Classifications

• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
  • C09D11/00—Inks
  • C09D11/30—Inkjet printing inks
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/14—Macromolecular materials
  • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
  • A61L27/225—Fibrin; Fibrinogen
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J3/00—Processes of treating or compounding macromolecular substances
  • C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J5/00—Manufacture of articles or shaped materials containing macromolecular substances
  • C08J5/18—Manufacture of films or sheets
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
  • C08J9/0066—Use of inorganic compounding ingredients
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
  • C08J9/26—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof by elimination of a solid phase from a macromolecular composition or article, e.g. leaching out
• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
  • C09D11/00—Inks
  • C09D11/02—Printing inks
  • C09D11/08—Printing inks based on natural resins
• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
  • C09D7/00—Features of coating compositions, not provided for in group C09D5/00; Processes for incorporating ingredients in coating compositions
  • C09D7/40—Additives
  • C09D7/65—Additives macromolecular
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J2201/00—Foams characterised by the foaming process
  • C08J2201/02—Foams characterised by the foaming process characterised by mechanical pre- or post-treatments
  • C08J2201/024—Preparation or use of a blowing agent concentrate, i.e. masterbatch in a foamable composition
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J2201/00—Foams characterised by the foaming process
  • C08J2201/04—Foams characterised by the foaming process characterised by the elimination of a liquid or solid component, e.g. precipitation, leaching out, evaporation
  • C08J2201/044—Elimination of an inorganic solid phase
  • C08J2201/0444—Salts
  • C08J2201/0446—Elimination of NaCl only
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J2201/00—Foams characterised by the foaming process
  • C08J2201/04—Foams characterised by the foaming process characterised by the elimination of a liquid or solid component, e.g. precipitation, leaching out, evaporation
  • C08J2201/046—Elimination of a polymeric phase
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J2207/00—Foams characterised by their intended use
  • C08J2207/10—Medical applications, e.g. biocompatible scaffolds
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J2300/00—Characterised by the use of unspecified polymers
  • C08J2300/16—Biodegradable polymers
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J2389/00—Characterised by the use of proteins; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
  • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
  • C08L89/00—Compositions of proteins; Compositions of derivatives thereof

Definitions

• Fibrin elastomers were invented in the 1940's as part of a U.S. defense sponsored research program to develop medical strategies for wounded military personnel. Fibrin elastomers developed out of the human plasma program led by Edwin Cohn at Harvard University. John Ferry, then at Woods Hole, led the group that was involved in developing fibrin elastomers. As a result of this work, elastomeric sheet forms of fibrin were developed and used successfully in neurosurgical applications, burn treatments, and peripheral nerve regeneration. See, for example, Ferry, J.D. etal, CUn. Invest. 23:566-572 (1944); Bailey, O.T. et al, J. Clin. Invest.
• thermoplastic materials such as biological response modifiers and drugs
• compositions that have the ability to respond to the local cellular milieu are needed.
• Methods to create spatial patterns of such molecules in materials are also needed.
• fabrication methods are needed that can be used to control properties of manufactured articles including for example the density, porosity, and mechanical properties, especially with regard to biopolymers.
• methods of manufacturing biocompatible materials with anisotropic properties are needed, especially with regard to extrusion or directed strain and/or printing technologies to impart such anisotropic properties.
• the constructs disclosed herein constitute biocompatible materials which can be degraded in response to the host tissues' proteolytic processes. Processing of native extracellular matrix (ECM) molecules, such as fibrinogen, into biopolymers, such as structural elastomeric and/or pliant films, grafts, and scaffolds for tissue regeneration applications, are readily applicable to orthopedics, neurosurgery, and maxillofacial surgery, ⁇ prosthetic tissue interface, as well as other clinical disciplines. Disclosed herein are methods of manufacture; including novel compositions comprising biopolymers.
• ECM extracellular matrix
• incorporation of biological response modifiers, antigens, drugs, hormones, tracers, labeled compounds, particulates (e.g., calcium phosphate, and bioglass) and other clinically relevant materials into the materials disclosed herein may be performed.
• spatial patterns such of growth factors, hormones, and other constituents may be used to alter biomechanical properties and bioresorption rates.
• compositions and methods comprising processing of polymeric materials, as well as applications and use of such materials in biological systems, for example.
• Polymeric materials include those that are biocompatible, including, for example, polymeric sugars, such as polysaccharides (e.g., chitosan) and glycosaminoglycans, (e.g., hyaluronan, chondroitin sulphate, dermatan sulphate, keratan sulphate, heparan sulphate, and heparin) and polymeric proteins, such as fibrin, collagen, fibronectin, larninin, and gelatin.
• the polymers may further comprise additional molecules of biological relevance that are placed on or in a polymeric matrix.
• Molecules of interest include, but are not limited to, biological response modifiers, antigens, drugs, hormones, tracers, labeled compounds, and others.
• the polymeric materials are plastic and, in certain embodiments, capable of deformation. Such plastics may be hard or soft plastic, depending on intended use. These polymers may be shaped, machined, formed, molded, extruded, etc., into desirable shapes depending on the intended uses. The polymers may be used to form matrices for bio-compatible scaffolds capable of being implanted and resorbed. In certain embodiments the surfaces of a structure may be further processed in any manner including milling, maching roughening, porating, etc. to promote attachements and migration of cells for example.
• cells may be seeded onto or into a scaffold, for example.
• porosity of such materials may be modified by any number of methods including introduction of a porogen which may be intercalated into the polymer matrix until removed by such means as solvation and sublimation, for example.
• the hydration of polymers including, for example, hydro gels, may be adjusted in any manner, including, but not limited to, removal of water by evaporation, osmosis, or any other method. Such procedures may be performed for a time, temperature, and/or pressure suitable for the intended application. Thus, in some embodiments, low temperature manufacturing processes are presented.
• compositions and methods are provided relating to protein-based biopolymers and plastics.
• an article of manufacture comprising a biopolymer is provided where the article is dehydrated.
• an article of manufacture comprising dehydrated biopolymers such as a protein, a fibrin, a fibrinogen, a collagen, a gelatin, an elastin, an extracellular matrix constituent, a polysaccharide, a hylauronic acid, and combinations thereof.
• an article of manufacture comprising a biopolymer where the article is dehydrated by means of a vacuum; the article forms a film; the article is elastic; the article is pliant; the article comprises disulfide bonds; the article comprises isopeptidic bonds; the article comprises monoaldehyde or polyaldehyde cross- linked amines; the article comprises pyran cross-linked amines; or the article is cross-linked with a cross-linking agent such as an iridoid derivative, genipin, a diimidate, a dione, a carbodiimide, an acrylamide, N,N'methylenebisacrylamide, a sugar, ribose, Factor XIII, fructose, l-ethyl-[3- (dimethylaminopropyl)] carbodiimide, 2,5-hexanedione, dimethylsuberimite, an aldehyde, ghitaraldehyde,
• an article of manufacture comprising a dehydrated biopolymer where the article comprises a compound such as a biological response modifier, an antigen, a drug, a hormone, a tracer, RNA, DNA, and a labeled compound, where the tracer is a quantum dot and the biological response modifier is a bone morphogenic protein.
• a compound such as a biological response modifier, an antigen, a drug, a hormone, a tracer, RNA, DNA, and a labeled compound, where the tracer is a quantum dot and the biological response modifier is a bone morphogenic protein.
• an article of manufacture comprising a biopolymer film where a biological response modifier, an antigen, a drug, a hormone, a tracer, RNA, DNA, or a labeled compound is deposited on a surface of the film or a biological response modifier, an antigen, a drug, a hormone, a tracer, RNA, DNA, or a labeled compound is incorporated into the polymer matrix of the film.
• an article of manufacture is provided comprising a biopolymer film further comprising a particulate such as hydroxyapatite, tricalcium phosphate, calcium phosphate, and calcium sulfate.
• an article of manufacture comprising a biopolymer film, where the film forms a laminated structure, and where the laminated structure is formed from a stack of sheets, a tubular roll, or combination thereof.
• structures disclosed herein are seeded with cells, such as stem cells.
• an article of manufacture comprising a biopolymer, where the article is compressed.
• an article of manufacture comprising a compressed biopolymer, where the biopolymer is, for example, a protein, a fibrin, a fibrinogen, a collagen, a gelatin, an elastin, an extracellular matrix constituent, a polysaccharide, a hylauronic acid, and combinations thereof.
• the biopolymer is, for example, a protein, a fibrin, a fibrinogen, a collagen, a gelatin, an elastin, an extracellular matrix constituent, a polysaccharide, a hylauronic acid, and combinations thereof.
• an article of manufacture comprising a compressed biopolymer, where the article is compressed at a pressure and a temperature and for a time sufficient to form a polymer matrix, and the article is formed in an extrusion die, a compression mold, or an injection mold.
• an article of manufacture comprising a compressed biopolymer, where the article comprises a filler or a plasticizer, where the plasticizer is, for example, a phthalate plasticizer, an adipate plasticizer, a trimellitate plasticizer, a maleate plasticizer, a sebacate plasticizer, a benzoate plasticizer, an epoxidized vegetable oil, a sulfonamide plasticizer, a phosphate plasticizer, water, a polyalcohol, a glycol, a glycerin, a glycerol, a polyether, an acetylated monoglyceride, an alkyl citrate, a polymeric plasticizer, and combinations thereof
• the plasticizer is, for example, a phthalate plasticizer, an adipate plasticizer, a trimellitate plasticizer, a maleate plasticizer, a sebacate plasticizer, a benzoate plasticizer, an epoxidized vegetable oil, a sulfonamide plasticizer,
• an article of manufacture comprising a compressed biopolymer, where the article is cross-linked with a cross-linking agent such as an iridoid derivative, genipin, a diimidate, a dione, a carbodiimide, an acrylamide, N,N'methylenebisacrylamide, a sugar, ribose, Factor XIII, fructose, l-ethyl-[3- (dimethylaminopropyl)] carbodiimide, 2,5-hexanedione, dimethylsuberimite, an aldehyde, glutaraldehyde, formaldehyde, NHS carboxylic acid ester, and combinations thereof.
• a cross-linking agent such as an iridoid derivative, genipin, a diimidate, a dione, a carbodiimide, an acrylamide, N,N'methylenebisacrylamide, a sugar, ribose, Factor X
• an article of manufacture comprising a compressed biopolymer, where the article comprises pores.
• an article of manufacture comprising a compressed biopolymer, where the article comprises a compound such as a biological response modifier, an antigen, a drug, a hormone, a tracer, and a labeled compound.
• a method for manufacturing polymer films comprising providing a hydrogel; and vacuum drying the hydrogel at a temperature and a pressure, and for a time, to form a dehydrated film, where the temperature is less than SO 0 C, the pressure is less than 20 millibars, and the hydrogel is formed from a polymer such as a protein, a fibrin, a fibrinogen, a collagen, a gelatin, an elastin, an extracellular matrix, constituent, a polysaccharide, a hylauronic acid, and combinations thereof.
• a polymer such as a protein, a fibrin, a fibrinogen, a collagen, a gelatin, an elastin, an extracellular matrix, constituent, a polysaccharide, a hylauronic acid, and combinations thereof.
• a method for manufacturing polymer films comprising vacuum drying a hydrogel at a temperature and a pressure, and for a time, to form a dehydrated film
• the hydrogel comprises a plasticizer such as a phthalate plasticizer, an adipate plasticizer, a trimellitate plasticizer, a maleate plasticizer, a sebacate plasticizer, a benzoate plasticizer, an epoxidized vegetable oil, a sulfonamide plasticizer, a phosphate plasticizer, water, a polyalcohol, a glycol, a glycerin, a glycerol, a polyether, an acetylated monoglyceride, an alkyl citrate, a polymeric plasticizer, and combinations thereof.
• a plasticizer such as a phthalate plasticizer, an adipate plasticizer, a trimellitate plasticizer, a maleate plasticizer, a sebacate plasticizer, a benzoate plasticizer, an epoxidized vegetable oil
• a method for manufacturing polymer films comprising vacuum drying a hydrogel at a temperature and a pressure, and for a time, to form a dehydrated film, where the hydrogel is cross-linked with a cross-linking agent such as an iridoid derivative, genipin, a diimidate, a dione, a carbodiimide, an acrylamide, NjN'methylenebisacrylamide, a sugar, ribose, Factor XIII, fructose, l-ethyl-3- (dimethylaminopropyl) carbodiimide, 2,5-hexanedione, dimethylsuberimiate, an aldehyde, glutaraldehyde, formaldehyde, and combinations thereof; the hydrogel comprises a compound such as biological response modifiers, an antigen, a drug, a hormone, a tracer, RNA, DNA, a labeled compound, and combinations thereof; and/or the hydrogel
• a method for manufacturing a plastic comprising admixing a biopolymer with a compound to create an admixture; and compressing the admixture at a pressure and a temperature to form a biopolymer matrix.
• a method for manufacturing a plastic comprising admixing a biopolymer with a compound to create an admixture; and compressing the 5 admixture at a pressure and a temperature to form a biopolymer matrix, where the temperature is less than 8O 0 C, the pressure is less than 6000 pounds, and the admixture is formed in an extrusion die, a compression mold, or an injection mold.
• a method for manufacturing a plastic comprising admixing a biopolymer with a compound and compressing the admixture at a pressure and a
• biopolymer matrix is formed from polymers such as a protein, a polysaccharide, a fibrin, a fibrinogen, a gelatin, a hylauronic acid, a collagen, an extracellular matrix constituent, an elastin, and combinations thereof; and where the biopolymer matrix comprises a plasticizer or a filler or combinations thereof, where the plasticizer is, for example, a phthalate plasticizer, an adipate plasticizer, a
• L 5 trimellitate plasticizer a maleate plasticizer, a sebacate plasticizer, a benzoate plasticizer, an epoxidized vegetable oil, a sulfonamide plasticizer, a phosphate plasticizer, water, a polyalcohol, a glycol, a glycerin, a glycerol, a polyether, an acetyl ated monoglyceride, an alkyl citrate, a polymeric plasticizer, and combinations thereof.
• a method for manufacturing a plastic comprising -0 admixing a biopolymer with a compound to create an admixture; and compressing the admixture at a pressure and a temperature to form a biopolymer matrix, where the biopolymer matrix is cross-linked with a cross-linking agent such as an iridoid derivative, genipin, a diimidate, a dione, a carbodiimide, an acrylamide, N,N'methylenebisacrylamide, a sugar, ribose, Factor XIII, fructose, l-ethyl-3- (dimethylaminopropyl) carbodiimide, 2,5- 25 hexanedione, dimethylsuberimiate, an aldehyde, glutaraldehyde, formaldehyde, and combinations thereof; and where the compound is, for example, a biological response modifier, an antigen, a drug, a hormone
• a method for manufacturing a porous plastic comprising admixing a biopolymer with a porogen; forming a biopolymer matrix comprising the porogen; and removing the porogen from the biopolymer matrix, where the biopolymer matrix is formed from polymers such as a protein, a polysaccharide, a fibrin, a fibrinogen, a gelatin, a hylauronic acid, a collagen, an extracellular matrix constituent, an elastin, and combinations thereof; the biopolymer matrix comprises a filler; or the biopolymer matrix comprises a plasticizer, where the plasticizer is, for example, a phthalate plasticizer, an adipate plasticizer, a trimellitate plasticizer, a maleate plasticizer, a sebacate plasticizer, a benzoate plasticizer, an epoxidized vegetable oil, a sulfonamide plasticizer, a phosphate plasticizer, water, a polyalcohol,
• a method for manufacturing a porous plastic comprising admixing a biopolymer with a porogen; forming a biopolymer matrix comprising the porogen; and removing the porogen from the biopolymer matrix, where the biopolymer matrix is cross-linked with a cross-linking agent such as an iridoid derivative, genipin, a diimidate, a dione, a carbodiimide, an acrylamide, N,N'methylenebisacrylamide, a sugar, ribose, Factor XIII, fructose, l-ethyl-3- (dimethylaminopropyl) carbodiimide, 2,5- hexanedione, dimethylsuberimiate, an aldehyde, glutaraldehyde, formaldehyde, and combinations thereof; and the biopolymer matrix comprises a compound such as a biological response modifier, an antigen, a drug, a hormone,
• a method for manufacturing a porous plastic comprising admixing a biopolymer with a porogen; forming a biopolymer matrix comprising the porogen; and removing the porogen from the biopolymer matrix, where the porogen is a solvation porogen; the porogen is soluble in an organic phase; the porogen is, for example, polyurethane, polylactic acid, polyglycolic acid, polylactic-co-glycolic acid, and polycaprolactone; the porogen is soluble in an aqueous phase; the porogen is sodium
• the porogen is a sublimation porogen; or the porogen is, for example, ammonium acetate, ammonium chloride, ammonium sulfate, ammonium bicarbonate, ammonium carbonate, and pyridmium trifluoroacetate.
• a method for cross-linking a polymer comprising providing a solid polymer powder admixed with a solid cross-linking agent capable of being activated by a solvent to form an admixture; forming a polymer matrix from the admixture comprising the solid cross-linking agent; and contacting the structure with a solvent which activates the cross-linking agent, where the cross-linking agent comprises a pyran moiety, the cross-linking agent is an iridoid derivative, or the cross-linking agent is genipin.
• a method for cross-linking a polymer comprising providing a solid polymer powder admixed with a solid cross-linking agent capable of being activated by a solvent to form an admixture; and forming a polymer matrix from the admixture comprising the solid cross-linking agent, where the polymer is a biopolymer such as a protein, a fibrin, a fibrinogen, a collagen, a gelatin, an elastin, an extracellular matrix constituent, a polysaccharide, a hylauronic acid, and combinations thereof; and where the admixture comprises a plasticizer such as a phthalate plasticizer, an adipate plasticizer, a trimellitate plasticizer, a maleate plasticizer, a sebacate plasticizer, a benzoate plasticizer, an epoxidized vegetable oil, a sulfonamide plasticizer, a phosphate plasticizer, polyalcohol, glycol, glycer
• a method for cross-linking a polymer comprising providing a solid polymer powder admixed with a solid cross-linking agent capable of being activated by a solvent to form an admixture; and forming a polymer matrix from the admixture comprising the solid cross-linking agent, where the admixture comprises a porogen, where the porogen is a solvation porogen or a sublimation porogen
• a method for cross-linking a polymer comprising providing a solid polymer powder admixed with a solid cross-linking agent capable of being activated by a solvent to form an admixture; and forming a polymer matrix from the admixture comprising the solid cross-linking agent, where the admixture further comprises a compound such as a biological response modifier, an antigen, a drug, a hormone, a tracer, RNA, DNA, a labeled compound, and combinations thereof.
