Connect public, paid and private patent data with Google Patents Public Datasets

Methods and compositions for treating intervertebral disc degeneration

Info

Publication number
CA2400826C
CA2400826C CA 2400826 CA2400826A CA2400826C CA 2400826 C CA2400826 C CA 2400826C CA 2400826 CA2400826 CA 2400826 CA 2400826 A CA2400826 A CA 2400826A CA 2400826 C CA2400826 C CA 2400826C
Authority
CA
Grant status
Grant
Patent type
Prior art keywords
matrix
disc
cells
nucleus
growth
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 - Fee Related
Application number
CA 2400826
Other languages
French (fr)
Other versions
CA2400826A1 (en )
Inventor
Jeffrey William Moehlenbruck
John Paul Ranieri
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zimmer Orthobiologics Inc
Original Assignee
Zimmer Orthobiologics Inc
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Grant date

Links

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3612Cartilage, synovial fluid
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3641Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
    • A61L27/3645Connective tissue
    • A61L27/3654Cartilage, e.g. meniscus
    • A61L27/3658Intervertebral discs
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3687Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3817Cartilage-forming cells, e.g. pre-chondrocytes
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A61L27/3843Connective tissue
    • A61L27/3852Cartilage, e.g. meniscus
    • A61L27/3856Intervertebral discs
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3895Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61FFILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, E.G. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02Prostheses implantable into the body
    • A61F2/30Joints
    • A61F2/44Joints for the spine, e.g. vertebrae, spinal discs
    • A61F2/442Intervertebral or spinal discs, e.g. resilient
    • A61F2002/444Intervertebral or spinal discs, e.g. resilient for replacing the nucleus pulposus
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L2400/00Materials characterised by their function or physical properties
    • A61L2400/06Flowable or injectable implant compositions
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L2430/00Materials or treatment for tissue regeneration
    • A61L2430/38Materials or treatment for tissue regeneration for reconstruction of the spine, vertebrae or intervertebral discs

Abstract

A fluid matrix comprising cross-linked remodelable collagen from a donor vertebrate animal is useful for regener-ating hydrodynamic function in damaged intervertebral discs in vivo. The matrix may be injectable and may comprise cells and a plurality of purified cell growth factors. The matrix promotes cell growth and elaboration of proteoglycans to facilitate regeneration of native tissues. The collagen in the matrix may be cross-linked using photooxidative catalysis and visible light, and purified cell growth factors are preferably at least partly bone-derived.

Description

Methods And Compositions For Treating Intervertebral Disc Degeneration Description Background Art This invention relates generally to methods and compositions useful in treating intervertebral disc impairment in humans and other mammals. More particularly, this invention concerns compositions useful in restoring hydrodynamic function and stimulating cell proliferation and extracellular matrix production in intervertebral discs that have been compromised by injury, degenerative disease, congenital abnormalities, and/or the aging process.
Compositions of the invention may be injectable, and may include growth factors, bioactive agents, and living cells. The compositions are useful for restoring, improving, or augmenting hydrodynamic function of the intervertebral disc, increasing intervertebral disc height, and stimulating cell proliferation and/or extracellular matrix production in intervertebral discs.

The human vertebral column (spine) comprises a plurality of articulating bony elements (vertebrae) separated by soft tissue intervertebral discs. The intervertebral discs are flexible joints which provide for flexion, extension, and rotation of the vertebrae relative to one another, thus contributing to the stability and mobility of the spine within the axial skeleton.
The intervertebral disc is comprised of a central, inner portion of soft, amorphous mucoid material, the nucleus pulposus, which is peripherally surrounded by an annular ring of layers of tough, fibrous material known as the annulus fibrosus. The nucleus pulposus and the annulus fibrosus together are bounded on their upper and lower ends (i.e., cranially and caudally) by vertebral end plates located at the lower and upper ends of adjacent vertebrae. These end plates, which are composed of a thin layer of hyaline cartilage, are directly connected at their peripheries to the lamellae of the inner portions of the annulus fibrosus. The lamellae of the outer portions of the annulus fibrosus connect directly to the bone at the outer edges of the adjacent vertebrae.
The soft, mucoid nucleus pulposus contains chondrocytes, which produce fibrils of collagen (primarily Type II collagen, but also Types IX, XI, and others) and large molecules of negatively charged, sulfated proteoglycans, as depicted in Figure 1. The term matrix as used herein refers to a coinposition which provides structural support for, and which facilitates respiration and movement of nutrients and water to and from, an intervertebral disc. The collagenous components of the nucleus pulposus extracellular matrix comprise a scaffold that provides for normal cell (i.e., chondrocyte) attachment and cell proliferation. The negatively charged proteoglycan component of the nucleus pulposus extracellular matrix attracts water to form a hydrated gel, which envelops the collagen fibrils and chondrocyte cells. In the normal healthy nucleus pulposus, water comprises between 50-90 % of the total weight.
The nucleus pulposus. thus plays a central role in maintaining normal disc hydrodynamic function. The large molecular weight proteoglycans are contained within the nucleus pulposus by the annulus fibrosus and by the vertebral end plates, and they attract water into the nucleus through sieve-like pores in the end plates. The resulting osmotic pressure within each disc tends to expand it axially (i.e., vertically), driving the adjacent vertebrae further apart. On the other hand, mechanical movements resulting in axial compression, flexion, and rotation of the vertebrae exert forces on the intervertebral discs, which tends to drive water out of the nucleus pulposus. Water movements into and out of an intervertebral disc under the combined influence of osmotic gradients and mechanical forces constitute hydrodynamic functions important for maintaining disc health.
Movement of solutes in the water passing between discs and vertebrae during normal hydrodynamic function facilitates chondrocyte respiration and nutrition within the discs. This function is critical to chondrocyte survival since nucleus pulposus tissues of intervertebral discs are avascular (the largest such avascular structures in the human body).
Maintaining sufficient water content in the nucleus pulposus is also important for absorbing high mechanical (shock) loads, for resisting herniation of nucleus pulposus matter under such loads, and for hydrating the annulus fibrosus to maintain the flexibility and strength needed for spine stability.
Normal hydrodynamic functions are compromised in degenerative disc disease (DDD).
DDD involves deterioration in the structure and function of one or more intervertebral discs and is commonly associated with aging and spinal trauma. Although the etiology of DDD is not well understood, one consistent alteration seen in degenerative discs is an overall decrease in proteoglycan content witliin the nucleus pulposus and the annulus fibrosus.
The loss in proteoglycan content results in a concomitant loss of disc water content.
Reduced hydration of disc structures may weaken the annulus fibrosus, predisposing the disc to herniation. Herniation frequently results in extruded nucleus pulposus material impinging on the spinal cord or nerves, causing pain, weakness, and in some cases permanent disability.
Because adequate disc hydration is important for stability and normal mobility of the spine, effective treatment of DDD would ideally restore the disc's natural self-sustaining hydrodynamic function. Such disc regeneration therapy may require substantial restoration of cellular proteoglycan synthesis within the disc to maintain the hydrated extracellular matrix in the nucleus pulposus. Improved hydrodynamic function in such a regenerated disc may result in restoration and reestablishment of intervertebral disc height. It may also provide for hnproved hydration of the annulus fibrosus, making subsequent herniation less likely.
Prior art approaches to intervertebral disc problems fail to restore normal self-sustaining hydrodynamic function, and thus may not restore normal spinal stability and/or mobility under high loads. One approach to reforming intervertebral discs using a combination of intervertebral disc cells and a bioactive, biodegradable substrate is described in U.S.
patent number 5,964,807 to Gan et al. The biodegradable substrate disclosed in Gan et al., including bioactive glass, polymer foam, and polymer foam coated with sol gel bioactive material, is intended to enhance cell function, cell growth and cell differentiation. The bioactive glass contains oxides of silicon, sodium, calcium and phosphorus. The polymer foam is described as biocompatible and 'uicludes polyglycolide (PGA), poly (D,L-lactide) (D,L-PLA), poly(L-lactide) (L-PLA), poly(D,L-lactide-co-glycolide) (D,L-PLGA), poly(L-lactide-co-glycolide) (L-PLGA), polycaprolactone (PCL), polydioxanone, polyesteramides, copolyoxalates, and polycarbonates. Gan et al. describes application of this approach to intervertebral disc reformation in mature New Zealand rabbits, concluding with ingrowth of cells and concurrent degradation of implanted material with little or no inflannnation. However, degradation of portions of the implanted material, such as acidic breakdown of PLAs, PGAs and PLGAs, may adversely affect cell growth, cell function and/or cell differentiation.
A somewhat analogous disclosure relating to tissues for grafting describes matrix particulates comprising growth factors that may be seeded with cells; see U.S.
patent number 5,800,537 to Bell. The matrix and cells are applied to scaffolds, which include biodegradable polymers, microparticulates, and collagen which has been cross-linked by exposure to ultraviolet radiation and formed to produce solids of foam, thread, fabric or film. The matrix particulates are derived from tissue from which cells and cell remnants have been removed without removing factors necessary for cell growth, morphogenesis and differentiation. Bell specifically avoids the use of reagents like high salt, or deliysidation reagents such as butanol/ether or detergents. Such reagents are unfavorably characterized as being responsible for removing from the source tissue factors essential for stimulatting repair and remodeling processes. Alternative approaches, in which such factors are obtained from other sources rather than being retained in the tissue, are not addressed.
Still another disclosure related to regeneration of cartilage is found in.
U.S. patent number 5,837,235 to Mueller et al. Mueller et al. describes comminuting small particles of autologous omentum or other fatty tissue for use as a carrier, and adding to the carrier growth factors such as Transforming Growth Factor Beta and Bone Morphogenic Protein. Mueller et al. does not teach cross-linking tissues to create a cross-linked matrix.
The Gan et al. patent above is representative of past attempts to restore or regenerate substantially natural hydrodynamic disc function to intervertebral discs, but such techniques have not been proven in clinical trials. Similarly, the approaches of Bell and Mueller et al. have not been widely adapted for disc regeneration, and better approaches are still needed because low back pain sufficient to prevent the patient from working is said to affect 60 % to 85 % of all people at some time in their life. In the absence of safer and more efficacious treatment, an estimated 700,000 discectomies and 550,000 spinal fusions are performed worldwide each year to treat these conditions. Several prosthetic devices and compositions employing synthetic components have also been proposed for replacement of degenerated discs or portions thereof. See, for example, U.S. patent numbers 4,772,287, 4,904,260, 5,047,055, 5,171,280, 5,171,281, 5,192,326, 5,458,643, 5,514,180, 5,534,028, 5,645,597, 5,674,295, 5,800,549, 5,824,093, 5,922,028, 5,976,186, and 6,022,376.
A portion of the disc prostheses referenced above comprise hydrogels which are intended to facilitate hydrodynamic function similar in some respects to that of healthy natural discs. See, for example, U.S. patent Number 6,022,376 (Assell et al.). These prosthetic hydrogels, however, are not renewed through cellular activity within the discs. Thus, any improvement in disc hydrodynamic function would not be self-sustaining and would decliine over time with degradation of the prosthetic hydrogel. Healthy intervertebral discs, in contrast, retain their ability to hydrodynamically cushion axial compressive forces in the spine over extended periods because living cells within the discs renew the natural hydrogel (i.e., extracellular matrix) component.
Restoration of a clinically useful degree of normal hydrodynamic function in degenerated intervertebral discs is an object of the present invention, and the methods and compositions described herein have been shown to induce and/or enhance such regeneration.
Disclosure of Invention The present invention comprises methods and compositions for intervertebral disc regeneration. In preferred embodiments, the compositions comprise a three-dimensional fluid matrix of digestion-resistant, cross-linked nucleus pulposus tissue from a donor vertebrate. The donor may be the patient or another animal of the same or different species.
Cross-linking of donor nucleus pulposus tissue for the present invention is preferably achieved through use of one or more photooxidative catalysts which selectively absorb visible light. See U.S. patent Nos.
5,147,514, 5,332,475, 5,817,153, and 5,854,397, Other cross-linking approaches may be used without departing from the scope of the invention, however.

Prior to cross-linking the tissues, chondrocytes of the donor vertebrate are preferably destroyed, fragmented, and/or removed (i.e., decellularized). A preferred decellularization approach involves soaking the tissue in a solution having high concentrations of salt (preferably NaC1) and sugar (preferably sucrose). Such high-salt, high-sugar solutions are referred to as HSHS solutions. Other decellularization approaches may be used, however. After the tissues are decellularized and cross-linked, the resulting fluid matrix may be lyophilized for sterilization and storage, and then rehydrated prior to use. Figure 2 illustrates a process for producing a preferred embodiment of the fluid matrix of the present invention.
The fluid matrix of the present invention is biocompatible, substantially non-immunogenic, and resistant to degradation in vivo. As such, it is capable of providing important internal structural support for an intervertebral disc undergoing regeneration during a period of accelerated proteoglycan synthesis. The cross-linked matrix nlay be delivered to the intervertebral disc space by injection through a syringe (as depicted in Figure 2), via a catheter, or other methods known in the art.
The three-dimensional fluid matrix of the present invention may be used alone or in coinbination with growth factors and/or living cells to facilitate regeneration of the structures of a degenerated disc. In patients having sufficient viable endogenous disc cells (chondrocytes) and cell growth factors, the three-dimensional cross-linked matrix alone may substantially contribute to the regeneration of hydrodynamic function in an intervertebral disc in vivo by providing improved mechanical stability of the disc and a more favorable environment for cellular growth and/or metabolism. Conversely, in another embodiment of the invention, a combination of the three-dimensional matrix and one or more purified, preferably bone-derived, cell growth factors may also be used to treat DDD in discs containing viable chondrocytes in a depleted proteoglycan hydrogel matrix. In this case, the cross-linked collagen provides an expanded remodelable three-dimensional matrix for the existing (native) chondrocytes within a disc, while the cell growth factors induce accelerated proteoglycan production to restore the hydrogel matrix of the patient.
The combination of the three-dimensional matrix and one or more purified cell growth factors is referred to as a cell growth medium. The present invention may also comprise an injectable cell growth medium. Individual purified cell growth factors may be obtained by recombinant techniques known to those skilled in the art, but a preferred plurality of bone-derived purified cell growth factors for the present invention is disclosed in U.S. patent numbers 5,290,763, 5,371,191 and 5,563,124. Bone-derived cell growth factors produced according to these patents are hereinafter referred to as "BP. "
Disc regeneration occurs as the cross-linked collagen and proteoglycan matrix supports living cells (which may include exogenous cells as well as native disc or other autologous cells) having inherent capability to synthesize Type II collagen fibrils and proteoglycans in vivo, among other extracellular matrix molecules. Where the patient's native disc cells have been removed or are otherwise insufficient to cause such proliferation, living cells may be added to the three-dimensional matrix of cross-linked nucleus pulposus material to further promote disc regeneration. Accordingly, in another embodiment, the present invention comprises a three-dimensional matrix of cross-linked nucleus pulposus tissue to which exogenous and/or autologous living cells have been added. The injectable combination of three-d'nnensional matrix material and exogenous and/or autologous living cells is termed herein an injectable cell matrix. Suitable cells for such an injectable cell matrix may be obtained, for example, from the nucleus pulposus of a manunalian vertebral disc, from cartilage, from fatty tissue, from muscle tissue, from bone marrow, or from bone material (i.e., mesenchymal stem cells), but are not limited to these tissue types. These cells are preferably cultured in vitro to confirm their viability and, optionally, to increase the cells' proliferation and synthesis responses using cell growth factors.
Growth factors may optionally be added to cell cultures to stimulate cellular development and elaboration of Type II collagen fibrils and proteoglycans suitable for maintaining an effective disc hydrogel matrix in vivo. An injectable fluid combining purified cell growth factors and a plurality of living cells is termed an injectable cell suspension, and is useful in treating DDD.
While an injectable cell matrix alone (i.e., without growth factors) may substantially regenerate liydrodynamic function in an intervertebral disc in vivo if sufficient native cell growth factors are present in the disc, purified (exogenous) cell growth factors may be added to an injectable cell matrix of the present invention to form yet another embodiment of the present invention.
Brief DescriQtion of Drawings Figure 1 is a diagram illustrating components of healthy nucleus pulposus tissue in a vertebrate.
Figure 2 is a diagram illustrating a process for preparation and use of a cross-linked matrix of porcine nucleus pulposus tissue in a preferred embodiment of the invention.
Figure 3 is a photographic reproduction of an SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis comparing the amount of proteins extracted from a cross-linked matrix of the present invention with a non-cross-linked control.

