Classifications

• • 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
  • A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
  • A61L33/06—Use of macromolecular materials
  • A61L33/12—Polypeptides, proteins or derivatives thereof, e.g. degradation products thereof
• • 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
• • 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
  • A61L27/28—Materials for coating prostheses
• • 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
  • A61L27/28—Materials for coating prostheses
  • A61L27/30—Inorganic materials
• • 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
  • A61L27/28—Materials for coating prostheses
  • A61L27/30—Inorganic materials
  • A61L27/306—Other specific inorganic materials not covered by A61L27/303 - A61L27/32
• • 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
  • A61L27/28—Materials for coating prostheses
  • A61L27/34—Macromolecular materials
• • 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
  • 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
  • A61L27/56—Porous materials, e.g. foams or sponges
• • 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

• Implantable prosthetic devices have been used in the surgical repair or replacement of internal tissue for many years. The efficacy of many types of implants is primarily dependent- upon the surrounding tissue's adaptive reformation around and ability to bond to the implant surface. In orthopedic implants in particular, the geometry and the quality of bone reformation determines how much load the bone can resist. Orthopedic implants include a wide variety of devices, each suited to fulfill particular medical needs. Examples of such devices are hip joint replacement devices, knee joint replacement devices, shoulder joint replacement devices, and pins, braces and plates used to set fractured bones. Some contemporary orthopedic implants, including hip and knee components, use high performance metals such as cobalt-chrome and titanium alloy to achieve high strength. These materials are readily fabricated into the complex shapes typical of these devices using mature metal working techniques including casting and machining.
• At least two other methods are currently employed for bone and joint replacement and repair. Those methods include: (1) the use of grouting materials such as poly(methyl methacrylate) (PMMA) as bone cement between the bone and the prosthesis; and (2) direct opposition of bone tissue onto porous and non-porous implant surfaces.
• PMMA poly(methyl methacrylate)
• the latter method is known as the "cementless implant method.”
• a prosthesis is coated with hydroxyapatite which is a major inorganic component of bone.
• the hydroxyapatite-coated prosthesis is then implanted in the bone cavity.
• the hydroxyapatite which is a calcium salt, is believed to facilitate osteointegration with the bone tissues.
• layers of hydroxyapatite can be detected between the prosthesis and the bone tissues.
• the invention is an improved implantable prosthetic device coated with a bioactive molecule.
• the prosthetic device provided according to the invention is convenient and simple to prepare.
• the bioactive molecules are directly coupled to the prosthetic device surface through a gold-sulfide bond using simple solution chemistry techniques.
• Prior art methods for modifying the surface of biomaterials were complex and cumbersome. For instance, in order to conjugate a molecule to a polymeric surface, the surface would first have to be modified to add a functional group to which the molecule could bind. In some cases the molecule would require the addition of a linking group which is capable of reacting with the functional group.
• the invention is a prosthetic device including a shaped substrate having a substrate surface, for implantation in a mammal, a layer of gold attached to the substrate surface and defining a tissue contacting surface, and a bioactive molecule bound to the gold layer.
• the shaped substrate can be, for example, a polymer, a metal, a plastic, a fabric, a ceramic, a biological material, or a composite of two or more materials.
• the gold layer may be any thickness but preferably the gold layer has a thickness of about 10 to 1000 Angstroms.
• the bioactive molecule in turn can form a monolayer on the surface of the gold which, depending on the size of the bioactive molecule, is about 1 to 500 Angstroms in thickness.
• the bioactive molecule can be virtually any molecule which can be attached to the gold layer and which can affect favorably the implant in its local environment once implanted.
• the bioactive molecule therefore, can be natural or synthetic including a protein, a peptide, a protein analog, a sugar, a lipid, a glycol protein, a glycolipid or a nucleic acid.
• the bioactive molecule is selected from the group consisting of a cell modulating molecule, a chemotactic molecule, an anticoagulant moleucle, an antithrombotic molecule, an anti-tumor molecule, an anti-infectious molecule, a growth potentiating molecule, and an anti-inflammatory molecule.
• the cell modulating molecule is selected from the group consisting of an anti-integrin antibody, a bone morphogenic protein, an integrin binding protein, and a cadherin binding protein.
• the chemotactic molecule is an extracellular matrix molecule selected from the group consisting of collagen, fibronectin, laminin, and proetoglycan.
• the anti-tumor molecule is selected from the group consisting of methotrexate, adriamycin, cyclophosphamide, and taxol.
• the anti-infectious molecule is selected from the group consisting of antibiotics such as penicillin according to another embodiment.
• the growth potentiating molecule is selected from the group consisting of growth factors such as PDGF, EGF, FGF, TGF, NGF, CNTF, and GDNF.
• the anti-inflammatory molecule is selected from the group consisting of steroidal and non-steroidal compounds.
• the layer of gold can be attached directly to the substrate surface.
• the layer of gold is attached to the substrate surface via attachment to an intermediate layer, such as a layer of titanium intermediate the gold layer and the substrate surface.
• the surface of the prosthetic device is formed of a porous material, wherein the layer of gold creates a gold surface that has projections and indentations and wherein the layer of gold has an approximately uniform thickness across the surface of the porous material.
• the invention is a prosthetic device including a shaped substrate having a substrate surface, for implantation in a mammal, a layer of gold attached to the substrate surface and defining a tissue contacting surface, and a bioactive peptide bound to the gold layer.
• the shaped substrate can be, for example, a polymer, a metal, a plastic, a fabric, a ceramic, a biological material, or a composite of two or more materials.
• the gold layer may be any thickness but preferably the gold layer has a thickness of about 10 to 1000 Angstroms.
• the bioactive peptide forms a monolayer on the surface of the gold which, depending on the size of the peptide, is about 1 to 500 Angstroms in thickness.
• the bioactive peptide can be any peptide which can be attached to the gold layer and which can affect favorably the implant in its local environment. It can be natural or synthetic.
• the bioactive peptide is selected from the group consisting of a cell modulating peptide, a chemotactic peptide, an anticoagulant peptide, an antithrombotic peptide, an anti- tumor peptide, an anti-infectious peptide, a growth potentiating peptide, and an anti- inflammatory peptide.
• the cell modulating peptide is selected from the group consisting of an anti-integrin antibody fragment, a cadherin binding peptide, a bone morphogenic protein fragment, and an integrin binding peptide.
• the cell modulating peptide is a integrin binding peptide which is selected from the group consisting of RGDC, RGEC, RGDT, DGEA, DGEAGC, EPRGDNYR, RGDS, EILDV, REDV, YIGSR, SIKVAV, RGD, RGDV , HRNRKGV, KKGHV, XPQPNPSPASPVVVGGGASLPEFXY, and ASPVVVGGGASLPEFX.
• the peptides also may be any functionally active fragment of the proteins disclosed herein as being bioactive molecules useful according to the invention.
• the chemotactic peptide is selected from the group consisting of functionally active fragments of collagen, fibronectin, laminin, and proteoglycan.
• the anti-tumor peptide is selected from the group consisting of functionally active fragments of protein anti- tumor agents.
• the anti-infectious peptide is selected from the group consisting of functionally active fragments of the protein anti-infectious agents according to another embodiment.
• the growth potentiating peptide is selected from the group consisting of functionally active fragments of PDGF, EGF, FGF, TGF, NGF, CNTF, GDNF, and type I collagen related peptides.
• the anti-inflammatory peptide is selected from the group consisting of functionally active fragments of anti-inflammatory agents.
• the layer of gold can be attached directly to the substrate surface.
• the layer of gold is attached to the substrate surface via attachment to an intermediate layer, such as a layer of titanium intermediate the gold layer and the substrate surface.
