US20030012818A1 - Drug delivery matrices to enhance wound healing - Google Patents

Drug delivery matrices to enhance wound healing Download PDF

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US20030012818A1
US20030012818A1 US10/132,619 US13261902A US2003012818A1 US 20030012818 A1 US20030012818 A1 US 20030012818A1 US 13261902 A US13261902 A US 13261902A US 2003012818 A1 US2003012818 A1 US 2003012818A1
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composition
matrix
bmp
kit
bioactive molecule
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Jason Schense
Hugo Schmoekel
Jeffrey Hubbell
Franz Weber
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Eidgenoessische Technische Hochschule Zurich ETHZ
Universitaet Zuerich
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Eidgenoessische Technische Hochschule Zurich ETHZ
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Assigned to EIDGENOSSISCHE TECHNISCHE HOCHSCHULE ZURICH, UNIVERSITAT ZURICH reassignment EIDGENOSSISCHE TECHNISCHE HOCHSCHULE ZURICH ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HUBBELL, JEFFREY A., SCHMOEKEL, HUGO, WEBER, FRANZ, SCHENSE, JASON C.
Publication of US20030012818A1 publication Critical patent/US20030012818A1/en
Priority to US11/739,607 priority patent/US20070202178A1/en
Priority to US12/845,354 priority patent/US8309518B2/en
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    • 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
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/04—Macromolecular materials
    • A61L31/043—Proteins; Polypeptides; Degradation products thereof
    • A61L31/046—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
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001—Use of materials characterised by their function or physical properties
    • A61L24/0015—Medicaments; Biocides
    • 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
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/10—Polypeptides; Proteins
    • A61L24/106—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
    • A61L27/14—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
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • 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
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/23—Carbohydrates
    • A61L2300/236—Glycosaminoglycans, e.g. heparin, hyaluronic acid, chondroitin
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A61L2300/414—Growth factors
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/45—Mixtures of two or more drugs, e.g. synergistic mixtures

Definitions

  • This invention is generally in the field of drug delivery and more specifically in the area of fibrin and synthetic matrices to enhance wound healing.
  • Fibrin matrices are present naturally in the body and serve as the initial matrix for wound healing.
  • fibrinogen When an injury occurs to tissue, blood vessels are compromised, allowing the precursor molecule, fibrinogen, to invade the wound.
  • the fibrinogen is then enzymatically cleaved and self-catalyzed into a loosely formed gel.
  • the gel is then covalently crosslinked through the action of the transglutaminase, factor XIIIa, resulting in a stable matrix. Pisano, Finlayson and Peyton, Science, 160, 892-893 (1968).
  • the final fibrin matrix includes various proteins in addition to fibrinogen, such as serum proteins present during the coagulation process, for example fibronectin and ⁇ 2-plasmin inhibitor.
  • Factor XIIIa can covalently crosslink these serum proteins to the fibrin matrix, which can then add additional bioactivities to the matrix that can modify the ability of cells to infiltrate and degrade the matrix.
  • serum proteins present during the coagulation process for example fibronectin and ⁇ 2-plasmin inhibitor.
  • Factor XIIIa can covalently crosslink these serum proteins to the fibrin matrix, which can then add additional bioactivities to the matrix that can modify the ability of cells to infiltrate and degrade the matrix.
  • Tamaki and Aoki J Biol Chem, 257, 14767-14772 (1982).
  • These matrices also contain many blood cells, which become entrapped inside the matrix during coagulation, further modifying the biochemical character of the matrix.
  • One major cell type is the platelet, a cell rich with natural supplies of potentially
  • fibrin is a matrix that is strongly conductive for cells, allowing them to easily infiltrate the wound site.
  • the process employed involves two key features.
  • the matrix contains adhesion sites, allowing the cells to attach and migrate into the gel. Additionally, the matrix is responsive to cell-derived proteolytic activity. This allows the matrix to be degraded locally, allowing the cells to migrate into the matrix uninhibited but preventing global degradation of the matrix. Herbert, Bittner and Hubbell, J Compar Neuro, 365, 380-391 (1996); Pittman and Buettner, Dev Neuro, 11, 361-375 (1989). Therefore, the natural matrix remains at the site of injury until it is infiltrated by cells, at which time it is degraded during this process leading to regenerated tissue.
  • BMP bone morphogenetic protein
  • Some growth factor receptors must be occupied for at least 12 hours to produce a maximal biologically effect. Therefore, a prolonged contact caused by a small but constant stream of growth factor near the site of need is very favorable for a healing response. At the same constant release rate, as the initial concentration of growth factor retained in the matrix increases, the time period for release from the matrix increases.
  • a further object of the present invention is to provide a method to decrease the solubility of a growth factor in a matrix made from fibrin or synthetic polymers.
  • compositions and methods for making compositions to improve wound healing are still a further object of the present invention.
  • Bioactive molecules are entrapped within a matrix for the controlled delivery of these compounds for therapeutic healing applications.
  • the matrix may be formed of natural or synthetic compounds.
  • the primary method of entrapment of the bioactive molecule is through precipitation of the bioactive molecule during gelation of the matrix, either in vitro or in vivo.
