CA2169191C - Neural regeneration using human bone morphogenetic proteins - Google Patents
Neural regeneration using human bone morphogenetic proteins Download PDFInfo
- Publication number
- CA2169191C CA2169191C CA002169191A CA2169191A CA2169191C CA 2169191 C CA2169191 C CA 2169191C CA 002169191 A CA002169191 A CA 002169191A CA 2169191 A CA2169191 A CA 2169191A CA 2169191 C CA2169191 C CA 2169191C
- Authority
- CA
- Canada
- Prior art keywords
- bmp
- bone morphogenetic
- morphogenetic protein
- cells
- nerve
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
-
- 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
-
- 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
- A61L31/047—Other specific proteins or polypeptides not covered by A61L31/044 - A61L31/046
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- 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
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction
Abstract
Methods and devices are disclosed for inducing growth of neural cells, and repairing neural defects in a mammal. The method comprises administering to said mammal at the site of neural defect, damage or depletion, an effective amount of a bone morphogenetic protein, either in admixture with a pharmaceutically acceptable vehicle, or adsorbed to a suitable matrix. The device comprises bone morphogenetic protein, optionally in combination with other factors, adsorbe d on a suitable matrix and contained within an artificial nerve replacement vessel.
Description
=..~.
NEURAL REGENERATION USING HUMAN BONE MORPHOGENETIC PROTEINS
The present invention relates to pharmaceutical uses of bone morphogenetic proteins (BMPs) for proliferation of neural cells and for regeneration of nerve tissue.
More particularly, the subject invention relates to the use of BMPs, preferably, BMP-2 through 10 for the treatment of central and peripheral nervous system diseases, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue.
BACKGROUND
Bone morphogenetic proteins 2 through 10 are members of the transforming growth factor-/i (TGF-f3) superfamily. The BMPs were originally discovered as osteogenic proteins capable of inducing bone formation in vivo. The transforming growth factors were initially identified on the basis of their ability to induce phenotypic transformation of mammalian cells grown in tissue culture, a phenomenon which has traditionally been associated with in vivo changes from normal to tumor cell growth.
Astrocytes are a type of glial cell found in the nervous system which function in axonal guidance, stimulation of neurite outgrowth, neuron inorphogenesis and migration. Astrocytes have also been implicated in induction of the vascular endothelial blood-brain barrier and transport of blood to the neurons. Astrocytes express an intermediate filament protein of the cytoskeleton, glial fibrillary acidic protein (GFAP), a very specific marker of astrocytes.
Two types of astrocytes have been classically described by location and niorphology.
Protoplasmic astrocytes are typically found in gray matter and have thick extensively branched processes, while fibrous astrocytes found in the white matter have long straight processes.
Astrocytes isolated from the optic nerve have been described antigenically as Type 1 and Type 2 on the basis of their staining for GFAP and the surface marker A2B5.
Originating from two different developmental lineages and at separate times, Type 1 astrocytes only stain for GFAP, while Type 2 astrocytes stain for both A2B5 and GFAP. Astrocytes provide a conducive environment for axon growth, which is an important aspect of nerve regeneration. Thus, the survival and differentiation of astrocytes are important factors in the ability of neural cells and tissue to survive and regenerate. Silver et al., United States Patent 5,202,120, describe a SUBSTITUTE SHEET (RULE 26) method using activated astrocytes to promote regeneration of axons. However, this method is disadvantageous in that it requires a supply of astrocytes, such as by autologous transplant.
SUMMARY OF THE INVENTION
It is one object of the present invention to provide methods and compositions capable of inducing the growth of neural cells. It is another object of the present invention to provide methods and compositions suitable for the generation of nerve cells and nerve tissue, and for the repair of neural defects.
In one embodiment, the present invention provides a method of inducing growth of neural cells which comprises administering to a mammal at a site of neural depletion, damage or defect, an effective amount of a recombinant human BMP (rhBMP) in admixture with a pharmaceutically acceptable vehicle. The BMP is preferably selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7 and heterodimers of BMP-2/6 and BMP-2/7.
In another embodiment, the present invention comprises a method of treating a mammal having a neural defect, neural damage or a neural condition, which method comprises administering to said mammal at a site of neural depletion, defect or damage, a nerve-regenerating amount of rhBMP in combination with a suitable matrix. The BMP is preferably selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7 and heterodimers of BMP-2/6 and BMP-2/7. Most preferred are BMP-2, BMP-4 and BMP-2/6 and BMP-2/7 heterodimers.
In a preferred embodiment, the present invention comprises a device for nerve replacement. The device preferably employs a matrix or carrier capable of maintaining the BMP
in a desired location and orientation to allow regeneration of neural tissue.
The BMP is adsorbed onto the matrix. The matrix may be made of any suitable carrier material known in the art. Preferably, the matrix is comprised of a suitable material selected from the group consisting of collagen, fibrin tissue adhesives, and components of normal endoneurial sheaths.
These components include laminin, hyalauronic acid and chondroitin sulfate proteoglycans, including versican. Tona et al., J. Histochemistry and Cytochemistrv, 41:593-599 (1993). In the most preferred embodiment, the matrix is comprised of cross-linked collagen. The collagen may be in any suitable form, but is preferably in the form of a sponge. The collagen may be shaped into a suitable shape for regeneration of nerve tissue. The BMP-adsorbed matrix may SUBSTITUTE SHEET (RULE 26) be applied to an artificial nerve replacement vessel which contains the matrix and BMP. The artificial nerve replacement vessel is preferably in the form of tubing or stent, such as vented silastic tubing.
DETAILED DESCRIPTION OF THE INVENTION
The present inventors have surprisingly found that the BMPs, particularly BMP-2, BMP-4 and heterodimers of BMP-2/6 and BMP-2/7 may be used to enhance nerve regeneration.
Nerve cells do not ordinarily proliferate after injury, and physiologic repair using microsurgical techniques often result in imperfect functional results, despite optimal care.
Nerve tissue must become neovascularized prior to repair. However, neovascularization occurs much later in nerves than in other biologic systems, slowing initial axonal repair, and often facilitating irreparable and time-dependent motor endplate atrophy. Further, the faster forming fibrotic scar tissue may prevent the success of naturally occuring nerve regeneration.
Consequently, the use of BMPs to enhance or accelerate nerve repair provides a method for improving nerve repair where it might not otherwise occur.
The DNA sequences of BMPs are known and have been described as follows: BMP-2 (sometimes referred to as BMP-2A) and BMP-4 (sometimes referred to as BMP-2B), U.S. Patent No. 5,013,649; BMP-3 U.S. Patent No. 5,116,738; BMP-5, U.S. Patent No.
5,106,748; BMP-6, U.S. Patent No. 5,187,076; BMP-7, U.S. Patent No. 5,141,905; BMP-8, PCT
Publication No. W093/00432; BMP-9, U.S. Patent No. 5,661,007; BMP-10, PCT Publication 3. Heterodimers are described in Unites States Patent No. 5,866,364.
Recombinant human BMP, such as rhBMP-2, may be made for use in the method of the invention by expressing the DNA sequences encoding a BMP in a suitable transformed host cell.
For example, using known methods, the DNA encoding BMP-2 may be linked to an expression vector such as pED (Kaufman et al., Nucleic Acids Res. 19, 4484-4490 (1991)), transformed into a host cell, and protein expression may be induced and maximized. Of course, degenerate DNA sequences encoding human BMP may also be employed to produce rhBMP, as can DNA
sequences encoding allelic variants of BMP.
Any suitable expression vector may be employed to produce rhBMP, such as rhBMP-2, SUBSTITUTE SHEET (RULE 26) for use in the present invention. For mammalian expression, numerous expression vectors are known in addition to the pED vector mentioned above, such as pEF-BOS
(Mizushima et al., Nucleic Acids Res. 18, 5322 (1990)); pXM, pJL3 and pJL4 (Gough et al., EMBO J.
NEURAL REGENERATION USING HUMAN BONE MORPHOGENETIC PROTEINS
The present invention relates to pharmaceutical uses of bone morphogenetic proteins (BMPs) for proliferation of neural cells and for regeneration of nerve tissue.
More particularly, the subject invention relates to the use of BMPs, preferably, BMP-2 through 10 for the treatment of central and peripheral nervous system diseases, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue.
BACKGROUND
Bone morphogenetic proteins 2 through 10 are members of the transforming growth factor-/i (TGF-f3) superfamily. The BMPs were originally discovered as osteogenic proteins capable of inducing bone formation in vivo. The transforming growth factors were initially identified on the basis of their ability to induce phenotypic transformation of mammalian cells grown in tissue culture, a phenomenon which has traditionally been associated with in vivo changes from normal to tumor cell growth.
Astrocytes are a type of glial cell found in the nervous system which function in axonal guidance, stimulation of neurite outgrowth, neuron inorphogenesis and migration. Astrocytes have also been implicated in induction of the vascular endothelial blood-brain barrier and transport of blood to the neurons. Astrocytes express an intermediate filament protein of the cytoskeleton, glial fibrillary acidic protein (GFAP), a very specific marker of astrocytes.
Two types of astrocytes have been classically described by location and niorphology.
Protoplasmic astrocytes are typically found in gray matter and have thick extensively branched processes, while fibrous astrocytes found in the white matter have long straight processes.
Astrocytes isolated from the optic nerve have been described antigenically as Type 1 and Type 2 on the basis of their staining for GFAP and the surface marker A2B5.
Originating from two different developmental lineages and at separate times, Type 1 astrocytes only stain for GFAP, while Type 2 astrocytes stain for both A2B5 and GFAP. Astrocytes provide a conducive environment for axon growth, which is an important aspect of nerve regeneration. Thus, the survival and differentiation of astrocytes are important factors in the ability of neural cells and tissue to survive and regenerate. Silver et al., United States Patent 5,202,120, describe a SUBSTITUTE SHEET (RULE 26) method using activated astrocytes to promote regeneration of axons. However, this method is disadvantageous in that it requires a supply of astrocytes, such as by autologous transplant.
SUMMARY OF THE INVENTION
It is one object of the present invention to provide methods and compositions capable of inducing the growth of neural cells. It is another object of the present invention to provide methods and compositions suitable for the generation of nerve cells and nerve tissue, and for the repair of neural defects.
In one embodiment, the present invention provides a method of inducing growth of neural cells which comprises administering to a mammal at a site of neural depletion, damage or defect, an effective amount of a recombinant human BMP (rhBMP) in admixture with a pharmaceutically acceptable vehicle. The BMP is preferably selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7 and heterodimers of BMP-2/6 and BMP-2/7.
In another embodiment, the present invention comprises a method of treating a mammal having a neural defect, neural damage or a neural condition, which method comprises administering to said mammal at a site of neural depletion, defect or damage, a nerve-regenerating amount of rhBMP in combination with a suitable matrix. The BMP is preferably selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7 and heterodimers of BMP-2/6 and BMP-2/7. Most preferred are BMP-2, BMP-4 and BMP-2/6 and BMP-2/7 heterodimers.
In a preferred embodiment, the present invention comprises a device for nerve replacement. The device preferably employs a matrix or carrier capable of maintaining the BMP
in a desired location and orientation to allow regeneration of neural tissue.
The BMP is adsorbed onto the matrix. The matrix may be made of any suitable carrier material known in the art. Preferably, the matrix is comprised of a suitable material selected from the group consisting of collagen, fibrin tissue adhesives, and components of normal endoneurial sheaths.
These components include laminin, hyalauronic acid and chondroitin sulfate proteoglycans, including versican. Tona et al., J. Histochemistry and Cytochemistrv, 41:593-599 (1993). In the most preferred embodiment, the matrix is comprised of cross-linked collagen. The collagen may be in any suitable form, but is preferably in the form of a sponge. The collagen may be shaped into a suitable shape for regeneration of nerve tissue. The BMP-adsorbed matrix may SUBSTITUTE SHEET (RULE 26) be applied to an artificial nerve replacement vessel which contains the matrix and BMP. The artificial nerve replacement vessel is preferably in the form of tubing or stent, such as vented silastic tubing.
DETAILED DESCRIPTION OF THE INVENTION
The present inventors have surprisingly found that the BMPs, particularly BMP-2, BMP-4 and heterodimers of BMP-2/6 and BMP-2/7 may be used to enhance nerve regeneration.
Nerve cells do not ordinarily proliferate after injury, and physiologic repair using microsurgical techniques often result in imperfect functional results, despite optimal care.
Nerve tissue must become neovascularized prior to repair. However, neovascularization occurs much later in nerves than in other biologic systems, slowing initial axonal repair, and often facilitating irreparable and time-dependent motor endplate atrophy. Further, the faster forming fibrotic scar tissue may prevent the success of naturally occuring nerve regeneration.
Consequently, the use of BMPs to enhance or accelerate nerve repair provides a method for improving nerve repair where it might not otherwise occur.
The DNA sequences of BMPs are known and have been described as follows: BMP-2 (sometimes referred to as BMP-2A) and BMP-4 (sometimes referred to as BMP-2B), U.S. Patent No. 5,013,649; BMP-3 U.S. Patent No. 5,116,738; BMP-5, U.S. Patent No.