• the surface of the prosthetic device is formed of a porous material, wherein the layer of gold creates a gold surface that has projections and indentations and wherein the layer of gold has an approximately uniform thickness across the surface of the porous material.
• the invention in another aspect is a prosthetic device including a shaped substrate formed of a textured material having a substrate surface with first projections and first indentations and a layer of gold attached to the substrate surface of the textured material, wherein the layer of gold creates a gold surface that has second projections and second indentations corresponding to the first projections and first indentations.
• the layer of gold has an approximately uniform thickness across the substrate surface of the textured material.
• the textured material is a porous material such as a porous titanium material, a porous polymer, or any other non-fabric porous material.
• the textured material is a polymer.
• the gold layer has a thickness of about 10 to 1000 Angstroms.
• the prosthetic device also includes a layer of bioactive peptide attached to the gold surface through a gold-sulfide bond.
• the invention is a prosthetic device including a shaped substrate having a substrate surface, a layer of gold attached to the substrate surface, and an RGDC peptide attached to the gold layer through a gold-sulfide bond.
• the shaped substrate is a polymer, a metal, a plastic, a fabric, a ceramic, a biological material, or a composite of two or more materials.
• the gold layer has a thickness of about 10 to 1000 Angstroms.
• the bioactive peptide forms a layer about 1 to 500 Angstroms in thickness.
• the layer of gold is attached directly to the substrate surface in one embodiment.
• the layer of gold is attached to the substrate surface via attachment to a layer of titanium intermediate the gold layer and the substrate surface.
• the surface of the prosthetic device is formed of a porous material, wherein the layer of gold creates a gold surface that has projections and indentations.
• the layer of gold has an approximately uniform thickness across the surface of the porous material.
• Figure 1 is a graph depicting the observed reflectivity change upon incubation of a clean gold surface with a 0.2 mM solution of the RGDC peptide
• Figure 2 is a graph depicting the SPR spectra taken in an air ambient before and after adsorption of the RGDC peptide layer; and Figure 3 is a graph depicting alkaline phosphatase activity from osteoblasts cultured on
• an implantable device could be coated with a bioactive molecule by first coating a substrate with a gold layer and then attaching the bioactive molecule through a simple reaction to the gold layer by forming a gold-sulfide bond.
• Prior art methods for attaching molecules to the surface of materials are cumbersome.
• the surface of the prosthetic device would have to be modified and would most likely require the addition of coupling reagents, making the preparation of such devices expensive, time consuming, and impractical.
• the preparation of metal implants having molecules attached to surfaces of the implants has been a difficult challenge in the prior art because most metal surfaces have oxide layers which make binding of coupling agents difficult.
• the implantable prosthetic device coated with bioactive molecules disclosed herein is prepared by a simple technique for coupling bioactive molecules to biomaterial surfaces.
• bioactive molecule is any biologically active molecule which includes a sulfhydryl group or to which a sulfhydryl group can be attached directly or indirectly. Examples are a peptide, protein (e.g., apoprotein, glycoprotein, antigen and antibody), a protein analog containing at least one non-peptide linkage in place of a peptide linkage, a nucleic acid, etc.
• Nucleic acids include nucleotides; oligonucleotides; and their art-recognized and biologically functional analogs and derivatives including, for example, oligonucleotide analogs having phosphorothioate linkages.
• Preferred bioactive molecules include a cell modulating molecule, a chemotactic molecule, anticoagulant moleucle, antithrombotic molecule, an anti-tumor molecule, an anti- infectious molecule, a growth potentiating molecule, and an anti-inflammatory molecule.
• a cell modulating molecule as used herein is a molecule that interacts with a cell and modifies the cell in any way e.g. alters gene expression, such as bone morphogenic protein, anti- integrin antibodies, integrin binding protein, and cadherin binding protein.
• a chemotactic molecule as used herein is a molecule which attracts cells to a surface or aids in a cell's attachment to a surface and includes extracellular matrix proteins such as collagen, fibronectin, laminin, and proetoglycan.
• An anti-tumor molecule as used herein is a molecule which decreases or prevents a further increase in growth of a tumor and includes anti-cancer agents such as Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adriamycin; Adozelesin; Aldesleukin;
• anti-cancer agents such as Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adriamycin; Adozelesin; Aldesleukin;
• Anthramycin Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat;
• Benzodepa Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone;
• Caracemide Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin;
• Cedefingol Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate;
• Cyclophosphamide Cytarabine; dacarbazine; Dactinomycin; Daunorubicin Hydrochloride;
• Fazarabine Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine;
• Interferon Alfa-2a Interferon Alfa-2b; Interferon Alfa-nl; Interferon Alfa-n3; Interferon Beta- 1 a; Interferon Gamma- 1 b; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole;
• Leuprolide Acetate Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate;
• Mitomalcin Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid;
• Nocodazole Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone
• Procarbazine Hydrochloride Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine;
• Temoporfin Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa;
• Vincristine Sulfate Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate;
• An anti-infectious molecule as used herein is a molecule which reduces the activity of or kills a microorganism and includes Aztreonam; Chlorhexidine Gluconate; Imidurea; Lycetamine;
• Nibroxane Pirazmonam Sodium; Propionic Acid ; Pyrithione Sodium; Sanguinarium Chloride ;
• Carbenicillin Potassium Carumonam Sodium; Cefaclor; Cefadroxil; Cefamandole; Cefamandole
• Cefmenoxime Hydrochloride Cefmetazole; Cefmetazole Sodium; Cefonicid Monosodium;
• Cefonicid Sodium Cefoperazone Sodium; Ceforanide; Cefotaxime Sodium; Cefotetan;
• Cephaloglycin Cephaloridine; Cephalothin Sodium; Cephapirin Sodium; Cephradine;
• Cetocycline Hydrochloride Cetophenicol; Chloramphenicol ; Chloramphenicol Palmitate ; Chloramphenicol Pantothenate Complex ; Chloramphenicol Sodium Succinate; Chlorhexidine
• Dicloxacillin Sodium Dihydrostreptomycin Sulfate; Dipyrithione; Dirithromycin; Doxycycline;
• Enoxacin Epicillin; Epitetracycline Hydrochloride; Erythromycin; Erythromycin Acistrate;
• Erythromycin Estolate Erythromycin Ethylsuccinate; Erythromycin Gluceptate; Erythromycin Lactobionate; Erythromycin Propionate; Erythromycin Stearate; Ethambutol Hydrochloride;
• Tromethamine Fumoxicillin; Furazolium Chloride; Furazolium Tartrate; Fusidate Sodium;
• Fusidic Acid Gentamicin Sulfate; Gloximonam; Gramicidin; Haloprogin; Hetacillin; Hetacillin
• Lincomycin Lincomycin Hydrochloride; Lomefloxacin; Lomefloxacin Hydrochloride;
• Lomefloxacin Mesylate Loracarbef; Mafenide; Meclocycline; Meclocycline Sulfosalicylate;
• Penicillin V Penamecillin; Penicillin G Benzathine; Penicillin G Potassium; Penicillin G Procaine; Penicillin G Sodium; Penicillin V; Penicillin V Benzathine; Penicillin V Hydrabamine; Penicillin V
• Ramoplanin Ranimycin; Relomycin; Repromicin; Rifabutin; Rifametane; Rifamexil; Rifamide;
• Sulfamerazine Sulfameter; Sulfamethazine; Sulfamethizole; Sulfamethoxazole;
• Sulfathiazole Sulfazamet; Sulfisoxazole; Sulfisoxazole Acetyl; Sulfisoxazole Diolamine;
• Tetracycline Phosphate Complex Tetroxoprim; Thiamphenicol; Thiphencillin Potassium;
• Trisulfapyrimidines Trisulfapyrimidines; Troleandomycin; Trospectomycin Sulfate; Tyrothricin; Vancomycin; Vancomycin Hydrochloride; Virginiamycin ; Zorbamycin; Difloxacin Hydrochloride ; Lauryl
• a growth potentiating molecule as used herein is a molecule which stimulates growth of a cell and includes growth factors such as PDGF, EGF, FGF, TGF, NGF, CNTF, and GDNF.