  • the bioactive molecule may be modified to reduce its effective solubility in the matrix to retain it more effectively within the matrix, such as through the deglycosylation of members within the cystine knot growth factor superfamily and particularly within the TGF ⁇ superfamily.
  • the matrix may be modified to include sites with binding affinity for different bioactive molecules, for example, for heparin binding. When these different bioactive molecules are added to the matrix, the bioactive molecules are bound to the matrix both by precipitation within the matrix and by binding to the sites in the matrix, thereby providing enhanced controlled delivery to a patient.
  • FIGS. 1A and 1B are graphs measuring the incorporation of a factor XIIIa substrate peptide into fibrin.
  • Fibrin gels were synthesized at 8 mg/mL from pre-diluted TissucolTM Kits (Baxter) (FIG. 1A) which were diluted by a factor of 2 ( ⁇ ) and 10 ( ⁇ ) or from purified fibrinogen either with ( ⁇ ) or without ( ⁇ ) 1 U/mL of exogenous factor XIIIa added to the prepolymerization mixture (FIG. 1B).
  • FIG. 2 is a graph measuring the retention of bioactive molecules in a fibrin matrix after washing.
  • Two separate bioactive molecules one water soluble molecule, heparin( ⁇ ), and one with low solubility at physiological pH, recombinant human bone morphogenetic protein (rh-BMP-2) ( ⁇ ), were added to the fibrin during polymerization and were repeatedly washed in phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • FIGS. 3A and 3B show the retention of rh-BMP-2 in fibrin gels.
  • the fibrin gels were polymerized with 10 ( ⁇ ), 20 ( ⁇ ), 100 ( ⁇ ) and 200 ( ⁇ ) ⁇ g/mL of non-glycosylated rh-BMP-2 present during polymerization of the gel and the percent of rh-BMP-2 remaining in the gel was determined after 10, 20, 30, 40, and 50 wash volumes in PBS.
  • FIG. 3A the fibrin gels were polymerized with 10 ( ⁇ ), 20 ( ⁇ ), 100 ( ⁇ ) and 200 ( ⁇ ) ⁇ g/mL of non-glycosylated rh-BMP-2 present during polymerization of the gel and the percent of rh-BMP-2 remaining in the gel was determined after 10, 20, 30, 40, and 50 wash volumes in PBS.
  • FIGS. 4A and 4B show healing levels of critical size rat calvarial defects.
  • the healing efficacy of fibrin gels with various glycosylated and non-glycosylated rh-BMP-2 formulations mixed within the gel were measured.
  • non-glycosylated rh-BMP-2 was mixed within the fibrin gel in concentrations of 0 (column I), 1 (column II), 5 (column III), and 20 (column IV) ⁇ g/mL.
  • FIG. 4A non-glycosylated rh-BMP-2 was mixed within the fibrin gel in concentrations of 0 (column I), 1 (column II), 5 (column III), and 20 (column IV) ⁇ g/mL.
  • fibrin gel (column I), fibrin gel mixed with the same heparin level (column V), fibrin gel with 1 ⁇ g/mL non-glycosylated rh-BMP-2 (column II), fibrin gel with 1.0 ⁇ g/mL glycosylated rh-BMP-2 with a transglutaminase domain to covalently link it to the fibrin gel (as described in U.S. Pat. No.
  • FIG. 5 is a bar graph of the radiologic healing of the canine pancarpal arthrodesis.
  • the efficacy of using non-glycosylated rh-BMP-2 in fibrin matrices was tested in this defect and compared to the clinical standard of cancellous autograft (Spongiosa autograft).
  • the mean of the healing scores obtained at four, eight and twelve weeks for the autograft and the non-glycosylated rh-BMP-2 in a fibrin gel were calculated. In the scoring system, 0 corresponded to no mineralization being visible, 1 corresponded to some mineralization being visible, 2 corresponded to a defect that is completely mineralized, and 3 corresponded to healed and remodeled defects.
  • Nine dogs were used to test the non-glycosylated rh-BMP-2 in a fibrin gel and 17 dogs were used for the control, spongiosa autograft.
  • a bioactive factor is “precipitated” if the concentration of bioactive factor exceeds the concentration limit that is soluble in the respective vehicle at a predefined pH and temperature.
  • precipitation also can encompass retention due to any physical interaction between the bioactive molecule and the matrix, i.e. adsorption, electrostatic forces, affinity precipitation, co-precipitation etc.
  • the terms “entrapment”, “inclusion” and “precipitation” are used synonymously as ways to achieve retention.
  • Microx means a three-dimensional network which can act as a scaffold for cell ingrowth and for bioactive molecules over a certain period of time.
  • “Deglycosylated bioactive molecules” means bioactive molecules which are not glycosolated, though, when found in nature, they are glycosylated at one or more sites of the molecule. In these molecules, the glycosylation has been removed from the molecule by chemical or enzymatic methods or by producing it as a non-glycosylated molecule.
  • a “deglycosylated growth factor” is a growth factor that can be glycosylated when expressed in a eukaryotic cell and where the polysaccaride sequence or glycosaminoglycans has been either clipped off after expression or the method of expression is such that the growth factor is not glycosylated. The latter happens for example if the growth factor is expressed in a prokaryotic cell.
