a bone extension model (H-DO model) that extends the bone at twice the normal speed was prepared, SDF-1 protein was administered to the bone extension site of the model, and the tissue regeneration promoting effect was verified. Finally, changes in blood flow at the extended site were analyzed using a two-dimensional laser blood flow meter.

  As a result of the examination, it was revealed that the number of Sca1-positive cells increased about 4 times in the middle stage of bone elongation compared to bone marrow without bone elongation. In addition, it was revealed that the majority of the Sca1-positive cells gathered were fractions of vascular endothelial progenitor cells and mesenchymal stem cells. In vivo imager analysis revealed that bone marrow mononuclear cells labeled with fluorescent dyes accumulated in the extension. On the other hand, it was found that the expression of SDF-1 and its receptors (CXCR4, CXCR7) increased during the bone extension period. Experiments with SDF-1 inhibitors showed that inhibition of SDF-1 suppressed cell migration activity and that inhibition of SDF-1 had little effect on the bone differentiation ability of bone marrow cells. . In addition, the inhibition of SDF-1 markedly suppressed callus formation and the extension was filled with chondrocytes (ie, cartilage formation). Furthermore, it was suggested that SDF-1 is not necessary for accumulation of mesenchymal stem cells and hematopoietic stem cells, but functions as an accumulation factor specific to vascular endothelial progenitor cells. In the analysis using the H-DO model, SDF-1 overexpression confirmed a significant effect of callus formation and a large number of mature blood vessels were observed. In addition, an increase in blood flow was observed due to overexpression of SDF-1.

  Taken together, the fact that SDF-1 functions as a specific accumulation factor of vascular endothelial progenitor cells, as well as regeneration of cartilage if an environment in which mesenchymal stem cells are supplied while inhibiting SDF-1 is formed. You can see that The latter fact is that SDF-1 inhibitor is administered and cells that are expected to be directly involved in cartilage tissue regeneration (such as mesenchymal stem cells) are supplied to the target site for cartilage tissue regeneration. It is extremely effective as a strategy.

As described above, as a result of intensive studies by the present inventors, an effective strategy for efficient regeneration of cartilage tissue has been found. The present invention described below is mainly based on the above results or knowledge.
[1] A composition for cartilage tissue regeneration containing an SDF-1 inhibitor.
[2] The composition for cartilage tissue regeneration according to [1], comprising one or more cells selected from the group consisting of mesenchymal stem cells, chondroblasts, chondrocytes and dental pulp stem cells .
[3] The SDF-1 inhibitor and one or more cells selected from the group consisting of mesenchymal stem cells, chondroblasts, chondrocytes, and dental pulp stem cells, Composition for cartilage regeneration
[4] A first component containing an SDF-1 inhibitor, and a second component containing one or more cells selected from the group consisting of mesenchymal stem cells, chondroblasts, chondrocytes, and dental pulp stem cells The composition for cartilage tissue regeneration according to [2], which is a kit comprising:
[5] It contains an SDF-1 inhibitor, and at the time of administration, one or more cells selected from the group consisting of mesenchymal stem cells, chondroblasts, chondrocytes and dental pulp stem cells are also administered. The composition for bone tissue regeneration according to [2].
[6] The method according to any one of [1] to [5], wherein the SDF-1 inhibitor exhibits inhibitory activity against both SDF-1 and CXCR4 binding and SDF-1 and CXCR7 binding. Composition for cartilage regeneration
[7] The composition for cartilage tissue regeneration according to any one of [1] to [5], wherein the SDF-1 inhibitor is AMD3100 or an anti-SDF-1 antibody.
[8] The SDF-1 inhibitor is locally or systemically administered to the target site, and one or more cells selected from the group consisting of mesenchymal stem cells, chondroblasts, chondrocytes, and dental pulp stem cells are locally applied to the target site. A method for regenerating cartilage tissue, characterized by administration or systemic administration.

