TW505518B - Compositions for the treatment and repair of defects or lesions in cartilage or bone using functional barrier - Google Patents

Abstract

Methods and compositions are provided for the treatment and repair of defects in the cartilage or bone of humans and other animals as in full-thickness defects in joints. To induce cartilage formation, a defect in cartilage is filled with a matrix having pores sufficiently large to allow cartilage repair cells to populate the matrix. The matrix contains an anti-angiogenic agent that serves as a functional barrier to prevent vascularization and bone growth into the cartilage area. The matrix filling the defect in cartilage may also contain a proliferation agent and a chemotactic agent, and a transforming factor in an appropriate delivery system. A functional barrier between the bone and cartilage areas of a full-thickness defect may also be created by heat-treating the areas of bleeding to form a transient tissue barrier which prevents blood vessels and associated cells from penetrating from the bone area into the cartilage area. If desired, the bone portion of the full-thickness defect may be filled with a matrix having pores large enough to allow cells to populate the matrix and to form blood vessels. The matrix filling the bone defect may contain an angiogenic factor and an osteogenic factor in an appropriate delivery system. Methods and compositions are also provided for assisted bone and connective tissue regeneration for dental and other applications.

Description

Printed by the Employees' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs -4- 505518 5. Description of the Invention (Technical Field of the Invention The present invention relates to the treatment and repair of patellar defects or money and cartilage and bone alterations. In particular, the invention Methods for treating cartilage and = defects or lesions (interchangeable in the text) and cartilage repair composition 'includes a matrix containing an anti-angiogenic agent as a functional barrier to prevent vascular growth of the bone matrix New cartilage tissue. The cartilage repair composition 3F may contain-or multiple proliferating agents and transforming factors to promote the growth and transformation of cartilage repair cells to form new stable cartilage tissue. The bone repair composition contains 3 The matrix of angiogenic factors that stimulate blood formation and bone growth factors that stimulate bone formation and can also be used to treat full-thickness bone defects. The compositions and methods of the present invention are particularly useful in the treatment of severe osteoarthritis, and others Full-layer defects seen in diseases or trauma that cause cartilage or bone damage. If it is desired to inhibit or delay the growth of certain tissues, other compositions and methods of the present invention (using other anti-groups Weaving factor as a functional barrier) can be used to treat other injuries and defects such as periodontal disease. Technical background Joints are one of the common bone connection methods in bones. The ends of normal bones cover joint cartilage tissue, which makes bone and bone Only the frictional movement can be carried out between the two sides [Edited by Weiss, Cell and TiSSue Biology (Munchen · Urban and Schwarzenburg, 1988) p.274] 0 The characteristic of articular cartilage is a specific structural organization It is composed of specialized cells (chondrocytes) buried in an intercellular substance (commonly referred to as " chondrocyte matrix " in the literature). This intercellular substance is rich in proteoglycan, which is mainly type II. Collagen microfibers, other proteins, and water [Bulkwater's paper size applies Chinese National Standard (CNS) A4 specifications (21〇 > < 297mm) (Please read the precautions on the back before filling this page)

Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 505518 A7 B7 V. Invention Description (2) (Buckwalter) and others, " Articular Cartilage: Injury and Repair, " in Injury and Repair of the Musculoskeletal Soft Tissues (Park Ridge, 111 .: American Academy Of Orthpaedic Surgons Symposium, 1987) p. 465] 0 Cartilage tissue has neither nerve distribution nor vascular or lymphatic system penetration. However, in the mature joints of adults, the subchondral bone tissue, which is located below, forms a narrow, continuous plate between bone tissue and cartilage, with nerve distribution and blood vessels. Below this bone plate, bone tissue forms a trabecula containing bone marrow. In immature joints, there are only primary trabeculae below the articular cartilage. A part of the meniscus tissue in the joint is also composed of cartilage, which is similar to articular cartilage [Beaupre, A. et al., Clin Orthot). Rel. Res. 72-76171986,] 〇 Joint Two types of defects can be identified on the surface, namely full-layer defects and shallow defects. These defects not only have different degrees of physical damage to cartilage, but also the nature of the repair response caused by various types of damage. Defects in the full-thickness joint surface include damage to hyaline cartilage, tortuous cartilage layers, and subchondral bone tissue containing its blood vessels and bone marrow. Damage to bone tissue can range from fissures or fissures to larger fissures in bone tissue. Full-thickness defects can cause severe pain because the bone plate contains sensory nerve endings. Such defects are usually caused by severe trauma or degenerative joint disease such as post-osteoarthritis. Occasionally, full-layer defects can cause bleeding and defamatory repair of subchondral bone. [Buckwalter et al., &Quot; Articular Cartilage: Composition, Structure, Response to Injury, and Methods of Facilitating Repair ", Articular Cartilage and Knee Joint Function Basic Science and Arthroscopy (New York: Raven Press, 1990) Article _____ This paper standard applies to China National Standard (CNS) A4 Specifications (210X297 mm) (Please read the notes on the back before filling out this page) Order 505518 A7 __________B7_ V. Description of the Invention (3) 19-56]. The resulting repair tissue is a fibrous type of cartilage filled with blood vessels', which is insufficiently biomechanical to sustain [previously, Buckwalter et al.]. Superficial defects in articular cartilage tissue are limited to the cartilage tissue itself. Such defects are notorious because they fail to heal and have no tendency to repair. Superficial defects can show cracks, fragments, or nicks on the surface of the joint, or appear as "crab meat" in the affected area. They do not contain bleeding vessels (blood spots) as seen in full_thickness defects. The cause of superficial defects may be unknown 'but often the result of mechanical chaos leading to abrasion of worn cartilage tissue. Mechanical disturbances can be caused by joint trauma, for example, replacement of ruptured meniscus tissue into the joint, joint meniscectomy, loosening of the joint due to rupture of the ligament 'joint arrangement disorder, fracture, or genetic disease. Superficial defects are also an early feature of degenerative joint disease such as osteoarthritis. There is no nerve distribution in the cartilage tissue [Ham ’s Histology (9th ed.) (Philadephia: J.B. Lippincott Co. 1987), pp. 266-272], and the surface defect is not painful. However, although painless, shallow defects do not heal and often degenerate into full-thickness defects. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling out this page) General letter because the articular cartilage lacks blood vessel distribution, the damaged cartilage tissue cannot receive sufficient or appropriate stimulation to defy the repair response [Webber, et al., &Quot; Intrinsic Repair Capabilities of Rabbit Meniscal Fibrocartilage: A Cell Culture Model ", (30th Ann. Orthop. Res. Soc ·, Atlanta, Feb. 1984; Webber, et al., J. Orthop. Res., 1, pp. 36-42 (1985)]. Theoretically, chondrocytes in cartilage tissue do not normally contact a sufficient amount of repair stimuli, such as those that are generally found in vascular distribution. -6_ Paper size Applicable to China National Standard (CNS) A4 specification (210X 297 mm) 505518 A7 B7 V. Description of the invention (4) Growth factor and fibrin clot in the tissues to be drawn. It is used to contact the damaged cartilage tissue to repair One method of stimulus is to drill or cut through the subchondral bone through cartilage to cause bleeding [previously, Buckwalter et al. (199)]. Unfortunately, tissues have been surgically invasive with this type of surgery. The injury < repair response is usually equivalent to the naturally occurring response in a full-thickness defect that causes bleeding, that is, the formation of fiber-type cartilage with insufficient biomechanical health. The method is long-lasting [previously, BuCkwalter ) Et al. (1990) have isolated many growth factors and are now available for research or biomedical applications [see, for example, Rizzin. A Dev. Biol. 130, pp. 411-422 (1988)]. Some of these growth factors, such as transforming growth factor Lu (TGF-Lu) have been reported to promote the formation of cartilage-specific molecules in mouse embryonic mesenchymal cells in vitro, such as type ^ collagen and cartilage-specific proteoglycans [ For example, Seyedin et al., Proc. Natl. Acad. Sci TTSA, pp. 2267-71 (1985); Seyedin et al., J. Biol rhem 261 · 5693_95 (1986); West Yeye (Seyedin) et al., J. Biol 262 · 1946-1949 (1987)]. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the notes on the back before filling this page). In addition, many obvious A protein factor that stimulates bone formation. Promoters include bone morphogenic proteins, osteogenin, bone osteogenic protein (the BOP), bone slander TGF_y5 s, the recombinant protein of hair. Millions of patients have been diagnosed with osteoarthritis, that is, degenerative defects or damage to their articular cartilage. Nevertheless, despite the various methods claiming to be able to provoke a repair response in damaged cartilage, there is still no widely used treatment [previously, Buckwalter (1990) et al., The Chinese paper standard is applicable to the standard of this paper ( CNS) A4 specification (210X297 mm) 505518 A7 B7 V. Description of the invention (5) Kuntson et al., J. Bone and Joint Surg. 68, pp. 795 (1986), Kuntson et al. J. Bone and Joint Surg. 67-B. 47 (1985); Kuntson et al., Clin. Orthop. 191. 202 (1984); Marquet, Clin, Orthop . > 146 · p. 102 (1980)]. Such treatments usually provide only temporary relief. Systemic use of "cartilage protectants" also claims to suppress the progression of osteoarthritis and relieve pain. However, such agents have not been shown to promote repair of damage or defects in cartilage tissue. At present, the treatment of patients with osteoarthritis mostly uses analgesics and anti-inflammatory agents to relieve symptoms. The treatment of repairing the surface defects of the articular cartilage will not wear off the cartilage to the subchondral bone plate. At this stage of disease, that is, when severe osteoarthritis, the nature and function of pain are not significantly affected, often showing that the entire joint needs to be removed, and replaced with metal and / or plastic artificial joints. At present, 500,000 operations are performed on the knees and hips every year, including joint removal and artificial rhyme replacement. [See Graves, EJ ·, "1988 Summary; National Hospital Discharge Survey ", Advanced Data from Vital and Health Statistics. 185. pp. 1-12 (June 19, 1990)] 〇 Employees of the Central Standards Bureau of the Ministry of Economic Affairs Printed by a consumer cooperative (please read the precautions on the back before filling out this page). Therefore, it is necessary to treat cartilage and bone tissue in superficial cartilage defects and cartilage and bone tissue in full-layer defects (if found in severe arthritis) reliably. Abstract of the Invention The solution of the present invention to the aforementioned problems is to provide effective treatment methods and compositions to slap repair of cartilage or bone damage in humans and other animals. Using the methods and compositions of the present invention can also promote traumatic The damage and various forms of osteoarthritis are cured, otherwise the latter will lead to the loss of effective joint function, resulting in ___-_ 8 ^ _ This paper size is applicable to the Chinese National Standard (CNS) A4 specification (210 × 297 mm) 505518 6 Description of the invention (阙Section may be removed and replaced .: In general, treatment of superficial bone defects or full-thickness defects = ,: = Includes filling of the defective cartilage with blood suppression Tube = agent of cartilage repair matrix, the angiogenic agent is a protease inhibitor or an antibody that antagonizes the angiogenic factor. The cartilage repair matrix can be combined with human and animal tissues; it can also contain proliferative agents and According to WJ and transformation factors, the cartilage repair matrix of the present invention is particularly suitable for the treatment of full-layer defects and significant shallow cartilage defects, and there may be cracks or fissures in one side. 〃 In another embodiment, the method of the present invention It includes heat-treating the area of the full-layer defect, where bleeding occurs to create a temporary tissue barrier, and the defect is filled with cartilage to repair the matrix after cooking. In this specific embodiment, the anti-angiogenic agent can be deleted from the matrix. Methods for protecting full-thickness defects of the joints also include filling the bone defects of the full-thickness defect with bone matrix to the bone-sacral interface stage I due to epitaxial bone damage or as needed. The matrix can be incorporated into animal tissue and is usually Biodegradable. Bone repair matrix can contain angiogenic factors and osteogenic factors. Defective residual cartilage can contain antiangiogenic agents that inhibit angiogenesis. Central Ministry of Economic Affairs standard Printed by the Consumer Bureau of the Prospective Bureau < Cartilage repair matrix fills the top of the cartilage surface. The cartilage repair matrix can also contain proliferative agents and transforming factors. Can be examined by arthroscopy, open surgery or percutaneous treatment steps The method of the present invention is used to treat superficial and full-thickness defects. According to some methods of the present invention, after the full-layer defect is identified, the defect can be treated by the following steps: (1) using a package containing angiogenic factors and packaging Appropriate conveying system • 9 paper standards are applicable to Chinese National Standards (CNS) μ Regulation Father 297 Public Manager 505518 V. Description of the invention (7) (for example, microlipids) osteogenic factor parent material composition filling defects Bone component; and (2) filling the defective cartilage portion with a composition containing a parent material, which is preferably biodegradable, contains an antiangiogenic agent and a transforming factor, and is packaged in a suitable delivery system in. In the second step, for example, an adhesion promoting factor such as transglutaminase can be used to bind the mother substance to the surface of the fully-recessed cartilage portion. Wang's can also help the periodontal disease with the method and composition of the present invention. For example, in the treatment of diseases such as periodontal degeneration, in which the periodontal ligament has subsided and the roots are exposed, the periodontal node can be filled with a mother substance containing one or more periodontal tissue formation factors and one or more epithelial formation inhibitors. The tissue space assists the regeneration of dental tissue. π = The method and composition of the present invention can be used to assist bone regeneration after tooth loss. In detail, the zygomatic and metatarsal ridges can be reconstructed using bone-degradable biodegradable matrices to fill the defect area. This matrix contains angiogenic osteogenic factors and the transfer of connective groups, 哉, 、, and celestial cells. And / or inhibitors of proliferation and / or differentiation to assist bone tissue 1 regrowth. In the periodontal connective tissue area, a matrix containing one or more factors that stimulate periodontal tissue formation can be used. Central Economic Standard h The method and composition printed by the staff consumer cooperative can also be used to prevent special joint calcification and calcification (such as joint replacement) and prevent foot bones from growing into cartilage in young animals and children with distal fractures Growing plate. In detail, the surrounding joint connective tissue space or f-bone growth space can be filled with matrix containing anti-angiogenic factors or anti-bone factors to prevent calcification and bone tissue formation. As detailed as possible, the present invention is more fully understood, and the following detailed description is provided. -10-This paper ruler Yan Caisong National Standard (CNS) A4 Specification 505518 A7 B7 V. Description of Invention (8) Use the following terms. ikcamp generating factor-as used herein means any peptide, polypeptide, protein or any other peptide that can suffocate or stimulate blood vessels and related cells (such as endothelial, peripheral blood vessels, mesenchymal and smooth muscle cells) and blood vessel related basement membrane Compound or composition. In vivo and in vitro assays for angiogenic factors are well known in the art [eg, Gibrone, M.A. et al., J. Natl. Career Inst ·, 12, ρρ · 413-419 (1974); Klagsbrun, M. et al., Cancer Res., 36 · ρρ · 110-113 (1976); Gross et al., Proc. Natl. Acad. Sci. (USAV 80 · pp. 2623 -2627 (1983); Gospodarowicz et al., Proc. Natl. Acad. M OJSA) Zl, pp. 4120-4124 (1976); Folkman et al., Proc. Natl. Acad. Sci. (USAV 76. pp. 5217-5221 (1979); Zetter, BR, Nature (London) 285 · pp. 41-43 (1980); Azizkhan, RG, etc. People, J. Exp. Med ·, 152. pp. 931-944 (1980)] 〇 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) Anti-angiogenic agents- As used herein, it refers to any compound or composition whose biological activity prevents the vascular growth of bone tissue to cartilage tissue such as anti-invasion factors, derived cartilage angiogenesis inhibitors, angiostatin, A protease inhibitor, antibodies against angiogenic factors (including bFGF and endothelial cell stimulating angiogenic factor (ESAF)), Suramin (Germanin®, Bayer Co., Germany), fumagillin, fumagillin analogs and AGM-1470 [ Peacock, DJ, et al., Cellular Immunology, 160, pp. 178_84 (1995)]. The in vitro and in vivo analysis of antiangiogenic agents is a known technique [eg, Moses, M.A., Clinical _-Jjj_ This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) 505518 A7 B7 V. Description of the invention (9) & Exptl. Rheumatology. 1 KSuppl. 8) · pp 567-69 (1993); Moses, M A. et al., J. Cell Bio .. 1190. pp. 475-82 (1992); Moses, M.A. et al., Science. 248. pp. 1408-10 (1990); Ingber, D. et al. People, Nature 34816). Pp. 555-57 (1990)]. Anti-tissue factor-as used herein, relates to any compound or composition whose biological activity selectively prevents unwanted growth of specific tissues. For example, anti-epithelial factors that selectively inhibit the formation of epithelium include anti-epithelial antibodies, vitamin A inhibitors, anti-retinol, anti-basement membrane antibodies, epithelial growth factor inhibitors, matrixes with fibrin-reinforced proteins, and inhibition of epithelium Cell proliferation, growth or differentiation or any other factor of epithelial formation [see eg Adams, JC and Watt, FM,, fFibronectin inhibits the terminal differentiation of human keratinocytes ", Nature, 340 · pp. 307-09 (1989)] 0 select Anti-connective tissue factors that sexually inhibit connective tissue formation include anti-connective tissue antibodies, antibodies against connective tissue-specific growth factors, antibodies against mesenchymal cell surface proteins, and factors that inhibit mesenchymal cell proliferation. Anti-bone factors that selectively inhibit the formation of bone tissue include anti-angiogenic factors and single or multiple strains of antibodies against members of the TGF_B superfamily, or combinations thereof. Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling out this page) Arthroscopy-as used here refers to the use of arthroscopy to inspect or perform joint surgery. -As used herein, it refers to a calcified connective tissue, which mainly contains a network of calcium and phosphate deposited in the form of hydroxide apatite, collagen (mainly type I collagen) and bone cells, such as Osteoblasts and osteoclasts. Bone repair cells-as used herein refers to a cell that, when exposed to an appropriate stimulus, will differentiate and transform into bone cells, such as osteoblasts or bone cells, but ____- 12- This paper size applies to Chinese national standards ( CNS) A4 size (210 X 297 mm) 505518 A7 B7 V. Description of the invention (10) (Please read the precautions on the back before filling this page) before forming bone. Bone repair cells include perivascular cells, mesenchymal cells, fibroblasts, fibroblast-like cells, and dedifferentiated chondrocytes. Cartilage--as used herein, refers to a connective tissue containing chondrocytes embedded in an intercellular substance (often referred to as cartilage matrix). This intercellular substance contains collagen microfibers (mainly type II collagen Plus other minor species, such as 'types IX and XI)' various proteoglycans (eg, chondroitin sulfate-, keratin sulfate-, and dermatan sulfate proteoglycans), other proteins and water . The cartilage used here includes articular cartilage and meniscal cartilage. Articular cartilage covers the surface of the bone at the joint, so that the bone does not come into direct contact with the bone when the joint is moved, so it avoids abrasion and damage to the opposite bone surface. Most normal healthy articular cartilage is also known as, vitreous ", which has the characteristic of frosty glass appearance. Meniscal cartilage usually appears in joints affected by vibration and movement. Locations of such meniscal cartilage include temporal-bone-mandible, sternum-clavicle, shoulder-clavicle, wrist and knee joints "Grav's Anatomy (New York: Bounty Books, 1977)]. # 1 Bone repair _ color straight_ As used herein refers to a cell that, when exposed to appropriate stimulation, will 'differentiate and transform into chondrocytes. Cartilage repair cells include mesenchymal cells' fibroblasts, fibroblast-like cells, Giant gift cells and dedifferentiated chondrocytes. Factors-as used herein refers to any compound or composition that regulates the adhesion of cells to extracellular shellfish, including fibronectin and other peptides as small as tetrapeptides, Which includes the tripeptide Arg_