  • the terms “deglycosylated”, “non-glycosylated” and “not glycosylated” are used synonymously herein.
  • Retention means that at least 10% of the initially applied concentration of bioactive molecule, preferably at least 60% and even more preferably at least 80%, is still present in the matrix after 10 wash volumes.
  • Ten wash volumes refers to placing the matrix in a solution resulting in a volume ratio of 1 part matrix to 10 parts of phosphate buffered saline (PBS 0.01M; pH 7.4) for at least 12 hours at 37° C.
  • Retention can be achieved, for example, by precipitation of the bioactive molecule.
  • Retainable concentration means that percentage of the initial concentration which is retained according to the manner described above.
  • Controlled release is due not only to slow and steady disintegration of the growth factor and its subsequent diffusion from the matrix, but is also due to the disintegration and enzymatic cleavage of the matrix.
  • “Gelation” means the formation of a three-dimensional network and thus the transition from a liquid composition to a viscous composition.
  • gel and “matrix” are used synonymously throughout the application. An in situ formation of the gel or matrix is due to the transition from a liquid state to a solid state at the site of application in the body.
  • “Hydrogel” means a class of polymeric materials which are extensively swollen in an aqueous medium, but which do not dissolve in water.
  • “Michael addition” or “Michael-type addition reaction” is the 1,4 addition reaction of a nucleophile to a conjugate unsaturated system under basic conditions.
  • the addition mechanism can be purely polar, or proceed through one or more radical-like intermediate state(s).
  • Lewis bases or appropriately designed hydrogen bonding species can act as catalysts.
  • conjugation can refer both to alternation of carbon-carbon, carbon-heteroatom or heteroatom-heteroatom multiple bonds with single bonds, or to the linking of a functional group to a macromolecule, such as a synthetic polymer or a protein. Double bonds spaced by a CH or CH 2 unit are referred to as homoconjugated double bonds.
  • Michael-type addition to conjugated unsaturated groups can take place in substantially quantitative yields at physiological temperatures, in particular at body temperature, but also at lower and higher temperatures. These reactions take place in mild conditions with a wide variety of nucleophiles, like amines and thiols.
  • the reaction as described herein is self-selective.
  • the first precursor component of the reaction reacts much faster with the second precursor component of the reaction than with other compounds present in the mixture at the site of the reaction; and the second precursor component reacts much faster with the first precursor component than with other compounds present in the mixture at the site of the reaction.
  • a nucleophile preferentially binds to a conjugated unsaturated group, rather than to other biological compounds, and a conjugated unsaturated group preferentially binds to a nucleophile rather than to other biological compounds.
  • Polymeric network means a structure in which substantially all of the monomers, oligomers or polymers present in the structure are bound by intermolecular covalent linkages through their available functional groups to result in one large molecule.
  • “In situ formation” refers to formation at a physiological temperature and at the site of injection in the body. This term is typically used to describe the formation of covalent linkages between precursor molecules, which are substantially not crosslinked prior to and at the time of injection.
  • “Polymerization” and “cross-linking” are used to indicate the linking of multiple precursor molecules, which results in a substantial increase in the molecular weight of the resulting molecule. “Cross-linking” further indicates branching, typically to yield a polymer network.
  • “Functionalize” means to modify in a manner that results in the attachment of a functional group or moiety.
  • a molecule may be functionalized by the introduction of a molecule which makes the molecule a strong nucleophile or a conjugated unsaturation.
  • a molecule for example PEG, is functionalized to become a thiol, amine, acrylate, or quinone.
  • “Functionality” refers to the number of reactive sites on a molecule.
  • the functionality of a strong nucleophile and a conjugated unsaturation will each be at least two. Mixing two components, for example a strong nucleophile and a conjugated unsaturation, with functionalities of two each, will result in a linear polymeric biomaterial. If at least one component has a functionality that is greater than two, upon mixing a cross-linked biomaterial will be formed.
  • Regularize means to grow back a portion or all of a tissue.
  • methods of regenerating bone following trauma, tumor removal, or spinal fusion, or for regenerating skin to aid in the healing of diabetic foot ulcers, pressure sores, and venous insufficiency are described herein.
  • Tissues which may be regenerated include, but are not limited to, skin, bone, nerve, blood vessel, and cartilage tissue.
  • peptide and protein are differentiated by their chain length.
  • Peptide means polyaminoacids containing up to 30 amino acids, preferably from about 10 to 20 amino acids.
  • Proteins are polyaminoacids containing more than 30 amino acids.
  • compositions are formed of a natural or synthetic matrix and a bioactive molecule, in particular a growth factor, which can be administered to a patient to improve wound healing.
  • the bioactive compound is released in a controlled manner from the matrix.
  • the bioactive molecule is preferably a deglycosylated member of the cystine knot growth factor superfamily, most preferably a non-glycosylated member of the TGF ⁇ superfamily.
  • the matrices may be biodegradable or nondegradable.
  • the matrices may be made of synthetic polymers, natural polymers, oligomers, or monomers. Synthetic polymers, oligomers, and monomers include those derived from polyalkyleneoxide precursor molecules, such as poly(ethylene oxide) (PEO), poly(ethylene glycol) (PEG) and copolymers with poly(propylene oxide) (PEG-co-PPO), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(ethyloxazoline) (PEOX), polyaminoacids, and pseudopolyamino acids, and copolymers of these polymers.