• a compound such as a biological response modifier, an antigen, a drug, a hormone, a tracer, RNA, DNA, a labeled compound, and combinations thereof.
• a method for cross-linking a polymer comprising providing a solid polymer powder admixed with a solid cross-linking agent capable of being activated by water, where the solid polymer contains free hydroxyl groups; and incubating the admixture for a sufficient time for cross-linking to occur.
• Figure IA shows a thin planar structure that has been patterned with bioactive materials, in this case growth factors.
• Figure IB shows the same planar structure after it has been rolled into a rod configuration with a gradient of growth factors that increases radially as one approaches the axis of the cylinder.
• Figure 2 illustrates a tubular mold that can be used to cast tubular structures of materials made in accordance with the present disclosure.
• Figure 3 shows a hydrogel that has been inserted into or formed within an extruder and is subsequently extruded by forcing the piston downward so that the hydrogel exits the extruder through the die at the bottom.
• Figure 4 shows a fibrin elastomer on which cy3-labeled bone morphogenetic protein-2 has been imprinted. Four concentrations are shown after seven days in cell culture conditions.
• Figure 5 shows six SEM images, three of fibrin film surfaces (FIG. 5 A-C) and three of fibrin film cross-sections (FIG. 5D-F).
• the film surfaces show the presence of fibrin fibrils (FIG. 5 A-C), which are confirmed in the cross-section images (FIG. 5D).
• Cross- sections also show the high density of the fibrin (FIG. 5E-F).
• Figure 6 shows a TEM image of a fibrin film. The lack of distinct patterns in the image demonstrates the random packing of fibrin fibrils within the firm.
• Figure 7 shows a time-pressure and a time-temperature profile for a 20 minute operation cycle for gelatin plastic pellet formation in a compaction press.
• the illustrated pressure and temperature data over time is representative of a 20 minute pellet compaction system.
• Figure 8 shows a time-pressure and a time-temperature profile for a 60 minute operation cycle for gelatin plastic pellet formation in a compaction press.
• the illustrated pressure and temperature data over time is representative of a 60 minute pellet compaction system.
• Figure 9 shows an SEM image of the pores formed in the matrix of a gelatin plastic by chloroform leaching of polylactic acid porogen from the gelatin matrix.
• Figure 10 shows SEM images of the machined surfaces of post- compressed gelatin plastics with glycerin plasticizer at 3OX magnification (FIG. 10A) and 6OX magnification (FIG. 10B).
• Figure 11 shows SEM images of the machined surfaces of post- compressed gelatin plastics with no glycerin plasticizer at 33X magnification (FTG. 1 IA), 50X magnification (FIG. HB) 3 and 10OX magnification (FIG. HC).
• Figure 12 shows plastics with incorporated quantum dots and bone morphogenetic protein (BMP-2) viewed under fluorescence.
• Samples labeled A are control groups containing no quantum dots.
• Samples labeled B and C contain quantum dots and BMP-2.
• Samples labeled D are cross-sectional sheets cut from the plastics labeled C.
• the samples labeled E are the same gelatin samples presented in D, only visualized without fluorescence.
• Figure 13 shows representative fluorescence images of the vascularization on each of the three types of protein-based plastics with incorporated quantum dots and BMP-2 in a chick chorioallantoic membrane (CAM) assay.
• CAM chick chorioallantoic membrane
• Figure 14 is a graph presenting the time-dependent percentage mass remaining during vacuum exposure of gelatin plastics with ammonium salt porogens incorporated into the gelatin matrix and subject to sublimation under vacuum conditions.
• Figure 15 is an SEM image of the porous matrix formed in a gelatin plastic that had ammonium acetate incorporated into the matrix and was removed by sublimation under vacuum. The resulting interconnected and extensive microporosity of the gelatin plastic post-sublimation is readily identifiable.
• Figure 16 is a set of graphs presenting the results from in vitro degradation experiments on gelatin plastic samples in serum-containing media at 37°C. The degradation was quantified as mean percent area (FIG. 16A) and mean percent mass (FIG. 16B) remaining. Samples were either not cross-linked or cross-linked with 0.6 % gluteraldehyde (GA) or 0.6 % genipin (GP). Symbols represent the mean + SD for triplicate determinations.
• Figure 17 is a graph presenting the results from in vitro degradation experiments on fibrin and urinary bladder ECM (UBECM) plastic samples in serum containing media at 37°C shown as mean percent area of sample remaining. Samples were either not cross-linked or cross-linked with 0.6 % gluteraldehyde (GA) or 0.6 % genipin (GP). Symbols represent the mean +.SD of triplicate determinations.
• Figure 18 is a graph presenting the results from in vitro degradation experiments on fibrin and urinary bladder ECM (UBECM) plastic samples in serum containing media at 37 0 C shown as mean percent mass of sample remaining. Samples were either not cross-linked or cross-linked with 0.6 % gluteraldehyde (GA) or 0.6 % genipin (GP). Symbols represent the mean +.SD of triplicate determinations.
• Figure 19 is a set of graphs presenting the results from in vitro degradation experiments on gelatin plastic samples in serum containing media at 37 0 C, where the gelatin plastic was cross-linked with genipin (2% w/w) during plastic formation.
• the graph in FIG. 19A shows mean percent area of sample remaining and the graph in FIG. 19B shows mean percent mass of sample remaining. Symbols represent the mean +_SD of triplicate determinations.
• Figure 20 is a set of graphs presenting the results from in vitro degradation experiments on fibrin plastic samples in serum containing media at 37 0 C, where the fibrin plastic was cross-linked with genipin (2% w/w) during plastic formation.
• the graph in FIG. 2OA shows mean percent area of sample remaining and the graph in FIG. 2OB shows mean percent mass of sample remaining. Symbols represent the mean +_SD of triplicate determinations. DETAILED DESCRIPTION
• each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains error necessarily resulting from the deviation found in its underlying respective testing measurements. Furthermore, when numerical ranges are set forth herein, these ranges are inclusive of the recited range end points (i.e., end points may be used). Also, it should be understood that any numerical range recited herein is intended to include all sub-ranges subsumed therein.
• a range of "1 to 10" is intended to include all sub-ranges between (and including) the recited minimum value of 1 and the recited maximum value of 10, that is, having a minimum value equal to or greater than 1 and a maximum value of equal to or less than 10.
• compositions within the present invention are generally described in the form of biopolymers for use in medical and biological systems. It will be understood, however, that the present invention may be embodied in forms and applied to end uses that are not specifically and expressly described herein. For example, one skilled in the art will appreciate that compositions and methods comprising plastics have application in many industries, as well as the medical arts.
• a component means one or more components, and thus, possibly, more than one component is contemplated and may be employed or used.
• compositions and methods of the invention include, but are not limited to, biocompatibility of the materials with the host; the ability of the materials to degrade in register to tissue regeneration; the binding of growth factors to the materials disclosed herein, which thereby helps minimize the dosages needed to produce therapeutic results; the ability to easily engineer the mechanical properties (e.g., ranging from elastic to rubbery to hard) of the materials; the ability to easily store the materials for off-the-shelf usage; the ability to easily shape the materials at a time and a place that where the materials will be used (e.g., the operating room, the battlefield) etc.; the ability of the structure to resisting tissue prolapse at the implantation site; and, the ability to modulate the physiological response to the implanted materials by incorporating other materials into the base material.
• biocompatibility of the materials with the host the ability of the materials to degrade in register to tissue regeneration; the binding of growth factors to the materials disclosed herein, which thereby helps minimize the dosages needed to produce therapeutic results
• compositions and methods of the invention include, but are not limited to, the use of proteins to create biopolymers.
• other naturally occurring materials such as the polysaccharides, such as chitosan or glycosaminoglycans, such as hyaluronic acids, as well as extracellular matrix constituents, such as fibrous proteins obtained by processes such as those disclosed in U.S.
• polymer refers to natural and synthetic molecules with repeating structural units including, but not limited to, molecules comprising gels and plastics.
• matrix refers to a network of linked subunits.
• polymer matrix refers to a network of linked polymer subunits and, thus, comprises the interior space as opposed to the surface of a polymer, or structure formed therefrom.
• biocompatible refers to the absence of stimulation of a severe, long-lived or escalating biological response to an implant or coating, and is distinguished from a mild, transient inflammation which typically accompanies surgery or implantation of foreign objects into a living organism.
• biocompatible, non-biodegradable polymers include, but are not limited to, polyethylenes, polyvinyl chlorides, polyamides, such as nylons, polyesters, rayons, polypropylenes, polyacrylonitriles, acrylics, polyisoprenes, polybutadienes and polybutadiene-polyisoprene copolymers, neoprenes and nitrile rubbers, polyisobutylenes, olefinic rubbers, such as ethylene-propylene rubbers, ethylene-propylene-diene monomer rubbers, and polyurethane elastomers, silicone rubbers, fluoroelastomers and fluorosilicone rubbers, homopolymers and copolymers of vinyl acetates, such as ethylene vinyl acetate copolymer, homopolymers and copolymers of acrylates, such as polymethylmethacrylate, polyethylmethacrylate, polymethacrylate, ethylene glycol dimeth
• biocompatible non-degradable polymers that are useful in accordance with the present disclosure include polymers comprising biocompatible metal ions or ionic coatings which can interact with DNA.
• gold and silver ions may be used, for example, for inhibiting inflammation, binding DNA, and inhibiting infection and thrombosis.
• biocompatible, biodegradable polymers include, but are not limited to, polylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-glycolytic acid (PLGA), polycaprolactone, and copolymers thereof, polyesters, such as polyglycolides, polyanhydrides, polyacrylates, polyalkyl cyanoacrylates, such as n-butyl cyanoacrylate and isopropyl cyanoacrylate, polyacrylamides, polyorthoesters, polyphosphazenes, polypeptides, polyurethanes, polystyrenes, polystyrene sulfonic acid, polystyrene carboxylic acid, polyalkylene oxides, alginates, agaroses, dextrins, dextrans, and polyanhydrides.
• PLA polylactic acid
• PGA polyglycolic acid
• PLGA polylactic-co-glycolytic acid
• polycaprolactone and copolymers thereof
• biopolymer refers to a biocompatible polymers comprising polymers that can be found naturally in organisms, as well as chemical and physical modifications of such polymers, and include, but are not limited to, proteins, fibrins, fibrinogen, collagens, gelatins, elastins, lamnin, fibronectin, extracellular matrix constituents, glycosaminoglycans, hylauronic acid, albumin, alginates, chitosans, cellulose, thrombin, heparin, polysaccharides, synthetic polyamino acids, prolamines, combinations thereof, and other such molecules.
• Biocompatible polymers include, but not limited to, biopolymers that can be, but are not necessarily, biodegradable.
• the term "native" when describing a substance refers to a purified form of the substance which is chemically identical to the substance as found in nature, such as, for example, fibrin, elastin, etc.
• the term "admixture” refers to a mixture of a base material and one or more additional materials.
• the base material of the admixture is a polymer, such as, for example, a biopolymer
• the additional material is a filler, a particulate, a porogen, a biological response modifier, an antigen, a drug, a hormone, a tracer,
• the admixture may be a slurry.
• slurry refers to an an admixture suspended in a liquid, which includes, but is not limited to plasticizers such as those disclosed herein and known in the art, and other such agents.
• biodegradable and bioerodible refer to the dissolution of a substance, such as implant or coating, into constituent parts that may be metabolized or excreted, under the conditions normally present in a living tissue.
• rate and/or extent of biodegradation or bioerosion may be controlled in a predictable manner.
• biological response modifier refers to any protein, glycoprotein, sugar, polysaccharide, lipid, DNA, RNA, aptamer, peptide, hormone, vitamin and other such substance, which when introduced into a host organism is capable of eliciting a biological response, and includes, but is not limited to, cytokines, growth factors, protein hormones, genes, or genetically modified organisms, such as viruses and bacteria, and the like.
• biological response modifiers include, but are not limited to, the interleukins (IL), such as IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, BL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, isoforms thereof and others; the interferons such as interferon alpha, beta, gamma and others; the growth factors, such as platelet derived growth factor (PDGF), acidic and basic fibroblast growth factor including FGF-I and FGF-2, transformation growth factor beta (TGF -beta, e.g.
• IL interleukins
• PDGF platelet derived growth factor
• TGF -beta transformation growth factor beta
• TGF-beta-1 and TGF-beta-2 insulin like growth factor (IGF, e.g., including IGF-I and IGF-II), epidermal growth factor (EGF, e.g., EGF and heparin binding EGF), tumor necrosis factor-alpha (TNF- ⁇ ), tumor necrosis factor- beta (TNF- ⁇ ), vascular endothelial growth factor (VEGF), isoforms thereof and others; antibodies; bone morphogenetic proteins(BMPs), including but not limited to BMP-2, BMP -4, and BMP-7, metalloproteases or prometalloproteases and inhibitors thereof, angiotensin converting enzyme inhibitors; plasminogen and tissue plasminogen activator (TPA) 5 including anisoylated plasminogen activator (TPA) and anisoylated plasminogen-streptokinase activator complex (APSAC) and inhibitors therof; RNA and DNA in its various forms to modify
• antigen refers to any molecule capable of eliciting an immunological response, including, but not limited to, immunological memory responses, T-cell responses, B cell responses, allergy, a vaccine response, inflammation, immunological tolerance, and the like.
• antigenic compound refers to any organism or substance comprising an antigen, and includes, but is not limited to, whole viruses, bacteria, tissue, and derivatives, modifications, and products thereof, capable of eliciting an immune response. Specific examples of antigens include, but are not limited to, protein antigens, polysaccharide antigens, haptens, tumor antigens, blood antigens, and the like.
• drug refers to a substance used as a medication or in the preparation of medication, including, but not limited to, a substance intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease.
• a drug may include, but is not limited to, small organic molecules, complex organic molecules, inorganic elements and molecules, and the like.
• drug encompasses for example, fungicides, antibiotics and other molecules.
• tracer refers to any molecule that is introduced into an organism or construct and capable of being detected.
• tracers include, but are not limited to, radioactive compounds, contrast agents, light-emitting molecules, quantum dots, fluorescent molecules, dyes, biomarkers, molecular tracers for imaging purposes (including fluorescence markers, radioactive markers, contrast agents for CT, microCT, MRI or forms of bio- imaging, and immunospeciflc markers), and others.
• a "labeled compound” refers to any substance modified such that it (or its metabolites, such as degradation products) is detectable by any means.
• a labeled compound may be labeled in any manner including attachment (e.g., covalent or non-covalent) of tracers to the molecule of interest.
• hormone refers to any molecule which acts as a biochemical messenger that regulates physiological events in living organisms.
• specific examples of hormones include, but are not limited to, steroid hormones, such as estrogen, pregnenolone, aldosterone, estradiol, Cortisol, testosterone, progesterone, etc.; peptide hormones, such as luteinizing hormone (LH), adrenocorticotropic hormone (ACTH), follicle stimulating hormone (FSH), and angiotensin WTlI; synthetic steroids including, but not limited to, glucocorticoids, such as prednisone, dexamethasone, triamcinolone, etc., mineralocorticoids, such as fludrocortisone, Vitamin D derivatives, such as dihydrotachysterol, synthetic androgens, such as oxandrolone, decadurabolin, etc., synthetic estrogens such as diethylstilbestrol (DES); synthetic progestin
• filler refers to any substance incorporated into the polymer in order to provide additional structural or mechanical properties to the compositions disclosed herein, and include, but are not limited to, particulates as disclosed herein including, but not limited to, calcium phosphate, hydroxyapatite, etc., excipients (e.g., inert compounds acting as bulking agents, such as carboxymethylcellulose), synthetic and/or naturally occurring substances, such as polysacharrides and proteins (e.g., fibrous or globular proteins), which can be, for example, inert or biologically active.
• excipients e.g., inert compounds acting as bulking agents, such as carboxymethylcellulose
• synthetic and/or naturally occurring substances such as polysacharrides and proteins (e.g., fibrous or globular proteins), which can be, for example, inert or biologically active.
• the term “heat-sensitive” refers to any compound which when heated beyond 80 0 C, becomes inactive.
• the term “heat-sensitive” compound encompasses any compound, such as a biological response modifiers, antigens, drugs, hormones, tracers, labeled compounds, which lose biological activity at a temperature greater than 80 0 C, by any means including melting, decomposition, denaturation. etc.
• bio-ink is intended to include any material, whether liquid, solid or semisolid, that is suitable for deposition as part of the construction of a scaffold and may comprise, for example, a biological response modifier, antigen, drug, hormone, tracer, RNA, DNA, labeled compound, combinations thereof, and other such substances. Any material that is biocompatible or biodegradable is suitable for use as a bio-ink in accordance with the present disclosure.
• scaffold includes essentially any assembly of materials that is designed to imitate a biological structure, such as, for example, by imitating an aspect of fine structure (e.g., pore size and/or abundance) or by imitating the ability to support adhesion and/or growth of at least one appropriate cell type.
• biomimetic extracellular matrix refers to a structure, such as a scaffold, comprised of extracellular matrix constituents.
• co-depositing describes the placement of two or more substances, usually bio-inks, at the same position in, for example, a scaffold. Substances may be co-deposited simultaneously or non-simultaneously (for example, sequentially).
• a “concentration gradient” is one or more dimensions (whether in space or time) along which the concentration and/or accessibility of one or more substances may vary.
• the term is intended to include gradients in which the concentration is uniform throughout (i.e., a flat line gradient) as well as gradients in which the concentration varies.
• Concentration gradients include both linear gradients (i.e., gradients which increase or decrease at a continuous rate) and non-linear gradients.
• a “spatial concentration gradient” is a concentration gradient in which the concentration may vary along one or more spatial dimensions.
• a “temporal concentration gradient” is a concentration gradient in which the concentration may vary over time.
• a “3-D concentration gradient” is a set of three orthogonal spatial dimensions in which the concentration of one or more substances may vary independently along each dimension.
• Cross-linking is the formation of a covalent attachment between two entities, typically polymer subunits.