Figure 4 is a photographic comparison of an H & E (hematoxylin and eosin) stained section of fresh porcine nucleus pulposus tissue with a cross-linked matrix of the present invention, both at 300X magnification.
Figure 5 is a photographic reproduction of a stained nitrocellulose membrane comparing the reactivity of Type II collagen digested from a cross-linked matrix of the present invention and a non cross-linked control.
Figure 6 is a comparison graph of the hydraulic/swelling capacity of a cross-linked matrix of the present invention and a non-crosslinked control.
Figure 7 is a diagram of an experimental process used to demonstrate stunulation of sheep cell ingrowth, proliferation, and new matrix synthesis in an embodiment of the present invention comprising a cross-linked matrix combined with bone protein growth factors (BP).
Figure 8 is a graph and a photograph indicating the results of an Alcian blue assay for matrix production in sheep nucleus pulposus cells stimulated by growth factors. -Figure 9 is a graph indicating the results of immunogenicity tests for a cross-linked matrix of the present invention in rabbit immunizations and sheep serum.
Figure 10 is a diagram of the protocol for an in vivo study of a matrix and growth factor combination of the present invention.
Figure 11 is a radiograph of a vertebral column from a sheep sacrificed at 2 months after an injection of a matrix and growth factor combination in an in vivo study of an embodiment of the present invention.
Figure 12 is a photographic reproduction of histology slides of vertebral discs of a sheep sacrificed at 2 months after an injection of a matrix and growth factor combination of the present invention.
Figure 13 is a radiograph of a vertebral column of a sheep sacrificed at 4 months after an injection of a matrix and growth factor combination in an in vivo study of the present invention.
Figure 14 is a photographic reproduction of histology slides of vertebral discs of a sheep sacrificed at 4 months after an injection of a matrix and growth factor combination of the present invention.
Figure 15 is a graph representing the results of an ELISA performed to measure the synthesis of Type II collagen and Chondroitin-6-sulfate under growth factor stimulation Figure 16a is a graph indicating the results of an Alcian blue assay for proteoglycan synthesis in human intervertebral disc cells stimulated by growth factor.
Figure 16b is a graph indicating the results of an Alcian blue assay for proteoglycan synthesis in another human intervertebral disc cells stimulated by growth factor.

WO 01/76654 PCT/USOl/11576 Figure 17 is a graph depicting the results of an Alcian blue assay for proteoglycan synthesis in baboon intervertebral disc cells stimulated by growth factor.
Figure 18 is an SDS-PAGE gel of HPLC fractions 27-16 from a sample of BP.
Figure 19 is an SDS-PAGE gel of HPLC fractions 27-16 with identified bands indicated according to the legend of Figure 20.
Figure 20 is an SDS-PAGE gel of BP witli identified bands indicated.
Figure 21 is a 2-D (two-dimensional) SDS-PAGE gel with internal standards indicated by arrows.
Figure 22 is a 2-D SDS-PAGE gel with circled proteins (growth factors) identified as in legend.
Figures 23A-230 are Mass Spectrometer results for tryptic fragments.
Figure 24 is a 2-D gel Western blot with anti-phosphotyrosine antibody.
Figures 25A-25D are 2-D gel Western blots with antibodies for the indicated proteins.
For Figure 25A, the growth factors are BMP-3 and BMP-2; for Figure 25B the growth factors are BMP-3 and BMP-7; for Figure 25C the growth factors are BMP-7 and BMP-2; and for Figure 25D the growth factors are BMP-3 and TGF-(31.
Figure 26 is a PAS (periodic acid schiff) stained SDS-PAGE gel of HPLC
fractions.
Figure 27 is an anti-BMP-7 stained SDS-PAGE gel of PNGase F treated BP.
Figure 28 is an anti-BMP-2 stained SDS-PAGE gel of PNGase F treated BP.
Figures 29A-29B are bar charts showing explant mass of glycosylated BP samples (Figure 29A) and ALP Score (Figure 29B) of the same samples.
Figures 30A-30B are an SDS-gel of BP (Figure 30B) and a scanning densitometer scan (Figure 30A).
Tables Table 1 is a chart showing antibody listing and reactivity.
Tables 2A-2B together comprise a chart showing tryptic fragment sequencing data.
Tables 3A-3F together comprise a chart showing tryptic fragment mass spectrometry data.
Table 4 is a chart illustrating the relative mass of major components of BP.
Best Mode for Carrying Out the Invention In a preferred embodiment, the invention comprises a biodegradable matrix, which is delivered as an incompressible fluid to induce and/or enhance regeneration or repair of tissues in the intervertebral disc. The biodegradable matrix comprises hydrophilic molecules, which will maintain and/or increase the "captured" water content in intervertebral disc tissues.
The biodegradable matrix may also serve as a carrier substrate for added growth factors and/or appropriate living cell types.

Since the biodegradable matrix of the present invention is a viscous fluid, it furnishes incompressible support when delivered within a closed, secure disc space.
Moreover, because it is distributed uniformly within a disc, the present fluid matrix has a force distribution effect, hydraulically transmitting forces evenly inside the disc. The matrix thus provides resistance against axial compression and annulus collapse, whereas otlier matrix materials (for example, polymer sponges and collagen sponges) will rapidly collapse under the axial compressive forces within the disc. Solid matrix materials, in contrast, will concentrate forces from end plates directly onto implants, leading to rapid deterioration of implants and/or end plates.
In a preferred embodiment, the biodegradable matrix of the present invention is injectable. Clinical application to a patient can thus be accomplished using minimally invasive techniques, significantly reducing both the cost of treatment and the likelihood of complications relative to procedures such as partial discectomy or vertebral fusion.
Sunilarly, the present invention avoids the requirement for boring a hole into the annulus to implant a prostlietic replacement nucleus pulposus device, such as a relatively solid biodegradable matrix, or to evacuate nucleus tissue to create space for an implanted biodegradable substrate.
The matrix of the present invention is a natural material, preferably prepared from normal, healthy nucleus tissue of animals and/or humans. Accordingly, the matrix is comprised of proteins and matrix molecules especially adapted for efficient hydrodynamic function in intervertebral discs. Such a matrix remains biodegradable under normal circumstances in the presence of specific cellular enzymes, albeit at a slower rate than endogenous disc matrix. It is an important feature of the invention that matrix breakdown products associated with the present invention are digestible by disc cells. In comparison, some matrix materials previously taught (e.g. polyvinyl alcohol) do not break down by physiological processes. In addition, some synthetic polymer substrates create acidic degradation byproducts, in particular PGA and PLA.
Immediate (substantially homogeneous) dispersion of cells within the present matrix is another advantage of the invention. The viscous fluid formulation preferred for injection can be mixed directly with cells of the appropriate type(s) and then delivered immediately to treat an intervertebral disc. In the matrix of the present invention it is not necessary to culture cells and matrix together for some days or weeks before implantation, as it is for certain matrix materials such as PGA and collagen sponges.

The matrix of the present invention is an appropriate substrate for cells, uniquely suited to the ingrowth, proliferation, and residence of intervertebral disc cells.
Intervertebral disc cells preferentially grow into and survive in the matrix of the present invention, compared to type I
collagen sponges fixed with formalin or glutaraldehyde.

The following examples illustrate the preparation of preferred embodiments of the invention and demonstrate its non-immunogenic and disc regenerative properties.
EXAMPLE 1: Preparation of a Cross-Linked, Fluid Matrix Suitable for Treatment of Degenerative Disc Disease A three-dimensional fluid matrix of cross-linked nucleus pulposus tissue in accordance with an embodiment of the present invention may be prepared from donor vertebrates. Although porcine donors were used in a particularly preferred einbodiment, nucleus pulposus tissues from other vertebrates may also be used, although mammalian vertebrates are preferred (e.g., human, porcine, bovine, ovine, etc.).
Although nucleus pulposus tissues may be harvested by a variety of methods from many vertebral donors, in a preferred embodiment nucleus pulposus tissues were dissected aseptically from spinal intervertebral discs of pigs. In a sterile environment (i.e., a laminar flow hood), the annulus fibrosus of porcine donors was sliced radially and the vertebral end plates separated to expose the nucleus pulposus. The latter material was curetted out of the central portion of the disc, devoid of annulus and end plate tissues.
The nucleus pulposus tissues thus harvested were inserted into sterile dialysis (filter) tubing having a preferred molecular weight cutoff of about 3500 Daltons to substantially prevent loss of low molecular weight proteoglycans from the tissues while substantially reducing bacterial or other contamination. Other semipermeable membranes or filtering membrane types may be used to perform these functions.
The nucleus pulposus tissues to be cross-linked are also preferably treated to destroy and remove donor cells and/or cell fragments. To this end, dialysis tubing containing nucleus pulposus tissues was submerged in a high-salt, high-sucrose (HSHS) solution of about 2.2%:
8.4% w/v (respectively) for about 48 hours. Concentration ranges for the HSHS
solution may be from 1 % to 50%, but a preferred HSHS solution contains 220 grams NaCI and 837.5 grams of sucrose in 10L water. Preferred HSHS incubation times are from about 24 to about 72 hours, although shorter or longer times may also advantageously be used. Exposure to this HSHS
solution results in osmotic destruction and fragmentation of native cliondrocyte cells (decellularization), and further results in denaturation of soluble cellular proteins and nucleic acids. The HSHS solution may also contain other reagents which further degrade nucleic acids (including but not limited to sulfones and nucleases), and other reagents which can extract membrane lipids (including but not limited to alcohol, chloroform, and methanol). Although native cells of the donor may be retained in other embodiments of the invention, decellularization and denaturation are preferred where exogenous (particularly xenogeneic) tissues are used, so as to reduce the potential for immunogenic responses. Processes other than exposure to HSHS
solutions may be used for his purpose.
5. Cross-linking of the nucleus pulposus tissues is preferably accomplished by a photo-mediated process in accordance with U.S. patent Nos. 5,147,514, 5,332,475, 5,817,153, and/or 5,854,397. In one such process, a photoactive dye (methylene blue) was dissolved in the HSHS
solution at a preferred dye concentration of about 20 mg/liter. The photoactive dye was allowed to permeate the nucleus tissues within the dialysis tubing during the initial storage/decellularization process in HSHS. A wide range of photoactive dyes and concentrations, as taught in the foregoing patents, may be used to obtain cross-linked fluid matrices suitable for use in regenerating mammalian disc tissues. Preferred dyes include methylene blue and methylene green at concentrations of about 0.001 % to about 1.0 % w/v.
To cross-link the collagen within the nucleus tissues, the dialysis tubing containing the dye-permeated nucleus tissues was placed in a photooxidation chamber and exposed to broad-spectrum visible light for 48 hours. In preferred embodiments of the invention, the tissues may be cross-linked from about 24 to about 72 hours. A solution of methylene blue in phosphate buffered saline (PBS) was maintained under controlled temperature at 10 C and circulated around the dialysis tubing within the photooxidation chamber to provide substantially constant temperature regulation of the nucleus tissues. Precise temperature control is not critical to the practice of the invention; however, maintaining a relatively cooler temperature is preferred to avoid damaging the tissues. Following photo-crosslinking of the collagen, the treated nucleus tissues were collected, lyophilized in a vacuum under centrifugation, and finely pulverized in a freezer-mill under liquid nitrogen. The cross-linked matrix product thus prepared can be sterilized using ganuna radiation, ethylene oxide (or other sterilants) and stored at -80 C until rehydrated for use. A preferred process for preparing a matrix according to the present invention is illustrated in Figure 2.
In addition to preparation of the cross-linked matrix, control (non-crosslinked) tissues were prepared following the above procedures, except that they were not exposed to light. These control, non-crosslinked tissues were used for comparison purposes.
To investigate the swelling capacity of cross-linked matrix versus non-crosslinked control, lyophilized samples of cross-linked matrix and non-crosslinked control were suspended in water and the increase in weight due to water absorption was measured at various times from 0 to 96 hours. As illustrated in Figure 6, the cross-linked matrix retained 95 %
of the hydraulic capacity of the non-crosslinked control.

EXAMPLE 2: Testing of Fluid Matrix to Evaluate Protein Modification Induced by the Cross-Linking Process One half gram of the matrix material obtained prior to the lyophilization step of EXAMPLE 1 was placed in 15 mis of a solution of 4M guanidine hydrochloride and agitated on a shaker for 24 llours to solubilize proteoglycans. After centrifugation, the supernatant was discarded and the pellet washed in distilled water 3 times for 5 minutes each.
The pelleted matrix material was then removed and blot-dried on filter paper.
One hundred mg of the blot-dried matrix was placed in a 1.5 ml microcentrifuge tube with 1000 l of 1% sodium dodecyl sulfate (SDS) containing 5% beta-inercaptoethanol (BME).
The matrix in SDS/BME was boiled for one hour to extract proteins (e.g., collagens). Samples were then centrifuged at 12000 rpm for 1 hour and aliquots of the supernatant were subjected to electrophoresis in gradient polyacrylamide gels.
Gels were stained with Coomassie blue or silver to visualize proteins extracted by the SDS/BME and heat treatment. As illustrated in Figure 3, collagen bands stained prominently in control, non-crosslinked tissues but exhibited only faint staining in cross-linked matrix. These results demonstrated that in the cross-linked matrix material, collagen proteins were not easily extracted by the above treatment, indicating that crosslinking had occurred.
In contrast, stained gels of the control tissues demonstrated that collagen proteins were readily extracted from non-crosslinked material by the above treatment. See Figure 3.
EXAMPLE 3: Matrix Histology to Evaluate Cellular Debris and Residual Membranous Material Cross-linked matrix material obtained prior to the lyophilization step of Example 1 was placed in 4% paraformaldehyde for tissue fixation. Standard histology techniques of embedding, sectioning, and staining of sections with hematoxylin & eosin dyes were performed.
Visualization of cross-linked matrix in H & E-stained sections demonstrated that the matrix preparation process facilitates destruction of cellular membranes and intracellular elements, with minimal membrane material remaining as compared to fresh porcine nucleus pulposus material as well as non-crosslinked tissue decellularized by HSHS treatment, freeze-thaw cycles, and HSHS
treatment plus freeze-thaw cycles. See Figure 4.
EXAMPLE 4: Evaluation of Matrix Antigenic Reactivi Using Monoclonal Antibodies to Type II Collagen Cross-linked matrix material obtained prior to the lyophilization step of Example 1 was also subjected to pepsin digestion to cleave Type II collagen proteins. The protein digests were run on SDS/PAGE and then transferred to a nitrocellulose membrane. Total protein transferred to the membrane was visualized using Colloidal Gold.
. The visualized nitrocellulose membranes were incubated with a mouse monoclonal antibody to Type II collagen and a secondary antibody (anti-mouse) conjugated with alkaline phosphatase. The antibody reactivity was visualized through addition of alkaline phosphatase substrate. As depicted in Figure 5, the antibodies toward Type II collagen did not react with pepsin digests of the cross-linked matrix as much as with the pepsin digests of the non-crosslinked control tissue. The results indicate that the matrix of the invention may have reduced antigenic epitopes for Type II collagen, and thus have less immunogenicity than non-crosslinked tissues.
See Figure 5.
EXAMPLE 5: Evaluation of Matrix Immunogenicity in Rabbit Antisera Production One grain of the lyophilized and pulverized matrix material prepared according to EXAMPLE 1 was dispersed in PBS (i.e., rehydrated) and centrifuged. The protein concentration of the supernatant was then determined using the BCA assay and the supernatant was diluted with PBS to a final concentration of 200 g of protein per ml of PBS. The diluted supernatant was then sterilized for injection protocols. Three rabbits were immunized with 100 g of protein from the sterilized supernatant. Each rabbit received 9 immunizations over a 14 week period and sera was collected from the rabbits on a regular schedule.
Antisera production against the protein extract was measured using an enzyme-linked immunosorbent assay (ELISA). Type II collagen was included as a positive control in the ELISA. Colorimetric evaluation of antisera directed against the matrix material demonstrated very low immunogenicity in rabbits. See Figure 9.
EXAMPLE 6: Matrix Formulation Including Serum and Other Fluids For Inlections And Delivery One gram of the lyophilized and pulverized matrix material prepared according to EXAMPLE 1 was sterilized with 70% etlianol and the ethanol was removed by successive PBS
rinses. The dispersed matrix was centrifuged and the pellet was suspended in heat-inactivated sheep serum at a ratio of 0.5g lyopliilized matrix to 1 ml serum to prepare a viscous fluid matrix which can be loaded into a standard syringe and delivered via a small gauge needle. In preferred embodiments of the invention, the serum is collected from the vertebrate animal or human patient to be treated, heat-inactivated to destroy unwanted protein components (complement proteins), and passed through a 0.2 micron sterile filtration unit. Different matrix/serum ratios may also be advantageously employed. Ratios ranging from 0.1g to 2.0 g of lyophilized matrix to 1 ml of serum are preferred.

