Scaffold with adhesive for articular cartilage repair

Abstract

To provide a method for treating an injury or defect in articular cartilage.SOLUTION: The present invention provides a method comprising the steps for: a) preparing a matrix implant; b) applying a barrier composition comprising a polymer to the bottom of the cartilage lesion; and c) implanting the implant onto the applied barrier composition.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 683,358, filed June 11, 2018, the disclosure of which is incorporated herein by reference in its entirety.

[0002] FIELD OF THEINVENTION A matrix implant is embedded into the articular cartilage lesion on a barrier composition effective to block the migration of cells, blood, and other materials from the subchondral region into the lesion. [Background technology]

[0003] background Articular cartilage consists of chondrocytes embedded in a large extracellular matrix composed of water molecules, collagen fibers, and proteoglycans. Damage to articular cartilage occurs in active individuals and the elderly as a result of acute or repetitive traumatic injuries or aging. Such damage can result in pain, affect mobility, and cause disability. Many treatment methods are currently in use. Current surgical procedures include microfracture, irrigation, debridement, drilling, and abrasion chondroplasty.

[0004] Irrigation involves irrigation of the joint with a solution of sodium chloride, Ringer's, or Ringer's and lactate. Debridement involves smoothing the rough surfaces of the cartilage and removing loose portions of the meniscus. These techniques provide temporary pain relief but have little or no potential for further healing. The temporary pain relief is believed to be due to removal of degenerated cartilage debris, proteolytic enzymes, and inflammatory mediators.

[0005] Microfracture involves removing damaged articular cartilage followed by physical injury to the underlying subchondral bone to expose bone marrow and bleed. Microfracture is performed by drilling small holes into the subchondral bone to allow migration of bone marrow-derived stem cells into the cartilage defect site. The surgery is performed by arthroscopy after cleaning of the cartilage defect. The surgeon can use an awl to create multiple small fractures in the subchondral bone. Blood and bone marrow containing stem cells ooze out of the fracture, creating a blood clot that releases cartilage-building cells. The body responds to the microfracture as it responds to injury, which results in the formation of new replacement cartilage. The blood clot introduces inflammatory cytokines, growth factors, and mesenchymal stem cells (MSCs) to fill the defect. These factors, especially stem cells, allow the production of new cartilage.

[0006] Communication between repair tissue in cartilage lesions and the subchondral bone plate can promote cartilage repair. Vasara, AI et al., OsteoArthritis and Cartilage, 2006, 14:1066-1074. Microfracture can provide long-term improvement for 7-17 years. Steadman, JR et al., Arthroscopy, 2003, 9(5):477-484. At the same time, microfracture promotes the formation of fibrocartilage rather than hyaline cartilage. Microfracture is also less effective in treating older patients, overweight patients, and patients with cartilage lesions larger than 2.5 cm. These patients may experience recurrence of symptoms as little as 1-2 years after surgery as the fibrocartilage wears away. At that point, such patients may have to re-engage in articular cartilage repair surgery.

[0007] Other options include osteochondral autograft transplantation (OAT) and osteochondral allograft transplantation (OCA). However, OAT is limited by donor site morbidity and the inability to treat large lesions, and OCA carries the risk of disease transmission and subchondral bone collapse. Direct transplantation of cells or tissue into defects and replacement of defects with biological or artificial substitutes currently account for only a small percentage of surgical interventions. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Vasara, AI et al., OsteoArthritis and Cartilage, 2006, 14:1066-1074 [Non-Patent Document 2] Steadman, JR et al., Arthroscopy, 2003, 9(5):477-484 Summary of the Invention

[0009] In one aspect, a method for treating damage or defects in articular cartilage is provided. The method includes the steps of preparing a matrix implant, applying a barrier composition comprising a polymer to the bottom of the cartilage lesion, and embedding the implant over the applied barrier composition. In some embodiments, the barrier composition is applied to the subchondral bone.

[0010] In some embodiments, the barrier composition is effective to block the migration of cells, blood, or other materials from the subchondral region into the cartilage lesion.

[0011] In some embodiments, the matrix implant is an acellular matrix implant. In some embodiments, the acellular matrix implant is selected from the group consisting of collagen type I, collagen type II, collagen type IV, collagen containing proteoglycans, collagen containing glycosaminoglycans, collagen containing glycoproteins, polymers of aromatic organic acids, gelatin, agarose, hyaluronin, fibronectin, laminin, bioactive peptide growth factors, cytokines, elastin, fibrin, polymers made from polylactic acid, polymers made from polyglycolic acid, poly(ε-caprolactone), poly(vinyl alcohol), poly(sebacic acid), poly(α-caprolactone ... Poly(lactic acid-co-glycolic acid), poly(lactic acid-co-ε-caprolactone), poly(lactic acid-co-vinyl alcohol), poly(lactic acid-co-sebacic acid), poly(glycolic acid-co-ε-caprolactone), poly(glycolic acid-co-vinyl alcohol), poly(glycolic acid-co-sebacic acid), poly(ε-caprolactone-co-vinyl alcohol), poly(ε-caprolactone-co-sebacic acid), poly(vinyl alcohol-co-sebacic acid), polyamino acids, hydroxypolyamides, polyamides, and polypeptide gels. Exemplary hydroxypolyamides are described in U.S. Patent Nos. 8,623,943; 9,315,624; and 9,505,882, all of which are incorporated herein by reference.

[0012] In some embodiments, the barrier composition comprises one or more of the following or polymerization products formed from the following: gelatin, type I collagen, periodate oxidized gelatin, photopolymerizable polyethylene glycol-co-poly(α-hydroxy acid) diacrylate macromer, four-arm polyethylene glycol derivatized with N-(acyloxy)succinimide and thiols plus methylated collagen, derivatized polyethylene glycol (PEG) crosslinked with alkylated collagen, tetra-N-hydroxysuccinimidyl or tetrathiol derivatized PEG (e.g., SprayGel Adhesion Barrier System from Covidien, or CoSeal™ from Baxter Healthcare), and crosslinked PEG with methylated collagen.

[0013] In some embodiments, the barrier composition comprises a sealant, hi some embodiments, the sealant forms a hydrogel after the barrier composition is applied to the subchondral bone.

[0014] In some embodiments, the barrier composition or sealant comprises a polymer. In some embodiments, the polymer is selected from the group consisting of gelatin, polyethylene glycol (PEG), derivatized PEG, poly(cyanoacrylate), polyurethane, poly(methylidene malonate), polyvinyl alcohol, polyamide, hydroxypolyamide, derivatized polyvinyl alcohol, acrylic polymer, fibrin, gelatin, polystyrene with catechol side chains, polyester, polypeptides containing dihydroxytyrosine, poly(α-amino carboxylic acid) with catechol side chains, Phragmatopoma californica (Phragmatopoma californica), and the like. californica, copolymers of polyethylene glycol and polylactide, copolymers of polyethylene glycol and polyglycolide, polyethers, polysaccharides, oxidized polysaccharides, polycationic polyamines, polyanions, poly(ester ureas), copolymers of polyethylene glycol and polylactide or polyglycolide, 4-arm pentaerythritol thiol and polyethylene glycol diacrylate, 4-arm tetra-N-hydroxysuccinimidyl ester or tetrathiol derivatized PEG, polymers formed from gelatin and oxidized starch, polymers formed from photopolymerizable polyethylene glycol-co-poly(a-hydroxy acid) diacrylate macromers, periodate oxidized gelatin, bifunctional polyethylene glycol and serum albumin derivatized with maleimidyl, succinimidyl, phthalimidyl and related active groups, and 4-arm polyethylene glycol derivatized with succinimidyl ester and thiol, and methylated collagen. In some embodiments, the polymer is gelatin or fibrin and the barrier composition comprises thrombin or a crosslinking agent.

[0015] In some embodiments, the barrier composition includes a viscosity adjusting component.

[0016] In some embodiments, the barrier composition comprises a stabilizer.

[0017] In some embodiments, the barrier composition comprises an enzyme effective to increase the degradation rate of the barrier composition.

[0018] In some embodiments, the barrier composition further comprises a structural material, hi some embodiments, the structural material comprises one or more of fibers, fibrin, alginate, hyaluronic acid, gelatin, cellulose, or collagen.

