WO2008121331A1

WO2008121331A1 - Materials and methods for treating nerve damage and promoting nerve repair and regeneration

Info

Publication number: WO2008121331A1
Application number: PCT/US2008/004073
Authority: WO
Inventors: Helen Marie Nugent; Yin Shan Ng; James Richard Birkhead; Desmond Anthony White
Original assignee: Pervasis Therapeutics, Inc.
Priority date: 2007-03-30
Filing date: 2008-03-28
Publication date: 2008-10-09

Abstract

Disclosed herein are materials and methods suitable for treating damaged or diseased nerves. Nerve sites can be treated by contacting a non-luminal surface of a nerve structure at or adjacent or in the vicinity of an area of damage or disease with an implantable material. The implantable material comprises a biocompatible matrix and cells and is in an amount effective to treat the nerve site. A composition comprising a biocompatible matrix and cells engrafted therein or thereon can be used to treat the nerve site. The composition can be a flexible planar material or a flowable composition.

Description

MATERIALS AND METHODS FOR TREATING NERVE DAMAGE AND PROMOTING NERVE REPAIR AND REGENERATION

Background of the Invention

[0001] Nerve and nervous system disorders include injuries, diseases, disorders or grafts that cause deviation from or interruption of the normal structure, function or connectivity of nerves and other components of the nervous system. Nerve and nervous system disorders can cause pain, discomfort or other problems, and many lead to serious medical conditions such as uncontrollable pain and paralysis.

[0002] Current treatments for injured, damaged or diseased nerves and other components of the nervous system are limited and often have adverse consequences. Treatment options vary with age, health, and the severity of the injury or disease. One objective of the present invention is to provide methods and materials for the treatment of injured, damaged or diseased nerves and to promote repair and regeneration of nerve tissue.

Summary of the Invention

[0003] The present invention exploits the discovery that injured, damaged or diseased nerves and other components of the nervous system can be treated effectively by administration of a cell-based therapy to a surface of a site of nerve injury, damage or disease. As disclosed herein, an implantable material comprising cells, preferably endothelial cells or cells having an endothelial-like phenotype, can be used to treat and manage injured, damaged or diseased nerves and cerebral aneurysms when the material is situated at or near the surface of an injured, damaged or diseased nerve fiber or bundle or near the surface of a cerebral aneurysm. This discovery permits the clinician to intervene in the treatment of an injured, damaged or diseased nerve and cerebral aneurysms for which there have heretofore been limited treatment options. [0004] One aspect of the present invention is a method of treating an injured, damaged or diseased nerve in an individual in need thereof, the method comprising contacting with an implantable material a surface of an injured, damaged, diseased or graft nerve structure at or adjacent to or in the vicinity of the site in need of treatment, wherein said implantable material comprises a biocompatible matrix and cells and further wherein said implantable material is in an amount effective to treat the injured, damaged or diseased nerve in said individual.

[0005] According to various embodiments, the biocompatible matrix can be a flexible planar material or a flowable composition and the cells can be endothelial, endothelial-like, non-endothelial cells, analogs thereof or co-cultures of endothelial cells and non-endothelial cells.

[0006] According to additional embodiments, the nerve is a peripheral nerve, a dorsal root or a spinal cord. The damage or disease can be a transected nerve, a partially transected nerve, a nerve graft, a crushed nerve, a pinched nerve, an inflamed nerve, an infection, an autoimmune disease, a dorsal root disorder, a peripheral nerve disorder, a demyelinating disorder such as multiple sclerosis, or a spinal cord disorder. According to one embodiment, the implantable material is applied to the exterior surface of the nerve or nerve graft.

[0007] According to various embodiments, the implantable material controls inflammation, controls fibroblast migration and proliferation, inhibits negative tissue remodeling, promotes positive tissue remodeling, promotes nerve fiber proliferation, promotes axon outgrowth, promotes neuronal connectivity, promotes myelin migration and proliferation, or promotes maturation and patency of nerve anastomosis. [0008] In another aspect, the invention is a composition suitable for the treatment or management of a nerve, the composition comprising a biocompatible matrix and cells, wherein the composition is in an amount effective to treat or manage the nerve.

[0009] According to various embodiments, the biocompatible matrix can be a flexible planar material or a flowable composition and the flowable composition can further comprise an attachment peptide and the cells are engrafted on or to the attachment peptide. In another embodiment, the cells can be endothelial, endothelial-like, non-endothelial cells or analogs thereof.

[0010] According to additional embodiments, the composition can further comprise a second therapeutic agent, an agent that inhibits infection, an anti- inflammatory agent, an agent that promotes axon outgrowth, for example, a nerve growth factor (NGF), or an attachment peptide.

[0011] In another aspect, the invention is a method of treating a cerebral aneurysm in an individual in need thereof. The method includes contacting with an implantable material a surface of a cerebral artery at or adjacent to or in the vicinity of the aneurysm site in need of treatment. The implantable material comprises a biocompatible matrix and cells and further wherein the implantable material is in an amount effective to treat the cerebral aneurysm in the individual. In a further aspect, the invention is a composition suitable for treatment or management of a cerebral aneurysm, the composition comprising a biocompatible matrix and cells, wherein the composition is in an amount effective to treat or manage the cerebral aneurysm.

[0012] In another aspect, the invention is a method of treating a nerve in an individual in need thereof. The method comprises applying an implantable material between one or more ends of a transected nerve. The implantable material comprises a biocompatible matrix and cells. The implantable material acts as a structural and biological support for nerve growth and the implantable material is provided in an amount effective to treat the nerve in said individual.

Brief Description of the Drawings

[0013] Figures IA and IB are representative cell growth curves according to an illustrative embodiment of the invention.

[0014] Figure 2 depicts the results of the PC 12 survival assay according to an illustrative embodiment of the invention. Detailed Description of the Invention

[0015] As explained herein, the invention is based on the discovery that a cell- based therapy can be used to treat, ameliorate, manage and/or reduce the effects of injured, damaged or diseased nerves, cerebral arteries or other components of the nervous system including neurons, myelin sheath, axons, peripheral nerves, dorsal root, spinal cord, the brain, and the neural circuits of these components. The cell- based therapy can also be used in conjunction with treatments currently in use to treat injury, damage or diseased nerves, such as nerve autografts or artificial grafts. The teachings presented below provide sufficient guidance to make and use the materials and methods of the present invention, and further provide sufficient guidance to identify suitable criteria and subjects for testing, measuring, and monitoring the performance of the materials and methods of the present invention.

[0016] When used in an effective amount, the cell-based therapy of the present invention, an implantable material comprising cells engrafted on, in and/or within a biocompatible matrix and having a preferred phenotype, produces factors positively associated with neuron, axon, and myelin regeneration, neuron and axon outgrowth, neuron survival, and neural connectivity. For example, when used in an effective amount, the cells of the implantable material, when engrafted in or within a biocompatible matrix and having a preferred phenotype, can produce quantifiable amounts of heparan sulfate (HS), heparan sulfate proteoglycans (HSPGs), nitric oxide (NO), transforming growth factor-beta (TGF-β), fibroblast growth factors (FGFs) including basic fibroblast growth factor (bFGF), matrix metalloproteinases (MMPs) and/or tissue inhibitors of matrix metalloproteinases (TIMPs).

[0017] For example, heparan sulfate proteoglycans and their associated heparan sulfate polymers are involved in neuron migration, axon outgrowth and guidance, and synapse formation. Additionally, transforming growth factor-beta (TGF-β) and basic fibroblast growth factor (bFGF) are involved in neuroprotection, neuroma formation and regulating collagen accumulation following injury or disease, resulting in axon sprouting and eventual nerve regeneration. Further, matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) and the maintenance of an appropriate balance of the two factors are involved in the processes of nerve regeneration and axon regrowth. Additionally, nitric oxide (NO) is involved in axon regeneration.

[0018] Accordingly, administration of an effective amount of the implantable material of the present invention can be used to treat, ameliorate, manage and/or reduce the effects of injured, damaged or diseased nerves, cerebral arteries, or other components of the nervous system by providing a targeted supply of therapeutic factors in vivo in an amount sufficient to induce and/or manage, for example, neuron, axon and/or myelin regeneration and/or connectivity.

[0019] Nerves can be damaged by physical injury or progressive disease. Exemplary causes of nerve damage include cuts or incisions resulting in damage to or severing of a nerve fiber, a nerve bundle, the myelin sheath surrounding a nerve or nerve bundle and/or an individual axon; swelling of surrounding tissue resulting in pressure on a nerve, also called a pinched nerve; autoimmune diseases targeting nerve fibers, the myelin sheath, or other component of the nervous system; infection; diabetes; and de-vascularization or other interruption of the supply of necessary nutrients to the nerve or other components of the nervous system. These injuries, alone or in combination, result in and/or result from more complex pathologies including for example, but not limited to, nerve transection or axotomy, peripheral nervous system disorders, demyelinating disorders, dorsal root disorder and/or spinal cord disorders.

[0020] Additionally, nerves and nerve bundles are often located adjacent to and/or traverse an adjacent path to arteries and veins, often traveling together in larger bundles of companion structures along the axes of the body. For example, the femoral nerve, femoral artery and femoral vein intersect at the femoral triangle and descend together into the leg. Accordingly, nerves are often in contact with the components of the vasculature, including its endothelial cells. Further, damage or injury to vascular structures during trauma, surgery or other interventions often results in concurrent damage or injury to adjacent nerve structures, resulting in unanticipated nerve damage. [0021] Transected Nerve Fibers: Injured or partially or completely transected nerve fibers or nerve bundles can be managed and repair promoted with the implantable material of the present invention. Transection of a nerve fiber, also called axotomy, results in degeneration of the axon distal to the site of injury, subsequent degeneration of the myelin sheath distal to the site of injury, and digestion of the sheath by proliferation Schwann cells. These distal changes are accompanied by proximal changes known collectively as retrograde degeneration and include but are not limited to retraction of the axon from the point of injury, chromatolysis and swelling of the neuron soma. Retrograde degeneration helps to prepare the injured neuron for subsequent regeneration. If regeneration occurs, new fibers sprout from the tips of the proximal axons as growth cones. After sprouting, the fibers reach the site of injury in about one day. However, fibrosis and collagen accumulation at the site of injury result in scar tissue formation that can slow or stop the growth of the new nerve fibers. If the new nerve fibers are able to penetrate the scar tissue, they will grow at a slow rate of about 0.25 mm per day. Once the new nerve fibers have past the site of scar tissue, they can continue to grow at a rate of approximately 4 mm per day. In the case of injured myelinated nerve bundles, the remaining endoneurial tube and Schwann cell bodies provide a guide for the regenerating nerve fibers. However, even unmyelinated peripheral nerve fibers are able to regenerate.

[0022] Central axons, often found near the spinal cord, are much less able to regenerate than peripheral axons. For example, following a spinal cord injury, regenerating nerve tract fibers often fail to cross the site of injury because of the development of dense scar tissue at the site of injury which forms a barrier to the advancement of the regenerating nerve fiber.

[0023] Nerve Grafts: When an injured or transected nerve fails to recover properly, a gap remains between the ends of the nerve and fibers at either end of the nerve may collect to form a painful lump called a neuroma. To attempt to repair the injured or transected nerve, a graft operation may be performed in which the neuroma, if present, is surgically removed, and an autograft (nerve or muscle) or artificial graft is sewn into the gap using microsurgical techniques. A nerve graft is typically obtained from the sural nerve, which is on the outer part of the leg. While this type of graft usually provides the best return of nerve function, it can cause loss of feeling in the area from which the donor nerve was obtained. In addition, the cut end of the donor nerve often forms a neuroma which may have to be surgically corrected.