Serum is a preferred fluid for mixture and delivery of the cross-linked matrix of the present invention because it contains various intrinsic growth factors that are beneficial to intervertebral disc cells. Serum also serves as a suitable carrier for extrinsic protein growth factors and/or small molecules. The beneficial effects of extrinsic growth factors on intervertebral disc cells are enhanced by the addition of serum.
Other fluids are also suitable for mixture and delivery of the viscous fluid matrix. For example, sterile saline or sterile water may also be used. The examples herein are not meant to be limiting as to the variety of carrier fluids which may be used to mix and deliver the matrix in the present invention.
EXAMPLE 7: Injection of Matrix Forinulation To Intervertebral Discs Using Pressure-Mediated S rin e Matrix material was prepared according to EXAMPLE 6 (mixed with serum) to form a viscous fluid and loaded into a standard syringe having a small gauge needle (e.g., 18-31 gauge) attached. Syringe injection pressure can be controlled simply by the fingers of the hand. In other embodiments of the invention, pressure to inject the viscous fluid can be controlled by an external device which concomitantly measures (e.g., via a pressure transducer) and delivers (e.g., by compressed air) a predetermined force to the syringe plunger.
In one preferred embodiment of this device, a thermal element is included in the needle.
By providing a needle having a thermal element, it is possible to deliver heat to the outer layers of the annulus fibrosus at the end of the treatment and during removal of the syringe needle in order to shrink collagen fibers around the needle and effectively seal the site of needle penetration.
It is further contemplated that the matrix of the present invention can be delivered to the disc space of a patient transpedicularly (i.e., through the pedicle of the vertebrae). In particular, the cross-linked matrix can be administered percutaneously via a biopsy cannula inserted through a channel in the pedicle. After delivery of the matrix, the channel can then be filled with bone cement or other like material to seal the channel.
EXAMPLE 8: Isolation of Human, Sheep, and Baboon Intervertebral Disc Nucleus Pulposus Cells Human intervertebral nucleus pulposus tissues were collected during surgery, suspended in Dulbecco's Modified Eagle Medium/ Nutrient Mixture F-12 (DMEM/F-12) in a 1:1 v/v mixture supplemented with antibiotics. The tissues were kept on ice until dissection, at which time they were rinsed 2-3 times in sterile Dulbecco's Phosphate Buffer Saline (DPBS) to remove any blood. In a laminar flow hood, the nucleus tissues were isolated and diced into small (2 mm) cubes, and then placed in a Tissue Culture Medium (hereinafter referred to as "TCM") comprising DMEM/F-12 culture media supplemented with 10% heat inactivated fetal bovine serum, 0.25% penicillin, 0.4% streptomycin, 0.001% amphotericin B, and 50 g/ml ascorbic acid. Only tissues clear of blood and other anomalous elements were used.
Placed on a shaker at 37oC, the tissues were digested with 0.01 % hyaluronidase (Calbiochem) in TCM
for 2 hours, 0.01 % protease (Sigma) in TCM for 1 hour, and 0.1% collagenase Type II
(Sigma) in TCM
overnight to obtain a suspension of human intervertebral disc nucleus pulposus cells.
The foregoing procedure was also applied to sheep and baboon intervertebral disc nucleus pulposus tissues to obtain suspensions of sheep and baboon intervertebral disc nucleus pulposus cells, respectively.
EXAMPLE 9: Primary Culture and Expansion of Human, Sheep, and Baboon Intervertebral Disc Nucleus Pulposus Cells Human intervertebral disc nucleus pulposus cells from EXAMPLE 8 were expanded by culturing in TCM at 37oC in 5% C02 atmosphere and 95% relative humidity. The TCM was changed every 2-3 days and the cells were passaged with trypsin to another container, when 80-90% confluent, for continued expansion.
The foregoing procedure was also applied to sheep and baboon intervertebral disc nucleus pulposus tissues to obtain an expanded supply of sheep and baboon intervertebral disc nucleus pulposus cells.
EXAMPLE 10: Alcian Blue Assay of Disc Cell Matrix Production in Human, Sheep, and Baboon Intervertebral Disc Nucleus Pulposus Cells Human intervertebral disc cells from EXAMPLE 9 were seeded and grown in 24 well plates in TCM in the presence or absence of exogenous growth factors. At various time points, TCM was aspirated out from the wells and the wells washed 3 times with PBS.
The cells were then fixed with 4% paraformaldehyde (pH 7.4) for 10min. The fixed cells were washed 2 times with PBS and then stained overnight with 0.5% Alcian blue in 0.1N hydrochloric acid (pH 1.5).
After overnight staining, excess stain was rinsed out with 3 rinses of PBS.
The remaining Alcian blue stain (bound to proteoglycans) was dissolved overnight into 6M guanidine hydrochloride and the absorbance at 630nm was measured using a spectrophotometer, providing an indication of the induction of matrix production by exogenous growth factors in human nucleus pulposus cells.
The foregoing procedure was also applied to sheep and baboon intervertebral nucleus pulposus cells from EXAMPLE 9 to obtain an indication of the induction of matrix production by exogenous growth factors in sheep and baboon nucleus pulposus cells.

EXAMPLE 11: Enzyme Linked Immunosorbent Assay (ELISA) on Ovine Intervertebral Disc Nucleus Pulposus Cells To detect specific antigenic epitopes in the synthesized matrix, sheep intervertebral nucleus pulposus cells from EXAMPLE 9, seeded and grown in monolayer, were fixed in 2%
glutaraldehyde for 1 hour at room temperature. The fixed cells were washed 3 times with TBS
for 5 min. each. To block non-specific antibody binding, the cells were incubated in a solution of Tris buffered saline (TBS) containing 1mM ethylene-dianune-tetraacetic acid (EDTA), 0.05%
Tween-20T"', and 0.25% bovine serum albumin for lhour. The blocking step was followed by 3 washes with TBS for 5 min. each. The cells were incubated with the primary antibody at room temperature for 2.5 hours, and the excess primary antibody was removed by 3 washes with TBS for 5 min. each. A second incubation with blocking buffer was performed for 10 min., followed by 3 washes with TBS. The cells were then incubated with the secondary antibody, which was conjugated with alkaline phosphates enzyme, for 3 hours at room temperature. The unbound secondary antibodies were removed by 3 washes of TBS for 5 min. each. The bound primary and secondary antibodies were detected by addition of an enzyme-specific substrate which produced a colored reaction. The colorimetric measurement was performed using a spectrophotometer, providing a quantitative measure of the presence of the bound antibodies.
EXAMPLE 12: Effect of Exogenous Growth Factors on Proteoglycan Svnthesis in Ovine Intervertebral Disc Nucleus Pulposus Cells Transforming growth factor- l (TGF(31) and a mixture of bone-derived protein growth factors (BP) produced according to U.S. patent Nos. 5,290,763, 5,371,191 and 5,563,124, were tested for their effects on stimulation of proteoglycan synthesis in ovine nucleus pulposus cells.
Sheep intervertebral disc nucleus cells were collected and cultured as described in EXAMPLES 8 and 9. Sheep cells were seeded in micromass (200,000) into the wells of a 24 well plate:
Growth factor dilutions were prepared in TCM supplemented with 0.5% heat inactivated fetal bovine serum. TGF(31 and BP were both tested at 10 ng/ml; BP was also tested at a concentration of 10 g/ml. Control wells without growth factors contained TCM
supplemented with 0.5% and 10% heat-inactivated fetal bovine serum. The cells were incubated in continuous exposure to the various growth factors for 7 and 10 days. At these time points, the cells were fixed and the amount of proteoglycan synthesis was measured by the Alcian blue assay as described in EXAMPLE 10.
At both 7 and 10 day time points, proteoglycan synthesis was significantly greater in the 10% fetal bovine serum control cultures than in the 0.5% fetal bovine serum control cultures. At the 7 day time point, BP at the higher 10 glml concentration produced a significant (93%) increase in proteoglycan synthesis above the level in 10% serum control culture and a greater (197 %) increase above the 0.5 % serum control. Slight increases in proteoglycan synthesis above the 0.5% serum control were observed in the 10 ng/ml TGF(31 and BP cultures, but these increases were not significant.
At the 10 day time point (Figure 8), 10 g/ml BP produced a significant increase (132%) in proteoglycan synthesis over the 10% serum control, while 10 ng/ml TGF(31 produced a significant increase (52%) above the 0.5% serum control. At 10 ng/ml, BP
exhibited a modest 20% increase in proteoglycan synthesis over the 0.5% serum control, while at the 10 g/ml concentration, BP produced an 890 % increase above the 0.5 % serum control.
EXAMPLE 13: Effect of Exogenous Growth Factors on Type 11 Collagen and Chondroitin-6-Sulfate Produced by Ovine Intervertebral Disc Nucleus Pulposus Cells TGF(31 and BP were tested for their effects on stimulation of Type II collagen and chondroitin-6-sulfate synthesis in sheep intervertebral disc nucleus pulposus cells. The cells were obtained and cultured as described in EXAMPLES 8 and 9 and seeded into tissue culture dishes.
The TGF(31 and BP growth factors were prepared in TCM supplemented with 0.5%
heat inactivated fetal bovine serum. TGF(31 was tested at a concentration of 10 ng/ml; BP was tested at a concentration of 10 pg/ml. Control cultures were incubated in TCM
supplemented with 0.5% serum alone.

After incubation with growth factors for 7 days, cell cultures were fixed in 2%
glutaraldehyde and the quantity of Type II collagen and chondroitin-6-sulfate produced in the cell cultures was detected by ELISA according to the procedure described in EXAMPLE
11. The primary antibodies used were mouse anti-human Type II collagen and mouse anti-human chondroitin-6-sulfate.
At 7 days, cell cultures incubated with 10 u.g/ml BP produced 324% more Type II
collagen and 1780% more chondroitin-6-sulfate than control cultures. 10 ng/ml TGF(31 increased production of Type II collagen by 115% and chondroitin-6-sulfate by 800% over controls. See Figure 15.
EXAMPLE 14: Effect of Exogenous Growth Factors on Proteoglycan Synthesis in Human Intervertebral Disc Nucleus Pulposus Cells TGF(31 and BP were tested for their effects on stimulation of proteoglycan synthesis in human nucleus cells. Human intervertebral disc nucleus pulposus cells obtained from Disc L5-S1 of a 40yr old female patient were cultured as described in EXAMPLES 8 and 9 and seeded into 24 well plates. After the cells adhered to the well surface, multiple dilutions of different growth factors were added. The concentrations of growth factors tested were 10 ng/ml TGF(31, and 10 and 20 gg/ml of BP. The dilutions were prepared in TCM. The cells were fixed after 5 and 8 days of continuous exposure to growth factors and proteoglycans synthesized were detected by the Alcian blue assay as described in EXAMPLE 10.
At 5 days only BP produced a significant increase in Alcian blue staining over controls.
At 10 gg/ml BP there was a 34 % increase over the control while at 20 g/ml there was a 23 %
increase over the control. The difference between the averages of 10 and 20 gg/ml BP was not significant.
At 8 days (Figure 16a), both growth factors exhibited a significant increase in Alcian blue staining over the control. TGFP1 at 10ng/ml had a 42% increase over the control. BP had a 60 % increase at 10 g/ml and 66 % increase at 20[tg/ml over the control.
EXAMPLE 15: Effect of Exogenous Growth Factors on Proteoglycan Synthesis in Human Intervertebral Disc Nucleus Pulposus Cells A second experiment to test the effects of TGF(31 and BP on proteoglycan synthesis was performed on a different liuinan patient from that described in EXAMPLE 14.
Human intervertebral disc cells obtained from another 40-year-old female patient were cultured as described in EXAMPLES 8 and 9 and seeded into 24 well plates. Growth factors were added after the cells were allowed to adhere overnight. TGF(31 was tested at a concentration of 10 ng/ml; BP was tested at 10 g/ml. After 6 and 9 days the cells were fixed and the amount of proteoglycans synthesized was measured by the Alcian blue assay as described in EXAMPLE 10.
At 6 days cells stimulated with 10 ng/ml TGFp1 produced 54% more proteoglycans than control, and 10 gg/ml BP increased production by 104% over the control. At 9 days (Figure 16b), 10 ng/ml TGFP1 increased production by 74% over controls, and 10 gg/ml BP increased production by 171 % over the control.
EXAMPLE 16: Effect of Exogenous Growth Factors on Proteoglycan Synthesis in Baboon Intervertebral Disc Nucleus Pulposus Cells TGF(31 and BP were tested for their effects on stimulation of proteoglycan synthesis in baboon nucleus cells. Baboon intervertebral disc nucleus pulposus cells were obtained from a 7 year old male baboon, cultured as described in EXAMPLES 8 and 9, and seeded into a 24 well plate. The cells were allowed to adhere to the well surface before the addition of growth factors.
The concentrations of growth factors tested were 10 g/ml BP and 10 ng/ml TGF(31. The dilutions were prepared in TCM. The cells were fixed after 4 and 8 days of continuous exposure to growth factors, and proteoglycan synthesis was detected by the Alcian blue assay as described in EXAMPLE 10.