• An anti-inflammatory molecule as used herein is a molecule which reduces an inflammatory response and includes steroidal and non-steroidal compounds;Alclofenac;
• Alclometasone Dipropionate Algestone Acetonide; Alpha Amylase; Amcinafal; Amcinafide;
• Amfenac Sodium Amiprilose Hydrochloride; Anakinra; Anirolac ; Anitrazafen; Apazone;
• Clobetasone Butyrate Clopirac
• Cloticasone Propionate Cormethasone Acetate
• Cortodoxone
• Deflazacort Desonide; Desoximetasone; Dexamethasone Dipropionate; Diclofenac Potassium; Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium; Difiunisal ; Difluprednate;
• Halcinonide Halobetasol Propionate; Halopredone Acetate; Ibufenac ; Ibuprofen; Ibuprofen
• Ibuprofen Piconol Aluminum
• Ilonidap Indomethacin
• Indomethacin Sodium Indoprofen ;
• Indoxole Intrazole; Isoflupredone Acetate; Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride ; Lornoxicam ; Loteprednol Etabonate; Meclofenamate Sodium; Meclofenamic
• Naproxol Naproxol ; Nimazone; Olsalazine Sodium; Orgotein ; O ⁇ anoxin; Oxaprozin; Oxyphenbutazone;
• Salcolex Salnacedin; Salsalate ; Sanguinarium Chloride ; Seclazone ; Sermetacin; Sudoxicam;
• An anticoagulant moleucle as used herien is a molceule that prevents clotting of blood and includes but is not limited to Ancrod; Anticoagulant Citrate Dextrose Solution ;
• Heparin Calcium Heparin Sodium; Lyapolate Sodium; Nafamostat Mesylate ; Phenprocoumon;
• Tinzaparin Sodium Warfarin Sodium.
• An antithrombotic moleucle as used herien is a molceule that prevents formation of a thrombus and includes but is not limited to Anagrelide Hydrochloride; Bivalirudin ; Dalteparin Sodium ; Danaparoid Sodium; Dazoxiben Hydrochloride; Efegatran Sulfate; Enoxaparin
• the bioactive molecule is a bioactive peptide.
• a "bioactive peptide” as used herein refers to oligopeptides having a chain of less than or equal to fifty amino acids and which is capable of performing a desired biological function.
• the bioactive molecule includes a cell modulating peptide, a chemotactic peptide, an anticoagulant peptide, an antithrombotic peptide, an anti-tumor peptide, an anti-infectious peptide, a growth potentiating peptide, and an anti-inflammatory peptide.
• a cell modulating peptide includes, for example, an antibody fragment or an integrin binding peptide.
• Bioactive peptides include peptide fragments of the proteins which are bioactive molecules disclosed herein and having the functional properties of those proteins.
• a preferred use for the peptide-coated implantable device of the invention is for enhancing and/or accelerating bone growth in areas of damaged bone or in bone replacement surgery.
• Bone and joint replacement surgeries are commonly used, for instance, to relieve pain, improve function, and enhance the quality of life for patients with medical conditions caused by osteoarthritis, rheumatoid arthritis, post-traumatic degeneration, avascular necrosis, and other aging-related conditions.
• the prosthetic device of the invention which is coated with bioactive peptides that enhance or accelerate bone growth significantly improve the ability of an implant to remain attached to the bone surface.
• Preferred integrin binding peptides which perform this function are RGDC, RGEC, RGDT, DGEA, DGEAGC, EPRGDNYR, RGDS, EILDV, REDV, YIGSR, SIKVAV, RGD, RGDV, and HRNRKGV.
• Anti-infectious peptides include include antibiotic peptides such as those disclosed in U.S. Patent No. 5,602,097. Anti-tumor and anti-infectious peptides are also disclosed in U.S. Patent No. 5,516,755.
• U.S. Patent No. 5,484,885 discloses chemotactic, antibiotic, and lipopolysaccharide binding peptide fragments of CAP37 protein. These peptide sequences are approximately five consecutive amino acids long.
• US Pat No. 5,354,736 discloses several collagen type I related peptides which are useful for promoting growth. Growth potentiating peptides also include low molecular weight tibial growth potentiating peptides such as those disclosed in U.S. Patent No. 5,576,301. These peptides are useful for potentiating tibial growth. These peptides have the following sequences: XPQPNPSPASPVVVGGGASLPEFXY and ASPVVVGGGAS
• Bioactive peptides such as those disclosed above are well known in the art.
• Other bioactive peptides useful according to the invention may be identified through the use of synthetic peptide combinatorial libraries such as those disclosed in Houghton et al., Biotechniques, 13(3):412-421 (1992) and Houghton et al, Nature, 354:84-86 (1991) or using phage display procedures such as those described in Hart, et al., J Biol. Chem. 269:12468 (1994).
• Hart et al. report a filamentous phage display library for identifying novel peptide ligands for mammalian cell receptors.
• phage display libraries using, e.g., Ml 3 or fd phage, are prepared using conventional procedures such as those described in the foregoing reference.
• the libraries display inserts containing from 4 to 80 amino acid residues.
• the inserts optionally represent a completely degenerate or a biased array of peptides.
• Ligands that bind selectively to a specific molecule such as a cell surface receptor are obtained by selecting those phages which express on their surface a ligand that binds to the specific molecule.
• Ligands that possess a desired biological activity can be screened in known biological activity assays and selected on that basis.
• phages that exhibit the binding characteristics are further characterized by nucleic acid analysis to identify the particular amino acid sequences of the peptides expressed on the phage surface and the optimum length of the expressed peptide to achieve optimum biological activity.
• peptides can be selected from combinatorial libraries of peptides containing one or more amino acids. Such libraries can further be synthesized which contain non-peptide synthetic moieties which are less subject to enzymatic degradation compared to their naturally-occurring counte ⁇ arts.
• U.S. Patent No. 5,591,646 discloses methods and apparatuses for biomolecular libraries which are useful for screening and identifying bioactive peptides. Methods for screening peptides libraries are also disclosed in U.S. Patent No. 5,565,325.
• Peptides obtained from combinatorial libraries or other sources can be screened for functional activity by methods known in the art. For instance when the peptide is a cell modulating peptide, and in particular an integrin binding peptide, one of ordinary skill in the art can easily determine whether the peptide will modulate bone cell activity by performing the in vitro studies set forth in example 2 to measure osteoblast differentiation. Likewise, similar experiments can be conducted for other types of cells using cell specific markers of differentiation or growth.
• the type of assay of course, used for a particular peptide depends on the source of the peptide. For instance if a peptide is a fragment of an anti -tumor molecule, the peptide should be tested for functional activity in an anti-tumor assay. Those of skill in the art can easily choose an appropriate assay for testing functionality of a particular peptide.
• bioactive molecules useful according to the invention are commercially available frorn many sources and methods for making these molecules also are well known in the art.
• Bioactive peptides and proteins may easily be synthesized or produced by recombinant means. Such methods are well known to those of ordinary skill in the art.
• Peptides and proteins can be synthesized for example, using automated peptide synthesizers which are commercially available.
