Cell composites for regenerating chondrocytes or chondrocyte-like cells

Abstract

This disclosure relates to the repair and / or regeneration of intervertebral discs in individuals that require it. To provide methods and compositions. [Solution] In a particular embodiment, for example, it is known that the intervertebral disc has degenerated tissue. Individuals who have, are suspected of having, or are at risk of having nucleus pulposus cells, nucleus pulposus-derived cells One or more components, a therapeutically effective amount of a conditioned medium produced from nucleus pulposus cells, are provided. In addition, fibroblasts, platelet-rich plasma (PRP), SOX9 (protein or nucleic acid) and / Alternatively, Tie2+ cells may be provided.

Description

Technical Field

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 278,635, filed on January 14, 2016, and U.S. Provisional Patent Application No. 62 / 413,587, filed on October 27, 2016, and both applications are hereby incorporated by reference in their entirety.

[0002] The present invention generally relates to at least the fields of medicine, surgery, anatomy, biology, cell biology, and / or molecular biology. In certain aspects, the present invention relates to the field of spinal disc repair. More specifically, the field of the present invention relates to the use of cell therapy to regenerate chondrocytes or other chondrocyte-like cells.

Background Art

[0003] Generally, cartilage is a tissue that does not naturally regenerate once damaged. In recent years, attempts have been made in laboratories to reconstruct damaged biological tissues by regenerating a part of the damaged tissue. This technique, defined as "tissue engineering," has received significant attention.

[0004] Tissue engineering involves developing biocompatible materials that can specifically interact with biological tissues to produce functional tissue equivalents. Tissue engineering involves the basic concept of collecting desired tissues from an individual, isolating cells from tissue specimens, proliferating cells, and reintroducing those cells into the same or different individuals. In other aspects, gene therapy, which has the ability to attract or generate desired cells in vivo, is utilized.

[0005] Fibroblasts have been used for the regeneration of chondrocytes or chondrocyte-like cells. For example, fibroblasts

[0006] Cells can induce differentiation into chondrocytes in a hypoxic environment under mechanical stress. It has been proven. However, current research supports the use of additional cell types in this mixture. There is currently no scientific evidence to support this.

[0006] intervertebral disc degeneration More than 65 million Americans suffer from back pain every year. By age 50, 85% of the population will show signs of intervertebral disc degeneration. (Degenerative disc disease of the spine (DDD)) Degeneration of the intervertebral discs, often called degenerative spondylosis, is a common disorder of the lower spine. Yes. Intervertebral disc degeneration can cause lumbar spinal stenosis (narrowing of the spinal canal that houses the spinal cord and nerve roots), and spinal Anterior spondylolisthesis (slippage of the intervertebral disc and vertebrae forward) and posterior spondylolisthesis (slippage of the intervertebral disc forward) DDD can lead to disorders such as posterior displacement of the vertebrae. DDD is painful and significantly impacts quality of life. This is a denatured state that can have a significant impact.

[0007] Aging is the most common cause of intervertebral disc degeneration. As the body ages, the vertebrae in the spine... The intervertebral disc loses important cells that make it viable, loses moisture, or dries out between the vertebrae. It loses its ability to act as a shock absorber. The bones and ligaments that make up the spine also lose their flexibility. It thickens. Unlike muscles, the intervertebral discs receive very little blood, so the intervertebral discs... It lacks the ability to heal or repair itself. For example, DDD sometimes occurs around the waist. Chronic lower back pain that radiates, or a throbbing pain in the buttocks or thighs when walking. There is.

[0008] It is unclear why some degenerated discs cause pain while others do not. This means that the nerve endings penetrate deeper into the annulus fibrosus or the outer layer of the intervertebral disc than in other individuals. This makes the intervertebral discs more susceptible to pain. This is commonly known as sciatica or lower back pain. Pain radiating to the lower extremities is caused by inflammatory substances compressing the internal intervertebral disc material or nucleus pulposus, or the nerves. These are the consequences of encountering nerve roots. These conditions include severe lower limb pain, difficulty standing, and It can cause symptoms such as difficulty walking and weakness or paralysis of the lower limbs. DDD is This can lead to a chronic state of debilitation, which can have a significant negative impact on the quality of life. D In severe cases of DD, conventional non-surgical treatments are often ineffective.

[0009] Restoring complete tissue function in a damaged or diseased spinal disc is difficult. The topic is: Although there are conventional methods such as artificial disc replacement or nuclear replacement, these treatments Many weaknesses still exist in the therapy. For example, artificial disc replacement has a cascade effect and It is known that mechanical stress may tend to move and transfer to other vertebrae above and below the affected area. ru. [Overview of the project] [Problems that the invention aims to solve]

[0010] This disclosure provides, for example, an effective and simple method for successfully repairing and / or regenerating a spinal disc. This relates to pure methods and compositions. [Means for solving the problem]

[0011] For example, embodiments of the present disclosure relate to mammalian individuals such as humans, dogs, cats, or horses. The present invention relates to a method and composition for repairing joints. The joint may be of any kind. However, in certain embodiments, the joint is an intervertebral disc, provided that it is in a mammalian individual Multiple intervertebral discs may be treated simultaneously or at different times. The methods and compositions of the present disclosure utilize, for example, notochord cells, small chondrocyte-like cells, collagen (such as type II collagen and / or collagen types I, V, VI, IX, and XII) fibrils, and / or proteoglycans (such as aggrecan) and other one or more nucleus pulposus components to repair and / or regenerate joint tissue or other objects