Gly-Asp '[Rooslathi et al., Cell 44, pp. 517-518 (1986)]. -13- Tong Guang ▼ * Public 7 9. 2 505518 Printed by A7 B7, Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of Invention (") Chemotactic Agents-as used herein refers to chemotaxis detection in standard in vitro Any compound or composition that attracts cells in a method, including peptides, proteins, glucoproteins, glucosamine polysaccharide chains. [Eg, Wahl et al., Proc. Natl · Acad. Sci. USA, M, 5788_92 (1987); Postkewaite et al., J. Exp. Med. 165 251-56 (1987); Moore et al., Int. J. Tiss. Reac .. XI, 301-07 (1989)] Chondrocytes-as used herein refers to cells that produce components of cartilage tissue, such as type II chondrogenic microfibers and fibers and proteoglycans. Fibroblast Growth Factor (FGF) —Any member of the FGF polypeptide family [Gimenez-Gallego et al., Biochem. Biophvs. Res. Commun. 135. pp. 541-548 (1986); Thomas Et al., Trends Biochem. Sci. · 11 pp. 81-84 (1986)] or derivatives thereof, may be derived from natural, synthetic or recombinant Ability to stimulate DNA synthesis and cell differentiation of various cells in vitro [Detection, see, for example, Gimenez-Gallego et al., Supra; Canalis et al., J. Clin. Invest .. 1572-1577 (1988)], including primary fibroblasts, chondrocytes, blood vessels and corneal endothelial cells, osteoblasts, myoblasts, smooth muscle and glial cells [Thomas et al., 1986 Based on their isoelectric point (pi), FGF can be classified as acidic (aFGF) or basic (bFGF) FGF. Parent material-as used herein refers to a porous composition, solid or semi-solid material, with A pore or space that is large enough for the cell population to occupy it. The term "maternal material" includes the material that forms the parent material, that is, the material that forms the parent material in the cartilage or bone defect. The parent material requires the addition of a polymerizing agent to Forming mother ____ -14-_ This paper size is applicable to China National Leather (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before filling this page) 505518 A7 B7 V. Description of the invention (12) ( Please read the notes on the back before filling Page) endoplasmic, such as adding thrombin to a blood-containing solution of fibrinogen to form fibrin blood parent material. Other parent materials include collagen, a combination of collagen and fibrin, agarose (ie, Sepharose®) and gelatin. Phosphate dance such as tricalcium phosphate, hydroxide apatite, or other calcium salts that form a solid parent material can Used alone or in combination with other parent materials for the treatment of bone defects. Membrane-, as used herein, means that it can be placed between the full-thickness defect and the cartilage defect, and can prevent bone Any material in which the cells of the defective portion migrate or infiltrate the cartilage defect portion of the full-thickness defect. The present invention provides a biofilm for use in compositions and methods for repairing full-thickness defects. Degradability is better. Osteogenic factor-as used herein refers to any peptide, peptide protein, or any other compound or composition that can stigmatize or stimulate bone formation. Osteogenic factor spit bone repair cells to differentiate into osteocytes , Such as osteoblasts or bone cells. This process can be reached through the intermediate state of cartilage tissue. Bone tissue formed from bone cells can contain bone-specific substances, such as type I collagen microfibers, phosphate hydroxide Stone minerals and various glycoproteins and a small amount of bone proteoglycans. The proliferative (mitotic) agent printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs-as used herein refers to any chemical compound or composition that can stimulate cell proliferation in vitro. Including peptides, proteins, and glucoproteins. In vitro assays for determining the proliferative (mitotic) activity of peptides, peptides, and other compounds are well known in the art [see Canalis et al., J. Clin · Invest ·, ρρ · 1572-77 (1988); Gimenez-Gallego, Biochem. Biophvs. Res. Commun · 135. ρρ · 541-548 (1986); Rizzino ) " Soft Agar Growth Assays for Transforming Growth Factors and Mitogenic -15- This paper size is applicable to Chinese National Standard (CNS) A4 specification (2i × 297mm) 505518 A7 B7 V. Description of the invention (13)

"Peptides", in Methods Enzymol · 146A (New York: Academic Press, 1987), pp · 341-52; Dickson et al., &Quot; Assay of Mitogen-Induced Effects on Cellular Incorporation of Precursors for Scavengers, de Novo · and Net DNA Synthesis ", in Methods Enzymol · 146A (New York: Academic Press, 1987), pp. 329-40]. A standard method for determining the proliferative (mitotic) activity of a compound or composition is in vitro detection Its ability to blame the non-transformed cell's anchor-independent growth in soft agar [eg, Rizzino, 1987, supra]. Other mitotic detection systems are also known [eg, (Gimenez-Gallego, et al., 1986, supra; Canalis, et al., 1988, supra; Dickson, et al., 1987, supra). The mitotic effect of reagents is often very dependent on concentration changes, and The effect can be reversed when the concentration of Fanyuan is lower or higher than the most appropriate concentration of mitotic effect. Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please first (Please read the notes on the reverse side and fill in this page) Transformation factor- • As used herein refers to any skin, polypeptide, protein or any other compound or composition that can spit cartilage repair cells and differentiate into chondrocytes. The ability of the compound or composition to stigmatize or stimulate cells to produce chondrocyte-specific proteoglycan and type II collagen in vitro is known in vitro [West Leaf; Ding (Seyedin) et al., Proc. Natl. Acad. Sci USA. 82 pp. 2267-71 (1985); Seyedin et al., Path. Immunol Res Z, pp. 38_42 (1987)]. Transforming growth factor yg (TGF · /?)-TGF -Any of the peptide families [Derynck, R. et al., Nature, 316, pp. 701-705 (1985); Roberts et al., &Quot; The transforming growth -16- This paper Standards are applicable to China National Standard (CNS) A4 (210X297 mm) 505518 A7 B7 V. Description of the invention (14) factor- / ?, s ", In Peptide growth factors and their receptors I (Berlin: Springer Verlag, 1990 ), P. 419)] or its derivatives, which are natural, synthetic or heavy Groups, which have the TGF- /? Characteristic ability to stimulate normal mouse kidney (NRK) cell growth and colony formation in soft agar assay [Roberts et al., &Quot; Purification of Type Transforming Growth Factors From Nonneoplastic Tissues " , In Methods for Preparation of Media, Supplements, and Substrata for Serum-Free Animal Cell Culture (New York: Alan R. Liss, Inc., 1984)] and sacrifice or stimulate cells to produce cartilage-specific proteoglycans and The ability of type II collagen can prove that it can spit cartilage repair cells into chondrocytes, [Seyedin et al., 1985, supra]. The present invention relates to compositions and methods for treating cartilage or bone defects or damage. The composition of the present invention contains a matrix material having pores of a size sufficient to allow a cell population to reside in the matrix material. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) When cartilage used to repair surface defects or full-thickness defects, the matrix contains anti-angiogenesis Agent, its biological activity prevents the vascular long cartilage to prevent bone formation and improper repair of cartilage tissue. The matrix also contains a proliferative agent to stimulate the growth of cartilage repair cells in the matrix. Proliferators also act as chemotactic agents to attract cartilage to repair cells to the parent material. In addition, in addition to the proliferative agent, the matrix can also contain chemotactic agents. In a preferred embodiment of the present invention, the parent material also contains a transforming factor at an appropriate concentration. The transforming factor is contained in or in combination with a transport system. This transport system releases the transforming factor at an appropriate time to proliferate the matrix. Cartilage repair cells are transformed into cartilage that produces stable cartilage tissue-17- This paper size applies to Chinese National Standard (CNS) A4 (21 × 297 mm) Α7 Β7 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (15 cells. The matrix can also contain cell adhesion promoting factors. 万 Repair of the matrix with cartilage with defects of the king layer and the king layer, without the need for chemotaxis or proliferators and also: the release of conversion factors needs to be substantially delayed. In the full-thickness defect, Tongdang allows the repairing cells to be stored under the bone and does not need to recover synovial fluid for this purpose. The area may contain proliferative agents and chemokines. Since the repaired cells will quickly cluster the defects, the transformation factor may substantially delay the exposure. However, the surface defects of the human and the induced proliferative repaired cells will be worse. It is important. However, if the defect area is large, the transformation factor can be separated to ensure that sufficient repair cells can proliferate before crossing the defect area and the exposure of the transformation factor. In addition, in the treatment of full-thickness defects, The parent material of the delivery system contains anti-angiogenic agents and turning agents to provide a continuous concentration over a period of time. "Monthly" In the case of a full-thickness defect extending to the bone, the defective bone portion is preferably filled with defects. The cartilage component is filled with the cartilage repair matrix of the present invention to fill the bone repair matrix. The matrix materials that can be used in the method or composition for filling or filling cartilage or bone defects of the present invention include fibrinogen (activated by thrombin) , Forming fibrin at the defect or damage), collagen, agarose, gelatin and any other biodegradable substances, the matrix formed has pores, the size of which is large enough for cartilage or bone repair cells to reside in this matrix Hyperplasia, and can be broken down and replaced by cartilage or bone during repair. In some cases, calcium phosphate-containing compounds such as tricalcium phosphate and hydroxide apatite can be used alone And other calcium salts that form solid parent materials or with other biodegradable parent materials and are used to treat bone defects. -18-Each paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) (please read the back first) (Notes for filling in this page)