5,106,748; BMP-6, U.S. Patent No. 5,187,076; BMP-7, U.S. Patent No. 5,141,905; BMP-8, PCT
Publication No. W093/00432; BMP-9, U.S. Patent No. 5,661,007; BMP-10, PCT Publication 3. Heterodimers are described in Unites States Patent No. 5,866,364.
Recombinant human BMP, such as rhBMP-2, may be made for use in the method of the invention by expressing the DNA sequences encoding a BMP in a suitable transformed host cell.
For example, using known methods, the DNA encoding BMP-2 may be linked to an expression vector such as pED (Kaufman et al., Nucleic Acids Res. 19, 4484-4490 (1991)), transformed into a host cell, and protein expression may be induced and maximized. Of course, degenerate DNA sequences encoding human BMP may also be employed to produce rhBMP, as can DNA
sequences encoding allelic variants of BMP.
Any suitable expression vector may be employed to produce rhBMP, such as rhBMP-2, SUBSTITUTE SHEET (RULE 26) for use in the present invention. For mammalian expression, numerous expression vectors are known in addition to the pED vector mentioned above, such as pEF-BOS
(Mizushima et al., Nucleic Acids Res. 18, 5322 (1990)); pXM, pJL3 and pJL4 (Gough et al., EMBO J.
4, 645-653 (1985)); and pMT2 (derived from pMT2-VWF, A.T.C.C. #67122; see PCT/US87?00033).
Suitable expression vectors for use in yeast, insect, and bacterial cells are also known.
Construction and use of such expression vectors is well within the level of skill in the art.
Recombinant BMP, such as rhBMP-2, may also be produced using a chimeric DNA
sequence which encodes for a mature BMP operably linked to a propeptide from a different BMP. For example, see U.S. 5,168,050.
Suitable host cells for production of BMPs useful in the present invention include, for example, mammalian cells such as Chinese hamster ovary (CHO) cells, monkey COS
cells, mouse 3T3 cells, mouse L cells, myeloma cells such as NSO (Galfre and Milstein, Methods in Enzymology 13, 3-46 (1981)), and the like. RhBMP may also be produced by transformation of yeast, insect, and bacterial cells with DNA sequences encoding BMP, induction and amplification of protein expression, using known methods. When produced in bacterial cells, it may be necessary to solubilize the bone morphogenetic protein.
Recombinantly produced BMP, such as rhBMP-2, must be purified from culture medium or cell extracts for use in the present invention. Culture medium or cell extracts containing rhBMP may be concentrated using a commercially available protein concentration filter, for 2 0 example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a purification matrix such as a gel filtration medium.
Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylamioethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification.
Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. The purification of BMP from culture supernatant may also include one or more column steps over such affinity resins ds lectin-agarose, heparin-toyopearl or Cibacrom blue 3GA Sepharose ; or by hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or by immunoaffinity chromatography. Finally, one or more reverse-phase high performance liquid * Trade-mark SUBSTITUTE SHEET (RULE 26) chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify BMP for use in the present methods. Some or all of the foregoing purification steps, in various combinations, can be employed to provide a substantially homogeneous isolated recombinant protein.
BMPs, such as rhBMP-2, can be used in the method of the invention for the in vivo treatment of mammals by physicians in a variety of disease conditions. These conditions include diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. BMPs may be used to increase the regeneration of nerve cells and nerve tissue in order to enhance or accelerate the healing of such disorders.
In accordance with the method of the invention, BMP, such as rhBMP-2, may be administered alone, in combination with other BMPs, or in combination with other therapies.
For example, rhBMP-2 may be efficaciously combined with a cytokine, lymphokine, growth factor, or colony stimulating factor, in the treatment of neural diseases.
Exemplary cytokines, lymphokines, growth factors, and colony stimulating factors for use in combination with BMP
in accordance with the method of the invention include, without limitation, EGF, FGF, interleukins 1 through 12, M-CSF, G-CSF, GM-CSF, stem cell factor, erythropoietin, and the like. In addition, the BMPs may be combined with neurotrophic factors such as CNTF, LIF, IL-6 and insulin-like growth factors [IGFs]. Additionally, proteins normally found in the neural environment may be added to the BMPs in accordance with the present invention.
These may include laminin, hyalauronic acid and chondroitin sulfate proteoglycans, including versican.
The BMP of the present invention may be administered employing a matrix capable of maintaining the BMP in a desired location and orientation to allow regeneration of neural tissue.
The BMP may preferably be adsorbed onto the matrix. The matrix may be made of any suitable material known in the art. Such materials include a suitable materials selected from the group consisting of collagen, fibrin tissue adhesives and components of normal endoneurial sheaths, SUBSTITUTE SHEET (RULE 26) 21691,91 including laminin, hyalauronic acid and chondroitin sulfate proteoglycans, including versican.
The matrix may preferably be porous, so as to allow the influx, migration, differentiation and proliferation of cells need for regeneration of neural tissue. In one preferred embodiment, the matrix is comprised of cross-linked collagen. The collagen may be in any suitable form, but is preferably in the form of a sponge. The collagen may be shaped into a suitable shape for regeneration of nerve tissue. In another preferred embodiment, the matrix comprises bioerodible particles, such as polymers of lactic acid (PLA), polymers of glycolic acid (PGA), and co-polymers of lactic acid and glycolic acid (PLGA). Also useful as the matrix are polymers of polyorthoesters. The matrix may comprise materials to promote the formation of neural tissue, such as fibrin, or vein graft.
The BMP-adsorbed matrix is then applied to an artificial nerve replacement vessel, preferably in the form of tubing or stent, such as vented silastic tubing. The artificial nerve replacement vessel may be comprised of any material which will hold the BMP-adsorbed matrix in place and allow for regeneration of nerve tissue. In one embodiment, autologous vein graft may be used as the nerve replacement vessel. The artificial nerve replacement vessel may comprise a resorbable material, such as polymers. In some preferred embodiments, the matrix may also serve as the artificial nerve replacement vessel.
Pharmaceutical compositions suitable for use in the method of the present invention may contain, in addition to the BMP, pharmaceutically acceptable carriers, diluents, fillers, salts, buffers, stabilizers, and/or other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier or other material will depend on the route of administration.
Administration of BMP, such as rhBMP-2, in the method of the invention can be carried out in a variety of conventional ways. For regeneration of nerve tissues, treatment of neural defect or nerve damage, topical administration of BMP is preferred. In the most preferred mode of administration, BMP is adsorbed to a biocompatible matrix and applied to an artificial nerve replacement vessel. The biocompatible matrix is preferably made of collagen, and may be in the form of a sponge, sheets or mats, or closely packed particles. The artificial nerve replacement vessel may be in the form of a tube or stent. Other materials suitable for artificial SUBSTITUTE SHEET (RULE 26) '..
nerve replacement vessel will be apparent to those skilled in the art. In a preferred embodiment, the artificial nerve replacement vessel comprises vented silastic tubing containing the BMP-adsorbed matrix. In another preferred embodiment, the artificial nerve replacement vessel comprises autologous vein graft. In some preferred embodiments, the same material may serve as both the matrix and the artificial nerve replacement vessel.
The amount of BMP useful in the method of the present invention will depend upon the = nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of BMP
with which to treat each individual patient. It is contemplated that the various pharmaceutical compositions of the present invention should contain about 0.1 g to about 100 mg, preferably about 0.1 g to 100 g of BMP per kg body weight. The actual dosing regimen will be determined by the attending physician considering various factors which modify the action of drugs, e.g., the condition, body weight, sex and diet of the patient, the severity of the condition, time and method of administration and other clinical factors.
In practicing the method of treatment of this invention, a therapeutically effective amount of BMP is administered to a mammal having such a disease state. The term "therapeutically effective amount" means the total amount of each active component of the method that is sufficient to show a meaningful patient benefit, i.e., healing of chronic conditions or increase in rate of healing. For example, a nerve-regenerating amount of a bone morphogenetic protein is that amount of protein which, when adsorbed to a suitable matrix carrier and implanted at a site of nerve damage, defect or depletion, will allow the regeneration of nerve tissue and/or amelioration of the neural damage, defect or depletion. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. A
therapeutically effective dose of BMP for practice of the method of this invention is contemplated to be in the range of about 0.1 g to about 100 mg per kg body weight per application. Generally, administration will be initiated at the low end of the dosing range initially, and the dose will be increased over a preselected time course until a positive effect is observed. Subsequently, incremental increases in dosage will be made limiting such incremental SUBSTITUTE SHEET (RULE 26) WO 95/05846 21 69 1 C~ ~ PCTIUS94/09330 increases to such levels that produce a corresponding increase in effect, while taking into account any adverse affects that may appear.
The duration of intravenous therapy using the method of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that the duration of each application of the BMP will be in the range of 12 to 24 hours of continuous administration.
Ultimately the attending physician will decide on the appropriate duration of therapy using the method of the present invention.
In accordance with the method of the invention, neural regeneration may be achieved in mammals by administration of a nerve-regenerating amount of BMP, such as rhBMP-2, in admixture with a pharmaceutically acceptable vehicle. For the purposes of the present invention, a nerve-regenerating amount of BMP, such as rhBMP-2, in accordance with the present invention is that amount of the protein necessary to cause regeneration of nerve. The nerve regeneration may be measured by weiglit or volume of the nerve tissue present.
It is contemplated that suitable host cells, transformed to express BMP, may also be administered to the patient in order to improve the growth or survival of neural cells or tissue.
The following examples are illustrative of the present invention, and are not limiting in any manner.
Parenteral formulations of BMP will be in the form of pyrogen-free, parenterally acceptable aqueous solutions. The preparation of such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A
preferred parenteral formulation should contain, in addition to BMP, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition according to the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additive known to those of skill in the art.
When administered topically, the BMP of the present invention may be in the form of a pyrogen-free, topically acceptable liquid or semi-solid formulation such as an ointment, cream, lotion, foam or gel. The preparation of suc~i topically applied formulations is within the skill in the art.
Suitable expression vectors for use in yeast, insect, and bacterial cells are also known.
Construction and use of such expression vectors is well within the level of skill in the art.
Recombinant BMP, such as rhBMP-2, may also be produced using a chimeric DNA
sequence which encodes for a mature BMP operably linked to a propeptide from a different BMP. For example, see U.S. 5,168,050.
Suitable host cells for production of BMPs useful in the present invention include, for example, mammalian cells such as Chinese hamster ovary (CHO) cells, monkey COS
cells, mouse 3T3 cells, mouse L cells, myeloma cells such as NSO (Galfre and Milstein, Methods in Enzymology 13, 3-46 (1981)), and the like. RhBMP may also be produced by transformation of yeast, insect, and bacterial cells with DNA sequences encoding BMP, induction and amplification of protein expression, using known methods. When produced in bacterial cells, it may be necessary to solubilize the bone morphogenetic protein.
Recombinantly produced BMP, such as rhBMP-2, must be purified from culture medium or cell extracts for use in the present invention. Culture medium or cell extracts containing rhBMP may be concentrated using a commercially available protein concentration filter, for 2 0 example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a purification matrix such as a gel filtration medium.
Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylamioethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification.
Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. The purification of BMP from culture supernatant may also include one or more column steps over such affinity resins ds lectin-agarose, heparin-toyopearl or Cibacrom blue 3GA Sepharose ; or by hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or by immunoaffinity chromatography. Finally, one or more reverse-phase high performance liquid * Trade-mark SUBSTITUTE SHEET (RULE 26) chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify BMP for use in the present methods. Some or all of the foregoing purification steps, in various combinations, can be employed to provide a substantially homogeneous isolated recombinant protein.
BMPs, such as rhBMP-2, can be used in the method of the invention for the in vivo treatment of mammals by physicians in a variety of disease conditions. These conditions include diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. BMPs may be used to increase the regeneration of nerve cells and nerve tissue in order to enhance or accelerate the healing of such disorders.
In accordance with the method of the invention, BMP, such as rhBMP-2, may be administered alone, in combination with other BMPs, or in combination with other therapies.
For example, rhBMP-2 may be efficaciously combined with a cytokine, lymphokine, growth factor, or colony stimulating factor, in the treatment of neural diseases.
Exemplary cytokines, lymphokines, growth factors, and colony stimulating factors for use in combination with BMP
in accordance with the method of the invention include, without limitation, EGF, FGF, interleukins 1 through 12, M-CSF, G-CSF, GM-CSF, stem cell factor, erythropoietin, and the like. In addition, the BMPs may be combined with neurotrophic factors such as CNTF, LIF, IL-6 and insulin-like growth factors [IGFs]. Additionally, proteins normally found in the neural environment may be added to the BMPs in accordance with the present invention.
These may include laminin, hyalauronic acid and chondroitin sulfate proteoglycans, including versican.
The BMP of the present invention may be administered employing a matrix capable of maintaining the BMP in a desired location and orientation to allow regeneration of neural tissue.
The BMP may preferably be adsorbed onto the matrix. The matrix may be made of any suitable material known in the art. Such materials include a suitable materials selected from the group consisting of collagen, fibrin tissue adhesives and components of normal endoneurial sheaths, SUBSTITUTE SHEET (RULE 26) 21691,91 including laminin, hyalauronic acid and chondroitin sulfate proteoglycans, including versican.
