KR102167440B1 - Methods of generating, repairing and/or maintaining connective tissue in vivo - Google Patents

Abstract

The present invention relates to a method for producing, repairing and/or maintaining connective tissue in a subject. In one embodiment, the invention relates to a method of producing, treating and/or maintaining cartilage tissue in a subject. The present invention also relates to a method for treating and/or preventing a disease caused by degradation or inflammation of connective tissue in a subject.

Description

How to create, repair and/or maintain connective tissue in vivo {METHODS OF GENERATING, REPAIRING AND/OR MAINTAINING CONNECTIVE TISSUE IN VIVO}

The present invention relates to a method for producing, repairing and/or maintaining connective tissue in a subject. In addition, the present invention relates to a method for treating and/or preventing diseases caused by decomposition or inflammation of connective tissue in a subject.

Non-hematopoietic progenitor cells reside in the body and, upon isolation, become pluripotent cells called mesenchymal progenitor cells (MPCs). More specifically, purified MPCs are capable of forming a wide variety of pluripotent cell colonies.

Simmons et al. (1994) describe a method of enriching MPCs from newly harvested bone marrow by selecting cells expressing the STRO-1 cell surface marker. As noted by the authors on pages 272-273, bone marrow cells are known to contain significant amounts of MPC, which can produce CFU-F. This CFU-F can then generate a wide variety of fully differentiated connective tissues such as cartilage, bone, adipose tissue, fibrous tissue and myelosupportive stroma under appropriate conditions.

MPC and CFU-F are typically present in very low concentrations in bone marrow cells (typically 0.05%-0.001%), so this rarity has been a major limitation of studies on them in the past. In the discussion of Simmons et al. (1994), it was confirmed that it is possible to enrich these MPCs to some extent from newly isolated bone marrow cells by selecting STRO-1 positive cells. In particular, MPC (and generated CFU-F) in which hematopoietic progenitor cells were not mixed could be isolated by selection of STRO-1 positive cells.

In WO 01/04268, more important progress has been made in MPC enrichment by identifying subpopulations in the STRO-1 positive cell fraction containing MPC. In particular, in WO 01/04268, the STRO-1 positive cell population was classified into three subgroups: STRO-1 ^dull , STRO-1 ^intermediate and STRO-1 ^bright . Through the colonogenic assay of CFU-F for various subgroups, it was proved that most of the MPCs were contained in the STRO-1 ^bright fraction.

WO 2004/085630 (Gronthos et al ) describes for the first time that MPCs exist in perivascular tissues. One of the benefits of this discovery is that it can greatly expand the range of feed tissues from which MPCs can be isolated or enriched, and that MPC sources can no longer be limited to bone marrow. According to the method of WO 2004/085630, tissues from which MPCs can be separated are human bone marrow, dental pulp, adipose tissue, skin, spleen, pancreas, brain, kidney, liver and heart. MPCs isolated from perivascular tissues are positive for 3G5, a cell surface marker. Thus, this is through enrichment of cells carrying the 3G5 marker, through enrichment of early-occurring surface markers present in perivascular cells such as CD146 (MUC18), VCAM-I, or through enrichment of the cell surface marker STRO-1. It can be separated through high expression thickening.

Avascular connective tissues are usually located in anatomical areas in the musculoskeletal system that require significant movement. Freely moving joints make up the nodes of most mammals. In synovial joints, the contact surfaces of the two opposing bones are effectively each other due to the presence of a low friction lubricant in the joint synovial fluid produced by cells lining the inside of the joint capsule that connects and covers the long bone. It is covered with gliding hyaline cartilage. In the spinal node, the joint is made by connecting rigid spinal bones with a flexible fibrocartilage ring (ring of fibroblasts) containing a hydrated gelatinous material (nucleus) in which chondrocytes similar to chondrocytes present in vitreous cartilage are densely distributed. Regardless of the type and location of the avascular connective tissue, both of them absorb water together with fibrous protein and type 2 collagen to synthesize an extracellular matrix containing a large amount of high negatively charged proteoglycans giving high tensile strength. It contains cells.

Avascular connective tissues, such as vitreous cartilage, internal meniscus 2/3, and intervertebral discs, have limited resilience and, when damaged, respond by functionally producing subfibrous cartilage scar tissue. Due to a plurality of factors dominant in aging, heredity, hormonal status, and physical injury, such avascular association cannot be achieved, leading to various clinical problems such as disc degeneration, back pain and osteoarthritis.

Current medical treatments commonly used to treat symptoms caused by failure of these connective tissues hardly resolve the underlying condition that causes the symptoms to occur, and in many cases, structural components of the extracellular matrix of tissues The problem is further exacerbated by degrading the performance of the synthetic resident cells. Ideally, the therapeutic treatment should at least protect cartilage, while improving the biosynthesis of the matrix and providing an environment effective in repairing and repairing damaged connective tissue.

Aggarwal et al. (2005) Blood. 105: 1815-1822. Ahrens et al. (1993) DNA Cell Biol. 12: 871-880. Allcock et al. (1977) Macromolecule 10:824. Anseth et al. (2002) J. Control Release 78:199-209. Appleyard et al. (1999) Osteoarthritis and Cartilage 7:281-294. Appleyard et al. (2003) Osteoarthritis Cartilage 11: 65-77. Arnoczky et al. In: Injury and repair of the musculoskeletal soft tissues. Eds, Woo S L-Y and Buckwalter. American Academy of Orthopaedic Surgeons, Park Ridge, Ill: 1988, pp. 487-37. Bellamy et al. (2006) Cochrane Database Syst Rev 2006(2):CD005321 Brandt (1993) Agents and Actions 40: 232-234. Brandt (1993a) Rheumatic Disease Clinics of North America. 19:29-44. Brandt et al. (2000) Arthritis Rheum. 43: 1192-1203. Bregni et al., Blood 80:1418-22. Burkhardt et al. (2001) Osteoarthritis Cartilage 9: 238-47. Cake et al. (2000) Osteoarthritis Cartilage 8: 404-411. Cake et al. (2003) Osteoarthritis and Cartilage 11: 872-8. Cake et al. (2004) Osteoarthritis and Cartilage 12: 974-81. Cake et al. (2005) Osteoarthritis Cartilage 13: 1066-75. Cake et al. Clinical and Experimental Rheumatology 2008 (in press). Dumond et al. (2003) Arthritis Rheum. 48: 3009-12. Englund (2004) Arthritis Rheum 50:2811-9. Ghosh et al. (1983) J Orthop Res I:153-173. Ghosh et al. (1983a) J Surg Res 35:461-473. Ghosh (1988) In: Bailliere's Clinical Rheumatology, International Practice and Research. Ed. P Brooks, Tindall, Saunders, London, UK, pp 309-338. Ghosh et al. (1990) Clin. Orthop. 252: 101-113. Ghosh (1991) J Rheumatol. 18:143-6. Ghosh et al. (1993) Curr. Ther. Res. 54: 703-713. Ghosh et al. (1993a) Semin. Arthritis Rheum. 22 (Suppl. 1): 18-30. Ghosh et al. (1993b) Semin Arthritis Rheum 22 (Suppl 1): 31-42. Ghosh et al. (1993c) Agents Actions Suppl 39: 89-93. Ghosh et al (2002) Semin. Arthritis Rheum. 32: 10-37. Gronthos et al. (1995). Blood 85:929-940. Gronthos et al. (2003) Journal of Cell Science 116: 827-1835. Huskisson et al. (1995) Journal of Rheumatology 22:1941-6. Hwa et al. (2001) J. Rheumatol. 28: 825-834. Jorgensen et al. (1987) J Bone Joint Surg Br. 69: 80-3. Little et al. (1997) J Rheumatol. 24: 2199-2209. Lo et al. (2003). JAMA. 290:3115-3127. Masuda et al. (2004) Spine 30:5-14. McKenzie et al. (1976) Ann. Rheum. Dis. 35: 487-497. McNicholas et al. (2000) J Bone Joint Surg Br. 82: 217-21. Melrose et al. (2003) J Histochem Cytochem 5:1331-1341. Melrose et al. (2002) Spine 27:1756-1764. Melrose et al. (2000) Histochem Cell Biol 114:137-46. Melrose et al. (2002a) Histochem Cell Biol 117:327-33. Melrose et al. (1998) J Vasc Surg 28:676-686. Moon et al. (1984) Clin Orthop Related Res 182:264-9. Nevitt et al. (1996) Arch Intern Med 156: 2073-80. Oakley et al. (2004) Osteoarthritis Cartilage 12: 667-79. Parker et al. (2003) J. Rheumatol. 6(2): 116-127. Pelletier et al. (2007) Arthritis Res Ther. 9: R74. Pfirrmann et al. (2001) Spine 26:1873-1878. Panjabi et al. (1985) J Orthop Res 3:292-300. Race et al. (2000) Spine 25:662-9. Richette and Bardin (2004) Joint Bone Spine 71:8-23. Roos et al. (1998) Arthritis Rheum 41: 687-93. Roos et al. (2001) Osteoarthritis and Cart. 9: 316-24. Sakai et al. (2005). Spine 30:2379-87. Simmons et al. (1994) Advances in Bone Marrow Purging and Processing: Fourth International Symposium, 271-280. Smit (2002) Eur Spine J 11:137-44. Smith and Ghosh (2001) Experimental models of osteoarthritis in: Osteoarthritis, Eds: Moskowitz et al. WB Saunders Company; pp171-199. Smith et al. (1997) Pathol. Biol. [Paris]. 45: 313-320. Teichtahl et al. (2005) Medical Hypothesis 65: 312-12. Wang et al. (2003) Biomaterials 24:3969-3980. Wilke et al. (1999) Spine 24:755-62. Zannettino et al. (1998) Blood 92:2613-2628.

Summary of the invention

The present inventors have confirmed the surprising fact that the intra-articular administration of MPC provides a cartilage-protective effect to existing joints with osteoarthritis, and induces the production and growth of cartilage tissue in the synovial joint and the nucleus of the intervertebral disc. This fact means that MPCs or their progeny, or supernatants or soluble factors derived from these MPCs can be used to protect or repair damaged connective tissue, as well as to create new functional tissue at the site of degeneration or lesion. do.

Accordingly, the present invention provides a method for treating and/or preventing a disease caused by decomposition or inflammation of connective tissue in a subject, the method comprising MPC and/or its progeny cells and/or a soluble factor derived therefrom. And administering to the subject.

In one embodiment of the invention, the connective tissue is rich in proteoglycans. The connective tissue may be cartilage, such as vitreous cartilage. In another embodiment, the disease is caused by a cartilage defect.

In another embodiment, the method comprises administering to the subject an MPC and/or progeny cell thereof and/or a soluble factor derived therefrom, wherein the MPC and/or progeny cell and/or soluble factor is at the site of the defect. It is not administered directly.

For example, administration may be made in the joint space to treat or prevent a defect in cartilage on the joint surface of the bone forming the joint. Similarly, administration can be made in the intervertebral disc space to treat or prevent defects in the peripheral disc. In another embodiment, the administration is intravenously at the site of the connection defect.

MPC and/or progeny cells and/or soluble factors can be administered by intraarticular injection. Intra-articular injection can be made to any joint in the body adjacent to the site of a cartilage defect or a site of a possible cartilage defect. For example, intraarticular injection can be made to the knee joint, hip joint, ankle joint, shoulder joint, elbow joint, wrist joint, hand or finger joint, foot joint or intervertebral disc joint.

In another embodiment of the invention, administration of MPC and/or progeny cells and/or soluble factors results in the maintenance or production of cartilage rich in proteoglycans and type 2 collagen. An example of cartilage rich in proteoglycans and type 2 collagen is vitreous cartilage. Preferably, the cartilage maintained or produced by the present invention is rich in type 1 collagen, the content of type 2 collagen is very small, and the content of proteoglycan is lower than that of vitreous cartilage, and is not fibrocartilage.

In embodiments of the disease "caused by decomposition and/or inflammation of connective tissue", tendinitis, back pain, rotary cuff tendon degradation, Carpal tunnel syndrome, DeQuervain's syndrome ), degenerative cervical and/or lumber discs, intersection syndrome, reflex sympathetic dystrophy syndrome (RSDS), stenosing tenosynovitis, phase There are epicondylitis, tenosynovitis, thoracic outlet syndrome, ulnar nerve entrapment, radial tunnel syndrome, and repetitive strain injury (RSI). It is not limited to these. Examples of diseases associated with decomposition and/or inflammation of vitreous cartilage include, but are limited to, osteoarthritis, rheumatoid arthritis, arthritis such as psoriatic arthritis and seronegative arthritis, inflammatory bowel disease or arthritis associated with ankylosing spondylitis, and intervertebral disc degeneration disorders. It is not.

In another preferred embodiment, the method further comprises administering hyaluronic acid (HA). HA can be administered in the same or different composition as the cells, supernatant and/or factor(s).

In addition, the present invention provides a composition comprising MPC and/or its progeny cells and/or hyaluronic acid.

The results presented herein indicate for the first time that soluble factors secreted from transplanted cultured MPCs support the protection, production and growth of connective tissue.

That is, the present invention provides a composition comprising the following ingredients:

i) a supernatant, or one or more soluble factors, derived from mesenchymal progenitor cells (MPC) and/or progeny cells thereof, and

ii) hyaluronic acid.

In another aspect, the present invention is a supernatant derived from mesenchymal progenitor cells (MPC) and/or progeny cells thereof, for the treatment and/or prevention of diseases caused by degradation and/or inflammation of connective tissue in a subject. , Or one or more soluble factors.