• the peptides and proteins can be produced by recombinant techniques by inco ⁇ orating the DNA expressing the peptide into an expression vector and transforming cells with the expression vector to produce the peptide.
• bioactive molecule is bound to a gold surface.
• many attempts have been made in the prior art to coat peptides, proteins and other biomaterials on various surfaces, each of these techniques has required the use of complex coupling techniques and surface modification including the use of coupling agents and linkers.
• bioactive molecules can be attached to a prosthetic device via a gold surface, through a simple technique that results in the formation of a bond between a gold and a sulfhydryl group.
• the bond that forms between a sulfhydryl group and gold only requires the interaction between the sulfhydryl group and the gold in a solution. The interaction does not require coupling agents or linkers or surface activation or modification of the gold.
• the molecule is added to the gold surface using simple solution chemistry techniques, e.g., simply exposing the gold surface to a solution of molecule in a solvent such as ethanohwater.
• This approach is simple and is non-line of sight dependent.
• a technique which is line of sight dependent only coats an external surface and does not coat internal pores or interstices.
• Non-line of sight dependent methods are capable of coating the internal surface area such as pores. This technique produces an evenly coated layer of molecule on any type of device, even those having a porous, spongy, or textured surface.
• Bioactive molecules can be attached to gold surfaces directly or via spacers. If direct, then bioactive molecules must have (or must be modified to have) a sulfhydryl group. If indirect, the bioactive molecule may or may not have sulfhydryl, but the spacer will have a sulfhydryl. In this instance the spacer is attached to the gold surface and the bioactive molecule is attached to the spacer, before or after attaching of the spacer to the gold surface. Proteins or peptides having endogenous cysteine groups already have a cysteine within the molecule and do not require the addition of another sulfhydryl group.
• a protein or peptide has more than one cysteine and those cysteines have formed di-sulfide bridges the molecule can be subjected to reducing agents to ensure that the sulfhydryl group is free and available.
• Proteins or peptides without endogenous cysteine groups can easily be manipulated to inco ⁇ orate a sulfhydryl group. For instance, peptides and proteins can be subjected to site directed mutagenesis to prepare a cysteine containing protein or peptide.
• a cysteine can be added to either the N-terminal or C-terminal of the peptide or protein or inco ⁇ orated within the peptide or protein or within a branch of the peptide or protein.
• a cysteine may be added anywhere in the peptide or protein that does not affect the biological activity of the peptide or protein. This is demonstrated schematically as follows:
• C wherein X, Y, and Z are any amino acid and C is cysteine.
• a cysteine group is added to either the C-terminal or the N-terminal of the peptide. More preferably, the cysteine group is on the C terminal region of the peptide.
• Proteins or peptides without endogenous cysteine and other non-sulfhydryl containing molecules can easily be manipulated to inco ⁇ orate a non-cysteine sulfhydryl group.
• sulfhydryl groups can be introduced into the molecules having a primary amine (or modified to have a primary amine) by reaction of the primary amine in the molecule with 2- iminothiolanc or Traut's reagent, or other commercially available reagents.
• a variety of commercially available reagents for coupling sulfliydryl groups to molecules are available from Pierce Chemical, Co ⁇ ., such as Traut's reagent (Product No. 26101), SATA (Product No.
• Traut's reagent is a water soluble reagent which reacts with primary amines at pH 7-10 to introduce sulfliydryl groups, as disclosed in Schram and Dulffer, Physiol. Chem., 358, 137-139 (1977). Traut's reagent has the following structure:
• SATA is a reagent which adds protected sulfhydryls to molecules by reacting with primary amines.
• SATA has the following chemical structure:
• SPDP which includes LC-SPDP and Sulfo-LC-SPDP also is capable of adding a sulfhydryl group to have the following structures:
• the oact ve molecule is prepared with a sulfhydryl group at, for example, the carboxyl (C) or amino (N) terminus and then is coupled to the gold surface.
• a spacer is synthesized with a sulfhydryl group, preferably at or near one end, and then this spacer is attached at this end to the gold surface and via a different functional group to the bioactive molecule.
• the spacer molecule may be coupled for example to the terminal amine group or carboxyl group of the bioactive peptide or protein.
• Spacer molecules can be selected, for example, which contain (or which can be modified to contain) a functional group that is reactive with the peptide or protein N-terminal amine group and allowing the functional group and the peptide or protein N-terminal amine to form a linkage in accordance with art-recognized procedures. See, e.g., March, J., Advanced Organic Chemistry, 4th Ed., New York, NY, Wiley and Sons, 1985), pp.326-1120.
• the spacer molecule may be coupled to a reactive group in the C-terminus of the bioactive peptide or protein. Additionally the spacer molecule may be coupled to a branch of a molecule or an internally active portion of a molecule or any end group.
• Thiol or amide groups may be added at any nucleotide of a nucleic acid.
• the amine group may be added so as to provide a point of attachment for a sulfhydryl group by the above- described reagents.
• Nucleic acids may also be synthesized with groups such as amine groups.
• the bioactive molecule is bound to a layer of gold which is attached to a substrate surface of a shaped substrate.
• the layer of gold covers all or part of the prosthetic device to define a tissue contacting surface.
• the tissue contacting surface is the surface of the gold to which the molecules are bound.
• the layer of gold may be extremely thin or it may be thick.
• the layer of gold may actually be the entire prosthetic device. In this case the layer of gold would encompass the shaped substrate as well.
• the layer of gold is thin because of the high cost of gold.
• the layer of gold is attached to the shaped substrate surface by any means known in the art. For instance, the gold layer can be added to the implant using evaporation, electroplating, sputtering or electrodeposition.
• the gold can be applied in a thin layer to the surface of the implant.
• the gold is attached to the substrate by electroplating or evaporation. Electroplating produces a gold layer which is non-line of site dependent. Using electroplating, therefore, a gold layer can be produced on an uneven surface such that the uneven nature of the surface is maintained.
• a shaped substrate as used herein is a material which has the shape of an implantable prosthetic.
• the selection of the shape of the prosthetic is governed by the physical requirements of space, geometry and function at the region where the implant is to be positioned in the body. Implants can be made available in a range of sizes to fit the varying sizes in the patient population.
• the bioactive molecule coating is on and within the pores of an implantable prosthesis of the type where tissue ingrowth is contemplated, wherein the bioactive molecule encourages the ingrowth of the tissue into the pores or facilitates attachment of tissue to the prosthetic.
• the coating is on a typical prosthesis or on a 'temporary implant', such as a long term but temporary catheter, and the coating is of an antibacterial agent to prevent colonization upon the prosthesis or catheter.
• the invention is useful in connection with prosthetic devices such as bone or joint replacement or repair prosthetics, vascular prostheses, including woven prostheses, catheters for implantation and the like. Virtually any implantable tissue contacting surface may be modified as described herein.
• the shaped substrate may be made from any material ordinarily used to prepare implants.
• the shaped substrate may be made from any of a wide variety of metals, such as, pure titanium, titanium alloy, stainless steel, cobalt-chrome alloy, and gold.
• the shaped substrate may also be made from polymeric matrix composites, such as continuous filament carbon, graphite, glass and aramid fibers embedded within a polymer matrix, such as polysulfone, polyether-ether-ketone, polyether-ketone-ketone, polyimide, epoxy or polycyanate, polymers including polyethylene, polyetheretherketone (PEEK), polypropylene, polymethylmethacrylate, polyamides, and polyester.
• polymeric matrix composites include but are not limited to polyethylene films, ultra-high molecular weight polyethylene films and fibers, polyvinylidene fluoride films, poly (methyl methacrylate) films, polystyrene films, nylon 12 films and fibers, various polyesters and polyacrylates, polyetherethereketones, aromatic polyamides, polyethylene terephthalate fibers and films, poly (tetramethylene terephthalate) films, and polyether-esters of poly (tetramethylene terephthalate).