[0012] The present disclosure relates to delivering allogeneic, autologous or xenogeneic cells (such as nucleus pulposus cells), and / or their conditioned media for treatment to a joint (including an intervertebral disc) of a mammalian individual that needs it (e.g., by injection or open application through a surgical site). In some embodiments the nucleus pulposus cells (in addition to, or alternatively, cells that can differentiate into nucleus pulposus cells) are provided with one or more other therapeutic agents. The one or more other therapeutic agents may be of any kind but in certain embodiments, the agents are cells, proteins, nucleic acids (including coding sequences, miRNA, mRNA, DNA, shRNA, siRNA, combinations thereof, etc.), small molecules or combinations thereof. The one or more other therapeutic agents may or may not be nucleus pulposus derived components

[0013] In certain embodiments, the therapeutic agent provided to the joint (in addition to one or more nucleus pulposus cells or nucleus pulposus-derived components) is a small molecule, an expressible nucleic acid, a peptide, a protein or a combination thereof Various nucleic acids, peptides and / or proteins are provided together with the cells ​​​​However, in certain embodiments, nucleic acids, peptides, or proteins may be Sox9 and / or platelet-rich plasma (PRP) and / or cartilage repair and / or regeneration Contains other nucleic acids, peptides, or proteins that aid in life. In certain embodiments, the marrow Cells that can differentiate into nuclear cells and / or nucleus pulposus cells have a nutrient matrix (nutr Including the presence or absence of blood vessels, one or more components of NP (notochord cells) It is provided in addition to (and / or Tie2+ cells, etc.).

[0014] In a particular embodiment, the present disclosure uses the delivery of at least one cartilage-forming mixture. For example, to biologically repair cartilage in the spinal disc (such as within the joint) or other cartilage. This relates to a method and composition for cartilage restoration. In a particular embodiment, this treatment is for cartilage restoration. It can function as an in vivo workstation in some cases. One or more cells and / or other therapeutic agents are provided together with an inactive device. The inert device may be of any type, but in certain embodiments, it is inactive. Sexual devices include a structure comprising two generally concentric, air-inflatable membranes. Or multiple cells and / or other therapeutic agents may be provided to the organism along with a scaffold. It doesn't have to be done.

[0015] This disclosure provides an effective amount of a cell mixture derived from healthy intervertebral discs, thereby enabling individuals to It includes methods to improve the condition of aging intervertebral discs. Also, to become younger and healthier. By providing an effective amount of cell mixtures that may be found in intervertebral discs, The present disclosure includes a method for improving the condition of aged intervertebral discs. The effective amount of cellular composites found in the intervertebral discs of humans who have not developed the disease is compared to the effective amount of aged intervertebral discs. By providing this to individuals with discs, the condition of aged intervertebral discs in those individuals can be improved. This includes a method. One embodiment of the present disclosure involves one or more human beings who have not developed a degenerative process. The effective amount of cell mixture derived from or found within one or more intervertebral discs of aging By providing this to individuals with aged intervertebral discs, the condition of the aged intervertebral discs in the individual can be improved. Includes methods to improve the degenerative process in one or more people who have not developed the disease. Cellular mixtures found in multiple intervertebral discs include nucleus pulposus cells, chondrocytes, and fibroblasts (bone, cartilage). and / or at least dermal fibroblasts or threads derived from the connective tissue of the body, including muscles. It may contain effective amounts of stem cells and / or adipocytes (including fibroblasts). The mixture may include, for example, collagen fibrils, proteoglycans, and / or aggrecans. It may also contain non-cellular components.

[0016] As described herein, the onset of intervertebral disc degeneration involves the presence of one or more painful symptoms. In some cases, degenerated intervertebral discs and / or disc degeneration within the spine can lead to The resulting weakness or paralysis is due to the loss of fluid in the intervertebral disc and / or the outer layer of the intervertebral disc. Small tears or cracks in the ring or capsule (in some cases, within the intervertebral disc) The nucleus is pushed out through a tear or crack in the capsule, thereby dislodging the intervertebral disc. It swells up, breaks open and exposes its contents (bursts), or breaks down into fragments. This may include damage to one or more vertebral discs.

[0017] Any intervertebral disc within the spine can be treated in the manner of the present disclosure, however in certain cases, the intervertebral disc The board is located in the lumbar region (lower back area) and / or neck region (cervical area).

[0018] In certain cases, this method utilizes scaffolding in conjunction with one or more compositions. Yes, it is possible. Scaffolds used in the regeneration of biological tissues allow cells to adhere to the surface of a material, creating three-dimensional tissue. It can be composed of materials that act as a matrix that can be formed. In certain embodiments This material may be non-toxic, biocompatible, and / or biodegradable. For example, as described above. The most widely used biodegradable polymers that meet physical requirements include polyglycolic acid ( PGA), lactic acid-glycolic acid copolymer (PLGA), poly-ε-caprolactone (PC L) Organic polymers such as polyamino acids, polyanhydrides, and polyorthoesters; collagen , natural hydrogels such as hyaluronic acid, alginic acid, agarose, and chitosan; poly(oxidized) Ethylene (PEO), poly(vinyl alcohol) (PVA), poly(acrylic acid) (P AA), poly(propylene fumarate-ethylene glycol) copolymer [P(PF-co Synthetic hydrogels include those such as -EG) and its copolymers.