The present invention is applicable to various categories of animals. For example, the subject may be a mammal such as a human, dog, cat, horse, cow or sheep. In one example, the subject is a human.

Throughout this specification, variations such as the terms "comprises" or "comprising" or "comprising" include the inclusion of the recited element, integer or step, elements, integers or groups of steps, and other elements, It will be understood that it does not exclude an integer, step, or group of elements, integers or steps.

The present invention is described below with reference to the following non-limiting examples and the accompanying drawings.

General description and optional definition

Except where specifically stated, all technical and scientific terms used herein are in the art (e.g., cell culture, stem cell biology, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry). It should be understood to have the same meaning as commonly understood by one of ordinary skill in the art.

Unless otherwise stated, the recombinant proteins, cell culture and immunological techniques used in the present invention are standard procedures well known to those skilled in the art. These techniques were described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), TA Brown (editor). , Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), DM Glover and BD Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and FM Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and JE Coligan et al . (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

As used herein, the terms “treating”, “treating” or “treatment” refer to administration of a therapeutically effective amount of a supernatant, soluble factor and/or cells according to the invention sufficient to reduce or eliminate one or more symptoms of a particular condition. Include.

As used herein, the term “preventing”, “preventing” or “preventing” refers to a therapeutically effective amount of a supernatant, soluble factor and/or cell according to the invention sufficient to stop or prevent the progression of one or more symptoms of a particular condition. Includes administration.

As used herein, the term “derived from mesenchymal progenitor cells” refers to a supernatant produced by culturing mesenchymal progenitor cells and/or progeny cells thereof in vitro, and/or one or more soluble factors.

As used herein, the term “supernatant” refers to a non-cellular material produced by culturing mesenchymal progenitor cells, and or progeny cells thereof, in vitro in an appropriate medium, preferably a liquid medium. Typically, the supernatant is prepared by culturing cells in a medium for appropriate conditions and time, and then removing cellular material by a process such as centrifugation. The supernatant may or may not have undergone an additional purification step prior to administration. In a preferred embodiment, the supernatant contains less than 10 ⁵ , more preferably less than 10 ⁴ , even more preferably less than 10 ³ , or even more preferably no living cells.

As used herein, the term “one or more soluble” factors refers to molecules, typically proteins, that are secreted from MPC and/or its progeny cells during cultivation.

Mesenchymal progenitor cells ( MPC ) or progeny cells, and derived from them An upper layer of water or one or more soluble factors

As used herein, “MPC” is a non-hematopoietic STRO-1 ⁺ progenitor cell capable of forming multiple pluripotent cell colonies.

Mesenchymal progenitor cells (MPC) are bone marrow, blood, dental pulp cells, adipose tissue, skin, spleen, pancreas, brain, kidney, liver, heart, retina, brain, hair follicle, intestine, lung, and lymph nodes. , Thymus, bones, ligaments, tendons, skeletal muscle, dermis and periosteum as cells found in, which can differentiate into various germ lines such as mesoderm, endoderm and ectoderm. Thus, MPCs are capable of differentiating into numerous cell types, including, but not limited to, adipose tissue, bone tissue, cartilage tissue, elastic tissue, muscle tissue and fibrous connective tissue. The specific lineage-commitment and differentiation pathways through which these cells enter are derived from endogenous bioactive factors such as mechanical influences and/or local microenvironmental conditions established by growth factors, cytokines and/or host tissue. It depends on various influences. Mesenchymal progenitor cells are, therefore, non-hematopoietic progenitor cells that become daughter cells, which are progenitor cells or stem cells that divide and soon become irreversibly differentiated to become phenotypic cells.

In a preferred example, a sample taken from a subject is enriched with mesenchymal progenitor cells (MPC). The terms'enriched','enriched' or variations thereof herein refer to a population of cells in which the proportion of one specific cell type or of multiple specific cell types is increased compared to the untreated population.

In a preferred embodiment, the cells used in the present invention are TNAP ⁺ , VCAM-1 ⁺ , THY-1 ⁺ , STRO-2 ⁺ , CD45 ⁺ , CD146 ⁺ , 3G5 ⁺ or a combination thereof. Preferably, the STRO-1 ⁺ cells are STRO-1 ^bright . Preferably, the STRO-1 ^bright cells are additionally one or more of VCAM-1 ⁺ , THY-1 ⁺ , STRO-2 ⁺ and/or CD146 ⁺ .

In one embodiment, the mesenchymal progenitor cells are perivascular mesenchymal precursor cells as described in WO 2004/85630.

When a cell is referred to as "positive" for a corresponding marker, it may be either a low (lo or dim) or high (bright, bri) expression of the marker depending on the degree of the marker present on the cell surface, and the term Is related to the intensity of fluorescence or other colors used in the cell's color classification process. The distinction between lo (or dim or dull) and bri will be understood in terms of the markers used to classify specific cell populations. When referring to a cell as “negative” for that marker, it does not mean that the marker is not expressed at all in the cell. This means that the marker is expressed at a relatively very low level in cells, and forms a very low signal when the marker is detectably labeled.

As used herein, “bright” refers to a marker on the cell surface that forms a relatively strong signal when detectably labeled. Without being bound by theory, it is suggested that “bright” cells express more of the target marker protein (eg, antigen recognized by STRO-1) than other cells in the sample. For example, STRO-1 ^bri cells form a stronger fluorescence signal than non-bright cells (STRO-1 ^dull/dim ) when labeled with FITC-conjugated STRO-1 antibody, as determined by FACS analysis. Preferably, the “bright” cells comprise at least about 0.1% of the brightest labeled bone marrow monocytes contained in the starting sample. In other examples, “bright” cells comprise at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, or at least about 2% of the brightest labeled bone marrow monocyte cells contained in the starting sample. In a preferred example, STRO-1 ^bright Cells show 2 log high STRO-1 surface expression. This "background", that is, STRO-1 ^- is calculated relative to the cells. In comparison, STRO-1 ^dim and/or STRO-1 ^intermediate cells show less than 2 log of STRO-1 surface expression, typically less than about 1 log than “background”.

The term "TNAP" as used herein is intended to encompass all isoforms of tissue non-specific alkaline phosphatase. For example, the term includes liver isoform (LAP), bone isoform (BAP) and kidney isoform (KAP). In a preferred embodiment, the TNAP is BAP. Particularly preferably, the TNAP used herein is a molecule capable of binding to the STRO-3 antibody produced in the hybridoma cell line deposited with the ATCC as accession number PTA-7282 on December 19, 2005 under the provisions of the Budapest Treaty. It is called.

In addition, in a preferred embodiment, the MPC can grow as clonogenic CFU-F.

Preferably, a significant proportion of the pluripotent cells are capable of differentiating into at least two or more different germ cells. In a non-limiting embodiment of a lineage in which pluripotent cells can be designated, bone progenitor cells; Pluripotent hepatocyte progenitors, which can become bile duct epithelial cells and hepatic cells; Oligodendrocytes and neural-defining cells capable of producing glial progenitor cells that proceed to astrocytes; Neural progenitor cells that proceed to nerves; There is a progenitor cell for cardiac muscle and cardiomyocytes, a glucose-responsive insulin secreting pancreatic β cell line. Other lineages include, but are not limited to, odontoblasts, dentin-producing cells and chondrocytes, and progenitor cells of the following cells: skin cells such as retinal pigment epithelial cells, fibroblasts, keratinocytes, Dendritic cells, hair follicle cells, renal duct epithelial cells, smooth muscle and skeletal muscle cells, testicular striatum, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, marrow stroma, heart Muscle, smooth muscle, skeletal muscle, pericyte, blood vessel, epithelium, glue, nerve, astrocyte and oligodendrocyte.

In other embodiments, MPCs are unable to become hematopoietic cells in culture.

In addition, the present invention relates to the use of a supernatant or soluble factor obtained from MPCs and/or their progeny cells (the latter also referred to as expanded cells) produced through in vitro culture. The amplified cells of the present invention may have a wide variety of phenotypes depending on culture conditions (including the number and/or type of stimulating factors in the culture medium), the number of passages, and the like. In certain embodiments, progeny cells are obtained after about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 passages from the parent population. However, progeny cells can also be obtained after culturing an arbitrary number of times from the parent population.

Progeny cells can be obtained by culturing in any suitable medium. The term "medium" as used herein for cell culture includes the environmental components surrounding the cell. The medium may be a solid, liquid, gas or mixture of phases and materials. The medium includes not only a liquid growth medium but also a liquid medium that does not support cell growth. In addition, the medium includes a gelatinous medium such as agar, agarose, gelatin and collagen substrate. Embodiments of the gas medium include a gas phase to which cells growing on a petri dish or other solid or semi-solid support are exposed. The term "medium" also refers to a substance intended to be used in cell culture, even if it is not in contact with the cells. That is, a nutrient rich liquid prepared for culturing bacteria is also a medium. Similarly, powder mixtures that, when mixed with water or other liquids, become suitable for cell culture may be referred to as “powder medium”.

In an embodiment, the progeny cells usable in the method of the present invention were amplified by culturing the isolated cells after separating TNAP+ MPC from bone marrow using magnetic beads labeled with STRO-3 antibody (appropriate culture conditions In an embodiment of Gronthos et al. (1995)).

In one embodiment, such amplified (offspring) cells (a minimum of five passages) is TNAP-, CC9 ^+, HLA class I ^+, HLA class ^{II -, CD14 -, CD19 -} , CD3 -, CD11a-c - , CD31 ^-, CD86 ^- and / or CD80 ^- can be. However, the expression of various markers may be changed under culture conditions different from those described above. Also, while cells of this phenotype may dominate in the amplified cell population, this does not mean that the proportion of cells that do not have this phenotype(s) is small (e.g., a small proportion in amplified cells is CC9- Can be). In one preferred embodiment, the amplified cells still have the ability to differentiate into several cell types.

In one embodiment, the amplified cell population used to obtain the supernatant or soluble factor, or the cells themselves, comprises cells in which at least 25% of the cells are, more preferably at least 50%, are CC9+.

In another embodiment, the amplified cell population used to obtain the supernatant or soluble factor or cells themselves comprises cells in which at least 40% of the cells are, more preferably at least 45%, are STRO-1 ⁺ .

In another embodiment, the progeny cells are LFA-3, THY-1, VCAM-1, ICAM-1, PECAM-1, P-selectin, L-selectin, 3G5, CD49a/CD49b/CD29, CD49c/CD29, CD49d/ CD29, CD90, CD29, CD18, CD61, Integrin β, 6-19, thrombomodulin, CD10, CD13, SCF, PDGF-R, EGF-R, IGF1-R, NGF-R, FGF-R , Leptin-R, (STRO-2 = Leptin-R), RANKL, STRO-1 ^bright, and a marker selected from the group consisting of CD146, or a combination of these markers.

In one embodiment, the progeny cell is a pluripotent amplified MPC progeny (MEMP) as defined in WO 2006/032092. Methods of preparing enriched populations of MPCs from which offspring can be derived are described in WO 01/04268 and WO 2004/085630. In vitro, MPCs are difficult to exist as absolutely pure preparations and will generally exist with other cells, which are tissue specific committed cells (TSCCs). WO 01/04268 describes a method for obtaining bone marrow-derived cells with a purity of about 0.1% to 90%. The population containing the MPC from which the progeny are derived may be harvested directly from a tissue source, or may be a population already amplified ex vivo.

For example, progeny are harvested, unamplified, substantially purified, accounting for at least about 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80 or 95% of the total cells of the population in which they are present. Can be obtained from a population of MPCs. This level can be achieved, for example, by selecting cells that are positive for one or more markers selected from the group consisting of TNAP, STRO-1 ^bright , 3G5 ⁺ , VCAM-1, THY-1, CD146 and STRO-2.

MEMP can be distinguished from just recovered MPC in that it is positive for the marker STRO-1 ^bri and negative for the marker alkaline phosphatase (ALP). On the other hand, membrane-isolated MPC is positive for both STRO-1 ^bri and ALP. In a preferred embodiment of the present invention, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% or more of the administered cells are the phenotype STRO-1 ^bri , ALP ^- it has a. In a more preferred embodiment, the MEMP is positive for one or more of the markers Ki67, CD44 and/or CD49c/CD29, VLA-3, α3β1. In another preferred embodiment, the MEMP does not exhibit TERT activity and/or is negative for marker CD18.

The MPC starting population is any one or more tissue types described in WO 01/04268 or WO 2004/085630, i.e. bone marrow, dental pulp, adipose tissue and skin, or more broadly adipose tissue, teeth, pulp, It can be derived from skin, liver, kidney, heart, retina, brain, hair follicle, intestine, lung, spleen, lymph nodes, thymus, pancreas, bone, ligaments, bone marrow, tendons and skeletal muscles.

In the practice of the present invention, cells carrying any given cell surface marker can be isolated in a number of different ways, but the preferred method is dependent on the binding of the binding agent to the marker of interest, and high levels of binding, low levels. It is separated from those that show or have no binding. The most common binding agents are antibodies or antibody-based molecules, preferably monoclonal antibodies or based on monoclonal antibodies because of the specificity of these latter substances. Antibodies can be used in two steps, but since other substances can also be used, ligands for these markers can also be adopted to enrich cells with or without them.

The antibody or ligand can be attached to a solid support to allow for crude separation. The separation technique is, preferably, a method of maximizing the viability maintenance of the collected fractions. A variety of techniques of varying efficacy can be employed to properly obtain crude isolates. The specific technique applied will depend on the efficiency of the separation, the cytotoxicity involved, the ease and speed of performance, and the need for sophisticated equipment and/or technical capabilities. The separation process includes, but is not limited to, magnetic separation using antibody-coated magnetic beads, affinity chromatography, and “panning” using an antibody attached to a solid matrix. FACS is a technique that provides accurate separation, but is not limited thereto.