[0019] In some embodiments, the method further comprises the step of introducing a top protective biodegradable polymer layer over the matrix implant.

[0020] In some embodiments, the matrix composition includes components that enhance cell attachment and / or proliferation. [The present invention 1001] (a) preparing a matrix implant; (b) applying a barrier composition comprising a polymer to the bottom of the cartilage lesion; and (c) embedding the implant onto the applied barrier composition. 23. A method for treating damage or defects in articular cartilage comprising: [The present invention 1002] The method of claim 1001, wherein the barrier composition is applied to the subchondral bone. [The present invention 1003] The method of claim 1001, wherein the barrier composition is effective to block the migration of cells, blood, or other materials from the subchondral bone into the cartilage lesion. [The present invention 1004] The method of any one of claims 1001 to 1003, wherein the matrix implant is an acellular matrix implant. [The present invention 1005] The method of the present invention 1004, wherein the acellular matrix implant comprises one or more of type I collagen, type II collagen, type IV collagen, collagen containing proteoglycans, collagen containing glycosaminoglycans, collagen containing glycoproteins, polymers of aromatic organic acids, gelatin, agarose, hyaluronan, fibronectin, laminin, bioactive peptide growth factors, cytokines, elastin, fibrin, polymers made from polylactic acid, polymers made from polyglycolic acid, poly(ε-caprolactone), polyamino acids, polypeptide gels, and polymeric thermoreversible gelling hydrogels (TRGH). [The present invention 1006] The method of any of claims 1001-1005, wherein the barrier composition comprises one or more of gelatin, type I collagen, periodate oxidized gelatin, photopolymerizable polyethylene glycol-co-poly(α-hydroxy acid) diacrylate macromer, four-arm polyethylene glycol derivatized with N-(acyloxy)succinimide and thiol plus methylated collagen, derivatized polyethylene glycol (PEG) crosslinked with alkylated collagen, tetra-N-hydroxysuccinimidyl or tetrathiol derivatized PEG, and crosslinked PEG with methylated collagen. [The present invention 1007] The method of any one of claims 1001 to 1006, wherein the barrier composition comprises a sealant. [The present invention 1008] The method of claim 1007, wherein the sealant forms a hydrogel after the barrier composition is applied to the subchondral bone. [The present invention 1009] The method of any one of claims 1001-1008, wherein the barrier composition or sealant comprises a polymer. [The present invention 1010] Polymers include gelatin, polyethylene glycol (PEG), derivatized PEG, cyanoacrylate, polyurethane, poly(methylidene malonate), derivatized polyvinyl alcohol, acrylic polymers, fibrin, gelatin, polystyrene with catechol side chains, polyester, Phragmatopoma californica (Phragmatopoma californica), copolymers of polyethylene glycol and polylactide, copolymers of polyethylene glycol and polyglycolide, polyethers, polysaccharides, oxidized polysaccharides, polycationic polyamines, polyanions, poly(ester ureas), copolymers of polyethylene glycol and polylactide or polyglycolide, 4-arm pentaerythritol thiol and polyethylene glycol diacrylate, 4-arm tetra-N-hydroxysuccinimidyl ester or tetrathiol derivatized PEG, polymers formed from gelatin and oxidized starch, polymers formed from photopolymerizable polyethylene glycol-co-poly(a-hydroxy acid) diacrylate macromers, periodate oxidized gelatin, bifunctional polyethylene glycols and serum albumin derivatized with maleimidyl, succinimidyl, phthalimidyl and related active groups, and 4-arm polyethylene glycols derivatized with succinimidyl esters and thiols, and methylated collagen. [The present invention 1011] The method of claim 10, wherein the polymer is gelatin or fibrin and the barrier composition comprises thrombin or a cross-linking agent. [The present invention 1012] The method of any one of claims 1001 to 1011, wherein the barrier composition comprises a component that adjusts viscosity. [The present invention 1013] The method of any one of claims 1001 to 1012, wherein the barrier composition comprises a stabilizer. [The present invention 1014] The method of any of claims 1001 to 1013, wherein the barrier composition comprises an enzyme effective to increase the degradation rate of the barrier composition. [The present invention 1015] The method of any one of claims 1001 to 1014, wherein the barrier composition comprises a structural material. [The present invention 1016] The method of claim 1015, wherein the structural material comprises one or more of fibers, fibrin, alginate, hyaluronic acid, gelatin, cellulose, or collagen. [The present invention 1017] The method of any one of claims 1001 to 1016, further comprising the step of introducing a protective biodegradable polymer onto the matrix implant. [Brief description of the drawings]

[0021] [Figure 1] 1 shows a histological analysis of the implant on the subchondral bone, with a portion of the image in the top panel being enlarged in the bottom panel. The left panel shows the tissue after the suture cell construct was implanted on the bone, where adhesive was applied underneath the cell construct. The center panel shows the tissue after the cell construct was implanted on the bone, where adhesive was applied underneath the cell construct. The right panel shows the tissue after the suture cell construct was implanted on the bone without adhesive. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Detailed Description Definition: Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0023] The terms "a," "an," and "the" do not denote a limitation of quantity, but rather denote the presence of "at least one" of the referenced element.

[0024] The term "cartilage" as used herein refers to a special type of connective tissue that contains chondrocytes embedded in an extracellular matrix.The biochemical composition of cartilage varies by type, but generally contains collagen, mainly type II cartilage with other minor types (e.g., types IX and XI), proteoglycans, other proteins, and water.Several types of cartilage are recognized in the art, including, for example, hyaline cartilage, articular cartilage, costal cartilage, fibrocartilage, meniscal cartilage, elastic cartilage, auricular cartilage, and yellow cartilage.

[0025] The term "chondrocyte" as used herein refers to a cell capable of producing the components of cartilage tissue.

[0026] The term "support matrix" refers to a biologically acceptable sol-gel or collagenous sponge, scaffold, honeycomb, hydrogel, biologically acceptable material suitable for receiving activated migrating chondrocytes or bone cells that provides structural support for the growth and three-dimensional proliferation of chondrocytes and for the construction of new hyaline cartilage or for the migration of osteochondrocytes into a bone lesion.

[0027] The inventors have found that the formation of healthy hyaline cartilage, but not fibrocartilage, is promoted by placing a biodegradable acellular matrix implant into a cartilage lesion on at least one layer of a barrier composition placed on the subchondral bone. Without wishing to be bound by theory, the barrier composition is effective to block the migration of cells, blood or fluid from the subchondral region into the lesion, any of which may tend to promote fibrocartilage formation within the lesion. The inventors have found that the blocking may allow chondrocytes derived from the implant, surrounding healthy cartilage tissue, and synovial stem cells present in the synovial fluid or synovium to develop cartilage in the implant. Cells migrating from the synovium and other adjacent tissues may produce cartilaginous sulfated glycosaminoglycans. The sealant and adhesive components in the barrier composition may prevent penetration of the subchondral bone, prevent bone edema, and allow the subchondral bone to heal completely independent of the implant. Bone edema is a source of pain and degeneration leading to osteoarthritis. The methods described herein offer advantages over microfracture, including reducing the risk of bone edema and promoting the formation of hyaline cartilage rather than fibrocartilage.

[0028] matrix A matrix implant for use in treating damage or defects in articular cartilage is provided, the matrix implant being configured to be placed on a barrier composition comprising a polymer that is applied to the bottom of a cartilage lesion, for example to the subchondral bone.

[0029] In various embodiments, the matrix is ​​a two- or three-dimensional structured composition or a composition that can be converted into a two- or three-dimensional structure, hi some embodiments, the matrix is ​​a sponge-like structure or a honeycomb-like lattice.