[0024] A second type of autograft is a muscle graft. In this case, a small piece of muscle tissue is snap frozen to kill the tissue and sewn to either end of the transected nerve. Although the muscle tissue is dead, the structure remains as a guide and structural support to allow nerve fibers to grow across the gap. While fewer complications with the donor site occur when using a muscle graft (no loss of feeling or neuroma formation), the recovery of nerve function to the injured nerve is less than that obtained using nerve grafts. [0025] An artificial graft may also be used. These typically consist of a tubal structure made from plastic or foam, which may be seeded with Schwann cells and/or covered with substances that promote axonal growth and elongation such as extracellular matrix (ECM) components, nerve growth factor (NGF), and/or other neurotrophic agents. Artificial grafts have been used with some success, but only for short gap distances (approximately 3 cm or less).

[0026] Nerve Pain: Acute and chronic nerve pain resulting from injury and/or disease of pain receptors and nerve fibers can be managed or repair promoted with the implantable material of the present invention. Acute pain, which frequently occurs in response to tissue injury, results from activation of peripheral pain receptors (nociceptors) and their specific sensory nerve fibers (A delta fibers and C fibers). Chronic pain, which often relates to ongoing tissue injury, is thought to be caused by persistent activation of the peripheral pain receptor fibers. Chronic pain may also result from neuropathic pain, pain caused by damage or dysfunction of the peripheral or central nervous system. Nerve pain may also result from nerve edema, or the buildup of endoneurial fluid pressure following compression or injury to the nerve structure. Nerve edema often presents as local compressive effects and associated pain that persist following a compression or injury to the nerve. Nerve edema also can affect the nerve microenvironment, resulting in chronic pain or further nerve injury. [0027] Nociceptive pain often results from injury or disease and may be somatic or visceral. Stimulation of somatic pain receptors, located in the skin, subcutaneous tissues, fascia, other connective tissues, periosteum, endosteum, and joint capsules, produces sharp or dull localized pain. Stimulation of visceral pain receptors, located in most viscera and the surrounding connective tissue, may result in a localized, deep aching or cramping, which may be referred to remote cutaneous sites, in the case of injury of a hollow organ, or more localized and sharp pain, in the case of injury of organ capsules or other deep connective tissues. Pain fibers enter the spinal cord at the dorsal root ganglia, travel up the lateral columns to the thalamus and then to the cerebral cortex. The pain signal is modulated along this pathway by excitatory and inhibitory nerve impulses and various neurochemical mediators.

[0028] Peripheral Nerve Disorders: Peripheral nervous system disorders can be managed and repair promoted with the implantable material of the present invention. Peripheral nervous system disorders often result from damage to or dysfunction of the nerve cell body, myelin sheath, axons, or neuromuscular junction. Peripheral nervous system disorders can be genetic or acquired in conjunction with toxic, metabolic, traumatic, infectious, or inflammatory conditions. Peripheral neuropathies may affect one nerve (mononeuropathy), several discrete nerves (multiple mononeuropathy, or mononeuritis multiplex), or multiple nerves diffusely (polyneuropathy). Some peripheral nervous system disorders involve a plexus (plexopathy) or nerve root (radiculopathy) and may involve more than one site.

[0029] Because sensory and motor cell bodies have different compositions in different locations, a nerve cell body disorder typically affects either the sensory or motor component but rarely both. Damage to the myelin sheath, or demyelination, slows nerve conduction and affects predominantly heavily myelinated fibers. Demyelination causes large-fiber sensory dysfunction (buzzing and tingling sensations), motor weakness, and diminished reflexes. Acquired demyelinating polyneuropathy often presents with profound motor weakness and minimal atrophy.

[0030] Because the vasa nervorum supplying a nerve do not reach the center of the nerve, the centrally located fascicles of the nerve are most vulnerable to vascular disorders, for example, vasculitis and ischemia. These disorders result in small-fiber sensory dysfunction, typified by sharp pain and burning sensations, motor weakness proportional to atrophy, and reflex abnormalities. Most often affecting the distal portion of a limb, initial peripheral nervous system disorder deficits tend to be asymmetric because the vasculitic or ischemic process is random. However, multiple infarcts may later coalesce, causing symmetric deficits, or multiple mononeuropathy. Toxic-metabolic or genetic disorders usually begin symmetrically whereas immune-mediated processes may be symmetric or, early in rapidly evolving processes, asymmetric.

[0031] Damage to the axon transport system for cellular constituents, especially microtubules and microfilaments, causes significant axon dysfunction. The smaller fibers and the most distal part of the nerve are affected first in part because they have greater metabolic requirements. Following initial injury, axonal degeneration slowly ascends, producing a distal-to-proximal pattern of symptoms, for example, stocking-glove sensory loss and weakness.

[0032] Peripheral nervous system disorders further include, but are not limited to, cervical spondylosis, disorders of neuromuscular transmission, Guillain-Barre syndrome (GBS), hereditary neuropathies, motor neuron disorders, myasthenia gravis, nerve root disorders, peripheral neuropathy, plexus disorders, spinal muscular atrophies, and thoracic outlet compression syndromes.

[0033] Demyelinatinp Disorders: Demyelinating disorders, for example, multiple sclerosis, can be managed and repair promoted with the implantable material of the present invention. Demyelination of peripheral nerve fibers may lead to slowing or failure of spike conduction, hyperexcitability, and interaction between nerve fibers. Slowed conduction occurs only at the site of demyelination, whereas the rest of the fiber has a normal conduction velocity. Unequal changes in conduction velocities in different fibers may contribute to altered sensation if the sensation requires the synchronous activity of many fibers in the nerve. Either spontaneous activity due to hyperexcitability or the cross-excitation of one demyelinated fiber by another could contribute to paresthesias, in which false peripheral events are signaled by inappropriately active nerves. [0034] Multiple sclerosis is characterized by patches of demyelination in the brain and spinal cord. Common symptoms include visual and oculomotor abnormalities, paresthesias, weakness, spasticity, urinary dysfunction, and mild cognitive impairment. Diagnosis is often made by evidence of a history of remissions and exacerbations in addition to objective demonstration of at least two separate neurological abnormalities by clinical signs or test results, MRI lesions, or other criteria, depending on symptoms. Current treatments for multiple sclerosis include corticosteroids for acute exacerbations, immunomodulatory drugs to prevent exacerbations, and supportive measures.

[0035] Multiple sclerosis is characterized by localized areas of demyelination and plaque formation, destruction of oligodendroglia, perivascular inflammation, and chemical changes in lipid and protein constituents of myelin in and around the plaques. Although axonal damage is possible, cell bodies and axons tend to be relatively preserved. Fibrous gliosis can develop in plaques disseminated throughout the central nervous system and are primarily found in white matter and particularly in the lateral and posterior columns, optic nerves, and periventricular areas. Tracts in the midbrain, pons, and cerebellum may also be affected. Gray matter in the cerebrum and spinal cord can be affected but often to a much lesser degree than white matter.

[0036] Multiple sclerosis is characterized by varied central nervous system deficits and by remissions and recurring exacerbations. Exacerbations average about three per year but frequency varies greatly. The most common initial symptoms of multiple sclerosis are paresthesias in one or more extremities, in the trunk, or on one side of the face; weakness or clumsiness of a leg or hand; and visual disturbances, for example, partial loss of vision and pain in one eye due to retrobulbar optic neuritis, diplopia due to ocular palsy, and scotomas. Other common early symptoms of multiple sclerosis include slight stiffness or unusual fatigability of a limb, minor gait disturbances, difficulty with bladder control, vertigo, and mild affective disturbances; all usually indicate scattered central nervous system involvement and may be subtle. Excess heat, for example, during warm weather, a hot bath, or fever, may temporarily exacerbate symptoms. [0037] Mild cognitive impairment is common in multiple sclerosis patients as well as apathy, poor judgment, or inattention. Affective disturbances, including emotional lability, euphoria, or, most commonly, depression, are also common. Depression may be reactive or partly due to cerebral lesions of multiple sclerosis and a few patients have seizures.

[0038] Dorsal Root Disorders: Dorsal root injuries and disorders can be managed and repair promoted with the implantable material of the present invention. Peripheral afferent fibers enter the spinal cord via the dorsal root ganglia, which are collections of pseudounipolar neurons lying just outside the spinal cord. The distal axons of dorsal root ganglion cells are peripheral nerve fibers. The proximal axons, called dorsal root fibers, enter the spinal cord. Like peripheral nerves, dorsal roots can become injured or irritated due to compression, transection or disease. The effects of dorsal root injuries are generally similar to those produced by peripheral nerve injuries, but can differ in their severity and distribution.

[0039] A common form of dorsal root injury is compression from herniation of an intervertebral disc. This type of injury can produce irritation of the dorsal root, resulting in pain over all or part of the affected mytome, sclerotome, or dermatome. Irritation can also produce paresthesias or hyperesthetic regions. If several dorsal roots receive sufficient damage, segmental anesthesia can result. Dorsal root injuries can also be caused by traction, inflammatory processes and diseases and ischemia. [0040] Spinal Cord Disorders: Spinal cord disorders can be managed and repair promoted with the implantable material of the present invention. The majority of proximal axons from the dorsal root ganglia enter the dorsal horn of the spinal cord via the dorsal roots and contact secondary neurons within the same segment of the spinal cord, ascend or descend a few spinal segments before contacting secondary neurons and/or enter a major ascending spinal tract. Each of these dorsal root fibers has a characteristic distribution within a spinal cord segment. As dorsal root fibers enter the spinal cord, they divide roughly into a lateral aggregate of small, unmyelinated fibers and a more medial aggregate of larger myelinated fibers. This segmentation is the beginning of a sorting process that separates the fibers by size, and therefore by modality, since small fibers are largely nociceptive and thermoceptive, whereas the large fibers convey discriminative touch and proprioceptive information.

[0041] Spinal cord disorders can cause permanent severe neurological disability. However, for some patients, such disability can be avoided or minimized if evaluation and treatment are rapid. Spinal cord disorders include arteriovenous malformations, infections including bacterial, fungal, TB and syphilis, which can cause tabes dorsalis, multiple sclerosis, spondylitic myelopathy, trauma, vitamin Bn deficiency, which causes subacute combined degeneration, syrinx, transverse myelopathy, spinal cord compression, and spinal cord tumors. Specific cord syndromes include transverse sensorimotor myelopathy, Brown-Sequard syndrome, central cord syndrome, anterior cord syndrome, and conus medullaris syndrome.

[0042] Cerebral Aneurysm: Cerebral aneurysms can be managed and repair promoted with the implantable material of the present invention. An aneurysm is a bulge or dilation in the wall of an artery, for example, the cerebral artery supplying blood to the brain. The bulge usually occurs in a weak area of the artery's wall. The pressure of blood inside the artery forces the weak area to balloon outward. If untreated, an aneurysm may rupture, resulting in internal bleeding. Aneurysms may be round (saccular) although most aneurysms are tubelike (fusiform).

[0043] The most common cause of aortic aneurysms is atherosclerosis, which weakens the wall of the aorta. Less common causes include injuries, inflammatory diseases of the aorta called aortitis, hereditary connective-tissue disorders such as Marfan syndrome, and some infectious diseases such as syphilis. In older people, almost all aneurysms are associated with atherosclerosis. High blood pressure, which is common among older people, and cigarette smoking increase the risk of an aneurysm.