  • polyalkyleneoxide precursor molecules such as poly(ethylene oxide) (PEO), poly(ethylene glycol) (PEG) and copolymers with poly(propylene oxide) (PEG-co-PPO), poly(vinyl alcohol) (PVA), poly(vinylpyrroli
  • Copolymers may also be formed with other water-soluble polymers or water insoluble polymers, provided that the conjugate is water soluble.
  • An example of a water-soluble conjugate is a block copolymer of polyethylene glycol and polypropylene oxide, commercially available as a PluronicTM surfactant (BASF).
  • Natural polymers, oligomers and monomers include proteins, such as fibrinogen, fibrin, gelatin, collagen, elastin, zein, and albumin, whether produced from natural or recombinant sources, and polysaccharides, such as agarose, alginate, hyaluronic acid, chondroitin sulfate, dextran, dextran sulfate, heparin, heparin sulfate, heparan sulfate, chitosan, gellan gum, xanthan gum, guar gum, water soluble cellulose derivatives, and carrageen.
  • proteins such as fibrinogen, fibrin, gelatin, collagen, elastin, zein, and albumin, whether produced from natural or recombinant sources
  • polysaccharides such as agarose, alginate, hyaluronic acid, chondroitin sulfate, dextran, dextran sulfate, heparin,
  • the matrix is a fibrin gel, created from any source of fibrinogen.
  • a fibrin gel can be created at physiological conditions, which means at conditions as found in humans and animals.
  • fibrin gel formation can also occur outside the body in the presence of thrombin and calcium and mainly depends on temperature and pH.
  • the fibrin gel can be formed outside the body at a temperature range of between 25° C. to 40° C. and a pH range of between 7 to 8. If the bioactive molecule is not soluble at these conditions, it will precipitate during polymerization and become entrapped within the matrix.
  • Fibrin gels can be synthesized from autologous plasma, cryoprecipitated plasma (e.g. fibrin glue kits, which are available commercially), fibrinogen purified from plasma, and recombinant fibrinogen and factor XIIIa.
  • cryoprecipitated plasma e.g. fibrin glue kits, which are available commercially
  • fibrinogen purified from plasma e.g. fibrinogen purified from plasma
  • recombinant fibrinogen and factor XIIIa e.g. fibrinogen purified from plasma
  • recombinant fibrinogen and factor XIIIa recombinant fibrinogen and factor XIIIa.
  • Synthetic matrices are known in tissue regeneration and wound healing. These include macroporous sponges of degradable polymers such as polylactic acid and its copolymers as well as hydrogel matrices based on water-soluble polymers such as PEG.
  • PEG is a precursor molecule for the formation of an enzymatically degradable matrix.
  • PEG is functionalized with chemically reactive groups, such as acceptor groups in the form of conjugated unsaturated bonds, including acrylates, vinyl sulfones and acrylamides, for Michael-type addition reactions.
  • the PEG is a four-armed PEG with a weight average molecular weight of 15 to 25,000 kDa.
  • the PEG precursors are in solution and are mixed with a second precursor molecule, such as peptides.
  • the peptides are in solution and contain two or more reduced cystine residues (nucleophilic thiol groups), with protease substrate sites intervening between the cystine sites.
  • Peptides with multiple cystines are described in WO 00/44808 to Elbert et al., published Aug. 3, 2000, herein incorporated by reference.
  • a gel forms rapidly by a Michael-type addition reaction between the multi-thiol component (the second precursor) and the multi-acceptor component (the first precursor, a functionalized PEG), so long as the sum of the functionality of the multi-acceptor (the number of Michael acceptor groups (m) per molecule) and the functionality of the multi-thiol (number of thiol groups (n) per molecule) is greater than 5.
  • the Michael addition between the thiol and the acceptor groups works from pH 6.5 up to very basic conditions at a wide variety of temperatures. However, when the precursor components are injected into the body for an in situ formation of the matrix, the pH must be appropriate for the body. Therefore in a preferred embodiment, the pH is between 7 and 8. A preferred temperature range is between 25° C. to 40° C. when the gel is formed outside the body. Inside the body, the gel is formed at body temperature.
  • the peptide is designed to be a substrate for plasmin or a matrix metalloproteinase
  • the resulting synthetic gels degrade in response to the enzymatic matrix remodeling influence of cells.
  • the multi-thiol i.e. the nucleophilic precursor component
  • the nucleophilic precursor component can be a PEG.
  • the gel may further comprise cell attachment sites, such as RGD sequences, covalently bound to the matrix to promote ingrowth and attachment of cells into the matrix.
  • the cell attachment site can be bound to the matrix by Michael addition reaction.
  • the RGD is modified such that it contains free thiol or cystine groups for reaction with the conjugated unsaturated bond.
  • the matrices can be further modified by including bioactive molecules, often derived from development, to enhance the regeneration of the wounded tissue. Pandit et al., J Biomater Appl, 14, 229-42 (2000); Hildebrand et. al., Am J Sports Med, 26, 549-54 (1998); Quirinia A, Scand J Plast Reconstr Surg Hand Surg, 32, 9-18 (1998).