2 The parent f in the composition and method of the present invention may be formed in advance or may be J, for example, the compound and composition (such as fibrinogen) may be dimerized to form a fibrin matrix. Differentiable parent materials include collagen (collagen 4 and collagen tablets), chemically modified collagen, gelatin beads or sponge materials such as agarose 'and any other gelatinous or combination materials (this material is made from The cheekbones or bone repair fine lining resides in the matrix material of the matrix towel), or a mixture of the above. ′ In the specific example of this item, fibrinogen is used to form the parent material, and 2 is added with thrombin in front to start polymerization. A buffered aqueous solution with a fibrinogen concentration of 0.5 mg / ml can be used. The fibrinogen solution is preferably 1¾ g / ¾ liter buffered aqueous solution. The matrix produced by the polymerization of the fibrin solution in the defect area has a sufficiently large pore size (for example, about 50-200 microns), so that cartilage or bone repair cells can freely colonize and proliferate in the matrix to fill the matrix. Occupied defect volume. It is best to add a sufficient amount of thrombin to the fibrinogen solution immediately before administration, so that the surgeon has enough time to accumulate material in the defect area before completing the polymerization. In general, the concentration of thrombin should be achieved within a few (2_4) minutes, as long-term exposure of cartilage to the air has been found to cause injury [Mitchell et al., J ^ Bone Joint Snrg, 71A, 89- 95 (1989)]. Thrombin should not be used in excess, as thrombin has the ability to lyse growth factor molecules, rendering them inactive. A thrombin solution of 10-500 units (preferably 100 units) per ml of the buffered aqueous solution can be prepared and added to the fibrinogen solution. In a preferred embodiment of the present invention, approximately 200 seconds before filling the defect, each fibrinogen solution (1 mg / ml) and approximately 20 microliters of blood coagulation are used. ) A4 size (210X297mm) 505518 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (η) ΓΓΓ, ml) mixed. If a lower concentration of thrombin is added, the coagulation required for poly-polymerization will be as follows: r = Achieving the fibrin production, and the approximate number of thrombin, because it depends on the environment: Degree: temperature of blood clotting solution, blood fiber The protein solution temperature and so on. % Filled Defective Mother I ^ Mother Beth Thrombin Activated Polymerization It is easy to persuade the external blood fibers I, 六, r, 田田 观, Shirahara, and Gu Lu samples to be condensed by thrombin. Human role to monitor. In the composition and method of the present invention, fibrinogen i is preferably formed from autologous fibrinogen molecules, for example, fibrinogen molecules obtained from the same species as the species to be treated (blood of animal animals. Also Non-immune fibrinogen from other species can be used. Fibrin, prion, and collagen-containing matrixes are preferably fibrin and gelatin. Also used in the composition and method of the present invention. Collagen matrix can also be used For repairing cartilage defects, including full-layer defects. In the preferred embodiment of the present invention, more solid mother-of-pearl, such as those containing martensite or tricalcium phosphate, is used to repair deep full-layer defects. When collagen is used For matrix materials, a solution with sufficient viscosity can be used, for example, collagen-viiess ("tablets"), sponge streets, or gelatin blood mixtures, without the need for a polymerization agent. Collagen masterbatch It can also be used with a fibrinogen solution activated by a polymerizing agent to produce a combined parent material. When using other biodegradable compounds to form a parent material, it is also possible not to use a polymerizing agent For example, you can choose Sepharose® solution, which is a liquid mother solution at 39-42 ° C, and becomes solid (ie, gelatinous) at 35-38 ° C. The concentration of Sepharose should also make filling defects Bingqi Ning (Please read the precautions on the back before filling in the 'installation-this page} -Order -20- This paper size applies Chinese National Standards (CNS) μ specifications (21〇X297 mm) 505518 No. 0861 〇8885 Chinese Patent Application Specification Correction Page (June 91) V. Description of the invention () The size of the sieve of the glue enables bone or cartilage repair cells to freely colonize the parent material and defect areas. The invention for cartilage repair In the composition, one or more appropriate concentrations of an anti-angiogenic agent may be added to the mother solution to prevent blood vessels from growing into the cartilage tissue. Useful anti-angiogenic agents include any biological activity that prevents the blood vessels of the bone tissue from growing inward to the cartilage tissue. Agents. Some examples of anti-angiogenic agents can be used in the present invention. The anti-angiogenic agents should be freely available to provide immediate activity in the parent material and may also be present in sustained release dosage forms as follows Related delivery systems for prolonged activity. One or more proliferative (mitotic) agents may be added to the matrix solution for cartilage repair. The proliferative agent should be in a suitable concentration range in order to have proliferation of cartilage repair cells in the filling matrix of the defect Effect. This reagent should also have a chemotactic effect on cells (such as TGF-々); however, it is possible to use a factor that only has a proliferative effect. In addition, for the migration of chemotaxis cells, it can be used before the cell proliferation Two different agents are used, each of which has a specific effect (chemotaxis or proliferation). The proliferative (mitotic) agents used to stimulate the proliferation of cartilage repair cells in the composition or method of the present invention include transforming growth factors ["TGFs "], Such as TGF-α, TGF-Ling; insulin-like growth factor ("IGf I"); acidic or basic fibroblast growth factors (" FGFs "); platelet-derived growth factors (" PDGF "); Epidermal growth factor (" egf "); and hematopoietic growth factors, such as interleukin 3 (" IL-3") and bone shape Genes ("BMPs"), such as Bone Morphogenetic Protein-2 ("BMP-2") [Rizzino, 1987, supra; Canalis et al., Supra, 1988; Growth factors in biology and medicine, Ciba Foundation Symposium. -21-This paper size applies Chinese National Standard (CNS) A4 (210X297 mm) 505518 A7 B7 V. Description of the invention (19) 116 (New York; John Wiley & Sons, 1985); Baserga, R., ed., Cell growth and division (Oxford: IRL Press, 1985); Sporn, MA and Roberts, ed. AB, Peptide growth factors and their receptors , Vols. I and II (Berlin: Springer · Verlage, 1990)]. However, these specific examples are not restrictive. Any compound or composition that has been proven to stimulate cell proliferation by in vitro cell proliferation assays can be used as the proliferative agent of the present invention. These assays are well known in the relevant arts [for example, Canalis et al., 1988, supra; Gimenez-Gallego, et al., 1986, supra; Dickson et al. , 1987, former; Rizzino, 1987, former]. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the notes on the back before filling this page). Chemotactic agents used in the compositions and methods of the present invention to attract cartilage repair cells to cartilage defects include, for example, 'TGF- y9 s' FGFs (acidic or experimental), PDGF, tumor necrosis factor (eg, TNF-from, TNF-yS) and proteoglycan degradation products, such as glucosamine polysaccharide chains [eg Roberts et al., (1990 ), Above; Growth factors in biology and medicine. Ciba Foundation Symposium 116 (New York; John Wiley & Sons, 1985); R. Baserga, eds., Cell growth and division (Oxford · IRL Press , 1985)]. Assays for determining the chemotactic ability of peptides and other compounds are well known in the art [for example, Postkewaite et al., 1987, supra; Wahl et al., 1987, supra; Moore, etc. People, 1989, supra) 0

In a preferred embodiment of the present invention, the matrix used to repair cartilage contains TGF- / 5 as a proliferative agent and as a chemotactic agent. In particular, you can use TGF- I -22- This paper size is applicable to China National Standard (CNS) A4 (210X 297 mm) 505518 A7 _____B7 ------------------------- 〇) or TGF-AII as a proliferative agent and as a chemotactic agent. Others (such as TGF _ /? III, TGFjIV, TGFj v, etc.) or peptides with Qing 0 activity [see Roberts et al. (! &Quot; 〇), above], and the future to be detected Other types of substances, and other growth factors are also available for this purpose. When used as a proliferative agent and chemotactic agent, TGF • Lu molecules are dissolved or suspended in the parent material. The concentration is preferably 2-50 nanograms per liter of the parent solution. good. It is understood that the preferred concentration of TGF- /? That stimulates cartilage repair cell proliferation depends on the particular animal to be treated. The transformation factor can also be present in the matrix solution used to repair cartilage, so that after the cartilage repair cell population resides in the matrix, the transformation factor will be released into the defect site at a concentration sufficient to promote cartilage repair cell differentiation (i.e., (Transformation) into chondrocytes, forming new stable cartilage tissue. When the transformation factor can inhibit or interfere with the effectiveness of the proliferative agent, the proper release timing of the transformation factor is particularly important [see Roberts et al. (1990), supra]. Transformation factors printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs that can be used in the compositions and methods of the present invention to promote cartilage repair include cartilage repair cells that can differentiate into chondrocytes, produce cartilage-specific proteoglycans, and type II collagen Any peptide, polypeptide, protein or any redundant compound or composition thereof. The ability of a compound or composition to stigmatize or stimulate cells to produce cartilage-specific proteoglycans and type I collagen can be determined using assays well known in the art [Seyedin et al., 1985, supra; Seyedin) et al., 1987, supra]. Transformation factors used in the compositions and methods of the present invention include, for example, TGF / ?, TGF- ?, FGF (acid or alkaline). The transformation factor can be used alone or in combination. In addition, 'TGF- / 3 can be used in combination with EGF. -23- This paper size applies to Chinese National Standards (CNS) M specifications (BOO7 seams) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 505518 5. Description of the invention (21) If necessary, the transformation factors can be packed in appropriate conveyance Packaged in or with the system, releases the transformation factor when appropriate. The delivery system that can be used in the present invention and the method includes microfat particles, biodegradable polymers, carbohydrate-based particles, water-in-oil emulsions, fiber mouth (such as collagen), and heparin sulfate Salt proteoglycans or other molecules that can spontaneously bind to transforming factors are chemically linked and osmotically pumped. Conveying systems such as microlipids, biodegradable polymers, fibers that incorporate transforming factors, and microparticles containing transformants that are primarily carbohydrates can be mixed with the mother liquid that fills defect I. These systems are known and can be obtained in related art [see P. Johnson and J G. Lords, edited,

Diu ^ livery Systems (Chichester, England; Ellis Horwood Ltd., I987)]. Microlipids can be prepared according to the procedures of Kim et al., Biochem. Biophys 4cta ?, pp. 339-348 (1983). Other microlipid preparation steps can also be used. Other factors that stimulate chondrocytes to synthesize cartilage tissue components can be included in the delivery system with transforming factors. Additional factors that stimulate chondrocytes to synthesize cartilage tissue components can include transforming factors in the delivery system. The timing of the transformation factor should be matched with the speed, in which the repaired cells will proliferate and fill the defects to be treated. If a substantial delay in the release of the transformation factor is not required, a transformation factor can be included in the free matrix form of the matrix and the matrix associated with the delivery system to provide continuous release at a suitable concentration. In the specific example of Chevujia of the present invention, the matrix used for repairing cartilage contains Tgf-spots as a proliferative agent and a chemotactic agent, and contains TGF-lu packaging in a delivery system as a turning agent. In particular, TGF-A I or TGF-II can be used as a proliferative agent and a chemotactic agent and as a transformation factor. Other TGF • cold type (such as TGF • point -24 wood paper standard suitable for financial rewards (CNS> Α4ϋ_ · (2—Jade ·· (Please read the precautions on the back before filling in this page)