Overview of mouse bone extension (Distraction Osteogenesis: DO) model. The treatment method (top), extension schedule (bottom left), and X-ray images (bottom right) of samples at the end of extension and 14 days after extension are shown. Accumulation of Sca1 (Stem Cell common antigen) positive cells in the extended gap. The state of the extended gap is shown schematically (top). Moreover, the result of the immunostaining with respect to the tissue sample of DO model is shown (below). A type of bone marrow stem cell that accumulates in the extended gap. Cells that accumulated in the extended gap were identified using a marker. The result of fluorescent staining with each marker (lower left) and a graph of the ratio of each cell (lower right) are shown. EPCs: vascular endothelial progenitor cells, HSCs: hematopoietic stem cells, MSCs: mesenchymal stem cells. Bone marrow cell accumulation by bone elongation. Using an in vivo imager, cell movement in the bone marrow was analyzed over time. An analysis method (left), an image (upper right), and a temporal change in fluorescence intensity (lower right) are shown. Gene expression of SDF-1 (ligand) and CXCR4 / CXCR7 (receptor). The gene expression of each molecule in bone extension was examined by real-time PCR (right). For comparison, the expression of VEGFR1, VEGFR2, PDGFRa and PDGFRb was also analyzed (left). Cell migration inhibitory effect by SDF-1 inhibitor. The effect of AMD3100, an SDF-1 inhibitor, was examined by an in vitro transwell migration assay (top). In addition, the effect on bone differentiation ability was confirmed (bottom). Callus formation inhibition by suppressing the function of SDF-1. Tissue staining images were compared between the control group (C-DO), the SDF-1 inhibitor AMD3100 group (+ AMD3100), and the anti-SDF-1 antibody group (+ anti-SDF-1 mAb) . HE: Hematoxylin and eosin staining SDF-1 function inhibition suppresses the accumulation of vascular endothelial progenitor cells. The fluorescent staining images (CD31, SDF-1) of the control group and the group administered with AMD3100 were compared (left). On the right is a comparison of the number of CD31 positive cells and the number of SDF-1 positive cells. Behavioral analysis of mesenchymal stem cells and hematopoietic stem cells. The fluorescent staining images (CD45, Sca1) of the control group and the group administered with AMD3100 were compared (top). The lower left is a comparison of Sca1-positive areas. The lower right is a breakdown of the constituent cells. High-speed DO model (H-DO) extension schedule (top) and extension image (bottom). The H-DO model without administration of SDF-1 (H-DO + vehicle) and the H-DO model with administration of SDF-1 (H-DO + SDF-1) were compared. SDF-1 promotes angiogenesis and restores blood flow. Fluorescence staining image of the extended metaphase gap between the H-DO model not administered SDF-1 (H-DO + vehicle) and the H-DO model administered SDF-1 (H-DO + SDF-1) (CD31) Were compared (top). Using a two-dimensional laser blood flow meter, control (C-DO model), H-DO model without SDF-1 administration (H-DO + vehicle), and H-DO model with SDF-1 administration (H-DO + SDF) -1) blood flow was compared (bottom). Angiogenesis promoting effect of SDF-1. Fluorescent staining images (CD31, αSMA) were compared between the H-DO model that did not receive SDF-1 (H-DO + vehicle) and the H-DO model that received SDF-1 (H-DO + SDF-1) . αSMA is a marker for pericytes and CD31 is a marker for endothelial cells (lower left).

  The first aspect of the present invention relates to a composition for regenerating cartilage tissue. In this specification, the term “cartilage tissue” is used in a broad sense, and includes various parts (for example, articular cartilage, costal cartilage, thyroid cartilage, tracheal cartilage, joint meniscus, joint disc, intervertebral disc, pubic connection, epiglottis Cartilage tissues such as cartilage, ear canal cartilage, and auricular cartilage. In the present specification, the “composition for cartilage tissue regeneration” refers to a composition used for regeneration (reconstruction) of cartilage tissue. The composition for regenerating cartilage tissue of the present invention exhibits cartilage tissue regenerating ability at the local site (target site) to which it is applied, and promotes repair and reconstruction of cartilage tissue. The composition of the present invention contains an SDF-1 inhibitor as an essential component. SDF-1 is one of chemokine ligands and is also called CXCL-12 or PBSF. SDF-1 specifically binds to CXCR4 and CXCR7 and exerts its physiological activity. The presence of multiple isoforms (SDF-1α, SDF-1β, SDF-1γ, SDF-1δ, SDF-1ε, etc.) has been confirmed in SDF-1.

