WO2003026489A2 - Compositions de biopolymere et de biopolymere-cellule permettant de reparer un tissu nerveux - Google Patents

Compositions de biopolymere et de biopolymere-cellule permettant de reparer un tissu nerveux Download PDF

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WO2003026489A2
WO2003026489A2 PCT/US2002/030900 US0230900W WO03026489A2 WO 2003026489 A2 WO2003026489 A2 WO 2003026489A2 US 0230900 W US0230900 W US 0230900W WO 03026489 A2 WO03026489 A2 WO 03026489A2
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composition
microglia
compositions
foams
microporous
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PCT/US2002/030900
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WO2003026489A3 (fr
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University Of Florida
Goldberg, Eugene, P.
Streit, Jacob, Wolfgang
Stopek, Joshua, B.
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Publication of WO2003026489A3 publication Critical patent/WO2003026489A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A61L27/3878—Nerve tissue, brain, spinal cord, nerves, dura mater
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction

Definitions

  • neurons of the mammalian central nervous system have a poor capacity for axonal regeneration.
  • neurons of the mammalian peripheral nervous system have a substantially greater capacity for axonal regeneration.
  • macrophage-derived cytokines may promote formation of glial scars and thereby inhibit axonal regeneration (Khan and Wigley, NeuroReport 5:1381-1385 (1994); Vick et al., J. Neurotrauma 9:S93-S103 (1992)).
  • Laminin and thrombospondin both extracellular matrix components, have been shown to promote neuronal regeneration. It has also been demonstrated that microglia derived IL-I and TGF- have been shown to induce NGF production in astrocytes and Schwann cells, demonstrating an indirect neurotrophic role for these cytokines.
  • the cell culture studies have also led to a number of in vivo transplantation experiments involving microglia or peripheral macrophages. Microglia transplanted into the injured spinal cord in conjunction with fetal neural transplants have been shown to increase regeneration of DRG sensory fibers. Regeneration of descending tracts accompanied by improved locomotor function was observed when macrophages alone were implanted into the transected spinal cord.
  • ECM-derived proteins are potent promoters of neurite outgrowth (Archibald et al., J. Comp. Neurol. 4:685-696 (1991); Webb et al, Biomaterials. 22(10): 1017-1028 (2001)).
  • Novel methods of depositing surfactant- immobilized fibronectin have been reported to enhance bioactivity and sensory neurite outgrowth in vitro (Biran et al., J. Biomed. Mater. Res. 55(1): 1-12 (2001); Webb et al, J. Biomed. Mater. Res. 54(4): 509-518 (2001)).
  • Collagen devices/matrices carrying large payloads of Schwann cells or growth factors have been grafted into the lesioned adult spinal cord and a substantial amount of nerve fiber in-growth, both myelinated and unmyelinated, has been noted immunohistochemically (Paino et al., J. Neurocytol. 23(7): 433-452 (1994); Joosten et al., J. Neuroscience. 69(2):619-26 (1995); Liu et al., J. Neurosci. Res. 51(6):723- 34 (1998)).
  • NT-3 corticospinal tract fibers (CST), although no CST fibers grew into areas caudal to the collagen implant. Despite this, functional recovery has been observed (Houweling et al., Exp Neurol. 153(1): 49-59 (1998)).
  • a variety of neurotrophins including brain derived neurotrophic factor (BDNF), nerve growth factor (NGF), NT-3 and NT4/5 have been delivered in like fashions, most commonly in cell culture experiments.
  • Extracellular matrix or ECM-like polysaccharides have been of interest in tissue repair and wound healing, including the use of hyaluronic acid (HA), the alginates and agarose.
  • Hyaluronic acid is a natural component of mammalian ECM, and like alginate, carboxymethyl cellulose (CMC), chitosan or agarose, can be readily prepared as a viscous solution, microporous foam, film or gel, and easily crosslinked (Seeger et al., J. Surg. Res. 68:63-66(1996); Burns et al., JSurg. Res. 59:644-652(1995); Goldberg et al, Prog Clin. Biol. Res. 381:191-204 (1993); Yaacobi et al., J. Swrg. Re*. 55:422 (1993); Yaacobi et al., Ophthalmic Surg.