[0019] In certain embodiments, for example, chondrogenesis involves autologous, allogeneic, or heterogeneous nucleus pulposus cells. Cells (or mixtures thereof), chondrocytes, fibroblasts (including bone, cartilage, and / or muscle cells) (including at least cutaneous fibroblasts or fibroblasts derived from the body's connective tissue), stem cells Using cells and / or adipocytes, at least in part, we start in vitro. It is possible that any cell or mixture of cells may be present in or near a joint. It can then be delivered. In embodiments in which fibroblasts are present in the mixture, a specific implementation Morphologically, fibroblasts attract other fibroblasts that are not captured during the cartilage regeneration process. It is possible. In certain embodiments, a mixture of cells or cell types may be one or It is delivered to the individual along with several other therapeutic agents. The cellular component of the delivery is delivered simultaneously with the non-cellular component. It can happen at one or at a different time.

[0020] In a particular embodiment, the matrix is ​​used for gene therapy and / or fibroblasts. In vitro or in vivo, together with cells and / or chondrocytes It can be introduced and then seeded to provide structure for the cartilage regeneration process.

[0021] Cell differentiation into chondrocytes or chondrocyte-like cells occurs a) before the cells are delivered to the organism. Cell differentiation in vitro; b) further differentiation in vitro before delivering cells to an organism Cell differentiation in vivo after delivery; and / or c) in vivo after delivery of cells Differentiation in ivo can occur in any appropriate manner, including differentiation in ivo. Delivery is to one or multiple cells. The transplantation of several compositions may be included. In some cases, the cells have an open distal end. It is provided to the individual with a needle-shaped instrument that has a long tube.

[0022] Embodiments of this disclosure describe the nucleus pulposus (N) of the same individual or another individual of the same or different species. The process includes providing an effective amount of a component derived from P) to the degenerated intervertebral disc of an individual, for the cartilage of an individual. The method includes generating cells or chondrocyte-like cells. In certain embodiments, NP-derived cells The components include notochord cells, small chondrocyte-like cells, collagen fibrils, proteoglycans and / or containing aggrecan. In some cases nucleic acids, peptides, proteins, small molecules or One or more therapeutic agents including that combination, or one or more therapeutic agents, can also cause changes in the individual. It can be provided to the affected intervertebral disc. Alternatively, one or more therapeutic agents can be mixed with cells in an individual. The substance is provided to the cell mixture in vitro before delivery. In certain embodiments Furthermore, nucleic acids, peptides, and / or polypeptides, which are therapeutic agents in themselves, are supplied to individuals. It is delivered to the cells to which it is intended.

[0023] In certain embodiments, this method involves the following cells in an organism: a) fibroblasts; b) notochord cells; c) T ie2+ cells; d) Tie2 gene product; e) Platelet-rich plasma (PRP); f) Sox9 gene Genetic products; g) Transformation growth factor beta-1 (TGFB1); h) Connective tissue growth factor (CT) GF); i) WNT1-induced signaling pathway protein 1 (WISP1), and / or The present invention provides one or more compositions containing one or more effective amounts of WISP2. This includes the process of providing NP cells to an organism. In at least certain cases, NP cells are e x Modified in vivo. Includes fibroblasts, chord cells and / or Tie2+ cells. The cells used in this disclosure may be modified ex vivo. In this process, the cells are subjected to hypoxia, mechanical stress (strain), or a combination thereof before the serving process. They are exposed together.

[0024] The present disclosure modifies specific cells to produce specific products, such as the Tie2 gene product, which are then produced by the cells. and / or produces the Sox9 gene product. In certain embodiments, it is delivered to an individual. The cells that are affected are COL1A1, COL1A2, COL2A1, COL3A1, and COL4A1. COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5 A1, COL5A2, COL5A3, COL6A1, COL6A2, COL6A3, CO L6A4, COL6A5, COL7A1, COL8A1, COL8A2, COL9A1 COL9A2, COL9A3, COL10A1, COL11A1, COL11A2, CO L12A1, COL13A1, COL14A1, COL15A1, COL16A1, CO L17A1, COL18A1, COL19A1, COL20A1, COL21A1, CO L22A1, COL23A1, COL24A1, COL25A1, COL26A1, CO L27A1, COL28A1, Gata4, Mef2C, Tbx5, Sox5, Sox6 Sox9, FGFR2, VEGF, MMP14, CD10, MMP1 3. WNT11, BAPX1, IL-1R1, IGFBP5, MMP16, BMP2, A LK1, BMP5, IGF1, MMP13, ADAMTS5, BCL10, MCOLN2 LRRC8C, PTGFR, RLF, MATN1, PDPN, TNFRSF18, IT GA10, THBS3, SCYL1BP1, KCNT2, 244533_at, ARF1 222348_at, SLC4A5, HSPC159, RHOQ, MATN3, SUL T1C2、236289_at、BCL2L11、FLJ16008、KLF7、NRP 2, SERPINE2, FN1, B3GNT7, ADAMTS9, ANKRD28, GA LNTL2, IRAK2, SETD5, FNDC3B, B3GNT5, CYTL1, IB SP、229221_at、PET112L、EDNRA、1563414_at、OS MR, C1QTNF3, ZFYVE16, 225611_at, MAST4, EDIL3 、230204_at, 230895_at, HAPLN1, PDLIM4, cr5q3 5 SQSTM1, SCUBE3, CMAH, 236685_at, BMP6, ULBP 2, LRP11, SOD2, SYNJ2, WTAP, HIG2, KIAA1718, FA M62B, UBE3C, TNFRSF10D, SLC25A37, ChGn, RB1CC 1, C8orf72, EIF2C2, HAS2, TRPS1, WISP1, 235821 _at, PTK2, ZCCHC7, RPS6, GLIS3, SLC28A3, 15558 41_at, MGC17337, EDG2, 229242_at, ITGB1, C10o rf49, YME1L1, AKR1C2, CHST3, LOXL4, SFXN3, 228 910_at, CD44, FOSL1, RELA, MMP12, MMP13, MMP3, KIAA0999, ASAM, LOC399959, ETNK1, SOX5, CHST1 1, ATF1, SRGAP1, DSPG3, LOC338758, KIAA0701, S LC41A2, RHOF, FZD10, NUPL1, USP12, UFM1, LECT1 , GPC6, ERO1L, BDKRB1, SEMA6D, LACTB, ARIH1, CS PG4, AGC1, LOC283824, VASN, WWP2, NOS2A, LOC20 1181, MSI2, PITPNC1, TGIF, 1552288_at, 155228 9_a_at, ZNF146, RELB, MIA, ZNF160, SNX5, BMP2, RNF24, HSUP1, MATN4, BIC, RUNX1, LIF, RP4-756G 23.1, RPS6KA3, TNMD, RP6-213H19.1, and their combinations? It contains or is modified to express a gene product selected from a group of genes.