[0030] In some embodiments, the matrix is ​​a support matrix. In some embodiments, the support matrix is ​​selected from the group consisting of collagen type I, collagen type II, collagen type IV, gelatin, agarose, collagen containing proteoglycans, glycosaminoglycans or glycoproteins, polymers of aromatic organic acids, fibronectin, laminin, bioactive peptide growth factors, cytokines, elastin, fibrin, polymers made from polyacids such as polylactic acid, polyglycolic acid or polyamino acids, polycaprolactone, polymers made from polylactic acid, polymers made from polyglycolic acid, poly(ε-caprolactone), poly(vinyl alcohol), poly(sebacic acid), poly(lactic acid-co The gel solution matrix may be prepared from one or more of the following: poly(lactic acid-co-ε-caprolactone), poly(lactic acid-co-vinyl alcohol), poly(lactic acid-co-sebacic acid), poly(glycolic acid-co-ε-caprolactone), poly(glycolic acid-co-vinyl alcohol), poly(glycolic acid-co-sebacic acid), poly(ε-caprolactone-co-vinyl alcohol), poly(ε-caprolactone-co-sebacic acid), polyamino acids, hydroxypolyamides, polyamides, absorbable ε-caprolactone polymers, polypeptide gels, copolymers thereof, and combinations thereof. The gel solution matrix may be a polymeric thermoreversible gelling hydrogel. The support matrix may have one or more of the following properties: biocompatible, biodegradable, hydrophilic, non-reactive, neutral charge, and defined structure.

[0031] In some embodiments, the matrix is ​​prepared by incubating or entangling a polysaccharide with a polyester comprising polylactic acid, polyglycolic acid, or a copolymer comprising polylactic acid, polyglycolic acid, polyethylene glycol, polyvinyl alcohol, and poly(sebacic acid). The polysaccharide may be oxidized.

[0032] In some embodiments, the matrix comprises one or more of collagen, hyaluronan, and chondroitin sulfate.

[0033] In some embodiments, the barrier composition comprises one or more of the following or polymerization products formed from the following: gelatin, type I collagen, periodate oxidized gelatin, photopolymerizable polyethylene glycol-co-poly(α-hydroxy acid) diacrylate macromer, four-arm polyethylene glycol derivatized with N-(acyloxy)succinimide and thiols plus methylated collagen, derivatized polyethylene glycol (PEG) crosslinked with alkylated collagen, tetra-N-hydroxysuccinimidyl or tetrathiol derivatized PEG (e.g., SprayGel Adhesion Barrier System from Covidien, or CoSeal™ from Baxter Healthcare), and crosslinked PEG with methylated collagen.

[0034] In some embodiments, the barrier composition comprises a sealant, hi some embodiments, the sealant forms a hydrogel after the barrier composition is applied to the subchondral bone.

[0035] In some embodiments, the barrier composition or sealant comprises a polymer. In some embodiments, the polymer is selected from the group consisting of gelatin, polyethylene glycol (PEG), derivatized PEG, poly(cyanoacrylate), polyurethane, poly(methylidene malonate), polyvinyl alcohol, polyamide, hydroxypolyamide, derivatized polyvinyl alcohol, acrylic polymer, fibrin, gelatin, polystyrene with catechol side chains, polyester, polypeptides containing dihydroxytyrosine, poly(α-amino carboxylic acid) with catechol side chains, polymers secreted by Phragmatopoma californica, copolymers of polyethylene glycol and polylactide, copolymers of polyethylene glycol and polyglycolide, polyethers, polysaccharides, oxidized polysaccharides, polycations polyamines, polyanions, poly(esters urine). In some embodiments, the barrier composition is a polymer that is a 4-armed poly(ethylene glycol) and polylactide or polyglycolide copolymer, a 4-armed pentaerythritol thiol and polyethylene glycol diacrylate, a 4-armed tetra-N-hydroxysuccinimidyl ester or tetrathiol derivatized PEG, a polymer formed from gelatin and oxidized starch, a polymer formed from a photopolymerizable polyethylene glycol-co-poly(a-hydroxy acid) diacrylate macromer, periodate oxidized gelatin, bifunctional polyethylene glycol and serum albumin derivatized with maleimidyl, succinimidyl, phthalimidyl and related active groups, and a 4-armed polyethylene glycol derivatized with succinimidyl ester and thiol, and a methylated collagen. In some embodiments, the polymer is gelatin or fibrin and the barrier composition comprises thrombin or a crosslinking agent.

[0036] In some embodiments, the matrix composition includes components that enhance cell attachment and / or proliferation.

[0037] In various aspects, the matrix is ​​a three-dimensional cell scaffold comprising a biocompatible polymer formed from a plurality of fibers configured to form a nonwoven three-dimensional open-celled matrix. The open-celled matrix can have a predetermined shape. The open-celled matrix can have a predetermined pore volume fraction. The open-celled matrix can have a predetermined pore shape. For example, the pores in the matrix can form a honeycomb lattice. The open-celled matrix can have a predetermined pore size.

[0038] In various embodiments, the matrix or support matrix has a defined pore size. Different pore sizes allow faster or slower infiltration of chondrocytes into the matrix, faster or slower growth and proliferation of cells, and ultimately higher or lower density of cells in the neo-cartilage construct, as described, for example, in U.S. Patent No. 8,906,686, which is incorporated herein by reference. Matrix pore size can be adjusted, for example, by varying the pH of the gel solution, collagen concentration, and freeze-drying conditions. The pore size of the matrix can be 50-500 μm, 100-300 μm, or 150-250 μm.

[0039] In various embodiments, the matrix may be porous or non-porous and may be applied as a caulk. Such a matrix may include a polymeric thermoreversible gelling hydrogel (TRGH). The caulk may be reconstituted using the patient's own synovial fluid, which may allow the matrix to be seeded with cells. Sponge-like materials may also be soaked with synovial fluid prior to implantation. The matrix may be allowed to harden after application.

[0040] In some embodiments, the matrix includes at least one therapeutic agent, which can be, for example, an anti-infective agent, an analgesic agent, an analgesic, or an anti-inflammatory agent, and an immunosuppressant.

[0041] Barrier Composition Provided is a barrier composition for use in treating damage or defects in articular cartilage. The barrier composition is applied to the base of the cartilage lesion, for example to the subchondral bone. A matrix implant is placed on the barrier composition. In some embodiments, a top protective biodegradable polymer is placed on the matrix implant.

[0042] Placement of a barrier composition onto the subchondral bone as described herein may allow for preservation of the integrity of the lesion following cleaning during surgery and may prevent migration of subchondral cells and cell products into the site of the cartilage defect. Without wishing to be bound by theory, preventing such migration creates an environment for healthy hyaline cartilage to form, while also preventing the formation of fibrocartilage by stem cells migrating from bone marrow into the matrix via the subchondral bone.

[0043] The barrier composition may include a sealant. A sealant is a biologically acceptable, typically fast-gelling formulation with a certain range of adhesive and cohesive properties. A sealant may be a biologically acceptable, rapidly gelling synthetic compound with adhesive and / or cohesive properties. In various embodiments, the sealant is a hydrogel, such as a derivatized polyethylene glycol (PEG), which is preferably crosslinked with a collagen compound, typically an alkylated collagen. A hydrogel may be formed after the barrier composition is applied to the subchondral bone. Examples of sealants include, but are not limited to, tetra-N-hydroxysuccinimidyl or tetrathiol derivatized PEG, or combinations thereof, commercially available from Cohesion Technologies, Palo Alto, Calif. under the trademark CoSeal™ (J. Biomed. Mater. Res Appl. Biomater., 58:545-555 (2001)); two-part polymeric compositions that rapidly form a matrix, where at least one of the compounds is polymeric, e.g., polyamino acids, polysaccharides, polyalkylene oxides or polyethylene glycols, and the two parts are linked by a covalent bond (U.S. Pat. No. 6,312,725, incorporated herein by reference); and cross-linked PEG with methyl collagen, e.g., cross-linked polyethylene glycol hydrogels with methyl-collagen. The sealants may rapidly gel or bond upon contact with tissue, particularly subchondral bone.

[0044] In various embodiments, the barrier composition comprises polymer.The exemplary polymer in the barrier composition includes, but is not limited to, gelatin and oxidized starch, 4-arm pentaerythritol tetrathiol and polyethylene glycol diacrylate, polymer formed from photopolymerizable polyethylene glycol-co-poly(a-hydroxy acid) diacrylate macromer, periodate oxidized gelatin, bifunctional polyethylene glycol and serum albumin derivatized with maleimidyl, succinimidyl, phthalimidyl and related active groups, and 4-arm polyethylene glycol derivatized with succinimidyl ester and thiol, and methylated collagen.