The matrix may preferably be porous, so as to allow the influx, migration, differentiation and proliferation of cells need for regeneration of neural tissue. In one preferred embodiment, the matrix is comprised of cross-linked collagen. The collagen may be in any suitable form, but is preferably in the form of a sponge. The collagen may be shaped into a suitable shape for regeneration of nerve tissue. In another preferred embodiment, the matrix comprises bioerodible particles, such as polymers of lactic acid (PLA), polymers of glycolic acid (PGA), and co-polymers of lactic acid and glycolic acid (PLGA). Also useful as the matrix are polymers of polyorthoesters. The matrix may comprise materials to promote the formation of neural tissue, such as fibrin, or vein graft.
The BMP-adsorbed matrix is then applied to an artificial nerve replacement vessel, preferably in the form of tubing or stent, such as vented silastic tubing. The artificial nerve replacement vessel may be comprised of any material which will hold the BMP-adsorbed matrix in place and allow for regeneration of nerve tissue. In one embodiment, autologous vein graft may be used as the nerve replacement vessel. The artificial nerve replacement vessel may comprise a resorbable material, such as polymers. In some preferred embodiments, the matrix may also serve as the artificial nerve replacement vessel.
Pharmaceutical compositions suitable for use in the method of the present invention may contain, in addition to the BMP, pharmaceutically acceptable carriers, diluents, fillers, salts, buffers, stabilizers, and/or other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier or other material will depend on the route of administration.
Administration of BMP, such as rhBMP-2, in the method of the invention can be carried out in a variety of conventional ways. For regeneration of nerve tissues, treatment of neural defect or nerve damage, topical administration of BMP is preferred. In the most preferred mode of administration, BMP is adsorbed to a biocompatible matrix and applied to an artificial nerve replacement vessel. The biocompatible matrix is preferably made of collagen, and may be in the form of a sponge, sheets or mats, or closely packed particles. The artificial nerve replacement vessel may be in the form of a tube or stent. Other materials suitable for artificial SUBSTITUTE SHEET (RULE 26) '..
nerve replacement vessel will be apparent to those skilled in the art. In a preferred embodiment, the artificial nerve replacement vessel comprises vented silastic tubing containing the BMP-adsorbed matrix. In another preferred embodiment, the artificial nerve replacement vessel comprises autologous vein graft. In some preferred embodiments, the same material may serve as both the matrix and the artificial nerve replacement vessel.
The amount of BMP useful in the method of the present invention will depend upon the = nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of BMP
with which to treat each individual patient. It is contemplated that the various pharmaceutical compositions of the present invention should contain about 0.1 g to about 100 mg, preferably about 0.1 g to 100 g of BMP per kg body weight. The actual dosing regimen will be determined by the attending physician considering various factors which modify the action of drugs, e.g., the condition, body weight, sex and diet of the patient, the severity of the condition, time and method of administration and other clinical factors.
In practicing the method of treatment of this invention, a therapeutically effective amount of BMP is administered to a mammal having such a disease state. The term "therapeutically effective amount" means the total amount of each active component of the method that is sufficient to show a meaningful patient benefit, i.e., healing of chronic conditions or increase in rate of healing. For example, a nerve-regenerating amount of a bone morphogenetic protein is that amount of protein which, when adsorbed to a suitable matrix carrier and implanted at a site of nerve damage, defect or depletion, will allow the regeneration of nerve tissue and/or amelioration of the neural damage, defect or depletion. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. A
therapeutically effective dose of BMP for practice of the method of this invention is contemplated to be in the range of about 0.1 g to about 100 mg per kg body weight per application. Generally, administration will be initiated at the low end of the dosing range initially, and the dose will be increased over a preselected time course until a positive effect is observed. Subsequently, incremental increases in dosage will be made limiting such incremental SUBSTITUTE SHEET (RULE 26) WO 95/05846 21 69 1 C~ ~ PCTIUS94/09330 increases to such levels that produce a corresponding increase in effect, while taking into account any adverse affects that may appear.
The duration of intravenous therapy using the method of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that the duration of each application of the BMP will be in the range of 12 to 24 hours of continuous administration.
Ultimately the attending physician will decide on the appropriate duration of therapy using the method of the present invention.
In accordance with the method of the invention, neural regeneration may be achieved in mammals by administration of a nerve-regenerating amount of BMP, such as rhBMP-2, in admixture with a pharmaceutically acceptable vehicle. For the purposes of the present invention, a nerve-regenerating amount of BMP, such as rhBMP-2, in accordance with the present invention is that amount of the protein necessary to cause regeneration of nerve. The nerve regeneration may be measured by weiglit or volume of the nerve tissue present.
It is contemplated that suitable host cells, transformed to express BMP, may also be administered to the patient in order to improve the growth or survival of neural cells or tissue.
The following examples are illustrative of the present invention, and are not limiting in any manner.
Parenteral formulations of BMP will be in the form of pyrogen-free, parenterally acceptable aqueous solutions. The preparation of such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A
preferred parenteral formulation should contain, in addition to BMP, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition according to the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additive known to those of skill in the art.
When administered topically, the BMP of the present invention may be in the form of a pyrogen-free, topically acceptable liquid or semi-solid formulation such as an ointment, cream, lotion, foam or gel. The preparation of suc~i topically applied formulations is within the skill in the art.
SUBSTITUTE SHEET (RULE 26) EXAMPLE I. BMP EFFECTS ON NEURAL CELLS
Example IA. CELL CULTURE
Balb c/SFME (Serum-Free Mouse Embryo) cells were obtained from the American Type Culture Collection (CRL 9392) and were grown as previously described (Sakai et al., PNAS:USA, 87:8378-8382 (1990)) in DME/F12 (1:1) medium containing bovine insulin, 10 g/ml (Eli Lilly); human transferrin, 25 g/ml (Collaborative Research); human high density lipoprotein, 20 g/ml (sigma); human epidermal growth factor, 100 ng/ml (PeproTech); bovine plasma fibronectin, 20 g/ml (GIBCO); sodium selenite, IOnM (GIBCO);
penicillin-streptomycin (10 U/ml), 1-glutamine (4mM) and 4-(2-hydroxy-ethyl) - piperazine-ethanesulfonic acid, pH 7.4 (15 mM).
Cells were passaged using trypsin/EDTA and soybean trypsin inhibitor (1 mg/ml) in a volume ratio of 1:2 and used between passages 19 and 50. Cells were counted, unless otherwise stated, with a Coulter Diagnostics counter.
Example IB. GROWTH AND DIFFERENTIATION FACTORS:
All recombinant human proteins used were of greater than 90% purify. EGF was purchased from PeproTech (NJ); recombinant human Activin-A was the generous gift of Helen New; TGF-fl l was purchased from R&D Systems; BMPs were purified from CHO
conditioned media through several purification steps at Genetics Institute.
Example IC. DIFFERENTIATION STUDIES:
For all immunofluorescence and FACS analysis, unless otherwise stated, cells were plated at 2.5 - 5 x 10 /cm2 and BMP-2 was added at 16-20 hrs. at the concentrations and for the length of time indicated.
Example ID. SURVIVAL STUDIES:
Cells were washed twice in medium without EGF and then plated at 0.8 - I x 105/cm2 into the same medium supplemented with various growth factors. These included BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-2/6 heterodimer, BMP-2/7 heterodimer and TGF-/31.
Percent viability and cell number were determined in duplicate by trypan blue dye exclusion with a SUBSTITUTE SHEET (RULE 26) WO 95/05846 2! 6919j PCT/US94/09330 hemocytometer at 44-48 hrs., counting a minimum of 400 cells per sample.
Percent viability was calculated as total live cells divided by the total number of cells at the endpoint since cell proliferation was seen in some conditions.
Example IE. IMMUNOFLUORESCENT ANTIBODY STAINING:
Media was removed from cells in four-well glass or plastic chamber slides (Lab Tek) and they were washed twice with PBS- Ca+2, Mg+2 free (CMF). For surface staining with the antibody A2B5 (Boehringer-Mannheim) the cells were initially fixed with 4% paraformaldehyde for 10 minutes, washed with PBS and then incubated with 1% rabbit serum in PBS
to block nonspecific binding. The antibody was diluted in 1% rabbit serum in PBS and incubated for 1 hour then the cells were washed in 1% rabbit serum in PBS before detection with a biotinylated rabbit anti-mouse antibody followed by a streptavidin-FITC (Zymed) conjugate.
For further double staining experiments or single internal staining for GFAP, cells were fixed in acetone/methanol (50:50) at -20C for 10 minutes. Permeabilization and blocking was performed with 0.2% Triton X-100 and 1% of either goat or rabbit serum in PBS depending on the second step reagent. Primary antibodies were either rabbit polyclonals (1:200) or mouse monoclonals (5 g/ml) as indicated, diluted in either 1% goat or rabbit serum in PBS, respectively, and incubated for one hour. Detection was with either a secondary biotinylated antibody (Zymed) and streptavidin phycoerythrin (PE) (Zymed) or a conjugated antilgGl-PE
antibody (Zymed).
Cells were examined with either a Zeiss Axiophot or an Olympus BH2-RFC
microscope equipped with epifluorescent optics and photographed with Ektachrome 1600 ASA
film.
Example IF. FACS ANALYSIS:
For these experiments, the cells were washed once with PBS and once with EDTA/salts before incubation in EDTA/salts for 20 minutes at room temperature. Cells were then removed by gentle pipetting on the surface of the plates. The plates were washed with EDTA/salts and combined with the cells which were then spun down, washed once more with PBS
and then counted. 1 x 106 cells were used for each antibody incubation. For A2B5 surface staining at 4C, the cell pellet was first incubated with 50 l heat inactivated rabbit serum to block nonspecific binding and then with the A2B5 antibody (Boehringer-Mannheim) or control class SUBSTITUTE SHEET (RULE 26) ~ t specific IgM at 5 g/ml diluted in I % rabbit serum/PBS for one hour.
Detection was with a directly conjugated anti-IgM-PE (Zymed). For further double staining experiments or single internal staining for GFAP, the cells were fixed in 0.25% paraformaldehyde at 4 C for one hour, spun down and then resuspended in 1 ml of 0.2% Tween 20 in PBS/Azide and incubated at 37 C for 15 minutes. 1 ml of 2% heat inactivated rabbit serum/PBS was added and the cells were spun down. The pellet was resuspended in 50 l of rabbit serum and then the primary antibodies and class-specific controls were diluted in 100 Yl of 1,% ,rabbit serum in PBS at 5 ug/ml. Final detection was with directly conjugated anti-IgGl-FITC antibody (Zymed, Fisher *
Biotech). Cells were washed with 1% rabbit serum, 0.2% Tween 20 in PBS/Azide, then with PBS and then finally restispended in 1% paraformaldehyde. FACS analysis was performed on a FACScan (Becton Dickinson, San Jose, Ca) using a 15mw, 488 nm air-cooled argon ion laser for fluorochrome excitation. Fltiorescence emission was measured in the standard FACScan configuration: 530 nm (FITC_, 585 nm (PE) and >650 nm (red fluorescence)..
Data was acquired and analyzed on a Hewlett Packard 340C computer system, using LYSYS 11 software (Becton Dickinson, San Jose, Ca). Isotype controls were run for eacii sample and gates were set for single staining experiments such that they included no more than 3% of the cells.
Example IG. WESTERN ANALYSIS:
Cells were plated into duplicate wells of a 6-well dish at 2.5 x I0'/cm' and the appropriate BMP or TGF-01 was added at 1, 10 and 100 ng/ml at 16 hours. After 44 hours the cells were harvested. One well was trypsinized and counted and the second was washed with PBS, the cells scraped into ice-cold PBS containing 1 mM Pefabloc (water-soluble protease inhibitor from Boehringer-Mannheini) and centrifuged at 400 x G. 1-2 volunies of 0.1 % Triton X-100, 1 mM Pefabloc, 0.125 M T ris base, pH 6.8, DNAse at 250 U/ml was added to the cell pellets and mixed. Finally 0.5% SDS and 20mM DTT were added to each. Based on the=cell counts of the duplicate wells, the equivalent volume containing 5 x 105 cells of each condition was loaded in each lane of a 12%, 1 nim Laemlli mini-gel (Novex). Bovine GFAP
was also loaded at 10 and 100 ng. After running, the gel was transferred at 300 mAinps x 1 hour in the presence of 0.05 % SDS to 0.45 Ic nitrocellulose. The blot was air dyed, fixed in 1% KOH, * Trade-mark 11 SUBSTITUTE SHEET (RULE 26) ...r washed and blocked in 0.5% Tween 20 in TBS (20 mM Tris, 500 mM NaCI, pH 8.5) then incubated in a 1:1000 dilution of GFAP antiserum (BTI) overnight. After washing in 0.5%
Tween 20 in TBS, the blot was incubated in a 1:3000 dilution of goat anti-rabbit HRP x 1 hour and then developed by Enhanced Chemiluminescence (Amersham kit). Briefly, the blot was washed in TBS-Tw20 followed by TBS, incubated in a 1:1 mixture of reagents A
and B for 1 minute and then exposed to film and developed.
RESULTS Treatment of SFME cells with TGF-/31 or serum resulted in distinct morphological changes accompanied by expression of the astrocyte-specific differentiation marker GFAP (Sakai et al., su ra). TGF-/31 treatment resulted in an elongated bipolar cell type with cytoplasmic processes at both ends which stained for GFAP. By contrast, fetal calf serum (FCS)-treated cells were larger in size, with a highly branched filament network which stained very strongly for GFAP.