[0044] A blood clot or thrombus often develops in the aneurysm because blood flow inside the aneurysm is sluggish. The clot may extend along the entire wall of the aneurysm. A blood clot may break loose becoming an embolus, travel through the bloodstream, and block arteries. Occasionally, calcium is gradually deposited in the wall of an aneurysm. [0045] Rupture of a cerebral aneurysm may cause bleeding into the brain tissue, also called an intracerebral hemorrhage, resulting in a stroke. Because cerebral aneurysms are near the brain and are usually small, their diagnosis and treatment differ from those of other aneurysms. Infected aneurysms of the cerebral arteries are particularly dangerous, making early treatment important. Treatment of cerebral aneurysms often involves surgical repair.

[0046] Diagnosis: Nerve and cerebral artery injuries, damage and disease can be identified using neurological diagnostic procedures know to those of skill in the field of neurology. Preliminary diagnostic procedures include a patient history and neurologic examination, including examination of motor system, muscle strength, gait, stance and coordination, sensation, reflexes and autonomic nervous system response. Additional diagnostic procedures include lumbar puncture, also known as a spinal tap, computed tomography (CT), magnetic resonance imaging (MRI), duplex Doppler ultrasonography, myelography, measurement of evoked responses (potentials) and electromyography and nerve conduction velocity studies.

[0047] The materials and methods of the present invention can be used in connection with any of the above-described injuries, damage and diseases, or numerous other nerve or cerebral artery interventions. In addition, the materials and methods of the present invention can be used in connection with any nerve or other surgical intervention resulting in damage to a nerve or other component of the nervous system and requiring surgery to improve surgical success and promote healing. Exemplary surgical interventions include, but are not limited to, repair by partial suture or complete anastomosis of a transected nerve or a cerebral aneurysm. The materials and methods of the present invention can be used in conjunction with these or other surgeries to increase effectiveness and promote healing.

Implantable Material

[0048] General Considerations: The implantable material of the present invention comprises cells engrafted on, in and/or within a biocompatible matrix. Engrafted means securedly attached via cell to cell and/or cell to matrix interactions such that the cells meet the functional or phenotypical criteria set forth herein and withstand the rigors of the preparatory manipulations disclosed herein. As explained elsewhere herein, an operative embodiment of implantable material comprises a population of cells associated with a supporting substratum, preferably a differentiated cell population and/or a near-confluent, confluent or post-confluent cell population, having a preferred functionality and/or phenotype.

[0049] Complex substrate specific interactions regulate the intercellular morphology and secretion of the cells and, accordingly, also regulate the functionality and phenotype of the cells associated with the supporting substratum. Cells associated with certain preferred biocompatible matrices, contemplated herein, may grow and conform to the architecture and surface of the local struts of matrix pores with less straining as they mold to the matrix. Also, the individual cells of a population of cells associated with a matrix retain distinct morphology and secretory ability even without complete contiguity between the cells. Further, cells associated with a biocompatible matrix may not exhibit planar restraint, as compared to similar cells grow as a monolayer on a tissue culture plate.

[0050] It is understood that embodiments of implantable material likely shed cells during preparatory manipulations and/or that certain cells are not as securely attached as are other cells. All that is required is that implantable material comprises cells associated with a supporting substratum that meet the functional or phenotypical criteria set forth herein.

[0051] That is, interaction between the cells and the matrix during the various phases of the cells' growth cycle can influence the cells' phenotype, with the preferred inhibitory phenotype described elsewhere herein correlating with quiescent cells (i.e., cells which are not in an exponential growth cycle). As explained elsewhere herein, it is understood that, while a quiescent cell typifies a population of cells which are near-confluent, confluent or post-confluent, the inhibitory phenotype associated with such a cell can be replicated by manipulating or influencing the interaction between a cell and a matrix so as to render a cell quiescent-like.

[0052] The implantable material of the present invention was developed on the principals of tissue engineering and represents a novel approach to addressing the above-described clinical needs. The implantable material of the present invention is unique in that the viable cells engrafted on, in and/or within the biocompatible matrix are able to supply to the nerve multiple cell-based products in physiological proportions under physiological feed-back control. As described elsewhere herein, the cells suitable for use with the implantable material include endothelial, endothelial-like, non-endothelial cells, analogs thereof or co-cultures of any of the foregoing and another cells type. Local delivery of multiple compounds by these cells in a physiologically-dynamic dosing provide more effective regulation of the processes responsible for maintaining functional nerve structures and diminishing the clinical sequel associated with nerve injury, damage or disease.

[0053] The implantable material of the present invention, when wrapped, deposited or otherwise contacted with the surface of a injured, damaged or diseased nerve site serves to reestablish homeostasis. That is, the implantable material of the present invention can provide an environment which mimics supportive physiology and is conducive to manage and/or promote healing a site of nerve injury, damage or disease.

[0054] For purposes of the present invention, contacting means directly or indirectly interacting with an exterior surface of a nerve structure or between two or more transected nerve endings, as defined elsewhere herein. In the case of certain preferred embodiments, actual physical contact is not required for effectiveness. In other embodiments, actual physical contact is preferred. AU that is required to practice the present invention is exterior deposition of an implantable material at, adjacent to or in the vicinity of an injured, diseased or damaged nerve site in an amount effective to treat the injured or diseased site. In the case of certain diseases or injuries, a diseased or injured site can clinically manifest on an interior surface. In the case of other diseases or injuries, a diseased or injured site can clinically manifest on an exterior surface of the structure. In some diseases or injuries, a diseased or injured site can clinically manifest on both an interior surface and an exterior surface of the structure. The present invention is effective to treat any of the foregoing clinical manifestations. [0055] For example, endothelial cells can release a wide variety of agents that in combination can inhibit or mitigate adverse physiological conditions associated with acute complications due to injury, damage or disease to nerves. As exemplified herein, a composition and method of use that recapitulates normal physiology and dosing is useful to treat and manage nerve healing. Typically, treatment includes placing the implantable material of the present invention at, adjacent to or in the vicinity of the injured, damaged or diseased nerve. When wrapped, wrapped around, deposited, or otherwise contacting a nerve structure, the cells of the implantable material can provide growth regulatory compounds to the nerve structure, for example within the nerve fiber or nerve bundle. It is contemplated that, while outside the nerve structure, the implantable material of the present invention comprising a biocompatible matrix or particle with engrafted cells provides a continuous supply of multiple regulatory and therapeutic compounds from the engrafted cells to the nerve structure.

[0056] Cell Source: As described herein, the implantable material of the present invention comprises cells. Cells can be allogeneic, xenogeneic or autologous. In certain embodiments, a source of living cells can be derived from a suitable donor. In certain other embodiments, a source of cells can be derived from a cadaver or from a cell bank.

[0057] In one currently preferred embodiment, cells are endothelial cells. In a particularly preferred embodiment, such endothelial cells are obtained from vascular tissue, preferably but not limited to arterial tissue. As exemplified below, one type of vascular endothelial cell suitable for use is an aortic endothelial cell. Another type of vascular endothelial cell suitable for use is umbilical cord vein endothelial cells. And, another type of vascular endothelial cell suitable for use is coronary artery endothelial cells. Yet another type of vascular endothelial cell suitable for use is saphenous vein endothelial cells. Yet other types of vascular endothelial cells suitable for use with the present invention include pulmonary artery endothelial cells and iliac artery endothelial cells.

[0058] In another currently preferred embodiment, suitable endothelial cells can be obtained from non-vascular tissue. Non-vascular tissue can be derived from any anatomical structure or can be derived from any non-vascular tissue or organ. Non-vascular tissue can be derived from any nerve or other tissue type. Exemplary anatomical structures include structures of the vascular system, the renal system, the reproductive system, the genitourinary system, the gastrointestinal system, the pulmonary system, the respiratory system and the ventricular system of the brain and spinal cord. [0059] In another embodiment, endothelial cells can be derived from endothelial progenitor cells or stem cells. In yet another embodiment, endothelial cells can be derived from neural progenitor cells or stem cells. In still another embodiment, endothelial cells can be derived from progenitor cells or stem cells generally, hi other preferred embodiments, cells can be non-endothelial cells that are allogeneic, xenogeneic or autologous and can be derived from vascular, neural or other tissue or organ. Cells can be selected on the basis of their tissue source and/or their immunogenicity. Exemplary non-endothelial cells include epithelial cells, neural cells, astrocytes, keratinacytes, Schwann cells, glial cells, secretory cells, smooth muscle cells, fibroblasts, stem cells, nerve stem cells, endothelial progenitor cells, cardiomyocytes, secretory and ciliated cells. The present invention also contemplates any of the foregoing which are genetically altered, modified or engineered.

[0060] In a further embodiment, two or more types of cells are co-cultured to prepare the present composition. For example, a first cell can be introduced into the biocompatible implantable material and cultured until confluent. The first cell type can include, for example, epithelial cells, neural cells, astrocytes, keratinacytes, Schwann cells, glial cells, secretory cells, smooth muscle cells, fibroblasts, stem cells, nerve stem cells, endothelial progenitor cells, a combination of smooth muscle cells and fibroblasts, any other desired cell type or a combination of desired cell types suitable to create an environment conducive to growth of the second cell type. Once the first cell type has reached confluence, a second cell type is seeded on top of the first confluent cell type in, on or within the biocompatible matrix and cultured until both the first cell type and second cell type have reached confluence. The second cell type may include, for example, epithelial cells, neural cells, astrocytes, keratinacytes, Schwann cells, glial cells, secretory cells, smooth muscle cells, fibroblasts, stem cells, nerve stem cells, endothelial cells, endothelial progenitor cells, or any other desired cell type or combination of cell types. It is contemplated that the first and second cell types can be introduced step wise, or as a single mixture. It is also contemplated that cell density can be modified to alter the ratio of the first cell type to the second cell type.

[0061] To prevent over-proliferation of smooth muscle cells or another cell type prone to excessive proliferation, the culture procedure and timing can be modified. For example, following confluence of the first cell type, the culture can be coated with an attachment factor suitable for the second cell type prior to introduction of the second cell type. Exemplary attachment factors include coating the culture with gelatin to improve attachment of endothelial cells. According to another embodiment, heparin can be added to the culture media during culture of the second cell type to reduce the proliferation of the first cell type and to optimize the desired first cell type to second cell type ratio. For example, after an initial growth of smooth muscle cells, heparin can be administered to control smooth muscle cell growth to achieve a greater ratio of endothelial cells to smooth muscle cells. [0062] In a preferred embodiment, a co-culture is created by first seeding a biocompatible implantable material with neural cells to create nerve fiber or nerve bundle structures, for example, but not limited to, structures that mimic the size and/or shape of the nerve structure. Once the neural cells have reached confluence, Schwann cells, astrocytes, keratinacytes or endothelial cells are seeded on top of the cultured neural cells on the implantable material to create a simulated structure.

[0063] All that is required of the cells of the present composition is that they exhibit one or more preferred phenotypes or functional properties. As described earlier herein, the present invention is based on the discovery that a cell having a readily identifiable phenotype when associated with a preferred matrix (described elsewhere herein) can facilitate, restore and/or otherwise modulate cell physiology and/or nerve homeostasis associated with the treatment of nerve disorders generally.

[0064] For purposes of the present invention, one such preferred, readily identifiable phenotype typical of cells of the present invention is an ability to inhibit or otherwise interfere with smooth muscle cell proliferation as measured by the in vitro assays described below. This is referred to herein as the inhibitory phenotype. [0065] One other readily identifiable phenotype exhibited by cells of the present composition is that they are able to control fibroblast proliferation and/or migration. Fibroblast activity can be determined using an in vitro fibroblast migration assay, described below. [0066] Another readily identifiable phenotype exhibited by cells of the present composition is that they are anti-thrombotic or are able to inhibit platelet adhesion and aggregation. Anti-thrombotic activity can be determined using an in vitro heparan sulfate assay and/or an in vitro platelet aggregation assay, described below.