It is preferable that the MPC separation method includes, for example, a solid phase classification step of the first step utilizing MACS that recognizes high level expression of STRO-1. Thereafter, a second step may be involved, and as disclosed in patent WO 01/14268, it is desirable to further increase the expression level of progenitor cells. This second sorting step may include using two or more markers.

The method of obtaining MPC may also include harvesting a source of cells by known techniques prior to the first enrichment step. Thus, the tissue will be surgically excised. Cells containing the source tissue will then be separated into so-called single cell suspensions. This separation can be accomplished in a physical and/or enzymatic manner.

Once a suitable MPC population has been obtained, it can be cultured or amplified in any suitable manner to obtain MEMPs.

In one embodiment, the cells are collected from the subject to be treated, cultured in vitro using a standard technique, and then used as a supernatant or soluble factor for administration to the subject as an autologous or allogeneic composition. Alternatively, an amplified cell is obtained. In another embodiment, cells of one or more established human cell lines are used to obtain a supernatant or soluble factor. In another useful embodiment of the invention, cells from non-human animals (or if the patient is not human and of a different species) are used to obtain the supernatant or soluble factor.

The present invention is derived from all animal species other than humans, such as cells of primates except humans, ungulate, canine, feline, lagomorph, rodent, bird, and fish cells It can be carried out using cells of. The primate cells that can be used in the present invention include, but are not limited to, chimpanzee, baboon, cynomolgus monkey, and any other cells of New World or Old World monkeys. The ungulate cells that can be used in the present invention include, but are not limited to, cells of cows, pigs, sheep, goats, horses, buffalo and bison. Rodent cells that can be used in the present invention include mice, rats, guinea pigs, hamsters, and gerbils, but are not limited thereto. Examples of rabbits that can be used in the present invention include rabbits, jack rabbits, hares, white-tailed rabbits, snowshoe rabbits, and pika, It is not limited to these. Chicken ( Gallus gallus ) is an example of algae that can be used in the present invention.

Cells usable in the method of the present invention can be stored prior to use or prior to obtaining a supernatant or soluble factor. Methods and protocols for preservation and storage of eukaryotic cells, particularly mammalian cells, are well known in the art (comparatively, eg, Pollard, JW and Walker, JM (1997) Basic Cell Culture Protocols, Second Edition, Humana Press, Totowa, NJ; Freshney, RI (2000) Culture of Animal Cells, Fourth Edition, Wiley-Liss, Hoboken, NJ). Any method of maintaining the biological activity of isolated stem cells such as mesenchymal stem/progenitor cells or progeny thereof can be applied to the present invention. In one preferred embodiment, the cells are maintained and stored using cryo-preservation.

Administration and composition

Supernatant or soluble factor

The method of the present invention may comprise applying the MPC-derived supernatant or soluble factors topically, systemically or locally, such as inside an implant or device.

In one specific embodiment, the present invention comprises systemic administration of an MPC-derived supernatant or soluble factor to a subject. For example, the supernatant or soluble factor can be administered by subcutaneous or intramuscular injection.

This embodiment of the present invention may be useful for the treatment of systemic degenerative diseases in which the production or repair of specific tissues is desirable. Examples of systemic degenerative diseases treatable in this way include osteoporosis or fractures, or degenerative diseases of cartilage.

In addition, MPC-derived supernatant or soluble factor is used to provide cosmetic functions, such as stretching the surface or other parts of the body, or to repair or replace cartilage tissue in which disease or trauma has occurred, or tissue that has not occurred normally. Patients who need this can be treated. Treatment may involve the creation of new cartilage tissue and/or the use of supernatants or soluble factors to maintain existing cartilage tissue. For example, MPc-derived supernatants or soluble factors can be used to treat cartilage conditions such as rheumatoid arthritis or osteoarthritis, or trauma or surgical wounds of cartilage.

MPC-derived supernatants or suspensions containing soluble factors can be formulated as oily suspensions suitable for injection. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil; Synthetic fatty acid esters such as ethyl oleate or triglycerides; Or liposomes. In addition, the suspension used for injection may contain substances that increase the viscosity of the suspension such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or substances that increase the solubility of the compound to enable preparation of highly concentrated solutions.

Sterile injectable solutions can be prepared by mixing the requisite amount of the supernatant or soluble factor with one or a combination of the above-listed components in a suitable solvent, and then sterilizing by filtration, if necessary. In general, dispersions are prepared by incorporating a supernatant or soluble factor into a sterile vehicle containing a basic dispersion medium and essential ingredients other than those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is vacuum drying and freeze drying, making a powder of the active ingredient and the desired additional ingredients from an already sterile-filtered solution. In another aspect of the invention, the supernatant or solubility factor may be formulated with one or more additional compounds that increase its solubility.

Cellular composition

In one embodiment, the cellular composition of the invention is administered as undifferentiated cells, such as cultured in a culture medium. As another example, the cellular composition may be administered after culture.

The cellular composition useful in the present invention can be administered alone or in combination with other cells. Cells that can be administered with the composition of the present invention include other multipotent or pluripotent cells, or chondrocytes, chondrocytes, bone cells, osteoblasts, osteoclasts, bone lining cells, stem cells, or Bone marrow cells, but are not limited to these. Various types of cells can be mixed with the composition of the present invention immediately before or immediately prior to administration, or co-cultured together for a period of time prior to administration.

In some embodiments of the invention, prior to initiating treatment with the cell composition, it may not be necessary or desirable to suppress the patient's immunity. Thus, transplantation of allogeneic or heterologous MEMPs or their progeny may in some cases be acceptable.

However, in other cases, it may be desirable or appropriate to pharmacologically suppress the patient's immunity prior to initiating cell therapy. This can be accomplished using systemic or local immunosuppressants, or by delivering the cells through an encapsulated device. Cells can be encapsulated in capsules, which are permeable to nutrients, oxygen, and therapeutic factors that the cells need, while immune humoral factors and cells are impermeable. Preferably, the encapsulant is hypoallergenic, can be easily and stably positioned in the target tissue, and provides additional protection to the implanted structure. These and other methods are known in the art for reducing or eliminating the immune response to transplanted cells. As another example, cells can be genetically modified to lower their immunogenicity.

General

"Therapeutically effective amount" means an amount effective to achieve the desired effect during the dosage and requisite time period.

"Prophylactically effective amount" means an amount effective to achieve the desired prophylactic outcome, such as preventing or inhibiting apoptosis or tissue damage, during the dosage and the required time period.

The supernatant administered, the amount of soluble factor, or MPC or progeny thereof, may vary depending on factors such as the disease state, age, sex and weight of the individual. The dosing regimen can be adjusted to provide an optimal therapeutic response. For example, a bolus may be administered once, dividedly administered several times over a certain period of time, or the dosage may be proportionally reduced or increased according to the urgency of the treatment condition. It may be beneficial to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. “Dosage unit form” means a physically discrete unit controlled as a single dosage to the subject to be treated herein, each unit comprising a predetermined amount of active compound calculated to form the desired therapeutic effect in combination with the required pharmaceutical carrier. Include.

It will be appreciated that the supernatant, amount of soluble factor or MPC or progeny thereof can be administered in the form of a composition comprising a pharmaceutically acceptable carrier or excipient.

As used herein, “pharmaceutically acceptable carrier” or “excipient” includes any and all solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic agents, absorption delaying agents, and the like, which are physiologically usable. In one embodiment, it is suitable for parenteral administration. As another example, the carrier may be suitable for intravenous, intraperitoneal, intramuscular, sublingual or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile powders for preparing sterile injectable solutions or dispersions on the fly. The use of such media and substances for pharmaceutically active substances is well known in the art. Except where any conventional medium or agent is not compatible with the active compound, their use in the pharmaceutical compositions of the present invention is included. In addition, supplementary active compounds can also be incorporated into the composition.

The therapeutic composition should be sterile and stable, typically under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome or other advanced structure. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyol (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Proper flowability can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be desirable to include isotonic substances such as sugars, polyalcohols such as mannitol, sorbitol or sodium chloride in the composition. Prolonged absorption of the composition for injection can be achieved by including in the composition substances that delay absorption, such as monostearate salts and gelatin. In addition, the stimulating factor can be administered in a time release formulation, such as a composition comprising a sustained release polymer. The active compounds may be formulated with carriers that protect the compound from rapid release, such as controlled release formulations, such as implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester, polylactic acid and polylactic, polyglycolic copolymer (PLG) can be used. A number of methods for preparing such formulations have been patented and are generally known to those skilled in the art.

The supernatant, soluble factor or cellular composition can be administered in combination with a suitable matrix, such as to provide a slow release of the soluble factor.

The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface characteristics. The matrices usable for the composition are biodegradable and can be chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid and polyanhydride. Other possible substances are biodegradable and well-defined biologically, such as bone or dermal collagen. The additional matrix consists of pure protein or extracellular matrix components. Other possible matrices are non-biodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminate or other ceramics. The matrix may be composed of any combination of materials of the type described above, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. Bioceramics can be modified in a composition such as calcium-aluminate-phosphate, and processed to change pore size, particle size, particle shape, and biodegradability.

The MPC-derived supernatant or soluble factor, MPC or its progeny, is surgically implanted, injected, or delivered (e.g., using a catheter or needle) to a site in need of repair or enlargement, or otherwise It can be administered directly or indirectly. Routes of administration of the MPC-derived supernatant or soluble factor include intramuscular, ocular, parenteral (including intravenous), intraarterial, subcutaneous, oral and nasal administration. Specific routes of parenteral administration include intramuscular, subcutaneous, intraperitoneal, intracranial, intraventricular (intraventricular), intracerebroventricular, intrathecal, intrathecal, intracisternal, intrathecal and/or peripheral-spine routes of administration. However, it is not limited thereto.

In some embodiments of the present invention, the formulation is described in US Patent Publication No. 2002/0022676; Anseth et al. (2002) and Wang et al. (2003), including in situ polymerizable gels.

In some embodiments, the polymer is dissolved at least in part in an aqueous solution, such as water, a buffered salt solution, or an aqueous alcohol solution, with charged side groups or monovalent ionic salts thereof. Examples of polymers with acidic side groups that can react with cations are poly(phosphagen), poly(acrylic acid), poly(methacrylic acid), copolymers of acrylic acid and methacrylic acid, poly(vinyl acetate) and sulfonated Sulfonated polymers such as polystyrene. A copolymer having an acidic side group formed by the reaction of acrylic or methacrylic acid with a vinyl ether monomer or polymer may also be used. Examples of acidic groups include carboxylic acid groups, sulfonic acid groups, halogenated (preferably fluorinated) alcohol groups, OH groups of phenols, and acidic OH groups.

Examples of polymers having basic side groups capable of reacting with anions include poly(vinyl amine), poly(vinyl pyridine), poly(vinyl imidazole), and some imino substituted polyphosphazenes. The ammonium or quaternary salt of the polymer can be formed from the backbone nitrogen or accessory imino groups. Examples of basic side groups are amino and imino groups.

Alginate can form a hydrogel matrix by ionic crosslinking in water, at room temperature, with divalent cations. Because of these mild conditions, alginates are the most commonly used polymers for encapsulation of hybridoma cells, such as disclosed in US Pat. No. 4,352,883. In the method disclosed in U.S. Patent No. 4,352,883, an aqueous solution containing a biological material to be encapsulated is suspended in an aqueous polymer solution, the suspension is made into droplets, and then converted into individual microcapsules by contact with a polyvalent cation. , The surface of the microcapsules is crosslinked with polyamino acids to form a semipermeable film around the encapsulated material.

Polyphosphagens are polymers with a backbone composed of nitrogen and phosphorus separated by cross-linking single and double bonds. Each phosphorus atom is covalently bonded to two side chains.

Polyphosphagens suitable for crosslinking, most of them can form salt bridges with divalent or trivalent cations, and have acidic side chain groups. Examples of preferred acidic side groups are carboxylic acid groups and sulfonic acid groups. Polyphosphagens stable to hydrolysis are formed of monomers having carboxylic acid side groups crosslinked by divalent or trivalent cations such as Ca ^{2 +} or Al ³⁺ . By incorporating monomers having imidazole, amino acid esters or glycerol side groups, a polymer that is decomposed by hydrolysis can be synthesized. For example, polycationic poly[bis(carboxylatophenoxy)]phosphazene (PCPP) can be synthesized, which is crosslinked with polyvalent cations dissolved in an aqueous medium at room temperature or at a temperature lower than room temperature, thereby forming a hydrogel matrix. To form.

Biodegradable polyphosphagens include two or more different types of side chains, acidic groups capable of forming salt bridges with polyvalent cations, and side groups that hydrolyze such as imidazole groups, amino acid esters, glycerol and glucosyl in in vivo conditions. Have

Hydrolysis of the side chain causes the polymer to erosion. Examples of hydrolyzable side chains are unsubstituted and substituted imidazoles, and amino acid esters in which a group is attached to a phosphorus atom via an amino linkage (a polyphosphazene polymer with both R groups attached in this way is a polyaminophosphagen). . In the case of polyimidazole phosphazene, some of the "R" groups on the polyphosphagen backbone are imidazole rings that are attached to the phosphorus of the vector through the nitrogen atom of the ring. Other "R" groups are methyl phenoxy groups or Allcock et al. It may be an organic moiety that is not involved in hydrolysis, such as other groups described in the scientific paper of (1977). Methods for synthesizing hydrogel materials and methods for preparing such hydrogels are known in the art.