Equipment · Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs 505518 A7 ______ B7__ V. Description of the Invention (22) III, TGF- /? IV, TGF_y5 V, etc.) or active peptides (see Robert, 1990, supra ) And other forms of the substance to be detected in the future, and other growth factors are also available for this purpose. The antiangiogenic agent is preferably contained in a transforming concentration containing TGF-L or BMP and a delivery system in a free state in the matrix. In the preferred embodiment of cartilage repair, the concentration of TGF_ is preferably from 2 to 50 nanograms per milliliter of the parental solution, and most preferably 2 to 10 micrograms per milliliter of the parental solution. It can be used as a proliferative agent and chemotactic agent. . Very high concentrations of TGF- can also be present in successive releases in the matrix composition as a turning agent. The continuation of TGF-y5; the best degree is more than 200 nanograms per milliliter of parent material, and the best is more than 500 milligrams per milliliter of parent material. In addition, the preferred concentration of BMP as a transforming factor is 100-2000 nanograms / ml. It is understood that the preferred concentration of TGF_Θ or BMP that differentiates cartilage repair cells depends on the particular animal to be treated. It is necessary to alternately contact cartilage repair cells with tGF in two concentration ranges. Because the TGF · concentrations (e.g., more than 200 nanograms per milliliter of mother solution) at relatively high concentrations, not only convert cartilage repair cells into chondrocytes, Inhibits the chemotactic attraction of cartilage repair cells; and at a relatively low concentration (for example, 2-10 nanograms / ml), TGF · /? Attracts cartilage repair cells and stimulates their proliferation, but it cannot defy the cartilage repair cells to transform into Chondrocytes that produce cartilage tissue. In the preferred embodiment of the present invention, in order to obtain chemotactic, proliferative, followed by transformation, the "human sequence" TGF-main free 'unembedded form and the embedded, or cryptic form exists in the matrix. In order to attract and stigmatize the proliferation of patella repair cells in the matrix and defective areas, TGF · yS molecules are dissolved or suspended in the matrix compared to ____ -25- This paper is a national standard (CNS) A4 specification (210X297) (Mm) " " '! — Clothing — I f Please read the precautions on the back before filling this page]-, il mf 1--⑽518 A7 B7 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs Explanation (23 Jia 'concentration is 2-10 nanograms per Φ liter of mother material solution. In order to promote the cartilage repair cells in the mother material to transform into chondrocytes, TGF-A molecules are also present in the mother shell, according to Kim (Kim) Et al., 1983, the method described above is hidden in polycystic microlipids with a concentration of more than 200 μg per ml of the parental solution, preferably more than 500-800 ng / ml. When repaired by attracted cartilage When the cells are colonized in the parent material and have started to degrade the parent material, the microlipids carrying TGF_ 0 will disintegrate and begin to decompose the parent material. During the decomposition of the parent material, the cartilage repair cells digest and / or decompose the microlipids, causing TGF_ Lu Yi is enough to spit the cartilage repair cells into chondrocytes. The two-stage delivery required for tgf-point chemotaxis and proliferative effects relative to the TGF- /? Transformation concentration can also be achieved by combining the TGF-Lu transformation concentration with the bioerodible polymer. In addition, a pump can be used, and the implanted osmotic pump is better to control the defect and the concentration of TGF_々 in the matrix. In this specific example of the present invention, the pump controls the concentration of TGF_0 in the matrix, that is, This pump releases TGF-0, which is initially chemotactic and proliferative-stimulated concentration, and then the transformation concentration. The transformation concentration of TGF · /? Is preferably transported by the pump about 1 to 2 weeks after the operation. It is better that the factor is delivered to the defect area to be confined to the matrix of the defect. The proliferative agent in the composition of the present invention and the transformation factor when used are applied to the defect site in the matrix. Its existence is therefore limited to very local This method is to prevent them from being freely injected or perfused into the joint cavity. This free perfusion can cause the adverse effects of stimulating synovial cells to produce joint exudate. The delay exposure of the transformation factor is not required. Many full-layer defects are compared with shallow defects (multiple of them (please read the precautions on the back before filling out this page). Binding · Book -26-505518 V. Description of the invention (24 Need to attract and proliferate repair cells) The repair cells are suitably passed and the delay in the exposure of the transforming factor is less important. However, in deep defects, it is desirable or delayed to expose the repairing factor to allow the repairing cells to cluster all the defects. The present invention is used in a bone repair composition, Add one or more angiogenic factors to the feed solution to stimulate blood vessels and related cells (such as endothelium, peripheral blood vessels' mesenchymal and smooth muscle cells), and basement membrane formation and endogenesis of bone defect areas. It can be used in the composition and method of the present invention. The angiogenic factors that stimulate blood vessel formation in the entire maternal deposition area in the bone defect area include bFGF, Dingbang_, PDGF, TNF · ", angiopoietin or angiotrophin 丨 angiotropm It has been found that heparin sulfate can enhance the angiogenic activity of bFGF. In a preferred embodiment of the present invention, heparin sulfate is dissolved, suspended or bound in the matrix, and the concentration is about 5_1 per milliliter of the matrix solution. Nanograms. The preferred concentration of angiopoietin is: TGF-々, 5 耄 micrograms per milliliter of mother solution; TGF_, 毫升 亳 micrograms per milliliter of mother solution; fDGF, 1 liter of mother solution 0 nanograms. But among the angiogenic factors mentioned above, bFGF combined with heparin sulfate is the best angiogenic factor. The bone factor printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs can also be used to repair bone In the mother matter solution, after the blood vessels and related cell populations reside in the mother matter, when the mother matter decomposes, osteogenic factors are released into the bone defect site, and the radon concentration is sufficient to promote the development of osteoblasts. The process of osteocytes. The osteogenic factor is secreted or packed in a suitable delivery system in the matrix, which is released when the matrix is decomposed. The transport system used in the cartilage repair composition can be used in the bone repair composition of the present invention, For example, microfat or carbohydrate-based microparticles (see above). A preferred embodiment of the present invention-27 The paper size is applicable to China National Standard (CNS) A4 (2Η) x 297 mm 505518 A7 __B7 _ 5. Description of the invention (25) In the example, the matrix used to repair the bone contains TGF- /? Packed in a delivery system as an osteogenic factor, with a concentration of 100 nanograms per milliliter of the matrix solution. Can be used Lower or higher TGF- concentration. In another embodiment, the parent material for bone repair contains BMp-2, which is packaged in the delivery system as a bone growth factor, and its preferred concentration is 100 · 2000 ng / Milliliter of parent material solution. In another embodiment, the parent material contains a suitable concentration of FGF and is packaged in a delivery system as a bone growth factor. The osteogenic factors used in the bone repair composition of the present invention include flammable hair. Bone repair cells differentiate into bone A cell, such as an osteoblast or osteocyte, produces any peptide, polypeptide, protein, or any other compound or composition of bone tissue. Osteogenic factors used in the present invention include proteins such as TGF- [Sampath, TR Et al., J. Biol. Chem. 265 (22), 13198-13205 (1990)], osteogenin [Luyten, FP et al., L_Biol. Chem .. 264 (15 13377-80 (1989)], Bone Morphogenetic Protein (BMP) [Wang, E. et al., Proc. Natl. Acad. Sci. USA, 82, 2220-24 (1990)], and TGF · /? Combined with epidermal growth factor (EGF). Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling out this page). The differentiation of mesenchymal cells, which is stigmatized by osteogenic factors, includes the formation of intermediate tissues such as fibrous, vitreous, and calcified Cartilage; and endogenous ossification of cartilage, which results in the formation of braided bone tissue that becomes reshaped and transformed into mature plate-shaped bone tissue. In some cases, bone can be generated directly from mesenchymal cells without the presence of intermediate tissue. In the parent material, bone tissue is usually formed 3 to 4 weeks after the blood vessels are formed in response to the angiogenic factors contained in the parent material and the mother material is infiltrated. Although in the absence of added angiogenesis and bone growth factors (at least the parent material is best used for large defects), the bone still grows to bone defects. The use of this factor-28- This paper size applies to China National Standard (CNS) A4 specifications (210X297 mm) One 505518 A7 ___ B7 _ V. Description of the invention (26) Speed up the repair process substantially. The parent material composition for repairing the bone portion of the full-thickness defect in the joint according to the present invention can also be used to treat any defect in the bone tissue if necessary. Such defects include fracture 'joint fragmentation' incomplete and delayed joints, epidermal arthrodesis, pseudoarthrosis, and congenital defects, trauma, tumor infections, degenerative diseases and other causes of loss of bone tissue Caused by bone defects. The bone repair matrix composition can also be used for prosthetic limb implantation and promote the stability of prosthetic limbs, promote the osseointegration of implant materials used in the internal fixation step, the stabilization of dental implant materials, and accelerate the healing effect of eruption of eruption, Spinal or other joint fusion steps. Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) Fibronectin or other compounds, including peptides as small as tetrapeptides, containing amino acid sequence Arg-Gly -Asp, which can be used as a cell adhesion promoter [Ruoslathi et al., Cell 44 · 517-18 (1986)] to promote the deposition of cartilage or bone repair cells and the matrix Sticky. Fibrin and certain collagen matrices already contain this sequence [Ruoslathi et al., 1986, supra]. When using other biodegradable masterbatches, such cell adhesion promoters can be mixed with the parent material before filling or covering defects with the parent material. Arg-Gly-Asp-containing peptides can also be chemically coupled to the parent material (for example, its fibers or screens) or to compounds (such as albumin) added to the parent material. The foregoing composition can be used in a method for slandering cartilage or osteogenesis in a specific defect site of animal cartilage or bone tissue. The method of the present invention can treat cartilage and bone defects in animals, including humans. This method is simple to apply and limited to the affected joint area.全 治 -29- This paper is again applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) 5. Description of the invention (27 The treatment can be performed by close-end endoscopy, open surgery or percutaneous procedures. In the method for treating cartilage or bone defect or damage of the present invention, the defect or damage is identified, and it is prepared and filled with the matrix composition according to the present invention. When binding = cartilage supplement, the matrix composition may contain anti-angiogenesis Agent to prevent blood organ ^ endogenous. Proliferation (filament ash) agent can also be stored in the mother matrix composition at appropriate concentration to stimulate the proliferation of matrix and cartilage repair cells in defects or damage. The same preparation at this concentration It can also be used as a chemotactic agent to attract cartilage repair cells. The limitation is that the factors used must have a combined effect on proliferation and chemotaxis (such as TGF- / ?, 2 to 10 nanograms per milliliter of parent material). Alternatively, the parent material may contain two different preparations, one with specific proliferation and the other with specific chemotactic effect. In another specific example, after supplementing the defective area with% parent material, the proliferation can be directly performed. And if necessary, the chemotactic agent is injected into the defect area filling the mother material. The injection is limited to the mother material and fills the defect area to avoid the exposure of synovial cells to growth factors that will cause cell proliferation and joint exudate. After the parent material composition (and, in the case of fibrin parent material, the parent material has been solidified), an angiogenic agent or a proliferative agent may be injected as needed to fill the defect of the parent material, and the joint capsule and skin incision may be closed and the joint arthroscopy may be terminated. Microscopic examination or open surgery. In the next cartilage repair step, the cartilage repair cells in the mother material are exposed to a transforming agent at an appropriate time, and the concentration of the transforming agent is sufficient to transform the cartilage repairing cells into chondrocytes that can produce stable cartilage tissue. This process can reach -30 by including the appropriate transfer system containing the transformation factor in the above-mentioned parent material composition-this paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm), invention description (28 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs ^ ° or 'π will be re-directed directly at the appropriate time ==: r II delivery. Yu did not make the bones soft In the case of deficiency, the cells should reach the transformation concentration after the initial implantation of the parent material into the defective area], and the factor can be added to the delivery system or directly inserted in order to promote the synthesis of the cartilage parent component at this point in time. In addition, additional anti-angiogenic agents can be included in the delivery system or directly injected. Cartilage or bone defects of animals can be visually confirmed during arthroscopy of joints, or simple inspection of damage # or defects during open surgery. Cartilage or Bone can also be extrapolated using computer-assisted local radiography (CAT scan) X-rays, synovial fluid or serum-labeled magnetic resonance imaging (MRi) analysis, or any other step known in the relevant art. Once confirmed Defects can be repaired surgically to enhance the defect's ability to physically retain the solution and parent material added to the treatment described in the article. It is best to replace it with a flat or shallow double concave shape, and the defect is shaped to have vertical edges or undercuts to retain the solution and parent material added to the treatment method described herein. According to the method of the present invention, the bone defect site of the full-layer defect can be filled with a bone repair matrix composition to the calcified cartilage layer at the interface between bone and cartilage so that a flat surface is formed. Filling a bone defect with a bone repair matrix is particularly useful when the defect is a few centimeters or deeper. The bone repair matrix composition can include an angiogenic factor and a bone generating factor in a convenient delivery system. The remaining cartilage portion of the defect is completely filled with a matrix composition that stimulates cartilage repair. The cartilage repair composition includes a parent material containing an antiangiogenic agent and, if necessary, a proliferative agent and a chemotactic agent. Anti-angiogenic agents for use in the compositions and methods of the invention include any agent capable of inhibiting the biological activity of blood vessels. Please read the back note of this invention