  • the type of molecule that is entrapped can be any of a large list of possible bioactive molecules, including growth factors, peptides, enzymes, protease inhibitors, antibiotics, synthetic homologues and other assorted molecules.
  • Preferred bioactive molecules have reduced solubility at physiological pHs.
  • Growth factors are particularly useful because they provide a well-characterized chemical entity that has been shown to play an important role in wound healing, and are often naturally present at the site of injury. Additionally, growth factors are pluripotent molecules, allowing them to activate many different cell types and induce a complicated healing response.
  • TGF ⁇ 2 transforming growth factor-beta 2
  • PDGF platelet derived growth factor
  • NEF nerve growth factor
  • hCG human chorionic ganodotropin
  • Cystines [II to V] and [III to VI] form a ring of eight amino acids through which the remaining disulphide bond (Cys [I to IV]) penetrates the molecule.
  • cystine knots fall into two structural classes: growth factor-type and inhibitor-like cystine knots.
  • cystine knot growth factor superfamily include the platelet derived growth factor (PDGF) superfamily, the transforming growth factor beta (TGF ⁇ ) superfamily and the glycoproteins alpha family.
  • PDGF platelet derived growth factor
  • TGF ⁇ transforming growth factor beta
  • BMPs BMPs
  • PDGFs transforming growth factor beta
  • TGF betas tumor necrosine growth factor beta
  • Not all of the growth factors are glycosylated when expressed by eukaryotic cells; for example, TGF beta 1, 2, and 3 are never glycosylated, irrespective of the expression system used.
  • BMP bone morphogenetic protein
  • BMP-2 and BMP-7 both have heparin binding affinity, are soluble at low pHs and are strong inducers of bone healing.
  • Wozney JM Prog Growth Factor Res, 1,267-80 (1989); Wozneyet al., J Cell Sci Suppl, 13, 149-56 (1990).
  • rh-BMP-2 has demonstrated the greatest healing potential and is even able to induce bone formation at an ectopic site. Jin et. al., J Biomed Mat Res, 52, 841 (2000). Since the solubility of rh-BMP-2 at physiological conditions is low, it can precipitate within a matrix. Thus, this molecule fits the characteristics necessary for delivery.
  • the BMPs are themselves members of the transforming growth factor beta (TGF ⁇ ) superfamily, and the structural homology between the members of the TGF ⁇ superfamily is also high. As such, results obtained with BMP-2 can be expected to be obtained with other members of the TGF ⁇ superfamily and members of the cystine knot growth factor superfamily.
  • the precipitation of growth factors that are members of the TGF superfamily and their prolonged release is further improved by using recombinant forms that are not glycosylated and are therefore less soluble.
  • Deglycosylated versions of BMP and other growth factors can be obtained using a number of techniques.
  • Several methods of deglycosylation are available in common practice, both chemical and biological.
  • One chemical method occurs through the use of hydrogen fluoride. Briefly, glycosylated proteins are mixed with polyhydrogen fluoride, pyridine and a scavanger. This leads to essentially complete deglycosylation without modification of the protein itself.
  • Biological methods center around the use of enzymes to cleave the glycosaminoglycans from the protein or expression in bacteria. Two examples are N-glycanase (Lin, Zhang et al.
  • glycopeptidase F Chopeptidase F
  • eukaryotic source i.e. glycosylated
  • solubility of the glycosylated rh-BMP-2 can be made to mimic that of non-glycosylated rh-BMP-2.
  • excipients to reduce the solubility of proteins, e.g. polymers of opposite charge to reduce the net charge of the protein.
  • biochemical methods Two primary methods for delivering bioactive factors are through biochemical and physical methods.
  • biochemical methods matrices are created which have a chemical affinity for the bioactive factor of interest. When the matrix is mixed with the bioactive molecule, the release of the molecule can be delayed or eliminated.
  • Physical methods which may be used to retain the bioactive molecules in the matrix, include precipitation, co-precipitation, affinity precipitation, and physical entrapment.
  • the bioactive molecules are precipitated inside a fibrin matrix to improve retention. This matrix, which contains the precipitated molecules, has a significant potential for wound healing.
  • Precipitation can be combined with other retention methods to produce biomaterials which improve wound healing.
  • modified biomaterials which contain sites with binding affinity for bioactive molecules.
  • the bioactive molecule is bound to the matrix, enhancing retention of the bioactive molecule in the matrix.
  • a matrix can be modified to include binding sites with heparin affinity and heparin can be added to bind with the matrix. Then, if the bioactive molecule has heparin binding affinity, the bioactive molecule will bind with the heparin and thus be retained in the matrix. This method can be performed in conjunction with the use of precipitation for slower release kinetics.
  • the retention of rh-BMP-2 is enhanced by binding to heparin which is bound to a modified fibrin matrix.
  • deglycosylated rh-BMP-2 is retained in the matrix because it is precipitated within the matrix due to its poor solubility and bound to heparin due to its heparin binding affinity.
  • these matrices provide a wide range of patients with therapies for healing bony defects.