  As an SDF-1 inhibitor, a substance that inhibits the binding between SDF-1 and a receptor (CRCX4 or CRCX7) by binding to SDF-1, a receptor (CRCX4 or CRCX7) that binds to SDF-1 and a receptor (CRXX4 or CRCX7) A substance that inhibits binding to CRCX4 or CRCX7), a substance that competes with SDF-1 for binding to a receptor (CRCX4 or CRCX7), and the like. More specifically, a protein having a structure similar to SDF-1 or a partial peptide thereof, a low molecular weight compound having a structure similar to the binding site of SDF-1, an anti-SDF-1 antibody (for example, JP 2010-500005 Or a functional fragment thereof, a low molecular compound that binds to the receptor binding site of SDF-1, an anti-CXCR4 antibody or a functional fragment thereof, a low molecular compound that binds to the SDF-1 binding site of CXCR4, an anti-CXCR7 antibody or a thereof Examples thereof include functional fragments, low molecular weight compounds that bind to the SDF-1 binding site of CXCR7, soluble CXCR4, and soluble CXCR7.

  Specific examples of SDF-1 inhibitors include AMD3100 (J. Exp. Med., 186, 1383-1388 (1997); Nat. Med., 4, 72-77 (1998)), AMD3465 (S. Hatase et al Biochem Pharmacl 70: 752-761 (2005), AMD11070 (Wong et al., Mol Pharmacol 74: 1485-1495 (2008)) T22 (T. Murakami, et al. J. Exp. Med., 186, 1389 -1393 (1997), ALX40-4C (J. Exp. Med., 186, 1395-1400 (1997), TF14016 (S. Mikami et al., J Pharmacol Exp Ther., 327 (2) 383-392, ( 2008)) and the like (see J. Exp. Med., 186, 1189-1191 (1997)).

  Among SDF-1 inhibitors, it is preferable to use those that show inhibitory activity for both the binding of SDF-1 and CRCX4 and the binding of SDF-1 and CRCX7. An example of such is AMD3100 (Sigma).

  Antibodies that can be used as SDF-1 inhibitors (anti-SDF-1 antibodies, anti-CRCX4 antibodies, anti-CRCX7 antibodies) can be prepared using immunological techniques, phage display methods, ribosome display methods, and the like. Preparation of a polyclonal antibody by an immunological technique can be performed by the following procedure. An antigen is prepared and used to immunize animals such as rabbits. In addition to antigens prepared from living organisms, recombinant antigens may be used. In order to enhance the immunity-inducing action, an antigen bound with a carrier protein may be used. As the carrier protein, KLH (Keyhole Limpet Hemocyanin), BSA (Bovine Serum Albumin), OVA (Ovalbumin) and the like are used. A carbodiimide method, a glutaraldehyde method, a diazo condensation method, an MBS (maleimidobenzoyloxysuccinimide) method, or the like can be used for carrier protein binding. On the other hand, an antigen expressed as a fusion protein can also be used. Such a fusion protein can be easily purified by a general method. Immunization is repeated as necessary, and blood is collected when the antibody titer sufficiently increases, and serum is obtained by centrifugation or the like. The obtained antiserum is affinity purified to obtain a polyclonal antibody. On the other hand, a monoclonal antibody can be prepared by the following procedure. First, an immunization operation is performed in the same procedure as described above. Immunization is repeated as necessary, and antibody-producing cells are removed from the immunized animal when the antibody titer sufficiently increases. Next, the obtained antibody-producing cells and myeloma cells are fused to obtain a hybridoma. Subsequently, after this hybridoma is monoclonalized, a clone that produces an antibody having high specificity for the target protein is selected. The target antibody can be obtained by purifying the culture medium of the selected clone. On the other hand, the desired antibody can be obtained by growing the hybridoma to a desired number or more, then transplanting it into the abdominal cavity of an animal (for example, a mouse), growing it in ascites, and purifying the ascites. For purification of the culture medium or ascites, affinity chromatography using protein G, protein A or the like is preferably used. Alternatively, affinity chromatography in which an antigen is immobilized may be used. Furthermore, methods such as ion exchange chromatography, gel filtration chromatography, ammonium sulfate fractionation, and centrifugation can also be used. These methods can be used alone or in any combination.

  One aspect of the composition of the present invention is characterized in that the SDF-1 inhibitor is combined with one or more cells selected from the group consisting of mesenchymal stem cells, chondroblasts, chondrocytes and dental pulp stem cells. . That is, SDF-1 inhibitor and specific cells are used in combination. In this embodiment, cartilage formation at the target site is promoted by the action of cells supplied from the outside and the action of the SDF-1 inhibitor.