[0045] In some embodiments, the barrier composition comprises polyethylene glycol, a polyethylene glycol-based material, or cross-linked polyethylene glycol. Exemplary polyethylene glycol (PEG)-based materials include, but are not limited to, CT-3, Coseal® (Baxter), Adherus® (Hyperbranch Medical Technology), and Resure® (Ocular Therapeutics). In some embodiments, the barrier composition comprises cross-linked polyethylene glycol and methylated collagen, e.g., CT-3. In some embodiments, the barrier composition is non-toxic to cells.

[0046] In some embodiments, the barrier composition comprises a cyanoacrylate or cyanoacrylate adhesive. Examples of cyanoacrylate adhesives include, but are not limited to, Dermabond® (Ethicon), Integuseal® (Kimberly Clark), Surgiseal® (Adhezion), Histoacryl® (Aesculap), Actabond™ (Bergen), and Indermil® (Covidien). Cyanoacrylates can bond to subchondral bone in the presence of water or moisture. Cyanoacrylates can have various chain lengths, which can affect the degree of bonding and biodegradability. In various embodiments, the barrier composition can be applied quickly. In various embodiments, the cyanoacrylate or cyanoacrylate adhesive allows the barrier composition to resist infection.

[0047] In some embodiments, the barrier composition comprises a polyurethane or polyurethane-based adhesive. Examples of polyurethane-based adhesives include, but are not limited to, TissuGlu® (Cohera). In some embodiments, polyurethane and polyurethane-based adhesives have enhanced biodegradability, for example, by modifying castor oil with isophorone diisocyanate or by reacting polycaprolactone diol and hexamethylene diisocyanate. Polyurethanes can be based on polycaprolactone diol.

[0048] In some embodiments, the barrier composition comprises poly(methylidene malonate) or a poly(methylidene malonate)-based adhesive. An example of a poly(methylidene malonate)-based adhesive includes, but is not limited to, Bondease® (Optmed). The barrier composition may be pasted onto the subchondral bone or applied to the subchondral bone using an applicator. The barrier composition comprising poly(methylidene malonate) or a poly(methylidene malonate)-based adhesive may have a quick drying time after the adhesive is set.

[0049] In some embodiments, the barrier composition comprises a derivatized polyvinyl alcohol or a derivatized polyvinyl alcohol-based material. An example of a derivatized polyvinyl alcohol-based material is Aeriseal® (Pulmonx). The derivatized polyvinyl alcohol can be formulated as a hydrogel, such as by adding water. Such a derivatized polyvinyl alcohol-based hydrogel can have properties similar to hyaline cartilage, such that application of the barrier composition can contribute to reducing pain and improving joint function.

[0050] In some embodiments, the barrier composition comprises an acrylic or acrylic-based material.

[0051] In some embodiments, the barrier composition comprises fibrin or a fibrin-based sealant. Examples of fibrin-based sealants include, but are not limited to, Tisseel® (Baxter) and Evicel® (Ethicon). The barrier composition may be formed of a mixture of two separate compositions, such as a fibrinogen-based composition and a thrombin-based composition, where fibrin forms upon mixing. Mixing and application may be facilitated by the use of an applicator or syringe with two or more chambers. The fibrin-based sealant may have low toxicity compared to other types of sealants. The fibrin-based sealant may be more biodegradable and biocompatible compared to other types of sealants. In various embodiments, the fibrin-based sealant is sterilized to remove viruses and other pathogens. In various embodiments, the barrier composition comprising fibrin or a fibrin-based sealant may be pasted or sprayed onto the exposed subchondral bone.

[0052] In some embodiments, the barrier composition comprises gelatin and thrombin, or a mixture of gelatin and thrombin. The barrier composition may further comprise fibrin. Examples of such barrier compositions include, but are not limited to, Surgiflo® (Ethicon) and Floseal® (Baxter). The barrier composition may be formed of a mixture of two separate compositions, for example, a composition comprising flowable gelatin and fibrinogen and a composition comprising thrombin. Mixing and application may be facilitated by the use of an applicator or syringe with two or more chambers. In some embodiments, 90% of the fibrin-based sealant degrades within 8 weeks. In various embodiments, the fibrin-based sealant is sterilized to remove viruses and other pathogens. In various embodiments, the barrier composition may be pasted or sprayed onto the exposed subchondral bone.

[0053] In some embodiments, the barrier composition comprises albumin together with one or more chemical crosslinkers. The barrier composition may be formed by mixing two separate compositions, for example, a composition comprising albumin and a composition comprising a chemical crosslinker (e.g., glutaraldehyde). Mixing and application may be facilitated by the use of an applicator or syringe with two or more chambers. Examples of such barrier compositions include, but are not limited to, Bioglue® (Cryolife), Progel™ (Neomend), and Preveleak® (Mallinckrodt Pharma).

[0054] In various embodiments, the barrier composition comprises a polymer described in any of U.S. Patent Nos. 6,312,725 and 6,624,245, as well as Wallace, DG, et al., J. Biomed. Mater. Res., 2001, 58:545-555, Hill, A. et al., J. Biomed. Mater. Res., 2001, 58:308-312, and Wise, PE et al., The American Surgeon, 68:553-562 (2002), all of which are incorporated herein by reference.For example, the CT-3 polymer is described in U.S. Patent No. 6,312,725.

[0055] In some embodiments, the barrier composition comprises a polystyrene with catechol side chains, for example, as described in US Patent Publication No. 2009 / 0036611, which is incorporated by reference in its entirety.

[0056] In some embodiments, the barrier composition comprises a polyester-based sealant or polyester.An example of a polyester-based sealant is poly(glycerol sebacate acrylate), as described in Mahdavi et al., Proc. Natl. Acad. Sci. USA, 2008, vol. 105, p. 2307.To enhance adhesion, poly(glycerol-co-sebacate acrylate) can be molded with a pattern based on the adhesive surface found on gecko feet, as described by Mahdavi et al.

[0057] In some embodiments, the barrier composition comprises sandcastle worm glue, for example, as described in U.S. Patent Publication No. 2016 / 0206300. Sandcastle worms (Phragmatopoma californica) can synthesize a polymeric adhesive liquid that hardens to form an adhesive within hours. The sandcastle worm glue can include polyphenolic proteins.

[0058] In some embodiments, the barrier composition comprises the KRYPTONITE™ bone matrix product, as described in US Pat. No. 7,964,207, which is incorporated herein by reference in its entirety.

[0059] In some embodiments, the barrier composition comprises a polymer prepared from a gel comprising gelatin and oxidized starch formed by mixing aqueous solutions of gelatin and oxidized starch. The gel can bond to tissue through the reaction of aldehyde groups on the starch molecules with amino groups on proteins of the tissue. In some embodiments, the adhesive bond strength is about 100 N / m. In some embodiments, the elastic modulus is about 8 x 10 6 Pa. The gelling sealant is degraded by enzymes that cleave the peptide bonds in gelatin and the glycosidic bonds in starch. In some embodiments, 90% of the barrier composition degrades in 14 days.

[0060] In some embodiments, the barrier composition comprises a polymer made from a copolymer of polyethylene glycol and polylactide or polyglycolide, further containing acrylate side chains, and which is photogelled in the presence of certain activating molecules.

[0061] In some embodiments, the barrier composition comprises a polymer comprising a water-soluble polymeric region. Exemplary polymers include polyethers, such as polyalkylene oxides, such as polyethylene glycol ("PEG"), polyethylene oxide ("PEO"), polyethylene oxide-co-polypropylene oxide ("PPO"), co-polyethylene oxide block or random copolymers, and polyvinyl alcohol ("PVA"), poly(vinylpyrrolidinone) ("PVP"), poly(amino acids), poly(saccharides), such as dextran, chitosan, alginate, carboxymethylcellulose, oxidized cellulose, hydroxyethylcellulose and / or hydroxymethylcellulose, hyaluronic acid, and proteins, such as albumin, collagen, casein, and gelatin. The water-soluble region of the macromer (e.g., PEG) can have an average molecular weight of about 3,500 daltons to about 40,000 daltons (e.g., about 3,500 daltons to about 35,000 daltons, or about 3,500 daltons to about 30,000 daltons, or about 3,500 daltons to about 25,000 daltons). In some embodiments, the PEG has an average molecular weight of about 3,500 daltons to about 20,000 daltons (e.g., about 3,500 to about 15,000 daltons, or about 3,500 daltons to about 10,000 daltons, or about 3,500 daltons to about 5,000 daltons). For example, the PEG can have an average molecular weight of about 35,000 daltons or about 25,000 daltons. In some embodiments, the PEG can have an average molecular weight of about 3,500 daltons to about 40,000 daltons. For example, the PEG may have an average molecular weight of about 25,000 Daltons. In other embodiments, the PEG may have an average molecular weight of about 35,000 Daltons.