The MPC-derived supernatant or soluble factor, MPC or progeny thereof, can be administered with other beneficial drugs or biological molecules (growth factors, trophic factors). When administered together with other substances, they can be administered together as a single pharmaceutical composition, or simultaneously or sequentially (before or after administration of other substances) with other substances as separate pharmaceutical compositions. Bioactive factors that can be administered together include anti-apoptotic agents (eg EPO, EPO mimetibody, TPO, IGF-I and IGF-II, HGF, caspase inhibitors); Anti-inflammatory agents (e.g., p38 MAPK inhibitors, TGF-beta inhibitors, statins, IL-6 and IL-1 inhibitors, PEMIROLAST, TRANILAST, REMICADE, SIROLIMUS, and NSAIDs (nonsteroidal anti-inflammatory agents; e.g. TEPOXALIN, TOLMETIN, SUPROFEN); immunosuppressants/immunomodulators (e.g., cyclosporine, calcineurin inhibitors such as tacrolimus; mTOR inhibitors (e.g., SIROLIMUS, EVEROLIMUS); anti-proliferative agents (e.g., azathioprine, myco Phenolate mofetil); corticosteroids (e.g., prednisolone, hydrocortisone); monoclonal anti-IL-2Rα receptor antibodies, such as antibodies (e.g. basiliximab), Daclizumab), polyclonal anti-T-cell antibodies (eg, anti-thymocyte globulin (ATG); anti-lymphocyte globulin (ALG); monoclonal anti-T cell antibody OKT3)); anti- Anti-thrombogenic agents (e.g. heparin, heparin derivatives, urokinase, PPack (dextrophenylalanine proline arginine chloromethylketone), antithrombin compound, platelet receptor antagonist, anti-thrombin antibody, anti-platelet receptor antibody, aspirin , Dipyridamole, protamine, hirudin, prostaglandin inhibitors and platelet inhibitors); And anti-oxidants (e.g., probucol, vitamin A, ascorbic acid, tocopherol, coenzyme Q-1O, glutathione, L- Cysteine, N-acetylcysteine) as well as local anesthetics.In another example, the MPC-derived supernatant or soluble factor, MPC or its progeny, is combined with a scar inhibitory factor, as mentioned in US Pat. No. 5,827,735. It can be administered concurrently.

In the case of treating and/or preventing diseases caused by decomposition and/or inflammation of connective tissue, it is preferable to administer the supernatant, soluble factor or cells together with a chondroprotective agent. Examples thereof include pentosan polysulfate (SP54 and Cartrophen), glycosaminoglycan polysulfate ester (Arteparon), glycyamino-glycan-peptide complex (Rumalon) and hyaluronic acid (Hyalgan), but these It is not limited. Additional examples are mentioned in Verbruggen (2005) and Richette and Bardin (2004). In a preferred example, the chondroprotective agent is hyaluronic acid.

Fibrin Glue

Fibrin glue is a type of surgical suture that is used in a variety of clinical situations. As those skilled in the art will recognize, many sealants are useful in the methods defined herein. However, a preferred example of the present invention relates to the use of fibrin glue.

As used herein, the term “fibrin glue” refers to an insoluble matrix formed by crosslinking of a fibrin polymer in the presence of potassium ions. Fibrin glue can be made from fibrinogen or a derivative or metabolite thereof, fibrin (a water-soluble monomer or polymer) and/or a complex thereof, derived from a fluid or biological tissue forming a fibrin matrix. In addition, fibrin glue can be formed from fibrinogen, a derivative thereof or a metabolite thereof, or fibrin produced by recombinant DNA technology.

In addition, fibrin glue can be made by the interaction of fibrinogen with a fibrin glue formation catalyst (eg, thrombin and/or factor XIII). As will be apparent to those skilled in the art, fibrinogen is converted into fibrin monomer by cleaving the protein in the presence of a catalyst (eg, thrombin). The fibrin monomer can then be crosslinked to form a polymer capable of forming a fibrin glue matrix. Crosslinking of the fibrin polymer can be enhanced by the presence of a catalyst such as Factor XIII. The catalyst for fibrin glue formation may be derived from plasma, cryoprecipitate, or other plasma fractions containing fibrinogen or thrombin. Alternatively, the catalyst can be produced by recombinant DNA technology.

The rate of clot formation depends on the concentration of thrombin mixed with fibrinogen. As an enzyme-dependent reaction, the higher the temperature (up to 37°C), the faster the clot formation rate. The tensile strength of the clot is dependent on the concentration of fibrinogen used.

The use of fibrinogen glues and methods of making and using them are described in US Pat. No. 5,643,192. Hirsh describes a method for the extraction of fibrinogen and thrombin components from a single donor and a combination of only these components for use as fibrin glue. Marx U.S. Patent 5,651,982 describes different formulations and methods of use of fibrin glue. Marx provides dermal glues and liposomes for use in mammals as a national bird sealant. The preparation and use of topical fibrinogen complexes (TFC) for injury healing are known in the art. In International Patent Publication No. WO96/17633 of The American Red Cross, TFC formulations comprising fibrinogen, thrombin and calcium chloride are discussed.

Several publications disclose the use of fibrin glue for delivery of therapeutic agents. In an embodiment, U.S. Patent 4,983,393 discloses a composition for use as an intravaginal insert comprising agarose, agar, saline solution, glycosaminoglycan, collagen, fibrin and enzymes. In addition, U.S. Patent 3,089,815 discloses a pharmacological preparation for injection consisting of fibrinogen and thrombin, and U.S. Patent 6,468,527 discloses a fibrin glue that facilitates the delivery of various biological and non-biological agents to specific sites in the body. .

Production of genetically modified cells

In one embodiment, the cells used in the methods of the present invention, such as for the production of supernatants or soluble factors, are genetically modified. Preferably, the cell is genetically modified to produce a heterologous protein. Typically, the cell will be genetically modified so that the heterologous protein is released from the cell. However, in one embodiment, the cell can be modified to express a functional non-protein encoding a polynucleotide such as dsRNA (typically RNA silencing), antisense oligonucleotides or catalytic nucleic acids (e.g., ribozymes or DNAzyme). have.

Genetically modified cells can be cultured in the presence of one or more cytokines in an amount sufficient to support the growth of the modified cells. The genetically modified cells thus obtained can be used immediately (eg, for transplantation), amplified in culture and in vitro, or stored for later use. The modified cells can be stored by methods well known in the art, such as freezing in liquid nitrogen.

Genetic modification herein includes any method of genetic modification, including introduction of an exogenous or foreign polynucleotide into a cell described herein, or modification of an endogenous gene within a cell. Genetic modification includes transfection (transfer of host DNA from a virus-mediated host or donor to a receptor in vitro or in vivo), transfection (transformation of cells with an isolated viral DNA genome), liposome-mediated transfer, electrophoresis. Electroporation, calcium phosphate transfection or co-precipitation, and the like, but are not limited thereto. Transfection methods include direct co-culture of cells and producer cells (Bregni et al., 1992) or culture alone with viral supernatant or with appropriate growth factors and polycations.

The exogenous polynucleotide is introduced into the host cell, preferably in the form of a vector. The vector preferably contains elements essential for transcription and translation of the inserted coding sequence. Methods used to construct such vectors are well known in the art. For example, techniques for constructing suitable expression vectors are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y. (3rd Ed., 2000); And Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York (1999).

Vectors include viral vectors such as retrovirus, adenovirus, adeno-associated virus, and herpes simplex virus; Cosmid; Plasmid vector; Synthetic vectors; And other recombinant vehicles typically used in the art, but are not limited thereto. Vectors containing both a promoter and a cloning site capable of operably linking a polynucleotide are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from companies such as Stratagene (La Jolla, Calif.) and Promega Biotech (Madison, Wis.). Specific examples include Stratagene's pSG, pSV2CAT, pXtl; And pMSG, pSVL, pBPV and pSVK3 from Pharmacia.

Preferred vectors are retroviral vectors (see, Coffin et al., "Retroviruses", Chapter 9 pp; 437-473, Cold Springs Harbor Laboratory Press, 1997). Vectors usable in the present invention can be recombinantly prepared by methods well known in the art. For example, in WO94/29438, WO97/21824 and WO97/21825 the construction of retro packaging plasmids and packing cell lines is mentioned. Exemplary vectors are pCMV mammalian expression vectors such as pCMV6b and pCMV6c (Chiron Corp.), pSFFV-Neo and pBluescript-Sk+. Non-limiting examples of retroviral vectors that may be used are those derived from rodent, avian or primate retroviruses. As a typical retroviral vector, there is a vector (MoMLV-vector) based on the Moluni rodent leukemia virus. Other MoMLV-derived vectors include Lmily, LINGFER, MINGFR and MINT. Additional vectors include GALV (Gibbon ape leukemia virus), MOMSV (Moloney murine sarcoma virus) and SFFV (spleen focus forming virus) derived vectors. MESV-MiLy is a vector derived from MESV (murine stem cell virus). Further, the retroviral vector includes a vector based on a lentivirus, and non-limiting examples include a vector based on human immunodeficiency viruses (HIV-1 and HIV-2).

In the preparation of the retroviral vector construct, the gag, pol, and env sequences of the virus can be removed from the virus to make a space for inserting the foreign DNA sequence. Genes encoded by foreign DNA are generally expressed in long terminal repeats (LTRs) under the control of strong viral promoters. Selection of the appropriate regulatory sequence is dependent on the host cell used, and the selection is made within the capabilities of one of skill in the art. In addition to the promoter of LTR, a number of promoters are known. Non-limiting examples include phage lambda PL promoter, human cytomegalovirus (CMV) early promoter; The U3 region promoter of Moloney rodent sacoma virus (MMSV), Lewis sacoma virus (RSV) or spleen follicle forming virus (SFFV); Granzyme A promoter; And the Granzyme B promoter. Additionally, inducible and multiple regulatory elements can be used. Selection of a suitable promoter will be apparent to those skilled in the art.

These constructs can be efficiently packed into viral particles provided the gag, pol and env functions are provided in trans by the packing cell line. Therefore, when the vector construct is introduced into the packing cell, the gag-pol and env proteins produced in the cell are combined with the vector RNA to produce an infectious viron secreted into the culture medium. The virus thus prepared can infect the target cell and insert it into the DNA of the target cell, but it cannot form infectious virus particles because it does not have an essential packing sequence. Since most of the currently used packing cell lines are transfected with individual plasmids each containing one of the essential coding sequences, multiple recombination is essential before a replication competent virus can be produced. In another embodiment, the packing cell line has a provirus. Proviruses are flawed, they can produce all of the proteins needed for the infectious virus combination, but cannot pack their RNA into a virus. Instead, the RNA produced by the recombinant virus is packed. Therefore, the virus stock released from the packed cells contains only the recombinant virus. Non-limiting examples of retroviral packing lines include PA12, PA317, PE501, PG13, PSI.CRIP, RDI 14, GP7C-tTA-G10, ProPak-A (PPA-6), and PT67.

Other suitable vectors include adenovirus vectors (cf. WO 95/27071) and adeno-associated viral vectors. These vectors are described in the art, such as Stem Cell Biology and Gene Therapy, eds. Quesenberry et al., John Wiley & Sons, 1998; And U.S. Patent Nos. 5,693,531 and 5,691,176, are well known. The use of an adenovirus-derived vector may be useful under certain conditions because the vector cannot infect non-dividing cells. Unlike retroviral DNA, adenovirus DNA is not inserted into the genome of the target cell. In addition, adenovirus vectors are superior to retroviral vectors in their ability to transport foreign DNA. Adeno-associated viral vectors are another useful delivery system. The DNA of these viruses can be inserted into non-dividing cells, and multiple polynucleotides are successfully introduced into various cell types using adeno-associated viral vectors.

In some examples, the construct or vector will comprise two or more heterologous polynucleotide sequences. Preferably, the additional nucleic acid sequence is a polynucleotide encoding a selectable marker, structural gene, therapeutic gene, or cytokine/chemokine gene.

Selectable markers can be included in constructs or vectors to monitor successful genetic modification and to select cells into which DNA has been inserted. Non-limiting examples thereof are drug resistance markers such as G148 or hygromycin. Additionally, negative selection can be used, for example when the marker is the HSV-tk gene. This gene will make the cell sensitive to agents such as acyclovir and ganciclovir. Although the NeoR (neomycin/G148 resistance) gene is commonly used, all existing marker genes that do not exist in target cells for which the gene sequence is available can be used. Other non-limiting examples include low-affinity nerve growth factor (NGFR), enriched fluorescent green protein (EFGP), dihydrofolate reductase gene (DHFR), bacterial hisD gene, rodent CD24 (HSA), rodent CD8a (lyt), a bacterial gene that confers resistance to puromycin or pleomycin, and beta-galactosidase.

The additional polynucleotide sequence(s) may be introduced into the host cell on the same vector, or may be introduced into the host cell on a second vector. In a preferred embodiment, the selection marker will be included as a polynucleotide on the same vector.

In addition, the present invention includes genetic modification of the promoter region of the endogenous gene such that the expression of the endogenous gene is up-regulated resulting in increased production of the encoded protein compared to native cells.

The present invention relates to a method for producing, repairing and/or maintaining connective tissue in a subject. In addition, the present invention relates to a method for treating and/or preventing diseases caused by decomposition or inflammation of connective tissue in a subject.