  Necessary cells (mesenchymal stem cells, chondroblasts, chondrocytes, dental pulp stem cells) may be prepared by conventional methods. For example, mesenchymal stem cells can be separated and purified from bone marrow and fat. Chondroblasts and chondrocytes can be obtained, for example, by inducing differentiation of mesenchymal stem cells into chondrocytes. For example, dexamethasone (Dex) or a transforming growth factor is used for such differentiation induction (see, for example, B. Johnestone et al., Exp. Cell Res., 238, 265 (1998)). Specific examples of culture media that can be used for differentiation induction include 50 μg / ml L-ascorbic acid-2-phosphate, 40 μg / ml L-proline, 100 μg / ml sodium pyruvate, 0.1μM dexamethasone, 6.25μg / ml insulin, 6.25μg / ml transferrin, 6.25μg / ml selenous acid, 10ng / ml human MSCBM (mesenchymal stem cell basic medium) supplemented with TGF-β3 (human TGF-β3). Medium exchange is performed appropriately during differentiation induction. For example, the medium is changed once every three days. The culture for inducing differentiation is continued, for example, for 1 day to 14 days.

As dental pulp stem cells, permanent dental pulp stem cells or deciduous dental pulp stem cells can be used. Dental pulp stem cells can be collected and prepared by the following method, for example. In this collection / preparation method, (1) collection of dental pulp, (2) enzyme treatment, (3) cell culture, and (4) collection of cells are sequentially performed.
(1) Collection of dental pulp After naturally deciduous deciduous teeth (or extracted deciduous teeth or permanent teeth) are sterilized with a chlorohexidine or isodine solution, the crown portion is divided and the pulp tissue is collected with a dental reamer.
(2) Enzyme treatment The collected dental pulp tissue is suspended in a basic medium (10% bovine serum / antibiotic Dulbecco's modified Eagle medium) and treated with 2 mg / ml collagenase and dispase at 37 ° C. for 1 hour. The pulp cells after enzyme treatment are collected by centrifugation for 5 minutes (5000 rpm). In principle, cell sorting with a cell strainer is not used because it reduces the collection efficiency of neural stem cell fractions of SHED and DPSC.
(3) Cell culture Cells are resuspended in 4 cc basal medium and seeded in 6 cm diameter adherent cell culture dishes. After culturing in an incubator adjusted to 5% CO ₂ and 37 ° C. for 3 days, the adherent cells that formed colonies are treated with 0.05% trypsin · EDTA for 5 minutes at 37 ° C. The dental pulp cells detached from the dish are seeded in an adherent cell culture dish having a diameter of 10 cm and expanded. For example, when observing with the naked eye, it reaches the sub-confluent state (a state in which the cells occupy about 70% of the surface of the culture container) or confluent, and the cells are detached from the culture container and collected, and the culture is filled again with the culture solution. Seed in containers. Subculturing may be repeated. For example, the subculture is performed 1 to 8 times to grow to the required number of cells (for example, about 1 × 10 ⁷ cells / ml). The cell can be detached from the culture vessel by a conventional method such as trypsin treatment. After the above culture, the cells may be collected and stored (storage conditions are, for example, -198 ° C.). Cells collected from various donors may be stored in the form of dental pulp stem cell banks.
(4) Cell recovery Next, cells are recovered. The cells can be collected by centrifuging after detaching the cells from the culture vessel by trypsin treatment or the like. The composition of the present invention is prepared using the cells thus recovered.

  The composition of this embodiment is typically provided as a combination of the prepared cells and an SDF-1 inhibitor. For example, an SDF-1 inhibitor may be mixed with a cell suspension obtained by suspending cells to be used in physiological saline or an appropriate buffer (eg, phosphate buffer) (others that can be used in combination) These components will be described later). On the other hand, for example, the composition of the present invention can be provided in the form of a kit comprising a first component containing an SDF-1 inhibitor and a second component containing a predetermined cell. In this case, both elements are administered to the target site simultaneously or at a predetermined time interval. Preferably both elements will be administered simultaneously. “Simultaneous” here does not require strict simultaneity. Therefore, when both elements are administered after mixing both elements, such as when administered under conditions where there is no time difference, administration of both elements such as promptly administering the other after one of them is administered. The case of being carried out under a condition without substantial time difference is also included in the concept of “simultaneous” here. On the other hand, when the other is administered at a predetermined time difference after one administration, it is preferable to set the time difference short. For example, the other is administered within 15 minutes, preferably within 10 minutes, more preferably within 5 minutes after administration of one. The first component (containing SDF-1) is administered locally or systemically to the target site. Preferably, local administration is employed to obtain a direct and rapid effect. In principle, the second component (containing cells) is locally administered to the target site.