• a "cross-linking agent” refers to any agent capable of cross- linking two entities. Cross-linking agents may be physical or chemical. Chemical cross- linking agents include, but are not limited to, iridoid derivatives (such as, for example, genipin), diimidates, diones (e.g., 2,5-hexanedione), carbodiimides, (e.g., l-ethyl-[3- (dimethylaminopropyl)] carbodiimide) (abbr., EDC), acrylamides (e.g., N,N'methylenebisacrylamide), sugars (e.g., ribose and fructose), proteins (e.g., enzymes, such as transglutaminase Factor XIII), dimethylsuberimidates, aldehydes (e.g., glutaraldeh
• Chemical cross-linking agents can be solids (e.g., powders) or liquids.
• solid cross-linking agents include, but are not limited to, genipin, dihomo bifuntional NHS esters, and foraldehyde sodium bisulfite.
• liquid crosslinking agents include, but are not limited to, formaldehye, glutaraldehyde, etc.
• Physical cross-linking agents include, for example, electromagnetic radiation, such as ultraviolet light, heat, microwaves, etc.
• cross-linked amine refers to any bridging bond between two polymers comprising nitrogen, such as the product of aldehyde cross-linking (i.e., an imine or an eneamine), or product of an ester cross-link, or an amide, or any other similar bond. Cross-linking may occur before or after formation of a structure.
• gelation refers to the phase transition that a polymer undergoes when it increases in viscosity and transforms from a fluid state into a semi-solid material, or gel. At this transition point, the molecular weight (weight average) of the polymer matrix becomes "infinite” due to the formation of an essentially continuous matrix throughout the nascent gel. Polymerization can continue beyond the point of gelation through the incorporation of additional polymer units into the gel matrix.
• gel may include both the semi-solid gel state and the high viscosity state that exists above the gelation temperature.
• gelation temperature refers to the temperature at which a polymer undergoes reverse thermal gelation, i.e., the temperature below which the polymer is soluble in water and above which the polymer undergoes phase transition to increase in viscosity or to form a semi-solid gel. Because gelation does not involve any change in the chemical composition of the polymer, the gel may spontaneously reverse to the lower viscosity fluid form when cooled below the gelation temperature.
• the gelation temperature may also be referred to as the gel-solution (or gel-sol) transition temperature.
• a “hydrogel” is defined as a substance formed when a polymer (natural or synthetic) becomes a 3-D open-lattice structure that entraps solution molecules, typically water, to form a gel.
• a polymer may form a hydrogel by, for example, aggregation, coagulation, hydrophobic interactions, cross-linking, salt bridges, etc.
• the hydrogel should be non-toxic to the cells.
• dehydrated whether referring to a structure, such as a film, or a hydrogel includes any substance that has had water removed from it by any processes, and, thus, includes partially hydrated hydrogels, such as those described herein.
• hydrogel solution is a solute and a solvent comprising a substance that if subjected to the appropriate conditions, such as temperature, salt concentration, pH, the presence of a protease, the presence of a binding partner, etc., becomes a hydrogel or part of a hydrogel.
• solution in a hydrogel solution is intended to include true solutions, as well as suspensions, such as colloidal suspensions, and other fluid materials where one component is not truly solubilized
• a “mechanical property” refers to essentially any property that provides some description for how a substance responds to the application of an external force.
• Exemplary mechanical properties include tensile strength, compressional strength, flexural strength, impact strength, elongation, modulus, toughness, having mechanical properties similar to rubber (e.g., rubbery); etc.
• Mechanical properties include, for example, pliability (i.e., "pliant” is the ability of a polymer to bend or deform without breaking), elasticity (i.e., "elastomeric” is the ability of a polymer to a recover the original shape after deformation) and other such properties.
• a film can be, for example, both elastic and pliant, elastic without being pliant, or pliant without being elastic. Where a film is neither elastic nor pliant, it is referred to herein as "rigid.”
• a “film” refers to a thin sheet.
• a film can be a sheet up to 1 ⁇ m thickness, up to 100 ⁇ m thickness, up to 10 ⁇ m thickness, up to 1 ⁇ m thickness, up to 100 ⁇ m thickness, up to 10 nm thickness, up to lnm thickness, or any range therebetween.
• a film will have many mechanical properties, such as, for example, elasticity, non-elasticity, pliancy, rigidity, etc., depending on the formulation and shape.
• a “particulate” refers to a solid of sufficiently small size that it can be incorporated into a polymer matrix.
• Particulates include, but are not limited, to crystals, polymers, powders, ceramics, minerals, metal salts, calcium phosphates, including apatites (e.g., hydroxyapatite) and tricalcium phosphate, calcium sulphate, calcium phosphate, as well as other mineral combinations selected for inclusion to promote osteoconductivity for orthopaedic and other related applications, glasses, bioglasses, porogens, and the like.
• porogen refers to any particulate incorporated into a polymer matrix, wherein the particulate may be removed by any means including dissolution or sublimation of the porogen into a liquid or gas phase.
• a porogen may be soluble in the aqueous phase, the organic phase, or capable of sublimation into a gas.
• a porogen may also comprise an encapsulated gas (i.e., CO2, N, O, etc.) or substance capable of releasing a gas, upon decomposition, such as, for example, sodium bicarbonate releasing CO 2 upon contact with an acid.
• powders as used herein comprise particles having an average diameter of less than 30 mesh (i.e., 595 microns). In some embodiments of the invention, the average diameter of the particles is less than 35 mesh (i.e., 500 microns), less than 40 mesh (i.e., 400 microns), less than 45 mesh (i.e., 354 microns), less than 50 mesh (i.e., 297 microns), less than 60 mesh (i.e., 250 microns), less than 70 mesh (i.e., 210 microns), less than 80 mesh (i.e., 177 microns), less than 100 mesh 5 (i.e., 149 microns), less than 120 mesh (i.e., 125 microns), less than 140 mesh (i.e., 105 microns), less than 170 mesh (i.e., 88 microns), less than 200 mesh (i.e., 74 micron
• the particles have a size range from between 10 and 800 microns. In another embodiment, the particles have a size range from between 30 and 400 microns. In. yet other embodiments, the size range of the particles may be between 40 and 390 microns, between 50 and 380 microns, between 60 and 370 microns, between 70 and 360 microns, between 80 and 350 microns, between 90 and 340 microns,
• a powder can be formed by any means know in the art or disclosed herein including £0 milling, grinding, spray-drying, etc.
• plastic refers to any substance, such as organic, synthetic, and/or processed materials that comprise polymers and can be made into structures such as 3- dimensional constructs and 2-dimensional constructs, such as, for example, films, sheets, laminates, filaments, and similar structures. See, for example, U.S. Patent No. 6,143,293.
• hard plastic refers to a plastic that tends to break in response to sufficient deformation and, thus, has small plastic and/or elastic deformation range; whereas the term “soft plastic” refers to a plastic that readily deforms under stress without breaking, and, thus, has a large plastic and/or elastic deformation range.
• structures may be stacked upon each other. When such a stack is comprised of films or sheets, the structure is laminated.
• laminated refers to a structure having layers.
• MIS minimal-invasive surgery
• MIS refers to surgical procedures for treatment, diagnosis, and/or examination of one or more regions of a patient's body using surgical and diagnostic instruments specially developed to reduce the amount of physical trauma associated with the procedure.
• MIS involves instruments that may be passed through natural or surgically created openings of small diameter into a body to their location of use so that examinations and minor surgical interventions are possible with substantially less stress being imposed on the patient, for example, without general anesthesia.
• MIS may be accomplished using visualization methods, such as fiberoptic or microscopic means.
• Examples of MIS include, for example, arthoscopic surgery, laparoscopic surgery, endoscopic surgery, thoracic surgery, neurosurgery, bladder surgery, gastrointestinal tract surgery, etc.
• nucleic acid refers to a polymeric form of nucleotides, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide.
• the terms should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single- stranded (such as sense or antisense) and double-stranded polynucleotides.
• polymerize means to form an aggregate of multiple s ⁇ bunits, where the exact number of subunits in an aggregate is not precisely controlled by the properties of the aggregate itself.
• polymerize does not refer to the formation of a hexameric enzyme complex that is designed to be consistently hexameric.
• the formation of hexamers of, for example, fibrin or actin is a polymerization.
• Polymers are generally elongate, but may be of any shape, including a globular aggregate.
• polymerization may occur by any means including, for example, formation of peptide bonds among polymer similar subunits termed isopeptidic bonds (e.g., amide bond or Schiff base formation with lysine and/or primary amines in proteins), disulfide bond formation, or any other mechanism by which polymeric subunits may be linked.
• isopeptidic bonds e.g., amide bond or Schiff base formation with lysine and/or primary amines in proteins
• disulfide bond formation e.g., amide bond or Schiff base formation with lysine and/or primary amines in proteins
• polypeptide and the terms “protein” and “peptide” which are used interchangeably herein, refers to a polymer of amino acids.
• a “subject” is essentially any organism, although usually a vertebrate, and most typically a mammal, such as a human or a non-human mammal.
• therapeutically effective amount refers to that amount of a modulator, drug or other molecule that is sufficient to effect treatment when administered to a subject in need of such treatment.
• the therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
• tissue refers to an aggregation of similarly specialized cells united in the performance of a particular function. Tissue is intended to encompass all types of biological tissue including both hard and soft tissue, including connective tissue
• vector refers to a nucleic acid capable of transporting another nucleic acid to which it has been linked.
• One type of vector which may be used herein is an episome, i.e., a nucleic acid capable of extra-chromosomal replication.
• Other vectors include those capable of autonomous replication and expression of nucleic acids to which they are linked.
• Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors”.
• expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer to circular double stranded DNA molecules that, in their vector form are not bound to the chromosome.
• plasmid and “vector” are used interchangeably as the plasmid is the most commonly used form of vector.
• present dislcosure is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.
• compositions disclosed herein may comprise natural or synthetic organic polymers that can be gelled, or polymerized, or solidified (e.g., by aggregation, coagulation, hydrophobic interactions, or cross-linking) into a 3-D open-lattice structure that entraps water or other molecules, e.g., to form a hydrogel.
• Structures may comprise a single polymer or a mixture of two or more polymers. Additionally, two or more polymers maybe co-deposited or mixed so as to form a polymeric mixture.
• Polymers used may be biopolymers which can be biocompatible, biodegradable, and/or bioerodible and may act as adhesive substrates for cells.
• the polymers disclosed herein are easy to process into complex shapes and have a rigidity and mechanical strength suitable to maintain the desired shape under in vivo conditions.
• the structures disclosed here are formed from plastics that have had most of the water removed and have subsequently been treated with a plasticizing agent (e.g., glycerol).
• a plasticizing agent e.g., glycerol
• the plastic precursors to final structures can be created in a variety of manners including solid free-form fabrication, such as by ink-jet printing, molding, extrusion, or casting, such as by the methods disclosed herein and known in the art. It is also possible to form structures by extruding the plastic precursors through a die.
• the die may, without limitation, have a number of forms so that the extruded plastic is shaped like a tube, filament, rod, or sheet. Extrusion can be accomplished at relatively low pressures and temperatures; under certain processing conditions the plastic may be partially or completely dehydrated by the extrusion process. After sufficient dehydration, with or without the use of osmotic membranes and/or lyophilization, the extruded material may be plasticized and, optionally, cross-linked. It is expected that the extrusion will, in many instances, create an alignment, i.e., anisotropy, of the constituent molecules within the plastic and so impart certain properties, such as toughness, to the final elastomeric and/or pliant materials. If desired, and if patterning is unimportant, growth factors, drugs, antigens, tracers, or other such molecules may be added into the bulk plastic material, such as the admixture or slurry prior to processing.
• compositions of the invention may be produced either by using pressure to force the liquid from the hydrogel, or by the use of a concentration gradient and a semipermeable osmotic membrane to remove the liquid from the hydrogel.
• a plasticizer In some embodiments, it is possible to simultaneously remove water from and add plasticizer to the hydrogel, such as by the use of one or more osmotic membranes.
• Other processes may be used to form structures; for example, in one embodiment, the slurry can be extruded through a thin, optionally heated, slit; extrusion can additionally impart improved biomechanical properties by alignment of the molecules along the direction of the extrusion.
• the water content of a hydrogel can be controlled vacuum drying, i.e., by controlling vacuum and/or temperature so as to dehydrate the hydrogel.
• vacuum processing techniques are especially useful for large scale processing.
• the water content of the hydrogel is controlled by evaporating the water under normal atmospheric pressure. Evaporative processes may be performed at any temperature.
• the temperature used to evaporate the water is less than the temperature at which molecules incorporated into the polymer matrix would denature. This is referred to as the subcritical pressure and/or temperature for the inclusion present in a polymer matrix.
• a gel can be dehydrated at various temperatures that would prevent the degradation or denaturation of heat-sensitive chemicals and proteins, e.g., at a temperature of 75°C, 70 0 C, 65°C, 6O 0 C, 55°C, 50 0 C, 45°C, 40 0 C, 35 0 C, 30 0 C, 25°C, or room temperature, and, thus, within a range of less than 70 0 C, less than 65°C, less than 60 0 C, less than 55 0 C, less than 50 0 C, less than 45 0 C, less than 40 0 C, less than 35 0 C, less than 30 0 C, less than 25°C, or room temperature, or less.
• Vacuum pressure can be less than 100 millibars, less than 50 millibars, less than 25 millibars, less than 20 millibars, less than 15 millibars, less than 10 millibars, less than 5 millibars, less than 1 millibar, or even less.
• drying may occur over any time period, such as over 1 hour, 2 hours, 4 hours, 8 hours, 16 hours, 24 hours, or longer.
• the drying time can be varied to allow the gel to remain partially hydrated; i.e., wherein not all of the trapped water in the gel is removed.
• the gels can be dried on a substantially planar surface, thus creating a substantially planar film.
• gels may be placed in a frame, and/or compressed between sheets of material that preserve the forms, such as plastic sheets.
• the gel can be dried over a formed shape, thus creating a formed film that can be removed from the shape.
• the gel can be dried directly onto a structure or surface and not removed, thereby creating a film coating on the structure or surface.
• the processing steps can be performed under tensile load conditions to modify subsequent biomechanical properties of the material by aligning filaments of the component material, e.g., fibrin.
• a rectangular section of a hydro gel can be clamped on opposite sides and lyophilized, resulting in an orientation of the components of the film on the micro- and nano-scale.
• plasticizer is added to the resulting material the orientation of the components of material can exhibit improved mechanical properties for application as a graft substitutes for soft tissue repair including vascular, tendon, and ligament tissues.
• fibrous materials, such as 5 fibrin are particularly well-suited for use as forming biocompatible structures as orientation and/or entanglement of the fibers can provide desirable strength to compositions of the invention while maintaining flexibility of the material.
• a temporal concentration gradient may be created, for example, by capsules designed for timed release of one or more substances.
• a temporal concentration gradient may be created through spatial patterning or structural design of the scaffold.
• a temporal concentration gradient may be created by immobilizing (e.g., via absorption or chemical cross-linMng either directly or via an intermediate) one or more substances on the scaffold in a pattern. In this manner, the timing of interaction with the substances will be controlled based on the time it takes for a
• a temporal concentration gradient may be created in a scaffold having a fixed porosity by including one or more substances at a remote location on or within the scaffold. In this manner, interaction with the substances will be delayed during the period of time that it takes a cell to invade the scaffold and reach the remote location within the scaffold.
• a temporal gradient may be created in a scaffold using a variable porosity to control the rate of cell invasion into the scaffold. As cells encounter a higher porosity environment, the rate of invasion will be slowed, thus delaying interaction with one or more substances located in an area having a higher porosity.
• a temporal gradient may be created using biodegradable or bioresorbable scaffold.
• the porosity of the scaffold may decrease, thus permitting cell invasion at a more rapid rate.
• breakdown of the scaffold may expose a previously inaccessible area within the scaffold.
• solid free-form fabrication (SFF) processes and apparatus are used in a layering manufacturing process to build up shapes by incremental materials deposition and fusion of thin cross-sectional layers.
• the structures are created ex vivo and then administered to a patient (e.g., surgically implanted or attached to a host organism such as a wound, bone fracture, etc.).
• the articles disclosed herein further comprise kits for use and may further include packaging and directions for intended use of such kits.
• the structure maybe fabricated out of biocompatible materials which are designed for short-term, long-term or permanent implantation into a host organism.
• a graft may be used to repair or replace damaged tissue or an artificial organ may be used to replace a diseased or damaged organ (e.g., liver, bone, heart, etc.).
• structures maybe fabricated out of biodegradable materials to form temporary structures. For example, a bone fracture may be temporarily repaired with a biodegradable structure that will undergo controlled biodegradation occurring concomitantly with bioremodeling by the host's cells.
• a 3-D structure of the structure may be fabricated directly using SFF.
• SFF magnetic resonance imaging
• CAT computerized axial tomography
• CAD computer-aided-design
• CAM computer-aided- manufacturing
• the methods and apparatus disclosed herein may be used to produce a non-specific 3-D structure (e.g., a block or cube), which is then cut or molded into the desired shape (e.g., using a laser, saw, blade, etc.).
• the methods and apparatus disclosed herein may be used to create structures with specific microstructural organization such that the structure has the anatomical and biomechanical features of naturally occurring tissues, or engineering designs that are biologically inspired.
• the microstructural organization includes the spatial concentration of intercalated materials (e.g., biological response modifiers, antigens, drugs, hormones, tracers, or labeled compounds), the degree of porosity of the structure, and/or channels that run through the 3-D structure for improved cell invasion, vascularization, and nutrient diffusion.
• plasticizer can be added to the resulting material (and in certain other embodiments addition of water and plasticizer can occur simultaneously). In some embodiments, the addition of plasticizer can be accomplished by soaking the dehydrated material in a bath of the plasticizer.
• Biocompatible plasticizers that can be used for this purpose include, but are not limited to, polyalchohols, such as glycerol and mixtures of water and glycerol, as well as other plasticizers known in the art and disclosed herein.
• the strength and toughness of the material can be increased by cross-linking, either as an intermediate step or after forming the material into its final configuration. Any cross-linking agent known in the art or disclosed hereinmay be used.
• the material is primarily composed of a single precursor material (e.g., fibrin or chitosan)
• improved properties can be obtained by including other components, such as fibronectin, collagen, gelatin, hyaluronic acid, hormones, biological response modifiers, fillers, tracers, drugs, and/or particulates, such as calcium phosphate (e.g., hydroxyapatite), etc. to produce distinct and unique final materials.