[0062] In some embodiments, the barrier composition comprises a PEG-based material, such as Duraseal™ (Covidien), Coseal® (Cohesion Technologies), and AdvaSeal™ (Ethicon). Barrier compositions comprising PEG and barrier compositions comprising PEG-based materials can have high adhesive strength, biocompatibility with subchondral bone, and flexibility.

[0063] The barrier composition may comprise a polycationic polyamine and at least one polyanion, wherein the at least one biodegradable polycationic polyamine comprises modified gelatin, as described in U.S. Patent No. 8,283,384, which is incorporated herein by reference in its entirety. In some embodiments, the gelatin is modified with ethylenediamine. In some embodiments, the polyanion is a polyphosphate compound.

[0064] The barrier composition may include a poly(ester urea) (PEU)-based adhesive comprising a PEU polymer backbone having one or more side chains comprising phosphate groups, as described in International Patent Publication No. WO2017 / 189534, which is incorporated herein by reference in its entirety, and a crosslinker comprising a divalent metal salt. In some embodiments, the divalent metal salt comprises a salt of calcium, magnesium, strontium, barium, zinc, or any combination of calcium, magnesium, strontium, barium, and zinc.

[0065] In various embodiments, the barrier composition comprises chondroitin sulfate.Chondroitin sulfate can be modified to include functional groups, such as methacrylate groups and aldehyde groups.Chondroitin sulfate can be crosslinked to form hydrogels, for example, by UV crosslinking with a photoinitiator.

[0066] In various embodiments, the barrier composition comprises multiple different polymers, sealants and / or adhesives.The properties of each polymer, sealant or adhesive present in the barrier composition can compensate for the advantages and disadvantages of other polymers, sealants or adhesives present.For example, the barrier composition can be formulated with two polymers, one of which has a faster degradation and bioresorption rate but a lower adhesive strength when compared with the other polymer.Such a barrier composition can have acceptable degradation, bioresorption and adhesive strength.

[0067] In various embodiments, the barrier composition may be in the form of a hydrogel. The hydrogel may be of sufficient thickness such that the barrier composition effectively blocks the movement of cells, blood, debris, and fluids from the subchondral space. Exemplary hydrogels and their components are described in U.S. Patent No. 7,009,034, which is incorporated herein by reference in its entirety. The hydrogel may be formed by crosslinking PEG with chitosan. Another exemplary hydrogel may be synthesized by forming a thioester bond between a thiol residue of a dendron and a PEG macromer.

[0068] In some embodiments, the barrier composition comprises oxidized polysaccharides, such as dextran and / or chitosan. Dextran is a complex polysaccharide with several branched structures, and unlike chitosan, does not have reactive amino groups. Oxidized dextran reacts with chitosan hydrochloride to form a gel that can adhere to tissue. Oxidized polysaccharides can be crosslinked with or mixed together with various materials. Exemplary oxidized dextran-derived sealants are described in: Balakrishnan, et al., Acta Biomater. 2017, vol 53, p. 343; Lisman, et al., J. Biomater. Appl. 2014, vol. 28, p 1386; Araki, et al., J. Torac. Cardiovasc Surg. 2007, v. 134, p. 1241. Exemplary chitosan-derived sealants are described in Hoque, et al. Mol. Pharm. 2017, vol. 14, p. 1218; Nie, et al. Carbohydr. Polym. 2013, vol. 96, p. 342; Medina, et al., Otolaryngol. Head Neck Surg. 2012, vol. 147, p. 357. Examples of chondroitin sulfate-derived sealants are described in Elisseeff et al., Mil. Med. 2014, vol. 179, p. 686.

[0069] In various embodiments, the barrier composition includes a viscosity adjusting component. Such components may include, for example, glycosaminoglycans (e.g., hyaluronic acid), carboxymethylcellulose (CMC), diethylene glycol dimethyl ether ("diglyme"), dimethylformamide ("DMF"), dimethyl succinate, dimethyl glutarate, dimethyl adipate, dextran, dextran sulfate, polyvinylpyrrolidone (PVP), combinations thereof, and the like. Viscosifiers that may be used to adjust the viscosity of the compositions of the present disclosure include polycyanoacrylates, polylactic acids, polyglycolic acids, lactic acid-glycolic acid copolymers, poly-3-hydroxybutyric acid, polyorthoesters, polyanhydrides, pectins, combinations thereof, and the like.

[0070] In various embodiments, the barrier composition includes a stabilizer. Suitable stabilizers may include those that prevent premature polymerization, such as quinones, hydroquinones, hindered phenols, hydroquinone monomethyl ether, catechol, pyrogallol, benzoquinone, 2-hydroxybenzoquinone, p-methoxyphenol, t-butylcatechol, butylhydroxyanisole, butylhydroxytoluene, t-butylhydroquinone, combinations thereof, and the like. Suitable stabilizers may also include anhydrides, silyl esters, sultones (e.g., α-chloro-α-hydroxy-o-toluenesulfonic acid-γ-sultone), sulfur dioxide, sulfuric acid, sulfonic acid, sulfurous acid, lactones, boron trifluoride, organic acids, alkyl sulfates, alkyl sulfites, 3-sulfolenes, alkyl sulfones, alkyl sulfoxides, mercaptans, alkyl sulfides, combinations thereof, and the like. In some embodiments, anhydrides, such as maleic anhydride, sebacic anhydride, and / or azelaic anhydride, may be used as stabilizers. In other embodiments, antioxidants such as vitamin E, vitamin K1, cinnamic acid, and / or flavanones may be used as stabilizers.

[0071] In various embodiments, the stabilizer is present in an amount of about 0.01 to about 10 weight percent of the barrier composition, hi some embodiments, the stabilizer is present in an amount of about 0.1 to about 2 weight percent of the barrier composition.

[0072] In some embodiments, enzymes may be added to the barrier composition to increase its degradation rate. Suitable enzymes include, for example, peptide hydrolases such as elastase, cathepsin G, cathepsin E, cathepsin B, cathepsin H, cathepsin L, trypsin, pepsin, chymotrypsin, gamma-glutamyltransferase (gamma-GTP), etc.; carbohydrate hydrolases such as phosphorylase, neuraminidase, dextranase, amylase, lysozyme, oligosaccharase, etc.; oligonucleotide hydrolases such as alkaline phosphatase, endoribonuclease, endodeoxyribonuclease, etc. In some embodiments, when an enzyme is added, the enzyme may be included in a liposome or microsphere to control its release rate, thereby controlling the degradation rate of the barrier composition.

[0073] In various embodiments, the barrier composition further comprises collagen type 1. Without wishing to be bound by theory, collagen type 1 may allow cell migration on the surface of the barrier composition and may stimulate clotting of blood from the subchondral bone.

[0074] In various embodiments, the barrier composition is in a hydrated form.

[0075] In some embodiments, the barrier composition can enable the matrix implant to remain securely in the collagen lesion or defect after implantation. There may be no need to suture the matrix implant.

[0076] The matrix system can be an acellular matrix. The acellular matrix can be a tissue that has been decellularized such that nuclear and cellular components are removed from the structural extracellular matrix. The acellular matrix can be prepared from tissue, including organs or isolated parts of organs. Exemplary tissues include heart valves, small intestinal submucosa, dermis, amniotic membrane, urinary bladder, omentum, pericardium, ligaments, blood vessels, and the like. In one embodiment, the tissue includes, but is not limited to, omentum and dermis. In another embodiment, the tissue is dermis. The tissue may be obtained from a variety of mammalian sources, including, but not limited to, human, goat, pig, cow, sheep, horse, and the like. The tissue can be decellularized by conventional techniques, including steps such as tissue preservation, decellularization, cleaning, decontamination, and storage.