Treatment of SFME cells with BMP-2, 4, 5, 6, 7 and BMP-2/6 and 2/7 heterodimer at 10 ng/ml resulted in a dramatic morphological change in their appearance, accoinpanied by expression of GFAP. The cells acquired many long cytoplasmic processes typical of primary astrocytes in culture. Overall, the intensity of GFAP staining observed with BMPs and calf serum was much greater than that observed for TGF-0. BMP-2 and BMP 2/7 heterodimer induced a cell type with the larger morphology, similar to what was seen with calf serum, while BMP-7 induced a morphology which was more fibrous in nature. It is possible that these morphologies reflect either phenotypic differences in the induced cell type (Type 1 vs. Type 2 astrocytes) or varying levels of GFAP or other cytoskeletal proteins. Control cells have a fibroblast-like appearance and do not stain for GFAP.
In order to accurately measure the level of GFAP expression induced by BMP, as well as compare the activity to that of TGF-a, a quantitative assay by fluorescence activated cell sorting was established. The cells were treated with 10 ng/ml of each BMP, TGF-,li 1 and Activin and 10% calf serum. The data were analyzed by percent of the population responding and mean fluorescence intensity.
The percentage of the population responding is reflective of the number of cells expressing GFAP, independent of the level of expression. BMP-2, 4, 5, 6, 7, 2/6 heterodimer SUBSTITUTE SHEET (RULE 26) and 2/7 heterodimer, TGF-0 and calf serum significantly induce the expression of GFAP
compared with the control. Activin, another member of the TGF-0 superfamily, also has no effect. The BMP-2/6 and 2/7 heterodimers are most effective in this parameter, resulting in approximately 65 to 72% responsive cells. BMP-2, 4, TGF-/31 and fetal calf serum treatments result in approximately 53 to 58% responsive cells; BMP-5, 6 and 7 treatments result in approximately 30 to 40% responsive cells.
= Mean fluorescence intensity (MFI) is indicative of the level of GFAP
expression; the higher the mean fluorescence, the greater the level of GFAP expressed. BMP-2/6 and 2/7 heterodimer induced cells have a mean fluorescence approximately 8-fold greater than that of the TGF-/31 induced cells. BMP-2, 4, 5, 6, and 7 induced cells have a mean fluorescence approximately 2 to 4-fold greater than that of calf serum. TGF-0 and calf serum all give values significantly higher than the control.
In order to compare the ability of BMPs and TGF-01 to induce GFAP, BMP-2, BMP-6, BMP-2/6 heterodimer and TGF-01 were tested over a concentration range of 0-10 ng/ml and the FACS assay was used for quantitation of GFAP expression. The concentration at which each factor gave a GFAP mean fluorescence value of 5 (10-fold over the control of 0.5) was used to compare relative activities. In terms of relative activity compared to TGF-a1, heterodimer was approximately 18 fold more active and BMP-2 and BMP-6 were aproximately 3-4 fold more active. BMP-2 and BMP-2/6 induced detectable levels of GFAP in the 0-0.08 ng/ml range while the first detectable GFAP increase with TGF-0 1 is in the 0.4-2 ng/ml range.
Western analysis also confirmed the higher levels of GFAP produced by SFME
cells after exposure to BMPs. In BMP or TGF-al treated cellular extracts, the polyclonal GFAP antibody used for detection specifically recognizes a protein in the 40-50 kD range, which runs slightly below the 52 kD bovine GFAP standard. The broad molecular weight range observed is probably the result of proteolysis. There was a dose-dependent increase in protein levels with BMP-2 and BMP-6 treatment from 1-100 ng/ml. GFAP induced by TGF-0 treatment was maximal at a 10 ng/ml dose and is approximately equal to that seen with only 1 ng/ml of BMP-2. This level could not be increased even at a 100 ng/ml dose. BMPs induced higher levels of GFAP than TGF-(.i 1.
Treatment of SFME cells with BMPs results in conversion of the "fibroblast-like" cells SUBSTITUTE SHEET (RULE 26) ...~r into two distinct GFAP-positive morphologies. One large, flat cell type with few processes is reminiscent of a protoplasmic type of astrocyte; another with very long cytoplasmic processes is characteristic of a fibrous astrocyte.
These cells were further characterized by double immunofluorescent antibody staining for A2B5 and GFAP. In the BMP-2/6 population, both Type 1 and Type 2 astrocyte lineage cells were present. The majority of cells which stained for GFAP but not A2B5 were of the Type 1 astrocyte lineage while the cells which stained for both A2B5 and GFAP
were of Type 2 astrocyte lineage. Control cells stained for A2B5 on their surface, but did not stain for GFAP.
In order to quantitate the populations of cells seen by immunofluorescent staining, we employed double staining FACS analysis. The data in Table I is expressed as an average of at least three experiments standard deviation. Control cells were approximately 37% A2B5 positive. Control cells did not stain positively for the astrocyte lineage markers. BMP-2, 6 and 2/6 treated cells did not stain only for A2B5, but did consist of the two astrocyte lineage populations. Greater than 60% were positive for GFAP alone indicating that they were of Type 1 lineage and about 18% were positive for both A2B5 and GFAP, indicating that these were of Type 2 lineage. TGF-01 treatment also resulted in a similar size population of Type 2 lineage cells (approximately 14%), but only approximately 40% positive population of Type 1 lineage cells. There also remained a small population of cells (approximately 7%) which single stained for A2B5. Overall, treatment of SFME cells with either BMPs or TGF-01 resulted in the loss of expression of A2B5 which cannot be totally accounted for in the A2B5/GFAP
population.
SUBSTITU'fE SHEET (RULE 26) TABLE 1: ASTROCYTES
Control 37.13 18.66 0.15 0.19 1.18 0.67 BMP-2 0.39 0.54 60.49 3.71 19.89 3.31 BMP-6 0.25 0.43 59.28 1.28 17.89 3.19 BMP-2/6 0.42 0.38 65.07 7.33 18.37 5.49 TGF-~ 1 7.05 1.02 39.72 3.02 14.23 3.04 SFME Cell Survival Studv EGF is required for survival of SFME cells and other factors such as FGF and cannot substitute for it. In the absence of EGF, SFME cells were treated with BMP-2, 7, 2/7 heterodimer, TGF-01 and activin. Activin has been shown to be a nerve cell survival molecule for P19 cells. Schubert et al., Nature 344:868-870 (1990). After 48 hours in the absence of EGF, there were only about 30% surviving cells, and an overall decrease in cell number.
Addition of EGF resulted in approximately 95% cell survival rate accompanied by a 5-fold increase in cell number. Cells treated with BMP-2, BMP-7 and BMP-2/7 heterodimer maintained a cell nuinber approximately 70-80% of the seeding density. However the cells did not proliferate. BMP-2 treated cells not only survived but also apeared to have differentiated.
The survival rate was approximately 80-85%. Treatment of cells with either TGF-0 1 or Activin resulted in survival rates of < 10% and at least a 10-fold decrease in cell number. Higher concentrations of TGF-a 1 did not increase survival.
EXAMPLE II. PERIPHERAL NERVE REGENERATION IN MAMMALS USING BMP-2 A. Preparation of Collagen Sponge Collagen sponges (Collastat1z, Vitaphore Wound Healing, Inc.) were cut in approximately Sli9STITUTE SHEET (RULE 26) ~.r 2 x 2 x 18 mm lengths, washed extensively in sterile glass distilled water, lyophilized, ethylen oxide sterilized and degassed prior to addition of BMP-2.
0.5 g of BMP-2 in 45% Acetonitrile, 0.1 % trifluoroacetic acid was evenly distributed over the length of each prepared sponge. These were then placed in a tube, frozen in liquid nitrogen and lyophilized. Control implants were prepared the same way except with 45%
Acetonitrile, 0. 1 % Trifluoroacetic acid buffer without BMP-2.
After lyophilization, the BMP-2 loaded and control sponges were placed inside of approximately 1.6 x 20 mm lengths of sterile vented silastic tubing. All manipulations were performed under sterile conditions. Excess tubing at either end of the implant was removed in the operating room prior to surgery.
The sciatic nerve of 6 Lewis rats were severed. Vented silastic or biodegradable stents, 1.6 mm internal diameter x 17 mm long, were inserted. Stents contained collagen matrix carrier with or without rhBMP-2. The collagen matrix carrier was composed of collagen sponge (Collastat)(approximately 1.5 mm x 15 mm). Aninials with the sciatic nerve severed and tied back to prevent reattachment served as positive controls. The unoperated hind limb served as age-matched negative controls.
The stents were applied microscopically and anastomosed to the severed nerve endings of the sciatic nerve. The nerve endings were inserted into the stent for 1 mm at each end, leaving a 15 mm gap. Aniinals were tested for electrical return of function at 6, 8 and 12 weeks post implantation. Compound muscle action potentials (CMAP) were examined, which provided a reliable, reproducible, transcutaneous procedure which is an accurate for determining the degree of functional return. Amplitude and latency are age-dependent and directly proportional to the number of reinnervated axons/inotor endplates.
Animals were sacrificed for pathological exainination at 12 weeks PI. Stains included H&E, Silver, Luxol-fast blue and S100. Unbiased quantification of the proximal, central and distal elements within the stent were performed. Stents placed within the subcutaneous tissues of several rats served as controls for the stains.
Results showed good nerve regeneration across the 15 mm nerve defect in 4 of 6 animals treated with 0.5 ug per device of BMP-2 deposited on Collastat sponge after 12 weeks. The controls without BMP-2 revealed no growth across the 15 mm nerve defect.
SUBSTITUTE SHEET (RULE 26)
Example IA. CELL CULTURE
Balb c/SFME (Serum-Free Mouse Embryo) cells were obtained from the American Type Culture Collection (CRL 9392) and were grown as previously described (Sakai et al., PNAS:USA, 87:8378-8382 (1990)) in DME/F12 (1:1) medium containing bovine insulin, 10 g/ml (Eli Lilly); human transferrin, 25 g/ml (Collaborative Research); human high density lipoprotein, 20 g/ml (sigma); human epidermal growth factor, 100 ng/ml (PeproTech); bovine plasma fibronectin, 20 g/ml (GIBCO); sodium selenite, IOnM (GIBCO);
penicillin-streptomycin (10 U/ml), 1-glutamine (4mM) and 4-(2-hydroxy-ethyl) - piperazine-ethanesulfonic acid, pH 7.4 (15 mM).
Cells were passaged using trypsin/EDTA and soybean trypsin inhibitor (1 mg/ml) in a volume ratio of 1:2 and used between passages 19 and 50. Cells were counted, unless otherwise stated, with a Coulter Diagnostics counter.
Example IB. GROWTH AND DIFFERENTIATION FACTORS:
All recombinant human proteins used were of greater than 90% purify. EGF was purchased from PeproTech (NJ); recombinant human Activin-A was the generous gift of Helen New; TGF-fl l was purchased from R&D Systems; BMPs were purified from CHO
conditioned media through several purification steps at Genetics Institute.
Example IC. DIFFERENTIATION STUDIES:
For all immunofluorescence and FACS analysis, unless otherwise stated, cells were plated at 2.5 - 5 x 10 /cm2 and BMP-2 was added at 16-20 hrs. at the concentrations and for the length of time indicated.
Example ID. SURVIVAL STUDIES:
Cells were washed twice in medium without EGF and then plated at 0.8 - I x 105/cm2 into the same medium supplemented with various growth factors. These included BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-2/6 heterodimer, BMP-2/7 heterodimer and TGF-/31.
Percent viability and cell number were determined in duplicate by trypan blue dye exclusion with a SUBSTITUTE SHEET (RULE 26) WO 95/05846 2! 6919j PCT/US94/09330 hemocytometer at 44-48 hrs., counting a minimum of 400 cells per sample.
Percent viability was calculated as total live cells divided by the total number of cells at the endpoint since cell proliferation was seen in some conditions.
Example IE. IMMUNOFLUORESCENT ANTIBODY STAINING:
Media was removed from cells in four-well glass or plastic chamber slides (Lab Tek) and they were washed twice with PBS- Ca+2, Mg+2 free (CMF). For surface staining with the antibody A2B5 (Boehringer-Mannheim) the cells were initially fixed with 4% paraformaldehyde for 10 minutes, washed with PBS and then incubated with 1% rabbit serum in PBS
to block nonspecific binding. The antibody was diluted in 1% rabbit serum in PBS and incubated for 1 hour then the cells were washed in 1% rabbit serum in PBS before detection with a biotinylated rabbit anti-mouse antibody followed by a streptavidin-FITC (Zymed) conjugate.
For further double staining experiments or single internal staining for GFAP, cells were fixed in acetone/methanol (50:50) at -20C for 10 minutes. Permeabilization and blocking was performed with 0.2% Triton X-100 and 1% of either goat or rabbit serum in PBS depending on the second step reagent. Primary antibodies were either rabbit polyclonals (1:200) or mouse monoclonals (5 g/ml) as indicated, diluted in either 1% goat or rabbit serum in PBS, respectively, and incubated for one hour. Detection was with either a secondary biotinylated antibody (Zymed) and streptavidin phycoerythrin (PE) (Zymed) or a conjugated antilgGl-PE
antibody (Zymed).