At 4 days there was no significant increase in proteoglycan synthesis between the different growth factors and the control. At 8 days (Figure 17), TGFR 1 and BP
significantly increased proteoglycan synthesis over the control, but the increase was only marginal. In particular, TGFp 1 produced a 21 % increase over the control while BP produced a 22 % increase over the control.
EXAMPLE 17: Staining of Seeded Matrix Material with Phalloidin Cross-linked matrix seeded with living cells was stained with phalloidin to indicate the growth and proliferation of living cells into the matrix. The media was rinsed from the matrix with 3 PBS washes of 5 min each. The matrix was fixed for 1 hour at room temperature with 4%
paraformaldehyde. The 4% paraformaldehyde was washed off with 3 PBS rinses.
The matrix was treated with 0.1% Triton-X 100T" for 3min and then washed with 3 PBS
rinses. The matrix was then stained with phalloidin-conjugated rhodamine"', made up in PBS, for 45 min. Excess phalloidin was washed off with PBS.
The matrix was mounted on slides and viewed under fluorescence with filter of T, range 530-550 nm.
EXAMPLE 18: Growth and Proliferation of Sheep Intervertebral Disc Nucleus Pulposus Cells into Non-Homo~enized Matrix with BP Growth Factor Ingrowth and proliferation of growth factor stimulated sheep intervertebral disc nucleus pulposus cells into the matrix of the present invention was investigated.
Cross-linked matrix material obtained prior to the lyophilization step of Example 1 was cut into square pieces 75mm on each side and sterilized in 70% ethanol for 3 hours. Remaining steps in the protocol were performed under aseptic conditions.
Ethanol was removed from the matrix with two 1-hour washes in sterile PBS, followed by a one hour wash in TCM. The matrix pieces were then suspended overnight in TCM having BP concentrations of 20ng/ml and 20 g/ml. The control was cross-linked matrix suspended in 20 g/ml BSA (bovine serum albumin). Each matrix piece was then placed in a well of a 24 well plate and seeded with TCM containing sheep intervertebral disc nucleus cells at 40,000 cells/ml.
The cells were allowed to grow into the matrix and the TCM was changed every 2-3 days.
Sample matrix pieces were fixed at 3, 6 and 9 days and stained with phalloidin as.described in EXAMPLE 17. The process is illustrated in Figure 7.
Infiltration of sheep nucleus pulposus cells into the matrix was observed at all of the 3, 6 and 9 day timepoints, indicating that the matrix is biocompatible: The number of cells observed per field was higher at 6 and 9 days, indicating that the cells were proliferating into the matrix.
More cells were observed in matrix pieces that had been suspended in TCM
containing BP than in controls having no growth factor. BP at 20 g/ml produced the greatest infiltration and proliferation of cells into the matrix.
EXAMPLE 19: Growth and Proliferation of Sheep Intervertebral Disc Nucleus Pulposus Cells into Homogenized Matrix with BP Growth Factor A further investigation of the ingrowth and proliferation of growth factor stimulated sheep intervertebral disc nucleus pulposus cells into the matrix of the present invention was made using homogenized matrix, as opposed to the non-homogenized matrix in EXAMPLE
18. Cross-linked matrix material obtained prior to the lyophilization step of Example 1 was homogenized using a tissue homogenizer, and sterilized in 70% ethanol for 3 hours. All subsequent steps in the protocol were under aseptic conditions.
The homogenized matrix was centrifuged at 3200 rpm for 10 min and the supernatant was discarded. The pelleted matrix was rinsed with two 1-hour PBS washes, followed by a 1-hour TCM wash. Between each wash the matrix was centrifuged, and the supernatant was discarded.
The pelleted matrix was then suspended overnight in TCM having BP
concentrations of 20 ng/ml and 20 g/ml. The control was cross-linked matrix suspended in 20 g/ml BSA
The TCM/matrix mixture was then centrifuged and the supernatant was discarded.
The matrix pellet was resuspended in TCM containing sheep intervertebral disc nucleus cells, obtained according to the procedure in EXAMPLES 8 and 9. The matrix/cell suspension was pipetted into wells of a 24 well plate. The TCM was changed every 2-3 days. The homogenized matrix seeded with cells was fixed at 4 days and stained with phalloidin as described in EXAMPLE 17.
The process is illustrated in Figure 7.
After 4 days, the layer of cross-linked matrix soaked in 20 g/ml BP and seeded with cells had contracted to form a rounded clump of compact tissue. This tissue was comprised of both the original cross-linked matrix and the newly synthesized matrix produced by the infiltrated cells. There were very few cells adherent to the well surface, indicating that most cells had infiltrated the matrix. This conclusion was reinforced by the dense infiltration of cells into the matrix as visualized by phalloidin staining. The cells had assumed a rounded morphology which is characteristic of nucleus chondrocytic cells, indicating reversion to their original morphology.
Cells had also grown into matrix soaked in 20 ngfml BP by 4 days, but cell ingrowth was not as dense as in the matrix soaked in 20 g/ml BP.
The control matrix suspended in BSA also had cells infiltrating into it, but it was the least populated among the different dilutions.

EXAMPLE 20: In Vivo Evaluation of Cross-linked Matrix and Bone Protein (BP) Growth Factor for Nucleus Pulposus Regeneration in an Ovine Lumbar Spine Model Pilot studies were conducted to evaluate preparative and surgical methods for the implantation of the cross-linked matrix containing BP growth factors into the intervertebral disc space of the sheep lumbar spine, to evaluate whether iinplantation of the matrix with growth factors arrests degeneration and/or stimulates regeneration of nucleus pulposus in a sheep disc degeneration model over a period of six months, and to assess the antibody-and cell-mediated immune response in sheep to the matrix/BP combination.
Study #1 One-half gram (0.5 g) of cross-linked, lyophilized and pulverized matrix prepared as described in EXAMPLE 1 was rehydrated and sterilized by two 4 hour rinses in 70%
isopropanol. The matrix was centrifuged and pelleted, and then rinsed in sterile PBS three times for 2 hours each to remove the isopropanol. The rehydrated matrix was again centrifuged and pelleted.
Bone Protein (BP) prepared according to U.S. patent Nos. 5,290,763 and 5,371,191 was obtained from Sulzer Biologics, Inc. (Wheat Ridge, CO) in a lyophilized forin.
Two milligrams (2 mg) of BP was suspended in 100 (1 dilute 0.01M hydrochloric acid to produce a 20 mg/ml BP
stock solution. The BP stock solution was diluted to 100 g/ml in sheep serum and the BP/serum suspension was sterile-filtered through a 0.2 micron filter. Next, 1.0 ml of the sterile BP/serum suspension was added to 1.0 ml of the rehydrated matrix described above to obtain a final concentration of 50 g BP per ml of cross-linked, rehydrated matrix/serum suspension. At the time of surgery, one aliquot (0.5 ml) of the reliydrated matrix/BP/serum suspension was loaded into a sterile 3 ml pressure control syringe with an 18 or 20 gauge needle for injection.
Three sheep were anesthetized and the dorsolateral lumbar area prepared for surgery.
Blood was drawn from each sheep pre-operatively, centrifuged, and serum collected for irrnnunology studies. A ventrolateral, retroperitoneal approach was made through the oblique abdominal muscles to the plane ventral to the transverse processes of the lumbar spine. The annuli fibrosi of intervertebral discs L3-4, L4-5, and L5-6 were located, soft tissues retracted, and a discrete 5 mm deep by 5 mm long incision was made into both L3-4 and L5-6 discs. The intervening, middle L4-5 disc remained intact to serve as an intra-operative control. Following annulus stab procedures, the musculature and subcutaneous tissues were closed with absorbable suture. After postoperative recovery, sheep were allowed free range in the pasture.
Two months after the annulus stab surgical procedures, the sheep were operated upon a second time. After anesthesia and preparation for surgery, the three operated lumbar spine levels were again exposed. Two hundred microliters (200 l) of the prepared test material (i.e., rehydrated matrix/BP/serum suspension) was injected into the intradiscal space of one (L5-6) of the experimentally-damaged discs. The second operated disc (L3-4) served as a sham-treated degenerative disc; the syringe needle punctured the annulus but no material was injected. After disc treatments, the musculature and subcutaneous tissues were closed with absorbable suture.
Following postoperative recovery, sheep were allowed free range of movement.
The study design is diagrammatically represented in Figure 10.
The sheep were sacrificed at 2, 4, and 6 months after the second surgery. The radiograph from the 2 month sheep showed a degenerative appearance of the untreated disc but a normal appearance in the control and treated discs (Figure 11). Histological analysis of the 2 month sheep as illustrated in Figure 12 confirmed extensive degeneration within the sham-treated, stab-induced degenerative disc. In both the control disc and the matrix/BP-treated disc, a normal sized gelatinous nucleus and regular, compact annulus were observed. In the 4 month and 6 month sheep, no obvious changes were seen in the radiograph of the three discs. A radiograph of the 4 month sheep is shown in Figure 13. However, on gross dissection in the 4 month sheep, the sham-treated disc exhibited obvious gross degeneration while the control and treated discs were normal in appearance ( Figure 14). In the 6 month sheep, there were no gross differences between the sham-treated, control, and treated discs.
Although there was some variation in the rate of degeneration using the annulus stab technique (i.e., the absence of clear degeneration in the 6 month sheep), these results suggest that the cross-linked matrix/BP treatment may protect against or impede the progress of stab-induced degeneration in sheep intervertebral discs.
Study #2 For the second study, matrix material was rehydrated and combined with BP and serum to produce a matrix/BP/serum suspension as described in Study #1.
Twelve sheep were anesthetized and the dorsolateral lumbar area prepared for surgery.
Blood was drawn from each sheep pre-operatively, centrifuged, and serum collected for immunology studies. A ventrolateral, retroperitoneal approach was made through the oblique abdominal inuscles to the plane ventral to the transverse processes of the lumbar spine. The annuli fibrosi of intervertebral discs Ll-2, L2-3, L3-4, L4-5, and L5-6 were located, soft tissues retracted, and a small diameter hole punched through the annulus using a syringe needle in 4 of the 5 discs. A small curette was then placed through the hole into the intradiscal space to remove a discrete portion of nucleus pulposus from each of the four discs in each sheep. In 2 of the 4 damaged discs, 0.5 ml of the matrix/BP/serum suspension was injected into the intradiscal spaces and the needle punctures were sealed off with ligament sutured over tliem. The immediate injection of this suspension was considered an "acute" treatment protocol. The 2 other damaged discs were left untreated at that time but were sealed off with ligament sutured over the needle punctures. The intervening, middle L3-4 disc remained intact in all sheep spines to serve as an intra-operative control. Following these procedures, the musculature and subcutaneous tissues were closed with absorbable suture. After postoperative recovery, sheep were allowed free range.
Six weeks after the first surgery to remove portions of the nucleus pulposus, the sheep were operated upon a second time. After anesthesia and preparation for surgery, the five operated lumbar spine levels were again exposed. In one of the two remaining nontreated discs which liad been damaged six weeks before, 0.5 milliliters of the prepared test material (i.e., rehydrated matrix/BP/serum suspension) was injected into the intradiscal space of the disc. The injection of this suspension six weeks later into a damaged disc was considered a "delayed"
treatment protocol. The second nontreated damaged disc served as a sham-treated degenerative disc; the syringe needle punctured the annulus but no material was injected.
The treatment method used in each of the four experimentally-damaged discs was randomized for location within the spines. That is, except for the intact control disc (L3-4), the locations of an "acute"
treatment disc, a "delayed" treatment disc, or a nontreated, damaged disc, were randomly assigned to one of the four different lumbar disc levels. After disc treatments, the musculature and subcutaneous tissues were closed with absorbable suture. Following postoperative recovery, sheep were allowed free range.
The sheep were sacrificed at 2, 4, and 6 months after matrix/BP/serum injections and the spines were fixed for histology in formalin. Cross-sections were taken from plastic-embedded discs, stained with H & E and Saffranin-O'", and evaluated for chondrocyte proliferation (cloning), proteoglycan staining intensity, level of fibrosis, and level of ossification. An evaluation of the "acute"
treatment discs, "delayed" treatment discs, sham-treated, and control discs was made in a blinded fashion and ranked +1, +2, or +3 (low, medium, or high) for each parameter listed above. Semiquantitative evaluation of the histological results was compared in 2month, 4 month, and 6 month sheep for both the "acute" and "delayed" (6 week) treatments.
The results demonstrated overall that injected matrix + BP stimulated chondrocyte cloning and accumulation of Saffranin-O staining of glycosaminoglycans in the nucleus matrix of damaged discs. In particular, the extent of regenerative repair was much greater in both "acute"
treatment discs and "delayed" treatment discs, compared to that observed in non-treated, damaged discs. This greater level of repair in matrix/BP-treated discs was statistically significant at the 0.01 level of confidence. There was also less fibrosis and ossification seen in the acute and delayed treatment discs compared to the non-treated discs.
A significant difference was also noted between the "delayed" treatment discs and the "acute" treatment discs in the level of proteoglycan staining. For example, Saffranin-O staining as an index to proteoglycan synthesis and content in the nucleus matrix was greater in the "delayed" matrix/BP-treatment discs than in the "acute" matrix/BP-treatment discs. Additional benefits apparent in the histological evaluation, which were associated with "delayed" treatment with matrix/BP, were an overall lack of bony transformation (ossification) or fibrous tissue accumulation (fibrosis) within the treated discs compared to the non-treated, damaged discs. In general, the results in Study #2 support and elaborate earlier indications from Study #1 that treatment of datnaged discs with the cross-linked matrix/BP may protect against or impede the progress of degeneration in experimentally-damaged sheep intervertebral discs.
EXAMPLE 21: Characterization of BP
Specific growth factors present in the mixture of growth factors produced according to U.S. patent Nos. 5,290,763, 5,371,191, and 5,563,124 (i.e., BP) have been identified. BP has been partially characterized as follows: HPLC fractions have been denatured, reduced with DTT
(dithiothreitol), and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). One minute high performance liquid chromatography (HPLC) fractions taken at from 27 to 36 minutes are shown in Figure 18. Size standards (ST) of 14, 21, 31, 45, 68 and 97 kDa were obtained as Low Range size standards from BIORAD(tm) and are shown at either end of the Coomassie blue stained gel (Figures 18 and 19). In the usual protocol, HPLC fractions 29 through 34 are pooled to produce BP (see box in Figures 18 and 19), as shown in a similarly prepared SDS-PAGE gel in Figure 33B.
An SDS-PAGE gel of BP was also analyzed by Western immunoblot with a series of antibodies, as listed in Table 1. Visualization of antibody reactivity was by horse radish peroxidase conjugated to a second antibody and using a chemiluminescent substrate. The reactivities are as indicated in Table 1.
The BP was further characterized by 2-D (two dimensional) gel electrophoresis, as shown in Figures 21 and 22. The proteins are separated in horizontal direction according to charge (pI) and in the vertical directiori by size according to the method of O'Farrell et al. (Cell, 12:1133-1142, 1977).
Internal standards, specifically tropomyosin (33 kDa, pI 5.2) and lysozyme (14.4 kDa, pI 10.5-11.0), are included and the 2-D gel was visualized by Coomassie blue staining. Figure 21 shows the stained 2-D gel with size standards indicated on the left. Tropomyosin (left arrow) and lysozyme (right arrow) are also indicated.

WO 01/76654 PCT/USOl/11576 The same gel is shown in Figure 22 with several identified proteins indicated by numbered circles. The proteins were identified by mass spectrometry and amino acid sequencing of tryptic peptides, as described below. The identity of each of the labeled circles is provided in the legend of Figure 22.
The various components of the BP were characterized by mass spectrometry and anvno acid sequencing of tryptic fragments where there were sufficient levels of protein for analysis.
The major bands in the 1-D (one dimensional) gels were excised, eluted, subjected to tryptic digestion, purified by HPLC and sequenced by methods known in the art. The major bands are identified by band number, as shown in Figures 19 and 20. The sequence data was compared against know sequences, and the fragments are identified as shown in Tables 2A-2B. In some cases, the identification is tentative due to possible variation between the human and bovine sequences and/or possible post translational modifications, as discussed below.
The same tryptic protein fragments were analyzed by mass spectrometry and the mass spectrograms are shown in Figures 23A-230. The tabulated results are shown in the Table depicted in Tables 3A-3F, which provides identification for each of the indicated bands, as identified in Figures 19 and 20. As above, assignment of band identify may be tentative based on species differences and post translational modifications.
The identified components of BP were quantified as shown in Figures 30A
and 30B. Figure 30B is a stained SDS-PAGE gel of BP and Figure 30A represents a scanning densitometer trace of the same gel. The identified proteins were labeled and quantified by measuring the area under the curve. These results are presented in Table 4 as a percentage of the total peak area.
As Table 4 indicates, there are 11 major bands in the BP SDS-PAGE gel representing about 60% of the protein in BP. Further, TGF-81 was quantified using commercially pure TGF-31 as a standard, and was determined to represent less than 1% of the BP protein. The identified proteins fall roughly into three categories: the ribosomal proteins, the histones, and growth factors, including active growth factors comprising members of the TGF-B superfamily of growth factors, which includes the bone morphogenic proteins (BMPs). It is believed that the ribosomal proteins and histone proteins may be removed from the BP without loss of activity, and the specific activity is expected to increase correspondingly.
Because several of the proteins migrated at more than one size (e.g., BMP-3 migrating as 5 bands) investigations were undertaken to investigate the extent of post-translational modification of the BP components.
Phosphorylation was measured by anti-phosphotyrosine immunoblot and by phosphatase studies. Figure 24 shows a 2-D gel, electroblotted onto filter paper and probed with a phosphotyrosine mouse monoclonal antibody by SIGMA (# A-5964). Several proteins were tllus shown to be phosphorylated at one or more tyrosine residues.
Similar 2-D electroblots were probed with BP component specific antibodies, as shown in Figures 25A-D. The filters were probed with BMP2, BMP-3 (Fig. 25A), BMP-3, BMP-7 (Fig.
25B), BMP-7, BMP-2 (Fig. 25C), and BMP-3 and TGF-(31 (Fig. 25D). Each shows the characteristic, single-size band migrating at varying pI, as is typical of a protein existing in various phosphorylation states.