• Such formulations can he useful in altering the physical characteristics of the material, the mechanical properties, and biological properties (i.e., degradation rate, cell attachment, application to wound site), depending upon the materials that are added to the elastomeric and/or pliant materials.
• the additional bioactive materials can be added to the polymer material in bulk if patterning is not required.
• complex constructs including three-dimensional spatial patterns, such as gradients, can be created by the use of the bioprinting technology disclosed in U.S. Patent Application Serial No. 10/391,458 filed March 18, 2003 and U.S. Provisional Application for Patent Serial No. 60/619,192 filed October 15, 2004, both of which are incorporated herein by reference in their entirety, in order to create patterns of hormones or other bioactive materials upon prefabricated materials, or by printing the materials directly so that the bioactive agents are incorporated within the materials.
• Such a printing process can be used alone or further processing steps, such as rolling, folding, or stacking such printed elastomeric and/or pliant materials, may be employed in order to fabricate much more complex constructs
• Drugs, tracers, hormones, antigens, biological response modifiers, etc. are among the materials that can be incorporated into or onto the biocompatible polymer in order to provide additional control of biodegradation. It is noted that depending on the source of the biopolymer, endogenous factors may already be present in the material; however, in such instances, additional advantages may be obtained by adding exogenous materials to the biopolymer. Exemplary approaches include addition of protease inhibitors and in certain other embodiments immobilization of the inhibitors within the the biopolymer matrix. Non-immobilized forms of inhibitors, although functional, are susceptible to being extracted during dehydration of the hydrogel and/or once applied to tissue implantation site.
• Immobilization of inhibitors via basic native binding (i.e., plasminogen activator inhibitors) or engineering solid-phase inhibitors (i.e., aprotinin) is a method that can be used to improve retention of inhibitors. Such methods are disclosed herein and known in the art.
• tracers, and/or labeled compounds may be incorporated into the polymer matrix.
• the tracer and/or label is a fluorescent molecule, such as a fiuorophore or quantum dot. Where such compounds are used, the movement of the label or tracer can be detected, demonstrating for example, degradation, diffusion, etc, of the tracer or labeled compound from the structure.
• the fluorescent compound is a fluorescent dye, including, but not limited to, small molecule fluorophores, such as fluorescein, (e.g., fluorescein isothiocyanate (FITC)), Pacific Blue, Cascade Blue, cyanine dyes (e.g., Cy3, Cy5, Cy5.5, and Cy7), Alexa dyes, etc; large fluorescent proteins, such as phycobiliproteins, R-phycoerythrin (R-PE), green fluorescent protein, and allophycocyanin (APC); tandem dyes (i.e., small molecule fluorophores covalently linked to phycobiliproteins, such as PE-TR, PE-Cy5, PE-Cy5.5, PE-Cy7, APC-Cy7, PerCP, etc.); and substances chemically conjugated to such dyes.
• small molecule fluorophores such as fluorescein, (e.g., fluorescein isothiocyanate (FITC)), Pacific Blue, Cascade Blue, cyan
• quantum dots are used as a tracer for biodegradation of an implanted structure.
• Quantum dots are nanocrystal semiconductor materials encapsulated by an inorganic "shell" which increases aqueous solubility of the quantum dot. When excited by an energy source, such as a laser, the quantum dots fluoresce.
• Quantum dots are fluorophores but differ from traditional fluorophores, such as organic fluorescent dyes and naturally fluorescent proteins. They are nanometer-scale atom clusters, containing from a few hundred to a few thousand atoms of a semiconductor material (often, cadmium mixed with selenium, or tellurium), which has been coated with an additional semiconductor shell (e.g., zinc sulfide) to improve the optical properties of the material.
• an additional semiconductor shell e.g., zinc sulfide
• quantum dots Unlike, traditional fluorophores, there is no ⁇ ->x* electronic transitions, thus allowing the quantum dots to be "tuned” to fluoresce over any spectrum and abrogating the need for multiple lasers for multicolor detection studies.
• quantum dots fluoresce brightly over a long period of time making them useful for time-gated studies.
• quantum dots are conjugated to proteins to allow detection as in conventional dye conjugate systems but with improved performance characteristics. Incorporation of fluorophores into or on the composition of the invention allows monitoring of an implanted structure in vivo in real time without the need for surgery. Methods of detecting fluorescence and various photodetection devices are known in the art.
• Materials printed upon films as disclosed herein can persist on the printed material for at least one week. See Fig. 4.
• biocompat ⁇ bility and limited angiogenesis can be demonstrated using the standard chick CAM assay (see, for example, Ribatti, D. et al., Jnt. J. Dev. Biol., 40, 1189-1197. (1996) Ribatti, D et al, Pathol Res Pract, 192:1068-1076 (1996), and Ribatti, D. et al, Anat Rec, 264:317-324(2001), all of which are incorporated herein by reference).
• multiple layers of polymeric films are stacked atop one another.
• gradients of bioactive materials and/or pores can be created by creating layers of the films each comprising the desired amounts of the bioactive materials within or on the surface of each layer and then stacking the different layers as desired.
• Such structures can be created in the manner disclosed in U.S. Patent No. 6,165,486, issued December 26, 2000, which is incorporated herein by reference. Such configurations are particularly useful when creating structures to fill cranial voids, for example.
• polymeric films are formed into sheets, tubes, rods, or filaments.
• Such structures are particularly useful as replacements for tendon, bone, or ligament, for example, and have application in long bone and non-long bone repair. Further incorporating growth factors or anabolic hormones and/or drugs can improve the biological response associated with tissue repair.
• Tube based structures also find use for vascular grafts and nerve guides, for example.
• the films find use as barrier membranes to protect tissues and prevent tissue adhesion.
• the compositions disclosed herein, e.g., fibrin-based elastomeric films offer significant advantages in promoting tissue and wound repair.
• Methods of forming the structure, such as tubular structures include creating hydrogels which are then cast into molds, as shown in Figure 2. Water can then be removed from the hydrogel (e.g., after removing the tubular structure from the mold or by using osmotic membranes as surfaces of the mold) and replaced by plast ⁇ cizer. In alternative embodiments, sheets of the compositions disclosed herein are rolled, such as on a mandrel, to create hollow tubular structures. In other embodiments, the hydrogel precursors can be extruded into tubular configurations.
• a substantially planar composition as disclosed herein may be rolled up on itself, in which case no mandrel would be needed.
• the elastomer can be cross-linked to retain the tubular shape and/or stapled, heated to a fusing temperature, or otherwise held in the tubular configuration.
• the integrity of a structure, such as those disclosed herein can be increase by including a biocompatible mesh, such as titanium, NYLONTM, or DACRONTM. The mesh can be added as a layer of the substantially planar material prior to rolling it into a tubular structure or it can. form the outer layer of the tubular structure.
• the materials disclosed herein can be print, cast, or extruded onto
• structures may be formed from ionic hydrogels, for example, ionic polysaccharides, such as alginates or chitosan.
• Ionic hydrogels may be produced by
• LO soluble in aqueous solutions e.g., water, or aqueous alcohol solutions that have charged side groups, or a monovalent ionic salt thereof.
• aqueous solutions e.g., water, or aqueous alcohol solutions that have charged side groups, or a monovalent ionic salt thereof.
• polymers with acidic side groups that can be reacted with cations, e.g., poly(phosphazenes), poly(acrylic acids), and poly(methacrylic acids).
• acidic groups include carboxylic acid groups, sulfonic acid groups, and halogenated (preferably fluorinated) alcohol groups. Examples of
• Polymers with basic side groups that can react with anions are poly(vinyl amines), poly(vinyl pyridine), and poly(vinyl imidazole).
• Polyphosphazenes are polymers with backbones consisting of nitrogen and phosphorous atoms separated by alternating single and double bonds. Each phosphorous atom is covalently bonded to two side chains.
• Polyphosphazenes that can be used have a majority of side chains that are acidic and capable of forming salt bridges with di- or trivalent cations. Examples of acidic side chains are carboxylic acid groups and sulfonic acid groups.
• Bioerodible polyphosphazenes have at least two differing types of side chains, acidic side groups capable of forming salt bridges with multivalent cations, and side groups that hydrolyze under in vivo conditions, e.g., imidazole groups, amino acid esters, glycerol, and glucosyl groups.
• Bioerodible or biodegradable polymers i.e., polymers that dissolve or degrade within a period that is acceptable in the desired application (usually in vivo therapy), will degrade in less than about five years or in less than about one year, once exposed to a physiological solution of pH 6-8 having a temperature of between about 25°C and 38 0 C. Hydrolysis of the side chain results in erosion of the polymer. Examples of hydrolyzing side chains are uns ⁇ bstituted and substituted imidizoles and amino acid esters in which the side chain is bonded to the phosphorous atom through an amino linkage.
• biopolymer matrices may be manufactured by preparing solid components as an admixture and then subjecting the admixture to pressure to induce formation of a polymer matrix.
• biopolymers such as protein, fibrin, fibrinogen, collagen, gelatin, elastin, extracellular matrix constituents, polysaccharide, hylauronic acid, and similar such polymers are powdered (e.g., milled, ground, spray dried, etc.) and then compressed.
• the powder is admixed with a substance, such as a plasticizer, a cross-linking agent, a filler, a particulate, a porogen, a biological response modifier, an antigen, a drug, a hormone, a tracer, RNA, DNA, or a labeled compound, combinations thereof, and the like prior to pressing.
• a substance such as a plasticizer, a cross-linking agent, a filler, a particulate, a porogen, a biological response modifier, an antigen, a drug, a hormone, a tracer, RNA, DNA, or a labeled compound, combinations thereof, and the like prior to pressing.
• the admixture is compressed at a temperature in which a heat-sensitive compound such as a biological response modifier would not denature or lose bioactivity.
• heat-sensitive proteins can be compressed at temperatures below the denaturation temperature or melting point of the protein, thus preserving bioactivity in the polymer matrix after compression.
• Biopolymers may be made in any shape including 3 -dimensional structures and 2-dimensional structures, such as sheets, rods, and filaments.
• Biopolymer structures can be prepared using different approaches (e.g., casting, cold-pressing, injections molding, die extrusion etc.), wherein the amount of pressure applied controlles the thickness and density of the biopolymer structure.
• a structure such as a bioplastic pellet, is formed from biopolymers by grinding solidified biopolymer into a powder with particle sizes less than or equal to 150 microns (e.g., at least 100 mesh standard sieve).
• a plasticizer is added to the biopolymer, e.g., 12.5%, 19%, 21%, 25%, 35% and 50% plasticizer by weight.
• the powder (and any additional components in the admixture) are mixed with, for example, glycerin, and mixed until homogenous.
• the biopolymer compositions disclosed herein are then pressed.
• compression may be accomplished by any means including, for example, using a pellet press. Compression may occur at any suitable pressure such as lOOOlbs., 20001bs., 30001bs., 40001bs., 50001bs., 60001bs., 7000lbs., 80001bs. of pressure. Those of skill in the art recognize that such pressures can be reported in any manner including interconversion to PSI, torr, bars or any other suitable measurement scale.
• a mold release agent such as lecithin is used to facilitate removal of a compressed biopolymer from a press or mold.
• a biopolymer is compressed at a suitable temperature (e.g., 60°, 66°, 70°, 80 0 C) and at a suitable pressure (e.g., 5000 lbs to 7000 lbs) and for a suitable time (e.g., 1, 5, 10, 20, 30, 40, 50, 60 minutes or longer) to form a biopolymer matrix.
• a suitable temperature e.g., 60°, 66°, 70°, 80 0 C
• a suitable pressure e.g., 5000 lbs to 7000 lbs
• a suitable time e.g., 1, 5, 10, 20, 30, 40, 50, 60 minutes or longer
• the mold pressure is maximized and remains steady (e.g., at approximately 7000 lbs of pressure) for approximately 20 minutes.
• the mold temperature is decreased from an initial value of approximately 80 0 C until reaching a final steady value of approximately 27°C (room temperature).
• a biopolymer admixture may comprise additional components mixed with the polymer powder, such as a porogen.
• a porogen may be distributed homogenously within the biopolymer powder or non-homogenously.
• Non-homgenous distribution of porogen can be used to introduce a pore gradient and/or distribution within the final formed structure, to modulate the biodegrability, mecanical properties, etc., of the structure.
• other components can be incorporated in such a manner, homogenously or inhomogenously, to introduce for example, biochemical gradients of a substance within the polymer matrix.
• water soluble polymers with charged side groups can be cross- linked by reacting a polymer with an aqueous solution containing multivalent ions of the opposite charge, either multivalent cations if the polymer has acidic side groups, or multivalent anions if the polymer has basic side groups.
• Cations for cross-linking the polymers with acidic side groups to form a hydrogel include divalent and trivalent cations, such as copper, calcium, aluminum, magnesium, and strontium.
• Aqueous solutions of the salts of these cations can be added to the polymers to form soft, highly swollen hydrogels and membranes.
• Anions for cross-linking the polymers to form a hydrogel include divalent and trivalent anions such as low molecular weight dicarboxylate ions, terepthalate ions, sulfate ions, and carbonate ions.
• Aqueous solutions of the salts of these anions can be added to the polymers to form soft, highly swollen hydrogels and membranes, as described with respect to cations.
• polycations can be used to complex and thereby stabilize the polymer into a semi-permeable surface membrane.
• examples of polycations include poly-L- lysine, as well as natural polycations, such as the polysaccharide, chitosan.
• polymers such as those known in the art and disclosed herein, can be cross-linked using chemical cross-linking agents.
• the chemical cross-linking agent is a solid.
• the solid cross-linking agent becomes active in the presence of water.
• a solid cross- linking agent can be admixed with a dry polymer or otherwise incorporated into a dehydrated polymer matrix (e.g., fibrin, gelatin, etc.).
• a solid crosslinking agent can be active prior to hydration or in the alternative, upon exposure to water.
• the solid cross- linking agent, genipin is incorporated into the polymer admixture and subsequently allowed to be activated by water which is either already in the polymer and/or which is absorbed when the subsequently formed polymer matrix is placed in a water bath. Because water is either already in the matrix (e.g., because of compactions) or rapidly diffuses into a polymer matrix (e.g., a biopolymer matrix), the cross-linking occurs rapidly and uniformly thoughout the gel.
• a solid cross-linking agent such as genipin can be used.
• Genipin (Cyclopenta(c) pyran-4-carboxylic acid, l,4a-alpha,5,7a-alpha-tetrahydro-l- hydroxy-7-(hydroxymethyl)-, methyl ester 4 ) is a pyrari hydrolytic product of geniposide, and it capable of forming cross-links with amines. Genipin is a non-toxic cross-linking agent, and, thus, better suited for use in numerous biomedical applications, since many other cross- linking agents such as glutaraldehyde have been shown to be toxic to cells.
• the solid cross-liiiking agent is formaldehyde sodium bisulfite, for example.
• NHS-esters of carboxylic acids are used as a solid cross-linking agent.
• a useful permeability of the macromer/polymer is in the range of between 10,000 D and 30,000 D, for example. Smaller macromers result in polymer matrices of a higher density with lower molecular weight cut-offs.
• the speed of erosion of a scaffold produced from a bioerodible or biodegradable polymer is also related to the molecular weights of the polymer.
• Higher molecular weight polymers e.g., with average molecular weights of 90,000 or higher
• lower molecular weight polymers e.g., average molecular weights of 30,000 or less
• additional features such as roughened spots, pores, holes, etc, are introduced into the scaffolds by machining milling, grinding, etc. to promote osteoconductive growth. Cells readily migrate and attach upon such roughened surfaces.
• pores into the compositions of the invention may also be used to regulate permeability, degradation rate, and mechanical properties of the compositions disclosed herein.
• pores may be introduced mechanically or chemically into the polymer matrix.
• pores are introduced mechanically, such as by machining (e.g., punching) holes in a film that is subsequently stacked or rolled as described herein.
• pores are introduced chemically by incorporating a porogen into the polymer and subsequently removing it once the polymer matrix has formed, hi certain embodiments, the porogen is soluble in aqueous or organic solvents.
• the porogen diffuses out of the polymer, leaving pores, hi some embodiments, NaCl particles can be used as a porogen with water as the solvent.
• polyurethane, polylactic acid, polylactic-co-glycolic acid, or polycaprolactone can be used in conjunction with an organic solvent, such as chloroform, for example.
• organic-soluble porogens in a polymer matrix can be advantageous where the polymer matrix comprises additional water-soluble substances, such as biological response modifiers and the like, since these will not diffuse out of the polymer matrix when it is placed in the organic solvent.
• the organic phase system can operate more efficiently than aqueous phase systems, as it has been observed that in certain embodiments residual NaCl may remain in the polymer matrix after leaching.
• the porogen can be a sublimation porogen.
• a sublimation porogen will sublime directly into the gas phase under the appropriate temperature and pressure, thus obviating the need for solvent-leaching altogether, hi certain embodiments, the sublimation porogen can be ammonium acetate, ammonium chloride, ammonium bicarbonate, ammonium carbonate, or pyridiniurn trifluoroacetate, for example.
• the temperature and pressure at which a substance sublimes is known as the triple point from a phase diagram. Thus, to introduce pores using sublimation, the temperature and pressure must be below this triple point of the porogen.
• the sublimation porogen is bio-compatible, meaning that should any residual porogen remain in the polymer matrix, it would not be toxic to an organism upon implantation or use.
• the sublimation porogen is removed under sufficiently mild conditions (such as low temperature and pressure) that any additional substance included in the polymer matrix, such as heat-sensitive proteins or drugs are not denatured or degraded during the sublimation process; e.g.
• the sublimation porogen is removed by reducing pressure, such as removal using a vacuum.
• Vacuum pressure can be less than 100 millibars, less than 50 millibars, less than 25 millibars, less than 20 millibars, less than 15 millibars, less than 10 millibars, less than 5 millibars, less than 1 millibar, or less.
• the sublimation porogen is removed along with water, e.g., drying a gel under a vacuum as discussed supra, and, thus, removing both water and the porogen at the drying temperatures and pressures disclosed herein.
• compositions of the invention comprise interconnected pores.
• interconnected pores are advantageous for biological uses because porogen residue is less likely to be trapped within a polymer matrix, and, thus, the construct is more likely to be biocompatible.
• compositions disclosed herein may be temperature-dependent or thermosensitive hydrogels. These hydrogels must have so-called "reverse gelation" properties, i.e., they are liquids at or below room, temperature, and gel when warmed to higher temperatures, e.g., body temperature.