A composition containing an SDF-1 inhibitor may be used, and predetermined cells may be administered in combination at the time of administration. The timing of administration of the composition and the predetermined cells in this case is the same as in the case of the kit form described above. That is, both are preferably administered at the same time, but they may be administered at a predetermined time difference. Incidentally, the desired reproduction effects as exhibited, may be the batch of example 1x10 ⁷ cells ～5X10 ⁷ or the dose of cells.

  Another embodiment of the composition of the present invention is a technique for accumulating in vivo mesenchymal stem cells (hereinafter also referred to as “MSC”) at a target site (hereinafter, the technique is referred to as “MSC accumulation technique”). It is used together. When the composition of this embodiment is applied, in vivo (endogenous) MSC accumulates at the target site by the MSC accumulation procedure, and SDF-1 function inhibition by the SDF-1 inhibitor occurs, and cartilage at the target site Formation is encouraged. Thus, according to the said aspect, the treatment method which does not need to supply (transplant) a cell from the outside is realizable. Therefore, a high therapeutic effect can be obtained while solving various problems (invasiveness, transplantation safety, stability of cell quality, time and cost) associated with cell transplantation.

  For example, MSC can be accumulated at the target site by applying tension to the target site. A typical example of such a procedure is bone extension. In bone extension, a dedicated device called a fixation device (internal fixation type or external fixation type) or a bone extension device is used. The method of osteogenesis usually consists of the steps of osteotomy, waiting period, osteogenesis period and osteosclerosis period. The bone extension speed is set in consideration of the site to be treated, but is usually 0.5 mm / day to 2 mm / day. For example, ADVANCE SERIES II-9 Bone Lengthening: Recent Progress (edited by Katseido Publishing Co., Ltd., supervised by Hariki Kiyonori, edited by Hiragi Sugihara).

  When the cartilage tissue composition of the present invention is used in combination with bone extension, usually, the composition of the present invention is locally administered between the start of the waiting period and the end of the bone extension period. The frequency of administration is not particularly limited. For example, administration is performed 1 to 10 times.

  Also in the composition of the embodiment in which specific cells are used in combination, a technique for applying tension to the target site (typically osteogenesis) may be further used in combination.

  The composition of the present invention contains an SDF-1 inhibitor in an amount necessary to obtain the expected therapeutic effect (ie, a therapeutically effective amount). The amount of the active ingredient of the composition of the present invention varies depending on the subject to be treated, the concomitant ingredients, the dosage form, etc., but the amount of the SDF-1 inhibitor is, for example, about 0.001 wt% to about 95 wt% so that the desired dose can be achieved. Set within the range.

It does not preclude the additional use of other ingredients, provided that the therapeutic effect expected of the composition of the present invention is maintained. Ingredients that can be additionally used in the present invention, including materials for preparing gels, are listed below.
(1) Substrate component, organic bioabsorbable material Examples of the substrate component or organic bioabsorbable material include glucosaminoglycans such as chondroitin sulfate and keratan sulfate, collagen (particularly type II, type IX, etc.), hyaluronic acid Fibrinogen (for example, Bolheel (registered trademark)) and the like can be used.

(2) Gelling material It is preferable to use a gelling material having high biocompatibility, and hyaluronic acid, collagen, fibrin glue, or the like can be used. Various types of hyaluronic acid and collagen can be selected and used, but it is preferable to employ one suitable for the application purpose (target site) of the composition of the present invention. The collagen used is preferably soluble (acid-soluble collagen, alkali-soluble collagen, enzyme-soluble collagen, etc.).