• the prosthetic device of the invention is useful for implantation in mammals.
• Mammals herein means humans, cats, dogs, mice, hamsters, pigs, goats, primates, horses, cows, and sheep.
• a preferred prosthetic device of the invention is a shaped substrate having a substrate surface, a layer of gold attached to the substrate surface, and an RGDC peptide attached to the gold layer through a gold-sulfide bond.
• the RGD peptide is a peptide found in many extracellular matrix proteins which is known to bind ⁇ 5 ⁇ , and ⁇ v ⁇ 3 integrin receptors. RGD attached to surfaces has been demonstrated to increase osteoblast attachment to the surface. It is preferred that orthopedic prosthetic devices are coated with RGDC.
• the prosthetic device with the bioactive molecule attached to the surface has been found to be extremely stable and as a result can be stored for extended periods of time.
• the stability of the device is important because it enables the device to be prepared in advance and shipped to a medical institution where it can be stored for future implantation.
• medical institutions can store many prosthetic devices having various molecules already coated on the surface for various applications.
• the prosthetic device of the invention may also be prepared and stored without the bioactive molecule attached to the device.
• the bioactive molecule can then be added at a later time point prior to use.
• the step of adding the bioactive molecule to the gold surface is simple and quick and may easily be performed immediately prior to a surgical process.
• the prosthetic device of the invention also includes a shaped substrate formed of a textured material and having a gold layer attached to the surface. More specifically the shaped substrate has a substrate surface with first projections and first indentations and a layer of gold is attached to the substrate surface of the textured material such that the layer of gold creates a gold surface that has second projections and second indentations corresponding to the first projections and indentations.
• the layer of gold optionally has an approximately uniform thickness across the substrate surface of the textured material.
• a "textured material” as used herein is a non-fabric material having small (about 1-1000 microns in size) interstices throughout.
• the shaped substrate may be made entirely of a textured material or may optionally be made of a non-textured material but having a surface which is coated with a textured material to produce a textured surface.
• the textured material is a porous material such as a porous titanium material, a porous polymer, or any other non-fabric porous material. Porous metal surfaces have been created by plasma spraying (U.S. Pat. No.
• the shaped substrate may be made of a non-textured material but having a surface which is at least partially coated with a textured material to produce a partially textured surface.
• the invention also encompasses a prosthetic device having a shaped substrate made from a non-textured material but at least partially coated with a textured material on which a layer of gold is attached.
• the substrate surface of the textured material has projections and indentations.
• "Projections and indentations” as used herein are microscopic cavities on the surface of the substrate defining a 'rough' surface microscopically.
• a substrate surface is said to have projections and indentations if it has a substantial region that is mostly free of a flat smooth surface, but instead is characterized by numerous indentations and projections throughout the region, numerous cavities having a diameter between 1 micron and 1 millimeter, preferably between 20 microns and 900 microns.
• the gold layer attached to the textured material creates a gold surface that also has projections and indentations and that has an approximately uniform thickness across the substrate surface.
• the implantable prosthetic device of the invention has many advantages over uncoated implants and even over peptide-coated implants that do not have a gold surface.
• Peptides The peptides used in the following studies are set forth in Table I. All peptides were synthesized commercially (QCB, Hopkinton, MA) to a purity of 98% or greater by HPLC and mass spectrometry. Peptides being coupled to FEP (the control substrate) included G or GGGG spacer sequence on their N- or C-terminus. Peptides being coupled to gold coated surfaces included a CG or CGGG spacer sequence on their N- or C-terminus. Control peptides were fabricated using scrambled sequences or, if known, amino acid substitutions.
• the electron beam gun was activated and a 60 angstrom coating of Ti was put onto the cover slips.
• the Ti source was then rotated away as the gold source was rotated into place.
• a 500 angstrom layer of gold was applied.
• the samples were then removed from the system and kept under nitrogen or covered in Kimwipes and aluminum foil until ready for use.
• Cysteine terminated peptides were solubilized in a 1 :1 ethanol: distilled water solution at a concentration of 0.22 mM. The gold substrates were exposed to this solution for one hour. Plain gold controls were made by exposing samples to peptide-free ethanol rdistilled water for one hour. Reactions were carried out in the dark to protect the light-sensitive cysteine.
• FEP membranes with immobilized peptide are useful for comparison pu ⁇ oses.
• the FEP membranes were prepared using surface modification and coupling techniques.
• FEP films (Dupont) 25 micrometers thick were cut into discs with a lathe (1.76cm diameters) and cleaned by sonication in hexanes and methanol for 20 seconds each.
• Surface hydroxyl (OH) groups were added to cleaned FEP films by a radio frequency glow discharge (RFGD) process.
• RFGD radio frequency glow discharge
• the films were placed in a chamber and brought to a pressure of 100 millitorr.
• the chamber was filled with hydrogen and methanol vapor at 500 millitorr for 10 minutes.
• the pressure was again reduced to 100 millitorr and the radio frequency glow discharge was activated for 1 minute.
• the films were reacted with CDl (40mg/lml in DMSO) for 24 hours.
• CDl 40mg/lml in DMSO
• the solution was supplemented with N-Hydroxy-succinimide (NHS, Fluka) (1 mg/ml in DMSO) (Frost, 1981)
• NHS N-Hydroxy-succinimide
• the excess CDI NHS was rinsed off of the films with DMSO before applying the peptide solution.
• Films were placed in 0.22 M peptide in IM MES buffer (pH 5) for 48 hours (Hearn, 1987). Films were rinsed sequentially with IM MES buffer, IM NaCl, and distilled water. This stringent rinsing protocol was used to remove adsorbed vs. linked peptide from the surface.
• Contact Angle Contact angles were measured with ethylene glycol, glycerol, distilled water, and ethanol on a goniometer. Each fluid was placed on the substrate using a syringe with a 30 gauge needle. At least three measurements per drop were taken. The surface energy was calculated using E. Sacher's method (Ratner, 1988; Kaelble, 1974; Kaelble, 1970). Contact angle data provides information regarding the surface chemistry and surface energetics of the top 5 Angstroms of a polymer substrate. A bead of pure liquid with a known surface tension is placed on the polymer surface. The resulting bead angle is measured using a goniometer (an alternate technique is to use a Cahn microbalance).
• a hydrophobic surface causes liquid beading and a high contact angle while a more hydrophilic surface is wettable and a small contact angle is observed.
• a range of fluids with polar (i.e. water) to non-polar (i.e. decane) characteristics are tested.
• SPR Surface plasmon resonance
• TAMRA SE has the advantage of maintaining stability for weeks and is stable in pH's ranging from 4 to 9.
• the excitation and emission wavelengths of this compound are 546 ⁇ and 576 ⁇ , respectively.
• TAMRA SE is made up as a ImM solution in DMF. It is then mixed with a pH 8.5 sodium tetraborate buffer in a 1 : 1 ration for a final solution concentration of 0.5 mM. This is reacted with the samples on a stirrer plate for four hours. Rinsing is done overnight in 4 M urea + 0.6% Tween 60. b.
• peptide stability under physiologic conditions Various immobilized peptides, tagged with fluorescent probes, are exposed to tissue culture media, tissue culture media with 10% serum, and osteoblasts. After 1, 7, 14 and 28 days of culture, substrates are rinsed several times, and assessed for fluorescence using confocal microscopy.
• RFGD treated FEP showed a contact angle of 60-65° with water confirming the presence of polar hydroxyl groups.