  • CMC carboxymethyl cellulose
  • Polysaccharide hydrogel scaffolds have been engineered to stimulate and guide neuronal process extension in three dimensions in vitro and in vivo (Yu et al., Tissue Eng. 4:291-304 (1999)); Bellakonda et al., supra. Bellakonda et al. elucidated the importance of gel concentration and pore size.
  • Primary neural cells do not extend neurites above a threshold agarose gel concentration of 1.25 % (w/v). Yu has also shown that neurite extension on these surfaces can be significantly increased in vitro when laminin is covalently coupled to the scaffold.
  • NeurogelTM (Organogel, Canada Ltee) a hydrogel of poly[N-2-(hydroxypropyl) methacrylamide] (PHPMA), has been reported. The polymer has been reported to bridge tissue defects and interface with host tissues. Immobilization of neuronal and glial cells within the PHPMA, as well as the incorporation of the RGD sequence (Woerly et al., Biomaterials. 17(3): 301-310 (1996); Woerly et al., Neurosci. Letters. 205(3): 197-201 (1996); Woerly et al., Biomater Sci Polym Ed.9(l): 681-711 (1998); Woerly et al., Biomaterials.
  • Natural phospholipid bilayers tend to be mechanically weak and unstable. This makes the synthesis of phospholipid bilayers in vitro very difficult.
  • a large number of synthetic phospholipid analogues many with polymerizable groups, have been studied in the past 25 years.
  • the present inventors have developed novel methods for the surface modification of biopolymers with phospholipids utilizing gamma radiation initiation polymerization (HydrograftTM), radio frequency (RF) plasma polymerization, radical polymerization and electropolymerization.
  • HydrograftTM gamma radiation initiation polymerization
  • RF radio frequency
  • CNS cellular membranes are comprised of approximately equal amounts of protein and phospholipid. It has been demonstrated that axonal synthesis of phospholipids is required for axonal growth (Posse de Chaves et al., J Cell Biol. 128(5): 913- 918 (1995)). During injury, the bulk of phospholipids are synthesized in the cell body and have to be transported to growth cones at the proximal axon stump, which may be some distance away. Fast anterograde axonal transport is thought to be the primary mechanism of transport for membrane-associated proteins and phospholipids.
  • Lipids are also transported from axons to myelin, although the significance of this process for myelination is not completely understood (Vance et al., Biochimica et Biophysica Acta. 1486(l):84-96 (2000)).
  • alternative sources of lipids as membrane materials required for axon extension have been proposed (de Chaves et al., JBiol. Chem. 49:30766-30773 (1997)).
  • the use of phospholipid biopolymers has been primarily focused on reducing the thrombogenicity of blood contact devices.
  • Arterial recanalization for stented balloon angioplasty has demonstrated significant restoration of function to diseased coronary and peripheral arteries using catheter based techniques.
  • Dacron vascular grafts (2mm), coated with polyMPC demonstrated in vivo patency after 5 days in a rabbit model. A significant reduction in platelet adhesion was also observed (Furuzono et al., Biomaterials. 21:327-333 (2000)).
  • Natural phospholipid bilayers tend to be mechanically weak and unstable. This makes the synthesis of phospholipid bilayers in vitro very difficult.
  • a large number of synthetic phospholipid analogues many with polymerizable groups, have been studied in the past 25 years.
  • the present inventors have developed novel methods for the surface modification of biopolymers with phospholipids utilizing gamma radiation initiation polymerization (HydrograftTM), radio frequency (RF) plasma polymerization, radical polymerization and electropolymerization.
  • HydrograftTM gamma radiation initiation polymerization
  • RF radio frequency
  • CNS cellular membranes are comprised of approximately equal amounts of protein and phospholipid. It has been demonstrated that axonal synthesis of
  • One embodiment of the invention relates to an implantable composition adapted for the stimulation of the growth of and the guidance of neural tissue across a lesion site in nerve tissue of a human or non-human mammal comprising: a) a space filling, cell compatible, bioerodable material that allows the growth therein and/or thereon of neural tissue, and b) seeded on and/or in the material, at least one biologically active cell type, the biologically active cell type being capable of producing, upon implantation of the composition in the mammal, at least one neurotrophic factor, growth factor, cytokine, extracellular matrix molecule or mixture thereof that is effective to provide neurotrophic support to mammalian nerve tissue.