• these hydrogels can be easily applied at or below room temperature as a liquid and automatically form a semi-solid gel when warmed to body temperature.
• temperature-dependent hydrogels are PLURONICSTM (BASF-Wyandotte), such as polyoxyethylene-polyoxypropylene F-108, F-68, and F-127, poly (N-isopropylacrylamide), and N-isopropylacrylamide copolymers.
• polymers can be manipulated to affect their physical properties, such as porosity, rate of degradation, transition temperature, and degree of rigidity.
• LCST lower critical solution temperature
• the addition of low molecular weight saccharides in the presence and absence of salts affects the lower critical solution temperature (LCST) of typical thermosensitive polymers, hi addition, when these gels are prepared at concentrations ranging between 5 and 25% (W/V) by dispersion at 4 0 C, the viscosity and the gel-sol (gel- solution) transition temperature are affected, the gel-sol transition temperature being inversely related to the concentration.
• LCST critical solution temperature
• the structures disclosed herein may be pH-dependent hydrogels- These hydrogels are liquids at, below, or above specific pH values, and gel when exposed to specific pHs, e.g., 7.35 to 7.45, the normal pH range of extracellular fluids within the human body. Thus, these hydrogels can be easily applied in the body as a liquid and automatically form a semi-solid gel when exposed to body pH. Examples of such pH- dependent hydrogels are TETRONICSTM. (B ASF-Wyandotte) polyoxyethylene- polyoxypropylene polymers of ethylene diamine, poly(diethyl arninoethyl methacrylate-g- ethylene glycol), and ⁇ oly(2-hydroxymethyl methacrylate). Such copolymers can be manipulated by standard techniques to affect their physical properties.
• structures disclosed herein can comprise light solidified plastics, e.g., solidified by either visible or ultraviolet light.
• hydrogels are made of macromers including a water soluble region, a biodegradable region, and at least two polymerizable regions as described, for example, in U.S. Pat. No. 5,410,016.
• the hydrogel can begin with a biodegradable, polymerizable macromer including a core, an extension on each end of the core, and an end cap on each extension.
• the core is a hydrophilic polymer
• the extensions are biodegradable polymers
• the end caps are oligomers capable of cross-linking the macromers upon exposure to visible or ultraviolet light, e.g., long wavelength ultraviolet light.
• Examples of such light solidified polymers can include polyethylene oxide block copolymers, polyethylene glycol polylactic acid copolymers with acrylate end groups, and 10,000 D polyethylene glycol-glycolide copolymer capped by an acrylate at both ends.
• the copolymers comprising these hydrogels can be manipulated by techniques known to the skilled artisan to modify their physical properties such as rate of degradation, differences in crystallinity, and degree of rigidity.
• structures may be a bioerodible or biodegradable synthetic polymers.
• Suitable polymers include, for example, bioerodible polymers such as polylactic acid (PLA), polyglycolic acid (PGA) 5 polylactide-co-glycolide (PLGA), polycaprolactone, polycarbonates, polyamides, polyanhydrides, polyamino acids, polyortho esters, polyacetals, polycyanoacrylates and degradable polyurethanes, and non- erodible polymers, such as polyacrylates, ethylene- vinyl acetate polymers, and other acyl substituted cellulose acetates, and derivatives thereof, non-erodible polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinyl imidazole), chlorosulphonated polyolefms, polyethylene oxide, polyvinyl alcohol, TEFLONTM, and nylon.
• bioerodible polymers such as polylactic acid
• the scaffolds disclosed herein may be used to prepare a scaffold for any mammal in need thereof.
• Mammals of interest include humans, dogs, cows, pigs, cats, sheep, horses, and the like, preferably humans.
• the methods, compositions, and apparatus disclosed herein may be used to prepare a variety of scaffolds that may be utilized as xenografts, allografts, artificial organs, or other cellular transplantation therapeutics.
• the scaffolds may be used to repair and/or replace any damaged tissue associated with a host.
• the scaffolds dislcosed herein may also be suitable for other applications, such as for hormone producing or tissue producing implants for deficient individuals who suffer from conditions, such as diabetes, thyroid deficiency, growth hormone deficiency, congenital adrenal hyperplasia, Parkinson's disease, and the like.
• apparatus and methods disclosed herein may be useful for creating scaffolds suitable for therapeutic applications, including, for example, implantable delivery systems providing biologically active and gene therapy products.
• the scaffolds disclosed herein may be useful for the treatment of the central nervous system, to provide a source of cells secreting insulin for treatment of diabetes, cells secreting human nerve growth factors for preventing the loss of degenerating cholinergic neurons, satellite cells for myocardial regeneration, striatal brain tissue for Huntington's disease, liver cells, bone marrow cells, dopamine-rich brain tissue, and cells for Parkinson's disease, cholinergic-rich nervous system cell for Alzheimer's disease, adrenal chromaffin cells for delivering analgesics to the central nervous system, cultured epithelium for skin grafts, and cells releasing ciliary neurotrophic factor for amyotrophic lateral sclerosis, and the like.
• the scaffolds disclosed herein may be used to repair bone injuries and induce healing thereof by inducing vascularization to the site of injury.
• the methods, compositions, and apparatus disclosed herein may be used to create 3-D scaffolds capable of providing a spatial and/or temporally organized therapeutic to a host at a desired location.
• the scaffolds contain 3-D patterns of therapeutic and/or diagnostic agents, such as biological response modifiers, antigens, drugs, hormones, tracers, or labeled compounds that operate within the host in a predictable and organized manner.
• a scaffold may have gradients of one or more growth factors which vary throughout the structure, such as a concentration gradient that diminishes from the center of the structure to the periphery, a gradient from one side of the structure to the other, etc., in an infinite variety of possible configurations.
• temporal gradients may also be engineered using the time release mechanisms described above.
• organized doses of one or more therapeutic factors can be provided to an organism in need thereof.
• spatial and temporal therapeutics may be used to induce organized neovascularization in a host at a desired location.
• angiogenic factors are produced at the site of injury producing a concentration gradient which decreases away from the site of injury.
• traditional approaches to inducing angiogenesis involve uniform application of angiogenic factors which typically lead to unorganized vessel formations or angiomas.
• the scaffolds disclosed herein may be engineered so as to provide a concentration gradient of angiogenic factors in a 3-D spatial and/or temporal configuration that mimics the naturally occurring wound healing response signals resulting in formation of organized and directed neovascularization at a desired location in a host.
• scaffolds may contain a 3-D pattern of adhesion molecules specific for one or more cell types.
• a 3-D pattern of adhesion molecules may be configured so as to attract and adhere particular cell types to the scaffold in a desired 3-D architecture.
• These scaffolds can be used to induce a desired configuration of cell attachment/tissue formation at a specified location.
• the scaffold maybe a permanent or long-term implant or may degrade over time as the host's natural cells replace the scaffold.
• two or more adhesion molecules with different cell binding specificities are patterned on the scaffold so as to immobilize two or more desired cell types into a specific 3-D pattern.
• a variety of techniques can be used to foster selective cell adhesion of two or more cell types to the scaffold.
• adhesion proteins such as collagen, f ⁇ bronectin, gelatin, collagen type IV, larninin, entactin, and other basement proteins, including glycosaminoglycans, such as heparan sulfate, RGD peptides, ICAMs, E-cadherins, and antibodies that specifically bind a cell surface protein (for example, an integrin, ICAM, selection, or E-cadherin).
• the therapeutic bio-inks disclosed herein may be cells which may be used to directly seed a 3-D cellular architecture of one or more cell types. Combinations of these approaches are also envisioned, e.g., 3-D patterns of cells and growth factors.
• cells may be used to coat small or large surface areas of devices, wound dressings or areas of the body. Such coatings may be applied directly to the device and applied to a desired location.
• cells may be applied individually or as a population aliquot. Any structure disclosed herein may be seeded with cells of therapeutic use, including stem cells.
• IPNs interpenetrating polymer networks
• IPNs are blends or alloys of two or more polymer components, each of which is a cross-linked 3-D network.
• the individual polymer component networks are more or less physically entangled with, but not covalently bonded to, the other polymer network(s) in the IPN.
• a feature of EPNs is that they permit combining advantageous properties from each of two polymers which are normally incompatible. For example, in a hydrophobic-hydrophilic system, flexibility and structural integrity might be imparted by the hydrophobic polymer and lubriciousness might be imparted by the hydrophilic polymer.
• An IPN may be a bicontinuous system in which each of the polymers forms a continuous matrix throughout the network.
• a scaffold maybe created in situ, or may be pre-fabricated and implanted into a patient, at a desired location using minimally invasive techniques.
• minimally invasive surgical techniques may be used to provide tissue sealants at focused areas and/or to provide short term and/or long term administration of therapeutic agents, including for example, cells, polypeptides, polynucleotides, growth factors, drugs, etc.
• minimally invasive techniques may be used to provide scaffolds for repairing hyaline cartilage and/or fibrocartilage in diarthroidal and amphiarthroidal joints.
• a resorbable vascular wound dressing maybe delivered in association with angioplasty procedures to deliver or fabricate a scaffold to selected sites inside or outside a blood vessel.
• Vascular wound dressings may be tubular, compliant, self- expandable, low profile, biocompatible, hemocompatible, and/or bioresorbable. In certain embodiments, such wound dressings may prevent or substantially reduce the risk of post- angioplasty vessel reclosure.
• vascular wound dressings may be fabricated with a therapeutic agent for treatment of vessel wounds, including, for example, anti-platelet agents, such as aspirin and the like, anti-coagulant agents, such as Coumadin and the like, antibiotics, anti-thrombus deposition agents, such as polyanionic polysaccharides including heparin, chondroitin sulfates, hyaluronic acid and the like, urokinase, streptokinase, plasminogin activators and the like, wound healing agents, such as transforming growth factor beta (TGF beta) and the like, glycoproteins, such as laminin, fibronectin and the like, various types of collagens.
• anti-platelet agents such as aspirin and the like
• anti-coagulant agents such as Coumadin and the like
• antibiotics such as polyanionic polysaccharides including heparin, chondroitin sulfates, hyaluronic acid and the like, urokinas
• the apparatus, methods, compositions, and products disclosed herein may be used to create bioresorbable wound dressings or band-aids.
• Wound dressings may be used as a wound-healing dressing, a tissue sealant (i.e., sealing a tissue or organ to prevent exposure to a fluid or gas, such as blood, urine, air, etc., from or into a tissue or organ), and/or a cell-growth scaffold.
• the wound dressing may protect the injured tissue, maintain a moist environment, be water permeable, be easy to apply, not require frequent changes, be non-toxic, be non-antigenic, maintain microbial control, and/or deliver effective healing agents to the wound site.
• bioresorbable sealants and adhesives examples include, for example, FOCALSEALTM produced by Focal; BERIPLASTTM produced by Adventis-Bering; VIVOSTATTM produced by ConvaTec (Bristol-Meyers-Squibb); SEALAGENTM produced by Baxter; FIBRXTM produced by CyoLife; TISSEELTM and TISSUCOLTM produced by Baxter; QUDCILTM produced by Omrix Biopharm; a PEG-collagen conjugate produced by Cohesion (Collagen); HYSTOACRYL BLUETM produced by Davis & Geek; NEXACRYL, NEXABOND, NEXABOND S/CTM, and TRAUMASEALTM produced by Closure Medical (TriPoint Medical); OCTYL CNATM produced by Dermabond (Ethicon); TISSUEGLUTM produced by Medi-West Pharma; and VETBONDTM produced by 3M.
• FOCALSEALTM produced by Focal
• BERIPLASTTM produced by
• Wound dressings maybe used in conjunction with orthopedic applications, such as bone filling/fusion for osteoporosis and other bone diseases, cartilage repair for arthritis and other joint diseases, tendon repair; for soft tissue repair, including nerve repair, organ repair, skin repair, vascular repair, muscle repair, and ophthalmic applications.
• wound dressings may be used to treat a surface, such as, for example, a surface of the respiratory tract, the meninges, the synovial spaces of the body, the peritoneum, the pericardium, the synovia of the tendons and joints, the renal capsule and other serosae, the dermis and epidermis, the site of an anastomosis, a suture, a staple, a puncture, an incision, a laceration, or an apposition of tissue, a ureter or urethra, a bowel, the esophagus, the patella, a tendon or ligament, bone or cartilage, the stomach, the bile duct, the bladder, arteries, and veins.
• a surface such as, for example, a surface of the respiratory tract, the meninges, the synovial spaces of the body, the peritoneum, the pericardium, the synovia of the tendons and joints, the renal capsule and other serosa
• wound dressings may be used in association with any medical condition that requires coating or sealing of a tissue.
• lung tissue may be sealed against air leakage after surgery; leakage of blood, serum, urine, cerebrospinal fluid, air, mucus, tears, bowel contents, or other bodily fluids may be stopped or minimized; barriers may be applied to prevent post-surgical adhesions, including those of the pelvis and abdomen, pericardium, spinal cord and dura, tendon, and tendon sheath.
• Wound dressings may also be useful for treating exposed skin, in the repair or healing of incisions, abrasions, bums, inflammation, and other conditions requiring application of a coating to the outer surfaces of the body.
• Wound dressings may also be useful for applying coatings to other body surfaces, such as the interior or exterior of hollow organs, including blood vessels. Restenosis of blood vessels or other passages may also be treated.
• the range of uses for wound dressings also include cardiovascular surgery applications, prevention of bleeding from a vascular suture line; support of vascular graft attachment; enhancing preclotting of porous vascular grafts; stanching of diffuse non-specific bleeding; anastomoses of cardiac arteries, especially in bypass surgery; support of heart valve replacement; sealing of patches to correct septal defects; bleeding after sternotomy; and arterial plugging; thoracic surgery applications, including sealing of bronchopleural fistulas, reduction of mediastinal bleeding, sealing of esophageal anastomoses, and sealing of pulmonary staple or suture lines; neurosurgery applications, including dural repairs, microvascular surgery, and peripheral nerve repair; general surgery applications, including bowel anastomoses, liver resection, biliary duct repair, pancreatic surgery, lymph node resection, reduction of seroma and hematoma formation, endoscopy-induced bleeding, plugging or sealing of trocar incisions, and repair
• wound dressings may be fabricated with therapeutic bio-inks to provide delivery of a therapeutic agent at a site of injury, including, for example, anti- infectives, such as antibiotic, anti-fungal or anti-viral agents, anti-inflammatory agents, mitogens to stimulate cell growth and/or differentiation, agents to stimulate cell migration to the site of injury, growth factors, cells, such as osteoblasts, chondrocytes, keratinocytes, and hepatocytes, to restore or replace bone, cartilage, skin, and liver tissue, respectively, etc.
• anti- infectives such as antibiotic, anti-fungal or anti-viral agents, anti-inflammatory agents, mitogens to stimulate cell growth and/or differentiation
• mitogens to stimulate cell growth and/or differentiation
• agents to stimulate cell migration to the site of injury growth factors, cells, such as osteoblasts, chondrocytes, keratinocytes, and hepatocytes, to restore or replace bone, cartilage, skin, and liver tissue, respectively, etc.
• therapeutic agents may be added to the wound dressing after fabrication, e.g., by soaking, spraying, painting, or otherwise applying the therapeutic agent to the dressing.
• wound dressings may be fabricated directly at a desired location or may be pre-fabricated and applied to the wound.
• Wound dressings may be in the form of flat films that may be adhered to tissue to cover the site of an injury or may be in the form of 3-D structures, such as plugs or wedges.
• prosthetic tissue interfaces may be engineered which can be used, for example to put inside, around, beside another material such as metal to promote adhesion and ingrowth of tissue.
• Pre-fabricated wound dressings may be supplied in standard configurations suitable for application to a variety of wounds and may be applied as is or may be cut, molded, or otherwise shaped prior to application to a particular wound.
• pre-fabricated wound dressings may be produced in a configuration tailored to a specific wound or wound type.
• the wound dressing is supplied as a moist material that is ready for application to a wound.
• the wound dressing is supplied as a dried material which may be rehydrated upon or prior to application to a wound.
• the apparatus, methods, compositions, and products disclosed herein may be used to fabricate coatings for devices to be used in the body or in contact with bodily fluids, such as medical devices, surgical instruments, diagnostic instruments, drug delivery devices, and prosthetic implants.
• Coatings may be fabricated directly on such objects or maybe pre-fabricated in sheets, films, blocks, plugs, or other structures and applied/adhered to the device.
• Such coatings may be useful as a tissue-engineering scaffold, as a diffusion membrane, as a method to adhere the implant to a tissue, as a delivery method for a therapeutic agent, and/or as a method to prolong implant stability, e.g., by preventing or suppressing an immune response to the implant from the host.
• coatings may be applied to implantable devices, such as pacemakers, defibrillators, stents, orthopedic implants, urologiea! implants, dental implants, breast implants, tissue ' augmentations, heart valves, artificial corneas, bone reinforcements, and implants for maxillofacial reconstruction; devices such as percutaneous catheters (e.g., central venous catheters), percutaneous cannulae (e.g., for ventricular assist devices), catheters, urinary catheters, percutaneous electrical wires, ostomy appliances, electrodes (surface and implanted), and supporting materials, such as meshes used to seal or reconstruct openings; and other tissue-non-tissue interfaces.
• implantable devices such as pacemakers, defibrillators, stents, orthopedic implants, urologiea! implants, dental implants, breast implants, tissue ' augmentations, heart valves, artificial corneas, bone reinforcements, and implants for maxillofacial reconstruction
• devices such as percutaneous catheters (e.g., central venous
• a composition of the invention may be placed into a seeping wound to seal off the blood flow.
• wound plug or blood clotting applications may be particularly useful, for example, in battlefield applications.
• wound dressings may be fabricated to provide delivery of a therapeutic agent at a desired location.
• Therapeutic agents may be included in a coating as an ancillary to a medical treatment (for example, antibiotics) or as the primary objective of a treatment (for example, a gene to be locally delivered).
• a variety of therapeutic agents may be used, including passively functioning materials, such as hyaluronic acid, as well as active agents, such as growth hormones.
• a wide variety of therapeutic agents may be used, including, for example, cells, proteins (including enzymes, growth factors, hormones, and antibodies), peptides, organic synthetic molecules, inorganic compounds, natural extracts, nucleic acids (including genes, antisense nucleotides, ribozymes, and triplex forming agents), lipids and steroids, carbohydrates (including heparin), glycoproteins, and combinations thereof.