[0025] The cells of this disclosure may be autologous, allogeneic, or heterogeneic to an individual.

[0026] In some embodiments, this method involves the use of NPs in the intervertebral discs of individuals suspected of degeneration. Further includes detecting degenerated intervertebral discs by measuring the level of notochord cells. The intervertebral disc is detected structurally or non-structurally (in the intervertebral disc, cells including notochord cells are detected). Non-structural detection can be achieved by monitoring the cellular level, for example. This may include sub-measures. This may involve examining the condition of the degenerated intervertebral disc or the level of notochord cells in the intervertebral disc NPs. Discharge may occur before or after delivery of one or more compositions of the present disclosure to the intervertebral disc. You don't have to do that.

[0027] In some embodiments, cartilage is repaired in the spinal disc of an individual that requires it and / or a method of regeneration, a) in which the intervertebral disc of an individual contains fibroblasts, stem cells, adipocytes or a) the process of providing the combination; b) one or more nucleus pulposus (NP)-derived intervertebral discs of an individual The process of providing the components; c) into the intervertebral disc of an individual, 1) Tie2+ cells; 2) Tie2 genes 3) Sub-products; 4) Platelet-rich plasma (PRP); 5) PRP+ cells; 6) Sox9 gene product; 6 )Sox9+ gene product; 7)TGFB1 gene product; 8)TGFB1+ cells; 9)CT GF gene product; 10) CTGF+ cells; 11) WISP1 gene product; 12) WISP 1) Cells; 13) WISP2 gene product; and / or 14) Of the WISP2+ cells There exists a method that includes the step of providing one or more. In a particular embodiment, N One or more components of P are notochord cells, small chondrocyte-like cells, collagen fibrils and Includes and / or aggrecan. In certain cases, step a) is step b) and / or This is preceded by c).

[0028] In another embodiment, cartilage is repaired and / or a method of regeneration, one of the compositions listed in a), b), and / or c) or more of them, combined with another one or more of the compositions listed in a), b), and / or c). a) fibroblasts, stem cells, adipocytes or a combination thereof; b) nucleus pulposus (NP) c) 1) Tie2+ cells; 2) Tie2 gene products; 3) multiple 4) Platelet-plasma (PRP); 5) PRP+ cells; 6) Sox9 gene product; 7) Sox9+ cells Cells; 7) TGFB1 gene product; 8) TGFB1+ cells; 9) CTGF gene product; 10 )CTGF+ cells; 11)WISP1 gene product; 12)WISP1+ cells; 13)WI SP2 gene product; and / or 14) a mixture of one or more WISP2+ cells. There exists a method that includes the steps of manufacturing an object and providing a mixture to an object. In this case, the mixture is produced in vitro or in vivo. In certain cases, the mixture is delivered to the individual by injection or insertion via open incision. It will be done.

[0029] In certain embodiments, for example, the method disclosed herein includes individuals with degenerated intervertebral discs, To deliver a therapeutically effective amount of notochord cells and / or notochord cell-conditioned medium to individuals in need. The process includes, for example, a notochord cell conditioned medium or one derived therefrom. Alternatively, in addition to multiple components, the organism contains notochord cells, small chondrocyte-like cells, and collagen progenitor cells. Effective amounts of fiber, proteoglycans, and / or aggrecans are provided in chordal cell culture. In individuals, the earth regenerates degenerated intervertebral discs or regenerates part of them, or cartilage cells as well. The process involves generating chondrocyte-like cells. The step of providing a notochord cell-adapted medium to the individual is used for degenerated intervertebral discs. This treatment aims to retract the degenerated disc, prevent further degeneration, or worsen the degenerated disc. This can prevent corruption. In certain embodiments, the chord cell conditioned medium is serum-free. In certain embodiments, one or The multiple components include transforming growth factor beta-1 (TGFB1), connective tissue growth factor (CTGF) (Also known as CCN2), WNT1-induced signaling pathway protein 1 (WISP1) ) and / or WISP2. In some cases, individuals have notochord cells and / or In addition to or instead of delivering chord cell adaptation medium, it delivers protein morphology to the organism. Alternatively, TGFB1, CTGF, WISP1 and / or WIS in any of the nucleic acid forms. P2 can be delivered. Notochord cells and / or notochord cell conditioned medium and / or Or by delivery of TGFB1, CTGF, WISP1 and / or WISP2, Compared to the loss of chord cells and / or stem cells in the absence of such delivery, in the nucleus pulposus Notochord cells and / or stem cells are retained.