Cells were examined with either a Zeiss Axiophot or an Olympus BH2-RFC
microscope equipped with epifluorescent optics and photographed with Ektachrome 1600 ASA
film.
Example IF. FACS ANALYSIS:
For these experiments, the cells were washed once with PBS and once with EDTA/salts before incubation in EDTA/salts for 20 minutes at room temperature. Cells were then removed by gentle pipetting on the surface of the plates. The plates were washed with EDTA/salts and combined with the cells which were then spun down, washed once more with PBS
and then counted. 1 x 106 cells were used for each antibody incubation. For A2B5 surface staining at 4C, the cell pellet was first incubated with 50 l heat inactivated rabbit serum to block nonspecific binding and then with the A2B5 antibody (Boehringer-Mannheim) or control class SUBSTITUTE SHEET (RULE 26) ~ t specific IgM at 5 g/ml diluted in I % rabbit serum/PBS for one hour.
Detection was with a directly conjugated anti-IgM-PE (Zymed). For further double staining experiments or single internal staining for GFAP, the cells were fixed in 0.25% paraformaldehyde at 4 C for one hour, spun down and then resuspended in 1 ml of 0.2% Tween 20 in PBS/Azide and incubated at 37 C for 15 minutes. 1 ml of 2% heat inactivated rabbit serum/PBS was added and the cells were spun down. The pellet was resuspended in 50 l of rabbit serum and then the primary antibodies and class-specific controls were diluted in 100 Yl of 1,% ,rabbit serum in PBS at 5 ug/ml. Final detection was with directly conjugated anti-IgGl-FITC antibody (Zymed, Fisher *
Biotech). Cells were washed with 1% rabbit serum, 0.2% Tween 20 in PBS/Azide, then with PBS and then finally restispended in 1% paraformaldehyde. FACS analysis was performed on a FACScan (Becton Dickinson, San Jose, Ca) using a 15mw, 488 nm air-cooled argon ion laser for fluorochrome excitation. Fltiorescence emission was measured in the standard FACScan configuration: 530 nm (FITC_, 585 nm (PE) and >650 nm (red fluorescence)..
Data was acquired and analyzed on a Hewlett Packard 340C computer system, using LYSYS 11 software (Becton Dickinson, San Jose, Ca). Isotype controls were run for eacii sample and gates were set for single staining experiments such that they included no more than 3% of the cells.
Example IG. WESTERN ANALYSIS:
Cells were plated into duplicate wells of a 6-well dish at 2.5 x I0'/cm' and the appropriate BMP or TGF-01 was added at 1, 10 and 100 ng/ml at 16 hours. After 44 hours the cells were harvested. One well was trypsinized and counted and the second was washed with PBS, the cells scraped into ice-cold PBS containing 1 mM Pefabloc (water-soluble protease inhibitor from Boehringer-Mannheini) and centrifuged at 400 x G. 1-2 volunies of 0.1 % Triton X-100, 1 mM Pefabloc, 0.125 M T ris base, pH 6.8, DNAse at 250 U/ml was added to the cell pellets and mixed. Finally 0.5% SDS and 20mM DTT were added to each. Based on the=cell counts of the duplicate wells, the equivalent volume containing 5 x 105 cells of each condition was loaded in each lane of a 12%, 1 nim Laemlli mini-gel (Novex). Bovine GFAP
was also loaded at 10 and 100 ng. After running, the gel was transferred at 300 mAinps x 1 hour in the presence of 0.05 % SDS to 0.45 Ic nitrocellulose. The blot was air dyed, fixed in 1% KOH, * Trade-mark 11 SUBSTITUTE SHEET (RULE 26) ...r washed and blocked in 0.5% Tween 20 in TBS (20 mM Tris, 500 mM NaCI, pH 8.5) then incubated in a 1:1000 dilution of GFAP antiserum (BTI) overnight. After washing in 0.5%
Tween 20 in TBS, the blot was incubated in a 1:3000 dilution of goat anti-rabbit HRP x 1 hour and then developed by Enhanced Chemiluminescence (Amersham kit). Briefly, the blot was washed in TBS-Tw20 followed by TBS, incubated in a 1:1 mixture of reagents A
and B for 1 minute and then exposed to film and developed.
RESULTS Treatment of SFME cells with TGF-/31 or serum resulted in distinct morphological changes accompanied by expression of the astrocyte-specific differentiation marker GFAP (Sakai et al., su ra). TGF-/31 treatment resulted in an elongated bipolar cell type with cytoplasmic processes at both ends which stained for GFAP. By contrast, fetal calf serum (FCS)-treated cells were larger in size, with a highly branched filament network which stained very strongly for GFAP.
Treatment of SFME cells with BMP-2, 4, 5, 6, 7 and BMP-2/6 and 2/7 heterodimer at 10 ng/ml resulted in a dramatic morphological change in their appearance, accoinpanied by expression of GFAP. The cells acquired many long cytoplasmic processes typical of primary astrocytes in culture. Overall, the intensity of GFAP staining observed with BMPs and calf serum was much greater than that observed for TGF-0. BMP-2 and BMP 2/7 heterodimer induced a cell type with the larger morphology, similar to what was seen with calf serum, while BMP-7 induced a morphology which was more fibrous in nature. It is possible that these morphologies reflect either phenotypic differences in the induced cell type (Type 1 vs. Type 2 astrocytes) or varying levels of GFAP or other cytoskeletal proteins. Control cells have a fibroblast-like appearance and do not stain for GFAP.
In order to accurately measure the level of GFAP expression induced by BMP, as well as compare the activity to that of TGF-a, a quantitative assay by fluorescence activated cell sorting was established. The cells were treated with 10 ng/ml of each BMP, TGF-,li 1 and Activin and 10% calf serum. The data were analyzed by percent of the population responding and mean fluorescence intensity.
The percentage of the population responding is reflective of the number of cells expressing GFAP, independent of the level of expression. BMP-2, 4, 5, 6, 7, 2/6 heterodimer SUBSTITUTE SHEET (RULE 26) and 2/7 heterodimer, TGF-0 and calf serum significantly induce the expression of GFAP
compared with the control. Activin, another member of the TGF-0 superfamily, also has no effect. The BMP-2/6 and 2/7 heterodimers are most effective in this parameter, resulting in approximately 65 to 72% responsive cells. BMP-2, 4, TGF-/31 and fetal calf serum treatments result in approximately 53 to 58% responsive cells; BMP-5, 6 and 7 treatments result in approximately 30 to 40% responsive cells.
= Mean fluorescence intensity (MFI) is indicative of the level of GFAP
expression; the higher the mean fluorescence, the greater the level of GFAP expressed. BMP-2/6 and 2/7 heterodimer induced cells have a mean fluorescence approximately 8-fold greater than that of the TGF-/31 induced cells. BMP-2, 4, 5, 6, and 7 induced cells have a mean fluorescence approximately 2 to 4-fold greater than that of calf serum. TGF-0 and calf serum all give values significantly higher than the control.
In order to compare the ability of BMPs and TGF-01 to induce GFAP, BMP-2, BMP-6, BMP-2/6 heterodimer and TGF-01 were tested over a concentration range of 0-10 ng/ml and the FACS assay was used for quantitation of GFAP expression. The concentration at which each factor gave a GFAP mean fluorescence value of 5 (10-fold over the control of 0.5) was used to compare relative activities. In terms of relative activity compared to TGF-a1, heterodimer was approximately 18 fold more active and BMP-2 and BMP-6 were aproximately 3-4 fold more active. BMP-2 and BMP-2/6 induced detectable levels of GFAP in the 0-0.08 ng/ml range while the first detectable GFAP increase with TGF-0 1 is in the 0.4-2 ng/ml range.
Western analysis also confirmed the higher levels of GFAP produced by SFME
cells after exposure to BMPs. In BMP or TGF-al treated cellular extracts, the polyclonal GFAP antibody used for detection specifically recognizes a protein in the 40-50 kD range, which runs slightly below the 52 kD bovine GFAP standard. The broad molecular weight range observed is probably the result of proteolysis. There was a dose-dependent increase in protein levels with BMP-2 and BMP-6 treatment from 1-100 ng/ml. GFAP induced by TGF-0 treatment was maximal at a 10 ng/ml dose and is approximately equal to that seen with only 1 ng/ml of BMP-2. This level could not be increased even at a 100 ng/ml dose. BMPs induced higher levels of GFAP than TGF-(.i 1.
Treatment of SFME cells with BMPs results in conversion of the "fibroblast-like" cells SUBSTITUTE SHEET (RULE 26) ...~r into two distinct GFAP-positive morphologies. One large, flat cell type with few processes is reminiscent of a protoplasmic type of astrocyte; another with very long cytoplasmic processes is characteristic of a fibrous astrocyte.
These cells were further characterized by double immunofluorescent antibody staining for A2B5 and GFAP. In the BMP-2/6 population, both Type 1 and Type 2 astrocyte lineage cells were present. The majority of cells which stained for GFAP but not A2B5 were of the Type 1 astrocyte lineage while the cells which stained for both A2B5 and GFAP
were of Type 2 astrocyte lineage. Control cells stained for A2B5 on their surface, but did not stain for GFAP.
In order to quantitate the populations of cells seen by immunofluorescent staining, we employed double staining FACS analysis. The data in Table I is expressed as an average of at least three experiments standard deviation. Control cells were approximately 37% A2B5 positive. Control cells did not stain positively for the astrocyte lineage markers. BMP-2, 6 and 2/6 treated cells did not stain only for A2B5, but did consist of the two astrocyte lineage populations. Greater than 60% were positive for GFAP alone indicating that they were of Type 1 lineage and about 18% were positive for both A2B5 and GFAP, indicating that these were of Type 2 lineage. TGF-01 treatment also resulted in a similar size population of Type 2 lineage cells (approximately 14%), but only approximately 40% positive population of Type 1 lineage cells. There also remained a small population of cells (approximately 7%) which single stained for A2B5. Overall, treatment of SFME cells with either BMPs or TGF-01 resulted in the loss of expression of A2B5 which cannot be totally accounted for in the A2B5/GFAP
population.
SUBSTITU'fE SHEET (RULE 26) TABLE 1: ASTROCYTES
Control 37.13 18.66 0.15 0.19 1.18 0.67 BMP-2 0.39 0.54 60.49 3.71 19.89 3.31 BMP-6 0.25 0.43 59.28 1.28 17.89 3.19 BMP-2/6 0.42 0.38 65.07 7.33 18.37 5.49 TGF-~ 1 7.05 1.02 39.72 3.02 14.23 3.04 SFME Cell Survival Studv EGF is required for survival of SFME cells and other factors such as FGF and cannot substitute for it. In the absence of EGF, SFME cells were treated with BMP-2, 7, 2/7 heterodimer, TGF-01 and activin. Activin has been shown to be a nerve cell survival molecule for P19 cells. Schubert et al., Nature 344:868-870 (1990). After 48 hours in the absence of EGF, there were only about 30% surviving cells, and an overall decrease in cell number.
Addition of EGF resulted in approximately 95% cell survival rate accompanied by a 5-fold increase in cell number. Cells treated with BMP-2, BMP-7 and BMP-2/7 heterodimer maintained a cell nuinber approximately 70-80% of the seeding density. However the cells did not proliferate. BMP-2 treated cells not only survived but also apeared to have differentiated.
The survival rate was approximately 80-85%. Treatment of cells with either TGF-0 1 or Activin resulted in survival rates of < 10% and at least a 10-fold decrease in cell number. Higher concentrations of TGF-a 1 did not increase survival.
EXAMPLE II. PERIPHERAL NERVE REGENERATION IN MAMMALS USING BMP-2 A. Preparation of Collagen Sponge Collagen sponges (Collastat1z, Vitaphore Wound Healing, Inc.) were cut in approximately Sli9STITUTE SHEET (RULE 26) ~.r 2 x 2 x 18 mm lengths, washed extensively in sterile glass distilled water, lyophilized, ethylen oxide sterilized and degassed prior to addition of BMP-2.
0.5 g of BMP-2 in 45% Acetonitrile, 0.1 % trifluoroacetic acid was evenly distributed over the length of each prepared sponge. These were then placed in a tube, frozen in liquid nitrogen and lyophilized. Control implants were prepared the same way except with 45%
Acetonitrile, 0. 1 % Trifluoroacetic acid buffer without BMP-2.
After lyophilization, the BMP-2 loaded and control sponges were placed inside of approximately 1.6 x 20 mm lengths of sterile vented silastic tubing. All manipulations were performed under sterile conditions. Excess tubing at either end of the implant was removed in the operating room prior to surgery.
The sciatic nerve of 6 Lewis rats were severed. Vented silastic or biodegradable stents, 1.6 mm internal diameter x 17 mm long, were inserted. Stents contained collagen matrix carrier with or without rhBMP-2. The collagen matrix carrier was composed of collagen sponge (Collastat)(approximately 1.5 mm x 15 mm). Aninials with the sciatic nerve severed and tied back to prevent reattachment served as positive controls. The unoperated hind limb served as age-matched negative controls.