I

I sheet I order-31-This paper is again applicable to Chinese National Standards (CNs) a4 size (210X297 mm) 505518 5. Description of the invention (29 I sheet test anti-angiogenic agent may contain one or more anti-angiogenic agents Molecule. The composition used in this step may also contain a transforming factor packaged in the delivery system and, if appropriate, in a free state. In the best method of cartilage repair of the present invention, the matrix contains an anti-angiogenic factor (in a free state) And packaged in or related to a sustained release transmission system), proliferators, chemotactic agents (which may be the same as the proliferative agent), and transformation factors, which are packaged in or related to a delivery system, which is released over a period of time The transforming factor causes the repaired cells that have clustered to begin to reconstruct the intercellular substance. The preferred composition is as described above. As described in U.S. Patent No. 5,270,300, the full-thickness defect bone-cartilage interface can be physically deserted (most It is good for biodegradable membrane) to be separated, the membrane is impermeable to cells (for example, the pore diameter is less than 5 micrometers), which is better than the cartilage part filled with full-layer defects. The membrane is placed on the bone defect filled with maternal material and must be dense Film Margin to the periphery of the defect area of the cartilage-bone junction to prevent endovascularization to the cartilage defect area. In this method, repair cells in the bone area do not easily cluster cartilage defects and therefore require a proliferative agent and / or in the cartilage repair matrix Chemotactic agents attract and stimulate the repair of synovial membrane repair cells. In a preferred embodiment of the present invention for treating full-layer defects, no membrane is placed at the bone-cartilage interface. The bone portion of the defect may or may not be filled as desired Bone repair composition. Defective cartilage portion is filled with a parent material composition, which contains an antiangiogenic agent and a transforming factor and optionally contains a proliferative agent and / or a chemotactic agent as described above. The angiogenic agent acts as a functional barrier to prevent endogeneity of blood vessels and bone formation in cartilage areas, avoids the necessity of physical membranes and allows repair cells in bone areas to migrate. In another specific embodiment of the present invention for treating full-layer defects , Bone _ Cartilage Boundary -32- This paper ruler Zhongqing Family CNS) Erge (2iqxI97 Public Sauce Factory Central Standards Bureau Employees Consumer Cooperatives printed 5U5M8 A7 ---------- B7 _ V. Invention Ming (30) — --- The surface is treated with a heating device to create a temporary biological membrane at the bone-cartilage interface to separate it. The heating device can be applied to the bloodstream to coagulate the blood and form a layer of precipitated proteins for biological physics The barrier can prevent the formation of bone tissue in blood vessels and defects. The heating device includes, but is not limited to, heated scalpels, scissors or tweezers. The heating temperature is about 20 trc. The heating device should be applied to the full-featured substrate. In another specific embodiment, co2_N2 or neodymium 鈒 laser can be used for heating. The heat created between the bone and cartilage surface is a temporary biofilm that can be used as an antiangiogenic agent in cartilage repair matrix. When using heating In the treatment method, the cartilage repair matrix should include a proliferative agent and / or a chemotactic agent. Chemical measurements can enhance parent material bonding. This measurement includes degrading the superficial layer of cartilage proteoglycan on the defect surface of the exposed collagen microfibers of cartilage, so that they can communicate with the female shell I collagen microfibers (when collagen matrix is used) or the maternal fibrin microfibers (when blood fibers Protein outflow) interaction. Proteoglycans on the surface of cartilage not only prevent fibrin or other biodegradable matrix from adhering to cartilage, but also inhibit local thrombin activity. Advantageously, proteoglycan degradation products also have a chemotactic effect on repairing cells [Moore, A.R. et al., Int. J Jiss. Reac ... pp 301-307 (1989)]. In a specific embodiment, the area is dried by sucking the defect surface with sterilized absorbent tissue, and the defect volume is filled with a sterilizing enzyme solution for 2 to 10 minutes to degrade the proteoglycan on the surface of cartilage and is limited to about 1 to 2 microns of wood within the defect surface. Different enzymes can be used alone or in combination in a sterile buffered aqueous solution to degrade proteoglycans. The pH of the solution can be adjusted to the optimal enzyme activity. The proteoglycan-degrading enzymes in the method of the present invention include chondroitinase ABC, chondroitinase AC, hyaluronidase, pepsin, trypsin, -33 · This paper is suitable for (2H) X297 public director) (Please read the (Please fill in this page for attention)

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J. ^ Clothing ------ (Please read the precautions on the back before filling out this page), 1T ^ .....--II-1 Printed by the Staff Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 505518 A7- ----- M V. Description of the invention (31) =, ln, papain, streptase, and _h = protease. The syngas of a particular enzyme or a synthase varies with the activity of the enzyme solution. In a preferred embodiment of the present invention, the defect is filled with a sterilized solution of chondroitinase AC at a concentration of 1 1 / ml and digested for 4 minutes. The optimal concentration of chondroitinase was determined according to the procedure of the example. Any other concentration used will degrade only about 1 to 2 microns below the shallow proteoglycan in a concentration and time. The amount and time of the enzyme solution should be maintained at least so that the protein sugars in the repair area are preferentially degraded. For chondroitinase 8 6 (: or 80) at a concentration of 11T / ml, a digestion period greater than 10 minutes can lead to unnecessary and proteolytic degradation of proteoglycans outside the defective area. In addition, a digestion time greater than 10 minutes contributes to the entire process. The whole time of the procedure (especially during open osteotomy) should be as low as possible, because cartilage exposure to air can be harmful [Mitehell et al. '(1989), New York and New York based on these reasons, the specificity of the method of the present invention The example includes the step of digesting proteoglycan with an enzyme and digestion time less than 10 minutes is preferred and digestion time is less than 5 minutes is optimal. According to the method of the present invention, the enzyme has degraded the proteoglycan on the defective surface. Afterwards, the enzyme solution should be removed from the defective area. The enzyme solution should be removed with a cotton suction using an aspirator equipped with a suction tip. In addition, it can be removed with a cotton suction alone. Remove the enzyme solution. After removing the enzyme solution, wet the entire defect with sterilized physiological saline (such as 0.15M NaCl), preferably three times. Then the wet defect should be dried. It can be dried with sterile gauze or cotton. The trap. Available fibrin glue (i.e., blood factor XIII or fibrin stabilization factor) to strengthen

The size of this paper is toward the national standard (CNS) A4 (210X297 mm) of the paper. 505518 A7 B7 V. Description of the invention (32) The matrix is bonded to the defect cartilage to promote the chemical bonding of the filaments on the surface of the defect (crosslinking) ) To cartilage collagen fibrils [see Gibble et al., Transtusion, 30 (8), pp. 741-47 (1990)]. Transglutaminase can be used to achieve the same effect [see eg Ichinose et al., J. Biol. Chem., 265 (23), ρ · 13411-14 (1990); " Transglutaminase, "Eds: VA Najjar and L. Lorand, Martinus Nijhoff Publishers (Boston, 1984)] can also use other compounds that can promote the adhesion of extracellular substances. The method and composition of the present invention can also be used to help treat periodontal disease. For example, in the treatment of periodontitis, in which periodontal ligament fades And the exposure of the neck can assist the regeneration of periodontal connective tissue. In particular, periodontal connective tissue (ie, ligaments, spaces) is filled with biodegradable matrix, which contains one or more factors that stimulate periodontal tissue formation such as IGF-1 or FGF, and one or more anti-epithelial factors to inhibit epithelial formation such as epidermal growth factor (EGF), anti-basement membrane antibodies, vitamin A inhibitors, anti-retinol and fibrin-rich matrix. The anti-epithelial factor should be in a free state and packaged in or related to the delivery system to provide continuous release of the matrix-filled area. The preferred matrix material is a cross-linked gelatin or collagen matrix with sufficient ability to adhere to teeth and gums. Economy Central Printed by the Consumer Bureau of the Standards Bureau (please read the precautions on the back before filling this page) The method and composition of the present invention can be used to assist bone regeneration (such as after tooth loss). When the tooth is lost, the bone in which the tooth is fixed Parts often shrink and degenerate. This area (ie, the epicondyle and inferior zygomatic ridge) must be reconstructed and reshaped before the dental implant is positioned. However, bone regeneration usually prevents the rapid growth of connective tissue. Borrowing bone as described above Repair of the parent material for prevention and the parent material includes an effective amount of one or more inhibitors of migration and / or proliferation and / or differentiation of connective tissue. For example, osteogenic factors that contain angiogenic factors and delivery systems, anti-connective groups -35- This paper size is in accordance with Chinese National Standard (CNS) A4 specification (210X297 mm) 505518 5. Invention description (33) The parent material of weaving factors (such as factors that inhibit mesenchymal cell proliferation) can be placed in the bone group and reconstructed Area. In order to promote the reorganization of the connective tissue around the periodontal connective tissue, parent materials containing stimulation factors that stimulate the migration of connective tissue and precursor cells (such as with FGF) can be placed in the teeth. Internal tissue space. The connective tissue matrix also contains anti-epithelial factors. Another aspect of the present invention is to prevent the occurrence of calcification and ossification around the joints in the connective tissue portion of the bone, such as joint replacement (such as total hip replacement). The parent material with anti-bone factors (such as anti-angiogenic agents, antibodies to TGF-々 and BMP chelating substances or a combination thereof) can be used in a free state after being associated with a delivery system that provides continuous release or packaged in Provide a continuous release delivery system in the connective tissue space around the joint garden to prevent calcification and the formation of bone tissue. A further use of the present invention is to prevent the inward growth of bone tissue that will extend to young animals and children's cartilage growth disks. Fracture site, which occurs when the long bone is fractured. To prevent the ossification of the connective tissue part around the bone, the parent material containing anti-osteogenic factors (such as anti-hematopoietic factors, resistance to TGF_a and BMP compounds or a combination thereof) can be used to fill the fracture gap of the cartilage growth disc area "In order to better understand the invention described in the text, the following examples are listed. They should be printed and interpreted by the Central Economic Standards Cooperatives' These examples are for illustrative purposes only, and the invention is not constructed in any way and is limited. Example 1 Protein poly_removal In order to promote and improve the degeneration of maternal material along the defect surface of the articular cartilage group, enzymes can be used to remove protein polyacetic acid molecules in the superficial cartilage matrix to expose the glue -36- Zhang scale applicable to Chinese national standards B7. V7. Description of the invention (34) (Please read the notes on the back before filling this page) Apply fibril mesh to the outside to apply matrix and migrate to repair cells. Different proteases and glucosamine polymerases are suitable for use. For this purpose, but should be controlled to provide the maximum activity of each enzyme. In this example, we tested the activity of chondroitinase Abc (0.5-5U / ml) and trypsin (0.5-4%) for Remove with proteoglycans. Use rabbit knee joints from Butcher Gang j. The mechanically treated shallow cartilage defect was exposed to the enzyme-coated liquid for 4 minutes by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs. The solution was then removed with absorbent tissue and Rinse the imperfections with physiological saline. According to this procedure, the cartilage tissue is quickly fixed in a 2% (v / v) glutar solution (buffered with 0.05m sodium dimethylsulfonate, pH 7 4). 0.7% (w / v) hexaamine ruthenium trichloride (RHT) for tissue examination. After fixation, the matrix includes 1% RHT-rhenium oxide solution (buffered with 01M: sodium mequinate). The tissue was dehydrated and embedded in Ep0I1 812. Cut into thin sections, stained with uranium acetate and lead citrate, and examined with an electron microscope. These sections were 'fixed with RHT (ie, Shendian) proteoglycans as dark stained particles. The enzyme concentration that removes shallow proteoglycans in the thick layer not exceeding 1-2 microns is defined as the best (the deeper the enzyme penetration will affect the underlying chondrocytes). Chondroitin age AB C was found to have a concentration of about 1 U The best activity was found at 1 mL / ml. Trypsin concentration was found The activity is best at 2.5%. The optimal range of activity for other glucosamine polymerases or proteases can be determined in a similar manner. Any buffer can be used in combination with the enzyme but is non-toxic and its maximum buffering capacity occurs near the maximum required enzyme activity P Η 値 Example 2 Adhesion to the parent material of the shallow defect Promote the persistence of the parent material by the controlled enzymatic digestion of shallow cartilage proteoglycans -37- This paper is in accordance with the Chinese National Standard (CNS) A4 specification ( 210X297 mm) 丄 〇Α7