[0077] The acellular matrix layer can be obtained by dividing the acellular matrix into thin sheets, typically having a thickness of about 50 microns to about 200 microns.

[0078] The matrix may further comprise at least one growth factor, which may be epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-β), bone morphogenetic protein (BMP), growth differentiation factor, anti-dorsal morphogenetic protein 1 (ADMP-1), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF) hedgehog protein, insulin-like growth factor, platelet-derived growth factor (PDGF), interleukin (IL), colony-stimulating factor (CSF), and / or activin. In addition, the matrix of these embodiments may further comprise collagen.

[0079] In some embodiments, the matrix can be fixed to the subchondral bone. Examples of fasteners include, but are not limited to, staples, darts, pins, screws, sutures, glue, or tacks. In other aspects, the prosthesis can be a prosthetic plate.

[0080] In some embodiments, the matrix further comprises at least one therapeutic agent. In various embodiments, the therapeutic agent can be, but is not limited to, an anti-infective agent, an analgesic agent, an analgesic, or an anti-inflammatory agent, and an immunosuppressant.

[0081] In some embodiments, the anti-infective agent is an antibiotic, such as gentamicin, dibekacin, kanendomycin, lividomycin, tobramycin, amikacin, fradiomycin, sisomicin, tetracycline, hydrochloride, oxytetracycline, hydrochloride, rolitetracycline, doxycycline hydrochloride, ampicillin, piperacillin, ticarcillin, cephalothin, cephaloridine, cefotiam, cefsulodin, cefinenoxime, cefinetazole, cefazolin, cefotaxime, cefoperazone, ceftizoxime, moxolactam, latamoxef, thienamycin, sulfazecin, aztreonam or a combination thereof.

[0082] In some embodiments, the analgesic or analgesic agent is morphine, a nonsteroidal anti-inflammatory (NSAID) drug, oxycodone, morphine, fentanyl, hydrocodone, naproxyphene, codeine, acetaminophen, benzocaine, lidocaine, procaine, bupivacaine, ropivacaine, mepivacaine, chloroprocaine, tetracaine, cocaine, etidocaine, prilocaine, procaine, clonidine, xylazine, medetomidine, dexmedetomidine, or a VR1 antagonist.

[0083] In some embodiments, the barrier composition has a surface area of ​​20 to 400 gf / cm 2 In some embodiments, the barrier composition has an adhesive strength in the range of 20 to 100 gf / cm 2 , 40~120 gf / cm 2 , 60~150 gf / cm 2 , 80~200 gf / cm 2 , 100~300 gf / cm 2, 200~400 gf / cm 2 , 20~40 gf / cm 2 , 30~50 gf / cm 2 , 40~60 gf / cm 2 , 50~70 gf / cm 2 , 60~80 gf / cm 2 , 70~90 gf / cm 2 , 80~100 gf / cm 2 , 90~110 gf / cm 2 , 100~120 gf / cm 2 , 110~130 gf / cm 2 , 120~150 gf / cm 2 , 140~170 gf / cm 2 , 160~200 gf / cm 2 , 180~220 gf / cm 2 , 200~240 gf / cm 2 , 220~260 gf / cm 2 , 240~280 gf / cm 2 , 260~300 gf / cm 2 , 280~320 gf / cm 2 , 300~350 gf / cm 2 , 320~370 gf / cm 2 , or 350-400 gf / cm 2 The adhesive strength is in the range of

[0084] In various embodiments, one or both of the barrier composition and matrix can be injected or implanted into the site of the cartilage defect. In various configurations, the site in need of tissue growth can be, but is not limited to, the dermis, rotator cuff tendons, Achilles tendons, ligaments such as the anterior cruciate ligament (ACL), posterior cruciate ligament (PCL), medial collateral ligament, lateral collateral ligament or periodontal ligament, sphincters such as the anal sphincter, urethral sphincter, esophageal sphincter or sinus sphincter, prolapsed tissue such as abdominal hernia, Cooper's hernia, diaphragmatic hernia, epigastric hernia, femoral hernia, incisional hernia, inguinal hernia, intervertebral disc hernia, Littlé hernia, obturator hernia, pantaloon hernia, perineal hernia, preperitoneal hernia, Richter's hernia, ischial hernia, sliding hernia, Spigelian hernia, etc. or umbilical hernia, intervertebral disc nucleus, intervertebral disc annulus, periosteal tissue, nervous tissue, such as central nervous system tissue (including spinal cord tissue) and demyelinated nervous tissue, nerve tunnels, such as nerve tunnels traversing bone tissue, mitral valve, tricuspid valve, aortic valve, pulmonary valve, vascular tissue including stents, stenosed cardiovascular tissue, costal cartilage, meniscal cartilage, epiglottic cartilage, laryngeal cartilage, such as arytenoid cartilage, cricoid cartilage, cuneiform cartilage and cornicle cartilage, outer ear cartilage, or Eustachian tube cartilage. EXAMPLES

[0085] The present invention is further described and demonstrated by the following examples. However, the use of these and other examples anywhere in this specification is merely illustrative and in no way limits the scope and meaning of the present invention or any exemplified term. Similarly, the present invention is not limited to any particular preferred embodiment described herein. Indeed, many modifications and variations of the present invention may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the invention in spirit or scope. The present invention is therefore to be limited only by the appended claims, along with the full scope of equivalents to which such claims are entitled.

[0086] Example 1 The pigs are divided into at least two groups with at least one control group. Each test group receives a barrier composition and the control group does not receive a barrier composition. In all groups, cartilage defects are created in the weight-bearing region of the medial femoral condyle of the knee joint.

[0087] In each test group, a barrier composition is applied onto the subchondral bone. Multiple test groups can be created to test different barrier compositions. In all groups, the same matrix is ​​applied after application of any barrier composition. The conditions for application of the matrix and any top polymer barrier on the matrix should be the same between all test and control groups.

[0088] Tests are performed at one month, and for additional periods, to determine whether the barrier composition prevents migration of subchondral components, such as cells and fluids, into the cartilage lesion. Such tests may include histological analysis and assays of the extent of fibrocartilage formation.

[0089] Additional tests may be performed, such as evaluation of inflammation, histological grading, and measurement of the rate and extent of improvement in the animals' mobility following surgery.

[0090] Those barrier compositions that provide optimal prevention of migration of subchondral components, minimal inflammation, and highest relative formation of hyaline cartilage versus fibrocartilage are then further tested for clinical application in human and animal patients.

[0091] Example 2 Scaffolds were prepared for porcine testing as follows: A honeycomb-type porous collagen sponge (5 mm diameter and 1 mm thickness, Koken, Tokyo, Japan) was immersed in 25 μl of cold 0.3% neutralized collagen solution (Vitrogen, Cohesion Tech, Palo Alto, CA) and then incubated at 37° C. for 1 h. The neutralized collagen solution solidified to form an acellular scaffold composed of collagen gel within the sponge.

[0092] The engineered cell constructs to be embedded with adhesive and sutures were prepared by taking a biopsy from porcine articular cartilage, mincing the biopsy, and then digesting it in 1.5 mg / ml collagenase (CLS 1, Worthington, Freehold, NJ) dissolved in Ham's F-12 (F-12, Invitrogen) with 100 μg / ml penicillin and 100 units / ml streptomycin (P / S, Invitrogen) on a rotator at 37° C. for 18 hours. Undigested tissue was removed using a cell strainer (70 μm mesh, BD Biosciences, Franklin Lakes, NJ). The isolated porcine articular chondrocytes (pAC) were rinsed twice with PBS by centrifugation at 1000 rpm for 10 minutes. Viable and dead cells were counted using a hemocytometer and trypan blue dye exclusion method. Cell viability in each biopsy was greater than 95%.