The stents were applied microscopically and anastomosed to the severed nerve endings of the sciatic nerve. The nerve endings were inserted into the stent for 1 mm at each end, leaving a 15 mm gap. Aniinals were tested for electrical return of function at 6, 8 and 12 weeks post implantation. Compound muscle action potentials (CMAP) were examined, which provided a reliable, reproducible, transcutaneous procedure which is an accurate for determining the degree of functional return. Amplitude and latency are age-dependent and directly proportional to the number of reinnervated axons/inotor endplates.
Animals were sacrificed for pathological exainination at 12 weeks PI. Stains included H&E, Silver, Luxol-fast blue and S100. Unbiased quantification of the proximal, central and distal elements within the stent were performed. Stents placed within the subcutaneous tissues of several rats served as controls for the stains.
Results showed good nerve regeneration across the 15 mm nerve defect in 4 of 6 animals treated with 0.5 ug per device of BMP-2 deposited on Collastat sponge after 12 weeks. The controls without BMP-2 revealed no growth across the 15 mm nerve defect.
SUBSTITUTE SHEET (RULE 26)
Claims (18)
1. ~A nerve replacement device for promoting the growth of astrocytes comprising an artificial nerve replacement vessel which contains a composition comprising a bone morphogenetic protein to promote the growth of astrocytes and a suitable matrix carrier, wherein the bone morphogenetic protein is selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7 and heterodimers of BMP-2/6 and BMP-2/7.
2. ~The device of claim 1, wherein the bone morphogenetic protein is selected from the group consisting of BMP-2, BMP-4, BMP-2/6 heterodimers and BMP-2/7 heterodimers.
3. ~The device of claim 1, wherein the matrix carrier comprises a suitable material selected from to the group consisting of collagen, fibrin tissue adhesives, laminin, hyalauronic acid and chondroitin sulfate proteoglycans.
4. ~The device of claim 3 wherein the matrix carrier comprises collagen.
5. ~The device of claim 4, wherein the collagen is in the form of a sponge.
6. ~The device of claim 1, wherein the artificial nerve replacement vessel comprises vented silastic tubing.
7. ~A use of a bone morphogenetic protein in admixture with a pharmaceutically acceptable vehicle, for inducing growth or regeneration of astrocyte cells in a mammal at a site of a severed or damaged nerve, wherein the bone morphogenetic protein is selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7 and heterodimers of BMP-2/6 and BMP-2/7.
8. ~A use of a bone morphogenetic protein in admixture with a pharmaceutically acceptable vehicle, for the production of a medicament, for inducing growth or regeneration of astrocyte cells in a mammal at a site of a severed or damaged nerve, wherein the bone morphogenetic protein is selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7 and heterodimers of BMP-2/6 and BMP-2/7.
9. A use of a bone morphogenetic protein in combination with a suitable matrix, for inducing growth or regeneration of astrocyte cells in mammal at a site of a severed or damaged nerve, wherein the bone morphogenetic protein is selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7 and heterodimers of BMP-2/6 and BMP-2/7.
10. A use of a bone morphogenetic protein in combination with a suitable matrix, for the production of a medicament, for inducing growth or regeneration of astrocyte cells in a mammal at a site of a severed or damaged nerve, wherein the bone morphogenetic protein is selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7 and heterodimers of BMP-2/6 and BMP-2/7.
11. A use of a bone morphogenetic protein for preparation of a composition for induction of growth or regeneration of astrocyte cells, wherein the bone morphogenetic protein is selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7 and heterodimers of BMP-2/6 and BMP-2/7.
12. A pharmaceutical composition for inducing formation of astrocytes, which comprises a bone morphogenetic protein in combination with a pharmaceutically acceptable vehicle, wherein said bone morphogenetic protein is selected from the group consisting of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7 and heterodimers of BMP-2/6 and BMP-2/7.
13. The pharmaceutical composition of claim 12, wherein the bone morphogenetic protein is selected from the group consisting of BMP-2, BMP-4, BMP-2/6 heterodimers and BMP-2/7 heterodimers.
14. The pharmaceutical composition of claim 12, wherein the bone morphogenetic protein is adsorbed to a suitable matrix.
15. The pharmaceutical composition of claim 14, wherein the matrix comprises collagen in the form of a sponge.
16. The pharmaceutical of claim 14, wherein the matrix is contained within an artificial nerve replacement vessel.
17. The pharmaceutical composition of claim 12, wherein said bone morphogenetic protein is BMP-2.
18. The nerve replacement devise of claim 1 for implantation at a site in need of peripheral nerve repair.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US11249293A | 1993-08-26 | 1993-08-26 | |
| US08/112,492 | 1993-08-26 | ||
| PCT/US1994/009330 WO1995005846A1 (en) | 1993-08-26 | 1994-08-19 | Neural regeneration using human bone morphogenetic proteins |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2169191A1 CA2169191A1 (en) | 1995-03-02 |
| CA2169191C true CA2169191C (en) | 2008-01-15 |
Family
ID=22344181
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002169191A Expired - Lifetime CA2169191C (en) | 1993-08-26 | 1994-08-19 | Neural regeneration using human bone morphogenetic proteins |
Country Status (15)
| Country | Link |
|---|---|
| US (1) | US5756457A (en) |
| EP (2) | EP0716610B1 (en) |
| JP (3) | JPH09501932A (en) |
| KR (2) | KR100329409B1 (en) |
| AT (1) | ATE326234T1 (en) |
| AU (1) | AU677866B2 (en) |
| CA (1) | CA2169191C (en) |
| DE (1) | DE69434739T2 (en) |
| DK (1) | DK0716610T3 (en) |
| ES (1) | ES2265146T3 (en) |
| FI (1) | FI960809A (en) |
| NO (1) | NO960711L (en) |
| OA (1) | OA10358A (en) |
| PT (1) | PT716610E (en) |
| WO (1) | WO1995005846A1 (en) |
Families Citing this family (162)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6150328A (en) * | 1986-07-01 | 2000-11-21 | Genetics Institute, Inc. | BMP products |
| US5459047A (en) * | 1986-07-01 | 1995-10-17 | Genetics Institute, Inc. | BMP-6 proteins |
| US6495513B1 (en) | 1991-03-11 | 2002-12-17 | Curis, Inc. | Morphogen-enhanced survival and repair of neural cells |
| US6506729B1 (en) | 1991-03-11 | 2003-01-14 | Curis, Inc. | Methods and compositions for the treatment and prevention of Parkinson's disease |
| US6723698B2 (en) | 1991-03-11 | 2004-04-20 | Curis, Inc. | Methods and compositions for the treatment of motor neuron injury and neuropathy |
| US6800603B2 (en) | 1991-03-11 | 2004-10-05 | Curis, Inc. | Morphogen-induced neural cell adhesion |
| US20080070842A1 (en) * | 1991-11-04 | 2008-03-20 | David Israel | Recombinant bone morphogenetic protein heterodimers, compositions and methods of use |
| DE69233022T2 (en) * | 1991-11-04 | 2004-02-12 | Genetics Institute, LLC, Cambridge | RECOMBINANT BONE MORPHOGENETIC PROTEIN HETERODIMERS, COMPOSITIONS AND METHODS OF USE |
| JP3681069B2 (en) * | 1993-05-12 | 2005-08-10 | ジェネティックス・インスチチュート・リミテッド・ライアビリティ・カンパニー | BMP-11 composition |
| US6291206B1 (en) * | 1993-09-17 | 2001-09-18 | Genetics Institute, Inc. | BMP receptor proteins |
| CA2176942C (en) * | 1993-12-07 | 2011-11-01 | Anthony J. Celeste | Bmp-12, bmp-13 and tendon-inducing compositions thereof |
| WO1996030038A1 (en) * | 1995-03-29 | 1996-10-03 | The Rockefeller University | Peptide growth factor having epidermal inducing activity |
| US7026292B1 (en) | 1995-12-12 | 2006-04-11 | Stryker Corporation | Compositions and therapeutic methods using morphogenic proteins and stimulatory factors |
| US6048964A (en) * | 1995-12-12 | 2000-04-11 | Stryker Corporation | Compositions and therapeutic methods using morphogenic proteins and stimulatory factors |
| ATE493141T1 (en) † | 1996-03-22 | 2011-01-15 | Stryker Corp | METHOD FOR IMPROVED FUNCTIONAL RECOVERY OF MOTOR COORDINATION, LANGUAGE OR SENSORY PERCEPTION AFTER CNS TRAUMA OR ISCHEMIA |
| US5808011A (en) * | 1996-07-01 | 1998-09-15 | Biopure Corporation | Method for chromatographic removal of prions |
| US6037519A (en) * | 1997-10-20 | 2000-03-14 | Sdgi Holdings, Inc. | Ceramic fusion implants and compositions |
| US10028851B2 (en) | 1997-04-15 | 2018-07-24 | Advanced Cardiovascular Systems, Inc. | Coatings for controlling erosion of a substrate of an implantable medical device |
| US8172897B2 (en) | 1997-04-15 | 2012-05-08 | Advanced Cardiovascular Systems, Inc. | Polymer and metal composite implantable medical devices |
| US6240616B1 (en) | 1997-04-15 | 2001-06-05 | Advanced Cardiovascular Systems, Inc. | Method of manufacturing a medicated porous metal prosthesis |
| US20010007657A1 (en) * | 1997-08-04 | 2001-07-12 | Reid James Steven | Compositions and methods for manipulating glial progenitor cells and treating neurological deficits |
| IL134289A0 (en) * | 1997-08-04 | 2001-04-30 | Univ California | Methods for treating neurological deficits |
| US7795202B2 (en) | 1997-08-04 | 2010-09-14 | Neurorepair, Inc. | Methods for treating a neurological disorder by peripheral administration of a transforming growth factor alpha (TGF-a) |
| US6455283B1 (en) | 1998-03-17 | 2002-09-24 | Genentech, Inc. | Nucleic acids encoding vascular endothelial cell growth factor-E (VEGF-E) |
| AU1276399A (en) * | 1997-11-07 | 1999-05-31 | Genetics Institute Inc. | Neuronal uses of bmp-11 |
| US20030170213A1 (en) * | 1998-01-23 | 2003-09-11 | Marc F. Charette | Methods and compositions for enhancing cognitive function using morphogenic proteins |
| US5984926A (en) * | 1998-02-24 | 1999-11-16 | Jones; A. Alexander M. | Bone screw shimming and bone graft containment system and method |
| US6541623B1 (en) | 1998-04-03 | 2003-04-01 | Hyseq, Inc. | Interleukin—1 receptor antagonist and uses thereof |
| US6294655B1 (en) | 1998-04-03 | 2001-09-25 | Hyseq, Inc. | Anti-interleukin-1 receptor antagonist antibodies and uses thereof |
| US6426191B1 (en) | 1998-04-03 | 2002-07-30 | Hyseq, Inc. | Assays involving an IL-1 receptor antagonist |
| US7147839B2 (en) * | 1998-05-29 | 2006-12-12 | Curis, Inc. | Methods for evaluating tissue morphogenesis and activity |
| AU773012B2 (en) * | 1998-07-24 | 2004-05-13 | Carnegie Institution Of Washington | Method for maintenance and propagation of germline stem cells using members of the TGF-beta family of growth factors |
| US6727224B1 (en) * | 1999-02-01 | 2004-04-27 | Genetics Institute, Llc. | Methods and compositions for healing and repair of articular cartilage |
| DE19906172C1 (en) * | 1999-02-08 | 2000-07-13 | Ethicon Gmbh | Resorbable implant used for inducing tissue formation, especially in bone regeneration, has specific density and porosity properties |
| WO2000064460A2 (en) * | 1999-04-23 | 2000-11-02 | Sulzer Orthopedics Ltd. | Composition for enhancing functional recovery of a mammal from central and/or peripheral nervous system injury of traumatic or pathological origin |
| WO2000065027A2 (en) | 1999-04-28 | 2000-11-02 | Genetics Institute, Inc. | Human gil-19/ae289 proteins and polynucleotides encoding same |
| EP2067788B1 (en) | 1999-05-18 | 2015-07-22 | Dyax Corp. | Fab fragment libraries and methods for their use |
| JP2003508493A (en) * | 1999-09-08 | 2003-03-04 | ジェネティックス・インスチチュート・インコーポレーテッド | BMP-9 composition and method for inducing differentiation of cholinergic neurons |