  • FIG. 12 Further embodiments of the invention concern methods for the repair of injured neural tissue, e.g., axons, white matter lesions and injured peripheral nerves in human or non-human animals in need thereof comprising the implantation of the above-described composition in the animal near the site of injury between the proximal axon, white matter or peripheral nerve stumps and their respective distal segments.
  • injured neural tissue e.g., axons, white matter lesions and injured peripheral nerves in human or non-human animals in need thereof comprising the implantation of the above-described composition in the animal near the site of injury between the proximal axon, white matter or peripheral nerve stumps and their respective distal segments.
  • Another embodiment of the invention concerns a method of promoting neuronal regeneration in an injured spinal cord of a human or non-human animal comprising implanting the above composition into the spinal cord of the animal near the site of injury between the proximal axon stumps and their respective distal segments.
  • the implantable device comprises a solid, microporous and/or channeled bioerodable, space-filling, cell-compatible, polymer gel, film or foam scaffold, scaffold allowing the ingrowth on or through any pores, interstices or channels thereof of neural tissue, upon or into which the biologically active cell types, e.g., microglia/macrophages have been seeded.
  • the structure provides 1) scaffolding and guidance channels (e.g., microporous polysaccharide, protein, polymer or polynucleotide), 2) a favorable surface and 3) multiple trophic factors and/or pro-regenerative extracellular matrix (ECM) molecules, such as laminin and thrombospondin, which may be produced by the biologically active cell types or added thereto.
  • scaffolding and guidance channels e.g., microporous polysaccharide, protein, polymer or polynucleotide
  • ECM extracellular matrix
  • the crux of the invention resides in the unique multicomponent composition of biopolymer materials and biological (cellular) constituents.
  • the bulk of the proposed implants may be natural polysaccharides, proteins and/or polynucleotide polymers (i.e., hyaluronic acid, gelatin, RNA and/or DNA, all derived from vegetables, fruit, plants, bacteria and/or human and/or non-human animals) or synthetic biopolymer material.
  • biopolymer materials have been synthesized and/or are available in a variety of architectures, molecular weights and their bioerodability as implants may be controlled by their physical and chemical properties including molecular structure, crosslink density and type of crosslinker.
  • Such biopolymers have been shown to be safe and efficacious in a plethora of biomedical applications including ophthalmological, cardiovascular and other soft tissue devices.
  • HA hyaluronic acid
  • CMC carboxymethyl cellulose
  • the alginates have found applications in neural, orthopedic, dental and a variety of soft tissue implants, including cell immobilization and encapsulation.
  • poly(vinyl pyrrolidone) PVP
  • poly (methacrylic acid) PMAA
  • poly(2-hydroxyethyl) methacrylate pHEMA
  • PA poly(methacrylamide)
  • PMA poly(2-hydroxyethyl) methacrylamide
  • PPA poly(methacrylamide)
  • PPA poly(methacrylamide)
  • PPHEMAA poly(2-hydroxyethyl) methacrylamide
  • PMAmPCs poly(methacryoylaminoalkyl) phosphorylcholines
  • PSPAs poly(sulfopropyl) acrylates
  • PSPMAs poly (sulfopropyl) methacrylates)
  • PMAs poly(methacrylates) or copolymers thereof.
  • phospholipid biopolymers as polymers of methacryloyloxyethyl phosphorylcholine (MPC), methacryloyloxypropyl phosphorylcholine, methacryloyloxybutyl phosphorylcholine, methacryloylaminoalkyl phosphorylcholine or similar phosphorylcholine containing polymers may be employed.
  • MPC methacryloyloxyethyl phosphorylcholine
  • methacryloyloxypropyl phosphorylcholine methacryloyloxybutyl phosphorylcholine
  • methacryloylaminoalkyl phosphorylcholine or similar phosphorylcholine containing polymers may be employed.
  • mixtures of the various bio-polymeric materials may also be used in the practice of the invention.
  • the devices of the invention are made as microporous and/or channeled structural substrates.
  • Such compositions have been shown to preferably adsorb endogenous phospholipids, which play a critical role in wound healing.
  • a unique and important advantage of such compositions is also their ability to be loaded with a variety of drugs, growth factors, extracellular matrix molecules, cells or transgene-containing viral vectors.
  • the composition of the invention additionally includes at least one substance effective, upon implantation of the composition in the mammal, of providing neurotrophic support to mammalian nerve tissue.