[0093] pACs were seeded into monolayer culture dishes (100 mm diameter) and incubated in DMEM / F-12 supplemented with 10% fetal bovine serum (FBS, Invitrogen) and P / S for 5 days at 37°C and 5% CO2 in air. Prior to seeding pACs into collagen gel / sponge scaffolds, pACs were harvested from the culture dishes with 0.05% trypsin-EDTA (Invitrogen). A solution of 0.3% pepsin-digested acid-soluble collagen from bovine skin (Cohesion, Palo Alto, CA) was neutralized with 1 / 10 volumes of 10x PBS and 0.1 N NaOH. 300,000 pACs suspended in 25 μl of this neutralized collagen solution were placed on a Teflon dish (Saint-Gobain Performance Plastics, Courbevoie, France), and the high fluid surface tension maintained the cell suspension within the desired area. A round collagen sponge (5 mm in diameter and 1.5 mm in thickness, Koken, Tokyo, Japan) composed of honeycomb-shaped pores was placed on the cell suspension and allowed to absorb the solution. These cell constructs were incubated at 37°C for 1 hour to allow the collagen solution to solidify into a gel. Culture medium was then added to the dish.

[0094] After 12 h incubation in medium, the cell constructs were transferred to a pressure-resistant culture chamber (TEP-1, PURPOSE, Shizuoka, Japan) connected to a bioreactor and incubated for 7 days at 37°C, 5% CO2 in air, with cyclic hydrostatic pressure (HP) of 0-0.5 MPa, 0.5 Hz, and medium replenishment at 0.05 ml / min. The cell constructs were then transferred to a conventional 12-well culture plate (each well containing one cell construct in 2 ml of medium) and incubated for an additional 14 days at atmospheric pressure, 37°C, and 5% CO2 in air. The culture medium was changed twice weekly. The cell constructs containing surrogates were harvested on day 21 for implantation. The cell viability and cell type of the surrogate constructs were evaluated histologically.

[0095] Example 3 Five different engineered cell constructs were implanted into pigs and their properties compared: a) an empty defect control ("Empty"), b) an acellular scaffold control ("Scaffold") implanted with adhesive and sutures, c) an engineered cell construct implanted with adhesive and sutures ("Cell-Construct"), d) an engineered cell construct implanted with adhesive alone ("Adhesive-Cell-Construct"), and e) an engineered cell construct implanted with sutures alone onto the subchondral bone ("Suture-Cell-Construct").

[0096] Two surgeries were performed in pigs. The protocol for animal studies was approved by the Animal Care and Use Committee at Charles River Laboratories (Worcester, MA). Sixteen castrated male pigs (Micro-Yucatan, Charles River Laboratories), aged 12-15 months and weighing 30-45 kg, were acclimated for >1 week before the first surgery. Animal anesthesia was induced with an intramuscular injection of 0.04 mg / kg atropine sulfate (Patterson, Devens, MA), 0.55 mg / kg butorphanol tartrate (Patterson), 1.5 mg / kg xylazine (VEDCO, St. Joseph, MO), and 20 mg / kg ketamine hydrochloride (VEDCO) and maintained with inhalation anesthesia using isoflurane (Patterson). The right knee joint was opened anterolaterally and the patella was dislocated medially to expose the trochlea and medial condyle.

[0097] During the first surgery, cartilage fragments from a non-weight-bearing site of the right knee were harvested to generate the cell construct, and two full-thickness cartilage defects were created in the weight-bearing site. One part of the defect was left empty (to be used as an empty) and the other part was embedded with an acellular scaffold (to be used as a scaffold). Four weeks after harvest of cartilage from the same animal, during a second surgery, the engineered cell construct was embedded into a surgically created defect in a weight-bearing site of the left knee.

[0098] In pigs, eight knees had two empties created in each, eight knees had two scaffolds implanted in each, eight knees had two cell-constructs implanted in each, four knees had two glue-cell-constructs implanted in each, and four knees had two suture-cell-constructs implanted in each. Two weeks after each surgery for empties, scaffolds, and cell-construct implantation, arthroscopy was performed to evaluate the joint cavity, defect, and implant. Six months after implantation of engineered cell constructs (or seven months after biopsy for empty defect creation and acellular scaffold implantation), animals were euthanized for histological evaluation of the treatment site.

[0099] During the surgical procedure, a small piece of cartilage tissue (biopsy) was taken from the trochlear head. Approximately 40 mg of biopsy was obtained, containing P / S and Ca. 2+ and Mg 2+ The cells were maintained in sodium-free Dulbecco's phosphate-buffered saline (DPBS; Invitrogen, Carlsbad, Calif.).

[0100] Two full-thickness defects that were 5 mm in diameter were created at the weight-bearing sites of the medial and distal femoral condyles using a skin punch (5 mm), a beaver blade, and a curette, taking care to avoid damage to the subchondral bone. Four animals were designated as empty controls and four animals were designated as the acellular scaffold control group.

[0101] To implant the cell constructs, the left knee joint was opened anterolaterally and the patella was dislocated medially to expose the medial femoral condyle. Applying the same method used for the empty defect and the acellular scaffold control, two cartilage defects (diameter 5 mm) were made on the condyle. The cell constructs were placed in the defects, each construct was sutured with four absorbable and two non-absorbable colored sutures, and the constructs were covered with adhesive (CT-3). The patella was reduced and the wound was closed in layers with absorbable sutures (0 PDS-II). The animals were then allowed free cage activity.

[0102] For glue-cell-construct implantation, the cell-construct was placed into the glue-coated defect and the cell-construct was covered with glue alone. Each construct was sutured with two non-absorbable pigmented sutures for arthroscopic confirmation. The suture-cell-construct was placed into the defect without glue and each construct was sutured with four absorbable and two non-absorbable pigmented sutures.

[0103] Two acellular scaffolds were implanted in the right side of the knee of animals in the acellular scaffold control group. Briefly, the acellular scaffolds were implanted into the defect with polyethylene glycol (PEG) / collagen-based tissue adhesive (CT-3, Angiotech, Vancouver, Canada) and each scaffold was sutured with six stitches using four absorbable (8-0 Vicryl, Ethicon, Somerville, NY) and two non-absorbable blue sutures (8-0 Proline, Ethicon) for use as markers during arthroscopic evaluation. After suturing, the surface of the implant was covered with CT-3 adhesive. After reduction of the patella, the wound was closed in layers with absorbable sutures (0 PDS-II, polydioxanone, Ethicon).

[0104] The animals were individually housed in cages and allowed free cage activity. The floor in each cage was covered with a thick rubber sheet to prevent slipping and further trauma to the knee joint after surgery.

[0105] Two weeks after each open knee surgery involving the creation of an empty defect control, the implantation of a scaffold, the implantation of a cell-construct, the implantation of an adhesive-cell-construct, and the implantation of a suture-cell-construct, the cartilage surface was evaluated arthroscopically to ensure that the constructs remained within the defect. If no implants remained in the site, histology samples were excluded from the evaluation. Also, the defects observed were milky white in color and identical in appearance to the surface of the adjacent cartilage. In fact, no engineered constructs or implanted acellular constructs were found without color-labeled sutures, suggesting that the adhesives and constructs did not prevent cell migration from the adjacent tissue.

[0106] The efficacy of the surgical adhesive was analyzed by performing macroscopic and histological evaluations on the cell-construct, adhesive-cell-construct, and suture-cell-construct groups 6 months after implantation of the engineered cell construct (7 months after biopsy and acellular scaffold implantation). The animals used were euthanized prior to necropsy.

[0107] The articular surfaces were evaluated, where the location of the empty defect control, the implanted acellular scaffold, and the implanted engineered cell constructs were identified based on gross anatomical findings: visual characteristics of the filling tissue in the defect compared to the surrounding host cartilage, filling rate, color, and surface integration with the host tissue. Macroscopic images were recorded with a digital camera (Coolpix E-995, Nikon USA, NY). The repaired cartilage was then harvested together with the subchondral bone and adjacent cartilage, fixed in 4% paraformaldehyde (JT Baker, Phillipsburg, NJ), and dissolved in PBS (pH 7.4) for 7 days on a gentle rotator at 4°C. The fixed tissues were then decalcified in 5% formic acid and sodium citrate solution (Sigma-Aldrich) for 1-2 weeks and embedded in paraffin. Serial longitudinal sections 4 μm thick were cut and then stained with either hematoxylin and eosin (H&E) or Safranin O fast green.