| EP1762244A1 (en) * | 1999-09-08 | 2007-03-14 | Genetics Institute, LLC | Bmp-9 compositions for inducing differentiation of cholinergic neurons |
| US7687462B2 (en) | 1999-10-05 | 2010-03-30 | The Regents Of The University Of California | Composition for promoting cartilage formation or repair comprising a nell gene product and method of treating cartilage-related conditions using such composition |
| US6368787B1 (en) | 1999-12-16 | 2002-04-09 | Stryker Corporation | Methods and compositions for identifying morphogenic protein analogs using morphogenic protein responsive inhibitory elements |
| US8109994B2 (en) | 2003-01-10 | 2012-02-07 | Abbott Cardiovascular Systems, Inc. | Biodegradable drug delivery material for stent |
| US6527801B1 (en) * | 2000-04-13 | 2003-03-04 | Advanced Cardiovascular Systems, Inc. | Biodegradable drug delivery material for stent |
| US20040087016A1 (en) * | 2000-05-12 | 2004-05-06 | University Of Utah Research Foundation | Compositions and methods for cell dedifferentiation and tissue regeneration |
| EP1318833A2 (en) * | 2000-05-12 | 2003-06-18 | The University of Utah Research Foundation | Compositions and methods for cell dedifferentiation and tissue regeneration |
| CA2648048A1 (en) | 2000-06-23 | 2002-01-03 | Genentech, Inc. | Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis |
| EP2042597B1 (en) | 2000-06-23 | 2014-05-07 | Genentech, Inc. | Compositions and methods for the diagnosis and treatment of disorders involving angiogenesis |
| CZ301649B6 (en) * | 2000-06-28 | 2010-05-12 | Ed. Geistlich Soehne Ag Fur Chemische Industrie Incorporated Under The Laws Of Switzerland | Tube for regeneration of nerves and process for producing thereof |
| US20030082233A1 (en) * | 2000-12-01 | 2003-05-01 | Lyons Karen M. | Method and composition for modulating bone growth |
| US20020114795A1 (en) | 2000-12-22 | 2002-08-22 | Thorne Kevin J. | Composition and process for bone growth and repair |
| TWI329129B (en) | 2001-02-08 | 2010-08-21 | Wyeth Corp | Modified and stabilized gdf propeptides and uses thereof |
| JP3871525B2 (en) * | 2001-04-26 | 2007-01-24 | ニプロ株式会社 | Biological tissue or organ regeneration device |
| DE60226321T2 (en) * | 2001-06-01 | 2009-07-09 | Wyeth | COMPOSITIONS FOR THE SYSTEMIC ADMINISTRATION OF SEQUENCES THAT CODE FOR BONE MORPHOGENESIS PROTEINS |
| TWI267378B (en) * | 2001-06-08 | 2006-12-01 | Wyeth Corp | Calcium phosphate delivery vehicles for osteoinductive proteins |
| WO2003015612A2 (en) * | 2001-08-13 | 2003-02-27 | University Of Florida Research Foundation, Inc. | Materials and methods to promote repair of nerve tissue |
| US7285304B1 (en) | 2003-06-25 | 2007-10-23 | Advanced Cardiovascular Systems, Inc. | Fluid treatment of a polymeric coating on an implantable medical device |
| US7989018B2 (en) | 2001-09-17 | 2011-08-02 | Advanced Cardiovascular Systems, Inc. | Fluid treatment of a polymeric coating on an implantable medical device |
| US6863683B2 (en) | 2001-09-19 | 2005-03-08 | Abbott Laboratoris Vascular Entities Limited | Cold-molding process for loading a stent onto a stent delivery system |
| US7320789B2 (en) | 2001-09-26 | 2008-01-22 | Wyeth | Antibody inhibitors of GDF-8 and uses thereof |
| PL374966A1 (en) * | 2002-02-21 | 2005-11-14 | Wyeth | Follistatin domain containing proteins |
| CA2476887A1 (en) * | 2002-02-21 | 2003-09-04 | Wyeth | Gasp1:a follistatin domain containing protein |
| TWI282283B (en) * | 2002-05-17 | 2007-06-11 | Wyeth Corp | Injectable solid hyaluronic acid carriers for delivery of osteogenic proteins |
| AU2003298519A1 (en) * | 2002-06-27 | 2004-06-23 | Roberto Beretta | Methods for preparing a solid-fibrin web |
| EP1542711A4 (en) * | 2002-08-13 | 2009-07-01 | Wyeth Corp | PEPTIDES AS SOLUBILIZING EXCIPIENTS FOR TRANSFORMING GROWTH FACTOR s PROTEINS |
| ES2344734T3 (en) * | 2002-09-16 | 2010-09-06 | Johns Hopkins University | ACTIVATION OF MYOSTATIN BY METALOPROTEASE, AND METHODS TO MODULATE THE ACTIVITY OF MYOSTATIN. |
| AR047392A1 (en) | 2002-10-22 | 2006-01-18 | Wyeth Corp | NEUTRALIZATION OF ANTIBODIES AGAINST GDF 8 AND ITS USE FOR SUCH PURPOSES |
| US20040223966A1 (en) * | 2002-10-25 | 2004-11-11 | Wolfman Neil M. | ActRIIB fusion polypeptides and uses therefor |
| US7758881B2 (en) | 2004-06-30 | 2010-07-20 | Advanced Cardiovascular Systems, Inc. | Anti-proliferative and anti-inflammatory agent combination for treatment of vascular disorders with an implantable medical device |
| US8435550B2 (en) | 2002-12-16 | 2013-05-07 | Abbot Cardiovascular Systems Inc. | Anti-proliferative and anti-inflammatory agent combination for treatment of vascular disorders with an implantable medical device |
| EP1635870A2 (en) * | 2003-06-02 | 2006-03-22 | Wyeth | Use of myostatin (gdf8) inhibitors in conjunction with corticosteroids for treating neuromuscular disorders |
| ES2383328T3 (en) | 2003-07-25 | 2012-06-20 | Amgen, Inc | Methods related to LDCAM and CRTAM |
| ES2282904T3 (en) * | 2003-09-12 | 2007-10-16 | Wyeth | SOLID BARS OF INJECTABLE CALCIUM PHOSPHATE FOR THE SUPPLY OF OSTEOGENIC PROTEINS. |
| US7198675B2 (en) | 2003-09-30 | 2007-04-03 | Advanced Cardiovascular Systems | Stent mandrel fixture and method for selectively coating surfaces of a stent |
| US20080249038A1 (en) * | 2003-10-07 | 2008-10-09 | Quark Biotech, Inc. | Bone Morphogenetic Protein (Bmp) 2A and Uses Thereof |
| US20050214339A1 (en) * | 2004-03-29 | 2005-09-29 | Yiwen Tang | Biologically degradable compositions for medical applications |
| CA2571318C (en) * | 2004-06-23 | 2011-06-07 | Tissuegene, Inc. | Nerve regeneration by transplanting cells expressing recombinant transforming growth factor superfamily protein |
| US8568469B1 (en) | 2004-06-28 | 2013-10-29 | Advanced Cardiovascular Systems, Inc. | Stent locking element and a method of securing a stent on a delivery system |
| US8241554B1 (en) | 2004-06-29 | 2012-08-14 | Advanced Cardiovascular Systems, Inc. | Method of forming a stent pattern on a tube |
| US8747879B2 (en) | 2006-04-28 | 2014-06-10 | Advanced Cardiovascular Systems, Inc. | Method of fabricating an implantable medical device to reduce chance of late inflammatory response |
| US8747878B2 (en) | 2006-04-28 | 2014-06-10 | Advanced Cardiovascular Systems, Inc. | Method of fabricating an implantable medical device by controlling crystalline structure |
| US8778256B1 (en) | 2004-09-30 | 2014-07-15 | Advanced Cardiovascular Systems, Inc. | Deformation of a polymer tube in the fabrication of a medical article |
| US7731890B2 (en) | 2006-06-15 | 2010-06-08 | Advanced Cardiovascular Systems, Inc. | Methods of fabricating stents with enhanced fracture toughness |
| US7971333B2 (en) | 2006-05-30 | 2011-07-05 | Advanced Cardiovascular Systems, Inc. | Manufacturing process for polymetric stents |
| WO2006020884A2 (en) * | 2004-08-12 | 2006-02-23 | Wyeth | Combination therapy for diabetes, obesity, and cardiovascular diseases using gdf-8 inhibitors |
| US9283099B2 (en) | 2004-08-25 | 2016-03-15 | Advanced Cardiovascular Systems, Inc. | Stent-catheter assembly with a releasable connection for stent retention |
| US7229471B2 (en) | 2004-09-10 | 2007-06-12 | Advanced Cardiovascular Systems, Inc. | Compositions containing fast-leaching plasticizers for improved performance of medical devices |
| US8173062B1 (en) | 2004-09-30 | 2012-05-08 | Advanced Cardiovascular Systems, Inc. | Controlled deformation of a polymer tube in fabricating a medical article |
| US7875233B2 (en) | 2004-09-30 | 2011-01-25 | Advanced Cardiovascular Systems, Inc. | Method of fabricating a biaxially oriented implantable medical device |
| US8043553B1 (en) | 2004-09-30 | 2011-10-25 | Advanced Cardiovascular Systems, Inc. | Controlled deformation of a polymer tube with a restraining surface in fabricating a medical article |
| US20060166251A1 (en) * | 2005-01-26 | 2006-07-27 | Archambault Joanne M | Use of sFRPs as markers of BMP activity |
| CN101137906A (en) * | 2005-03-23 | 2008-03-05 | 惠氏公司 | Detection of an immune response to gdf-8 modulating agents |
| US20060229715A1 (en) * | 2005-03-29 | 2006-10-12 | Sdgi Holdings, Inc. | Implants incorporating nanotubes and methods for producing the same |
| AU2006230434A1 (en) * | 2005-03-30 | 2006-10-05 | Wyeth | Methods for stimulating hair growth by administering BMPs |
| US7381048B2 (en) | 2005-04-12 | 2008-06-03 | Advanced Cardiovascular Systems, Inc. | Stents with profiles for gripping a balloon catheter and molds for fabricating stents |
| US20060292690A1 (en) * | 2005-06-22 | 2006-12-28 | Cesco Bioengineering Co., Ltd. | Method of making cell growth surface |
| CN106177996A (en) * | 2005-06-23 | 2016-12-07 | 组织基因公司 | Neuroprotective compound |
| US7658880B2 (en) | 2005-07-29 | 2010-02-09 | Advanced Cardiovascular Systems, Inc. | Polymeric stent polishing method and apparatus |
| US9248034B2 (en) | 2005-08-23 | 2016-02-02 | Advanced Cardiovascular Systems, Inc. | Controlled disintegrating implantable medical devices |
| WO2007025306A1 (en) * | 2005-08-26 | 2007-03-01 | University Of Rochester | Transplantation of glial restricted precursor-derived astrocytes for promotion of axon growth |
| DE102005042455A1 (en) * | 2005-09-06 | 2007-04-12 | Medizinische Hochschule Hannover | neural implant |
| US7867547B2 (en) | 2005-12-19 | 2011-01-11 | Advanced Cardiovascular Systems, Inc. | Selectively coating luminal surfaces of stents |
| US20070156230A1 (en) | 2006-01-04 | 2007-07-05 | Dugan Stephen R | Stents with radiopaque markers |
| US7951185B1 (en) | 2006-01-06 | 2011-05-31 | Advanced Cardiovascular Systems, Inc. | Delivery of a stent at an elevated temperature |
| US7964210B2 (en) | 2006-03-31 | 2011-06-21 | Abbott Cardiovascular Systems Inc. | Degradable polymeric implantable medical devices with a continuous phase and discrete phase |
| US8069814B2 (en) | 2006-05-04 | 2011-12-06 | Advanced Cardiovascular Systems, Inc. | Stent support devices |
| US7761968B2 (en) | 2006-05-25 | 2010-07-27 | Advanced Cardiovascular Systems, Inc. | Method of crimping a polymeric stent |
| US7951194B2 (en) | 2006-05-26 | 2011-05-31 | Abbott Cardiovascular Sysetms Inc. | Bioabsorbable stent with radiopaque coating |
| US20130325105A1 (en) | 2006-05-26 | 2013-12-05 | Abbott Cardiovascular Systems Inc. | Stents With Radiopaque Markers |
| US8343530B2 (en) | 2006-05-30 | 2013-01-01 | Abbott Cardiovascular Systems Inc. | Polymer-and polymer blend-bioceramic composite implantable medical devices |
| US7959940B2 (en) | 2006-05-30 | 2011-06-14 | Advanced Cardiovascular Systems, Inc. | Polymer-bioceramic composite implantable medical devices |
| US7842737B2 (en) | 2006-09-29 | 2010-11-30 | Abbott Cardiovascular Systems Inc. | Polymer blend-bioceramic composite implantable medical devices |
| US8034287B2 (en) * | 2006-06-01 | 2011-10-11 | Abbott Cardiovascular Systems Inc. | Radiation sterilization of medical devices |
| US8486135B2 (en) | 2006-06-01 | 2013-07-16 | Abbott Cardiovascular Systems Inc. | Implantable medical devices fabricated from branched polymers |
| US8603530B2 (en) | 2006-06-14 | 2013-12-10 | Abbott Cardiovascular Systems Inc. | Nanoshell therapy |
| US8048448B2 (en) | 2006-06-15 | 2011-11-01 | Abbott Cardiovascular Systems Inc. | Nanoshells for drug delivery |
| US8535372B1 (en) | 2006-06-16 | 2013-09-17 | Abbott Cardiovascular Systems Inc. | Bioabsorbable stent with prohealing layer |
| US8333000B2 (en) | 2006-06-19 | 2012-12-18 | Advanced Cardiovascular Systems, Inc. | Methods for improving stent retention on a balloon catheter |