Native and phosphatase treated BP samples were also assayed for morphogenic activity by explant mass and ALP (alkaline phosphatase) score. The results showed that AcP treatment reduces the explant mass and ALP score from 100 % to about 60 %.
The BP was also analyzed for glycosylation. Figure 26 shows an SDS-PAGE gel stained with periodic acid schiff (PAS) - a non-specific carbohydrate stain, indicating that several of the BP components are glycosylated (starred protein identified as BMP-3). Figures 27 and 28 show two specific proteins (BMP-7, Figure 27 and BMP-2, Figure 28) treated with increasing levels of PNGase F (Peptide-N-Glycosidase F), and immunostained with the appropriate antibody. Both BMP-2 and BMP-7 show some degree of glycoslyation, but appear to have some level of protein that is resistant to PNGase F, as well (plus signs indicate increasing levels of enzyme).
Functional activity of PNGase F and sialadase treated samples were assayed by explant mass and by ALP score, as shown in Figure 29A and 29B, indicating that glycosylation is required for full activity.
In summary, BMPs 2, 3 and 7 are modified by phosphorylation ("33 %) and glycosylation (50%). These post-translation modifications do affect protein morphogenic activity.
Matrix compositions useful in treating intervertebral disc impairment in vertebrates, including humans, may be prepared according to the foregoing descriptions and examples. While various embodiments of the inventions have been described in detail, modifications and adaptations of those embodiments will be apparent to those of skill in the art in view of the present disclosure. However, such modifications and adaptations are within the spirit and scope of the present inventions, as set forth in the following claims.

Aiatibody Information Specificity Antigen Host Species PC/MC Source Catalog No.
TGF-01 (human) Protein Rabbit Polyclonal Promega G1221 TGF-02 (human) Peptide Rabbit Polyclonal Santa Cruz sc-90 Biotechnology TGF-(i3 (human) Peptide Rabbit Polyclonal I Santa Cruz sc-82 Biotechnology BMP-2 (human) Protein Rabbit Polyclonal Austral Biologics PA-513-9 BMP-3 (human) Peptide Chicken Polyclonal Research Genetics NA
B1VII'-4 (human) Peptide Goat Polyclonal Santa Cruz sc-6896 Biotechnology BMP-5 (human) Peptide Goat Polyclonal Santa Cruz sc-7405 Biotechnology BMP-6 (human) Peptide Mouse Monoclona Novocastra NCL-BMP6 1 Laboratories BMP-7 (human) Peptide Rabbit Polyclonal Research Genetics NA
FGF-1 (human) Peptide Goat Polyclonal Santa Cruz sc-1884 Biotechnology osteonectin Protein Mouse Monoclona DSHB AON-1 (bovine) 1 osteocalcin Protein Rabbit Polyclonal Accurate Chemicals A761(R1H
(bovine) serum albumin Protein Rabbit Polyclonal Chemicon International AB870 (bovine) transferrin Protein Chicken Polyclonal Chemicon International AB797 (human) apo-Al Protein Goat Polyclonal Chemicon International AB740 lipoprotein (human) M - r. :-. ' '- ' ~'-.. . =. =~ , =
N ro orn M in ~. ~i! r r ~y C~= a p~- o. KA OS V it) a+ ~= uf~ r e- r ~7 tA
tU t4' N tal N M- N t+J .(?I nf C7 th t" d d.c~ r.r ~. 10 CO

0 a M o. o= .o.: ~ o'.
~;: z o ~ ,- r =- a; - .-= a. a a a n0 +... p~ M^ M m M-- M~ go"~ M'-- M' M M v~ c9 Yp 0] i-a1 1~. fIl 1, - h., m 1~. a1 Of 01' q~
U" U o. tn ,rn U!~ .= r-- ~ r. N~
r-. Z ur M' fn. `- tn +S a~. ..~r~',r~~o-~~,~ ..~...~r~o: a.. a d C m.. G c C' - c c a~ ~ m e w = ' =~ = ~/! c4 = - tb =. t6 i co . t6 ctf = C6 W N m to ~ - .. o E E E E E 0 o= - 0 E
E
~ ' == ~ . =
n=21 ~. 9L.
n. Ll. p`.
cu E
~~ -r ~y= ~ ~ ~ ~~ ~h = ~VOi N
Q. ti O d~ O O O
IJ?y!a C ~El M M M M. ' . +- M.'M - f3 .G M
O - (~ C . Q L1; 11. '.G ~ a. - . ~ i= p o ~ ON~.=.~2 .~u g.. ' ~' O= O
MM4CtNh J IYl m fn m CD' ! (O J.SD ~.1 h bA
v ~ ~ . = t=r==. t=~=- r' - C'~V ~ ~- " ~ -. 0 ~
~ z=~- - 'e~-~ , e~- tf7 O. ~
CV Gil = CO d) V) 1n = ~r d v cli ~
u~ z z' :
a n _ z z u.. of . ~ : uJ' w w w Q. a. c Q a 1=i., cn .. . l.L- =z V u-_ -~ . Q =--~ . ~"= (~+ (~ = (n - . w (f U) J
~..~ ~- t~.= c~~t~". ~.. e.~ t- w ~' a~ `~ = -- w i-- c~ a =c~ a . . . r v a v~ uO- x rn' -a. (D 0 a n. n.. 'a . .
w a ..
a x 03 w 0 z Z. , . . .~
c~
w ~
W p,, d Y. W W= d W
q C]',~' J= - J J . M o . '= = >_ a .10 .'. = - .J c~ z -' >-j , . .. c~
at y Q > 0 u J J (7 . r.y . n. .. Cl Cl 9C'J

x = LLiLLLLLLL
. 7o ';l.. . .
, ~ ~ ~.C , ' ~ ~." : C ,~ ~. . = =. , ~ =O p~ , =.
- Q .-. O ' , -.~ . ~-, .. = S y. O .
rn- lti ~. -.n ~ tV ~ ua ~ t~ t~. ~= t~,,~ j~' .~ n ~-+ .- ao cp m N ~u tlYo tC. w -py f~ M. ~cI ~ dx.. GT
VYi:,d ~ tXq G M,C/~ My V~= N~
N. '.~i-=~' w~i.: u.W+ z. in w cqi ~`='~ .K.:` v V+: Z N' .~F.:' v'~f-: v W~."~r.r ~'r..~=' '~i.~ W: t' ~ CO ~ N M d ~. _ tfl =' , t-.; '. C~ O~

.~, = - =_ . , , .
= , .~ u~ t~ r. =~= .~ c~~i r- o .c~ `;~ . ' d~
M'm cv cu. cv c~v .^= eh co . ' .. r ' ~-. ,.~ . ^ =, = r~

ZM=~~Nh'n". a j maov~ ao~- = t.o~co_ ~ ~n ~~~.

Gf =L+ ;C , =~' = '- - _ ~ , .
~~ 't4 CC . lQ. C = . . .W. = ~.. C.:
.~ ~ . õG- ~ `~ = =~'.., '-. .O . . ~ ~ _ +C .c = a . r w J J 34 N
to 0.. .[l. [i.
~ ' i0 N ~y = ' _ _ O ' .
'~" "-' . ~ O ' . O . - ~ _ _~: = = .
t~,1 `'= ' . .
V ' N
. . L1 Q- N_ !C
p I O ~~ ~ O
ao ~o. cD
cr~
co o. rn cr- r ~ .~.
v2 V = a"~i >0 ~
~ ~ ~ -= ' ff`. a ~
.~. u,~ F- C9 = = ci t~ ~ : ' .
~ 0 t- ..j Z fJ9 . . ~ .
Q
} ' >
='aa . , o o Q ' ; ~. = Q =~
a m. a . ~= . ~. a ' ~
w a-. r :.~ . . ~j ' ,g?
.~ ' ! ~; ~ ~~ Q C7- ~,~'= ~ ==: ~ ~.. , (f. _ = p .
, .=~. a 'C1 ~ ~,' ' L] ' } U! =_ ~
=
yi . ; ~ = F , .
JL< .e-..
~. . .., = ed Cd .
~ ~ ~n: o~.= IF' N N c~

c cc ts 'vi =
~
c N
'C7 C p"- .
E.. c. : o'-o a'- : x-c m n .C.). . m =m .'.
p .
~.? Q- t, ;- C3 p Q -0) y .LC) Q U.) . . N
N ; . = ..

U cri N . t0. - . LV. - ~p N r 0 o . ' . = . . .

=L! - ' . r . 'W W tt *': .. ~. .V', 6~ - .M ~(Q r~ ~ O _ a) .CO :..
1 - Iw ~ ~7 ~ ;' : = 1 l- to' K~1 C~ tV 'CV v-=. y. r . YA ~' -Cf ~st, ~ O ft9 ~O O) tl~ r r . ~ ' ~
. -., O ~ =CO Cfl .M tG M" tn. t~ U. tlMD tOt7 ` ~ ~
V .. . .'..
G. r .. C co a0 ~ 4).- cO 'o ' c0 - bf ~p !i1 i~ 1~' N' co 1--fV ~ M , t] e-~ Ni N T 06 C? = N
~= O, ... O O C O-_-' ~~Q O-~ f7. ~ ~ O OO O. O O.G O , CO C

N r. 0) N . 0-- O. N pp (~ Of 1+ h. CO O
A- ' y M i+ OCl Cr!. M' "IY M(G lC1='!` -~ GO N~~ ..tD
O H v y Or- 04 O. ~ N 0) O4. il: ^'ci 00 - .C Q
Ul. a; m h. 14 'O a- h O P- N ~. M rl/ e- r- Op V - r- e-~ r U) k- d t!) 1+ .. - a= - =. MC'r) tt) 0) st C~ .'d lD
y_ t!1 -~ ~- . ...- e.- 'N '. C) N t- M T - N N

y h . . ~ h co t0 : N 'cy co ;- t0 ;c;7 ~.
v eti Q c4 d -n h.' ~~. .. i~ " . N'~
'~ =¾ ~ tV Csi 00 - M. = Oo N rn to 'ct' or). 0 'It. 1l-M O t ). 'c~ N'- = ' r. C'M9 CO 4~") ~O) 't m ;.= r~ . . r- 'i- r N. e- . ~. ~--~ fiV T- r, r M~-= N N
.a'. ' y~ - ' '= py -- ~ , 00 N.= O r 4; Z cG ~1 tD: tXt N.
. C V co U - . . , . ~ .U.. , = ~,. ~f1õ . . , ~ O
00 . N ? . ~ . . ..5~ - . , = . : Q.
4, O =. . , y. .
co . . co - . .. ' . .. .~ -. coc . .~
..' I.E
~. . Q
~~-=: y, Q-, h "~ .~G~ 5 .Y. ~y N,. ~ s.

Qj 0.'V ~'.. Yl'V O GURa ~ N: `r III

^ LL.' v7 Q
"OmN V~ N
N ~;~ " " +5"a =~+ 't9.
õ'õ ca ~ " . - " "=.' ~` c.
t U
0 E v- O.~ co E.N,s C C y , V
(D p ~".
~~' >p:~~
o~.M T1 fl O~ M
t~ L
L = , ~ - .~ . .. . ~
Q~ ' . " . . .. -. . . = .
> QI M ' . . . ' " ~ . ~.
C ~- . .~= . = . . r r V . Cd . ~ . . . - . . . - -Q = " " ' ' " - .
6~ AO I~. 1~ d p N N" _.Q. =. . C7' N 1~ õa.
tfl en to ' N c0' C). QD O) !t? CO tfT' N~
t7 sT M VJ M =N . t-. N M
~
(O ~(?. O~D tl M'. , ~ M- O~ i4 tb O
1- . W st 1. 1.'. .... " C 00 = N~.ti r W ~. ch M cn cq M'f!) N N r N M. et ti Q ~ r 'tA" O tn t~+1 P- (O "= ' qJ jw` GQ 0 a o 0 oi m Ci o .r v ." o 4 o o c s 'o o ti :-=. ' o o o c5 0 .o ~"w =_ .

d ~. Gl r CO l'D 'r ~N o7 aq . .. ~ ~M. N] ~A 1 y y, tn 'C~7 ' . ti) f~ C~ G
CO GO ,' I~ a- V 4 '. tn Q' y y~ Cc 00 dC =
CCI 0 ~+ .
i0N Q: "CD , tA
C) ,~' r d' tq .<D:.I"~' tl' C . . . M =O to CO
N~ ~ C9 tY M r r . S~_ .~ " . - ... ~ .
~'.
'et. QO e- CO'.. t0 N' M(0. 4(0 4) cfl to' to ~. N= C / = t p M ,C). N e0 Q' ~t; uq C~: c0 o6 =(b r ' : i d ' to "ct", . E 7 i - C V Ob' tD t= .-:~' dD t[7 (3) = f/~ 0 r ~ st' U) '(O 'f~ CO. NO' pat= R 4 ~ " r= r' r' r ~ a~l N"'- . M M
. .
. ~ .Z . M m " . " ,. = Ln in .
ii ~ ~o co :" ~.. . . ~ = . . ~.~- '= .
. . ' ' ~ - " ' = --O~
Y 3C7 x y . i 'n tn s~.-. '~ 4 L ¾~. aa ~ ~ a .''CL m m' = i" M

~ b `n . ', =" . co . =
F-H

'ya yCL
o-m~. om I ~r v- p ,~ . ~
N=W

E 'sN c~c ~' o m cm (D 'm ;:M
~ ' =, -- `~ G' (n- pp .~ -. ' . p .~: r . = .Lr, . . Q ~ r . . = ~ , _.

. . ' . . . . . . . . -M, -- - N = ~ ~ ' ~ ~ . .~rj. ~- m 4 . . .

!n .`ct CA. r co O. V O U>" OP - 1~ co '- - I ~ = ~. .Or. t ~ N CO N D. LO '. . ' - . 0 tfS u7 ~h( r pp . yn ~ .~; c? cV~ v ~c ~ .7 (O~''. . = M . . Cn M sr ~ -s'7 =
L - a <O - r f0 .r (v , . . ..i.' C] a (l~ OO ! . . W ~f7 o d : . ~ o, = . ~ lE1 O) 'f~ lm d) s- st co tp' M. cr! M iY . N
C - -,`r , 00 . ~ ~ 6> sb N a0. fV t+ O (O ~ " . ~ ~ 1`~ '- N t- C CD =1~ ' O O~ r M' O M.M C N
e^ C O M st Eõ~ Q~ O O O O O` O ~ O. O O ~' G} -' O 4= O, .C r 4.. N
O y ~
~'. ' . . - .. = .. . . . .
, Gl h= G co t0 O - r Op .r- 04 'C7 co tL1 r Cf f+
y V N r dN tO N M tl} O) O<0 . - M- 4) P~ CO r P~
v~+ p0 M C~f +3 f7i M' [7 r pi M ": '.CD' SU r r 1~
0) M`Q 2D Lll. e_- t0 !n O' O
~-= N O r~Q= ~ M Co r ~C) CO tf) sY .. r e- r N M

~ ~
ca L!) rn ~p a0 N 1- . V O 1` r t- st C~ N N' t0 M' r: tG I~ ~(O O) Q. ~ N) tV M S> ~. .. 'C!j~ .. CO (O e- r S70 -/-~ .. Q t!) - .Q) M~ d' (O .. . T. <D~ f.q C3 r e--- =- C~) (D 'tA O. O' .~.tl~ O' O r. d O e-. M fD 1~ st ` r st. ~[T t0 6) sY
e-y :
.~ C . N
y~ : Z n'~.' M m N y ~. M m =
w a': n ~n' ~ ~
Z
O N y co cu t> >.....' ,~ a~ . a _ . = =
~_ . y : s t s y ft fl m ~ O cv ~ ' ?S ~=~o~~.
dE, o.~cp--c~ +~ ao = . cn .