[0067] A further readily identifiable phenotype exhibited by cells of the present composition is that they are able to regulate survival and differentiation of neuronal cells. Neuronal cell survival and differentiation can be determined using an in vitro PC 12 cell survival assay and/or an in vitro PC 12 cell differentiation assay, described below.

[0068] A further readily identifiable phenotype exhibited by cells of the present composition is the ability to restore the proteolytic balance, the MMP-TIMP balance, the ability to decrease expression of MMPs relative to the expression of TIMPs, or the ability to increase expression of TIMPs relative to the expression of MMPs. Proteolytic balance activity can be determined using an in vitro TIMP assay and/or an in vitro MMP assay described below. [0069] In a typical operative embodiment of the present invention, cells need not exhibit more than one of the foregoing phenotypes. In certain embodiments, cells can exhibit more than one of the foregoing phenotypes.

[0070] While the foregoing phenotypes each typify a functional endothelial cell, such as but not limited to a vascular endothelial cell, a non-endothelial cell exhibiting such a phenotype(s) is considered endothelial-like for purposes of the present invention and thus suitable for use with the present invention. Cells that are endothelial-like are also referred to herein as functional analogs of endothelial cells; or functional mimics of endothelial cells. Thus, by way of example only, cells suitable for use with the materials and methods disclosed herein also include stem cells or progenitor cells that give rise to endothelial-like cells; cells that are non- endothelial cells in origin yet perform functionally like an endothelial cell using the parameters set forth herein; cells of any origin which are engineered or otherwise modified to have endothelial-like functionality using the parameters set forth herein.

[0071] Typically, cells of the present invention exhibit one or more of the aforementioned functionalities and/or phenotypes when present and associated with a supporting substratum, such as the biocompatible matrices described herein. It is understood that individual cells attached to a matrix and/or interacting with a specific supporting substratum exhibit an altered expression of functional molecules, resulting in a preferred functionality or phenotype when the cells are associated with a matrix or supporting substratum that is absent when the cells are not associated with a supporting substratum.

[0072] According to one embodiment, the cells exhibit a preferred phenotype when the basal layer of the cell is associated with a supporting substratum. According to another embodiment, the cells exhibit a preferred phenotype when present in confluent, near confluent or post-confluent populations. As will be appreciated by one of ordinary skill in the art, populations of cells, for example, substrate adherent cells, and confluent, near confluent and post-confluent populations of cells, are identifiable readily by a variety of techniques, the most common and widely accepted of which is direct microscopic examination. Others include evaluation of cell number per surface area using standard cell counting techniques such as but not limited to a hemacytometer or coulter counter.

[0073] Additionally, for purposes of the present invention, endothelial-like cells include but are not limited to cells which emulate or mimic functionally and phenotypically the preferred populations of cells set forth herein, including, for example, differentiated endothelial cells and confluent, near confluent or post- confluent endothelial cells, as measured by the parameters set forth herein.

[0074] Thus, using the detailed description and guidance set forth below, the practitioner of ordinary skill in the art will appreciate how to make, use, test and identify operative embodiments of the implantable material disclosed herein. That is, the teachings provided herein disclose all that is necessary to make and use the present invention's implantable materials. And further, the teachings provided herein disclose all that is necessary to identify, make and use operatively equivalent cell-containing compositions. At bottom, all that is required is that equivalent cell- containing compositions are effective to treat, manage, modulate and/or ameliorate a nerve site in accordance with the methods disclosed herein. As will be appreciated by the skilled practitioner, equivalent embodiments of the present composition can be identified using only routine experimentation together with the teachings provided herein.

[0075] In certain preferred embodiments, endothelial cells used in the implantable material of the present invention are isolated from the aorta of human cadaver donors. Each lot of cells is derived from a single donor or from multiple donors, tested extensively for endothelial cell purity, biological function, the presence of bacteria, fungi, known human pathogens and other adventitious agents. The cells are cryopreserved and banked using well-known techniques for later expansion in culture for subsequent formulation in biocompatible implantable materials.

[0076] Cell Preparation: As stated above, suitable cells can be obtained from a variety of tissue types and cell types. In certain preferred embodiments, human aortic endothelial cells used in the implantable material are isolated from the aorta of cadaver donors. In other embodiments, porcine aortic endothelial cells are isolated from normal porcine aorta by a similar procedure used to isolate human aortic endothelial cells. Each lot of cells can be derived from a single donor or from multiple donors, tested extensively for endothelial cell viability, purity, biological function, the presence of mycoplasma, bacteria, fungi, yeast, known human pathogens and other adventitious agents. The cells are further expanded, characterized and cryopreserved to form a working cell bank at the third to sixth passage using well-known techniques for later expansion in culture and for subsequent formulation in biocompatible implantable material.

[0077] The human or porcine aortic endothelial cells are prepared in T-75 flasks pre-treated by the addition of approximately 15 ml of endothelial cell growth media per flask. Human aortic endothelial cells are prepared in Endothelial Growth Media (EGM-2, Lonza Group Ltd, Basel, Switzerland). EGM-2 consists of Endothelial Cell Basal Media (EBM-2, Lonza Group Ltd, Basel, Switzerland) supplemented with EGM-2 singlequots, which contain 2% FBS. Porcine cells are prepared in EBM-2 supplemented with 5% FBS and 50 μg/ml gentamicin. The flasks are placed in an incubator maintained at approximately 37°C and 5% CO₂ / 95% air, 90% humidity for a minimum of 30 minutes. One or two vials of the cells are removed from the -160⁰C to -140⁰C freezer and thawed at approximately 37°C. Each vial of thawed cells is seeded into two T-75 flasks at a density of approximately 3 x 10³ cells per cm², preferably, but no less than 1.0 x 10³ and no more than 7.0 x 10³; and the flasks containing the cells are returned to the incubator. After about 8-24 hours, the spent media is removed and replaced with fresh media. The media is changed every two to three days, thereafter, until the cells reach approximately 85-100% confluence preferably, but no less than 60% and no more than 100%. When the implantable material is intended for clinical application, only antibiotic-free media is used in the post-thaw culture of human aortic endothelial cells and manufacture of the implantable material of the present invention.

[0078] The endothelial cell growth media is then removed, and the monolayer of cells is rinsed with 10 ml of HEPES buffered saline (HEPES). The HEPES is removed, and 2 ml of trypsin is added to detach the cells from the surface of the T- 75 flask. Once detachment has occurred, 3 ml of trypsin neutralizing solution (TNS) is added to stop the enzymatic reaction. An additional 5 ml of HEPES is added, and the cells are enumerated using a hemocytometer. The cell suspension is centrifuged and adjusted to a density of, in the case of human cells, approximately 2.0 - 1.75 x 10⁶ cells/ml using EGM-2 without antibiotics, or in the case of porcine cells, approximately 2.0 - 1.50 x 10⁶ cells/ml using EBM-2 supplemented with 5% FBS and 50 μg/ml gentamicin.

[0079] Biocompatible Matrix: According to the present invention, the implantable material comprises a biocompatible matrix. The matrix is permissive for cell growth and attachment to, on or within the matrix. The matrix is flexible and conformable. The matrix can be a solid, a semi-solid or flowable porous composition. For purposes of the present invention, flowable composition means a composition susceptible to administration using an injection or injection-type delivery device such as, but not limited to, a needle, a syringe or a catheter. Other delivery devices which employ extrusion, ejection or expulsion are also contemplated herein. Porous matrices are preferred. The matrix also can be in the form of a flexible planar form. The matrix also can be in the form of a gel, a foam, a suspension, a particle, a microcarrier, a microcapsule, or a fibrous structure. A preferred flowable composition is shape-retaining. A currently preferred matrix has a particulate form. The biocompatible matrix can comprise particles and/or microcaπϊers and the particles and/or microcarriers can further comprise gelatin, collagen, fibronectin, fibrin, laminin or an attachment peptide. One exemplary attachment peptide is a peptide of sequence arginine-glycine-aspartate (RGD). [0080] The matrix, when implanted on a surface of a nerve structure, can reside at the implantation site for at least about 7-90 days, preferably about at least 7-14 days, more preferably about at least 14-28 days, most preferably about at least 28-90 days before it bioerodes.

[0081] One preferred matrix is Gelfoam^® (Pfizer, Inc., New York, NY), an absorbable gelatin sponge (hereinafter "Gelfoam matrix"). Another preferred matrix is Surgifoam^® (Johnson & Johnson, New Brunswick, NJ), also an absorbable gelatin sponge. Gelfoam and Surgifoam matrices are porous and flexible surgical sponges prepared from a specially treated, purified porcine dermal gelatin solution.

[0082] According to another embodiment, the biocompatible matrix material can be a modified matrix material. Modifications to the matrix material can be selected to optimize and/or to control function of the cells, including the cells' phenotype (e.g., the inhibitory phenotype) as described above, when the cells are associated with the matrix. According to one embodiment, modifications to the matrix material include coating the matrix with attachment factors or adhesion peptides that enhance the ability of the cells to control smooth muscle cell and/or fibroblast proliferation and migration, to decrease abnormal collagen deposition, to decrease fibrosis, to increase TIMP production, to decrease inflammation, to increase heparan sulfate production, to increase prostacyclin production, and/or to increase bFGF, TGF-Bi and nitric oxide (NO) production. [0083] According to another embodiment, the matrix is a matrix other than

Gelfoam. Additional exemplary matrix materials include, for example, fibrin gel, alginate, gelatin bead microcarriers, polystyrene sodium sulfonate microcarriers, collagen coated dextran microcarriers, PLA/PGA and pHEMA/MMA copolymers (with polymer ratios ranging from 1-100% for each copolymer). According to one embodiment, a synthetic matrix material, for example, PLA/PGA, is treated with NaOH to increase the hydrophilicity of the material and, therefore, the ability of the cells to attach to the material. According to a preferred embodiment, these additional matrices are modified to include attachment factors or adhesion peptides, as recited and described above. Exemplary attachment factors include, for example, gelatin, collagen, fibronectin, fibrin gel, and covalently attached cell adhesion ligands (including for example RGD) utilizing standard aqueous carbodiimide chemistry. Additional cell adhesion ligands include peptides having cell adhesion recognition sequences, including but not limited to: RGDY, REDVY, GRGDF, GPDSGR, GRGDY and REDV.

[0084] That is, these types of modifications or alterations of a substrate influence the interaction between a cell and a matrix which, in turn, can mediate expression of the preferred inhibitory phenotype described elsewhere herein. It is contemplated that these types of modifications or alterations of a substrate can result in enhanced expression of an inhibitory phenotype; can result in prolonged or further sustained expression of an inhibitory phenotype; and/or can confer such a phenotype on a cell which otherwise in its natural state does not exhibit such a phenotype as in, for example but not limited to, an exponentially growing or non-quiescent cell. Moreover, in certain circumstances, it is preferable to prepare an implantable material of the present invention which comprises non-quiescent cells provided that the implantable material has an inhibitory phenotype in accordance with the requirements set forth elsewhere herein. As already explained, the source of cells, the origin of cells and/or types of cells useful with the present invention are not limited; all that is required is that the cells express an inhibitory phenotype.

[0085] Embodiments of Implantable Materials: As stated earlier, implantable material of the present invention can be a flexible planar form or a flowable composition. When in a flexible planar form, it can assume a variety of shapes and sizes, preferably a shape and size which conforms to a contoured exterior surface of a nerve structure when situated at or adjacent to or in the vicinity of an injured or diseased site. Examples of preferred configurations suitable for use in this manner are disclosed in co-owned international patent application PCT/US05/43967 filed on December 6, 2005 (also known as Attorney Docket No. ELV-002PC), the entire contents of which are herein incorporated by reference.