  • substances include, e.g., gene therapy agents, steroids, growth factors, cytokines, extracellular matrices and the like or mixture thereof.
  • the composition additionally contains a neurotrophic agent that promotes neural tissue regeneration, repair or both, such as, e.g., phospholipids, polymeric phospholipids, laminin, laminin-like IKVAV protein polymers, fibronectin, fibronectin-like RGD protein polymers, thrombospondin, hyaluronic acid, collagen and the like or mixtures thereof.
  • a neurotrophic agent that promotes neural tissue regeneration, repair or both, such as, e.g., phospholipids, polymeric phospholipids, laminin, laminin-like IKVAV protein polymers, fibronectin, fibronectin-like RGD protein polymers, thrombospondin, hyaluronic acid, collagen and the like or mixtures thereof.
  • compositions of this invention are uniquely suitable for the seeding of brain microglia and/or peripheral macrophages either onto their surfaces, or encapsulated within.
  • other cell types which have been shown to assist neural tissue repair, such as e.g., Schwann cells, neurons, ependymal cells, peripheral stem cells, neural stem cells, oligodendrocytes, astrocytes, T cells, fibroblasts or mixtures thereof may be employed.
  • Various combinations of microglia, peripheral macrophages, Schwann cells, astrocytes and stem cells may be also used to populate the substrate compositions to provide a combination of cell types that would optimally produce the complex array of neurotrophic factors to enhance nerve tissue regeneration.
  • these tissue repair devices are implantable structures comprised of biodegradable biopolymer compositions and structures incorporating
  • brain microglia peripheral macrophage and/or other neural and glial cells including Schwann cells and stem cells.
  • the biologically active cell types are capable of producing, upon implantation of the composition in the mammal, BDNF, NGF, NT-3, NT-4/5, TGF-beta, GM-CSF, TNF-alpha, VEGF, bFGF, IGF-a or mixtures thereof.
  • the biologically active cell types are also capable of producing, upon implantation, IL-1, IL-1 beta, IL-6, IL-8, IL-10, IL-12 or mixtures thereof.
  • the implantable devices may be used to repair white matter lesions resulting from autoimmune and/or degenerative conditions, such as multiple sclerosis, leukodystrophy, and leucoaraiosis, and for peripheral nerve repair, in particular, for the treatment of neuromas.
  • the compositions of the invention are surgically implantable and/or surgically/minimally invasively injectable.
  • microglia and/or macrophages After spinal cord injury, e.g., the implantation of microglia and/or macrophages into the lesion site can stimulate endogenous regenerative nerve fiber growth and the recruitment of other pro-regenerative cells such as Schwann cells.
  • the present invention provides a unique structural support and stable terrain for such cells in the form of a synthetic, channeled/microporous scaffold, which is designed to completely fill the injury/lesion site. These compositions have been designed to prevent or reverse the complications associated with cystic cavitation, which may jeopardize the regrowth of regenerating/surviving neural tissue.
  • the microporous implant compositions quickly recruit a dense cellular infiltrate, which forms an intimate interface between injured and healthy tissue, preventing additional tissue loss or damage.
  • the invention to provides a continuous cellular source of trophic factors and
  • biopolymer and biopolymer-cell compositions promote endogenous regeneration and wound healing in the repair of the injured rubrospinal tract of the adult rat spinal cord. It has been shown that these implant compositions fill the injury/lesion site and are non-inflammatory in nature.
  • the implant compositions of the invention support a large biologically active cell type infiltrate comprised preferably of microglia, macrophages, Schwann cells, neurons, ependymal cells, peripheral stem cells, neural stem cells, oligodendrocytes, astrocytes, T cells, fibroblasts or mixtures thereof.
  • NF-M Neurofilament
  • CGRP calcitonin gene related peptide
  • biopolymer-cell implant compositions recover greater coordinated motor function versus controls (lesion only).
  • compositions and structures of this invention are designed to promote the growth and regeneration of axons in the CNS, which have been severed by injury or otherwise destroyed by disease processes. Injured or damaged CNS axons do not normally regenerate or reconnect and, at best, there may only be aberrant, misguided, and/or stunted growth.
  • the reasons for poor axonal regeneration in the CNS are largely due to a growth-inhibiting environment in the CNS which is marked by glial scarring, presence of inhibitory myelin protein, and a weak neurotrophic response.
  • the devices of the present invention overcome these obstacles.