,II

~ ~.
O m N ' _ h- .
N " ~ ~ ~ . . ~, .
=+ - ~' ,~ ~' - ~ .. . .
C: U ~p N
N ~ lp ~ v . . . . . . -.
E o~~ ~ v~ .
m ~ ~.a'~ -. .
o>m ~ ~~
`
, ~,.
~
-L . ' ' õ - . . . .. . . , . `! -~ ' . . . .
O C!' ~ :.;. Qr uNy M
(.~ ~6 ' - . .
i,q,~ ~ ~ . . ~ = ' . ~ ~. ' . ~ " = ' p o , ~ CO ~t O N o0 CV. ~A 'C r'r e-. Q~ CO N M st OO h. C7 Q~p ' ~c'00 - -dN' CM CO= M r ' . - ~ N = . N ~N M~ . . Ci M M ~= '~ O t~ ~
N. . . ~- . i ~ O : ~ ~ = ~ ~ ~ N ~ N ~
~ ¾ e- O tb "c"! .Q <1 r- r N O 1+7. CA N b~= t~ 00 C¾= 64 Q (O e- V 1~ Q r- ~= fD <O O O r=r a- .
~T 1 M st. M[h N r r. . N N M" `V' M. ~' a~
~
~S. ~ ~ 00 00 sY 1~ 1`+ ~1'> ', N M . ~ lD (D ~~O M e~- f~.. (O `d' uY p.
C1['ay~a =~' R O e- ~Y ~A tA r L7 Q' O O O i+ N N CD O. O Q e-~_~ d, cioo-c~o'= tio cooo cooc~=- c~ooo _ 4~= UN ad. -~ ~ R. - .. :_ ~ ~ _ .
~ 01 ~' r N"~= 1~ M. -(p. .N ' iA OQ fD N O e- O tA "O .tt> 1~
tl. . . R --U ~ M . . 6~ ~O OO t~~ .N. , . 00 ..-' , - M- -t0 N ~ Rt t4 ~ LO ~
t0" W O -ff =
y= d~~ r d~I h~ -t7 " ~D: Q).. ~t7i CC1 r tp O i r 1~ C. CO
~'.+ R p.' R tJ 1 Ci N' O. ~!' N tfl i! 00 O- ' f~ st Ga .e- . d. et ~ ~ ~ t!) ~ e~- ~- ~ N c~q r '.. ~ ~ ' . ar- 1M-.~. er=- . - ~ ~ N N .r~-. . -.~~-- e0~. ~ N
~ ~
y i*> .' M 00 .eY d ar' a0 u7 .. cfl d O CO h'O t=7 13f d O f~
~ N C1 ~ N= .: L` tf) N. c~) M. ~ CCJ r M. 1~ N - .N- M!~ CS) ~ ~ tp r t}=
~ a:~ t ~ M'sY '00 L7. (O ~ tt) (O t-'=' ~f1 G' r C1 f`+ G C1i OD
, ~ ~ 4~ N O~. N~- ~. a0 O' . R tii' O~ e-. st ~ CO CO
:~ t!~ e- (O 1~ ~1 ~t ,:r. . SA O e- M e- r N (p d. et' 00 N
Da ' e- r t-. N M r e- r- r r r' t- .fV N r. '.~- e- r N
.C=-~ ' ' "y - . .. - . . . . .
,~~' . O - ~ ~ . ' ~ Op ~ - .. e+` ~ ' ._ N ~.. - , r, ~ , ~ .
i ~ Z . ~vy. m on0 N ~ C~ vyi "^+ : er- ~ p. ~. N p j.
,i' U.. N-'~ ~.. f~. ~ Q ap y`~ .~ ~ y~ ~.
'. i~i' V~ . ~~ - .. O~Q,,. ti':~Q.. ' O~~ ' ' NNa p.~ . ~ . ~, .. p .: p. .. . . a .. = .. : ~ ..
, aõ .
' o .
Vf C..
,G ~ c~a: t~tl h ~ ~ .
~G ~-' _'' : .. 'C '.` ~ ~ a :`. _ . d . .. d N ,.'~-.. " - . ~ . - ~ . ~ "..~+ J ' - - - ',zC . . _ - : ,.C - I
'i ~õ'O,~ fl,' m ~c ~ M ac ~ a~ ' ~Y". ,~ N~ ~,~ N. r ~ i Np" ~~~.. ~~o'.". ~~~... .' ~r4' .. a. ~" a v~-.. U
!' Q ~o- '.`nEm' u' E.~ ~ E:t- ~~u~i. ~
M .. . ~ .
'I ~ . O '. ,.. = ~
~ R == , ' aQ
, H m - . . .,- .
i I
I

S a . .. = ' c%j .= w - . = . .

III
C 0)~
.ci MQ
? m .0 $ S .
i+C9 ' - '~a=q~.~j , .

~ ` . = . . _ = . -. G/ - =
~
o . . .. ~ .. = , N 4Q ~ 49 e- ~Y ttr e- = - tC! N r t+3 ~ NM t~i Ci N~{ = c~1 A' N f~ da C7 d M c0 M M C7 ''tt N~

i-~ m tt] O t~ 1~ N st M tp fR r"O~ =
= F~ - .- = - ~'i N M M =c~ , c`~- ~. - =~ m Q ty. ry =~-= ~- o V Q O G'O O O'-.. O C] G Q O O O O G O r , d = .
m N N d r ~h O N'd 1~ Q; d m i- Iti N=
y st ;s1 ~O Of t0' = M~0. M,~ff Kf ~A c~X tA 1~= Q~ A
I d' y a~ ~ st e='r% i++ CO .. ~ st - N` O o0 ~~pp r-' t~
I y~ ~y Q~ C7 . 1~ CO V O . 0~
C. ff!
~ N C~1 ~? 'c~1 i- tD V
N - (O
= y V~ r ' a~- N C~1) a~.- ..- =+ r.' r e-- e- r e~ '. . ~-- r r- M
C/1 =`
tp M m~[) 'r O m ~- .~p 1~ tr1 c?~ N Gl m. .
41 ti m- R ` ~; `0; 07= uf ~ et.t -' aD O! a0 ao N Gf ~= e= 47 d<] e= ~- I~' nQ e- d N O 00= OD
RJ d h CO ~~ r= q~ d.. N Cfl. -C3 ~p M O
rL N~ e e- N(O d V N GV ~ M 1~ Q', t0 d D'- N rr ... r t- =a-1~
.y, _ _ . - . = - . . _ = , . . - " .

r ._ :. .. . ' d. _~-'' ' = '. , = . U: - - ' a CY
~., .
-o - . . .
o -~.-~ ~--- =, :~ - -,~-. . '~. ~=~~~~~- :. =.
cl .. y ~ , _= ' .
+.+ _ ~ . . - . .
d ' m. +~-=(V ~,, +~+r :....
I~ . 'tn t~ . c~a ~3 c~. ' . ~ = ~''3~ N . . . y~ T - ~ = = ' ~ rr .
(L) n.. .m ~
a . m .N N c "n CL a :m M. =g- -E . ..E - E~. ~.' = ~ . -.
H

: . . ~, a' = O m N

ci ~

, .

= ~ L _..._-_-...-__~ .___..._-_.' -- . -. _._- ......______ . . _...__-__-_--___.. .._._.___-_.- - .._.-__.__._-_ ...._._-._. _..__ U ~ ~ . .
.b .
d a~ =' 1~ 90 p~p M ul -, R~l M M=
~ CYO = .'CO~'. ~1~1d' ~ . ...
W ~ ch A. a) N
v E-1 y O : G C
O

i O. V/= C') =' tCJ. .1~. 1~.
M
Q ,tfl b N
rA
~ tf c*j V E h t~t ,~Q,= C7 , oD R(Qf!,'m ~' ..~.~ - r = -M
N = tr~-.~.'. .' r a~--' v<'~j h ~. . =Q =:~ et Z. . C9 m . - .

o ..
A'. yy c ~..
~ o'=
.a~ : .
d v 4d' o' a~ g ~ '.
. M " =~i': ~ -~ = ~.:.... N .
~ m .

TABLE 4 Quantitation of Identified BP proteins Identified Protein Percentage of Total Protein I.,ORP 2 BIvA'-3 11 BMP-3 and A2-MG 3 RL6 & BMP-3 4 Histone 3 Histone 3 Histone & BMP-3 4 RL32 & BMP-3 8 SPP24 & TGF-(i2 6 Total 58%

Claims (29)

1. A fluid matrix comprising nucleus pulposus tissue of a donor vertebrate, wherein at least a portion of the nucleus pulposus tissue is cross-linked, decellularized, denatured and/or rendered substantially non-immunogenic.
2. The fluid matrix of claim 1, wherein the donor vertebrate is a mammal.
3. The fluid matrix of claim 2, wherein the mammal is porcine, bovine or ovine.
4. The fluid matrix according to claim 1, 2 or 3, wherein at least a portion of the nucleus pulposus tissue is decellularized.
5. The fluid matrix according to any one of claims 1 to 4, wherein at least a portion of the nucleus pulposus tissue is denatured.
6. The fluid matrix according to any one of claims 1 to 5, wherein at least a portion of the nucleus pulposus tissue is rendered substantially non-immunogenic.
7. The fluid matrix according to any one of claims 1 to 6, wherein at least a portion of the nucleus pulposus tissue is cross-linked.
8. The fluid matrix of claim 6, wherein at least a portion of the nucleus pulposus tissue is rendered substantially non-immunogenic after removal from the donor.
9. The fluid matrix of claim 6, wherein at least a portion of the nucleus pulposus tissue is rendered substantially non-immunogenic by decellularizing, denaturing and/or cross-linking.
10. The fluid matrix of claim 9, wherein at least a portion of the nucleus pulposus tissue is rendered substantially non-immunogenic by decellularizing.
11. The fluid matrix of claim 7, wherein at least a portion of the nucleus pulposus tissue is cross-linked with a photoactive catalyst.
12. The fluid matrix of claim 11, wherein the photoactive catalyst comprises methylene blue.
13. The fluid matrix according to any one of claims 1 to 12, wherein the nucleus pulposus tissue is lyophilized and subsequently rehydrated.
14. The fluid matrix of claim 13, wherein the nucleus pulposus is rehydrated with serum.
15. The fluid matrix according to any one of claims 1 to 14, further comprising at least one living cell.
16. The fluid matrix of claim 15, wherein the at least one living cell comprises a chondrocyte.
17. The fluid matrix of claim 15, wherein the at least one living cell comprises a mesenchymal stem cell.
18. The fluid matrix of claims 15, 16 or 17, wherein the at least one living cell is human-derived.
19. The fluid matrix of claim 15, 16, 17 or 18, wherein the at least one living cell is cultured in vitro.
20. The fluid matrix of claim 19, wherein the at least one living cell is cultured with the nucleus pulposus tissue.
21. The fluid matrix of claim 20, wherein the at least one living cell infiltrates the nucleus pulposus tissue.
22. The fluid matrix according to any one of claims 1 to 21, further comprising at least one growth factor.
23. The fluid matrix of claim 22, wherein the at least one growth factor is bone-derived.
24. The fluid matrix of claim 22, wherein the at least one growth factor comprises a serum growth factor.
25. The fluid matrix of claims 22, 23 or 24, wherein the at least one growth factor comprises a member of the transforming growth factor-beta superfamily of growth factors.
26. The fluid matrix of claim 25, wherein the at least one growth factor comprises a transforming growth factor-beta.
27. The fluid matrix of claim 25, wherein the at least one growth factor comprises a bone morphogenic protein.
28. Use of the fluid matrix according to any one of claims 1 to 27 in the preparation of a medicament for the treatment of intervertebral disc disease.
29. Use of the fluid matrix according to any one of claims 1 to 28 for the treatment of intervertebral disc disease.
CA 2400826 2000-04-07 2001-04-09 Methods and compositions for treating intervertebral disc degeneration Expired - Fee Related CA2400826C (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US09545441 US6723335B1 (en) 2000-04-07 2000-04-07 Methods and compositions for treating intervertebral disc degeneration
US09/545,441 2000-04-07
PCT/US2001/011576 WO2001076654A1 (en) 2000-04-07 2001-04-09 Methods and compositions for treating intervertebral disc degeneration

Publications (2)

Publication Number Publication Date
CA2400826A1 true CA2400826A1 (en) 2001-10-18
CA2400826C true CA2400826C (en) 2010-02-23

Family

ID=24176265

Family Applications (1)

Application Number Title Priority Date Filing Date
CA 2400826 Expired - Fee Related CA2400826C (en) 2000-04-07 2001-04-09 Methods and compositions for treating intervertebral disc degeneration

Country Status (7)

Country Link
US (4) US6723335B1 (en)
JP (1) JP2003530364A (en)
CA (1) CA2400826C (en)
DE (2) DE60120667T2 (en)
EP (2) EP1707225A3 (en)
ES (1) ES2263612T3 (en)
WO (1) WO2001076654A1 (en)