[0086] Flowable Composition: In certain embodiments contemplated herein, the implantable material of the present invention is a flowable composition comprising a particulate biocompatible matrix which can be in the form of a gel, a foam, a suspension, a particle, a microcarrier, a microcapsule, macroporous beads, or other flowable material. The current invention contemplates any flowable composition that can be administered with an injection-type delivery device. For example, a delivery device such as a percutaneous injection-type delivery device is suitable for this purpose as described below. The flowable composition is preferably a shape-retaining composition. Thus, an implantable material comprising cells in, on or within a flowable-type particulate matrix as contemplated herein can be formulated for use with any injectable delivery device ranging in internal diameter from about 18 gauge to about 30 gauge and capable of delivering about 50 mg of flowable composition comprising particulate material containing preferably about 1 million cells in about 1 to about 3 ml of flowable composition. [0087] According to a currently preferred embodiment, the flowable composition comprises a biocompatible particulate matrix such as Gelfoam^® particles, Gelfoam^® powder, or pulverized Gelfoam^® (Pfizer Inc., New York, NY) (hereinafter "Gelfoam particles"), a product derived from porcine dermal gelatin. According to another embodiment, the particulate matrix is Surgifoam™ (Johnson & Johnson, New Brunswick, NJ) particles, comprised of absorbable gelatin powder. According to another embodiment, the particulate matrix is Cytodex-3 (Amersham Biosciences, Piscataway, NJ) microcarriers, comprised of denatured collagen coupled to a matrix of cross-linked dextran. According to a further embodiment, the particulate matrix is CultiSpher-G (Percell Biolytica AB, Astorp, Sweden) microcarrier, comprised of porcine gelatin. According to another embodiment, the particulate matrix is a macroporous material. According to one embodiment, the macroporous particulate matrix is CytoPore (Amersham Biosciences, Piscataway, NJ) microcarrier, comprised of cross-linked cellulose which is substituted with positively charged N,N,-diethylaminoethyl groups.

[0088] According to alternative embodiments, the biocompatible implantable particulate matrix is a modified biocompatible matrix. Modifications include those described above for an implantable matrix material.

[0089] Related flowable compositions suitable for use to manage the development and/or progression of healing in nerve sites in accordance with the present invention are disclosed in co-owned international patent application PCT/US05/43844 filed on December 6, 2005 (also known as Attorney Docket No. ELV-009PC), the entire contents of which are herein incorporated by reference.

[0090] Preparation of Implantable Material: Prior to cell seeding, the biocompatible matrix is re-hydrated by the addition of water, buffers and/or culture media such as EGM-2 without antibiotics at approximately 37°C and 5% CO₂ / 95% air for 12 to 24 hours. The implantable material is then removed from their re- hydration containers and placed in individual tissue culture dishes. The biocompatible matrix is seeded at a preferred density of approximately 1.5-2.0 x 10⁵ cells (1.25-1.66 x 10⁵ cells /cm³ of matrix) and placed in an incubator maintained at approximately 37°C and 5% CO₂ / 95% air, 90% humidity for 3-4 hours to 24 hours to facilitate cell attachment. The seeded matrix is then placed into individual containers (Evergreen, Los Angeles, CA) or tubes, each fitted with a cap containing a 0.2 urn filter with EGM-2 and incubated at approximately 37°C and 5% CO₂ / 95% air. Alternatively, three sponges can be placed into 150 ml bottles. The media is changed every two to three days, thereafter, until the cells have reached near- confluence, confluence or post-confluence. The cells in one preferred embodiment are preferably passage 6, but cells of fewer or more passages can be used.

[0091] Cell Growth Curve and Confluence: A sample of implantable material is removed on or around days 3 or 4, 6 or 7, 9 or 10, and 12 or 13, the cells are counted and assessed for viability, and a growth curve is constructed and evaluated in order to assess the growth characteristics and to determine whether confluence, near confluence or post-confluence has been achieved. Representative growth curves from two preparations of implantable material comprising porcine aortic endothelial cell implanted lots are presented in FIGS. IA and IB. In these examples, the implantable material is in a flexible planar form. Generally, one of ordinary skill will appreciate the indicia of acceptable cell growth at early, mid- and late time points, such as observation of an increase in cell number at the early time points (when referring to FIG. IA, between about days 2-6), followed by a near confluent phase (when referring to FIG. IA, between about days 6-8), followed by a plateau in cell number once the cells have reached confluence as indicated by a relatively constant cell number (when referring to FIG. IA, between about days 8- 10) and maintenance of the cell number when the cells are post-confluent (when referring to FIG. IA, between about days 10-14). For purposes of the present invention, cell populations which are in a plateau for at least 72 hours are preferred.

[0092] Cell counts are achieved by complete digestion of the aliquot of implantable material such as with a solution of 0.5 mg/ml collagenase in a CaCl₂ solution in the case of gelatin-based matrix materials. After measuring the volume of the digested implantable material, a known volume of the cell suspension is diluted with 0.4% trypan blue (4:1 cells to trypan blue) and viability assessed by trypan blue exclusion. Viable, non-viable and total cells are enumerated using a hemacytometer. Growth curves are constructed by plotting the number of viable cells versus the number of days in culture. Cells are shipped and implanted after reaching confluence.

[0093] For purposes of the present invention, confluence is defined as the presence of at least about 4 x 10⁵ cells/cm³ when in a flexible planar form of the implantable material (1.0 x 4.0 x 0.3 cm), and preferably about 7 x 10^s to 1 x 10⁶ total cells per aliquot (50-70 mg) when in a flowable composition. For both, cell viability is at least about 90% preferably but no less than 80%. If the cells are not confluent by day 12 or 13, the media is changed, and incubation is continued for an additional day. This process is continued until confluence is achieved or until 14 days post-seeding. On day 14, if the cells are not confluent, the lot is discarded. If the cells are determined to be confluent after performing in-process checks, a final media change is performed. This final media change is performed using EGM-2 without phenol red and without antibiotics. Immediately following the media change, the tubes are fitted with sterile plug seal caps for shipping. [0094] Evaluation of Functionality and Phenotvpe: For purposes of the invention described herein, the implantable material is further tested for indicia of functionality and phenotype prior to implantation. For example, conditioned media are collected during the culture period to ascertain levels of heparan sulfate, transforming growth factor-βi (TGF-βi), basic fibroblast growth factor (b-FGF), tissue inhibitors of matrix metalloproteinases (TIMP), and nitric oxide (NO) which are produced by the cultured endothelial cells. In certain preferred embodiments, the implantable material can be used for the purposes described herein when total cell number is at least about 2, preferably at least about 4 x 10⁵ cells/cm³ of implantable material; percentage of viable cells is at least about 80-90%, preferably >90%, most preferably at least about 90%; heparan sulfate in conditioned media is at least about 0.23-1.0, preferably at least about 0.5 microg/mL/day; TGF-βi in conditioned media is at least about 200-300 picog/mL/day, preferably at least about 300 picog/ml/day; b-FGF in conditioned media is below about 200 picog/ml, preferably no more than about 400 picog/ml; TIMP-2 in conditioned media is at least about 5.0 - 10.0 ng/mL/day, preferably at least about 8.0 ng/mL/day; NO in conditioned media is at least about 0.5 - 3.0 μmol/L/day, preferably at least about 2.0 μmol/L/day.

[0095] Heparan sulfate levels can be quantified using a routine dimethylmethylene blue-chondroitinase ABC digestion spectrophotometry assay. Total sulfated glycosaminoglycan (GAG) levels are determined using a dimethylmethylene blue (DMB) dye binding assay in which unknown samples are compared to a standard curve generated using known quantities of purified chondroitin sulfate diluted in collection media. Additional samples of conditioned media are mixed with chondroitinase ABC to digest chondroitin and dermatan sulfates prior to the addition of the DMB color reagent. All absorbances are determined at the maximum wavelength absorbance of the DMB dye mixed with the GAG standard, generally around 515-525 nm. The concentration of heparan sulfate per day is calculated by multiplying the percentage heparan sulfate calculated by enzymatic digestion by the total sulfated glycosaminoglycan concentration in conditioned media samples. Chondroitinase ABC activity is confirmed by digesting a sample of purified 100% chondroitin sulfate and a 50/50 mixture of purified heparan sulfate and chondroitin sulfate. Conditioned medium samples are corrected appropriately if less than 100% of the purified chondroitin sulfate is digested. Heparan sulfate levels may also be quantitated using an ELISA assay employing monoclonal antibodies. [0096] TGF-β, TIMP, and b-FGF levels can be quantified using an ELISA assay employing monoclonal or polyclonal antibodies, preferably polyclonal. Control collection media can also be quantitated using an ELISA assay and the samples corrected appropriately for TGF-β _1,TIMP, and b-FGF levels present in control media. [0097] Nitric oxide (NO) levels can be quantified using a standard Griess

Reaction assay. The transient and volatile nature of nitric oxide makes it unsuitable for most detection methods. However, two stable breakdown products of nitric oxide, nitrate (NO₃) and nitrite (NO₂), can be detected using routine photometric methods. The Griess Reaction assay enzymatically converts nitrate to nitrite in the presence of nitrate reductase. Nitrite is detected colorimetrically as a colored azo dye product, absorbing visible light in the range of about 540 nm. The level of nitric oxide present in the system is determined by converting all nitrate into nitrite, determining the total concentration of nitrite in the unknown samples, and then comparing the resulting concentration of nitrite to a standard curve generated using known quantities of nitrate converted to nitrite.

[0098] The earlier-described preferred inhibitory phenotype is assessed using the quantitative heparan sulfate, TGF-Bj, TIMP, NO and/or b-FGF assays described above, as well as quantitative in vitro assays of PC 12 cell differentiation, PC 12 cell survival, neural stem cell differentiation, neural stem cell survival, smooth muscle cell growth, fibroblast collagen deposition activity and inhibition of thrombosis as follows. For purposes of the present invention, implantable material is ready for implantation when one or more of these alternative in vitro assays confirm that the implantable material is exhibiting the preferred inhibitory phenotype.

[0099] To evaluate regulation of primary neuronal cells in vitro, the magnitude of differentiation of PC 12 cells in contact with the implantable material and/or a co- culture of PC 12 cells with the implantable material is determined. PC 12 cells are seeded at a density of about 50,000 cells/well in 12 transwell plates (Corning, Inc., Corning, NY) in growth medium (DMEM 4.5% glucose (ATCC; Rockville, MD), supplemented with 5% FBS (Hyclone Laboratories, Logan UT), 10% horse serum (Gibco Laboratories, Lawrence, MA) and Penicillin-Streptomycin lOμg/ml (Sigma Chemical Co., St. Louis, MO)). The cells are allowed to attach for 24 hours. The media is then replaced with assay media (DMEM 4.5% glucose supplemented with 3% FBS and Penicillin-Streptomycin), differentiation media (assay media supplemented with 100 ng/ml nerve growth factor (NGF) beta (ReproTech, Rocky Hill, NJ) as a positive control), or placed in contact with a co-culture of the implantable material placed in a net insert over cultured PC 12 cells (such that the PC 12 cells and the cells of the implantable material share the same 2.5 ml assay media). After seven days, images of the cells are taken using a phase contrast microscope and the number of cells and neurite outgrowth in each sample is determined. The effect of the co-culture of PC 12 cells and implantable material on PC 12 differentiation is determined by comparing the number of cells and the magnitude of neurite outgrowth immediately before the addition of implantable material to form the co-culture with that after seven days of exposure to the co- culture, and to control media (assay media with and without the addition of nerve growth factor (NGF)). The magnitude of differentiation associated with the co- culture samples are compared to the magnitude of differentiation associated with the positive control. According to a preferred embodiment, the implantable material is considered regulatory if the conditioned media and/or the co-culture with the implantable material enhances PC 12 cell differentiation by 20-100% of the positive control. [0100) In an illustrative embodiment of the invention, the effect of co-culture with the implantable material on PC 12 neuronal primary cell differentiation was determined by comparing the amount of axon outgrowth present when the PC 12 cells are co-cultured with the implantable material compared to the negative control (assay media only) and positive control (assay media plus 100 ng/mL NGF-β). According to this illustrative embodiment, axon outgrowth is present in both the co- culture and in the positive control, suggesting the implantable material is able to regulate the differentiation and neurite outgrowth of PC 12 and other neuronal primary cells.