Figure 1. Morphological scores of femur and tibial cartilage at 12 weeks after meniscal resection, mean + SD for joints injected with hyaluronan (HA) or HA + mesenchymal progenitor cells (MPC) at various concentrations.
Figure 2. Femoral and tibial osteophyte scores, mean + SD, for joints injected with hyaluronan (HA) or HA + various concentrations of mesenchymal progenitor cells (MPC) at 12 weeks after meniscal resection.
Fig. 3. [HA/(MPC+HA)] ratio of cartilage morphology joint scores in animals injected with mesenchymal progenitor cells (MPC) at various concentrations. Ratio = 1 means that the two treatments are equally effective, and ratio> 1 means that MPC+HA is more effective than HA.
Fig. 4. [HA/(MPC+HA)] ratio of osteoproliferative bodies in animals injected with MPC + HA at various concentrations compared to HA alone. Ratio = 1 means that the two treatments are equally effective, and ratio> 1 means that MPC+HA is more effective than HA.
Fig. 5. The thickness score of the area measured by histomorphometry at 12 weeks after meniscal chondrectomy for the cartilage of the joint injected with hyaluronan (HA) or 10 ⁸ MPC + HA, mean + SE. All femoral cartilage areas were HA + 10 ⁸ MPC> HA (p<0.05).
Figure 6. Mean histopathological total scores for Mankin's deformed joints + [HA/(MPC+HA)] ratio of SE of animals injected with various concentrations of mesenchymal progenitor cells (MPC). Ratio = 1 means that the two treatments are equally effective, and ratio> 1 means that MPC+HA is more effective than HA.
Fig. 7. Mean morphology scores of femoral and tibial cartilage + SD for joints injected with HA or 10 ⁸ MPC + HA at 12 weeks, 24 weeks and 52 weeks after meniscal resection.
Figure 8. Mean femoral and tibial osteoproliferative scores + SD for joints injected with HA or 10 ⁸ MPC + HA at 12 weeks, 24 weeks and 52 weeks after meniscal chondrectomy.
Fig. 9. [HA/(MPC+HA)] ratio of cartilage morphology joint scores for animals injected with mesenchymal progenitor cells (MPC) at 12 weeks, 24 weeks and 52 weeks after meniscal chondrectomy. Ratio = 1 means that the two treatments are equally effective, and ratio> 1 means that MPC+HA is more effective than HA.
Fig. 10. [HA/(MPC+HA)] ratio of osteoproliferative joint scores for animals injected with mesenchymal progenitor cells (MPC) at 12 weeks, 24 weeks and 52 weeks after meniscal chondrectomy. Ratio = 1 means that the two treatments are equally effective, and ratio> 1 means that MPC+HA is more effective than HA.
Figure 11. Mean histopathological score of cartilage for Mankin's deformed joint of animals injected with mesenchymal progenitor cells (MPC) at 12 weeks, 24 weeks and 52 weeks after meniscal chondrectomy + [HA/(MPC+HA) of SE ] Rain. Ratio = 1 means that the two treatments are equally effective, and ratio> 1 means that MPC+HA is more effective than HA.
Fig. 12. Mean stiffness of patella cartilage + SE of joints injected with hyaluronan (HA) or various concentrations of MPC + HA. *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001.
13. Hyaluronan (HA) or 10 ⁸ MPC + HA was injected, and sacrificed at 12, 24 and 52 weeks after meniscal resection, average patellar cartilage strength of the joint + SE. *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001.
Fig. 14. Phase lag of patella cartilage, mean + SE of joints injected with hyaluronan (HA) or various concentrations of MPC + HA. *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001.
Fig. 15. Phase lag of patella cartilage of the joint, mean + SE, which was injected with hyaluronan (HA) or 10 ⁸ MPC + HA and sacrificed at 12, 24 and 52 weeks after meniscal resection . *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001.
Fig. 16. A comparison of articular cartilage morphology scores of untreated castrated male sheep and ovarian resected female sheep, showing that the severity of OA lesions was remarkable in the female group.
Fig. 17. A comparison of the osteoproliferative body score of the joint 12 weeks after meniscal chondrectomy in untreated castrated male sheep and ovarian resected ewe, and the score was significantly higher in the female group.
Fig. 18. Histopathological score of modified Mankin's cartilage at 36 weeks after meniscal resection for ovarian resected ewe joints injected with hyaluronan (HA) or 10 ⁸ MPC + HA at 12 weeks after meniscal resection , Mean + SD. P value = HA versus MPC+HA. These results indicate that MPC alone injection lowered the abnormal histopathological score of femoral vitreous cartilage to a higher level than tibial cartilage for 6 months.
Fig. 19. [HA/(MPC+HA)] of the total modified Mankin's cartilage histopathological score at 36 weeks after meniscal chondrectomy for a joint of an ovarian resected ewe administered intraarticular injection at 12 weeks after meniscal chondrectomy. ratio. Ratio = 1 means that MPC+HA and HA are equally effective, and ratio >1 means that MPC+HA has a higher protective effect than HA alone. Data = mean + SEM. These results indicate that MPC alone injection lowered the abnormal histopathological score of femoral vitreous cartilage to a higher level than tibial cartilage for 6 months.
Fig. 20. For a joint of an ovarian resected ewe injected with hyaluronan (HA) or 10 ⁸ MPC + HA at 12 weeks after meniscal resection (MX), compared to a non-injected joint at 12 weeks after MX, meniscal cartilage Mean histopathological score of modified Mankin cartilage at 36 weeks after resection + SD. P value is treated versus 12 weeks NIL. This result suggests that MPC alone injection lowered the abnormal histopathological score for 6 months.
Fig. 21. Histomorphometric data of femoral cartilage at 36 weeks after meniscal cartilage resection for an ovarian resected female joint injected with hyaluronan (HA) or 10 ⁸ MPC + HA at 12 weeks after meniscal resection. Data are presented as mean + SEM. P value = HA versus MPC+HA. These results suggest that MPC alone injection produced more vitreous cartilage over 6 months than hyaluronic acid.
Fig. 22. Joint of an untreated ewe sacrificed at 12 weeks after meniscal resection, or hyaluronan (HA) or MPC + HA injected at 12 weeks after meniscal resection and then sacrificed at 12 or 24 weeks thereafter For, average thickness of femoral cartilage measured by histomorphometric method + SEM. Data are presented as mean + SEM. The P value is based on 12 weeks of NIL-free treatment. These results suggest that MPC alone injection increased the vitreous cartilage thickness for 6 weeks.
Fig. 23. Joints of untreated ewes sacrificed at 12 weeks after meniscal resection, or ewes sacrificed at 12 weeks or 24 weeks after injection of hyaluronan (HA) or MPC + HA at 12 weeks after meniscal resection For, the area of femoral cartilage measured by histomorphometric method. Data are presented as mean + SEM. The P value is based on 12 weeks of NIL-free treatment. These results suggest that MPC alone injection increased the vitreous cartilage thickness for 6 weeks.
Fig. 24. Joints of untreated ewes sacrificed at 12 weeks after meniscal resection, or ewes sacrificed at 12 weeks or 24 weeks after injection of hyaluronan (HA) or MPC + HA at 12 weeks after meniscal cartilage resection For, IGD (Integrated Grey-scale Density) measured by histomorphometric method as a measurement of total proteoglycan (PG) of femoral cartilage. Data are presented as mean + SEM. The P value is based on 12 weeks of NIL-free treatment. This result suggests that MPC alone injection produced significantly more proteoglycan-containing cartilage than hyaluronic acid injection for 6 weeks.
Fig. 25. A chart showing the level of lumbar spine treated with mesenchymal progenitor cells (MPC) in both groups.
Figure 26. Average recovery of disc height at 3 and 6 months after injection of MPC and HA into the nucleus of a positive degenerated disc

Example

Example 1: immunoselected MPC Increase and Upper layer collection

Bone marrow (BM) was collected from sheep under 2 years old. Briefly, 40 ml of BM was aspirated from the anterior iliac crest into a tube containing lithium-heparin anticoagulant. BMMNC was prepared by density gradient separation using Lymphoprep ^TM (Nycomed Pharma, Oslo, Norway) according to the previously disclosed bar (Zannettino et al., 1998). After centrifugation at 4°C for 30 minutes at 400 xg, the pale yellow membrane layer was removed with a transfer pipette, and HBBS (Hank's balanced salt containing 5% fetal bovine serum (FCS, CSL Limited, Victoria, Australia)) solution; Life Technologies, Gaithersburg, MD) was washed 3 times in "HHF".

As previously disclosed, TNAP+ was subsequently isolated by self-activated cell sorting (Gronthos et al., 2003; Gronthos et al., 1995). Briefly, approximately 1-3 x 10 ⁸ BMMNCs were incubated on ice in a blocking buffer consisting of 10% (v/v) normal rabbit serum in HHF. The cells were incubated on ice for 1 hour with 200 μl of a 10 μg/mL solution of STRO-3 mAb in blocking buffer. Subsequently, the cells were washed twice in HHF by centrifugation at 400 xg. A 1/50 dilution of goat anti-mouse γ-biotin (Southern Biotechnology Associates, Birmingham, UK) in HHF buffer was added, and the cells were incubated on ice for 1 hour. Cells were washed twice in MACS buffer (1% BSA, 5 mM EDTA and 0.01% sodium azide-added Ca ^{2 +} -and PBS without Mn ^{2 +} -) as described above and in a final volume of 0.9 mL MACS buffer. Resuspended.

100 μl of streptavidin microbeads (Miltenyi Biotec; Bergisch Gladbach, Germany) was added to the suspension and incubated for 15 minutes on ice. The cell suspension was washed twice, resuspended in 0.5 mL MACS buffer, subsequently loaded onto a mini MACS column (MS Columns, Miltenyi Biotec), washed three times with 0.5 mL MACS buffer, and STRO-3 mAb Cells that do not bind to (Attached to the American Type Culture Collection (ATCC) on December 19, 2005 as accession number PTA-7282, see WO 2006/108229) were obtained. After adding 1 ml of MACS buffer, the magnet was removed from the column, and TNAP-positive cells were separated by positive pressure. Some cells in each fraction can be stained with streptavidin-FITC, and purity can be measured by flow cytometry.

As previously disclosed (Gronthos et al., 1995), the primary culture was seeded in α-MEM added with 20% fetal bovine serum, 2 mM L-glutamine, and 100 μM L-ascorbate-2-phosphate, and MACS It was established from isolated TNAP+ cells.

Cells were cultured up to passage 5 so that the condition medium (supernatant) could be collected.

Example 2: On the dose-dependent intrachondral effect of autoimmune-selected mesenchymal progenitor cells (MPC) on cartilage integrity of a model of early OA caused by bilateral total median meniscal resection of castrated adult ram Research

The knee joint meniscus or meniscus is an important weight-bearing structure that improves joint cartilage lubrication and provides lateral stability during joint connection. Surgical removal of a ruptured or degenerated meniscus, i.e., meniscal chondrectomy, has been shown to increase the risk of developing osteoarthritis (OA) after several years, although a common orthopedic procedure (Englund, 2004). It is known that mechanical entrapment of the synovial membrane in the space where the meniscus was previously located as a surgery induces partial regeneration of meniscus replicas (Moon et al., 1984). However, as a result of the study of meniscal resection experiments in dogs, it was found that these alternative structures are essentially made of fibrous tissues with biomechanical properties that are much inferior to those of the original meniscus (Ghosh et al., 1983). Moreover, 6 months after meniscal resection, the incidence of osteoarthritis (OA) in the joints of the experimental animals was comparatively serious, indicating that the functional support provided by the regrown structure on the articular cartilage was very limited ( Ghosh et al. 1983a). Large and small animal models of osteoarthritis (OA) enabled longitudinal evaluation of spatial and temporal changes in joint tissue that occur during the onset of disease, which is difficult to obtain using human patients (Smith and Ghosh, 2001). In sheep Merino, lateral or median meniscal resection has been shown to re-create the biochemical, biomechanical and histopathological changes of typical OA (Smith and Ghosh, 2001). The sheep OA model has also been widely used to investigate the outcome of various modalities of postoperative treatment (Ghosh, 1991; Smith and Ghosh, 2001), but to date, regrowth of meniscus and progression of OA and how these events are It has never been used to evaluate whether it could be affected by intra-articular mesenchymal progenitor cell (MPC) therapy.

In our previous study, bilateral total medial meniscectomy (BTM) for sheep Merino causes pathological changes in articular cartilage (AC), subchondral and synovial tissues, and premature human osteoarthritis (OA). ) Has been found to stimulate the development of. We have previously used this animal model to evaluate disease-modulating OA drugs.

Way

BTM was performed on 36 merino castrated rams. Two weeks after BTM, the joints were randomly injected with 2 mL high molecular weight hyaluronan (HA) or 2 mL autologous Stro-3+ MPC suspended in 2 mL HA. Four dose groups of MPC were investigated: Group A = 10 milliones (mil) MPC [n=6]; Group B = 25 mil MPC [n=6]; Group C = 100 mil MPC [n=18] and Group D = 150 mil MPC [n=6]. Groups A, B and D were sacrificed 12 weeks after BTM, and group C was sacrificed after 12 weeks [n=6], 24 weeks [n=6] and 52 weeks [n=6] BTM.

At autopsy, both central compartments of the BTM joint were double-blinded for joint cartilage (AC) damage and osteoproliferative body (OP), and scored on a scale of 0-4. The synovial tissue and 5 mm wide coronal osteochondral slices were removed from the mid-line of the femur and tibia, treated and histopathological changes (Little et al., 1997) and tissue using the mentioned method. Scored for morphological analysis (Cake et al., 2003).

In order to measure the strength and phase lag of articular cartilage, the intact patella of all joints was treated with external and biomechanical indentation (Appleyard et al., 2003).