Families Citing this family (81)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE69714035D1 (en) * 1997-08-14 2002-08-22 Sulzer Innotec Ag Composition and apparatus for the repair of cartilage tissue in vivo comprising nanocapsules with osteoinductive and / or chondroinductive factors
US8128698B2 (en) 1999-10-20 2012-03-06 Anulex Technologies, Inc. Method and apparatus for the treatment of the intervertebral disc annulus
US7004970B2 (en) 1999-10-20 2006-02-28 Anulex Technologies, Inc. Methods and devices for spinal disc annulus reconstruction and repair
US7052516B2 (en) 1999-10-20 2006-05-30 Anulex Technologies, Inc. Spinal disc annulus reconstruction method and deformable spinal disc annulus stent
US6592625B2 (en) 1999-10-20 2003-07-15 Anulex Technologies, Inc. Spinal disc annulus reconstruction method and spinal disc annulus stent
US8163022B2 (en) 2008-10-14 2012-04-24 Anulex Technologies, Inc. Method and apparatus for the treatment of the intervertebral disc annulus
US8632590B2 (en) 1999-10-20 2014-01-21 Anulex Technologies, Inc. Apparatus and methods for the treatment of the intervertebral disc
US7951201B2 (en) 1999-10-20 2011-05-31 Anulex Technologies, Inc. Method and apparatus for the treatment of the intervertebral disc annulus
US7615076B2 (en) 1999-10-20 2009-11-10 Anulex Technologies, Inc. Method and apparatus for the treatment of the intervertebral disc annulus
US7935147B2 (en) 1999-10-20 2011-05-03 Anulex Technologies, Inc. Method and apparatus for enhanced delivery of treatment device to the intervertebral disc annulus
DE60033055D1 (en) 1999-12-06 2007-03-08 Warsaw Orthopedic Inc Intervertebral disc treatment device
US6805695B2 (en) 2000-04-04 2004-10-19 Spinalabs, Llc Devices and methods for annular repair of intervertebral discs
US6723335B1 (en) * 2000-04-07 2004-04-20 Jeffrey William Moehlenbruck Methods and compositions for treating intervertebral disc degeneration
FR2812186B1 (en) * 2000-07-25 2003-02-28 Spine Next Sa flexible connection piece for the stabilization of the spine
FR2812185B1 (en) 2000-07-25 2003-02-28 Spine Next Sa Rigid connecting piece for the stabilization of the spine
ES2358498T3 (en) 2000-10-24 2011-05-11 Cryolife, Inc. Bioprosthetic filling and methods, particularly for the in situ formation of intervertebral discs bioprosthesis.
US7226615B2 (en) * 2000-11-07 2007-06-05 Cryolife, Inc. Expandable foam-like biomaterials and methods
US20030069639A1 (en) * 2001-04-14 2003-04-10 Tom Sander Methods and compositions for repair or replacement of joints and soft tissues
JP4955388B2 (en) * 2001-08-31 2012-06-20 ユニヴァーシティー オブ サザン カリフォルニア Crosslinking reagents for the treatment of spinal disc disease
WO2003049669A3 (en) 2001-12-10 2004-03-11 Colbar R & D Ltd Methods, devices, and preparations for intervertebral disc treatment
WO2003066120A1 (en) * 2002-02-04 2003-08-14 Ferree Bret A Treating degenerative disc disease through transplantation of allograft disc
US6812211B2 (en) 2002-03-19 2004-11-02 Michael Andrew Slivka Method for nonsurgical treatment of the intervertebral disc and kit therefor
US7622562B2 (en) * 2002-06-26 2009-11-24 Zimmer Orthobiologics, Inc. Rapid isolation of osteoinductive protein mixtures from mammalian bone tissue
FR2842724B1 (en) 2002-07-23 2005-05-27 Spine Next Sa spinal fixation system
US20040054414A1 (en) 2002-09-18 2004-03-18 Trieu Hai H. Collagen-based materials and methods for augmenting intervertebral discs
JP2006515765A (en) * 2002-11-15 2006-06-08 エスディージーアイ・ホールディングス・インコーポレーテッド Collagen-based materials and methods for the treatment of synovial joint
US7744651B2 (en) * 2002-09-18 2010-06-29 Warsaw Orthopedic, Inc Compositions and methods for treating intervertebral discs with collagen-based materials
US20040186471A1 (en) * 2002-12-07 2004-09-23 Sdgi Holdings, Inc. Method and apparatus for intervertebral disc expansion
KR20050092795A (en) * 2003-01-31 2005-09-22 짐머 오르쏘바이올로직스 인코포레이티드 Hydrogel compositions comprisign nucleus pulposus tissue
CA2517108A1 (en) * 2003-02-25 2004-09-10 Tokai University Medium for stem cells in regeneration of intervertebral disc and regeneration of intervertebral disc using stem cells
US20040193274A1 (en) * 2003-03-28 2004-09-30 Trieu Hai H. Materials and methods for augmenting and/or repairing intervertebral discs
US7879102B2 (en) * 2003-09-30 2011-02-01 Depuy Acromed, Inc. Method for treatment of defects in the intervertebral disc
WO2005081870A3 (en) * 2004-02-20 2006-12-14 Iv H Davis Adkisson Intervertebral disc repair, methods and devices therefor
US20060275273A1 (en) * 2004-02-20 2006-12-07 Seyedin Mitchell S Intervertebral Disc Repair, Methods and Devices Therefor
WO2005107827A1 (en) * 2004-05-07 2005-11-17 Seikagaku Corporation Nucleus pulposus filler
FR2870718B1 (en) * 2004-05-25 2006-09-22 Spine Next Sa processing the complete degeneration of an intervertebral disc
US20070286881A1 (en) * 2005-07-14 2007-12-13 Brian Burkinshsw Method, composition and kit for treating degenerated disc disease and discogenic pain
US7393437B2 (en) * 2004-09-14 2008-07-01 The University Of Hong Kong Photochemically crosslinked collagen scaffolds and methods for their preparation
US8697139B2 (en) 2004-09-21 2014-04-15 Frank M. Phillips Method of intervertebral disc treatment using articular chondrocyte cells
US8206448B2 (en) 2004-10-29 2012-06-26 Spinal Restoration, Inc. Injection of fibrin sealant using reconstituted components in spinal applications
US7597687B2 (en) * 2004-10-29 2009-10-06 Spinal Restoration, Inc. Injection of fibrin sealant including an anesthetic in spinal applications
US20110213464A1 (en) * 2004-10-29 2011-09-01 Whitlock Steven I Injection of fibrin sealant in the absence of corticosteroids in spinal applications
CN100443064C (en) 2005-09-08 2008-12-17 吴忠仕;胡建国 Preparation process of biological valve-possessed duct for pulmonary artery vessel restoration or reconstruction
US20070073397A1 (en) * 2005-09-15 2007-03-29 Mckinley Laurence M Disc nucleus prosthesis and its method of insertion and revision
FR2890850B1 (en) 2005-09-20 2009-04-17 Abbott Spine Sa spinal fixation system
FR2890851B1 (en) * 2005-09-21 2008-06-20 Abbott Spine Sa Ancillary tensioning a flexible tie.
US20070213822A1 (en) * 2006-02-14 2007-09-13 Sdgi Holdings, Inc. Treatment of the vertebral column
CN101478934B (en) 2006-04-28 2012-08-08 香港大学 Bioengineered intervertebral discs and methods for their preparation
US20070258941A1 (en) * 2006-05-02 2007-11-08 Pfister Brian E Methods and compositions for remediation of disc herniation by modifying structure
US8118779B2 (en) * 2006-06-30 2012-02-21 Warsaw Orthopedic, Inc. Collagen delivery device
US8399619B2 (en) * 2006-06-30 2013-03-19 Warsaw Orthopedic, Inc. Injectable collagen material
US20080004431A1 (en) * 2006-06-30 2008-01-03 Warsaw Orthopedic Inc Method of manufacturing an injectable collagen material
US20080004703A1 (en) * 2006-06-30 2008-01-03 Warsaw Orthopedic, Inc. Method of treating a patient using a collagen material
US20080082170A1 (en) * 2006-09-29 2008-04-03 Peterman Marc M Apparatus and methods for surgical repair
US7824270B2 (en) * 2007-01-23 2010-11-02 C-Flex Bearing Co., Inc. Flexible coupling
US20080312694A1 (en) * 2007-06-15 2008-12-18 Peterman Marc M Dynamic stabilization rod for spinal implants and methods for manufacturing the same
US20090004455A1 (en) * 2007-06-27 2009-01-01 Philippe Gravagna Reinforced composite implant
EP2047813A1 (en) 2007-10-11 2009-04-15 Abbott Spine Bone fixing system and method of use
US8128635B2 (en) * 2007-10-23 2012-03-06 Zimmer Spine S.A.S. Bone fixation tensioning tool and method
US9308068B2 (en) * 2007-12-03 2016-04-12 Sofradim Production Implant for parastomal hernia
EP2111810B1 (en) * 2008-04-24 2011-07-06 Zimmer Spine System for stabilizing at least a portion of the spine
US9242026B2 (en) * 2008-06-27 2016-01-26 Sofradim Production Biosynthetic implant for soft tissue repair
US20100160968A1 (en) * 2008-12-19 2010-06-24 Abbott Spine Inc. Systems and methods for pedicle screw-based spine stabilization using flexible bands
US20100227697A1 (en) * 2009-03-04 2010-09-09 C-Flex Bearing Co., Inc. Flexible coupling
WO2010129692A1 (en) 2009-05-05 2010-11-11 Cornell University Composite tissue-engineered intervertebral disc with self-assembled annular alignment
US20110059178A1 (en) * 2009-09-08 2011-03-10 Musculoskeletal Transplant Foundation Inc. Tissue Engineered Meniscus Repair Composition
US20110060412A1 (en) * 2009-09-08 2011-03-10 Musculoskeletal Transplant Foundation Inc. Tissue Engineered Meniscus Repair Composition
US8652153B2 (en) 2010-01-11 2014-02-18 Anulex Technologies, Inc. Intervertebral disc annulus repair system and bone anchor delivery tool
US20140255507A9 (en) * 2010-10-14 2014-09-11 The Regents Of The University Of California Method for promoting the synthesis of collagen and proteoglycan in chondrocytes
US9358122B2 (en) 2011-01-07 2016-06-07 K2M, Inc. Interbody spacer
FR2972626B1 (en) 2011-03-16 2014-04-11 Sofradim Production Prosthesis comprising a knitted spacer fabric and openwork
FR2977790B1 (en) 2011-07-13 2013-07-19 Sofradim Production Prosthesis for umbilical hernia
US9526603B2 (en) 2011-09-30 2016-12-27 Covidien Lp Reversible stiffening of light weight mesh
FR2985271B1 (en) 2011-12-29 2014-01-24 Sofradim Production Tricot pins
FR2995779B1 (en) 2012-09-25 2015-09-25 Sofradim Production Prothese including a mesh and a consolidation means
FR2995788B1 (en) 2012-09-25 2014-09-26 Sofradim Production Hemostatic patch and method of preparation
FR2995778B1 (en) 2012-09-25 2015-06-26 Sofradim Production reinforcement prosthesis of the abdominal wall and method of manufacture
US9433404B2 (en) 2012-10-31 2016-09-06 Suture Concepts Inc. Method and apparatus for closing fissures in the annulus fibrosus
US9737294B2 (en) 2013-01-28 2017-08-22 Cartiva, Inc. Method and system for orthopedic repair
US20150290248A1 (en) * 2014-04-10 2015-10-15 Nanofiber Health, Inc. Fibrous component for health, performance, and aesthetic treatment
WO2017044570A1 (en) * 2015-09-08 2017-03-16 Clemson University Decellularized biomaterial and method for formation