(3) Solvent The composition of the present invention may contain an aqueous solvent. As the aqueous solvent, sterilized water, physiological saline, a buffer solution such as a phosphate solution, or the like can be used. The prepared cells can be suspended in physiological saline or PBS (phosphate buffered physiological saline) and SDF-1 can be added to obtain the composition of the present invention, which can be applied to the target site.

(4) Others In addition to the above components, the composition of the present invention comprises a carrier, excipient, disintegrant, buffer, emulsifier, suspension, soothing agent, stabilizer, preservative, preservative, cell protection. Agents (eg, dimethyl sulfoxide (DMSO) and serum albumin), antibiotics, pH adjusters, various components for the purpose of cell activation, proliferation or differentiation induction (vitamins, cytokines, growth factors, steroids, Osteoinductive factor (BMP) etc.) may be included.

  The final form of the composition of the present invention is not particularly limited. Examples of forms are liquid (liquid, gel, etc.) and solid (powder, fine granules, granules, etc.). Preferably, the composition of the present invention is prepared in a gel form for the purpose of improving operability and therapeutic effect. As used herein, the term “gel” refers to a state having an appropriate viscosity and high retention at a target site, such as fibrin gel or fibrin glue used for medical purposes. For example, a gel-like composition is formed by adding a gelling agent or a thickener, or adding fibrinogen and thrombin.

  The composition of the present invention is used for repair and reconstruction of cartilage tissue. For example, the composition of the present invention is used for treatment of osteoarthritis, cartilage dysplasia, degenerative disc disease, meniscal injuries, achondroplasia, isolated osteochondritis, etc. Can be applied.

  Subjects to which the composition of the present invention is administered include humans or non-human mammals (pet animals, domestic animals, laboratory animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats. Sheep, dogs, cats, etc.). Preferably, the composition of the present invention is used for humans.

  The composition of the present invention is locally administered to a target site by, for example, injection, implantation, filling, or application to a tissue defect. Alternatively, systemic administration (for example, intravenous injection, intraarterial injection, intraportal injection, intradermal injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection). If it is prepared in a gel form having an appropriate fluidity, it can be applied by a simple technique such as filling, pouring or coating. Moreover, if it is a gel, it can be easily inserted into the application site using an injection needle or the like (it can also be applied without opening the wound), and is pre-molded according to the shape of the tissue defect. The versatility is high.

  A person skilled in the art can set an appropriate dose in consideration of a treatment target, a target site, and the like. For example, for adults (weight approximately 60 kg), the dosage should be set so that the amount of SDF-1 inhibitor per dose is about 0.01 mg to 1 mg / kg, preferably about 0.1 mg to 0.5 mg / kg. Can do.

  The following studies were conducted with the aim of developing a new tissue regeneration therapy that controls the in vivo stem cell accumulation system.

1. Identification of tissue regeneration model in which stem / progenitor cells accumulate in vivo (1) Preparation of mouse tibial bone extension model (DO model) It was verified that bone extension is a treatment method that applied stem cell / progenitor cell accumulation. . A mouse tibia bone extension model (DO model) was prepared (FIG. 1). After clearly showing the tibia, the 27G needle was penetrated in two places at the top and bottom, and then connected and fixed to the extension device with an immediate polymerization resin. After the resin hardened, the bone was cut and closed. In the extension schedule, the waiting period after osteotomy was 5 days, the extension period was 8 days, the curing period was 14 days, and the extension speed was 0.2 mm / 12 hours. As shown in the lower right of FIG. 1, in the sample on the 27th day, bone regeneration was confirmed by X-ray photography.

(2) Accumulation of Sca1 (Stem Cell common antigen) positive cells in the extended gap
A DO model tissue sample was prepared, and immunostaining was performed targeting the stem cell common antigen Sca1. It was revealed that Sca1-positive cells increased about 4 times in the extended gap on the 9th day, which is the middle phase of extension, compared with the control (bone marrow without surgery) (FIG. 2).

(3) Types of bone marrow stem cells accumulated in the extended gap (gap) It was decided to identify the types of stem cells accumulated in the gap. When multiple staining was performed with CD31, a common marker for vascular endothelial cells and vascular endothelial progenitor cells (EPC), CD45, a blood cell marker, and Sca1, a stem cell marker, It became clear that the fraction of leaf stem cells (MSC) accounted for the majority (FIG. 3).