[0101] To evaluate regulation of primary neuronal cells in vitro, the magnitude of PC 12 cell survival in contact with the implantable material and/or media conditioned with the implantable material is determined. PC 12 cells are seeded at a density of about 50,000 cells/well in 12 transwell plates (Corning, Inc., Corning, NY) in growth medium (DMEM 4.5% glucose (ATCC; Rockville, MD), supplemented with 5% FBS (Hyclone Laboratories, Logan UT), 10% horse serum (Gibco Laboratories, Lawrence, MA) and Penicillin-Streptomycin lOμg/ml (Sigma Chemical Co., St. Louis, MO)). The cells are allowed to attach for 24 hours. The media is then replaced with assay media (DMEM 4.5% glucose supplemented with 3% FBS and Penicillin-Streptomycin), differentiation media (assay media supplemented with 100 ng/ml nerve growth factor (NGF) beta (ReproTech, Rocky Hill, NJ) as a positive control), or placed in contact with a co-culture of the implantable material placed in a net insert over cultured PC 12 cells (such that the PC 12 cells and the cells of the implantable material share the same 1.5 ml assay media. After seven days, the cells are washed twice in wash buffer (Phenol Red-free EBM supplemented with Gentamicin 0.1 mg/ml (both Lonza Group Ltd, Basel, Switzerland)) and incubated in 400 μl collection media (Phenol Red-free EBM supplemented with 0.5% FBS and Gentamicin 0.1 mg/ml (all Lonza Group Ltd,

Basel, Switzerland)) with lOOμl MTS (MTS cell proliferation assay, Promega Corp., Madison, WI) for 2 hours. 100 μl of media are removed from the treated cultures and untreated controls and placed into 96 well plates. The plates are read in a plate reader to determine the optical density (O.D.) of the cells at 490 nm. According to a preferred embodiment, the implantable material is considered to have a positive effect on PC 12 cell survival if the conditioned media results in viability of about 50- 100% of the positive control viability.

[0102] Figure 2 depicts the results of the PC 12 survival assay according to an illustrative embodiment of the invention. The survival of PC 12 neuronal primary cells is determined by comparing the metabolic activity of PC 12 cells when in contact with implantable material co-culture (right column) compared to the negative control (left column) and positive control (center column). According to this illustrative embodiment, PC 12 cell survival is enhanced in the presence of the implantable material. However, the survival was not as significant when the PC 12 cells are treated with only the conditioned media from the implantable material, suggesting that necessary factors produced by the implantable material are labile and/or inactive and/or degraded in conditioned media.

[0103] To evaluate regulation of primary neuronal cells in vitro, the magnitude of differentiation of neural stem cells in contact with the implantable material and/or a co-culture of neural stem cells with the implantable material is determined. Adult hippocampal arctic ground squirrel neural stem cells isolated from the hippocampus of adult arctic ground squirrels following hibernation are seeded at a density of about 50,000 cells/well in 12 transwell plates (Corning, Inc., Corning, NY) in supplemented growth medium. The cells are allowed to attach for 24 hours. The media is then replaced with assay media, differentiation media (as a positive control), or placed in contact with a co-culture of the implantable material placed in a net insert over cultured neural stem cells (such that the neural stem cells and the cells of the implantable material share the same 2.5 ml assay media). After seven days, images of the cells are taken using a phase contrast microscope and the number of cells and neurite outgrowth in each sample is determined. The effect of the co-culture of neural stem cells and implantable material on neural stem cell differentiation is determined by comparing the number of cells and the magnitude of neurite outgrowth immediately before the addition of implantable material to form the co-culture with that after seven days of exposure to the co-culture, and to control media (assay media with and without the addition of nerve growth factor (NGF)). The magnitude of differentiation associated with the co-culture samples are compared to the magnitude of differentiation associated with the positive control. According to a preferred embodiment, the implantable material is considered regulatory if the conditioned media and/or the co-culture with the implantable material enhances neural stem cell differentiation by 20-100% of the positive control. [0104] In an illustrative embodiment of the invention, the effect of co-culture with the implantable material on neural stem cell differentiation is determined by comparing the amount of axon outgrowth present when the neural stem cells are co- cultured with the implantable material compared to the negative control and positive control. According to this illustrative embodiment, it is expected that axon outgrowth will be present in both the co-culture and in the positive control, suggesting the implantable material is able to regulate the differentiation and neurite outgrowth of neural stem cells and other neuronal primary cells.

[0105] To evaluate regulation of primary neuronal cells in vitro, the magnitude of neural stem cell survival in contact with the implantable material and/or media conditioned with the implantable material is determined. Adult hippocampal arctic ground squirrel neural stem cells are seeded at a density of about 50,000 cells/well in 12 transwell plates (Corning, Inc., Corning, NY) in supplemented growth medium. The cells are allowed to attach for 24 hours. The media is then replaced with assay media, differentiation media (as a positive control), or placed in contact with a co- culture of the implantable material placed in a net insert over cultured neural stem cells (such that the neural stem cells and the cells of the implantable material share the same 1.5 ml assay media). After seven days, the cells are washed twice in wash buffer and incubated in 400 μl collection media with lOOμl MTS (MTS cell proliferation assay, Promega Corp., Madison, WI) for 2 hours. 100 μl of media are removed from the treated cultures and untreated controls and placed into 96 well plates. The plates are read in a plate reader to determine the optical density (O.D.) of the cells at 490 nm. According to a preferred embodiment, the implantable material is considered to have a positive effect on neural stem cell survival if the conditioned media results in viability of about 50-100% of the positive control viability.

[0106] To evaluate inhibition of smooth muscle cell growth in vitro, the magnitude of inhibition associated with cultured endothelial cells is determined. Porcine or human aortic smooth muscle cells are sparsely seeded in 24 well tissue culture plates in smooth muscle cell growth medium (SmGM-2, Lonza Group Ltd, Basel, Switzerland). The cells are allowed to attach for 24 hours. The media is then replaced with smooth muscle cell basal media (SmBM) containing 0.2% FBS for 48-72 hours to growth arrest the cells. Conditioned media is prepared from post- confluent endothelial cell cultures, diluted 1 : 1 with 2X SMC growth media and added to the cultures. A positive control for inhibition of smooth muscle cell growth is included in each assay. After three to four days, the number of cells in each sample is enumerated using a Coulter Counter or using a colorimetric assay after the addition of a dye. The effect of conditioned media on smooth muscle cell proliferation is determined by comparing the number of smooth muscle cells per well immediately before the addition of conditioned media with that after three to four days of exposure to conditioned media, and to control media (standard growth media with and without the addition of growth factors). The magnitude of inhibition associated with the conditioned media samples are compared to the magnitude of inhibition associated with the positive control. According to a preferred embodiment, the implantable material is considered inhibitory if the conditioned media inhibits about 20% of what the heparin control is able to inhibit.

[01071 To evaluate inhibition of thrombosis in vitro, the level of heparan sulfate associated with the cultured endothelial cells is determined. Heparan sulfate has both anti-proliferative and anti-thrombotic properties. Using either the routine dimethylmethylene blue-chondroitinase ABC digestion spectrophotometric assay or an ELISA assay, both assays are described in detail above, the concentration of heparan sulfate is calculated. The implantable material can be used for the purposes described herein when the heparan sulfate in the conditioned media is at least about 0.23-1.0, preferably at least about 0.5 microg/mL/day. [0108] Another method to evaluate inhibition of thrombosis involves determining the magnitude of inhibition of platelet aggregation in vitro associated with platelet rich-plasma or platelet concentrate (from Research Blood Components, Brighton, MA). Conditioned media is prepared from post-confluent endothelial cell cultures and added to aliquots of the platelet concentrate. A platelet aggregating agent (agonist) is added to the platelets seeded into 96 wells as control. Platelet agonists commonly include arachidonate, ADP, collagen type I, epinephrine, thrombin (Sigma-Aldrich Co., St. Louis, MO) or ristocetin (available from Sigma- Aldrich Co., St. Louis, MO). An additional well of platelets has no platelet agonist or conditioned media added, to assess for baseline spontaneous platelet aggregation. A positive control for inhibition of platelet aggregation is also included in each assay. Exemplary positive controls include aspirin, heparin, indomethacin (Sigma- Aldrich Co., St. Louis, MO), abciximab (ReoPro^®, Eli Lilly, Indianapolis, IN), tirofiban (Aggrastat^®, Merck & Co., Inc., Whitehouse Station, NJ) or eptifibatide (Integrilin^®, Millennium Pharmaceuticals, Inc., Cambridge, MA). The resulting platelet aggregation of all test conditions are then measured using a plate reader and absorbance read at 405 nm. The plate reader measures platelet aggregation by monitoring optical density. As platelets aggregate, more light can pass through the specimen. The plate reader reports results in absorbance, a function of the rate at which platelets aggregate. Aggregation is assessed as maximal aggregation at 6 to 12 minutes after the addition of the agonist. The effect of conditioned media on platelet aggregation is determined by comparing maximal agonist aggregation before the addition of conditioned medium with that after exposure of platelet concentrate to conditioned medium, and to the positive control. Results are expressed as a percentage of the baseline. The magnitude of inhibition associated with the conditioned media samples are compared to the magnitude of inhibition associated with the positive control. According to a preferred embodiment, the implantable material is considered inhibitory if the conditioned media inhibits about 20% of what the positive control is able to inhibit.

[0109] To evaluate fibroblast migration in vitro, the regulation of fibroblast migration associated with cultured endothelial cells is determined. Human foreskin fibroblast cells are sparsely seeded in 12 or 24 well tissue culture plates The cells are grown to confluence and the media is then replaced with Dulbecco's modified

Eagle's media (DMEM) containing 0.5% FBS and PS for 24 hours to growth arrest the cells. Cultures are scratched with a 250 μl sterile tip and washed twice with collection media. The following media conditions are then added to the wells: (1) Collection Media alone (CM) or conditioned media prepared from the implantable material (i.e., post-confluent endothelial cells grown on a matrix). After 16-24 hours, injury images are taken and the degree of migration determined by direct visualization. The effect of conditioned media on fibroblast migration is determined by comparing the number of fibroblasts within the scratch wound region per well immediately before the addition of conditioned media with that after 16-24 hours of exposure to conditioned media and to control media. According to a preferred embodiment, the implantable material is considered regulatory if the conditioned media inhibits or enhances fibroblast migration by at least about 20% of the control, more preferably by at least about 40% of the control, and most preferably by at least about 60% of the control.

[0110] When ready for implantation, the planar form of implantable material is supplied in final product containers, each preferably containing a 1 x 4 x 0.3 cm (1.2 cm³), sterile implantable material with preferably approximately 5-8 x 10⁵ or preferably at least about 4 x 10⁵ cells/cm³, and at least about 90% viable cells (for example, human aortic endothelial cells derived from a single cadaver donor) per cubic centimeter implantable material in approximately 45-60 ml, preferably about 50 ml, endothelial growth medium (for example, endothelial growth medium (EGM- 2), containing no phenol red and no antibiotics). When porcine aortic endothelial cells are used, the growth medium is also EBM-2 containing no phenol red, but supplemented with 5% FBS and 50 μg/ml gentamicin.