Family Cites Families (151)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5831210B2 (en) 1973-04-09 1983-07-05 Takeda Chemical Industries Ltd
US4172128A (en) 1975-03-26 1979-10-23 Erhard Thiele Process of degrading and regenerating bone and tooth material and products
US4761471A (en) 1980-08-04 1988-08-02 The Regents Of The University Of California Bone morphogenetic protein composition
JPS57144756A (en) 1981-03-04 1982-09-07 Koken Kk Impermeable laminated film
US4619989A (en) 1981-05-05 1986-10-28 The Regents Of The University Of Cal. Bone morphogenetic protein composition
US4455256A (en) 1981-05-05 1984-06-19 The Regents Of The University Of California Bone morphogenetic protein
US4529590A (en) 1982-12-27 1985-07-16 Leveen Robert F Production of angiogenetic factor
US4801299A (en) 1983-06-10 1989-01-31 University Patents, Inc. Body implants of extracellular matrix and means and methods of making and using such implants
US4596574A (en) 1984-05-14 1986-06-24 The Regents Of The University Of California Biodegradable porous ceramic delivery system for bone morphogenetic protein
US4620327A (en) 1984-07-05 1986-11-04 Caplan Arnold I Process of adapting soluble bone protein for use in stimulating osteoinduction
JPH0678460B2 (en) 1985-05-01 1994-10-05 株式会社バイオマテリアル・ユニバース The porous transparent polyvinyl Al Yule gel
US4678470A (en) 1985-05-29 1987-07-07 American Hospital Supply Corporation Bone-grafting material
US4834757A (en) 1987-01-22 1989-05-30 Brantigan John W Prosthetic implant
US4774227A (en) 1986-02-14 1988-09-27 Collagen Corporation Collagen compositions for bone repair containing autogeneic marrow
US5902741A (en) 1986-04-18 1999-05-11 Advanced Tissue Sciences, Inc. Three-dimensional cartilage cultures
US4902296A (en) 1986-10-29 1990-02-20 The University Of Virginia Alumni Patents Foundation Use of demineralized bone matrix in the repair of segmental defects
US4743259A (en) 1986-10-29 1988-05-10 The University Of Virginia Alumni Patents Foundation Use of demineralized bone matrix in the repair of segmental defects
US4952404A (en) 1987-06-19 1990-08-28 President And Fellows Of Harvard College Promotion of healing of meniscal tissue
US5108438A (en) 1989-03-02 1992-04-28 Regen Corporation Prosthetic intervertebral disc
US5681353A (en) 1987-07-20 1997-10-28 Regen Biologics, Inc. Meniscal augmentation device
US5258043A (en) 1987-07-20 1993-11-02 Regen Corporation Method for making a prosthetic intervertebral disc
DE69031483T2 (en) * 1989-08-02 1998-02-05 Univ North Carolina A process for cross-linking collagen thereby generated product
US4772287A (en) 1987-08-20 1988-09-20 Cedar Surgical, Inc. Prosthetic disc and method of implanting
US4863732A (en) 1987-12-16 1989-09-05 Collagen Corporation Injectable composition for inductive bone repair
GB8804640D0 (en) 1988-02-27 1988-03-30 Gkn Crompton Fastening system for window &c
US5545229A (en) 1988-08-18 1996-08-13 University Of Medicine And Dentistry Of Nj Functional and biocompatible intervertebral disc spacer containing elastomeric material of varying hardness
US5510418A (en) 1988-11-21 1996-04-23 Collagen Corporation Glycosaminoglycan-synthetic polymer conjugates
US5356630A (en) 1989-02-22 1994-10-18 Massachusetts Institute Of Technology Delivery system for controlled release of bioactive factors
US5062845A (en) 1989-05-10 1991-11-05 Spine-Tech, Inc. Method of making an intervertebral reamer
US5100422A (en) 1989-05-26 1992-03-31 Impra, Inc. Blood vessel patch
US5259043A (en) 1989-10-10 1993-11-02 Unisys Corporation Filtering illumination for image lift
FR2659226B1 (en) 1990-03-07 1992-05-29 Jbs Sa Prothese for intervertebral discs and instruments of implementation.
US5342394A (en) 1990-05-16 1994-08-30 Olympus Optical Co., Ltd. Apparatus for blocking a vein branch and method of blocking a vein branch
DE69111021T2 (en) 1990-10-31 1996-01-04 Gendler El A flexible membrane made from organic bone matrix for repairing and restoring bone.
US6054122A (en) 1990-11-27 2000-04-25 The American National Red Cross Supplemented and unsupplemented tissue sealants, methods of their production and use
CA2071912C (en) 1990-11-30 2002-10-15 Hanne Bentz Use of a bone morphogenetic protein in synergistic combination with tgf-beta for bone repair
US5192326A (en) 1990-12-21 1993-03-09 Pfizer Hospital Products Group, Inc. Hydrogel bead intervertebral disc nucleus
US5047055A (en) 1990-12-21 1991-09-10 Pfizer Hospital Products Group, Inc. Hydrogel intervertebral disc nucleus
US5206023A (en) 1991-01-31 1993-04-27 Robert F. Shaw Method and compositions for the treatment and repair of defects or lesions in cartilage
US5171278A (en) 1991-02-22 1992-12-15 Madhavan Pisharodi Middle expandable intervertebral disk implants
US5390683A (en) 1991-02-22 1995-02-21 Pisharodi; Madhavan Spinal implantation methods utilizing a middle expandable implant
US5290763A (en) 1991-04-22 1994-03-01 Intermedics Orthopedics/Denver, Inc. Osteoinductive protein mixtures and purification processes
US5563124A (en) 1991-04-22 1996-10-08 Intermedics Orthopedics/ Denver, Inc. Osteogenic product and process
US6107004A (en) 1991-09-05 2000-08-22 Intra Therapeutics, Inc. Method for making a tubular stent for use in medical applications
US5741429A (en) 1991-09-05 1998-04-21 Cardia Catheter Company Flexible tubular device for use in medical applications
US6027863A (en) 1991-09-05 2000-02-22 Intratherapeutics, Inc. Method for manufacturing a tubular medical device
CA2117088A1 (en) 1991-09-05 1993-03-18 David R. Holmes Flexible tubular device for use in medical applications
US5270300A (en) 1991-09-06 1993-12-14 Robert Francis Shaw Methods and compositions for the treatment and repair of defects or lesions in cartilage or bone
US5304194A (en) 1991-10-02 1994-04-19 Target Therapeutics Vasoocclusion coil with attached fibrous element(s)
US5147374A (en) 1991-12-05 1992-09-15 Alfredo Fernandez Prosthetic mesh patch for hernia repair
DE4210334A1 (en) 1992-03-30 1993-10-07 Stoess & Co Gelatine Biodegradable, water-resistant polymeric material
US5413571A (en) 1992-07-16 1995-05-09 Sherwood Medical Company Device for sealing hemostatic incisions
US5800537A (en) 1992-08-07 1998-09-01 Tissue Engineering, Inc. Method and construct for producing graft tissue from an extracellular matrix
US5437288A (en) 1992-09-04 1995-08-01 Mayo Foundation For Medical Education And Research Flexible catheter guidewire
US5478739A (en) 1992-10-23 1995-12-26 Advanced Tissue Sciences, Inc. Three-dimensional stromal cell and tissue culture system
DE4306850C1 (en) 1993-03-01 1994-08-18 Ethicon Gmbh Implant, especially for sealing trocar insertion points
US5709854A (en) * 1993-04-30 1998-01-20 Massachusetts Institute Of Technology Tissue formation by injecting a cell-polymeric solution that gels in vivo
US5425772A (en) 1993-09-20 1995-06-20 Brantigan; John W. Prosthetic implant for intervertebral spinal fusion
US5370660A (en) 1993-11-01 1994-12-06 Cordis Corporation Apparatus and method for delivering a vessel plug into the body of a patient
US5514180A (en) 1994-01-14 1996-05-07 Heggeness; Michael H. Prosthetic intervertebral devices
US5556429A (en) 1994-05-06 1996-09-17 Advanced Bio Surfaces, Inc. Joint resurfacing system
US6187048B1 (en) 1994-05-24 2001-02-13 Surgical Dynamics, Inc. Intervertebral disc implant
US5681310A (en) 1994-07-20 1997-10-28 Yuan; Hansen A. Vertebral auxiliary fixation device having holding capability
DE69522060D1 (en) 1994-09-08 2001-09-13 Stryker Technologies Corp Spinal disc nucleus of hydrogel
US5707962A (en) 1994-09-28 1998-01-13 Gensci Regeneration Sciences Inc. Compositions with enhanced osteogenic potential, method for making the same and therapeutic uses thereof
US5916225A (en) 1994-09-29 1999-06-29 Surgical Sense, Inc. Hernia mesh patch
US5824093A (en) 1994-10-17 1998-10-20 Raymedica, Inc. Prosthetic spinal disc nucleus
US5562736A (en) 1994-10-17 1996-10-08 Raymedica, Inc. Method for surgical implantation of a prosthetic spinal disc nucleus
US5674296A (en) 1994-11-14 1997-10-07 Spinal Dynamics Corporation Human spinal disc prosthesis
US5733337A (en) 1995-04-07 1998-03-31 Organogenesis, Inc. Tissue repair fabric
US5902785A (en) 1995-06-06 1999-05-11 Genetics Institute, Inc. Cartilage induction by bone morphogenetic proteins
DE19525197A1 (en) 1995-07-11 1997-01-16 Hoechst Ag Granular detergent builder
DE59509135D1 (en) * 1995-10-11 2001-05-03 Sulzer Markets & Technology Ag Method for a photo-oxidative treatment of tissues containing collagen
ES2211895T5 (en) 1995-11-08 2009-10-29 Zimmer Gmbh intervertebral prothesis.
DE69631490D1 (en) 1995-11-09 2004-03-11 Univ Massachusetts Boston Restoration of the tissue surface with compositions of hydrogel-cell
EP2111876B1 (en) * 1995-12-18 2011-09-07 AngioDevice International GmbH Crosslinked polymer compositions and methods for their use
US5645597A (en) 1995-12-29 1997-07-08 Krapiva; Pavel I. Disc replacement method and apparatus
US5749894A (en) 1996-01-18 1998-05-12 Target Therapeutics, Inc. Aneurysm closure method
US5800550A (en) 1996-03-13 1998-09-01 Sertich; Mario M. Interbody fusion cage
US5964807A (en) * 1996-08-08 1999-10-12 Trustees Of The University Of Pennsylvania Compositions and methods for intervertebral disc reformation
US6007570A (en) 1996-08-13 1999-12-28 Oratec Interventions, Inc. Apparatus with functional element for performing function upon intervertebral discs
US5716416A (en) 1996-09-10 1998-02-10 Lin; Chih-I Artificial intervertebral disk and method for implanting the same
US5911752A (en) 1996-09-13 1999-06-15 Intratherapeutics, Inc. Method for collapsing a stent
WO1998020939A3 (en) * 1996-11-15 1998-09-03 Advanced Bio Surfaces Inc Biomaterial system for in situ tissue repair
US5827328A (en) 1996-11-22 1998-10-27 Buttermann; Glenn R. Intervertebral prosthetic device
US5776142A (en) 1996-12-19 1998-07-07 Medtronic, Inc. Controllable stent delivery system and method
US5879366A (en) 1996-12-20 1999-03-09 W.L. Gore & Associates, Inc. Self-expanding defect closure device and method of making and using
US5815904A (en) 1997-03-13 1998-10-06 Intratherapeutics, Inc. Method for making a stent
US5800549A (en) 1997-04-30 1998-09-01 Howmedica Inc. Method and apparatus for injecting an elastic spinal implant
DE69714035D1 (en) * 1997-08-14 2002-08-22 Sulzer Innotec Ag Composition and apparatus for the repair of cartilage tissue in vivo comprising nanocapsules with osteoinductive and / or chondroinductive factors
US6511958B1 (en) * 1997-08-14 2003-01-28 Sulzer Biologics, Inc. Compositions for regeneration and repair of cartilage lesions
US6080579A (en) * 1997-11-26 2000-06-27 Charlotte-Mecklenburg Hospital Authority Method for producing human intervertebral disc cells
US6033394A (en) 1997-12-05 2000-03-07 Intratherapeutics, Inc. Catheter support structure
US6110164A (en) 1997-12-05 2000-08-29 Intratherapeutics, Inc. Guideless catheter segment
US6273876B1 (en) 1997-12-05 2001-08-14 Intratherapeutics, Inc. Catheter segments having circumferential supports with axial projection
US5976174A (en) 1997-12-15 1999-11-02 Ruiz; Carlos E. Medical hole closure device and methods of use
US6132461A (en) 1998-03-27 2000-10-17 Intratherapeutics, Inc. Stent with dual support structure
US6132460A (en) 1998-03-27 2000-10-17 Intratherapeutics, Inc. Stent
US7087577B2 (en) * 1998-10-16 2006-08-08 Zimmer Orthobiologies, Inc. Method of promoting natural bypass
US6203732B1 (en) 1998-07-02 2001-03-20 Intra Therapeutics, Inc. Method for manufacturing intraluminal device
US6022343A (en) 1998-09-03 2000-02-08 Intratherapeutics, Inc. Bridged coil catheter support structure
GB9819882D0 (en) * 1998-09-11 1998-11-04 Tissue Science Lab Limited Injectable collagenous tissue compositions
US6290692B1 (en) 1998-11-03 2001-09-18 Daniel J. Klima Catheter support structure
US6264659B1 (en) 1999-02-22 2001-07-24 Anthony C. Ross Method of treating an intervertebral disk
US6206921B1 (en) 1999-02-22 2001-03-27 Peter A. Guagliano Method of replacing nucleus pulposus and repairing the intervertebral disk
US6183518B1 (en) 1999-02-22 2001-02-06 Anthony C. Ross Method of replacing nucleus pulposus and repairing the intervertebral disk
US6428576B1 (en) * 1999-04-16 2002-08-06 Endospine, Ltd. System for repairing inter-vertebral discs
US20030040800A1 (en) * 2000-04-26 2003-02-27 Li Lehmann K. Apparatus and method for replacing the nucleus pulposus of an intervertebral disc or for replacing an entire intervertebral disc
US6245107B1 (en) 1999-05-28 2001-06-12 Bret A. Ferree Methods and apparatus for treating disc herniation
EP1203074A4 (en) * 1999-06-29 2003-09-10 J Alexander Marchosky Compositions and methods for forming and strengthening bone
US6344058B1 (en) * 1999-08-13 2002-02-05 Bret A. Ferree Treating degenerative disc disease through transplantation of allograft disc and vertebral endplates
US6371990B1 (en) * 1999-10-08 2002-04-16 Bret A. Ferree Annulus fibrosis augmentation methods and apparatus
US6419702B1 (en) * 1999-08-13 2002-07-16 Bret A. Ferree Treating degenerative disc disease through transplantation of the nucleus pulposis
US20030004574A1 (en) * 1999-10-08 2003-01-02 Ferree Bret A. Disc and annulus augmentation using biologic tissue
US6719797B1 (en) * 1999-08-13 2004-04-13 Bret A. Ferree Nucleus augmentation with in situ formed hydrogels
US20030026788A1 (en) * 1999-10-08 2003-02-06 Ferree Bret A. Use of extracellular matrix tissue to preserve cultured cell phenotype
US6352557B1 (en) * 1999-08-13 2002-03-05 Bret A. Ferree Treating degenerative disc disease through transplantion of extracellular nucleus pulposus matrix and autograft nucleus pulposus cells
US6340369B1 (en) * 1999-08-13 2002-01-22 Bret A. Ferree Treating degenerative disc disease with harvested disc cells and analogues of the extracellular matrix
US6508839B1 (en) * 1999-08-18 2003-01-21 Intrinsic Orthopedics, Inc. Devices and methods of vertebral disc augmentation
WO2002054978A3 (en) * 1999-08-18 2002-11-07 Intrinsic Therapeutics, Inc. Devices and method for nucleus pulposus augmentation and retention
US20040024465A1 (en) * 1999-08-18 2004-02-05 Gregory Lambrecht Devices and method for augmenting a vertebral disc
US7998213B2 (en) * 1999-08-18 2011-08-16 Intrinsic Therapeutics, Inc. Intervertebral disc herniation repair
US6264695B1 (en) * 1999-09-30 2001-07-24 Replication Medical, Inc. Spinal nucleus implant
EP1229873B1 (en) * 1999-10-29 2004-01-07 Drexel University Associating hydrogels for nucleus pulposus replacement in intervertebral discs
JP4711520B2 (en) * 2000-03-21 2011-06-29 日本ケミカルリサーチ株式会社 Physiologically active peptide-containing powder
US6723335B1 (en) * 2000-04-07 2004-04-20 Jeffrey William Moehlenbruck Methods and compositions for treating intervertebral disc degeneration
US20040083001A1 (en) * 2000-06-29 2004-04-29 Rita Kandel Intervertebral disc
US20020032155A1 (en) * 2000-06-30 2002-03-14 Ferree Bret A. Method of treating disc herniation and disc degeneration with concentrated growth and differentiation factors
US20020045942A1 (en) * 2000-10-16 2002-04-18 Ham Michael J. Procedure for repairing damaged discs
ES2358498T3 (en) * 2000-10-24 2011-05-11 Cryolife, Inc. Bioprosthetic filling and methods, particularly for the in situ formation of intervertebral discs bioprosthesis.
CA2429168C (en) * 2000-11-15 2010-06-08 Bio Syntech Canada Inc. Method for restoring a damaged or degenerated intervertebral disc
US20030069639A1 (en) * 2001-04-14 2003-04-10 Tom Sander Methods and compositions for repair or replacement of joints and soft tissues
US7156877B2 (en) * 2001-06-29 2007-01-02 The Regents Of The University Of California Biodegradable/bioactive nucleus pulposus implant and method for treating degenerated intervertebral discs
EP1437989A2 (en) * 2001-08-27 2004-07-21 James C. Thomas, Jr. Expandable implant for partial disc replacement and reinforcement of a disc partially removed in a discectomy and for reduction and maintenance of alignment of cancellous bone fractures and methods and apparatuses for same.
WO2003049669A3 (en) * 2001-12-10 2004-03-11 Colbar R & D Ltd Methods, devices, and preparations for intervertebral disc treatment
US6812211B2 (en) * 2002-03-19 2004-11-02 Michael Andrew Slivka Method for nonsurgical treatment of the intervertebral disc and kit therefor
US6706068B2 (en) * 2002-04-23 2004-03-16 Bret A. Ferree Artificial disc replacements with natural kinematics
US20040054413A1 (en) * 2002-09-16 2004-03-18 Howmedica Osteonics Corp. Radiovisible hydrogel intervertebral disc nucleus
KR101095771B1 (en) * 2002-09-18 2011-12-21 워쏘우 오르쏘페딕 인코포레이티드 Natural tissue devices and methods of implantation
US20040054414A1 (en) * 2002-09-18 2004-03-18 Trieu Hai H. Collagen-based materials and methods for augmenting intervertebral discs
KR20050092795A (en) * 2003-01-31 2005-09-22 짐머 오르쏘바이올로직스 인코포레이티드 Hydrogel compositions comprisign nucleus pulposus tissue
US7309359B2 (en) * 2003-08-21 2007-12-18 Warsaw Orthopedic, Inc. Allogenic/xenogenic implants and methods for augmenting or repairing intervertebral discs
WO2005034781A1 (en) * 2003-09-29 2005-04-21 Promethean Surgical Devices Llc Devices and methods for spine repair
US20050071012A1 (en) * 2003-09-30 2005-03-31 Hassan Serhan Methods and devices to replace spinal disc nucleus pulposus
US20050113923A1 (en) * 2003-10-03 2005-05-26 David Acker Prosthetic spinal disc nucleus
US7651682B2 (en) * 2003-10-29 2010-01-26 Gentis, Inc. Polymerizable emulsions for tissue engineering
WO2005113032A3 (en) * 2004-05-21 2006-05-04 Alastair J T Clemow Replacement of nucleus pulposus using a hydrogel
US7789913B2 (en) * 2004-06-29 2010-09-07 Spine Wave, Inc. Methods for injecting a curable biomaterial into an intervertebral space
EP1788986A1 (en) * 2004-08-30 2007-05-30 Spineovations, Inc. Method of treating spinal internal disk derangement
US20070005140A1 (en) * 2005-06-29 2007-01-04 Kim Daniel H Fabrication and use of biocompatible materials for treating and repairing herniated spinal discs
US20070093905A1 (en) * 2005-10-21 2007-04-26 O'neil Michael J Degenerative disc regeneration techniques

Also Published As

Publication number Publication date Type
EP1272236A1 (en) 2003-01-08 application
CA2400826A1 (en) 2001-10-18 application
ES2263612T3 (en) 2006-12-16 grant
US20050002909A1 (en) 2005-01-06 application
EP1707225A2 (en) 2006-10-04 application
US6723335B1 (en) 2004-04-20 grant
US7556649B2 (en) 2009-07-07 grant
US20110256106A1 (en) 2011-10-20 application
EP1707225A3 (en) 2011-08-10 application
DE60120667D1 (en) 2006-07-27 grant
JP2003530364A (en) 2003-10-14 application
DE60120667T2 (en) 2007-05-31 grant
EP1272236B1 (en) 2006-06-14 grant
WO2001076654A1 (en) 2001-10-18 application
US20100021439A1 (en) 2010-01-28 application

Similar Documents

Publication Publication Date Title
Ruszczak Effect of collagen matrices on dermal wound healing
US6933326B1 (en) Particulate acellular tissue matrix
US6387693B2 (en) Method for producing cartilage tissue and implants for repairing enchondral and osteochondral defects as well as arrangement for carrying out the method
US7824701B2 (en) Biocompatible scaffold for ligament or tendon repair
US6863694B1 (en) Osteogenic implants derived from bone
US6180606B1 (en) Compositions with enhanced osteogenic potential, methods for making the same and uses thereof
Glowacki et al. Collagen scaffolds for tissue engineering
US6808585B2 (en) Osteogenic implants derived from bone
US6306169B1 (en) Tissue implant
US7875296B2 (en) Conformable tissue repair implant capable of injection delivery
US5326357A (en) Reconstituted cartridge tissue
US7468192B2 (en) Method for repair of cartilage lesions
US20050251268A1 (en) Cartilage allograft plug
US20020082698A1 (en) Method for treating a patient using a cultured connective tissue construct
US20050074481A1 (en) Device for regeneration of articular cartilage and other tissue
US20030039695A1 (en) Collagen carrier of therapeutic genetic material, and method
Lynn et al. Antigenicity and immunogenicity of collagen
US5563124A (en) Osteogenic product and process
US20050071012A1 (en) Methods and devices to replace spinal disc nucleus pulposus
US6241981B1 (en) Composition and method for repairing neurological tissue
US5904716A (en) Method for reconstituting cartilage tissue using demineralized bone and product thereof
US20040034427A1 (en) Bioartificial intervertebral disc
US20110091517A1 (en) Biocompatible scaffolds with tissue fragments
Wang et al. Characterization of matrix-induced osteogenesis in rat calvarial bone defects: II. Origins of bone-forming cells
US20100036503A1 (en) Composition for a Tissue Repair Implant and Methods of Making the Same

Legal Events

Date Code Title Description
EEER Examination request
MKLA Lapsed