  • the modified fibrin or synthetic matrices serve as a replacement for bone grafts, and thus may be applied in many of the same indications. These indications include, but are not limited to, spinal fusion cages, healing of non-union defects, bone augmentation, and dental regeneration.
  • these matrices can be used in implant integration.
  • implants can be coated with a modified matrix, either natural or synthetic, inducing the neighboring bone area to grow into the surface of the implant and preventing loosening and other associated problems. These examples are merely illustrative and do not limit the number of possible indications for which the matrices described herein can be used.
  • growth factor-enriched matrices can be used for healing chronic wounds in skin
  • the material is applied to the wound area as a preformed matrix.
  • the material is gelled in situ in the body.
  • the matrix material can be made from synthetic or natural precursor components.
  • the precursor components should not be combined or come into contact with each other under conditions that allow polymerization of the components prior to application of the mixture to the body. This is achieved by a system which separates the first precursor composition from the second precursor composition, where the first and second precursor compositions comprise components that form a three dimensional network upon mixing under conditions that allow polymerization of the components. Additionally at least one of the precursor compositions may contain a biologically active molecule which is a deglycosylated member of the cystine knot growth factor superfamily. Depending on the precursor components and their concentration, gelling can occur quasi-instantaneously after mixing. Therefore, it is difficult to inject a gelled material through an injection needle.
  • the matrix is formed from fibrinogen. Fibrinogen, through a cascade of various reactions, gels to form a matrix when contacted with thrombin and a calcium source at appropriate temperature and pH. Therefore, during storage it is necessary to prevent the three components, fibrinogen, thrombin and a calcium source, from coming into contact with each other. As long as at least one of the three components is separate from the other two, the gel will not form.
  • fibrinogen which may additionally contain aprotinin or another protease inhibitor to increase stability, is dissolved in a buffer solution at physiological pH, ranging from pH 6.5 to 8.0, preferably from pH 7.0 to 7.5, and stored separately from a solution of thrombin in a calcium chloride buffer (with a concentration range of 40 to 50 mM).
  • the buffer solution for the fibrinogen can be a histidine buffer solution in a preferred concentration of 50 mM, which may additionally contain NaCl in a preferred concentration of 150 mM or Tris buffer saline, preferably at a concentration of 33 mM.
  • the bioactive molecule may be in either the fibrinogen or the thrombin solution.
  • the fibrinogen solution contains the bioactive molecule.
  • the fibrinogen and the thrombin solutions can optionally be stored frozen to enhance stability during storage. Prior to use, the frozen fibrinogen and the thrombin solutions are defrosted and mixed.
  • fibrinogen and thrombin are stored together, but separately from the calcium source.
  • the fibrinogen is stored with the calcium source and separately from the thrombin.
  • fibrinogen and thrombin are stored in separate containers in lyophilized form.
  • Either fibrinogen or thrombin can contain the bioactive molecule.
  • a Tris or histidine buffer solution is added to the lyophilized fibrinogen.
  • the buffer may additionally contain aprotinin.
  • the lyophilized thrombin is dissolved in the calcium chloride solution. Then the fibrinogen and thrombin solutions are mixed.
  • the mixing step preferably occurs by combining the separate containers, vials or syringes which contain each solution with a two-way connecting device having a needle attached at one side.
  • the containers, vials or syringes are bipartite with two chambers separated by an adjustable partition.
  • one of the chambers contains lyophilised fibrinogen, while the other chamber contains an appropriate buffer solution. If pressure is applied to one end of the syringe body, the partition moves and releases bulges in the syringe wall to transfer the buffer into the fibrinogen chamber and dissolve the fibrinogen.
  • a bipartite syringe body is used for storage and dissolution of the thrombin.
  • both bipartite syringe bodies are attached to the two way connecting device and the contents are mixed by squeezing them through the injection needle attached to the connecting device.
  • the connecting device may additionally comprise a static mixer to improve mixing of the contents.
  • the fibrinogen is diluted eight-fold and thrombin is diluted twenty-fold prior to mixing. This ratio results in a gelation time of approximately one minute.
  • the matrix is formed from synthetic precursor components capable of undergoing a Michael-type addition reaction.
  • the nucleophilic precursor component (the multi-thiol) only reacts with the multi-acceptor component (e.g. a conjugated unsaturated group) at basic pH. Therefore, the three components which must be separated prior to mixing are the base, the nucleophilic component and the multi-acceptor component.
  • Both the multi-acceptor and the multi-thiol component are stored as solutions in buffers. Both of the solutions can contain a cell attachment site and additionally a bioactive molecule.
  • the first solution of the system can contain the nucleophilic component and the second solution of the system can contain the multi-acceptor component. Either of the two solutions can contain the base; alternatively, the base can be present in both solutions.
  • the multi-acceptor and the multi-thiol can be mixed together in the first solution and the second composition can contain the base.
  • the bipartite syringe body is equally well-suited for the synthetic precursor components as it is for the natural precursor components.
  • the multi-acceptor and multi-thiol components are stored in pulverized form in one of the chambers and the basic buffer is in the second chamber.