• Figure 1 is a graph of the observed reflectivity change upon incubation of a clean gold surface.
• the SPR spectra of the RGDC layer was analyzed to obtain the thickness and refractive index of the peptide layer.
• Figure 2 depicts a theoretical curve for the RDGC layer which was generated using the above parameters.
• the film thickness value of 23-25 A indicates that the peptide molecules are in an upright orientation.
• Non-SH containing RGD failed to bind to the gold surface.
• Rat calvarial osteoblasts were used as a model system because they have been used extensively in in vitro for studies of bone cell differentiation. These cells undergo a predictable, temporal expression of biochemical and gene markers of the osteoblast phenotype over a three to four week period in culture (Aronow 1990, Harris 1994). Lian et al. have described three phases of osteoblast growth and differentiation in vitro (Breen 1994). The initial phase (days 1-6) involves active cell proliferation and increases in collagen type I gene expression. Matrix maturation occurs over the second week in culture and was accompanied by increased alkaline phosphatase mRNA expression and enzyme activity. The final phase involved cell aggregation into nodules with subsequent mineralization. This period included increased osteocalcin and osteopontin gene expression and protein synthesis. This standard sequence of osteoblast differentiation served as the reference by which experimental substrates were evaluated.
• Cells from the second and third digestions were pooled to form an osteoblast rich suspension. These cells were rinsed, pelleted and plated in MEM (Gibco) with 10% FBS (Hy clone) at a density of 6,510 cells per cm 2 (Lian, 1990). After confluence, the media was switched from MEM to a mineral rich BGJb media, to which 10% FBS, 50 mg/ml ascorbic acid, and lOmM Beta Glycerol Phosphate are added (Lian 1990). For sub-cultivated experiments, primary cells were expanded in T-75 flasks with MEM and 10% FBS.
• Peptides from Table I were synthesized as described above. Each peptide was coupled to a substrate at a concentration of 0.22M.
• Cell counting was performed by rinsing several times with DMEM, and using the MTT assay (see below) or by fixing with formalin and performing and performing counts in ten different high powered microscopic fields on each individual substrate.
• Cell Counting with MTT Assay Standard curves were prepared by plating rat calvarial osteoblasts at densities of 100,000, 50,000, 25,000, 10,000 and 5,000 cells/well. Cells were incubated in serum-free Dulbecco's Modified Eagle Media, i.e. DMEM (Gibco) for two hours. Then 3-[4.5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, i.e.
• MTT in media was added to a final concentration of 0.5 mg.ml.
• the plates were placed back in the incubator for three hours. Each well was then rinsed with Hanks' Balanced Salt and then 1 ml of 10% Sodium Dodecyl Sulfate, i.e. SDS (Sigma) was added. Cells were covered in aluminum foil to protect it from light and left at room temperature for 12 hours. The SDS solution was then removed, placed into cuvettes, and examined in a Beckman DU-65 spectrophotometer at a wavelength of 570nm.
• peptide-coated substrates For testing peptide-coated substrates, cells were plated at 50,000 cells per well plates. Attachment was evaluated at 20 minutes, 1 hour, 3 hours and 24 hours. At the conclusion of each time oint unattached cells were removed by rinsing three times with HBSS. MTT in serum- free media was added at a concentration of 0.5 mg/ml to perform cell counting and incubator for 3 hours to allow the cells to process the MTT. The plates were then removed and each well given a single HBSS rinse followed by addition of 1 ml of 10% SDS for cell lysis. After 12 hours the SDS solution was removed, placed into cuvettes, and examined in a Beckman DU-65 spectrophotometer at a wavelength of 570nm.
• Cells were harvested and specific radioactivity (cpm) measured using a scintillation counter. Briefly, cells were washed 3 times with ice-cold PBS to remove excess label, trypsinized, spun down into a pellet, and lysed with 120 ul phosphate buffered 1% nonidet P-40 (Sigma). One hundred microliters of each sample were mixed with 800 ul 0.2% BSA, 100 ul 75% trichloroacetic acid (TCA), and centrifuged. Supernatant was removed and the pellet was centrifuged again with 1 ml 7.5% TCA. The pellet was then solubilized in 900 ml 0.1 N NaOH at 37°C overnight and neutralized with 100 ul IN Hcl.
• TCA trichloroacetic acid
• Counts were normalized with DNA and expressed as cpm/ug DNA. All data was normalized using total DNA. A fluorometric DNA assay (Arronow, 1990) was performed on the remaining 20 ul aliquots of cell lysate using a TKO 100 mini-fluorometer (Hoefer, San Francisco, CA, USA) to normalize cell counts. Samples were incubated with benzimidazole (Hoechst 33258; Pharmacia Biotech, Piscataway, NJ) and fluorescence quantified. DNA content was obtained using a calf thymus DNA (Pharmacia Biotech) standard curve.
• Alkaline Phosphatase Activity Alkaline phosphatase (AP) activity of cell lysates was determined by an established enzymatic conversion assay using p-nitrophenol phosphate as a substrate (Spiess). The enzyme activity was expressed as nanomoles of p-nitrophenol produced per minute per milligram of protein (nmol/min mg protein). The protein content was determined using the Biorad protein assay kit (Biorad, Hercules, CA) using BSA as the standard.
• Alkaline Phosphatase Staining Osteoblasts were fixed in buffered 2% paraformaldehyde for 24 hours. Before staining, cells were rinsed in distilled water. Alkaline phosphatase staining was visualized by incubating the cells for 30 min in 0.1 M Tris HCL pH 8.5 containing 0.4 mg/ml naphthol AS-MX phosphate + 1 mg/ml Fast Blue BB salt. Cells were then rinsed in 1 M PBS and preserved in PBS glycerol. The intensity of osteoblasts stained with alkaline phosphatase was qualitatively assessed by counting the number of osteoblasts per lOx field.
• Osteocalcin RIA Osteocalcin levels were assessed after removing aliquots of conditioned media from cell cultures of experimental groups using a radioimmunoassay (RIA) for rat osteocalcin (rat osteocalcin kit, BTI, Stoughton, MA) according to a previously described method (Gundberg 1984). Purified rat osteocalcin, goat anti-rat osteocalcin antibody, normal goat nonimmune serum, donkey anti-goat 2nd antibody, RIA buffer and [1-125] rat osteocalcin were used as reagents for RIA as provided by BTI.
• RIA radioimmunoassay
• Extracellular Matrix Protein and Integrin Gene Expression by Northern Analysis During the course of bone development and metabolism, a variety of osteoblast growth and differentiation factors are known to be expressed in vitro (Ibaraki, 1992) and in vivo (Jingushi, 1991, Sandberg, 1993). Integrin gene expression is also modulated.
• RNA extraction RNAzolTM (Tel-Test, Friendswood, TX) reagent was added to the cell cultures removed of media and then shaken gently until a viscous, opaque liquid was seen. The contents were transferred to ice cold tubes to which chloroform was added and vortexed. After centrifugation for 10 min. at 10,000 ⁇ m, the top aqueous phase was re-extracted with fresh RNAzol and chloroform. After a series of re-extraction and centrifugations, the cell pellet was washed in 70% ethanol and resuspended in 50 ul TE buffer. The concentrations and purity of the RNA is measured with a spectrophotometer using the ratio of A 260 and A 280 .
• RNA gel RNA (15-20 ug) from specific experimental groups was separated on the basis of size with a denaturing 1.2% agarose (Formaldehyde) gel electrophoresis. All RNA gels were run for 3 hours at 100 V and photographed using an ultraviolet light source.
• RNA was then cross linked and baked on permanently onto the membrane (under a UV lamp and baked at 120° C for 15 minutes).