• the agents to be incorporated can have a variety of biological activities, such as vasoactive agents, neuroactive agents, hormones, anticoagulants, ⁇ mmunomodulating agents, cytotoxic agents, antibiotics, antivirals, or may have specific binding properties, such as antisense nucleic acids, antigens, antibodies, antibody fragments, or a receptor.
• the methods, compositions, and apparatus disclosed herein may be used to create 3-D capsules, tablets, structures, and matrices that deliver therapeutic agents including antibiotics, antivirals, antiinflammatories, both steroidal and non-steroidal, anti-neoplasties, anti-spasmodics, including channel blockers, modulators of cell-extracellular matrix interactions, including cell growth inhibitors and anti-adhesion molecules, enzymes and enzyme inhibitors, anticoagulants and/or antithrombotic agents, growth factors, DNA, RNA, inhibitors of DNA, RNA or protein synthesis, compounds modulating cell migration, proliferation and/or growth, vasodilating agents, and other drugs commonly used for the treatment of injury to tissue.
• therapeutic agents including antibiotics, antivirals, antiinflammatories, both steroidal and non-steroidal, anti-neoplasties, anti-spasmodics, including channel blockers, modulators of cell-extracellular matrix interactions, including cell growth inhibitors and anti-adhesion molecules, enzymes and enzyme inhibitors
• these compounds include angiotensin converting enzyme inhibitors, prostacyclin, heparin, salicylates, nitrates, calcium channel blocking drugs, streptokinase, urokinase, tissue plasminogen activator (TPA) and anisoylated plasminogen activator (TPA) and anisoylated plasminogen-streptokinase activator complex (APSAC), colchicine and alkylating agents, and aptamers.
• angiotensin converting enzyme inhibitors include angiotensin converting enzyme inhibitors, prostacyclin, heparin, salicylates, nitrates, calcium channel blocking drugs, streptokinase, urokinase, tissue plasminogen activator (TPA) and anisoylated plasminogen activator (TPA) and anisoylated plasminogen-streptokinase activator complex (APSAC), colchicine and alky
• modulators of cell interactions include interleukins, platelet derived growth factor, acidic and basic fibroblast growth factor (FGF), transformation growth factor beta (TGF-beta), epidermal growth factor (EGF), insulin-like growth factor, and antibodies thereto.
• FGF acidic and basic fibroblast growth factor
• TGF-beta transformation growth factor beta
• EGF epidermal growth factor
• nucleic acids include genes and cDNAs encoding proteins, expression vectors, antisense and other oligonucleotides, such as ribozymes which can be used to regulate or prevent gene expression.
• bioactive agents include modified extracellular matrix components or their receptors, and lipid and cholesterol sequestrants.
• therapeutic agents which may be used in conjunction the compositions and methods of the invention include proteins, such as biological response modifiers including cytokines, interferons and interleukins, proteins, and colony-stimulating factors.
• proteins such as biological response modifiers including cytokines, interferons and interleukins, proteins, and colony-stimulating factors.
• Carbohydrates including sialyl Lewis antigen which has been shown to bind to receptors for selectins to inhibit inflammation.
• a "deliverable growth factor equivalent” (DGFE), a growth factor for a cell or tissue, may be used, which is broadly construed as including growth factors, cytokines, interferons, interleukins, proteins, colony-stimulating factors, gibberellins, auxins, and vitamins; further including peptide fragments or other active fragments of the above; and further including vectors, i.e., nucleic acid constructs capable of synthesizing such factors in the target cells, whether by transformation or transient expression; and further including effectors which stimulate or depress the synthesis of such factors in the tissue, including natural signal molecules, antisense and triplex nucleic acids, and the like.
• DGFE deliveryable growth factor equivalent
• Exemplary DGFE's are VEGF, ECGF, bFGF, BMP, and PDGF 3 and DNA's encoding for them.
• Exemplary clot dissolving agents are tissue plasminogen activator, streptokinase, urokinase, and heparin.
• drugs having antioxidant activity may be used, which are useful, for example, in the prevention of adhesions.
• examples include superoxide dismutase, or other protein drugs include catalases, peroxidases, and general oxidases or oxidative enzymes, such as cytochrome P450, glutathione peroxidase, and other native or denatured hemoproteins.
• mammalian stress response proteins or heat shock proteins such as heat shock protein 70 (hsp 70) and hsp 90, or those stimuli which act to inhibit or reduce stress response proteins or heat shock protein expression, for example, flavonoids, also may be used.
• the pharmaceutical composition or the therapeutic agent for overactive bladder of the present invention may be used, administered or produced in a single preparation or a combination of preparations so far as the preparations are formulated so as to contain the respective active ingredients, Compound (I) or a pharmaceutically acceptable salt thereof and an anticholinergic agent.
• the pharmaceutical composition or the therapeutic agent for overactive bladder has a unit dose form suitable to oral administration, such as tablets or capsule, or has a unit dose form suitable to parenteral administration, such as injections.
• preparations are used or administered as a combination of preparations, they may be used or administered together or separately at an interval.
• the compositions of the invention comprise a polymer matrix that forms a tablet, capsule, or biodegrable implant suitable for oral or parenteral delivery of a therapeutic or diagnostic agent.
• a polymer matrix that forms a tablet, capsule, or biodegrable implant suitable for oral or parenteral delivery of a therapeutic or diagnostic agent.
• Such compositions are especially useful where degradation of the capsule or matrix is in response to the local cellular or physiological mileu, e.g., temperature, pH, sites of inflammation and/or necrosis, for example.
• the compositions of the invention may include a diluent, excipient, disintegrator, lubricant, binder, surfactant, water, physiological saline, vegetable oil solubilizer, isotonizing agent, preservatives, antioxidants etc., in addition to the therapeutic or diagnostic agent.
• Tablets and capsules may include excipients, such as lactose, disintegrators, such as starch, lubricants , such as magnesium stearate, binders, such as hydroxypropyl cellulose, surfactants, such as fatty acid ester, and plasticizers such as glycerin.
• excipients such as lactose, disintegrators, such as starch, lubricants , such as magnesium stearate, binders, such as hydroxypropyl cellulose, surfactants, such as fatty acid ester, and plasticizers such as glycerin.
• the structures disclosed herein may be characterized with respect to mechanical properties, such as tensile strength using an Instron tester, for polymer molecular weight by gel permeation chromatography (GPC), glass transition temperature by differential scanning calorimetry (DSC), and bond structure by infrared (IR) spectroscopy, with respect to toxicology by initial screening tests involving Ames assays and in vitro teratogenicity assays, and implantation studies in animals for immunogenicity, inflammation, release, and degradation studies.
• mechanical properties such as tensile strength using an Instron tester, for polymer molecular weight by gel permeation chromatography (GPC), glass transition temperature by differential scanning calorimetry (DSC), and bond structure by infrared (IR) spectroscopy, with respect to toxicology by initial screening tests involving Ames assays and in vitro teratogenicity assays, and implantation studies in animals for immunogenicity, inflammation, release, and degradation studies.
• the processes and compositions disclosed herein may be used in situ or ex vivo to manufacture a tissue engineered construct to control angiogenesis.
• the process may be used to fabricate a biomimetic extracellular matrix (bECM) of fibrin incorporating a recombinant human fibroblast growth factor-2 (FGF-2).
• bECM with FGF-2 may be used to induce controlled angiogenesis.
• a fibrin-based bECM design with or without gradients of FGF-2 targeted for angiogenesis is used in bone tissue engineering.
• Angiogenesis is a requisite for osteogenesis and successful incorporation of tissue engineered bone grafts.
• a broad range of biopolymer matrix materials and components targeted for different tissues may be applicable.
• a bECM delivery of growth factors such as the bone morphogenetic proteins, cells or both, provide structural support, cues, and surfaces for cell attachment.
• growth factors such as the bone morphogenetic proteins, cells or both
• examples include seeding and culturing bECMs with ECs and other cells, such as stem cells, in vitro seeding and culturing structured bECMs, which have networks of channels, with hepatocytes and other cell types in vitro seeding ECs and other cells into micromachined branched channels, cultured in vitro,, and the resulting layers are folded into 3-D structures; seeding bECMs with cells transfected with a recombinant retrovirus encoding VEGF; and incorporation of VEGF-A 165 or FGF-2 in bECMs by entrapment, adsorption, microcarriers or immobilized to matrices by covalent bonding.
• a process of forming a scaffold includes incorporating fibrin
• Cells, GFs, and an ECM are fundamental tissue building blocks. Functional roles for each of these building blocks in homeostasis and wound repair guide the tissue engineering designs.
• Angiogenesis is a reoccurring theme in homeostasis and wound repair. As a consequence of the powerful role angiogenesis has on wound repair, the apparatus, compositions, and methods disclosed herein provide for tissue-engineered therapies. Without angiogenesis, tissues with a volume exceeding_a few cubic millimeters cannot survive by diffusion of nutrients and oxygen.
• Angiogenesis occurs under specific spatial and temporal control. It has been suggested that temporal release of VEGF and platelet derived growth factor-BB (PDGF-BB) from a bECM effectively enhances neovessel formation. It is believed that VEGF promotes chemo attraction, mitogenesis, and differentiation of endothelial cells and that PDGF enhances smooth muscle cell development for neovessels.
• PDGF-BB platelet derived growth factor-BB
• a bECM may be constructed that delivers an angiogenic factor, and, thus, fulfills several biological criteria to support wound repair.
• the angiogenic factor can be spatially localized, protected, and delivered in a controllable and predictable manner by the bECM.
• Angiogenic factors such as VEGF, FGF-2 and PDGF are typically delivered endogenously in soluble forms. Moreover, diffusion and convective flow at the wound implant site which could "wash out” and dilute the factors should be avoided. Increasing the administered doses could mitigate such effects but would be problematic due to potential systemic side-effects. Therefore, factors should be tethered or entrapped within the bECM so that the pharmacokinetics will be sufficiently predictable angiogenesis and subsequent tissue repair.
• the bECM/FGF-2 developed in accordance with the methods, compositions, and apparatus disclosed herein provide an organized functional platform for normal vessel formation. Using gradients, pores, and structural forms disclosed herein the FGF-2 can be incorporated throughout the bECM, and the release of FGF-2 can be regulated by, for example, spatial concentration gradients, and/or released during cellular ingrowth and biodegradation. Therefore, neovessel formation is directed and organized.
• the methods, compositions, and apparatus disclosed herein may be used in bone tissue engineering. Since angiogenesis and osteogenesis are linked, there is a strong correlation between recipient site vascularity and bone graft viability. Recent studies with knockout mice for VEGF underscore the interrelationship between angiogenesis and bone.
• the initial phase of bone graft healing includes chemotactic and chemokinetic signals (e.g., VEGF, PDGF, FGF-2) directing angiogenesis within the fibrin clot.
• chemotactic and chemokinetic signals e.g., VEGF, PDGF, FGF-2
• spatial and temporal patterns of GFs required for angiogenesis and osteogenesis also are required to regulate mitogenesis, cell shape, movement differentiation, protein secretion, and apoptosis.
• the relatively predictable and organized set of cellular and molecular events during bone regeneration provide a mechanism for creating a controlled spatial gradient of an angiogenic factor in the bECM for bone tissue engineering.
• an angiogenic factor in the bECM for bone tissue engineering.
• ECs endothelial cells
• FGF-2 vascular endothelial growth factor
• a fibrin-based bECM may include two or more angiogenic molecules, including, for example, FGF-2 and PDGF.
• Such bECMs comprising FGF-2 and PDGF are useful, for example, to regenerate healing of critical-sized defects (CSD).
• a tissue engineered design of a calvarial CSD has a gradient that increases from the bottom to the top of the structure. When such structure is placed into a CSD defect, the gradient encourages migration of cells in an upward direction toward the region having a higher growth factor concentration. The ⁇ temporal migration of cells could also be controlled using a decreasing porosity gradient from the bottom to the top (e.g., the top is less porous than the bottom).
• a thick or non-porous layer in one or more areas of the scaffold to prevent cell migration in a certain direction.
• Such layers act as guides for the formation of tissue along predetermined shapes or axes.
• a tissue engineered design of a calvarial CSD has a gradient of immobilized FGF-2, with concentrations higher in the center of the bECM, gradually decreasing from the center to the periphery to optimize chemoattractant and mitogenic effects that guide controlled neovessel formation.
• the PDGF at the center of the bECM promotes recruitment of smooth muscle cells to stabilize the neovessels.
• temporal control may be achieved through a spatial arrangement of PDGF and FGF-2.
• spatial variations of fibrin porosity also modulate temporal patterns.
• the fibrin microstructure determines the torturosity of the 3-D matrix, and manipulation of torturosity affects the bECM mechanical properties, the rate of invading cell migration, proteolysis, and growth factor availability. An increase in the fibrin compliance promotes EC differentiation in vitro.
• the concentration range, direction, and shape for the gradient design may be determined by the biological properties of the wound. CSD studies have reported a significant quantitative difference in osteogenic cell sources for peripheral bone, dural, and subcutaneous cell sources.
• VEGF vascular endothelial growth factor
• FGF-2 may be used because it is angiogenic and osteogenic.
• FGFs are mitogenic polypeptides implicated in embryonic development, angiogenesis, regeneration, and wound healing.
• acidic and basic FGFs, FGF-I, and FGF-2 are used for therapeutic applications for angiogenesis and bone formation.
• these isoforms instigate a vasodilatory effect, mediated perhaps by an intracellular calcium-nitric oxide loop. This beneficial hemodynamic effect, as well, as the angiogenic capacity of FGFs merit enthusiasm as an angiogenic factor for a tissue engineered therapy.
• FGF-2 may be used for the positive affects of FGF-2 on bone formation and fracture healing.
• microencapsulation of biological factors by degradable polymer microspheres is a popular approach in tissue engineering. Accordingly, in certain exemplary embodiments, microencapsulation may be used to control the release of diffusible molecules over time, producing a transient diffusion gradient to regulate cell response. In other embodiments,
• FGF-2 may be immobilized with tissue transglutaminase (tTG).
• tissue transglutaminase tTG
• Specific binding of the FGF- 2 to the bECM i.e., FGF-2 in the solid-phase
• FGF-2 in the solid-phase provides maintenance of spatial patterns.
• Many GFs sustain residence in native ECMs through specific binding patterns.
• the methods disclosed herein provide bulk fabrication techniques, as well as, methods of spatial patterning. The binding interactions determine GF availability and influence receptor binding, and, therefore, significantly impact cell responses.
• hydrogels may be used as a precursor to form the structural scaffold.
• Suitable hydrogels include, for example, fibrin, chitosan, collagen, alginate, poly(N-isopropylacrylamide), and hyaluronate, which can be deposited and gelled with the aid of a second component that modulates cross-linking, pH, ionic concentration, or by photopolyrnerization, or temperature increase with body contact.
• fibrin may be used. During wound healing, fibrin provides a foundational substratum for wound healing and angiogenesis.
• Fibrin results when circulating plasma fibrinogen becomes localized in a wound and following a cascade of coagulation events is finally proteolytically cleaved by thrombin and self-assembles into an insoluble fibrin network. Following this gelation event, the interconnecting fibrin fibers become stabilized by interfibril cross-linking catalyzed by transglutaminase Factor XIII (FXIII). From the plasma and platelet degranulation, a range of GFs, cell attachment molecules, proteases, and blood cell components become immobilized and entrapped within the fibrin matrix. Fibrin properties can be controlled for degradation rate and porosity.
• a fibrin bECM can be modified with GFs, osteoconductive bioceramics, and plasmids, so as to expand clinical versatility.
• Fibrin is known to bind with high affinity to FGF-2. Fibrin has demonstrated excellent biocompatibility in clinical applications.
• other hydrogels or composites of these hydrogels may be used.
• Gelation rate, structure, and material properties of fibrin polymers are determined by relative concentrations of fibrinogen and thrombin, pH, ionic strength, and other biophysical parameters present during fibrin polymerization.
• fibrinogen concentration directly affects fibrin gel strength, as does cross-linking of the fibrin gel with FXIII, which also protects fibrin from plasmin proteolysis, thus, modulating bECM degradation.
• the resulting 3-D microstructural properties of the fibrin gel play a decisive role in EC migration, proliferation, and angiomorphogenesis.
• FGF-2 and VEGF stimulation of migration is enhanced by more rigid or less porous fibrin gels, whereas capillary morphogenesis is enhanced by less rigid or more porous gels.
• fibrinogen, thrombin, FGF-2, tissue transglutaminase (tTG), and dilutant buffers are combined to form the biopolymer matrix, hi certain embodiments, pH and ionic strength are held constant in 100 mM Tris buffer, pH 7.0, containing 150 mM NaCl, and 5 mM CaCl.
• biopolymer matrices in their simplest form consist of fibrinogen and thrombin. These two components form the base for both a native thrombus formation and commercial fibrin glue.
• the addition of TGs crosslinks fibrin fibrils can be used to stabilize the fibrin polymer, thereby improving mechanical properties.
• TGs are Ca 2+ -dependent enzymes that catalyze post-translational modification of proteins through the formation of ⁇ -glutamyl- ⁇ -lysine cross-links between polypeptide chains.
• Plasma FXIII is activated by thrombin and is primarily associated with the covalent cross- linking of fibrin fibrils. A stronger clot is produced with FXIII.
• tTG is widely distributed in cells and tissues and does not require proteolytic activation. TGs impart fibrolytic resistance by cross-linking ⁇ 2-antiplasmin to fibrin fibrils and by cross-linking the fibrin ⁇ -and/or ⁇ - chains.
• TGs have a broad range of substrate proteins including fibrinogen/fibrin, fibronectin, plasminogen activator inhibitor-2, ⁇ 2-antiplasmin, IGF binding protein-1, osteonectin, ⁇ - casein, collagen, laminin, and vitronectin.
• substrate proteins including fibrinogen/fibrin, fibronectin, plasminogen activator inhibitor-2, ⁇ 2-antiplasmin, IGF binding protein-1, osteonectin, ⁇ - casein, collagen, laminin, and vitronectin.
• tTG is preferred because it does not require thrombin activation, is readily available, and because it is a factor in osteogenesis.
• Human plasminogen- free fibrinogen and human thrombin may be purchased from Enzyme Systems Research Laboratories (South Bend, Ind.), tTG from Sigma (St Louis, Mo.), and human recombinant FGF-2 from ReproTech, Inc. (Rocky Hill, NJ.). Such materials are also available from GMP facilities and FDA approved sources.