  • the matrices typically contain a dosage of 0.01 to 5 mg/mL of bioactive molecule. This dosage range is in accordance with the levels of active protein used in other clinical trials. However, lower doses may also be used due to the improved delivery that the matrices provide. For example, when non-glycosylated rh-BMP-2 was used in healing non-union cranial defects in rats, very low doses of 1-10 ⁇ g/mL were effective. As such, when using precipitated growth factors, and especially advantageous forms such as non-glycosylated forms, significant reductions in dosing are possible. Thus less bioactive molecule is necessary to get the same result.
  • bioactive molecule is rleased completely within several weeks following administration. Within two to four weeks, it is likely that the original matrix has been completely remodeled and all of the bioactive molecules have been released.
  • a test measuring the native enzymatic activity of the coagulation enzyme, factor XIIIa was performed. This test was performed by measuring the ability of fibrin gel from two different sources to covalently incorporate a synthetic substrate during the coagulation process.
  • One source of the fibrin gel came from a fibrin glue kit, while the second source came from a purified fibrin gel.
  • Peptides derived from ⁇ 2-plasmin inhibitor can be covalently incorporated into fibrin gels through the action of factor XIIIa.
  • one method for testing the enzymatic activity in a fibrin gel or dilution thereof involves testing the ability of different fibrin sources to incorporate this same peptide.
  • the gels were synthesized with various amounts of fluorescently labeled peptide and washed with TBS (0.03M, pH 7.4) to remove free peptide from the matrix. The gels were then degraded with the minimum amount of plasmin necessary and analyzed with size exclusion chromatography. The amount of fluorescent signal (i.e. peptide) bound to the matrix was determined when various dilutions of fibrin glue kits or purified fibrin gels were employed. This result was correlated to the amount of crosslinking activity present in the matrix.
  • FIGS. 1A and 1B depict the results of this test.
  • the results of the test demonstrate that when similar concentrations of fibrin are tested, the level of incorporation is similar.
  • the biochemical enzymatic activity in a fibrin glue kit (FIG. 1A) proved to be similar to that in a purified fibrin gel (FIG. 1B).
  • higher protein (and factor XIIIa) concentrations lead to higher incorporation levels.
  • a non-glycosylated recombinant form of a bone morphogenetic protein which was prepared from prokaryotic cells ( E. coli ) (rh-BMP-2), was mixed in different fibrin gels, and the gels were tested in a rat femur defect. Because the protein was expressed in a prokaryotic system, it was not glycosylated.
  • Fibrin gels were synthesized from a variety of sources. Purified fibrinogen from Sigma Chemical and a blood bank were employed, as well as fibrin glues (Baxter) at several dilutions. These gels were loaded with rh-BMP-2 and placed in a critical size (5 mm full thickness) femur defect.
  • This in vitro assay involved comparing the release kinetics of the entrapped non-glycosylated rh-BMP-2 to the release kinetics of a molecule that is known to have high solubility at physiological pH.
  • Fibrin gels were polymerized using purified fibrinogen (Sigma) at 8 mg/mL and 2 U/mL thrombin at pH 7.4. Calcium was added so that the final concentration was 2.5 mM to increase the rate of gelation
  • Example 3 demonstrate the use of non-glycosylated rh-BMP-2 in bone regeneration in fibrin matrices.
  • Non-glycosylated rh-BMP-2 will likewise be advantageous for regeneration of bone, as well as other tissues, in matrices other than fibrin.
  • the results of this Example may be extended by structural similarity to other members of the BMP family, and by the same structural similarity to other members of the TGF ⁇ superfamily, including TGF ⁇ 1, TGF ⁇ 2, TGF ⁇ 3, and the numerous other members of the TGF ⁇ superfamily.
  • these results may also be extended to other wound healing situations, including healing of chronic wounds in the diabetic, in the venous insufficiency patient, and the pressure ulcer.
  • non-glycosylated members of the TGF ⁇ superfamily are broadly useful in the promotion of wound healing and tissue regeneration.
  • Examples 4 and 5 describe in vivo tests, in which the bioactivity of the precipitated non-glycosylated rh-BMP-2 was examined.
  • the in vivo assays used matrices with entrapped rh-BMP-2 in critical size bony defects in the rat. These defects do not spontaneously heal on their own. Therefore these models allow one to determine the osteogenic potential of a particular treatment. Schmitz JP, Clin Orthop 1986, 205, 299-308.
  • both a long bone model (5 mm full segmental femur defect) (Example 4) and a cranial model (8 mm defect) (Example 5) were employed. In each model, the healing potential of a fibrin matrix with rh-BMP-2 entrapped was compared to that for a fibrin matrix lacking rh-BMP-2.
  • Fibrin gels were polymerized using purified fibrinogen (Sigma) at 8 mg/mL and 2 U/mL thrombin at pH 7.4. Some of the gels included prokaryotic rh-BMP-2 mixed into the solution before gelation. Calcium was added to increase the rate of gelation.
  • Fibrin gels were polymerized using purified fibrinogen (Sigma) at 8 mg/mL and 2 U/mL thrombin at pH 7.4. Some of the gels included prokaryotic rh-BMP-2 mixed into the solution before gelation. Calcium was added to increase the rate of gelation.