A7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs

5. Description of the invention (37) fluid. In the case of agarose, the defects were filled with agarose solution at 39-42 ° C. Once cooled (3 5 -3 8 ° C), agarose masterbatch is formed at the defect. Thirty rabbits were used in this experiment (two for each matrix and growth factor). In each case, the mother matrix is still stuck to the defect, and becomes completely clustered by the repair cells like fibroblasts. This condition was found to be 8 to 10 days after the earliest operation. After 4 weeks of operation, the structure of the repaired tissue did not change further, but the biodegradable matrix was remodeled by the repaired cells and replaced with a cell matrix other than the loose connective tissue type. 'This tissue did not turn into cartilage. Example 5 The growth factor trapped in the biodegradable matrix was applied to the defect area to provide chemotactic stimulation of the repair cells to the defect area and the proliferation of the repair cells, and then the main release of the transforming factor was used to provide defects in the second stage. The zone is transformed into a transparent maggot. Observe that after the growth factor is applied, the defective matrix is completely filled with repair cells. These cells can reshape the stacked matrix (see Example 4) and promote the introduction of transforming factors (such as TGF- /?) Into the capsule. Investigation of the effects of forms (such as microlipids), in which when mothers completely use the repaired cell clusters that have reshaped the intercellular structure, they can be released due to the very low concentration of TGF-0 (such as 2 -1 0 nanograms). (Ml / ml) fibrinogen solution (1 mg / ml) for the purpose of promoting initial chemotactic and proliferative effects According to the method of Kim et al. (1983), microlipids were also encapsulated in these lipids containing TGF- To the same blood fiber at the appropriate concentration ^ = original solution to provide per litre of blood fiber when microlipids are ruptured and TGF- is released "-40- 505518 Printed by A7, Consumer Cooperative of the Central Standards Bureau, Ministry of Economic Affairs (38) White Plain High Degrees of 100 to 1000 nanograms of TGFj promote repair cells to transform into chondrocytes and to transform into cartilage via matrix filling defects when the repair cells of fibrin matrix clusters begin to reshape intercellular matter (second period). 10 only Rabbit (in which the superficial knee cartilage defect was prepared as described in Example 2) She encapsulated the fibrinogen mixture of TGF_Lu with free tincture and liposomes to the defect. In different experiments of this continuous experiment, free The concentration of the state t〇f_Lu is maintained at 2 to 10 nanograms / ml fibrinogen while the concentration of the encapsulated tgf_々 is different to provide (once aTGF_々 is released from the microlipids) the concentration is between 100 and 1000 nanograms of TGF-Lu / ml fibrinogen at 100 nanograms. In any case, the formation of hyaline cartilage tissue occurred in the treatment of Shao site. Obtaining better reproducible results, the concentration is greater than 200 nanograms of cystic TGF_Lu / Ml fibrinogen falling liquid, preferably more than 500 nanograms of TGF_Lu / ml fibrinogen solution. Example 6 Tissue transformation dark dark evening ice In this experiment, a group of 6 adult rabbits Undergo knee surgery to produce As in the shallow defect of Example 2. The entire process of applying the shallow defect repair is to treat with chondroitin S mother ABC (1 U / 耄, 4 minutes), followed by fibrin masterbatch (1 耄 g / 耄Lipofibrinogen solution, 20 μl 100u / 亳 L thrombin solution / ml fibrinogen solution) containing free form TGF- /? (2 to 10 nanograms / ml) and microlipid pack The capsule TGF-y? (800 ng / ml) filled the defect. Three rabbits were killed after 8, 10, and 12 days of operation, and the remaining were killed at 20, 24, and 28 days. In this animal mode In the middle of j 2 and 20 days, the original, fibroblast-like repair cell tissue was transformed into hyaline cartilage. This knot -41-This paper size is in accordance with Chinese National Standard (CNS) A4 specification (21〇 > < 297mm) ---- (Please read the precautions on the back before filling this page)

Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs, 1T 505518 A7 B7 V. Description of Invention (39) The results are determined by the organization inspection. At 8 to 12 days, the fibrous patchy cells remained (partially or completely remodeled with fibrinogen), however, at 20 and the following days, the defect space was partially or completely covered with hyaline cartilage tissue. filling. Example 7 Administration of the Cartilage Patch in Mini-Pig Mode The above experimental procedure for rabbit mode was applied to the larger animal mode, mini-pig. Use a planing knife to cut 4 adult mini pigs (2_4 years old, 80-110 pounds) into the skeletal sulcus and middle metatarsal soil to produce shallow defects (0.6 cm wide, 0.6 cm deep, and about 10 to 15 cm long). The defect was then treated with chondroitinase ABC (1U / ml 4 points, as described above for rabbits). The enzyme solution was removed, the defects were dried, rinsed with physiological saline, and then dried again. Then the fibrinogen solution was used to fill the defects. The fibrinogen used in this experiment contains 2 to 6 nanograms of free-form TGF · /? / Ml, and 1500 to 2000 nanograms of liposome-encapsulated TGF _ /? / Ml fibrinogen solution. Before filling the defects, thrombin was added to the mother solution as described above in the rabbit experiment. After 6 days of operation, the mini-pigs were killed, and the tissues were inspected for the location of maternal filling defects. Healing is indicated at all locations where hyaline cartilage tissue is formed at the treatment site. Example 8 Resistance to angiogenic agents in articular cartilage defects The full-thickness articular cartilage defects of 1 cm deep and 10 cm wide were produced in the middle condyle and sacrum groove of the knee joint of adult mini pigs. Five lesions were made in each of the two animals using a planing device. In each sacral sulcus, two defects were made in the skull, two defects were made in the tail, and one defect was made in the middle of the femur. Borrowed by blood-load --- (Please read the precautions on the back before filling out this page) —Order ------- J — '· -42- 505518 A7 B7 Printed by the Staff Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs Fifth, the description of the invention (40) Health giant vision controls each lesion to extend vertically to the spinal cord bone (including blood vessels and bone marrow cells) to ensure that the child's joint has made a full-thickness defect. The defect was then treated with chondroitinase ac (1 U / pen liter '4 points). The enzyme solution was removed, the defects were dried, rinsed with physiological saline, and then dried. The defect was then filled with cartilage repair matrix solution. The mother solution used in this experiment included a copolymer of gelatin (Gelfoam, Upjon) (using 100 mg / ml) and fibrinogen (using 20 mg / ml). Add thrombin (using 50 Ι · υ ·) to the top surface of the defect and place the matrix into the defect to allow it to diffuse into the matrix. The cartilage repair matrix contains free-form proliferative agent-like insulin growth factor _ 〖(IGF_ 1) ', the degree of maturity is about 40¾ micrograms per halo, such as transforming factor, and the matrix capacity is encapsulated in microlipids TGF- /? 1, the concentration of 500 nanograms per milliliter, such as the mother substance capacity of the transformation factor. In addition, to the same microlipids containing TGF-1, free lipid suramin at a concentration of i 0 milliram * grain volume and microlipids encapsulated at a concentration of 10 millimolar volume were added. In the control lesions, the lesions were treated in the same manner without the addition of suramin. About 8 weeks after the operation, the animals were killed and the tissues were examined for the location of the maternal filling defect. The defective space portion of the adjacent osteochondral tissue (that is, the area filled with the suramin matrix-containing composition) fills the articular cartilage tissue. The same portion of the defect space in the control lesion was filled with newly formed bone tissue without the suramin treatment. The above experiment was repeated with BMP-2 at a concentration of 1000 ng / ml of parent material volume instead of TGF-5 / 1. Got the same result. Example 9 Repair of full-layer defects in articular cartilage using thermal procedures. Made in the middle cheekbones and sacrum grooves of adult mini-pigs. 1 cm deep and 10 cm. 43- This paper size applies Chinese National Standard (CNS) A4 specifications ( 210X297 mm) JI. 丨, Approval-(Please read the precautions on the back before filling out this page) Order 505518 A7 B7 Printed by the Consumers' Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (41) Layer articular cartilage defect. Five lesions were found in every two mini pig knee joints. Blood flow occurred in each lesion, and the heating device was applied to the defect surface to solidify and form a biological physical barrier. We used a heated scalpel (heated to 22 (rc) to create a temporary tissue barrier. Filling the articular cartilage defects of any joint of a mini pig with cartilage repair matrix, which contains a concentration of about 40 nanograms per milliliter of mother Mass fraction 1 (^ -1 and a concentration of 500 nanograms per milliliter of microlipid capsule cysts (^-/? 3. In another joint defect, suramin is included in Example 8 Maternal matter. The defect of the joint of the second mini-pig is filled with cartilage for repairing the maternal material, which contains a concentration of about 40¾: microgram / ml of the mother substance volume of igf-1 and a concentration of 1000 nanograms / ml of the mother substance volume. Lipids encapsulate BMP-2. In another joint defect of the second minipig, suramin was included in the parent material described in Example 8. As in previous experiments, animals were killed and examined after 8 weeks of manipulation. Any animal There is no bone tissue formation in the defect space of adjacent joint cartilage tissue. However, the defect space is full of articular cartilage tissue. Example 10 The anti-angiogenic agent is used to deepen the articular cartilage of the potential; the full-thickness defect is supplemented to the adult mini pig knee joint Very deep full-thickness articular cartilage defect in the middle sacrum and skeletal groove (Depth to 5 cm). Use a planing device under normal anesthesia to maintain the animal's lesions. The defective bone part can be filled with the bone repair matrix composition described above. The defective bone part should be filled with cartilage into the cartilage. _Bone interface. The articular cartilage defect space can be filled with cartilage repair matrix, which contains the above-mentioned (for example, Example 8_9)-anti-angiogenic factor. J-Ί .----- 装 ------ ^- ----- (Please read the notes on the back before filling this page) 44-