(4) Analysis by in vivo imager
Using an in vivo imager, cell movement in the bone marrow was analyzed over time. First, a mouse bone marrow mononuclear cell fraction was collected and labeled with a near-infrared fluorescent dye DiR. The cells were transplanted into the bone marrow of the tibia mesial head during bone extension surgery. Fluorescence was detected for the sample on the 5th day before the start of the extension and the sample on the 13th day at the end of the extension. In the sample on the 5th day, a fluorescent signal localized in the transplanted part was detected, but on the 13th day, two peaks of the transplanted part and the extended part were formed (FIG. 4). That is, it became clear that the transplanted cells migrated toward the extension during the extension period.

  From the above examinations (1) to (4), it was shown that osteogenesis is a regenerative phenomenon applying stem cell / progenitor cell accumulation and is a model suitable for analysis of tissue regeneration by in vivo stem cell accumulation. .

2. Expression analysis of SDF-1 and its receptors (CXCR4, CXCR7) Gene expression of SDF-1 ligand, CXCR4 receptor and CXCR7 receptor during bone extension was analyzed by real-time RT-PCR. The results are shown in FIG. The expression level was expressed as a relative value when the gene expression level in the untied mouse tibia and bone was taken as 1. Consistent with previous reports, it was confirmed that the gene expression of angiogenic factors VEGF and PDGF receptor increased during the extension process (left of FIG. 5). On the other hand, the expression of SDF-1, CXCR4 and CXCR7 was shown to increase during the extended period (right of FIG. 5).

3. Functional analysis of SDF-1 (1) Transwell migration assay (in vitro transwell migration assay) and bone differentiation induction experiment First, a transwell migration assay (In vitro transwell migration assay) was performed. Bone marrow-derived mononuclear cells collected from ICR mice were placed in the upper chamber, and an agent for examining cell accumulation ability was placed in the lower chamber, and the number of cells that migrated to the lower chamber was counted 12 hours later. The cells were measured at 5 locations at random in a 10 × field of view, and the average value was obtained. A sample containing DMEM in the lower chamber was used as a negative control, and a sample containing DMEM containing 30% FBS was used as a positive control. In the group to which SDF-1 (concentration of 150 ng / ml) was added to DMEM, cell migration was approximately tripled. Cell migration was inhibited in the group in which SDF-1 and its inhibitor AMD3100 (Sigma, 5 μg / ml) were added to DMEM and in the group in which SDF-1 and anti-SDF-1 antibody were added to DEME ( FIG. 6 top). Thus, it was shown that SDF-1 protein promotes migration of bone marrow cells and that the migration activity is suppressed by SDF-1 inhibitors. On the other hand, bone differentiation induction experiment was performed, and it was confirmed that neither AMD3100 nor anti-SDF-1 antibody affected bone differentiation ability (bottom of FIG. 6).

(2) Role of SDF-1 in bone elongation 1
Next, in order to investigate the role of SDF-1 in bone elongation, AMD3100 (5 mg / kg) was administered every day until 1 day before the extension period in the DO model, or anti-SDF-1 antibody (dose 20 μg) was administered every other day. Was injected subcutaneously. In the control group, remarkable callus formation was confirmed at the end of extension, but in the group in which SDF-1 was inhibited, callus formation was significantly suppressed (FIG. 7). Formation of Alcian blue positive cartilage was confirmed in the extended gap. These results indicate that the SDF-1 molecule plays an essential role in callus formation of the bone extension.

(3) Role of SDF-1 in bone elongation 2
Clinically, it is known that a decrease in blood flow in the extension part inhibits callus formation. Therefore, we analyzed whether inhibition of SDF-1 function affects the accumulation of vascular endothelial progenitor cells in the extension. In the control, CD31 positive, vascular endothelial progenitor cells were accumulated at the extension site (FIG. 8). These cells express a lot of SDF-1. In the extension treated with AMD3100, the number of CD31 and SDF-1-positive vascular endothelial progenitor cells decreased to about 1/7 (FIG. 8). Thus, SDF-1 has been shown to play an essential role in the accumulation of vascular endothelial progenitor cells into extensions.