[0111] In other preferred embodiments, the flowable composition (for example, a particulate form biocompatible matrix) is supplied in final product containers, including, for example, sealed tissue culture containers modified with filter caps or pre-loaded syringes, each preferably containing about 50-60 mg of flowable composition comprising about 7 x 10⁵ to about 1 x 10⁶ total endothelial cells in about 45-60 ml, preferably about 50 ml, growth medium per aliquot.

[0112] Shelf-Life of Implantable Material: The implantable material of the present invention comprising a confluent, near-confluent or post-confluent population of cells can be maintained at room temperature in a stable and viable condition for at least two weeks. Preferably, such implantable material is maintained in about 45-60 ml, more preferably about 50 ml, of transport media with or without additional FBS or VEGF. Transport media comprises EGM-2 media without phenol red. FBS can be added to the volume of transport media up to about 10% FBS, or a total concentration of about 12% FBS. However, because FBS must be removed from the implantable material prior to implantation, it is preferred to limit the amount of FBS used in the transport media to reduce the length of rinse required prior to implantation. VEGF can be added to the volume of transport media up to a concentration of about 3-4 ng/ml. [0113] Crvopreservation of Implantable Material: The implantable material of the present invention can be cryopreserved for storage and/or transport to the implantation site without diminishing its clinical potency or integrity upon eventual thaw. Preferably, implantable material is cryopreserved in a 15 ml cryovial (Nalgene^®, Nalge Nunc Int'l, Rochester, NY) in a solution of about 5 ml CryoStor CS-IO solution (BioLife Solutions, Oswego, NY) containing about 10% DMSO, about 2-8% Dextran and about 20-75% FBS. Cryovials are placed in a cold iso- propanol water bath, transferred to an -80°C freezer for 4 hours, and subsequently transferred to liquid nitrogen (-150⁰C to -165°C). [0114] Cryopreserved aliquots of the implantable material are then slowly thawed at room temperature for about 15 minutes, followed by an additional approximately 15 minutes in a room temperature water bath. The material is then washed about 3 times in about 200 - 250 mL saline, lactated ringers or EBM. The three rinse procedures are conducted for about 5 minutes at room temperature. The material may then be implanted.

[0115] To determine the bioactivity of the thawed material, following the thaw and rinse procedures, the cryopreserved material is allowed to rest for about 48 hours in about 10 ml of recovery solution. For porcine endothelial cells, the recovery solution is EBM-2 supplemented with 5% FBS and 50 μg/ml gentamicin at 37°C in 5% CO₂; for human endothelial cells, the recovery solution is EGM-2 with or without antibiotics. Further post-thaw conditioning can be carried out for at least another 24 hours prior to use and/or packaging for storage or transport.

[0116] Immediately prior to implantation, the transport or cryopreservation medium is decanted and the implantable material is rinsed in about 250-500 ml sterile saline (USP). The medium in the final product contains a small amount of FBS to maintain cell viability during transport to a clinical site if necessary. The FBS has been tested extensively for the presence of bacteria, fungi and other viral agents according to Title 9 CFR: Animal and Animal Products. A rinsing procedure is employed just prior to implantation, which decreases the amount of FBS transferred preferably to between 0-60 ng per implant, but preferably no more than 1-2 μg per implant. [0117] The total cell load per human patient will be preferably approximately 1.6-2.6 x 10⁴ cells per kg body weight, but no less than about 2 x 10³ and no more than about 2 x 10⁶ cells per kg body weight.

[0118] Administration of Implantable Material: The implantable material of the present invention when in a flowable composition comprises a particulate biocompatible matrix and cells, preferably endothelial cells, more preferably vascular endothelial cells, which are about 90% viable at a preferred density of about 0.8 x 10⁴ cells/mg, more preferred of about 1.5 x 10⁴ cells/mg, most preferred of about 2 x 10⁴ cells/mg, and which can produce conditioned media containing heparan sulfate at least about 0.23-1.0, preferably at least about 0.5 microg/mL/day, TGF-βi at at least about 200-300 picog/ml/day, preferably at least about 300 picog/ml/day, and b-FGF below about 200 picog/ml and preferably no more than about 400 picog/ml; TIMP-2 in conditioned media is at least about 5.0 - 10.0 ng/mL/day, preferably at least about 8.0 ng/mL/day; NO in conditioned media is at least about 0.5 - 3.0 μmol/L/day, preferably at least about 2.0 umol/L/day; and, display the earlier-described inhibitory phenotype.

[0119] For purposes of the present invention generally, administration of the implantable particulate material is localized to a site in the vicinity of, adjacent or at a site of disease, damage or blockage of a nerve structure. The site of deposition of the implantable material is an exterior surface of a nerve structure. As contemplated herein, localized deposition can be accomplished as follows.

[0120] In a particularly preferred embodiment, the flowable composition is first administered percutaneously, entering the patient's body near the nerve structure and then deposited on an exterior surface of the nerve site using a suitable needle, catheter or other suitable percutaneous delivery device. Alternatively, the flowable composition is delivered percutaneously using a needle, catheter or other suitable delivery device in conjunction with an identifying step to facilitate delivery to a desired exterior surface of the nerve site. The identifying step can occur prior to or coincident with percutaneous delivery. The identifying step can be accomplished using physical examination, ultrasound, and/or CT scan, to name but a few. The identifying step is optionally performed and not required to practice the methods of the present invention.

[0121] Preferably, flowable composition is deposited on an exterior surface of a nerve structure, either at the site of disease or damage to be treated, or adjacent to or in the vicinity of the site of disease or damage. The composition can be deposited in a variety of locations relative to a nerve site, for example, at the site of damage or disease, surrounding the site of damage or disease or adjacent to the site of damage or disease. According to a preferred embodiment, an adjacent site is within about 0 mm to 20 mm of the nerve site. In another preferred embodiment, a site is within about 21 mm to 40 mm; in yet another preferred embodiment, a site is within about 41 mm to 60 mm. In another preferred embodiment, a site is within about 61 mm to 100 mm. Alternatively, an adjacent site is any other clinician-determined adjacent location where the deposited composition is capable of exhibiting a desired effect on a nerve site in the proximity of the nerve site. [0122] In another embodiment, the flowable composition is delivered directly to a surgically-exposed non-luminal surface at, adjacent to or in the vicinity of a nerve structure. In this case delivery is guided and directed by direct observation of the site. Also in this case, delivery can be aided by coincident use of an identifying step as described above. Again, the identifying step is optional. [0123] According to another embodiment of the invention, the flexible planar form of the implantable material is delivered locally to a surgically-exposed extraluminal, non-luminal, exterior site or interior cavity at, adjacent to or in the vicinity of a nerve site. In one case, at least one piece of the implantable material is applied to a desired site by passing one end of the implantable material under the nerve structure. The ends are then wrapped around the structure, keeping the implantable material centered. The ends overlap each other to secure the material in place. In other cases, the implantable material does not need to completely wrap around the circumference of the structure; it need only conform to and contact a surface of the structure and be implanted in an amount effective to treat a damaged or diseased site. Examples

1. Peripheral nerve damage resulting from nerve transection.

[0124] The rat model described by Turgut et al. (J. Clin. Neurosci., 2006 Aug;13(7):753-8.) will be studied to demonstrate treatment and management of nerve damage resulting from nerve transection. At least four rats will undergo surgical sciatic nerve transection and primary suture anastomosis. In each rat, both sciatic nerves will be exposed via a posterior thigh approach and isolated with a plastic sheet. Each nerve will be sharply transected using a steel scalpel, and the nerve ends approximated with four to six 10/0 nylon sutures placed around the nerve in the epineurium using standard microsurgical methods. Half of the rats will receive an effective amount of the implantable material at or near the nerve anastomosis post-transection where nerve damage is evident. The wound will be closed with 4/0 nylon sutures and the rats replaced unrestricted in their cages. Animals will be monitored for complications.

[0125] Two months post-surgery, animals will be sacrificed and unilateral sciatic nerve specimens including the anastomotic region will be carefully removed. The excised segments will be gently stretched on pieces of index card and processed (fixed and sectioned) for immunohistochemical analysis. Following processing, the sections will be stained with primary antibodies to either TGF-βl or bFGF and exposed to secondary antibodies conjugated to detectable labels using standard protocols. Following immunostaining, sections will be examined by light microscopy to see the extent of TGF-β or bFGF staining. The degree of staining of the endoneurium, perineurium, epineurium and perivascular region will be evaluated by researchers double-blinded to the experimental conditions on the following semi- quantitative scale. Intensity (I) will be scored on a scale from 1-4 (I: none, mild, moderate, strong) and distribution (D) on a scale from 1-4 (D: none, focal, patchy, diffuse). Tissues with I x D less than or equal to 4 will be considered 'weakly positive,' those with I x D greater than 4 as 'strongly positive' and those with no immunoreactivity as 'absent.' It is expected that rats treated with the implantable material will display increased levels of TGF-β and bFGF in at least the epineurium and perivascular regions, consistent with a role for TGF-β and bFGF in improved nerve fiber regeneration and myelin regeneration and altered collagen deposition.

2. Nerve graft.

[0126] The rat model described by Hadlock et al. (Microsurgery, 2001, 21 :96- 101) will be studied to demonstrate improvement in axonal growth as measured by histological and functional assays. At least twenty Fisher rats will undergo microsurgical removal of a section of the left sciatic nerve, followed by autograft repair. Anesthetized animals will each have their left sciatic nerve sharply transected in two places to leave a 7 mm gap between cut ends. The 7 mm section of nerve will be rotated such that the distal end of the graft is placed adjacent to the proximal stump and the proximal end of the graft is placed adjacent to the distal stump. The autografts will be sewn into place using two to three 10-0 nylon sutures at each end. Ten of the animals will receive an effective amount of the implantable material at or near the autograft, and the incision will be closed in layers. Animals will be allowed to recuperate and given food and water ad libidum.

[0127] Animals will be tested for return of motor function at regular intervals following the graft procedure. Return of function will be assessed in a blinded fashion, with standard sciatic function index (SFI) measurements (10.5 weeks) and extensor postural thrust testing (EPT), a method of assessing pure motor recovery (4-10.5 weeks). Following the 10.5-week survival time, animals will be sacrificed, and the grafts will be harvested, fixed and stained for electron microscopy analysis. A blinded assessment of axon measurements will be obtained, including fiber number, density and average diameter as well as percentage of neural tissue in the total cross section.

[0128] It is expected that animals treated with the implantable material will show greater return to function as assessed by SFI measurements and EPT testing. In addition, treated animals will show increased fiber number, density and average diameter as well as increased percentage of neural tissue in cross section, as compared to untreated controls. 3. Nerve damage resulting from crushed nerve.

[0129] The rat model described by Gantus et al. (Brain Res., 2006 Nov 29; 1122(l):36-46.) will be studied to demonstrate treatment and management of nerve damage resulting from crushed nerve. One hundred and ten rats will undergo surgical sural nerve crush, as near as possible to the sciatic ramification, using surgical fine tweezers and applying moderate pressure for 10 s. The surgery will be performed on each rat's right nerve and the contralateral intact nerve will be used as a control. Two groups of animals will be maintained similarly, except the treatment group will receive an effective amount of the implantable material at or near the nerve post-crush where nerve damage is evident. Animals will be sacrificed on day 2, day 7 or day 21 after surgery. Animals will be perfused with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 for 20 min. Afterward, the nerve will be dissected and the distal segment divided into three parts: the first part (0.2 mm long-near to the crush) will be discarded due to mechanical damage, the second part (0.3 mm long) will be used for transmission electron microscopy (TEM) and the third part (0.5 mm long) will be used for immunofluorescence.