The treatment effect was statistically analyzed using Kruskal-Wallis nonparametric analysis, and the Mann Whitney U nonparametric analysis with a level of significance of p<0.05 was used. Statistical analysis of group comparisons was performed.

In order to compare the group mean of the MPC + HA-injected joints and the HA-injected joints of each group, statistical analysis was performed using the equally variance two-tailed Student's T-test with a level of significance p<0.05.

To compare the patellar cartilage between the MPC + HA-injected (treated) and HA-injected joints of each group, statistical analysis was performed using an independent sample T-test with a significance level of p<0.05.

result

At 12 weeks after BTM, the gross morphological score showed MPC dose-dependent effects on AC conservation and OP formation: compared to HA alone injection, 100 million MPC was the most effective chondroprotective administration. Appears (Figs. 1 and 2). The OP ratio was 100 = 25>10>150 million MPC, but the total AC score ratio (HA+MPC)/(HA) was found to be 100>150>25=10 (Figs. 3 and 4). A statistically significant lower score was observed for total femur and tibial AC, and p = 0.052 for group C MPC femur cartilage compared to HA alone (FIG. 1 ).

Histological analysis of group C MPC+HA tibial plateau showed that AC was in the middle (p = 0.057) and lateral area (p = 0.028); It was found to be thicker compared to the corresponding HA-AC in all regions (p = 0.01) (Fig. 5). The average adjusted Mankin score for the AC section of the group C MPC+HA joint was smaller than the corresponding HA section, but was not statistically significant. In addition, when the total Mankin score ratio for the HA-injected joint and the contralateral HA+MPC-injected joint of each group was calculated, it was clearly shown that the administration of 100 million MPC was the most effective (FIG. 6 ).

To protect the integrity of articular cartilage, the question of the sustainability of administration of 100 million MPC was questioned 22 and 50 weeks after injection, i.e. 24 and 52 weeks after meniscal resection, the morphological, histological and biomechanical properties of the tissue. It was solved by studying. As is evident in Figures 7 and 8, the mean values of the morphological scores of HA and HA+100 million MPC decreased over time, but there is some evidence of therapeutic effect at week 24. This observation is supported by the HA/MPC+HA data, indicating a stronger effect of cells inhibiting osteoproliferative bodies for 52 weeks (Figures 10 and 11), whereas a curve similar to the Mankin histopathological score , After 52 weeks, the protective effect of MPC was lost (FIG. 11 ).

Biomechanical indentation studies of joint patellar cartilage in all animal groups are generally consistent with morphological and histological data. However, the strength of cartilage is affected by the thickness of the cartilage, and the cartilage thickness may become enlarged in the early stages of injury, but normalizes over time. This situation can occur in the current model, where the strength of patellar cartilage measured for the 25 and 100 millione MPC groups was significantly more significant than the 10 and 150 million MPC groups, which showed the most damaged tissue in other studies. It is because it is low (Figs. 12 and 13). This interpretation is supported by the phase delay data showing that the kneecaps in the 100 million MPC group were significantly lower compared to the HA-injected joint and 150 millione MPC administration (FIG. 14). Moreover, the mean phase delay value observed at 12 weeks was found to increase significantly after 24 and 52 weeks after meniscal resection, which means that the beneficial treatment effect of the injected MPC is lost after 6-12 months (Fig. 15). The phase delay reflects the molecular aggregation of the cartilage extracellular matrix and the lower the (phase) angle, the greater the elasticity, and thus the ability to recover from deformation (Cake et al., 2005).

The cartilage protection effect observed for the 100 mil MPC-injected joint decreased with time, and the positive effect observed at 12 and 24 weeks after BTM disappeared after 52 weeks.

There was no evidence for synovial histopathological control. The clinical and gross histopathology conducted on these animals showed no evidence of adverse systemic effects of MPC.

Sintering

To the best of the present inventors' knowledge, this is the first report on the beneficial therapeutic effect of autologous MPC on cartilage conservation in early OA models. MPCs release growth factors and cytokines, and are known to upregulate anti-inflammatory cytokines (eg, IL-4, IL-10), while inhibiting the production of TNF-α by other cells. The paracrine activity of MPCs can promote the biosynthesis of chondrocytes of the new matrix, but may also reduce the activity and local production of catabolic mediators. This finding that 100 million MCP has a cartilage protective effect in this study corresponds to the mechanism of action. The data obtained from the study of the sheep indicated that the duration of the cartilage protection effect by a single injection of 100 million MPC into the cartilage was 6-12 months after treatment, suggesting that multiple injections are required for long-term treatment of OA patients. do.

While intracartilage HA injection is widely used to treat knee arthritis, the evidence that the treatment has a cartilage protective effect is limited (Ghosh et al., 2002). However, HA treatment in cartilage slows the onset of OA and alleviates symptoms, but it is more persistent than corticosteroids in cartilage (Bellamy et al., 2006).

Example 3: In a severe osteoarthritis model caused by bilateral total medial meniscectom performed on the knee joint of an ovarian resected ewe, hyalunonan (HA) or 100 million mesenchymal progenitor cells (MPC ) + Relative therapeutic effect of injection of HA on cartilage conservation

The meniscus of the knee joint plays an important role in protecting the articular cartilage (AC) against damage in the case of normal joints (Arnoczky et al., 1988). Resection of the meniscus entirely or partially in the human body following damage to the meniscus generally causes early degeneration of AC and progression of osteoarthritis (OA) (Jorgensen et al., 1987; Roos et al., 1998 and McNicholas et al. al., 2000). In experimental studies, unilateral or bilateral total median meniscal resection of sheep has been shown to cause premature rupture of AC and early onset of OA (Ghosh et al, 1990; Appleyard et al., 1999 and Ghosh et al. , 1993c).

Since the AC damage in the joint with meniscal resection is a result of cutting stress and high focal burden on the cartilage, one side meniscal resection that can support the weight without pain by using the opposite hind limb without surgery In the case of bilateral meniscal resection, it was found that cartilage degeneration progressed more rapidly than in the case of (Ghosh et al., 1993a and 1993b; Appleyard et al., 2003; Little et al., 1997 and Oakley et al., 2004). Moreover, it was found that ovarian-resected ewe, which had undergone bilateral menisectomy, had more OA progression than that of castrated adult males (castrated rams) due to the cessation of the circulation of the chondroprotecting hormone, esterogen, usually (Parker et al., 2003). For this reason, as a large animal model for the study of disease-regulating anti-OA drugs, an ovarian resected, bilaterally removed meniscus cartilage is preferred (Ghosh et al., 1993; Smith et al., 1997; Burkhardt et al., 2001; Hwa et al., 2001 and Cake et al., 2000). Therefore, for this investigation, both models of ovarian resected/bilateral meniscal resected OA were selected, and the purpose of this investigation was that autologous mesenchymal progenitor cells (MPC) injected into cartilage (IA) were used for growth or regeneration of proteoglycan-rich cartilage. To evaluate the effect on induction and the effect on cartilage protection compared to the anti-OA treatment currently used, IA hyalunonan (HA).

Way

Total central meniscus (BTM) on both sides of 18 adult merino ewes, which had undergone ovariectomy 3 months ago by a known method, was performed (Cake et al., 1004). The surgical procedure and post-surgical regimen used in the BTM are the same as described for the BTM study of castrated rams described in Example 2.

At 12 weeks after BTM, 6 animals were sacrificed, and for the remaining 12 ewes whose meniscus was excised, 100 million MPCs suspended in 2 mL high molecular weight HA or 2 mL Profreeze ^® and 2 mL HA were randomly injected. . In Example 2, the administration of MPC+HA was shown to have the most beneficial cartilage protective effect in the BTM male sheep model. The meniscal resected and injected ewes were divided into two groups of 6 animals each, and sacrificed at 24 and 36 weeks after BTM, that is, at 12 and 24 weeks after injection into HA or MPC+HA cartilage. In order to measure the sex-dependent effect on the destabilization of the AC joint, BTM was performed on 6 untreated castrated rams, and sacrificed 12 weeks after menisectomy.

At autopsy, the joint was incised to remove the meniscus, and photographs were taken of the central femur and tibial plateau. In order to measure the total morphological change of cartilage, images recorded by double blinding using a 0-4 scale were scored. The synovial membrane of the folder above the patella and coronal osteochondral slices with a width of 5 mm were removed from the canonical line of the femur and tibia of each joint and processed for the preparation of histological sections. Using a modified Mankin scoring system as previously described, cartilage histopathology was evaluated by double blinding (Little et al., 1997). Synovial histopathology was scored according to the criteria recently described by Cake et al. (2008).

The same histological sequence of parts of the cartilage block used for Mankin scoring was also used for histological analysis as previously described (Caket et al., 2000; Cake et al., 2004). The method used a computer-assisted image analysis (ImagePro Plus v.3.0, Media Cybernetics) to obtain quantitative data on dimensions and an index of the proteoglycan content of AC stained with toluidine blue. Briefly, 1300 dpi mayikeurotek slide scanner having a resolution 35t plus (Microtek Model No. PTS-1950) back, J ^® software image from a personal computer, the obtained image of the stained section by (http: //rsb.info.nih. gov/ij/) was used. The digital images of the femur and tibial sections were subdivided into internal, central, and external regions, each of which occupied approximately 1/3 of the total area of the cartilage. The spatial correction of the system was achieved by scanning a high accuracy reticule of 10x10mm. The scale was used to measure the length (mm) and area (mm ² ) for each area of the acquired image. The average thickness of the portions was determined by dividing the area by the length. The optical density (OD) of the TB stained cartilage portion was obtained as the average gray value (MGV) (total gray value/number of pixels) and was taken as an index of the proteoglycan (PG) content. The integrated gray-scale density (IGD) was calculated as the area area of MGV x section. The gray scale system used was not independently calibrated for TB stained portions known as PG content, all histological sections were cut on the same microtome, had the same thickness, and were treated as groups using the same staining protocol. Therefore, differences in cartilage staining were relative rather than absolute. In order to measure the strength and phase delay of articular cartilage, intact patella of all joints were removed within 1 hour after sacrifice, immediately frozen, and stored until a local anatomical biomechanical indentation study (Appleyard et al., 2003).

Kruskal-Wallis nonparametric analysis (Kruskal-Wallis nonparametric method) to determine the difference between treatment results (HA vs. HA+MPC) measured by morphological and histological scoring systems or between untreated controls at 12 weeks after treatment vs. BTM. analysis), and statistical analysis was performed using the Mann Whitney U nonparametric analysis with a significance level of p<0.05 to confirm differences between group comparisons.

The data obtained by histomorphological analysis of the digitized histological part was evaluated using the equally variance bi-tailed Student T-test with a level of significance p<0.05. Statistical analysis of the biomechanical parameters of patella cartilage was calculated for various treatments and time after BTM using an independent sample T test with a significance level of p<0.05.

result

Through the overall morphological evaluation of intra-articular cartilage abrasion and osteoproliferative formation of ewe 12 weeks after BTM, the OA model is more aggressive and severe than male castrated meniscus resected by the same surgical procedure. It was confirmed that the disease was indicated. In the untreated female control group, the average cartilage morphological score was 87% of the maximum score that the femur was used to assess this parameter, and the tibia was 75%. After meniscal resection was performed by the same surgical procedure, the total morphology score obtained for the joint derived from an untreated castrated male 12 weeks later was significantly smaller than that of the oophorectomy female (FIGS. 16 and 14 ), These findings were consistent with previous studies using bilateral meniscal resection (Parker et al., 2003; Cake et al., 2004).

At 24 and 36 weeks after meniscal resection, both treatments resulted in a lower mean femoral morphological cartilage score than baseline in ewes that were not treated 12 weeks after meniscal resection (data not shown), whereas treatment with MPC and HA Little differences between the joints were observed. From this, the present inventors interpreted that in the serious OA model, due to the severity of the total morphological damage (wear and bone proliferative body score), the parameters became too sensitive to observe the therapeutic effect.

Modified Mankin histopathological scores for cartilage in the untreated group 12 weeks after BTM were found to be consistent with the degree of cartilage damage measured morphologically (FIGS. 16 and 17 ). Contrary to morphological parameters, the total mean modified Mankin score for the femoral cartilage of ovariectomized ewe, awarded MPC+HA, was found to be lower than the score corresponding to the joint awarded with HA alone, and was significantly lower than that of HA alone. In general, lower cell numbers (p = 0.01) and inner zone toluidine blue (IT TB) staining for proteoglycans showed a stronger tendency (p = 0.06) (FIG. 18 ). For tibial cartilage, this effect was less pronounced (FIG. 18 ).

At 36 weeks after meniscal resection, the modified Mankin histomorphological cartilage score of the MPC+HA injected joint measured lower compared to the HA injected joint was the ratio of the mean total modified Mankin score for the two intra-cartilage treatments. At the time of measurement, it was noticeable (Fig. 19). In the same animal, if each ratio obtained from joints treated by both methods is 1, it means that both methods are equally effective. However, if the ratio is greater than 1, it can be said that MPC+HA treatment is more beneficial. From Fig. 19, it is clear that the average of the ratios obtained for femoral cartilage is significantly higher than the unit after 36 weeks after BTM, but the tibia ratio (1.12) in the two treatments is more preferable in the group injected with MPC+HA. Is presented (Fig. 19).