[0030] Individuals can receive multiple doses of the therapeutic composition, with each dose being administered over several days or weeks. It can be delivered at any time interval, such as within a few months and / or several years. Individuals are different. Multiple therapeutic compositions of this disclosure can be administered via a certain route. In some cases, In this case, individuals who still have one or more symptoms of degenerated intervertebral discs are subject to the 1 of the Disclosure. One or more therapeutic compositions are provided. In a particular case, the individuals are 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 years old, and other specific ages. An effective amount of one or more therapeutic compositions of this disclosure is provided, starting from a certain age. In the case of the individual, whether or not one or more symptoms of degenerated intervertebral discs were detected. Regardless, individuals prone to degenerated intervertebral discs may be affected by one or more therapeutic compositions of this disclosure. An effective amount is provided. For example, individuals prone to degenerated intervertebral discs are those who engage in repetitive activities. Individuals with injuries to the body or spine, individuals participating in exercise (including high-contact exercise), and / or This includes individuals whose jobs require physical labor.

[0031] In some cases, the effective amount of one or more components derived from the nucleus pulposus (NP) was Provided to individuals that need to receive one or more of the components; in certain cases, for example If one or more components derived from NP are notochord cells, notochord cell-adapted medium, or small chondrocytes, The organism contains collagen fibrils, proteoglycans and / or aggrecans. Therapeutic agents may also be provided. In certain embodiments, one or more NP-derived substances are provided to an individual. For the purpose of specifically providing components, individuals are provided with one or more components derived from NPs. Before providing the body with one or more components derived from NPs, the individual may have one or more components derived from NPs. It may be determined that multiple components are required. In some cases, the individual is derived from NP. One or more components of the above are provided, but one or more other components derived from NP are also provided. Not provided. In certain cases, substantially all of the contents of NP are provided to the individual. or not provided. For example, in a particular embodiment, an individual has notochord cells, notochord cells Conditioned medium, small chondrocyte-like cells, collagen fibrils, proteoglycans and / or The essence includes one or more components derived from NP, such as one or more aggrecans. An effective amount of the same, or a composition consisting of the same, is provided.

[0032] The foregoing is a description of the features of the present invention, and is intended to help you better understand the detailed description of the present invention that follows. The technical advantages have been outlined to some extent. Additional features and advantages of the present invention are described below. The subject matter of the clear claims will be described below. The concepts and specifics disclosed Embodiments are a basis for modifying or designing other structures to carry out the same objective of the present invention. Those skilled in the art should recognize that it can be easily used as a foundation. The construction of such an equivalent is Those skilled in the art will understand that this does not deviate from the spirit and scope of the invention as set forth in the attached claims. It is so. When considered in relation to the attached figure, the novel features that are considered to be characteristic of the present invention are The characteristics, along with further objectives and advantages, are described below regarding both the organization and methods of work. It will be better understood from the beginning. However, each of the figures is an example and explanation. It is provided for its intended purpose only and is not intended to define the scope of the invention. This should be explicitly understood. This application refers to several references and documents. And all of that is incorporated into this specification in its entirety. [Modes for carrying out the invention]

[0033] As used herein, the designations, "a" or "an" mean one or more. It is possible. When used herein in a claim, it is used in combination with the word "includes". When used together, the words "a" or "an" refer to one or more words. It can mean that. As used herein, “another” means at least two It can mean one thing, or more. For example, in a particular embodiment, this An explicit aspect of the present invention is that one or more arrangements "essentially consist of" or "consist of" This can be done. Some embodiments of the present invention involve one or more elements, methods, and processes of the present invention. It may consist of or essentially consist of a process and / or a method. Any method or composition relating to any other method or composition described herein It is thought that this can be done.

[0034] U.S. Patent No. 7,850,983, U.S. Patent Application Publication No. 2014 / 0314726; United States Patent Application Publication No. 2014 / 0044682; U.S. Patent Application Publication No. 2014 / 03772 No. 31; and WO / 2015 / 035395 are incorporated herein by reference in their entirety. It gets crowded.

[0035] I. Definition

[0036] As used herein, the term "chondrocyte-like cell" refers to a stem cell, not a primary chondrocyte. Cells derived from cells (such as mesenchymal stem cells) or other lineages (such as fibroblasts) This refers to these chondrocyte-like cells. These chondrocyte-like cells have the phenotype of chondrocytes (cartilage cells). For example, This means that these cells have the shape of cartilage cells (for example, polygonal and / or rhomboid cells). In addition to having cells, it is also capable of aggregation, and contains sulfated proteoglycans and type II collagen. This means that it is possible to produce cartilage matrix components such as ions. Therefore, for example, cartilage cells Typical markers for cystoid cells are chondroitin sulfate and keratan sulfate proteoglycans. Aggrecan, type II collagen, Sox-9 protein, cartilage linkage protein, and also comprising one or more of the heparan sulfate proteoglycans, which are perlecans. .