[0130] Nine animals (three for each time point) from each group (treated and untreated) will be used in the electron microscopy study. The nerves will be fixed, stained, and sectioned. Semi-thin sections (500 nm) will be stained with toluidine blue and observed by light microscopy. Ultrathin sections (60-70 nm) will be stained with uranyl acetate and lead citrate for observation under the Transmission Electron Microscope (Zeiss EM-900). By day 2, the fibers of untreated animals are expected to show granular dissolution of the cytoskeleton that will persist until day 7, at which time myelin debris inside Schwann cells and macrophages will be seen. The beginning of axon sprouting and remyelination are also expected to be seen. By day 21, more extensive axon regeneration will be observed together with remyelination of axons, but some naked axon sprouts will continue to be seen. In contrast, treated animals are expected to regenerate axons and myelin sheaths at earlier time points and to a greater extent.

[0131] Nineteen animals from each group will be used in the immunofluorescence study. The nerve segments will be fixed and exposed to primary and secondary antibodies to detect extracellular matrix proteins such as laminin (α3 and α3 chains), type IV collagen, chondroitin sulfate proteoglycans (CSPGs), MMP-3 (matrix metalloproteinase 3), TIMP-I and TIMP-2 (tissue inhibitor of metalloproteinases 1 and 2). For negative controls, primary antibodies will be omitted. Sections will be observed and photographed using a microscope with fluorescence optics and image capture device. To quantify fluorescence levels, 15 (five for each time interval) of the 19 animals will be used. Immunofluorescence images will be analyzed with Image-Pro Plus (version 4.0), by evaluating the ratio between stained area and total field area for three to five images of each nerve cross- sectional area.

[0132] Additionally, the distal sural nerve segments of 36 animals from each group will be subjected to Western blot analysis to quantify MMP-3, MMP-7, TIMP-I and TIMP-2 expression. Nerve segments will be boiled for 10 min. and then centrifuged at 12,000 rpm for 10 min. The concentration of supernatant proteins will be determined using the Bradford method. 40 mg of protein will be run on 10% and 15% SDS-PAGE gels. Proteins will be transferred to PVDF (polyvinylidene fluoride) membranes using standard protocols. Membranes will be exposed to primary antibodies against each of the proteins, followed by secondary antibody staining, performed using standard protocols. Membranes will be reacted with an enhanced chemiluminescence Western blot analysis system (ECL-Plus kit, Amersham). The reaction image will be acquired and quantified, with values normalized to levels measured in unoperated nerves.

[0133] The amount of laminin (α3 and cc3 chains) expressed in untreated animals is expected to increase after nerve crush and return to normal levels over the course of the experiment, consistent with a role for laminin in axon regeneration and myelination. Treated animals are expected to show increased levels and/or temporal persistence of laminin, which may cause an increase in the level of axon regeneration and myelination.

[0134] Untreated animals are expected to show increased expression of type IV collagen following nerve crush, consistent with a role for collagen in the process of nerve repair. Treated animals are expected to show an optimal level of type IV collagen expression that promotes axon growth and remyelination, but that prevents accumulation of excess collagen at the site of nerve crush. The amount of MMP-3 expressed in the crushed nerve of untreated animals is expected to decrease over the course of the experiment, consistent with a role for MMP-3 in degrading type IV collagen. Treated animals are expected to show an optimal level of MMP-3 that will control the amount of collagen available for promoting axon growth and remyelination but prevent accumulation of excess collagen at the site of nerve crush.

[0135] Untreated animals are expected to show decreased levels of chondroitin sulfate proteoglycans (CSPGs) immediately after nerve crush. A decrease in CSPGs following nerve crush is consistent with a role for CSPGs in inhibiting neuronal growth. CSPG levels are expected to return to normal over the course of the experiment, however. Animals treated with the implantable material are expected to maintain lower levels of CSPGs for an extended period of time, allowing more complete nerve repair. Untreated animals are expected to show an increase in

MMP-7 expression at early time points following nerve crush, consistent with a role for MMP-7 in degrading CSPGs. MMPs may also release growth factors to promote neurite migration. Treated animals are expected to show higher levels of expression of MMP-7, allowing for increased degradation of the neural growth-inhibiting CSPGs and thus increased axon regeneration.

[0136] Untreated animals will likely show an increase in the levels of TIMP-I and -2, followed by a gradual return to normal levels over the course of the experiment. TIMP-2 has been reported to regulate negatively the progression of the cell cycle, maintaining the immaturity of axons to support peripheral nerve regeneration. Treated animals are expected to maintain higher TIMP-I and -2 levels to longer time periods, thus increasing the extent of axon regeneration and remyelination.

4. Treatment of human patients with nerve disorders.

[0137] Human patients that have been diagnosed with nerve injury, damage or disease will be studied to demonstrate treatment or management of nerve disorders. Patients will be examined to identify a nerve system injury or disease. Two groups of patients will be maintained similarly, except one group will receive an effective amount of the implantable material at or near the injured or diseased nerve structure. Reduction and/or amelioration of injury or disease of the affected nerve structure will be monitored over time by ultrasound, MRI, CT scan, physical exam, and other relevant procedures depending on the type of nerve disorder present in the patient. It is expected that patients treated with the implantable material will display return to function, reduction and/or amelioration of injury or disease to the treated nerve.

5. Surgical treatment of human patients with nerve disorders.

[0138] Human patients that have been diagnosed with nerve injury, damage or disease and who will be undergoing surgery for those disorders will be studied to demonstrate treatment and management of nerve disorders. Patients will be examined to determine the affected nerve structure and effective surgical treatment. Two groups of patients will be maintained similarly, except the treatment group will receive an effective amount of the implantable material in conjunction with surgery of the injured or diseased nerve structure. Reduction and/or amelioration of injury or disease of the affected nerve structure will be monitored over time by ultrasound, MRI, CT scan, physical exam, and other relevant procedures depending on the type of nerve disorder present in the patient. It is expected that patients treated with the implantable material will display return to function, reduction and/or amelioration of the nerve disorder at a higher level than the control group.

6. Surgical treatment of human patients with cerebral aneurysm.

[0139] Human patients that have been diagnosed with a cerebral aneurysm and who will be undergoing surgery for the disorder will be studied to demonstrate treatment and management of cerebral aneurysm. Patients will be examined to determine the affected cerebral artery and effective surgical treatment. Two groups of patients will be maintained similarly, except the treatment group will receive an effective amount of the implantable material in conjunction with surgery to reinforce or repair the cerebral aneurysm. Reduction and/or amelioration of the cerebral aneurysm will be monitored over time by ultrasound, MRI, CT scan, and other relevant procedures depending on the location and type of cerebral aneurysm present in the patient. It is expected that patients treated with the implantable material will display reduction and/or amelioration of the cerebral aneurysm at a higher level than the control group.

[0140] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

What is claimed is:

Claims

1. A method of treating a nerve in an individual in need thereof, the method comprising:

contacting with an implantable material a surface of a nerve at or adjacent to or in the vicinity of an area of damage, disease or graft, wherein said implantable material comprises a biocompatible matrix and cells and further wherein said implantable material is in an amount effective to treat the nerve in said individual.

2. The method of claim 1 wherein the biocompatible matrix is a flexible planar material.

3. The method of claim 1 wherein the biocompatible matrix is a flowable composition.

4. The method of claim 1 wherein the cells are endothelial, endothelial-like, non-endothelial cells, analogs thereof, or co-cultures of endothelial cells and non- endothelial cells.

5. The method of claim 1 wherein the nerve is a peripheral nerve.

6. The method of claim 1 wherein the nerve is a dorsal root.

7. The method of claim 1 wherein the nerve is a spinal cord.

8. The method of claim 1 wherein the damage or disease is a transected nerve.

9. The method of claim 8 wherein the damage or disease is a partially transected nerve.

10. The method of claim 1 wherein the damage or disease is a nerve graft.

11. The method of claim 1 wherein the damage or disease is a crushed nerve.

12. The method of claim 1 wherein the damage or disease is a pinched nerve.

13. The method of claim 1 wherein the damage or disease is an inflamed nerve.

14. The method of claim 1 wherein the damage or disease is an infection.

15. The method of claim 1 wherein the damage or disease is an autoimmune disease.

16. The method of claim 1 wherein the damage or disease is a dorsal root disorder.

17. The method of claim 1 wherein the damage or disease is a peripheral nerve disorder.

18. The method of claim 1 wherein the damage or disease is a demyelinating disorder.

19. The method of claim 18 wherein the damage or disease is multiple sclerosis.

20. The method of claim 1 wherein the damage or disease is a spinal cord disorder.

21. The method of claim 1 wherein the implantable material is applied to the exterior surface of the nerve or nerve graft.

22. The method of claim 1 wherein the implantable material controls inflammation.

23. The method of claim 1 wherein the implantable material controls fibroblast migration and proliferation.

24. The method of claim 1 wherein the implantable material inhibits negative tissue remodeling and/or promotes positive tissue remodeling.

25. The method of claim 1 wherein the implantable material promotes nerve fiber proliferation.

26. The method of claim 1 wherein the implantable material promotes axon outgrowth.

27. The method of claim 1 wherein the implantable material promotes neuronal connectivity.

28. The method of claim 1 wherein the implantable material promotes myelin migration and proliferation.

29. The method of claim 1 wherein the implantable material promotes maturation and patency of nerve anastomosis.

30. A composition suitable for the treatment or management of a nerve, the composition comprising a biocompatible matrix and cells, wherein said composition is in an amount effective to treat or manage the nerve.

31. The composition of claim 30 wherein the biocompatible matrix is a flexible planar material.

32. The composition of claim 30 wherein the biocompatible matrix is a flowable composition.

33. The composition of claim 32 wherein the flowable composition further comprises an attachment peptide and the cells are engrafted on or to the attachment peptide.

34. The composition of claim 30 wherein the cells are endothelial, endothelial- like, non-endothelial cells, analogs thereof or co-cultures of endothelial cells and non-endothelial cells.

35. The composition of claim 30 wherein the cells are selected from the group consisting of near-confluent cells, confluent cells, and post-confluent cells.

36. The composition of claim 30 wherein the cells are at least about 80% viable.

37. The composition of claim 30 wherein the cells are not exponentially growing.

38. The composition of claim 30 wherein the cells are engrafted to the biocompatible matrix via cell to matrix interactions.

39. The composition of claim 30 wherein the composition further comprises a second therapeutic agent.

40. The composition of claim 30 wherein the composition further comprises an agent that inhibits infection.

41. The composition of claim 30 wherein the composition further comprises an anti-inflammatory agent.

42. The composition of claim 30 wherein the composition further comprises an agent that promotes axon outgrowth.

43. The compositions of claim 42 wherein the agent is nerve growth factor (NGF).

44. The composition of claim 30 wherein the composition further comprises an attachment peptide.

45. A method of treating a cerebral aneurysm in an individual in need thereof, the method comprising:

contacting with an implantable material a surface of a cerebral artery at or adjacent to or in the vicinity of an area of damage or disease, wherein said implantable material comprises a biocompatible matrix and cells and further wherein said implantable material is in an amount effective to treat the cerebral aneurysm in said individual.

46. A composition suitable for the treatment or management of a cerebral aneurysm, the composition comprising a biocompatible matrix and cells, wherein said composition is in an amount effective to treat or manage the cerebral aneurysm.

47. A method of treating a nerve in an individual in need thereof, the method comprising:

applying an implantable material between one or more ends of a transected nerve, wherein said implantable material comprises a biocompatible matrix and cells and further wherein said implantable material acts as a structural and biological support for nerve growth and further wherein said implantable material is in an amount effective to treat the nerve in said individual.