• Hybridization & Detection cDNA probes for rat alkaline phosphatase and rat osteocalcin were kindly provided by Dr. J. Lian (University of Massachusetts, Worcester, MA).
• the cDNA probes for ⁇ 5 and ⁇ l integrins were provided by Dr. E. Ruoslahti (Cancer Institute, La Jolla, CA).
• the cDNA probe for bone sialoprotein was provided by Dr. J. Sodek (University of Toronto, Toronto, Canada), while the cDNA probe for collagen was provided by Dr. B. Kream (University of Connecticut, New London, CT).
• the cDNA probes for GAPHDH, beta-actin, human osteopontin, and human osteonectin were obtained from the American Type Culture Collection (ATCC).
• the membranes were treated with various buffers (5X SSPE, 50% formamide, 2% SDS and 10X Dengardt's solution).
• the hybridized probes were radioimmunodetected through the use of 32 P.
• the membranes were then reacted with the cDNA probes and hybridized with the probes at 68° C overnight and washed through a series of (2X SCC & 0.1% SDS) solutions. After the washing steps, the membrane were exposed with x-ray film.
• the mount of RNA was quantified through comparison with the known amount of RNA transcript that was loaded in the lane on the gel. The size of the RNA molecule was calculated by measuring the distance migrated and comparing it to the standard. All mRNA hybridization experiments were performed twice with each cDNA probe. All cDNA was normalized to GAPDH.
• RCOBs in DMEM with 10% fetal bovine serum were plated at 25,000 per square centimeter. Visual analysis revealed higher levels of attachment at 20 minutes on the RGDC treated substrates. This was quantitatively confirmed using an MTT assay which showed that at 20 minutes there was 100% greater attachment to the RGDC surface compared with gold and RGD treated surfaces. Similar to the gold surface RCOBs showed much greater attachment when cultured on RGDC-FEP modified surfaces than on unmodified FEP.
• Osteocalcin was evaluated since it is a marker of bone cell differentiation and because radioimmunoassays (RIA) are commercially available (Arono, 1990; Gundberg, 1984; Ibaraki. 1992).
• RGD-FEP coupled membranes induced significantly higher levels of osteocalcin synthesis compared with all other groups.
• the unique ability of RGD-FEP coated substrates to enhance osteocalcin synthesis is consistent with increased RCOB mRNA expression seen at day 14.
• the RGE is closest to RGD in stimulating osteocalcin synthesis.
• RAD peptide was similar to OH and TCP.
• Alkaline Phosphatase gene expression was observed on all substrates at minimal levels but was significantly higher on RGDC coated gold surfaces. No change over the time period studied in Alkaline Phosphatase levels was observed in the cells cultured on FEP surfaces.
• bone sialoprotein gene expression was much higher on RGDC coated gold surfaces than on gold surfaces alone or gold surfaces coated with a control peptide. Bone sialoprotein gene expression was not observed in cells cultured on FEP surfaces.
• integrin ⁇ integrin gene expression was observed on all substrates at minimal levels but was significantly higher on RGDC coated gold surfaces.
• the mRNA signal detected from cells cultured on RGDC coated gold surfaces had shifted from one band to two bands. This shift to two bands was not detected in RNA isolated from cells cultured on any of the other surfaces.
• ⁇ 5 integrin ⁇ 5 integrin gene expression was observed in cells cultured on RGDC coated gold surfaces but was not detected in cells cultured on any other surfaces. Similar to ⁇ , integrin, the expression pattern of ⁇ 5 integrin was observed to shift on day 14 from a single band to a double band.
• cytoplasmic domains of integrins are relatively short (approximately 50 amino acids), but are sufficiently long enough to interact with cytoskeletal proteins in focal contacts (or focal adhesions or adhesion plaques).
• Focal adhesions are connected to the nucleus via microspikes or bundles of actin filaments.
• fluorescence photobleaching integrins were fluorescently labeled, then overexposed to form a bleached spot. This bleached area did not move, showing the restricted mobility of integrins in focal contacts (Duband, 1986).
• Solowska (1989) showed that expression of a mutant form of avian integrin beta 1 subunit lacking the cytoplasmic domain produces hybrid heterodimers which, while efficiently exported to the cell surface and still capable of binding fibronectin, do not localize efficiently in focal adhesions. This further implicates the cytoplasmic domain of the beta 1 subunit in interactions required for cytoskeletal organization.
• cytoskeletal proteins present in focal adhesions are well-defined: vinculin, talin, and alpha actinin serve as links between integrins and the bundles of actin filaments (stress fibers) of the cytoskeleton.
• Evidence in the literature suggests that focal adhesions are required for signal transduction from the ECM to the nucleus of the cell.
• focal adhesion kinase FAK
• tyrosine kinase a tyrosine kinase
• Both fibronectin and type I collagen are present in the extracellular matrix.
• Fibronectin/RGDC Study Experimental groups included RGDC, RADC, fibronectin adsorbed to gold, plain gold, and plain glass surfaces.
• Gold substrates were manufactured by evaporating 80 angstroms of titanium to 12 mm glass coverslips (Fisher), followed by a 500 angstrom layer of gold.
• cysteine terminated peptides to the gold substrates, a 0.22 mM solution of the desired peptide was solubihzed in a 1 :1 mixture of distilled water and ethanol. These substrates were then incubated overnight. Plain gold controls were exposed to ethanol and distilled water as well.
• Fibronectin substrates were produced by incubating gold coverslips with 10 ⁇ g/ml of fibronectin (Collaborative Biomedical Products, Bedford, MA) for 60 minutes, followed by 10 mg/ml bovine serum albumin (BSA) (Sigma, St. Louis, MO) for 30 minutes to cover any non-specific binding sites. All coverslips were then washed three times in HBSS to remove any non-adsorbed protein (Puleo, 1991). Primary rat calvarial osteoblasts were isolated according to protocol and seeded for periods of 3 or 24 hours in serum free or serum conditions. At each time point cells were rinsed in warm PBS, fixed in 3.7 % paraformaldehyde for 30 minutes, and rinsed several times with HBSS.
• BSA bovine serum albumin
• Vinculin and actin were labeled via the following protocol: nonspecific sites were blocked in 5% BSA for 30 minutes, cells were then permeabilized with 0.2 % Triton X-100 (Fisher) for 10 minutes, incubated in a 1 :50 mouse anti- human vinculin antibody solution (Sigma St. Louis, MO), blocked for 30 minutes, and incubated with a anti-mouse rhodamine secondary antibody (1 :50) and FITC conjugated phalloidin (Molecular Probes).
• Type I Collagen/DGEAGC Experimental groups included DGEAGC and rat tail type
• Gold substrates were manufactured by evaporating 80 angstroms of titanium to 12 mm glass coverslips (Fisher), followed by a 500 angstrom layer of gold.
• a 0.22 mM solution of the desired peptide was solubihzed in a 1 :1 mixture of distilled water and ethanol. These substrates were then incubated overnight. Plain gold controls were exposed to ethanol and distilled water as well.
• Type I collagen substrates were produced by incubating gold coverslips with 10 ⁇ g/ml of collagen (Collaborative Biomedical Products, Bedford, MA) for 60 minutes, followed by 10 mg/ml bovine serum albumin (BSA) (Sigma, St. Louis, MO) for 30 minutes to cover any non-specific binding sites. All coverslips were then washed three times in HBSS to remove any non-adsorbed protein (Puleo, 1991). Primary rat calvarial osteoblasts were isolated according to protocol and seeded for periods of 3 or 24 hours in serum free or serum conditions. At each time point cells were rinsed in warm PBS, fixed in 3.7 % paraformaldehyde for 30 minutes, and rinsed several times with HBSS.