Then, the present inventors experimented with the treatment effect over time with the Mankin score. It was found that the effect on femoral cartilage with time was significantly different between the MPC + HA and HA alone treatment groups after meniscal resection, 24 and 36 weeks, that is, 12 and 24 weeks after injection (FIG. 20). In the group receiving MPC+HA, the mean score at Weeks 24 and 36 was progressively lower compared to the baseline at Week 12. This resulted in a decreased score and toluidine blue staining of the inner zone for 24 weeks of cell cloning (P = 0.01) and 36 weeks of cells (P = 0.04) and proteoglycans (PG) compared to the untreated group at 12 weeks (P = 0.04) increase (Fig. 20). This increase was not observed in the HA alone group. The synovial pathology scores for any group or intracartilage treatment were approximately the same.

Using histomorphological analysis method, for the 3 regions (internal, central, and external) of the phalanx of the joint injected 36 weeks after BTM, the area and intensity of TB staining as an index of cartilage thickness and PG content. The analysis is presented in Figure 21. It was clear that the treatment groups differed significantly from each other up to 36 weeks after BTM. The femoral cartilage of the MPC+HA-injected joint was thicker than the corresponding cartilage of the HA-injected joint (Fig. 21A), and occupied a significantly larger area (Fig. 21B). The femoral cartilage of the MPC+HA injected joint was larger in volume, and the proteoglycan content determined by the aggregated gray-scale density of the TB stained portion was also larger (FIG. 21C ).

In the time-based analysis, by comparing the parameters again, the effect of MPC+HA and HA alone treatment group on femoral cartilage over time at 24 and 36 weeks after meniscal resection, i.e., 12 and 24 weeks after injection. It was found that this was significantly different. Using the same histomorphologic methodology, the present inventors demonstrated that when MPC+HA injection was administered 12 weeks after meniscal chondrectomy, proteoglycans were administered at 12 and 24 weeks after HA alone (i.e. at 24 and 36 weeks after BTM). -It could be explained that the growth or regeneration of the rich femoral cartilage gradually increases further (Figs. 22 to 24). Therefore, when MPC+HA was injected into the ewe joint from which the meniscus was removed at 12 weeks after BTM, the femoral cartilage was adjusted to the baseline value of the untreated joint at 24 and 36 weeks after the BTM, and 12 weeks after the meniscal cartilage resection. Compared to, it was significantly thicker (Figure 22) and generally had a larger area (Figure 23). The area where the area of the femoral cartilage of the HA-injected joint was scanned showed no statistically significant change compared to the untreated control group at 12 weeks (FIGS. 22 and 23 ). The accumulated gray-scale density, which measured the PG content of the femoral cartilage portion, was significantly higher in both HA and MPC+HA-injected joints compared to the same cartilage area of the untreated group 12 weeks after BMT. Although high, the magnitude of the change caused by MPC+HA was significantly greater than that of HA alone (FIG. 24). The MPC+HA group developed nearly 60% more proteoglycan-rich femoral tissue at 36 weeks compared to baseline (p<0.001), and this rate of cartilage growth did not reach flat phase, whereas only HA was injected. One group reached the leveling phase, and only about 30% of the tissue increased further. This indicates that treatment with MPC+HA continued to significantly greater proteoglycan-rich cartilage (i.e., growth and/or cartilage growth) over a period of 24 weeks, compared to any temporal effect of both baseline and HA treatment alone. Regeneration).

In the injected joint, the results of the indentation study on the patellar cartilage did not show that the biomechanical properties were different between the two treatment groups, but the time course after meniscal resection and the group not treated at 12 weeks after BMT Confirm the change. At 24 weeks after meniscal resection, the strength of patellar cartilage in the MPC+HA group was significantly higher than at 12 weeks (P = 0.05) and 36 weeks (P <0.01) (data not shown). However, all treatment groups produced thicker patellar cartilage at 36 weeks compared to the low 24 weeks (P = 0.001) compared to the untreated control group (P = 0.01) at 12 weeks. At 24 and 36 weeks, the phase delay of patellar cartilage in both treatment groups was higher compared to the untreated 12 week control group (P = 0.001) (data not shown).

Review

The present study revealed that in ovarian resected ewe, bilateral median meniscal resection caused pathological changes in articular cartilage after 3 months, consistent with progressive and severe OA. Thus, the overall morphological score for the femur and tibial cartilage was 87-70% of the maximum scor. Interestingly, when castrated males were treated with the same surgical procedure and sacrificed at the same time period (12 weeks), the damage was observed to be less severe than that observed in ovarian resected females. The degree of cartilage pathology reflects the high overall modified Mankin histopathology score observed for this group, which is consistent with the value of early OA (Little et al., 1997). Previous studies have confirmed a strong association between menopause and OA in females, which is explained by the cessation of the estrogen cycle (Roos et al., 2001; Pelletier et al., 2007 and Nevitt et al., 1996). Although supported by studies of ovarian resected ewe (Parker et al., 2003 and Cake et al., 2004), a recent additional study showed that the fat-inducing hormone Leptin plays a more important role in mediating damaged cartilage and OA. (Dumond et al, 2003 and Teichtahl et al., 2005). Therefore, the 3-month period after BTM was adopted as a starting point for measuring the alleviating effect on the rate of progression of the cartilage pathology at 12 and 24 weeks after drug administration of HA or MPC+HA intraarticular injection.

The results of this study show that a single administration of 100 million MPC dispersed in 2 mL HA and 2 mL Profreeze ^® (commercial cryoprotectant) into the cartilage in the joint with established severe OA, slows the progression of joint pathology, and 2 mL HA. The degree of growth and/or regeneration of proteocan-rich cartilage could be further improved than that administered. Surprisingly, in all parameter experiments, the protective effects mediated by growth/regeneration and MPC were observed to be more significant after 12 and 24 weeks after administration, indicating a gradual effect that did not reach the plateau phase. . The cause of this phenomenon is currently unclear, but growth factors such as elements of the TGF-β superfamily, for example, BMP released by MPC (Ahrens et al., 1993; Aggarwal et al., 2005) It is possible to support the assimilation (compensation) stage of the cartilage for the deformed mechanical stress imposed on the joint by cartilage ablation. This view is supported by histomorphological data showing that the volume of proteoglycans and the degree of staining for them in the MPC-injected group were greater than when treatment was initiated 12 weeks after BTM. These matrix changes are consistent with an increase in chondrocyte biosynthesis. Importantly, the size of the anabolic parameters was generally shown to be larger in the cartilage of animals receiving MPC+HA than in HA alone. The efficacy of MPC to protect and even enhance the cartilage in response to mechanical load is a known inhibitory effect on the metabolism of chondrocytes mediated by many typical OA treatments, such as a number of steroids and non-steroidal anti-inflammatory drugs (NSAIDs). (McKenzie et al., 1976; Ghosh, 1988; Brandt, 1993 and 1993a; Huskisson et al., 1995).

Multiple intra-articular HA injections have been used as a treatment for the management of knee OA for over 30 years. While public opinion is that this type of treatment provides clinical relief of symptoms, recent reconsiderations and meta-analysis of published HA clinical treatments have shown that there is stronger associated with intra-articular injection, difficulty in blinded investigations, and bias in publication. The validity of this conclusion was questioned based on the placebo effect (Brandt et al., 2000; Lo et al., 2003). There is also debate about whether intra-articular HA exhibits cartilage protection or cartilage regeneration activity. However, by extensive animal investigation, in models of OA caused by unilateral and bilateral meniscal resection of rabbits and sheep and amputation of the anterior teen ligament in dogs, HA has an analgesic effect, anti-inflammatory effect and disease control effect. It turned out to be visible. The discussion of these data was reviewed with preclinical and experimental-based clinical studies on HA of various molecular weights (Ghosh et al., 2002).

In this study, it was evaluated for a single injection of intra-articular HA alone or in combination with MPC. Based on the data of this study, the present inventors concluded that the possible cartilage protective effects and long-lasting growth and regeneration effects by the MPC+HA combination are mediated by MPC. In this regard, the design of this study allowed each animal to act by self-regulating, by conferring HA to one joint and conferring an equal amount of HA and MPC in Profreeze ^® cryoprotectant to the opposite joint. It is important. In this study, both knee joints were surgically exacerbated and injected simultaneously, so that the present inventors were convinced that the magnitude and nature of the weight-bearing mechanical stress acting on the articular cartilage is the same in both joints.

From this study, the present inventors found that when a single intra-articular administration of MPC+HA in an amount of a previously present, severe OA intraarticular, increased cartilage after 24 weeks of treatment compared to the baseline before treatment and the control group injected with HA. It was concluded that the growth or regeneration of proteroglycan-rich cartilage is maximized by the extracellular matrix.

Example 4: immunoselected MPC A study on playing the used amount of disc

Way

36 matched Mayo castrated rams (aged approximately 18-24 months) were used in this study. To cut and remove the PG of NP, for all 36 sheep, 1.0 IU chondroitina in about 0.1 ml of sterile physiological saline was placed on three adjacent lumbar discs (L3-L4, L4-L5, L5-L6). ABC (Seikagaku Corporation, Japan) was administered. Chondroitinase ABC was not administered to the remaining lumbar discs (L1-L2 and L2-L3) and stored as a control. After 15 weeks (±3 weeks) after administration of chondroitinase ABC, MPC (0.5 x 10 ⁶ cells) in freezing medium (NAO) ProFreeze ^™ or ProFreeze ^™ alone An injection in which hyaluronic acid (Euflexxa ^® , Ferring Pharmaceuticals) in the same volume as NAO (Lonza Walkersville Ltd.) was mixed was directly into the nuclei pulposi of chondroitinase ABC-treated intervertebral discs shown schematically in FIG. 25 Was administered. Each experimental group was sacrificed 3 and 6 months later as summarized in Table 1.

Under induction anesthesia, lateral simple radiographs of the animal's lumbar spine were taken at the following time points: Day 0 (administration of chondroitinase ABC (Seikagaku Corporation, Japan)), day of test substance administration (lumbar disc degeneration) 15 weeks ± 3 weeks after induction), and 3 months and 6 months after test substance implantation. (Masuda et al., 2004), using the index of the intervertebral height (DHI) calculated by averaging the measurements of the anterior, central, and posterior parts of the IVD and dividing this by the average of the heights of the adjacent intervertebral parts. Radiographs were evaluated.

Under induction anesthesia, MRI of the lumbar spine was taken at the following time points: Day 0 (administration of chondroitinase ABC [Seikagaku Corporation, Japan]), the day of administration of the test substance (15 weeks after induction of lumbar disc degeneration±) 3 weeks), and 3 and 6 months after test substance implantation. Discs were graded from MRI scans using the Pfirrmann classification system (Pfirrmann et al., 2001). Spinal motion segments selected for histochemical and biochemical analysis were separated by cutting through the vertebrae of the skull and coccyx close to the cartilage end plate using a bone saw. The spinal surface was fixed in Histochoice ^® (Hewlett Packard, McMinnville, USA) for 56 hours in batch and demineralized several times for 2 weeks in 10% formic acid in 5% neutral buffered formalin with continued stirring until completely demineralized. And it was confirmed using a Faxitron HP43855A X-ray cabinet (Hewlett Packard, McMinnville, USA).

By standard histological methods, a plurality of sagittal slices (5 mm thick) of decalcified specimens were dehydrated with a graded ethanol solution and embedded in paraffin wax. Paraffin sections having a thickness of 4 μm were fixed on a Superfrost Plus glass microscope slide (Menzel-Glaser), dried at 85° C. for 30 minutes, and then dried at 55° C. overnight. The sections were deparaffinized in xylene (4 times x 2 min) and then rehydrated with tap water through a gradient ethanol wash (100-70% v/v). One section of all blocks prepared from sagittal slices was stained with hematocillin and eosin. Sections coded by an independent histopathologist were tested comparing the degree of histological properties between the enzyme-only group and the group to which MPC was administered after enzyme-injection. As shown in Table 2, a four-point semi-quantitative grading system was used to measure the microscopic properties of the whole disk. To indicate the degree of synthesis of the disc substrate, additional colored staining such as Alcian Blue (for general glycosanoglycan substances) and safranin O (particularly for chondroitin sulfate substances) was prepared. .

In addition, as previously described, immunohistochemistry was performed using a sequencer cassette and a disposable Coverplate immunostaining system (Melrose et al., 2003; Melrose et al. , 2002; Melrose et al., 2000; Melrose et al., 2002a; Melrose et al., 1998; Panjabi et al., 1985; Race et al., 2000; Smit, 2002). Intrinsic peroxidant activity was initially prevented by incubating tissue sections with 3% H ₂ O ₂ . Thereafter, chondroitinase ABC (0.25 U/ml) treatment in 20 mM Tris-acetate buffer pH 8.0 at 37° C. for 1 hour and bovine testis hyaluronid in phosphate buffer pH 5.0 at 37° C. for 1 hour The tissue sections were cut by combining the 1000 U/mL treatment, and then 3 with 0.5M NaCl (TBS) or proteinase-K (DAKO S3020) in 20 mM Tris-HCl pH 7.2 for 6 minutes at room temperature. It was washed twice and exposed to the antigenic epitope. Thereafter, the tissue was immersed in 20% normal pig serum for 1 hour, and large and small proteoglycans and collagen were probed with a number of first antibodies (Table 3). The negative control sections were also treated by omitting the first antibody or replacing the true first antibody of interest with an unrelated isotype matched first antibody. Commercial (DAKO) isotype matched mouse IgG (DAKO code X931) or IgM (DAKO code X942) control antibodies were used at this stage. The DAKO products X931 and X942 do not exist in mammalian tissues and are direct to Aspergillus niger glucose oxidant, an enzyme that is not induced, mouse monoclonal IgG ₁ (clone DAK-GO1) and monoclonal IgM (clone DAK-GO8). It was an antibody. 0.05% 3, 3'-diaminobenzidene dihydrochloride and 0.03% H ₂ O ₂ in TBS as substrate, Nova RED, nitro blue tetrazolium/5-bromo-4-chloro3-indolyl Horseradish peroxidation or second antibody conjugated alkaline phosphatase was used in the detection with phosphate/iodine nitrotetrazolium violet (NBT/BCIP/INT) or New Fuchsin. The stained slides were experimented with a bright-field microscope and photographed using a Leica MPS 60 photo microscope digital camera system.