[0108] In addition, immunostaining using type II collagen antibody was performed. For immunohistochemical analysis, sections were deparaffinized in xylene and rehydrated with graded ethanol and PBS. To efficiently expose epitopes, sections were incubated in 700 U / ml bovine testicular hyaluronidase (Sigma) and 2 U / ml pronase XIV (Sigma) for 1 hour at 37°C. Sections were then incubated in polyclonal antibody against type II collagen (Southern Biotech, Birmingham, AL).

[0109] Histology data is shown in Figure 1. The "cell construct adhesive + suture" and "cell construct adhesive alone" groups had an adhesive barrier (CT-3 sealant) during surgery, while the "cell construct suture alone" group had no sealant applied during surgery. In the "cell construct adhesive + suture" and "cell construct adhesive alone" groups, there was clear delineation of the subchondral bone from the implant, and healthy bone tissue appeared underneath the implant. In the "cell construct suture alone" group, there was significantly greater penetration of the suture cell implant into the bone when compared to the other groups.

[0110] Histologic findings were then scored using a modified version of the histologic grading scale developed by Sellers et al., J. Bone Joint Surg. Am., 1997, 79(10):1452-63. Three investigators blindly assessed the longitudinal sections using the following criteria: 1) filling of the defect, 2) integration with the host adjacent cartilage, 3) matrix staining with Safranin O fast green (metachromatic), 4) chondrocyte morphology, 5) structure within the entire defect, 6) surface structure, and 7) penetration. The cell-construct group had adhesive and sutures, the suture-cell-construct group had sutures only, and the adhesive-cell-construct group had adhesive only. Scores are shown in Table 1 below.

[0111] [Table 1] Data are presented as mean ± SD. a, p < 0.05 between the indicated groups; b, p < 0.05 between the indicated groups.

[0112] The number of gaps or lack of continuity between the regenerated tissue and the adjacent cartilage was counted and classified with respect to integration with the adjacent cartilage in Table 1. The regenerated tissue was integrated with the cell-construct with a significantly lower number of gaps compared to the gaps in the empty (P<0.05). The integration of the adhesive-construct and suture-construct was similar to the cell-construct in the number of gaps (Table 1).

[0113] Safranin O fast green staining indicated the quality of the sulfated cartilage matrix, the main component of articular cartilage. The regenerated tissue within the scaffold and cell-construct was slightly reduced in amount compared to the adjacent cartilage. Matrix staining of the adhesive-cell-construct and suture-cell-construct revealed that the quality of their sulfated cartilage matrix was similar to that of the cell-construct (Table 1).

[0114] Chondrocyte morphology within the regenerated tissue was analyzed as it exhibits healthy non-pyknotic nuclei, chondrocyte shape and extracellular matrix quality. Chondrocytes in the adhesive-cell-construct and suture-cell-construct were similar in morphology to the cell-construct (Table 1).

[0115] The "structure within the entire defect" classification in Table 1 is an assessment of the density of regenerated tissue, sometimes with a loose texture that appears as gaps or crevices. The adhesive-cell-construct and suture-cell-construct had similar density of regenerated tissue to the cell-construct.

[0116] In Table 1, the "surface structure at defect" classification represents the ability of the regenerated tissue surface to withstand weight-bearing and joint-loading stresses. The majority of the regenerated tissue surface in the cell construct was consistently covered with multilayered tissue, extending to the surface transition zone of the adjacent cartilage. The surface structures of the adhesive-cell-construct and suture-cell-construct were similar in grade to the cell-construct (Table 1).

[0117] The "penetration" classification in Table 1 represents edema formation in the subchondral bone and is important for determining complete healing of the defect. Penetration of the suture-cell-construct into the subchondral bone was significantly greater than that of the cell-construct and adhesive-cell-construct (P < 0.05, Table 1).

[0118] Type II collagen in the adhesive-cell-constructs and suture-cell-constructs was similar in strength to the cell-constructs.

[0119] Example 4 A human patient having a cartilage defect, damage to cartilage, or cartilage lesion in the knee undergoes surgery. If a cartilage lesion does not already exist, such a lesion can be created by removing cartilage from the site of the cartilage defect or damage to the cartilage.

[0120] A first layer of a barrier composition comprising polyethylene glycol is introduced into the lesion and placed at the base of the lesion, such as on the subchondral bone. The barrier composition is formulated so that it rapidly gels from a flowable liquid or paste to a load-bearing gel within 3-15 minutes. The barrier composition is allowed to harden or solidify so that it is effective to prevent subchondral cell entry and block migration of foreign components, such as blood-borne agents, cells and cellular debris, into the cavity.

[0121] The support matrix is ​​cut to fit the dimensions of the cartilage lesion. The support matrix is ​​then embedded into the cartilage lesion. No sutures are used. At least one layer of sealant is added onto the embedded support matrix. The wound is then sutured.

[0122] Patients are initially examined every two weeks to assess improvement in pain and mobility. * * *

[0123] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the present invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. It is further understood that all values ​​are approximate and are provided for illustrative purposes.

[0124] Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

Claims

1. 1. A pharmaceutical comprising a kit for use in the treatment of damage or defects in articular cartilage, the kit comprising: a) an acellular matrix implant, and b) a barrier composition comprising a gel and an enzyme, the enzyme being an enzyme that degrades about 90% of the barrier composition within 14 days after application; Including, the barrier composition is applied to the bottom of the articular cartilage; The acellular matrix implant is embedded onto the applied barrier composition. Medicine.

2. The pharmaceutical according to claim 1 , wherein the enzyme is capable of degrading a peptide bond, a glycosidic bond, or a combination thereof.

3. 3. The method of claim 1, wherein the barrier composition comprises a polymer formed from a copolymer of polyethylene glycol and polylactide or polyglycolide.

4. The method of claim 3 , wherein the polymer further comprises an acrylate side chain.

5. The pharmaceutical composition of any one of claims 1 to 4, wherein the barrier composition is gelled by light in the presence of an activating molecule before the acellular matrix implant is embedded onto the barrier composition.

6. The pharmaceutical according to any one of claims 1 to 4, wherein the enzyme comprises a peptide hydrolase, a sugar chain hydrolase, an oligonucleotide hydrolase, or a combination thereof.

7. The pharmaceutical of claim 6, wherein the peptide hydrolase comprises elastase, cathepsin G, cathepsin E, cathepsin B, cathepsin H, cathepsin L, trypsin, pepsin, chymotrypsin, gamma-glutamyltransferase (gamma-GTP), or a combination thereof.

8. The pharmaceutical composition according to claim 6 , wherein the glycan hydrolase comprises phosphorylase, neuraminidase, dextranase, amylase, lysozyme, oligosaccharase, or a combination thereof.

9. The pharmaceutical of claim 6 , wherein the oligonucleotide hydrolase comprises an alkaline phosphatase, an endoribonuclease, an endodeoxyribonuclease, or a combination thereof.

10. The pharmaceutical according to any one of claims 1 to 9, wherein the enzyme is present in a liposome, a microsphere, or a combination thereof.

11. The method of claim 10, wherein the liposomes, microspheres, or a combination thereof are used to regulate the release rate of an enzyme, thereby controlling the degradation rate of the barrier composition.

12. The method of any one of claims 1 to 11, wherein the matrix further comprises at least one growth factor.

13. 13. The pharmaceutical composition of claim 12, wherein the growth factor comprises epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-β), bone morphogenetic protein (BMP), growth differentiation factor, anti-dorsal morphogenetic protein 1 (ADMP-1), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), hedgehog protein, insulin-like growth factor, platelet-derived growth factor (PDGF), interleukin (IL), colony-stimulating factor (CSF), or activin, or a combination thereof.

14. The pharmaceutical composition of any one of claims 1 to 13, wherein the matrix further comprises at least one therapeutic agent.

15. The method of claim 14 , wherein the therapeutic agent comprises an anti-infective agent, an analgesic, an analgesic, or an anti-inflammatory agent, an immunosuppressant, or a combination thereof.

Images