• BSA bovine serum albumin
• Vinculin and actin were labeled via the following protocol: nonspecific sites were blocked in 5% BSA for 30 minutes, cells were then permeabilized with 0.2 % Triton X-100 (Fisher) for 10 minutes, incubated in a 1 :50 mouse anti-human vinculin antibody solution (Sigma St. Louis, MO), blocked for 30 minutes, and incubated with an anti-mouse rhodamine secondary antibody (1 :50) and FITC conjugated phalloidin (Molecular Probes).
• vinculin staining revealed the ability of RGDC peptide modified surfaces to support focal adhesion formation in the absence of serum. Fibronectin coated surfaces also supported focal adhesion formation. Cells on both surfaces tended to have vinculin staining located at the cell periphery in the form of distinct plaques at the cell tips. No vinculin staining was observed on cells plated on RADC, glass or plain gold.
• vinculin staining revealed the ability of DGEAGC peptide modified surfaces to support focal adhesion formation in the absence of serum. Collagen coated surfaces also supported focal adhesion formation. Cells on both surfaces tended to have the brightest vinculin staining located at the cell periphery in the form of either distinct plaques or groups of strands at the cell tips. No vinculin staining was observed on cells plated on glass or plain gold.
• peptide modified surfaces can influence short and long term cell responses like attachment, shape and function.
• Titanium rods were generously donated by Osteonics Corporation (NJ, USA). Rods were cleaned according to ASTM standards before coating them with a 500 layer of gold using an electron beam evaporator. Rods were immersed in a 0.22 M solution (1 :1 ethanol: water) of RGDC (American Peptide Company, Sunnyvale, CA) overnight at room temperature and stored in sterile PBS, using the techniques described above, until the time of surgery. FEP rods were coupled with peptides using the techniques described above. Un-coated titanium rods are used as a control.
• the materials were initially cleaned in a radio frequency glow discharge chamber using a flow-through system with an Argon atmosphere.
• the alloys were immediately transferred to a nitric acid bath for 30 min in order to passivate the surface according to ASTM standards (Puelo 1994).
• the samples were transferred to a gold evaporation chamber and reacted with peptides as described above. Characterization of gold coated titanium materials, FEP and titanium materials were performed as described above.
• Quantitative histomo ⁇ hometric analysis and pull-out biomechanical testing was conducted at 2 and 4 weeks on implants inserted bilaterally into the femoral canal of 20 adult Sprague Dawley rats. Parameters evaluated included the area and thickness of new bone formed around the implants, the percent of the implant covered by new bone, and the interfacial shear strength at the bone/implant interface.
• the distal rat femur provides a well-studied site for bone material interactions and offers a sufficient bony area to implant small specimens.
• Adult Sprague Dawley rats weighed an average of 415 ⁇ 12 g at the time of surgery.
• the rats were anesthetized using a 0.5 ml intraperitoneal injection of Nembutal and 0.1 ml of Cefazolin was injected intramuscularly at the surgical site.
• Reaming of the distal end of the femoral canal was done first by inserting an 18 gauge needle down the femoral shaft, followed by irrigation of the femur with sterile saline, reaming with a 1.5 mm drill bit using a hand held drill to prevent thermal necrosis, irrigation, reaming with a 16 gauge needle, irrigation, and final reaming of the outer cortex with a 14 gauge needle.
• the rod was then press fit into place with the outermost end below the condylar surface, in each case.
• RGDC coated rods were placed at random with one control rod and one experimental rod being placed bilaterally in each animal. Lateral and anterior-posterior X-rays were taken postoperatively to assess rod position. The fascia and skin are closed in standard fashion using 5-1 vicryl bioresorbable sutures.
• femurs were removed from the dental plaster and stored in phosphorous buffered saline for 24 hours until fixation in 3.7% paraformaldehyde for 48 hours at room temperature. Decalcification was performed according to a method described by Frank, et al, (1993). Briefly, bones were allowed to demineralize over the course of 2 weeks in 15% formic acid solution at 4 ⁇ C. Bones were rinsed and permbealized in 6.8% sucrose/PBS solution overnight. Dehydration of bones was conducted as follows: 20 minutes per ethanol concentration: 70, 80, 90, 95%.
• Bones were sectioned from the growth plate at 2, 5, 8, 12, and 15 mm and embedded in Historesin (Leica, Germany) for histological analysis. Briefly, bones were infiltrated for 48 hours at 4 ⁇ C and then embedded overnight. 5 ⁇ m sections were made. Specimens were stained using Hematoxylin and Eosin and GomoriOs trichrome stains. Quantitative histomo ⁇ hometrical analysis was conducted on bone cross sections sectioned at 5mm using IP Lab Software. Images of bone cross sections were captured by microscope and imported into a computer via a CCD camera.
• Biomechanical Pull-out Testing The biomechanical pull-out strength between the bone/RGDC and bone/Au was measured using the widely imployed pull-out test (Chae et al in 1992 and Tisdel et al in 1994 , Branemark & Berzin All testing was performed in a blinded fashion.
• An alignment jig was designed in order to insure a pure tensile force was applied to the rod.
• Dental plaster was used to hold the proximal portion of the femur in place during testing.
• Modified needle-drivers gripped the end of the implant as it was pulled from the bone at a crosshead speed of 5 mm/min. The force required to break the interface was recorded and the portion of the implant estimated to be contact bone was also recorded.
• Biomechanical No statistical differences were found between peptide modified and gold control rods for the interfacial shear strength at 2 and 4 weeks respectively. It should be noted however, that the mean of the peptide modified group at 4 weeks was 38% higher than the control group (Table 1).
• Histology Although there were not a significant differences in the pull-out forces between groups, there were significant differences in the amount of bone (thickness and area) formed around the implants at two and four weeks. There were no differences in the percent of the implant cross section covered by bone (76 ⁇ 14%, 74 ⁇ 5%) for the RGDC and Au groups respectively. At four weeks more of the implant was covered by bone but the percent of the implant cross section covered by bone for the RGDC and Au groups did not differ significantly (92 ⁇ 4 % vs. 90 ⁇ 7 %). The area of new bone formed around RGDC implants was not significantly more compared to Au controls at 2 weeks (0.108 ⁇ m 2 ⁇ 0.026 vs. 0.082 ⁇ m 2 ⁇
• Example 5 Peptides act synergistically to increase bone cell responsiveness.
• the response of bone cells to peptide combinations showing synergy or high individual levels of activity is evaluated in vitro and in vivo using methods described above with combinations of peptides rather than a single peptide.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Medicinal Chemistry (AREA)
• Epidemiology (AREA)
• Animal Behavior & Ethology (AREA)
• General Health & Medical Sciences (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Transplantation (AREA)
• Oral & Maxillofacial Surgery (AREA)
• Dermatology (AREA)
• Inorganic Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Organic Chemistry (AREA)
• Dispersion Chemistry (AREA)
• Polymers & Plastics (AREA)
• Biomedical Technology (AREA)
• Molecular Biology (AREA)
• Materials Engineering (AREA)
• Hematology (AREA)
• Surgery (AREA)
• Materials For Medical Uses (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=21971332

Family Applications (1)

Country Status (3)

Families Citing this family (24)

Family Cites Families (4)

• 1998
  • 1998-07-01 CA CA002301064A patent/CA2301064A1/en not_active Abandoned
  • 1998-07-01 EP EP98933098A patent/EP1006945A1/de not_active Withdrawn
  • 1998-07-01 WO PCT/US1998/013792 patent/WO1999001089A1/en not_active Application Discontinuation

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events