• fibrinogen is prepared at concentrations in the range of 4-75 mg/ml. Four mg/ml is the concentration of native fibrin clots, and up to 130 mg/ml is used in commercially available fibrin glue formulations, such as TIS SEELTM. Thrombin concentrations between 1 to 50 NIH units/ml can be tested to modify gelation time, fibrin fibrillar diameter and porosity.
• biopolymer may comprise of FGF-2 concentrations between 1-12 ng/ml.
• biopolymer formation can occur at 23 0 C.
• Protein-based compositions may be stored at -70 0 C or freeze-dried prior to use to maintain viability.
• Biopolymers can be formed using the methods as known in the art and described herein.
• the system can be validated using immunohistochemical staining and SEM and/or fluorescent microscopy.
• the persistence of FGF-2 can be validated using fluorescence-labeld FGF-2 and 125 I labled FGF-2 labeling incorporated into the biopolymer matrix.
• fibrinogen biopolymer can be conjugated to Cy5 and mixed with unlabeled fibrinogen (5% volrvol to unlabelled fibrinogen).
• FGF-2 can be conjugated to Cy3 and mixed with unlabeled FGF-2 (5% vol: vol to unlabelled FGF-2).
• Subsequent confocal microscopy can be performed using a Zeiss confocal LSMl 0 microscope equipped with 5 mW Ar 488/514 nm and a 5 mW He/Ne 633 nm lasers.
• a Zeiss Plan-Neofluar 20x0.5 NA water immersion objective can be used to image sections in l ⁇ m, or better, increments. Images can be processed using Zeiss LSM software.
• Persistence of FGF-2 can be measured by immediately fixing or placing the construct in excess phosphate buffered saline, pH 7.4 (PBS), containing 0.02% sodium azide for various times (0, 0.5, 1, 4, 8, 24, 72 hrs) at 23°C using time 0 as the control.
• PBS phosphate buffered saline
• radiolabeled 125 I-FGF-2 can be used.
• biopolymers comprising FGF-2 can be placed in 24 well tissue culture plates for 3 H-thymidine assay.
• Human umbilical ECs (HUVECs) can be purchased from Clonetics (BioWhittaker, Inc., Walkersville, Md.) and maintained according to supplier's instructions. Cells can then be grown 70% confluence and seeded onto the biopolymer at 20,000 cells/well in serum-free media. After 48 hr culture, 0.5 ⁇ Ci 3 H -thymidine can be added to the wells. After overnight culture, biopolymers can be trypsinized to dissolve the biopolymer matrix and cells can be washed with PBS. Subsequent 3 H-thymidine incorporation into the growing cells can be determined by standard protocols known in the art.
• Statistical analyses can be performed using multiple analysis of variance (ANOVA) and Tukey's post-hoc test for multiple comparison analysis, with a significance level of p ⁇ 0.05.
• microstructure porosity, fibril diameter
• SEM scanning electron microscopy
• fluorescence confocal microscopy Patterns and concentrations of therapeutic factors may be determined by fluorescence microscopy using direct fluorescent labeling and immunofluorescence.
• tTG cross- linking of FGF-2 can be used.
• a broad range of substrate proteins for FXIII and tTG have been identified, including fibrinogen/fibrin, fibronectin, plasminogen activator inhibitor-2, ⁇ 2-antiplasmin, IGF binding protein- 1, osteonectin, P-casein, collagen, laminin, and vitronectin.
• substrate proteins for FXIII and tTG include fibrinogen/fibrin, fibronectin, plasminogen activator inhibitor-2, ⁇ 2-antiplasmin, IGF binding protein- 1, osteonectin, P-casein, collagen, laminin, and vitronectin.
• different TGs or FXIII may be used.
• FGF-2 maybe cross-linked to a dilute solution of fibrinogen prior to formulation FGF-2 specifically binds fibrinogen via standard reaction using BS 3 (a water soluble bis(sulfosuccinimidyl) suberate) from Pierce (Rockford, 111.).
• This cross-linker may be used to immobilize IGF-T to metal surfaces and it is biocompatible.
• an oligoglutamine moiety maybe coupled onto FGF-2 via BS 3 .
• the exact nature of the binding region can be tailored to maximize its reactivity; for example, chain length and composition can be altered.
• Various oligopeptides can be synthesized which are rich in both glutamine and the facilitating amino acids.
• Cross-linking heparin to fibrinogen or fusion peptides using TG substrate sequences may be utilized.
• Engineered peptides, fusion proteins, and other such molecules may also be used to promote attachment of therapeutic agents, such as drugs, growth factors, etc., to matrix components either directly as a fusion protein (i.e., a growth factor with a TG substrate component without a protease cleavage site) or an engineered peptide (i.e., such as a heparin binding domain sequence with a TG substrate sequence that may be used to immobilize heparin to serve as a generic binder for proteins containing heparin binding domains).
• a fusion protein i.e., a growth factor with a TG substrate component without a protease cleavage site
• an engineered peptide i.e., such as a heparin binding domain sequence with a TG substrate sequence that may be used to immobilize heparin to serve
• structures can be assayed in vitro or via the CAM model by immediately placing the construct in serum-free media containing 50 ⁇ g/ml BSA (Insulin RIA grade, Sigma, St. Louis, Mo.) and 1 ⁇ g/ml aprotinin at 23 0 C. These samples can be incubated with media changes so as to remove unbound growth factors such as FGF-2. Holding the temperature at 23 0 C and the addition of a protease inhibitor, such as aprotinin, helps stabilize the biopolymer structure.
• BSA Insulin RIA grade, Sigma, St. Louis, Mo.
• tissue-engineered constructs The effectiveness of tissue-engineered constructs is often evaluated in the art in vitro prior to assessment in vivo. In vitro results may not directly translate to in vivo results. However, compared to in vivo experimentation, in vitro experimentation is associated with reduced expense, increased experimental turnover rates, and more selective control of associated variables. These considerations support in vitro experimentation in the tissue engineering design process.
• CAM chorioallantoic membrane
• Angiogenesis and the CAM have become an important in vivo biological assay to screen therapies for wound repair and blood vessel development.
• CAM will be used to assess angiogenesis in response to the fibrin /FGF- 2 designs.
• a cutting device is used to make a 17 mm diameter hole in the horizontal center of eggs.
• An optically clear plastic insert (15 mm OD xl 0 mm ID) can be used to create windows for focused treatment application and subsequent in situ assessment. Placing sample constructs of smaller size than the insert provides a border region surrounding the construct within the viewing window allows in situ observation of the directed vascular ingrowth.
• the CAM assay consists of incubating fertilized White Leghorn eggs at 37.8°C in 60% relative humidity. On day three, eggs are opened using a mid-horizontal orientation in the cutting device. Removal of 0.5 ml of albumin from the large end of the egg prior to cutting drops the embryo from the cutting site, protecting it from vibration and surgical trauma. Porous medical tape placed over the hole minimizes evaporative loss and prevents contamination. On day 8, window inserts are placed through the hole and rest directly on the CAM.
• a construct can be placed in the CAM on day 10. In situ imagining allows a digitally recording for image processing to be made from days one through eight.
• the construct placed into the CAM inserts can be recovered at this time and prepared for histological analyses of angiogenesis. Embryos, membranes, and the construct can be fixed in ovo in Bouin's fluid.
• the window/CAM area can then be removed, dehydrated, and embedded in paraffin.
• Serial sections can be made in a plane parallel to the CAM surface. Sections can be stained using 0.5% toluidene blue.
• Angiogenesis can be evaluated with a Zeiss Axiophot microscope interfaced with an image analysis system using Zeiss imaging software.
• Quantitative data can be analyzed by multiple analysis of variance (ANOVA) and Tukeys post-hoc test for multiple comparison analysis at a level of significance of p ⁇ 0.05.
• ANOVA analysis of variance
• Tukeys post-hoc test for multiple comparison analysis at a level of significance of p ⁇ 0.05.
• Example 1 Fibrin processing.
• a fibrinogen formulation for a 1 mL batch was prepared by mixing a solution comprising 250 ⁇ L of 40 mg/mL Aventis or diaPharma fibrinogen (commercially available from ZBL Behring, King of Prussia, PA; or diaPharma, West Chester, OH respectively), 230 ⁇ L of sterile water, 300 ⁇ L of IM NaCl (Sigma, St. Louis, MO), 200 ⁇ L of 200 mM bicine, pH 8.0 (Sigma), 10 ⁇ L of 100 U/mL Factor XIII (ZBL Behring, King of Prussia, PA), and 10 ⁇ L of 10 mg/mL bone morphogenetic protein 2 (BMP -2) (R&D Systems, Minneapolis, MN).
• BMP -2 bone morphogenetic protein 2
• a thrombin formulation for the 1 mL batch was prepared by mixing a separate solution comprising 100 ⁇ L of 100 U/mL thrombin (Enzyme Research Labs, South Bend, IN) and 10 ⁇ L of 500 mM CaCl 2 (Sigma).
• the fibrin gels were prepared by adding 15 ⁇ L of the thrombin solution to a 1.5 mL microfuge tube, adding 350 ⁇ L of the fibrinogen solution to the microfuge tube, and immediately transferring the mixed solution to a gel mold. The gel solution was then allowed to set for 20-30 minutes in the gel mold to allow for complete gelation. After 20-30 minutes when the gel solution has completely set, the molds were disassembled and the resulting fibrin films removed.
• the gel molds were constructed of four components: bottom pieces, 0.75mm top pieces, 1.5mm top pieces, and standard binder clips.
• Bottom pieces comprise 2.5cm x 2.5cm x 0.63 cm TEFLONTM segments
• 0.75mm top pieces comprise 2.5cm x 2.5cm x 0.075cm TEFLONTM square segments with 12.5mm x 12.5mm square segments cut out and removed from the center
• 1.5mm top pieces comprise 2.5cm x 2.5cm x 0.15cm TEFLONTM square segments with 12.5mm x 12.5mm square segments cut out and removed from the center.
• the gel molds were assembled by placing one 1.5 mm top piece on top of a bottom piece, and placing one 0.75 mm top piece on top of the one 1.5 mm top piece with the edges of the segments aligned. The segments were clamped together with the binder clips.
• the gel mold assemblies were placed inside a 100 mm Petri dish with a wet tissue (commercially available from Kimberly-Clark, Roswell, GA) and with a closed lid to create a humidified chamber. It was found that fibrin gel structure (i.e., fibrin fibril formation) is highly dependent on the pH, NaCl concentration, CaCl 2 concentration, and fibrinogen concentration. Therefore, modification of these concentrations and volumes in the initial gel formulation imparted different properties to the final film. Accordingly, on skilled in the art could modify the initial gel formulation to prepare films with structures and properties that are suitable for their particular applications while still being within the scope and spirit of the present invention.
• the fully-hydrated fibrin gels were lyophilized in order to remove water and reduce the thickness of the fibrin gel films from 2.25 mm to approximately 100 ⁇ m. Lyophilization was performed in a gel dryer (commercially available from BioRad, Hercules, CA). The lid of the gel dryer was opened and the silicon rubber gasket was peeled back. Spectrapor 1 dialysis tubing (6-8k MWCO, Spectrum Laboratories, Collinso Dominguez, CA) was soaked in PBS and cut along one side to allow the tubing to be opened up into a sheet. The opened tubing had 3cm x 3cm pieces cut into it. The resulting dialysis "sheets" were placed onto the gel dryer.
• the fibrin gel was placed in the center of the dialysis sheet.
• a 2.5cm x 2.5cm x 3cm TEFLONTM segment with a 20mm x 20mm square cut out of the center was placed around the gel.
• a 2.5cm X 2.5cm x 0.075cm Teflon segment was placed on top of the 2.5cm x 2.5cm x 3cm TEFLONTMsegment.
• the silicon rubber gasket was carefully repositioned and the gel dryer lid closed.
• a lyophilizer unit (Labconco, Kansas City, MO) was activated and allowed to reach reduced pressure and temperature. The lyophilizer unit was then connected to the gel dryer.
• the gel was dried into a film using one of two alternative methods: (1) the gel dryer was run at 50 0 C for 2-2.5 hours, or (2) the gel dryer was run at room temperature for 8-10 hours.
• the dialysis sheet with the attached fibrin film was carefully removed and immediately placed in a 6 well microwell plate containing 3 mL PBS with 10 units/mL penicillin G sodium, 10 ⁇ g/mL streptomycin sulfate solution (invitrogen, Carlsbad, CA), and 10 ⁇ M D-Phe-Pro-Axg-chloromethylketone (PPACK) thrombin inhibitor (BIOMOL
• Lyophilized film was allowed to sit for 24 hours at 4°C. Every 24 hours the buffer was replaced with fresh, sterile PBS, but only for a total of 2 buffer exchanges maximum. The films were stored at 4°C until further processing or analysis was performed.
• the lyophilization process produced films that were transparent to translucent in appearance and possessed elastomeric properties as demonstrated in FIG. IB.
• Example 2 An alternative method to that disclosed in Example 2 was also used to process the fibrin gels upon gelation and removal from the gel molds, m this method, an osmotic dehydration process was used (based upon the method of Muller and Ferry, US Patent No. 4,548,736).
• Fibrin gels were prepared the same as in the method of Example 1. The fibrin gels were placed on a coverslip inside a 60 mm Petri dish. Approximately 500 ⁇ L of a 35% high molecular weight polyvinyl alcohol solution was added to the inside of an approximately 3inch segment of SPECTRAPOR-1TM (6-8,000 MWCO) dialysis membrane tubing soaked in PBS. The ends of the tubing were clamped off.
• the tubing was placed on top of the gel making sure the polyvinyl alcohol solution rested directly on top of, and in complete contact with, the entire gel to ensure that water evenly diffused from the gel and entered into the tubing along the osmotic gradient.
• a lid was placed on the Petri dish and it was incubated at room temperature for 24 hours.
• the resulting fibrin film was removed from the dish and soaked in a 50% glycerol solution for 24 hours at room temperature followed by storage at 4°C in a PBS solution.
• This method involved longer processing time and occasional inhomogeneities in film thickness due to occasional lack of even osmosis. However, this method was advantageous with respect to the relatively mild treatment of the film and because the film never completely dried out.
• the fibrin films fabricated according to the method described in Example 1 and either of Examples 2 or 3 were transparent to translucent in appearance and possessed elastomeric properties.
• Example 4 Fibrin film biocompat ⁇ bility assay.
• Example 5 Fibrin film biocompatibility assay.
• Fibrin gels were prepared with 10 mg/mL diaPharma fibrinogen (commercially available from diaPharma, West Chester, OH), 300 mM NaCl 3 40 mM bicine pH 8.0, and further comprising 10 ng/mL fibroblast growth factor 2 (FGF-2) and processed into films as described in Example 1, and either of Examples 2 or 3. These films were placed on 10-day old chick CAMs. Twenty-four hours later bleeding could be seen around blood vessels under the films. Without being bound by theory, the observed bleeding was most likely due to the very high concentration of FGF-2 as a result of the approximately 20-fold decrease in gel thickness observed upon lyophilization. Final FGF-2 concentrations in the film were estimated to be approximately 200 ng/mL.
• VEGF 165 vascular endothelial growth factor
• Example 6 2-D inkjet tissue printing system and BMP -2 layering on fibrin films.
• Fibrin films were prepared with 11 mg/mL diaPharma fibrinogen, 1 U/mL Factor XlIl, 300 mM NaCl, and 40 mM bicine, pH 8.0.
• 4 square patterns of Cy3-labeled BMP -2 was printed on top of the film in a grid pattern as shown in FIG. 4.
• the film was submerged in PBS and stored at 4°C.
• the media was changed daily and the film imaged for Cy3 fluorescence over the course of one week using a M2BIO stereomicroscope with a 1.6X objective (IX zoom), a Retiga Exi CCD camera, and a standard Cy3 filter set.
• the film exhibited little loss of fluorescence over the 7 days of the evaluation indicating stable BMP-2 incorporation into the film.
• Example 7 Scanning electron, microscopy (SEM) of fibrin films.
• Fibrin gels were prepared with 11 mg/mL diaPharma fibrinogen, 1 U/mL Factor XIII, 300 mM NaCl 5 and 40 mM bicine, pH 8.0) and processed into films. These films were cut into quarters and processed for scanning electron microscopy (SEM). Specimens were fixed in 1% glutaraldehyde and 3% paraformaldehyde in PBS overnight. Subsequently, after 3 washes of PBS, the specimens were fixed for one hour in 1 % OsO 4 buffered with PBS.
• SEM scanning electron microscopy
• the Os ⁇ 4 was removed with three five-minute washes of ddHDjO, followed by dehydration in an ascending series of ethanol (50%, 70%, 80%, 90%, and 3 changes of 100%) The samples were held in each ethanol wash for 10 minutes and then held in 100% ethanol overnight.
• the specimens were dried in a Pelco CPD2 critical point dryer (Clovis, CA) using CO 2 at 1200 psi and 42°C. Dried specimens were attached to SEM stubs using double stick tape and coated with gold using a Pelco SC-6 sputter coater. Specimens were examined using a Hitachi 2460N Scanning Electron Microscope (Pleasanton, CA). Digital images were obtained using Quartz PCI Image software (Vancouver, BC, Canada).
• FIG. 5 shows SEM images of the film surface and cross-section.
• the surface views show a somewhat smooth surface with a dense fiber-like packing (FIG. 5B). Some surface cracks and wrinkles are induced by the fixation and critical point drying steps (FIG. 5A, 5C).
• the cross-sectional views confirm this. Fibrin fibrils were visible, indicating that film processing does not destroy native fibrin structure (FIG. 5D-F).
• the high density of fibrils was a result of the 20-fold reduction of fibrin hydrogel thickness as a result of the lyophilization or osmotic dehydration processing of Examples 2 or 3 respectively.
• Example 8 Transmission electron microscopy (TEM) of fibrin films.
• TEM Transmission electron microscopy
• Fibrin gels were prepared with 11 mg/mL diaPharma fibrinogen, 1 U/mL Factor XIII, 300 mM NaCl, and 40 mM bicine, pH 8.0) and processed into films. These films were cut into quarters and processed for transmission electron microscopy (TEM). Specimens were fixed in 1% glutaraldehyde and 3% paraformaldehyde in PBS overnight. After 3 washes of PBS, the specimens were fixed for one hour in 1% OsO 4 buffered with PBS. After osmium fixation, sections were washed with three changes of ddEbO and dehydrated with an ascending series of ethanol (50%, 70%, 80%, 90% and 100%).