[0037] As used herein, the term "hypoxic" refers to a deficiency of oxygen. This refers to the partial pressure of oxygen, which is approximately 20%, 15%, 10%, less than 5%, and others.

[0038] As used herein, the term “joint” refers to a region within the body where two bones of the skeleton connect. It refers to.

[0039] In certain embodiments, the term “sow” as used herein refers to scaffolding This refers to transplanting cells into the intervertebral disc, where the scaffold may be located within the intervertebral disc or in vi They may exist as tro. Cells attach to a scaffold, then grow and differentiate within the scaffold. In certain embodiments, the term “sow” means direct injection without scaffolding. This refers to the seeding of cells into the nucleus pulposus. This differentiation is performed in vitro and in vitro. This can happen in both vocal and vocal performance.

[0040] II. General Implementations

[0041] As a rapidly expanding field, in at least some cases, tissue engineering is biomimetic. Developing tissue substitutes provides an alternative solution for articular cartilage repair and regeneration. In some embodiments of this disclosure, the repair and / or regeneration of tissues including cartilage is involved. There are methods and compositions for this purpose. As used herein, the term "repair" refers to the absence of This refers to the restoration of normal cartilage function, regardless of the composition of new tissue to replace the damaged area. As used herein, “regeneration” refers to the normal function of damaged or diseased articular cartilage. Not only does it restore the cartilage, but it also creates new cartilage that is indistinguishable from or very similar to the original cartilage. It is defined as a process that also brings about the formation of weave.

[0042] In certain embodiments, nucleus pulposus cells and / or the conditioned medium produced therefrom are modified It is provided to individuals who need it, such as those with sexually transmitted disc disorders, degenerative discs, etc. This is used to treat or This can be prevented. For example, from the nucleus pulposus and / or the conditioned medium produced therefrom. Cells can be obtained from the individual being treated, such as from a healthy intervertebral disc of the same individual; It can be obtained from another individual of the same species; or from an individual of a different species. Nucleus pulposus Methods for isolating cells are well known in the art (e.g., Gruber et al., 20 (2006; Feng et al., 2013; Tang et al., 2014). Nucleus pulposus cells are commercially available. It is possible to do this. Methods for producing conditioned media from cells are known in the art. ru.

[0043] Cells derived from the nucleus pulposus (and / or any other cells delivered to an individual) require it. The cells may be exposed to one or more compositions before being delivered to the organism. For example, the cells To express one or more compositions that help the health and / or proliferation of cells They may be modified, and / or the cells may be used for cartilage repair and / or regeneration. It may be modified to express one or more compositions. In certain embodiments, By manipulating cells, specific nucleic acids for therapeutic purposes (the nucleic acid itself is therapeutic, or the nucleic acid is a therapeutic agent) Vectors that express the gene product (viral (adenovirus, adeno-associated virus, Possessing lentiviruses, retroviruses, or other nonviral (plasmids) To cause. In other embodiments, cells or other vectors (such as liposomes) are manipulated. , provides a specific protein or a functionally active peptide fragment thereof. The protein is, It may also be delivered as a fusion protein, for example, the target protein is used for cartilage repair and / or it may be fused with another protein or protein fragment that facilitates regeneration. Alternatively, the target protein may be another protein or protein such as a marker or label. It may be fused with fragments. Proteins or parts thereof that can be provided to an organism Examples of nucleic acids that code for the whole are: COL1A1, COL1A2, COL2A1, C OL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5 , COL4A6, COL5A1, COL5A2, COL5A3, COL6A1, COL6 A2, COL6A3, COL6A4, COL6A5, COL7A1, COL8A1, CO L8A2, COL9A1, COL9A2, COL9A3, COL10A1, COL11A 1, COL11A2, COL12A1, COL13A1, COL14A1, COL15A 1, COL16A1, COL17A1, COL18A1, COL19A1, COL20A 1, COL21A1, COL22A1, COL23A1, COL24A1, COL25A 1、COL26A1、COL27A1、COL28A1、Gata4、Mef2C、Tb x5、Sox5、Sox6、Sox9、FGFR2、VEGF、MMP14、フォークヘ ッド、CD10、MMP13、WNT11、BAPX1、IL-1R1、IGFBP5、 MMP16、BMP2、ALK1、BMP5、IGF1、MMP13、ADAMTS5、 BCL10、MCOLN2、LRRC8C、PTGFR、RLF、MATN1、PDPN 、TNFRSF18、ITGA10、THBS3、SCYL1BP1、KCNT2、24 4533_at、ARF1、222348_at、SLC4A5、HSPC159、RH OQ、MATN3、SULT1C2、236289_at、BCL2L11、FLJ16 008、KLF7、NRP2、SERPINE2、FN1、B3GNT7、ADAMTS 9、ANKRD28、GANLTL2、IRAK2、SETD5、FNDC3B、B3G NT5、CYTL1、IBSP、229221_at、PET112L、EDNRA、1 563414_at、OSMR、C1QTNF3、ZFYVE16、225611_at 、MAST4、EDIL3、230204_at、230895_at、HAPLN1、 PDLIM4、cr5q35 SQSTM1、SCUBE3、CMAH、236685_ at、BMP6、ULBP2、LRP11、SOD2、SYNJ2、WTAP、HIG2 KIAA1718, FAM62B, UBE3C, TNFRSF10D, SLC25A3 7、ChGn、RB1CC1、C8orf72、EIF2C2、HAS2、TRPS1、 WISP1、235821_at、PTK2、ZCCHC7、RPS6、GLIS3、S LC28A3, 1555841_at, MGC17337, EDG2, 229242_a t, ITGB1, C10orf49, YME1L1, AKR1C2, CHST3, LOX L4, SFXN3, 228910_at, CD44, FOSL1, RELA, MMP12 , MMP13, MMP3, KIAA0999, ASAM, LOC399959, ETNK 1, SOX5, CHST11, ATF1, SRGAP1, DSPG3, LOC33875 8, KIAA0701, SLC41A2, RHOF, FZD10, NUPL1, USP1 2, UFM1, LECT1, GPC6, ERO1L, BDKRB1, SEMA6D, LA CTB, ARIH1, CSPG4, AGC1, LOC283824, VASN, WWP2 , NOS2A, LOC201181, MSI2, PITPNC1, TGIF, 15522 88_at, 1552289_a_at, ZNF146, RELB, MIA, ZNF16 0, SNX5, BMP2, RNF24, HSUP1, MATN4, BIC, RUNX1, LIF, RP4-756G23.1, RPS6KA3, TNMD, RP6-213H19 1. The osmotic-sensitive transcription factor TonEBP and its combinations. The gene products mentioned above. It can be delivered as nucleic acid or protein.