| US8017237B2 (en) | 2006-06-23 | 2011-09-13 | Abbott Cardiovascular Systems, Inc. | Nanoshells on polymers |
| US9072820B2 (en) | 2006-06-26 | 2015-07-07 | Advanced Cardiovascular Systems, Inc. | Polymer composite stent with polymer particles |
| US8128688B2 (en) | 2006-06-27 | 2012-03-06 | Abbott Cardiovascular Systems Inc. | Carbon coating on an implantable device |
| US7794776B1 (en) | 2006-06-29 | 2010-09-14 | Abbott Cardiovascular Systems Inc. | Modification of polymer stents with radiation |
| US7740791B2 (en) | 2006-06-30 | 2010-06-22 | Advanced Cardiovascular Systems, Inc. | Method of fabricating a stent with features by blow molding |
| US7823263B2 (en) | 2006-07-11 | 2010-11-02 | Abbott Cardiovascular Systems Inc. | Method of removing stent islands from a stent |
| US7757543B2 (en) | 2006-07-13 | 2010-07-20 | Advanced Cardiovascular Systems, Inc. | Radio frequency identification monitoring of stents |
| US7998404B2 (en) | 2006-07-13 | 2011-08-16 | Advanced Cardiovascular Systems, Inc. | Reduced temperature sterilization of stents |
| US7794495B2 (en) | 2006-07-17 | 2010-09-14 | Advanced Cardiovascular Systems, Inc. | Controlled degradation of stents |
| US7886419B2 (en) | 2006-07-18 | 2011-02-15 | Advanced Cardiovascular Systems, Inc. | Stent crimping apparatus and method |
| US8101198B2 (en) | 2006-07-26 | 2012-01-24 | The Regents Of The University Of California | Osteogenic enhancer composition |
| US8016879B2 (en) | 2006-08-01 | 2011-09-13 | Abbott Cardiovascular Systems Inc. | Drug delivery after biodegradation of the stent scaffolding |
| JP4569543B2 (en) * | 2006-08-18 | 2010-10-27 | ニプロ株式会社 | Precursor for tissue regeneration device with swellable rod |
| US9173733B1 (en) | 2006-08-21 | 2015-11-03 | Abbott Cardiovascular Systems Inc. | Tracheobronchial implantable medical device and methods of use |
| WO2008029493A1 (en) * | 2006-09-08 | 2008-03-13 | Stelic Institute Of Regenerative Medicine, Stelic Institute & Co. | Nerve fiber degeneration inhibitor |
| US7923022B2 (en) | 2006-09-13 | 2011-04-12 | Advanced Cardiovascular Systems, Inc. | Degradable polymeric implantable medical devices with continuous phase and discrete phase |
| WO2008071226A1 (en) * | 2006-12-11 | 2008-06-19 | Medizinische Hochschule Hannover | Implant of cross-linked spider silk threads |
| US8099849B2 (en) | 2006-12-13 | 2012-01-24 | Abbott Cardiovascular Systems Inc. | Optimizing fracture toughness of polymeric stent |
| US7718616B2 (en) | 2006-12-21 | 2010-05-18 | Zimmer Orthobiologics, Inc. | Bone growth particles and osteoinductive composition thereof |
| US8262723B2 (en) | 2007-04-09 | 2012-09-11 | Abbott Cardiovascular Systems Inc. | Implantable medical devices fabricated from polymer blends with star-block copolymers |
| US7829008B2 (en) | 2007-05-30 | 2010-11-09 | Abbott Cardiovascular Systems Inc. | Fabricating a stent from a blow molded tube |
| US7959857B2 (en) | 2007-06-01 | 2011-06-14 | Abbott Cardiovascular Systems Inc. | Radiation sterilization of medical devices |
| US8202528B2 (en) | 2007-06-05 | 2012-06-19 | Abbott Cardiovascular Systems Inc. | Implantable medical devices with elastomeric block copolymer coatings |
| US8293260B2 (en) | 2007-06-05 | 2012-10-23 | Abbott Cardiovascular Systems Inc. | Elastomeric copolymer coatings containing poly (tetramethyl carbonate) for implantable medical devices |
| US8425591B1 (en) | 2007-06-11 | 2013-04-23 | Abbott Cardiovascular Systems Inc. | Methods of forming polymer-bioceramic composite medical devices with bioceramic particles |
| US8048441B2 (en) | 2007-06-25 | 2011-11-01 | Abbott Cardiovascular Systems, Inc. | Nanobead releasing medical devices |
| US7901452B2 (en) | 2007-06-27 | 2011-03-08 | Abbott Cardiovascular Systems Inc. | Method to fabricate a stent having selected morphology to reduce restenosis |
| US7955381B1 (en) | 2007-06-29 | 2011-06-07 | Advanced Cardiovascular Systems, Inc. | Polymer-bioceramic composite implantable medical device with different types of bioceramic particles |
| KR20120018202A (en) | 2009-05-26 | 2012-02-29 | 더 리젠츠 오브 더 유니버시티 오브 캘리포니아 | Fibromodulin peptide |
| US20110002897A1 (en) | 2009-06-11 | 2011-01-06 | Burnham Institute For Medical Research | Directed differentiation of stem cells |
| US8568471B2 (en) | 2010-01-30 | 2013-10-29 | Abbott Cardiovascular Systems Inc. | Crush recoverable polymer scaffolds |
| US8808353B2 (en) | 2010-01-30 | 2014-08-19 | Abbott Cardiovascular Systems Inc. | Crush recoverable polymer scaffolds having a low crossing profile |
| AU2011291537B2 (en) | 2010-08-19 | 2016-06-02 | The Regents Of The University Of California | Compositions comprising perivascular stem cells and Nell-1 protein |
| JPWO2012029148A1 (en) * | 2010-09-01 | 2013-10-28 | 株式会社オステオファーマ | Recombinant human bone morphogenetic protein-2 lyophilized formulation |
| CA2817584C (en) | 2010-11-15 | 2018-01-02 | Zimmer Orthobiologics, Inc. | Bone void fillers |
| US8726483B2 (en) | 2011-07-29 | 2014-05-20 | Abbott Cardiovascular Systems Inc. | Methods for uniform crimping and deployment of a polymer scaffold |
| US8956394B1 (en) | 2014-08-05 | 2015-02-17 | Woven Orthopedic Technologies, Llc | Woven retention devices, systems and methods |
| US9907593B2 (en) | 2014-08-05 | 2018-03-06 | Woven Orthopedic Technologies, Llc | Woven retention devices, systems and methods |
| US20160074071A1 (en) | 2014-09-16 | 2016-03-17 | Woven Orthopedic Technologies, Llc | Methods of using woven retention devices and systems |
| US9999527B2 (en) | 2015-02-11 | 2018-06-19 | Abbott Cardiovascular Systems Inc. | Scaffolds having radiopaque markers |
| KR20170138436A (en) * | 2015-03-17 | 2017-12-15 | 이스티튜토 바이오키미코 이탈리아노 지오바니 로렌치니 에스.피.에이. | Purification of osteogenic protein (BPM) |
| US9700443B2 (en) | 2015-06-12 | 2017-07-11 | Abbott Cardiovascular Systems Inc. | Methods for attaching a radiopaque marker to a scaffold |
| US10555758B2 (en) | 2015-08-05 | 2020-02-11 | Woven Orthopedic Technologies, Llc | Tapping devices, systems and methods for use in bone tissue |
| US11395681B2 (en) | 2016-12-09 | 2022-07-26 | Woven Orthopedic Technologies, Llc | Retention devices, lattices and related systems and methods |
| US11116876B2 (en) | 2018-02-20 | 2021-09-14 | Shu-Tung And Alice Li Foundation Inc. | Methods and devices for repair of severed peripheral nerves with erythropoietin |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4868161A (en) * | 1984-06-29 | 1989-09-19 | City Of Hope | Method for promoting nerve regeneration |
| US4955892A (en) * | 1988-10-24 | 1990-09-11 | Louisiana State University | Neural cell adhesion protein nerve prosthesis |
| US5162430A (en) * | 1988-11-21 | 1992-11-10 | Collagen Corporation | Collagen-polymer conjugates |
| US4920962A (en) * | 1988-11-23 | 1990-05-01 | Claude Proulx | Splint-like element for use in end-to-end nerve suture |
| US4963146A (en) * | 1989-04-20 | 1990-10-16 | Colla-Tec Incorporated | Multi-layered, semi-permeable conduit for nerve regeneration |
| DE69231946T2 (en) * | 1991-03-11 | 2002-04-04 | Curis Inc | PROTEIN-INDUCING MORPHOGENESIS |
| EP0592562B1 (en) * | 1991-06-25 | 1999-01-07 | Genetics Institute, Inc. | Bmp-9 compositions |
| US5306307A (en) * | 1991-07-22 | 1994-04-26 | Calcitek, Inc. | Spinal disk implant |
| ES2201059T5 (en) * | 1992-07-31 | 2007-11-01 | Curis, Inc. | REGENERATION AND REPAIR OF NERVES INDUCED BY MORPHOGENS. |
-
1994
- 1994-08-19 KR KR1020017013378A patent/KR100329409B1/en not_active IP Right Cessation
- 1994-08-19 WO PCT/US1994/009330 patent/WO1995005846A1/en active IP Right Grant
- 1994-08-19 AU AU78682/94A patent/AU677866B2/en not_active Expired
- 1994-08-19 EP EP94929728A patent/EP0716610B1/en not_active Expired - Lifetime
- 1994-08-19 KR KR1019960700909A patent/KR100328752B1/en not_active IP Right Cessation
- 1994-08-19 DE DE69434739T patent/DE69434739T2/en not_active Expired - Lifetime
- 1994-08-19 PT PT94929728T patent/PT716610E/en unknown
- 1994-08-19 EP EP06009976A patent/EP1716864A1/en not_active Withdrawn
- 1994-08-19 AT AT94929728T patent/ATE326234T1/en active
- 1994-08-19 JP JP7507663A patent/JPH09501932A/en active Pending
- 1994-08-19 CA CA002169191A patent/CA2169191C/en not_active Expired - Lifetime
- 1994-08-19 DK DK94929728T patent/DK0716610T3/en active
- 1994-08-19 ES ES94929728T patent/ES2265146T3/en not_active Expired - Lifetime
-
1995
- 1995-05-05 US US08/435,120 patent/US5756457A/en not_active Expired - Lifetime
-
1996
- 1996-02-22 NO NO960711A patent/NO960711L/en not_active Application Discontinuation
- 1996-02-22 FI FI960809A patent/FI960809A/en unknown
- 1996-02-23 OA OA60780A patent/OA10358A/en unknown
-
2004
- 2004-09-15 JP JP2004268730A patent/JP2005007196A/en not_active Withdrawn
-
2007
- 2007-02-02 JP JP2007024523A patent/JP2007130027A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| NO960711D0 (en) | 1996-02-22 |
| FI960809A0 (en) | 1996-02-22 |
| AU7868294A (en) | 1995-03-21 |
| JP2007130027A (en) | 2007-05-31 |
| ES2265146T3 (en) | 2007-02-01 |
| KR100328752B1 (en) | 2002-09-26 |
| EP0716610A1 (en) | 1996-06-19 |
| ATE326234T1 (en) | 2006-06-15 |
| WO1995005846A1 (en) | 1995-03-02 |
| DE69434739D1 (en) | 2006-06-22 |
| JP2005007196A (en) | 2005-01-13 |
| CA2169191A1 (en) | 1995-03-02 |
| JPH09501932A (en) | 1997-02-25 |
| EP0716610B1 (en) | 2006-05-17 |
| EP1716864A1 (en) | 2006-11-02 |
| DK0716610T3 (en) | 2006-09-04 |
| NO960711L (en) | 1996-02-22 |
| OA10358A (en) | 2001-10-22 |
| PT716610E (en) | 2006-08-31 |
| DE69434739T2 (en) | 2007-05-10 |
| US5756457A (en) | 1998-05-26 |
| KR960703615A (en) | 1996-08-31 |
| KR100329409B1 (en) | 2002-03-20 |
| AU677866B2 (en) | 1997-05-08 |
| FI960809A (en) | 1996-02-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2169191C (en) | Neural regeneration using human bone morphogenetic proteins | |
| Rasubala et al. | Platelet-derived growth factor and bone morphogenetic protein in the healing of mandibular fractures in rats | |
| Ten Dijke et al. | Growth factors for wound healing | |
| Wozney | The bone morphogenetic protein family and osteogenesis | |
| JP3706133B2 (en) | BMP-9 composition | |
| US7709442B2 (en) | In vivo synthesis of connective tissues | |
| US20140296325A1 (en) | Methods and compositions for inducing brown adipogenesis | |
| JP2011084574A (en) | Pharmaceutical composition for use of melanoma inhibiting activity factor (mia) for cartilage and bone repair | |
| Eppley et al. | Free bone graft reconstruction of irradiated facial tissue: experimental effects of basic fibroblast growth factor stimulation | |
| Kay et al. | Corneal endothelial modulation: a factor released by leukocytes induces basic fibroblast growth factor that modulates cell shape and collagen. | |
| US8513191B2 (en) | Therapeutic agent and therapeutic method for periodontal diseases and pulpal diseases | |
| Pajer et al. | Neuroectodermal stem cells grafted into the injured spinal cord induce both axonal regeneration and morphological restoration via multiple mechanisms | |
| Rasubala et al. | Comparison of the healing process in plated and non-plated fractures of the mandible in rats | |
| Kondo et al. | Morphology, Development, and Neurotrophic Regulation of Cochlear Afferent Innervation | |
| Ingolia et al. | The therapeutic potential of inducing molecules | |
| Axon | Damien P. Kuffler | |
| Hu | VEGF-Dependent Mechanisms Controlling Osteoblast Differentiation and Bone Formation During Bone Repair |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| MKEX | Expiry |
Effective date: 20140819 |