• TEM transmission electron microscopy
• Propylene oxide was used as a transitional solvent, and the specimens infiltrated with LR White (London Resin Company, Reading, Berkshire, England). The LR White was polymerized at 60 0 C for 48 hours. Thin (lOOnm) sections were cut using a diamond knife on a Reichert-Jung Ultracut E ultramicrotome (Leica, Wetzlar, Germany), placed on copper grids, and stained with uranyl acetate, and lead citrate. The grids were viewed on a Hitachi H-7100 transmission electron microscope (Pleasanton, CA) operating at 50 kV. Digital images were obtained using an AMT Advantage 10 CCD Camera System (Advanced Microscopy Techniques Corporation, Danvers, MA) and NIH Image software (Bethesda, MD).
• FIG. 6 shows a TEM micrograph of the fibrin film.
• the fibrin appears random and homogeneous. Pore size is very small and in the nanometer range, which is expected as a result of the high fibrin density within the film.
• Example 9 Fibrin film formulation further comprising tricalcium phosphate.
• Fibrin gel films were prepared by the method described in Examples 1 and 2, further comprising the addition of tricalcium phosphate (0.4 mg of TCP powder per 400 uL of fibrinogen solution). The films were qualitatively evaluated for handling properties and found to handle well. [Inventors: please describe the properties that were observed or tested] Thin films comprising TCP have potential for bone tissue engineering applications.
• Example 10 MD-63 Osteosarcoma cell seeding on fibrin films.
• Fibrin gel films were prepared with 11 mg/mL diaPharma fibrinogen, 1 U/mL Factor XIII, 300 mM NaCl, and 40 mM bicine, pH 8.0 and processed to films by the method described in Example 2.
• MG-63 osteosarcoma cells in modified Eagle's medium F-12 (MEM F-12) supplemented with serum did not adhere or spread out on these films. Cells were bound to the films but stayed rounded. Without being bound by theory, it was believed that thrombin remaining in the gel prior to dehydration was concentrated and actually 7 inhibited cell attachment through proteolytic activity once films were placed under in vitro conditions.
• Example 11 Extracellular matrix incorporation into fibrin films.
• Gels were prepared with 11 mg/mL diaPharma fibrinogen, 1 U/mL Factor XIII, 300 mM NaCl, and 40 mM bicine, pH 8.0 according to the method described in Examples 1 and 2, and further comprising lyophilized and powdered extracellular matrix (ECM) preparations.
• ECM extracellular matrix
• Gels were prepared with 11 mg/mL diaPharma fibrinogen, 1 U/mL Factor XIII 5 300 mM NaCl, and 40 mM bicine, pH 8.0 and processed into films according to the method described in Examples 1 and 2. Films were subsequently treated in one of the following ways: (1) 26 ⁇ M PPACK thrombin inhibitor or (2) 20% formamide solution containing 26 ⁇ M PPACK. These films were then submerged in PBS or submerged in 1 mg/mL fibrinogen followed by 1 U/mL thrombin to form an additional fibrin layer on the film surface. MG-63 osteosarcoma cells were placed on top of these films in 6 well microplates containing MEM- Fl 2 media supplemented with serum. The results were as follows:
• Example 13 Fibrinogen source testing.
• Fibrin gels were prepared with 10 mg/mL Aventis fibrinogen, 1 U/mL Factor XIII, 300 mM NaCl, 40 mM bicine, pH 8.0 and processed into films according to the method described in Examples 1 and 2. Films were then post-treated with either PBS alone or PBS containing 23 ⁇ M PPACK, 10 units/mL penicillin G sodium, and 10 ⁇ g/mL streptomycin. Films were further post-treated in either PBS or PBS containing 1 mg/nxL fibrinogen followed by 1 U/mL thrombin. MG-63 cells were placed on films in 6 well microplates containing MEM-F 12 supplemented with serum and antibiotic. Cells attached to, and spread on, both sets of films. Due to these results, combined with those of Examples 10 and 12, Aventis fibrinogen was determined to be the better source of fibrinogen and formamide was determined to be an optional post-treatment for these experiments.
• Example 14 Cell differentiation on seeded fibrin films.
• Fibrin gels were prepared with 10 mg/mL Aventis fibrinogen, 1 U/mL Factor XIII, 300 mM NaCl, 40 mM bicine, pH 8.0, and 2.5 ng/mL BMP-2. Gels were processed into films according to the method described in Examples 1 and 2. The final BMP-2 concentration was estimated at approximately 50 ng/mL. Films were also made without BMP-2. The films were placed in 6 well microplates. AU wells contained 3 mL Dulbecco's modified Eagle's medium (DMEM) supplemented with serum and antibiotic and aprotinin at 1 ⁇ g/mL. The wells were occupied as follows:
• DMEM Dulbecco's modified Eagle's medium
• Well 2 C2C12 mouse myoblast cells with 50 ng/mL BMP-2 added to the media
• Well 3 C2C12 mouse myoblast cells on the fibrin film not containing BMP-2
• Well 4 C2C12 mouse myoblast cells on the fibrin film containing BMP-2
• ALP alkaline phosphatase
• Example 15 Protein-based rubbery to hard plastic formulations.
• Gelatin, fibrin, fibrinogen and extracellular matrix plastics were fabricated in the form of approximately 0.5cm height x 1.3 cm diameter disks (hereinafter "pellets") using a commercially available press apparatus. In some experiments, the pellets were subsequently lyophilized under vacuum.
• the gelatin plastics comprised powdered gelatin, glycerin plasticizer, and in some experiments further comprised FGF-2, BMP-2, quantum dots, NaCl, polylactic acid, hydroxyapatite, ammonium acetate, ammonium bicarbonate, ammonium carbonate, and/or pyridinium TFA.
• the fibrin plastics comprised polymerized fibrin (ground to form a powder), glycerin plasticizer, and in some experiments further comprised FGF-2, BMP-2, andquantum dots.
• the fibrinogen plastics comprised powdered fibrinogen, glycerin plasticizer, and in some experiments further comprised water, FGF-2, BMP-2, quantum dots, NaCl, polylactic acid, hydroxyapatite, water, or ammonium acetate.
• the ECM plastics comprised powdered urinary bladder (UB) derived ECM, and in some experiments further comprised glycerin plasticizer, FGF-2, BMP-2, and quantum dots, as well as water in further experiments. The specific formulations were formed into homogeneous pellets that held together well and were used for additional post-processing and testing.
• Example 16 Gelatin plastic with glycerin as plasticizer - low heat compression pellet processing.
• Gelatin plastic pellets were prepared by compaction with glycerin as a plasticizer additive in concentrations of 0%, 12.5%, 19%, 21%, 25%, 36%, 40% and 50%. Percentages at about 40% provided particularly useful mechanical properties.
• the plastic pellets were formed by grinding solidified gelatin into a powder with particle sizes less than or equal to 150 microns (powders passing a 100 mesh standard sieve). A sample of the ground gelatin powder was mixed with glycerin plasticizer, and optionally with the substances described in Example 15 in quantities appropriate to achieve desired concentrations in the resulting plastic.
• the gelatin powder and any additional components were mixed with glycerin in 50 mL conical plastic centrifuge tubes and extensively manually mixed for 5- 10 minutes three times to ensure proper homogenization of the mixture, and left to incubate for at least 2 hours.
• the homogeneous mixture was then loaded into a standard pellet press.
• the press mold was twice coated with lecithin mold release agent prior to mixture loading in order to lubricate the inner surface to ensure easy removal of the pressed pellet from the mold without undesirable sticking.
• the press mold was loaded approximately % ⁇ full with the glycerin-wetted gelatin powder mixture.
• the press was then set to a temperature between 50 0 C and 125°C and a pressure ranging from 5000 lbs to 7000 lbs and the press operated for 20 to 60 minutes, where the specific operating parameters were set based on the desired properties of the resulting plastic.
• the press operating parameter profiles were over a 20 and a 60 minute operation, respectively, for a typical pellet formation.
• the mold pressure maximized and held steady at approximately 7000 lbs of pressure for approximately 20 minutes, and the mold temperature generally decreased from an initial value of approximately 80 0 C until reaching a final steady value of approximately 27 0 C (room temperature) at approximately 40 minutes into the 60 minute process.
• the resulting plastic pellet was removed from the mold, trimmed of excess plastic, and stored dry at 4 0 C.
• gelatin plastic formulations were formed into homogeneous pellets that held together well and were used for additional post-processing and testing. Hardness of the compacted plastic was a function of the amount of plasticizer added to milled powders prior to compaction. A more rubbery plastic resulted with increasing percent plasticizer.
• Example 17 Sodium chloride as a porogen in gelatin plastics.
• Solid sodium chloride (NaCl) was evaluated as a porogen in gelatin plastics prepared as described in Example 16.
• the solid NaCl crystals were added to the gelatin powders prior to compaction processing or introduced into the press molds either on either the top or the bottom of the gelatin pellet and physically pressed into the plastic.
• the embedded NaCl was removed by placing the pressed pellets in water and allowing the solid NaCl to dissolve into solution and leach out of the plastic leaving open pores. Neither the top pressed nor bottom pressed NaCl embedded porogen yielded even pores. Plastics formed with pre-mixed NaCl did not exhibit open pored structure.
• Example 18 NaCl as a porogen with gelatin plastic cross-linking.
• Example 19 Polylactic acid (PLA) as a porogen in gelatin plastics.
• FIG. 9 is a micrograph illustrating the formation of the pores in the gelatin matrix due to the leaching of the physically trapped PLA.
• Example 20 Gelatin plastic extrusion processing.
• Gelatin (19%vol:vol) plastic was prepared and processed as described in Example 16. The clear plastic pellets were further processed by extrusion. The gelatin plastic was extruded at a series of temperatures, ranging from 6O 0 C to 117°C (i.e., 6O 0 C, 70 0 C, 78 0 C, 80 °C, 90 0 C, 100 0 C, 110 0 C, and 117 0 C). Notwithstanding the prior plastic pressing steps, the extruded gelatin plastic formed bubbles and expanded after extrusion. Cold rolling of the extruded gelatin plastic resulted in cracking.
• Example 21 Hydroxyapatite incorporation into gelatin plastics.
• HA Hydroxyapatite
• Example 22 Machinability of gelatin plastics.
• the gelatin plasties prepared according to the methods described in Example 16 possess demonstrated machinability with varying concentrations of glycerin plasticizer.
• the relative hardness of the plastic determined its compatibility to machining techniques.
• the plastics generally exhibit excellent machinability as indicated by the precise formation of approximately 215 micron squares after machining, FIG. 10 and FIG. 11.
• the softer plastics that resulted from higher concentrations of glycerin plasticizer formed qualitatively rougher machined surfaces, FIG. 10, whereas the harder plastics that resulted from lower concentrations of glycerin plasticizer formed more clean machined surfaces, FIG. 11.
• Example 23 Pressing of gelatin plastic into sheets.
• the gelatin plastics prepared according to the method described in Example 16 possess excellent formability in terms of pressing thin sheets.
• the sheets formed from the gelatin plastic are thin, partially transparent, flexible, and tough.
• the resulting plastic shape was a function of the specific mold in which it was formed.
• the sheet comprised gelatin powder and 23% (vol:vol) glycerin pressed with a specially constructed rectangular shape mold.
• Example 24 Fibrin and fibrinogen pressed-plastics with glycerin plasticizers.
• the fibrin formulations described in Example 1 but using porcine fibrin were powdered and further modified by addition of glycerin and/or water plasticizers and processed into plastic disks as described in Example 16 (i.e, fibrinogen compacted at temperatures of 80 0 C, 87°C, and 121°C; and fibrin compacted at temperatures of 79°C and 101 0 C).
• the non-polymerized fibrinogen was mixed with glycerin and water and processed into plastic disks as described in Example 16.
• Example 25 ECM-based pressed-plastic fabrication.
• Powdered urinary bladder extracellular matrix (UB ECM) was pressed into plastic pellets as described in Example 16. Specifically, a first pellet was made without glycerin and processed at 102 0 C. A second pellet was made with 21% glycerin and processed at 77 0 C. A third pellet was made with 38% glycerin and processed at 100 0 C.
• Example 26 Growth factor inclusion and distribution visualization with quantum dots.
• FIG. 12 presents representative samples of plastics comprising quantum dots viewed under fluorescence.
• Samples labeled A were control groups not containing quantum dots.
• Samples labeled B and C contained quantum dots and BMP-2, and samples labeled D were cross- sectioned pellet sheets from samples C from indicated plastic.
• Samples labeled E is visual micrographs of cut films of gelatin plastic. Fluorescence was observed throughout the entire plastic sample for all formulations and processing parameters investigated, which indicated homogenous distribution of the quantum dots and BMP-2.
• Example 27 Quantum dot incorporation into ECM plastic to monitor degradation in vivo.
• fibrin-based plastics were prepared according to the method described in Example 16 and further comprised carboxyl coated (8.0 ⁇ M) 800nm quantum dots.
• the addition of the quantum dots permit the noninvasive monitoring of the in vivo degradation of the plastic, for example through the skin when implanted in a patient.
• the degradation of the plastic would be evaluated through the loss of fluorescence at the implant site.
• the in vitro results indicted retention of biological activity of BMP-2 incorporated into the plastics as demonstrated by the induction of extensive vascularization in a chick CAM model performed analogously to the method described in Example 4.
• FIG. 13 presents representative fluorescence images of the vascularization on each of the three types of protein-based plastics.
• Example 28 Ammonium compounds as porogens.
• ammonium compounds are solids at room temperature and atmospheric pressure, and are known to undergo sublimation to the gaseous phase at reduced pressures. Moreover, these ammonium salts are known in the art to be biocompatible. Commercially available compunds were, thus, tested for their ability to sublimation. Based on these data, gelatin plastics were processed according to the method described in Example 16, and comprised ammonium acetate. The ammonium acetate was physically incorporated into the plastic matrix and formed into pellets as described in Example 16. The pellets were then exposed to vacuum to sublime the ammonium acetate, thereby leaving open pores in the plastic matrix. Pore formation was quantified as percent remaining mass over the course of the vacuum treatment, FIG. 14.
• FIG. 15 presents an SEM image of the resulting microporosity of the gelatin plastic post-sublimation.
• the interconnected pores are readily identifiable and extend throughout the entire plastic pellet. It was further discovered that ammonium acetate provided for the most rapid sublimation and most efficient formation of pores.
• Plastic degradation testing was performed on non-cross-linked materials processed according to the method described in Example 16 and on identical plastics further processed by cross-linking with glutaraldehyde and genipin .
• the degradation testing was performed on gelatin, fibrin, and ECM-based plastic pellets in three solutions: PBS only ( ⁇ ), 0.6% genipin in PBS (GP), and 0.6% glutaraldehyde in PBS (GA).
• the solutions were prepared and transferred into three 15 ml tubes per solution for a total of nine tubes.
• the samples were arranged into sets of three, i.e. Gelatin ⁇ , GP, GA ⁇ , Fibrin ⁇ 0, GP, GA ⁇ , ECM ⁇ ⁇ , GP, GA).
• the appropriate plastic pellet was placed into each dish and allowed to cross-link at 23°C with rocking for 21.5 hours. It was observed that all three of the genipin-containing samples turned blue after only 50 minutes. After 21.5 hours, the solutions were vacuum aspirated from the wells and the samples rinsed three times with PBS. Each plastic pellet was measured for initial area and initial mass. The plastic samples were then transferred to new wells, submerged in DMEM-F 12 solution containg 0.1% Na azide, and 1% streptomycin/penicillin, sealed with laboratory film and incubated at 37°C and 5% CO2.
• the area of the plastic pellet was measured by arranging two rulers perpendicular to each other with the millimeter scales on the inside edges so that measurements were readily taken in mm by mm, and securing the rulers with tape.
• the scale was placed underneath a microscope lens so that the focus included as much of both axes as possible.
• the scale was fastened to the microscope stage. When measuring area, the pellet was left in solution as much as possible, preferably submerged in the well. This ensured that diffraction due to the solution was consistent for all measurements.
• the pellet was aligned so that two of its edges were flush with each axis of the rulers. If necessary, the pellet was moved uni-axially to measure a side, then the other edge was measured separately. During testing pipette tips were used as the tools for manipulating the pellets.
• the pellet and scale was viewed through the microscope objective and the size of the pellets read from the visual scales.
• the mass of the plastic pellets was measured using a standard laboratory scale of appropriate precision (at least accurate to 0.02 mg). A pellet was taken out of solution and blotted twice to remove excess solution. The mass was measured on the scale and the chip immediately returned to the well. Fresh media solution was added to each well during the area and mass measurements while the pellet was out of the well. Measurements were taken every 24 hours for up to 45 days and the pellets were incubated at 37 0 C and 5% CO 2 in the sealed multiwell dishes.
• Example 30 Plastic degradation testing with external cross-linking.
• FIG. 16, FIG. 17, and FIG. 18 illustrate these data graphically.
• Non-cross-linked gelatin plastic swelled approximately 6-fold as measured by both area (FIG. 16A) and mass (FIG. 16B), after which samples rapidly dissolved and were essential gone by 24 hrs post initiation of incubation.
• Cross- linked samples either glutaraldehyde (GA) cross-linked or genipin (GP) cross-linked, swelled approximately 2-fold, and then maintained both area and mass through the 45 day test period.
• Gluteraldehyde and genipin have similar cross-linking chemistries, but genipin is a plant-based molecule reported to have much less biotoxicity.
• Example 31 Plastic degradation testing with internal cross-linking.
• Example 30 However, no swelling occurred with the internally cross-linked GP gelatin samples compared to the approximately 200% swelling for externally cross-linked gelatin samples. Similarly to the externally cross-linked GP gelatin samples, internally cross-linked GP gelatin did not degrade. This represents a significant advance permitting not only control of degradation but also shape retention using internally GP cross-linked proteins.
• Example 32 Plastic degradation testing with internal cross-linking.
• FIG. 20 shows the change in mass and demonstrated that that internal GP cross-linked fibrin does not swell and does not degrade over time, making it particularly useful for biomedical applications such as for implants.

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• Compositions Of Macromolecular Compounds (AREA)
• Other Resins Obtained By Reactions Not Involving Carbon-To-Carbon Unsaturated Bonds (AREA)
• Processes Of Treating Macromolecular Substances (AREA)
• Manufacture Of Porous Articles, And Recovery And Treatment Of Waste Products (AREA)

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