[0044] In another embodiment, any cells delivered to an individual in need are used for cartilage regeneration. To provide treatment to an individual, such as providing, facilitating, or enhancing repair. Exposure to one or more conditions that cause, facilitate, or enhance certain conditions. In embodiments thereof, any cells of the present disclosure are subjected to hypoxic conditions (e.g., before being delivered to an organism) Exposure to low oxygen partial pressure (such as O2 below 5%), high osmotic pressure environments, mechanical stress, or a combination thereof. In other embodiments, cells can be delivered in vivo as Alternatively, they may be exposed to hypoxic conditions, mechanical loads, or a combination thereof. Examples include intermittent hydrostatic pressure, fluid shear stress, hypoxic partial pressure, direct pressurization, or a combination thereof. .

[0045] In certain embodiments, Tie2+ cells are provided to individuals that require them. ie2 is the angiopoietin-1 receptor; CD202B; hTIE2; p140 TE K; Soluble TIE2 Variant 1; Soluble TIE2 Variant 2; TEK; TEK Tyrosine Tyrosine kinase, endothelium; TIE-2; TIE2; intima endothelial cell kinase; tyrosine protein Tyrosine kinase receptor TEK; Tyrosine-protein kinase receptor TIE-2; VMCM; It is also sometimes called VMCM1. It codes for part or all of the Tie2 protein. By transforming or transfecting the cells in question with a vector containing nucleic acids, It can produce Tie2+ cells. Tie2+ cells are produced in the individual being treated. They can be isolated from other individuals, including other individuals of the same or different species. For example, Tie2+ cells can be conjugated with Tie2 antibodies or aptamers. The entities can be isolated using methods that are well known in the art.

[0046] Cells derived from the nucleus pulposus and / or the conditioned medium produced therefrom are provided to the individual, In certain embodiments, such cells are notochord cells, small chondrocyte-like cells or This may include a combination of the following: In addition to cell delivery, collagen fibrils and / or Providing individuals with glycans or other components derived from the nucleus pulposus, such as any proteoglycans, is possible. Yes, it is possible. Any composition for delivery may be labeled, and the composition may include an antibiotic, a buffer. It may also include salt, culture medium, and other substances.

[0047] In certain embodiments, the method of the present disclosure includes joint pathologies, including the joint being a spinal disc. The method includes the step of identifying a defect or deficiency. A method for determining intervertebral disc defects is in the art. Although it is publicly known, in certain embodiments, it includes CT scans and magnetic resonance imaging. These include discography, combinations thereof, and others. In certain cases, the intervertebral disc is formed by notochord cells. If it is determined that an individual has a decrease in [a certain characteristic], the individual may require treatment according to the Disclosure.

[0048] For example, an individual undergoing treatment may have intervertebral disc problems due to an unknown cause, The individual may be 50 years of age or older, or an athlete. Specific Embodiments In this case, the individuals are in their 20s, 30s, or 40s.

[0049] In certain embodiments, this disclosure relates to the contents of a cell and one or more younger cells. Cellular mixture containing several cells present in or isolated from stronger intervertebral discs The method includes a process for improving the environment of aged intervertebral discs. Cellular composites derived from or found within the intervertebral discs of humans who have not undergone the degenerative process. By providing an effective amount of this substance to individuals with aged intervertebral discs, aging occurs in the individual. It is included to improve the condition of the intervertebral disc. In certain embodiments, the cell mixture is linear Fibroblasts; Stem cells; Adipocytes; Notochord cells; Tie2+ cells; PRP+ cells; Sox9+ cells Cells; TGFB1+ cells; CTGF+ cells; WISP1+ cells, WISP2+ cells or so This includes combinations of the following: In certain cases, the individual is the Tie2 gene product; platelet-rich plasma (P RP); Sox9 gene product; TGFB1 gene product; CTGF gene product; WISP1 One or more of the following are provided: a gene product; a WISP2 gene product; or a combination thereof. ru. [Examples]

[0050] The following examples are included to demonstrate preferred embodiments of the present invention. The technology disclosed herein was discovered by the inventors and works well in practice with the present invention. It represents the technology and, therefore, is considered to constitute a preferred form for its practice. Those skilled in the art should recognize that this disclosure may be possible. However, those skilled in the art should recognize that this disclosure is not possible. In light of this, many of the disclosed embodiments do not depart from the spirit and scope of the invention. It should be recognized that by making changes, similar or comparable results can still be achieved. ru.