(4) Role of SDF-1 in bone extension 3
Next, the behavior of mesenchymal stem cells and hematopoietic stem cells was analyzed. AMD3100 treatment increased the number of Sca1-positive cells in the extension by a factor of 3 (FIG. 9). 20% of Sca1 positive cells were CD45 positive hematopoietic stem cells. 80% were CD45 negative and CD31 negative mesenchymal stem cells. These analysis results suggest that SDF-1 is not required for accumulation of mesenchymal stem cells and hematopoietic stem cells, but functions as a vascular endothelial progenitor cell-specific accumulation factor in the tissue regeneration process of bone elongation. It was done.

4). Examination with high-speed DO model While bone lengthening can obtain large tissue regeneration without cell transplantation, it has clinical problems (such as a long healing period). In order to shorten the treatment period, it is desirable to increase the extension rate, but rapid extension operation induces an extreme ischemic state, leading to atrophy of the extension site and scar formation. Aiming at shortening the treatment period in bone extension, a high-speed DO model (H-DO) (upper part of FIG. 10) was prepared that performs bone extension at twice the normal speed.

  200 ng of SDF-1 protein (R & D Systems) was mixed with type I collagen scaffold (Nitta Gelatin Co., Ltd.) and administered locally to the H-DO model every other day from the first day before extension (Day 4). (FIG. 10 top). As a result of the analysis by immunohistochemical staining, no callus was formed in the SDF-1 non-administered group (H-DO / vehicle), and cartilage tissue was observed extensively (bottom of FIG. 10). In contrast, in the SDF-1 administration group (H-DO / SDF-1), callus formation occurred at the end of the sclerosis period, and almost no cartilage tissue was observed (bottom of FIG. 10). Moreover, when the gap of the extended metaphase was observed by immunostaining, it became clear that the number of CD31 positive cells decreased in the H-DO group was significantly increased by the administration of SDF-1 (upper figure 11). . The H-DO group (H-DO + vehicle) did not detect vascular structure, but the SDF-1 administration group (H-DO + SDF-1) consisted of CD31-positive vascular endothelium and aSMA-positive vascular smooth muscle cells. Many mature blood vessels could be observed (FIG. 12). Furthermore, when blood flow was measured using a two-dimensional laser blood flow meter, the blood flow decreased at the extension in the H-DO model, and blood flow was restored by administration of SDF-1, as confirmed by the previous results. (Fig. 11 bottom).

The facts and findings revealed by this study are summarized below.
(1) In the process of extending bone, endogenous bone marrow stem cells accumulate in the affected area. These accumulated cells are thought to play an important role in tissue regeneration.
(2) Stem cells mainly accumulated by bone elongation are vascular endothelial precursor cells and mesenchymal stem cells. Bone elongation is a suitable model for analyzing the accumulation mechanism of these cells.
(3) SDF-1 is a specific accumulation factor of vascular endothelial progenitor cells. By inhibiting SDF-1, it is possible to manipulate the differentiation lineage of mesenchymal stem cells and promote the formation of cartilage tissue.

  The composition of the present invention promotes the regeneration of cartilage tissue at the target site. The composition of the present invention can treat various cartilage diseases (osteoarthritis, cartilage dysplasia, degenerative disc disease, meniscal injuries, achondroplasia, amputated osteochondritis, etc.) or cartilage augmentation, etc. Used for

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Claims (4)

Containing SDF-1 inhibitor consisting of AMD3100, AMD3465, AMD11070, T22, ALX40-4C, TF14016, anti-SDF-1 antibody, anti-CRCX4 antibody or anti-CRCX7 antibody ,
A composition for regenerating cartilage tissue comprising a combination of one or more cells selected from the group consisting of mesenchymal stem cells, chondroblasts, chondrocytes and dental pulp stem cells . The cartilage tissue according to claim 1 , comprising an SDF-1 inhibitor and one or more cells selected from the group consisting of mesenchymal stem cells, chondroblasts, chondrocytes, and dental pulp stem cells. A composition for regeneration. A kit comprising a first component containing an SDF-1 inhibitor and a second component containing one or more cells selected from the group consisting of mesenchymal stem cells, chondroblasts, chondrocytes and dental pulp stem cells The composition for cartilage tissue regeneration according to claim 1 , wherein Containing an SDF-1 inhibitor, and at the time of administration, one or more cells selected from the group consisting of mesenchymal stem cells, chondroblasts, chondrocytes and dental pulp stem cells are also administered, The composition for bone tissue regeneration according to claim 1 .

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