  • Fibrin gels which contained rh-BMP-2, contained either 1, 5 or 20 ⁇ g of rh-BMP-2 added to the polymerization mixture. These materials were explanted and tested at three weeks. All of the defects treated with 20 ⁇ g of precipitated rh-BMP-2 were completely filled with woven bone and bone marrow (see FIG. 4A, column IV). The defects with 5 ⁇ g of rh-BMP-2 had nearly complete healing, with 90% of the original defect area filled with calcified tissue (see FIG. 4A, column III). The defects with 1 ⁇ g of rh-BMP-2 showed very good healing as well, with 73% of the defect area filled with new, woven bone (see FIG. 4A, column II).
  • Enzymatically degradable synthetic matrices were tested in the same cranial defect model as the fibrin matrices described above.
  • the synthetic gels were formed by reacting a four-armed PEG-vinylsulfone having a weight average molecular weight of 20 kDa with crosslinking linear peptides, such as GCRPQGIWGQDRC (SEQ ID NO: 1), that contain multiple cystines at pH 7.5.
  • the PEG-vinylsulfone was dissolved in a TEOA buffer (0.3 M, pH 8.0) to forma 10% (wt/wt) solution.
  • the peptide was dissolved in the same buffer.
  • the components for the gels were prepared such that the final concentration obtained were 8 mg/ml fibrinogen, 2.5 mM Ca ++ , 10 NIH Units/ml of thrombin and 600 ⁇ g non-glycosylated rh-BMP-2/ml gel. Gelation began after mixing and injection of the components into the fracture site. Gelation time was 30-60 seconds. The contamination of the components with small amounts of blood in the wound did not influence the gelation properties.
  • 0 corresponded with no mineralized tissue in the joint gap visible
  • 1 corresponded with visible mineralized tissue in the joint gap
  • 2 corresponded with bony bridging of the joint gap
  • 3 corresponded with remodeled bony bridging with absent subchondral plate.
  • 59% of the spongiosa group reached a score of 2 or greater in all joints (the standard level indicating clinical healing), whereas 87.5% of the non-glycosylated rh-BMP-2 group reached a score of 2 or greater.
  • Dog 10 with the bilateral panarthrodesis had a post operative period without complications.
  • the first control radiograph after 4 weeks showed no visible difference in the bony healing of the two arthrodesis (score 1 for all joints).
  • the spongiosa-treated carpus had no improvement (score 1), whereas the non-glycosylated rh-BMP-2 treated carpus improved to a score of 2.
  • the score for the spongiosa treated leg was 2, and 2.33 for the non-glycosylated rh-BMP-2 treated arthrodesis.
  • the components for the gels were prepared such that the final concentration obtained were 8 mg/ml fibrinogen, 2.5 mM Ca ++ , 10 NIH Units/ml of thrombin and 600 ⁇ g non-glycosylated rh-BMP-2/ml gel. Gelation was allowed after mixing and injection of the components into the fracture site. Gelation time was 30-60 seconds. The contamination of the components with small amounts of blood in the wound did not influence the gelation properties.
  • Cat #3 suffered a very comminuted tibial fracture, which was stabilized by an external fixateur.
  • the tibia developed an atrophic nonunion with severe bone loss.
  • a 2.7 mm-plate was applied after shortening of the fibula to reduce the gap of the tibia.
  • the bone from the fibula was morselised and mixed in the fibrin with the non-glycosylated rh-BMP-2 to provide living cells to the fracture site.
  • the follow-up radiographs showed new bone formation and building up of a new cortex along the whole tibia.
  • the plate was removed, and 300 ⁇ g non-glycosylated rh-BMP-2 in fibrin was applied a second time.
  • the bone continued to augment, and six months after the first non-glycosylated rh-BMP-2 treatment, the fracture had healed.
  • Cat #4 had an open tibial fracture, which was stabilized by an external fixateur. After a mild, transient osteomyelitis, the bone of the tibia started to atrophy despite the stable conditions.
  • the fibrin/non-glycosylated rh-BMP-2 was applied through a stab incision in the fracture gap. After four weeks, no bony reaction was visible on the radiographs, but after seven weeks the fracture gap was smaller, and 4 months after the treatment the bone had bridged.
  • rh-PDGF-AB is known to contain a N-glycosylation site on the A chain, which is suggested to be used when the protein is expressed by a eukaryotic cell.
  • rh-PDGF-AB expressed in E. coli which is expected to be non-glycosylated, was used in this study, and is soluble in physiological pH up to 0.2 mg/mL.
  • Non-glycosylated PDGF-AB was tested at 2 ⁇ g in 50 ⁇ L of gel.
  • Fibrin gels were polymerised using a modified formulation of TissucolTM (Baxter), and the synthetic gel was formed of 4-armed PEG-acrylate cross-linked with 2-armed PEG-thiol.
  • the gels were washed in buffered saline (PBS 0.01 M, pH 7.4, with 0.1% BSA), and the wash was changed after 12 hours. The amount of therapeutic molecules released in the wash was then determined by ELISA.

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JP4566515B2 (ja) 2010-10-20
MXPA03009760A (es) 2005-10-05
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ATE420670T1 (de) 2009-01-15
ES2321068T3 (es) 2009-06-02
WO2002085422A1 (en) 2002-10-31
CA2681952A1 (en) 2002-10-31
DE60230873D1 (de) 2009-03-05

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