[0051] Example 1 One example of the method of this disclosure is allogeneic, heterogeneic, or autologous nucleus pulposus cells, and / or there The conditioned medium produced from, and / or other genes that aid in the production of Sox9 or cartilage. Genes and / or chord cells, and / or Tie2+ cells, and / or poly There is the introduction of platelet-plasma (PRP). In certain embodiments, such delivery is Delivering cells found in large quantities within healthy nuclei pulposus is beneficial to the differentiation process. A decrease in the amount of ox9 or other genes, notochord cells and / or Tie2+ is associated with degenerative intervertebral discs. They play an important role in the development of plate disease. These cells are transferred to new nuclei that arise from fibroblast differentiation. By adding back the cells, a stronger and more vibrant environment is created for the newly differentiated cells. This will result in... Cartilage generally has limited access to blood flow and nutrients, so in the body... It is considered to be the most difficult organization to regenerate. For this reason, Sox9 or other elements such as other genes, notochord cells, Tie2+ cells and / or PRP It can be added to a fibroblast mixture. This study shows that the reduction of these cell types is actually This suggests that it may be a trigger or origin of degenerative intervertebral disc disease.

[0052] References All patents and publications mentioned in this specification represent the level of expertise of those skilled in the art to whom this invention relates. To the same extent as when each individual publication is specifically and individually indicated by reference. All patents and publications are incorporated herein by reference in their entirety.

[0053] Patents and patent applications U.S. Patent No. 7,850,983, U.S. Patent Application Publication No. 2014 / 0314726; United States Patent Application Publication No. 2014 / 0044682; U.S. Patent Application Publication No. 2014 / 03772 Issue 31; and WO / 2015 / 035395

[0054] Publications Feng, G., L. Li, H. Liu, Y. Song, F. Huang, C. Tu, B. Shen, Q. Gong, T. Li, L. Liu, J. Zeng, Q. Kong, M. .Yi, M.Gupte, PXMa, F.Pei; "Hypoxia is a factor in 3D scaffolding." "Osteoarthrit" differentially regulates extracellular matrix production in nucleus pulposus and annular fibrous cells. is and Cartilage 21 (2013) pp. 582-588.

[0055] Gruber HE1, Hoelscher GL, Leslie K, Ingram JA, Hanley EN Jr. "Human in agarose or collagen sponge" "3D Culture of Intervertebral Disc Cells: Evaluation of Proteoglycan Production" Biomaterials.2 January 006;27(3):371~6, Epub August 11, 2005.

[0056] Lijie Zhang, Jerry Hu, Kyriacos A.Athanas iou "The Role of Tissue Engineering in Articular Cartilage Repair and Regeneration" Crit Rev Biom ed Eng. 2009;37(1~2):pp.1~57.

[0057] Majumdar MK, Wang E and Morris EA. "BMP-2 and BMP-9 promotes chondrogenesis and differentiation of human multipotent mesenchymal cells and inhibits IL-1. "Overcoming [the problem]" J.Cell.Physiol. 2001 (189): pp. 275-284 .

[0058] Seppa, N., "Cartilage creation. New joint tissue keeps people active, knee or hip." "May reduce the need for joint replacement," Science News 2012. 189#3):22 pages.

[0059] Tang, X. William J. Richardson, Robert D. Fi. tch, Christopher R. Brown, Robert E. Isaacs, "A novel non-enzymatic method for isolating human intervertebral disc cells preserves the phenotype of nucleus pulposus cells." Jun Chen, Cytotechnology (2014), 66:979~98 6 pages.

[0060] Wang SZ1, Rui YF, Lu J, Wang C. "Cells of intervertebral disc degeneration and "Molecular Biology: Current Understanding and Implications for Potential Therapeutic Strategies" Cell Prolif .2014 Oct;47(5):381~90, doi:10.1111 / cpr. 12121.Epub August 11, 2014.

[0061] Zaslav K. Cole B. et al.: The American Journal of Sports Medicine.2009;37(1):42-55.

[0062] Zaslav K. Cole B et al., "Patients who have failed with previous treatments for articular cartilage "Prospective Study of Autologous Chondrocyte Transplantation in the American Journal" f Sports Medicine.2009;37(1):42-55.

[0063] The present invention and its advantages have been described in detail, but the finer points of the present invention as defined in the appended claims are as follows: Various changes, substitutions, and modifications may be made herein without deviating from God and the scope. It should be understood that the scope of this application is limited to the processes, machines, and manufacturing processes described in the specification. The purpose is to limit the construction, composition of the object, means, methods and processes to specific embodiments. i. As described herein, it will be readily apparent to those skilled in the art from the disclosure of the present invention. Current versions that perform substantially the same functions as the corresponding embodiments, or achieve substantially the same results. This also includes existing or future-developed processes, machines, manufacturing processes, compositions of objects, means, and methods. The process may be utilized in accordance with the present invention. Accordingly, the appended claims may be used in accordance with the present invention. The scope of this includes processes, machinery, manufacturing, compositions of objects, means, methods, or steps. To target.

Claims

[Claim 1] The invention described in the specification.