Grading system for histological changes in the lower lumbar disc (BEP bone end plate, CEP cartilage end plate) rank Fiber ring Nucleus Cartilage end plates Marginal/subchondral One Intact lamellar
Narrow interlayer substrate
Intact lubricating adhesion
Vessels in the outer third only Uniformity
No gap Constant thickness
Intact adhesion to bone
Constant calcification of depth <1/5
Constant cell distribution Flat thickness BEP
Only the thoracic bone
Junction distinct from CEP
Penetration of several blood vessels into the CEP 2 Minor lamellar cracking and unstructured
Slight temperament expansion
Non-organization of adhesion outer ring lesions without recovery reaction Minor cracking
Mild cell necrosis
Yoon's slight posterior dislocation
Minor chondrone formation Mild cartilage thinning
Small transverse cracks
Irregular thickening of calcified areas
Several invasive vascular channels
Small chondrons BEP slightly not flat
Nodule shemo
Minimal remodeling of BEP
Small marginal osteoproliferative body 3 Moderate expansion of temperament
Moderate cracking of adhesions
No outer 1/3 minimum chondroid dysplasia in radioactively torn areas
Cystic degeneration
Vascular and minimal repair response to outer and middle third outer ring lesions Moderate crack
Moderate cell necrosis
Cystic degeneration
Rear potential inside the wheel
Collagen afferent expansion
Moderate chondron formation Marked cartilage thinning
Thickening of marked calcified areas
Many transverse cracks
Many vascular channels
Many chondrons BEP that is not moderately flat
Nodules vascularized shemo
Moderate thickening of the soju image
Defects in the bone lamellar
Minimal fibrotic tissue in the bone marrow space
Medium sized osteoproliferative body 4 Extensive Lamellar Unstructured
Radioactive tear extends to the outer third
Massive chondroid dysplasia
Blood vessels in all areas
Outer ring lesion with a marked recovery response Complete loss of the nucleus
Sagging body formation
Marked chondron formation Total loss of cartilage
Calcification of residual cartilage
Extensive crack BEP not very flat
Nodular ossified sheath
Large osteoproliferative body
Remarkable thickening of small limbs
Marked fibrosis of the bone space
Cartilage formation

Primary antibody against proteoglycan and collagen core protein epitope Primary antibody epitope Clone (isotype) References Big pg
Aggrecan
Versican
AD 11-2A9 (IgG)
12C5 (IgG)
26,30
26, 28 Collagen
Type 1
Type 2
I8H5 (IgG ₁ )
II-4CII (IgG ₁ )
23, 28
28 Primary antibody epitope Clone (isotype) References IV type
VI type
IX type CIV-22 (IgG ₁ )
Rabbit polyclonal
Mouse monoclonal D1-9 (IgG ₁ ), B3-1 (IgG _2b ) 28
28
35

Samples of the annulus and nucleus were excised in finely diced advanced blocks, and a part of the known wet weight tissue area was freeze-dried until the weight did not change. Three sets (1-2mg) of dried tissue were hydrated in 6M HCl for 16 hours at 110°C, and then hydroxyproline was analyzed as a measurement of tissue collagen content for the neutralized digestion fraction (Sakai et al. ., 2005). 3 sets (~2mg) of dried tissue were also digested with papain, and sulfated glycosaminoglycan was analyzed as a PG measurement using metachromatic pigment, 1,9-dimethylmethylene blue, in the aliquot of the dissolved tissue. (Sakai et al., 2005).

The motor segments were wrapped in gauze dipped in saline, sealed with a two-ply polyten bag, and frozen at -30°C until biomechanical testing was performed. It was confirmed that this treatment did not alter the biomechanical characteristics of the tissue (Panjabi et al., 1985). Biomechanical tests were performed to determine the strength of each disk against axial compression, flexion, extension, lateral bending and axial torsion under limited computer-controlled conditions close to physiological load (Panjabi et al., 1985; Race et al. ., 2000; Smit, 2002; Wilke et al., 1999). All details of the test protocol are described elsewhere (Panjabi et al., 1985; Race et al., 2000; Smit, 2002; Wilke et al., 1999). The specimen (functional spinal column unit, FSU) for testing consists of two adjacent vertebrae, intervening discs, and connecting ligaments. Three FSUs were tested per spinal column: Regel digested with C-ABC alone was digested with C-ABC and then treated with hyaluronic acid alone, intermediate levels with C-ABC, followed by hyaluronic acid and MPC. I did. Each FSU was placed in two aluminum alloy cups and fixed with three bolts and cold polymethyl methacrylate dental cement (Vertex SC Self Curing, Dentimex BV, Zeist, Holland). Measures were taken so that the middle of the intervertebral disc was positioned horizontally. The motor segment will be placed in the middle of the cup by nailing it through the spinal canal to the hole in one of the cups. All tests were performed in a brine bath maintained at 37°C. Prior to initiating the test, each FSU will be preloaded with 0.5 MPa stress until reproducible hydration is achieved. This was used as a baseline before each test. The preloaded 0.5 MPa stress stimulus relieves standing and was based on in vivo measurements of intervertebral pressure.

The mechanical test was performed using a Model 8511 dynamic servohydraulic material testing device (INSTRON Pty Ltd, High Wycombe, UK) equipped with a '6-step Freedom' load cell, and monitoring and force control were simultaneously performed on all three plans. Made it possible. The device was controlled with a personal computer and custom software for recording and analyzing data. Test data were obtained with stable hysteresis from five final sinusoidal 0.1 Hz load cycles in either axial load or torsion control. Tests were performed on pure axial compression, left and right lateral bending, flexion/extension combinations and pure axial torsion.

A pure axial compression of 200 N was applied to the FSU with little or no bending or bending accompanying the load. All compression tests were performed using point contact on the surface of the cranial cup. The neutral axis of bending (NAB) was measured by applying a cyclic load to the joint through a point on an aluminum alloy cup on which the specimen was fixed to form a very weak bending. These attempts and failures can be made as close to pure axial compression as possible using tight point load contact. Despite some variability between specimens, this point was found in the sagittal plane approximately 10 mm anterior to the spinal canal and slightly posterior to the central point of the disc. Marks were marked 10 mm anterior and posterior to the left and right of the NAB to indicate the offset load for the bending test. A maximum compressive load of 200N was applied to each point to form 2Nm bending and 200N axial compression.

Conservative bending and compressive loads were selected to confirm that the disc, posterior element, end plate and other ligament structures were not damaged. Pure bending was not created using this loading method. Instead, a combination of bending and axial compression was presented in the flex/extension combination and side bend tests. The inventors considered this to support that in vivo loads hardly form pure bending than the combination of compression and bending. In each load case, all loads were uniformly applied to each specimen, making the mechanical response directly comparable.

In the torsion test, a pure axial torsion of 5 Nm will be applied. This was within the expected physiological torque range applied in other experiments. Using a new custom designed torque test system, we will apply pure torque to each FSU. The system uses a ballscrew/thrust plate mechanism to convert the axial potential of an Instron actuator into pure rotation. The X-Y bearing table ensures that the FSU does not have a fixed center of rotation added to it during testing. This is an important point, as the center of rotation is not constant during the axis rotation. The lower cup was secured with a torque transducer, and the upper cup was secured to an X-Y suitable for table and ball screw/push plate mechanisms.

All tests were performed first in an intact FSU. After completion, the disk was cut through the neural foramen using a small smooth lead, and the entire posterior element was cut, and the posterior was cut and separated. The entire interphalangeal joint, interspinous and superfine ligaments are cut, and the intervertebral discs, posterior and anterior longitudinal ligaments remain intact. This incision was made in the form of a wedge that widens posteriorly to prevent contact between the interprotrusion joints. Then all tests were repeated with separate disks.

The data analysis included parameters such as stiffness in the linear region, hysteresis, and elongation energy and area in the neutral zone during the 5th loading cycle. Control level data were compared to denaturation/MPC-injected levels, and repeated analysis of variance measurements were performed for each of the biomechanical parameters.

result

All animals in the MPC injected group maintained normal body weight, but there was no evidence of adverse events during the experiment.

In chondroitinase-ABD injected discs, the depletion of PG by this enzyme reduced the disc height index (DHI) by 38% after 3 months in all of the injected discs. This reduction in disc height indicates a state of degeneration of the nucleus prior to treatment, and so far is referred to as pre-MPC DHI. HA or MPC + HA failed to achieve any significant increase in DHI compared to pre-MPC DHI 3 months after injection into the degeneration site (FIG. 26 ). However, up to 6 months after treatment, the discs injected with MPC + HA showed an average increase of 52% in DHI compared to the corresponding 3-month score (group 1) (Fig. 26 and Table 4). On the other hand, in the disk injected with only HA, an average improvement of the DHI score of 23.1% was observed during the same period (FIG. 26 and Table 4). Importantly, the average DHI of the discs injected with MPC + HA at low concentration was comparable to the DHA score for the control discs injected with non-chondrotinase ABC (i.e., no-degeneration) 6 months after treatment (Figure 26 ).

Table 4 shows the statistical analysis results of DHI at 6 months vs. 3 months after HA or MPC + HA injection.

As a result of administering a positive amount of MPC and an appropriate carrier such as high-weight hyaluronic acid (HA) to the nucleus of a degenerated IVD made experimentally, as a result of radiograph analysis by restoration of the disc height in the present invention, the extracellular matrix of the disc It has been found to promote regeneration. This analysis is based on the speculation that, in the loaded spinal column, the disk length is maintained by the presence of high concentrations of matrix proteoglycans in the NPs and inner-wheels, which impart a high expansion pressure to such structures along with the water molecules bound to them. . Indeed, the use of chondroitinase-ABC to induce disc degeneration at the beginning of this experiment depends on the ability of this enzyme to degrade and remove most of the proteoglycans from the NP extracellular matrix.

The data obtained so far suggest that the therapeutic effect mediated by MPC is a relatively slow process. In the current study, the MPC concentration of 0.5 x 10 ⁶ was particularly effective.

This experiment ended 6 months after the injection of MPC into the disk, but the level of recovery of disk height obtained after injection of MPC at a crude concentration was close to that observed in the internal control group injected with non-chondroitinase ABC. Confirmed, which means that the maximum level of NP reconstruction was achieved during this period.

Those skilled in the art will appreciate that numerous modifications and/or modifications can be made to the present invention, as shown in the specific embodiments, without departing from the spirit or scope of the present invention as broadly described. Accordingly, all aspects of the invention are considered to be illustrative and non-limiting.

All publications discussed and/or referenced herein are incorporated herein in their entirety.

All contents of documents, papers, materials, devices, articles, etc. included in this specification are only for the purpose of providing the contents of the present invention. Any or all of these matters exist before the priority date for each claim of the present application, form part of the prior art field, or are accepted as acknowledging that they are common knowledge in the field applicable to the present invention. Not.

Mesenchymal progenitor cells (MPC) + hyaluronan (HA) or HA alone was injected into a positive degenerated lumbar disc, then the degree of recovery of disc height at 3 and 6 months MPC Before injection DHI MPC After injection 3 months
DHI MPC After injection 6 months
DHI No injection cABC Exclusive cABC +HA cABC +HA+MPC No injection cABC Exclusive cABC +HA cABC +HA+MPC No injection cABC Exclusive cABC +HA cABC +HA+MPC Average 0.054 0.04 0.04 0.04 0.055 0.0416667 0.0433333 0.0383333 0.0566667 0.0516667 0.0533333 0.05833333 Standard Deviation 0.015 0.01 0.01 0.01 0.01 0.014 0.008 0.014 0.015 0.017 0.008 0.007 % Change from 3 months to 6 months 0.2 23.9 23.1 52.174 Statistical significance (P value) 0.83 0.31 0.059 0.0142 P <0.05 = significant DHI = Disk height index cABC = Chondroitinase ABC

Claims (15)

TNAP ⁺ mesenchymal progenitor cells (MPC) or progeny cells thereof, or both, comprising an enriched cell population,
A composition for treating or preventing osteoarthritis or intervertebral disc degeneration,
In the above, (i) progeny cells are obtainable by amplifying the cell population by culture, and (ii) progeny cells have the ability to differentiate into other cell types, the composition. The composition of claim 1, wherein the cell population is not formulated to be administered directly to a cartilage defect site. The composition of claim 1, wherein the cell population is formulated to be administered to a joint space. The composition according to claim 3, wherein the joint space is a knee joint, a hip joint, an ankle joint, a shoulder joint, an elbow joint, a wrist joint, a hand or finger joint, a foot joint, or an intervertebral disc joint. The composition of claim 3, wherein the cell population is formulated to be administered by intraarticular injection. The composition of claim 1, wherein administration of the cell population causes maintenance or production of proteoglycan-rich cartilage. The composition according to claim 6, wherein the proteoglycan-rich cartilage is vitreous cartilage. 8. The composition according to any one of the preceding claims, further comprising hyaluronic acid (HA). i) TNAP ⁺ mesenchymal progenitor cells (MPC) or progeny cells thereof or both, and
ii) containing hyaluronic acid,
Composition for treating or preventing osteoarthritis or disc degeneration. delete delete delete delete delete delete

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