Claims (1)

A8 B8 C8 D8 Patent Application No. 086108885 Chinese Patent Application Amendment (June 91) Patent Application Scope 1. A composition for treating cartilage defects, including: a biodegradable matrix or a matrix-forming material For coating cartilage defects or diseased areas; and an effective amount of anti-angiogenic agent to prevent blood vessels from growing into cartilage. 2. The composition according to item 1 of the scope of the patent application, wherein the matrix or the matrix-forming material further contains an effective amount of a transforming factor to repair the cells into cartilage cells. 3. The composition according to item 2 of the applied patent garden, wherein the matrix or the matrix-forming material further contains an effective amount of a proliferative agent to stimulate the proliferation of the repaired cells. 4. The composition according to item 3 of the scope of patent application, wherein the matrix or the matrix-forming material further contains an effective amount of a chemotactic agent to attract repair cells. 5. The composition according to item 2 of the scope of patent application, wherein the transformation factor is related to the delivery system. 6. The composition according to item 5 of the scope of patent application, wherein the anti-angiogenic agent is in a free form and is also related to the same delivery system. 7. A composition according to item 6 of the application, wherein the transforming factor and the anti-angiogenic agent are related to the same delivery system. 8. The composition according to item 7 of the scope of patent application, wherein the delivery system is used for the transfer of transformation factors and the anti-angiogenic agent is selected from the group consisting of microlipids, biodegradable polymers, collagen fibers, carbohydrate-based microparticles and oil-in-oil Emulsion of water. 9. The composition according to item 2 of the patent application, wherein the matrix is selected from the group consisting of fibrin, gelatin, gelatin, agar gum, and combinations thereof. 10. The composition according to item 6 of the scope of patent application, in which the anti-angiogenic agent The paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X297 public envy) D8 6. The scope of patent application is Suramin. 11. The composition according to item 5 of the scope of patent application, wherein the transforming factor is selected from the group consisting of transforming growth factor cold (TGF-cold) and BMP. 12. The composition according to item 1 of the scope of patent application, wherein the biodegradable matrix or the matrix-forming material is selected from the group consisting of: fibrinogen / thrombin, collagen, fibrin, collagen The combination of glutinous white and fibrin, agar and gelatin; and the anti-angiogenic agent selected from the group consisting of: angiostatin, metalloproteinase inhibitor, anti-angiogenic defamatory antibody , Suramin (Slxramin), fumagillin, fumagillin analogs and AGM-1470. 13. The composition according to item 1 of the scope of patent application, in which the biodegradable matrix or the matrix-forming material is selected from the group consisting of: fibrinogen / thrombin, collagen, fibrin , Collagen and fibrin combination, agar gelatin, and gelatin; and the anti-angiogenic agent is suramin at a concentration of 10 mM; also included in the delivery system as a transformation factor at a concentration of 500-800ng / ml Transforming Growth Factor / 5 (TGF- / 9). 14. A composition for promoting bone regeneration in an animal, comprising: containing an effective amount selected from b-fibroblast growth factor (bFGF), transforming growth factor (TGF-cold), platelet-derived growth factor (PDGF), tumor necrosis Factor a (TNF-a), angiopoietin, and angiotropin group of angiogenic factors, parent material; selected from transforming growth factor cold (TGF-cold), bone morphogenetic protein-2-present Paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) 6. Scope of patent application (BMP), bone morphogenetic protein_2 (BMp_2), osteopoietin, fibroblasts, protoblast growth factor (FGF) and Transforming Growth Factor 々 (TGF · Cold) An osteogenic agent combined with a group of fibroblast growth factor (EGF), combined with liposomes, bioerodible polymers, carbohydrate-based microparticles, water_ A delivery system consisting of an oil emulsion, fibers and osmotic pumps; and an antibody selected from anti-connective tissue antibodies, antibodies against connective tissue-specific growth factors, and antibodies against mesenchymal cell surface proteins And a group of anti-connective tissue factors that inhibit the proliferation of mesenchymal cells to inhibit connective tissue formation. 15 · —a set of methods for treating full-layer defects in animal cartilage, including: Chondral defects or damaged areas are selected from the group consisting of fibrin / thrombin, collagen, fibrin, collagen and fibrin, biodegradable matrix or matrix formation of agar gelatin and gelatin Materials; and an effective amount selected from angiostatic inhibitors, metalloproteinase inhibitors, antibodies against angiogenic defamatory factors, suramin, fumagillin, fumagillin analogs, and AG M-147 A group of anti-angiogenic agents to prevent blood vessels from growing inwardly into the cartilage. 16. The set according to item 15 of the scope of patent application, wherein the parent material additionally contains an effective amount selected from transforming growth factor cold (TGF · Cold ), Tumor necrosis factor Jiang (TNF-α), fibroblast growth factor (fgf) and combinations thereof, and the transformation factor of a group of transforming growth factor 々 (TGF-dots) combined with epidermal growth factor (egf) The cells are repaired to the chondrocytes by the transformation. This paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) 505518 A8 B8 C8 D8 6. Application scope of patent 17. According to the 16th set of application patent scope Group, in which the mother substance additionally contains an effective amount selected from the group consisting of transforming growth factor (TGF- / 5), epidermal growth factor (EGF), insulin-like growth factor (IGFE), platelet-derived growth factor (PDGF), and fibroblasts Growth factor (fgf), transforming growth factor a (TGF-α), human growth hormone and hematopoietic growth factor, a group of multiplication agents, in which the multiplication agent is present in an appropriate concentration to stimulate the proliferation of repaired cells. 18. The kit according to item 17 of the scope of the patent application, wherein the parent material additionally contains an effective amount selected from the group consisting of transforming growth factor cold (TGF-cold), epidermal growth factor (EGF), insulin-like growth factor (iGFE), hGH , Chemotactic agents composed of platelet-derived growth factor (PDGF), tumor necrosis factor a (TNF-a), tumor necrosis factor cold (TNF- / S) and proteoglycan products to attract repair cells. _ 19. The kit according to item 17 or 18 of the scope of the patent application, wherein the transformation factor is combined with a delivery system selected from the group consisting of: liposomes, bio-nameable polymers, chemically linked to sulfuric acid Heparin proteoglycan collagen fibers and carbohydrate-based microparticles release transforming factors at sufficient concentrations to transform cells to chondrocytes. 20. The set according to item 15 of the scope of patent application, comprising: a bone protein for filling defects is selected from the group consisting of fibrinogen / thrombin, collagen, fibrin, collagen and fibrin, agar The second biodegradable matrix or matrix-forming material of the group consisting of gelatin and gelatin; the second biodegradable matrix or matrix-forming material contains an effective amount of selection -4- G House Standard (CNS) A4 Specification (210X297 Public Director) 505518 Composed of b fibroblast growth factor (bFGF), transforming growth factor dots (TGF-Ling), platelet-derived growth factor (PDGF), tumor necrosis factor α (TNF-α) 'angiopoietin and angiopoietin Group of angiogenic factors to stimulate the formation of blood vessels and ingrowth; the second biodegradable matrix or matrix forming material also contains selective transforming growth factor TGF (TGF-N), bone morphogenic protein (BMp ), A group of bone morphogenetic protein-2 (BMP-2), osteopoietin, fibroblast growth factor (FGF) and transforming growth factor 々 (TGF- / S) combined with epidermal growth factor (EGF) An osteogenic agent, combined with a delivery system selected from the group consisting of liposomes, bioerodible polymers, sugar-based microparticles, water-oil emulsions, fibers, and osmotic pumps, is used to deliver bone repair The concentration of osteogenic agent that differentiates cells into bone cells that form bone. 21. The set according to item 15 or 20 of the scope of the patent application, further including a group selected from chondroitinase ABC, chondroitinase AC, hyaluronidase, papain, trypsin, chymotrypsin, streptomycin & amp A group of proteoglycan-degrading agents consisting of SUph V8 protease. 22. The set according to item 21 of the Chinese Patent Application 'additionally includes fibrin glue or transglutamic acid S, wherein the method further comprises the step of filling with fibrin glue or transfiber before filling the defect with a matrix forming material. The glutamase covers the defective surface. 23. A composition for promoting bone regeneration in an animal, comprising a parent material, which contains an effective amount of an angiogenic agent, an osteogenic agent related to a delivery system, and an anti-connective tissue factor that inhibits connective tissue formation. -5-