  • the polysaccharide foam structures help prevent scar formation
  • the macrophages seeded onto the foam polymer are phagocytic and remove myelin which contains inhibitory proteins and, at the same time, they produce an abundance of growth factors and other proregenerative molecules.
  • the implant is microporous and/or channeled similar to that of an oriented sponge
  • the pores/channels provide mechanical support and guidance for regenerating axons.
  • the bulk of the device is composed of the biopolymer, e.g. polysaccharide.
  • the bulk biopolymer component is bioerodable, dissolves over time and is excreted, enzymatically degraded or adsorbed to endogenous tissue.
  • a device of this type promotes regeneration and results in severed axons being re-connected with greatly enhanced functional neurological recovery.
  • Preferred products are implants used to bridge a spinal cord lesion. Chronic spinal cord injuries often produce a cavity which further impedes the already weak regenerative potential of injured axons.
  • the implants of this invention will temporarily fill the cavity providing a scaffold that is highly attractive for axons to grow on and to be guided through to the other side of the cavity. Additionally, the devices of the invention can be used for the
  • the device might be particularly useful for repairing peripheral nerves, since peripheral nerves do have a strong innate potential for regeneration.
  • An implant of the kind described here, placed into a peripheral nerve lesion offers the potential to prevent formation of post-traumatic neuromas which can be the cause of considerable pain and distress.
  • a major benefit of the present invention is clearly the opportunity for functional recovery from traumatic spinal cord injury.
  • spinal cord injury there is no cure for spinal cord injury, and typically spinal cord injury patients spend the rest of their lives in wheelchairs. This creates enormous heath care and rehabilitation costs which are in the billions annually in the United States alone.
  • the proposed implantable prosthesis would allow patients to regain partial or full neural and motor functions and in order to move and even walk again.
  • the present invention is predicated on the discovery that the combination of both neurobiology and material science enable the development of novel bioerodable, implantable devices that supply the necessary components to facilitate axonal regrowth across, e.g., a spinal cord lesion.
  • One prototypical implantable device of the invention comprises a microporous and/or channeled polysaccharide foam, on which microglia may be seeded.
  • the strategy is to provide essential components that will foster axon regeneration: (1) temporary structural support and guidance channels in the form of a polysaccharide scaffold, and (2) trophic and tropic support by microglia or other proregenerative cells and/or therapeutic agents.
  • Solutions of 2% (w/v) alginate were prepared using high-speed mechanical mixing and air pressure filtration techniques. Tris buffer was used as the solvent. Solutions were pressure filtered through 70 micron Spectra filters into sterile 500 ml glass screw top bottles. One ml of alginate solution was pressure injected into each well of a 12 well Falcon (Sigma) cell culture plate. The plate was sealed with parafilm, solution allowed to settle and frozen overnight in a - 20 °C freezer. The plate was loaded into a cold 900 ml Labconco flash freeze flask and lyophilized overnight (-10 ⁇ m Hg, -50 °C).
  • Alginate microporous foam samples were removed from the culture plates using microforceps and placed into sterile 50 ml centrifuge tubes. Twenty milliliters of 0.1 M CaCl crosslinking solution was pipetted into each tube. Alginate foams were allowed to incubate in the crosslinking solution for 30 minutes. The tubes were drained and foams repeatedly washed with nanopure water for 2 days. Foams were autoclaved fully hydrated, suspended in 30 ml nanopure water, on a programmed liquid cycle. These preparations were stored refrigerated until further biological use.
  • rat microglia were cultured from perinatal rat brains and isolated as follows. Briefly, rat whole brains were stripped of meninges while immersed in dissociation solution (D). Clean fragments were mechanically minced, transferred to a 50 ml conical tube, and incubated under bi-directional rotation in 0.05% trypsin in solution "D” for 20 minutes at 37°C. An equal volume of minimal essential medium (MEM) containing 10% (v/v) fetal
  • MEM minimal essential medium
  • sterile microporous alginate foams were cut to approximately lmm 3 and placed into sterile 500 ⁇ l microfuge tubes. Microglia were harvested as described above. Cell viability was confirmed by vital staining. A dense microglial cell suspension was micropipetted into the microfuge tube. The tube was placed on an orbital rotator in an incubator for one hour prior to implantation into the injured rat spinal cord. Immediately prior to surgery, tubes were placed on ice.
  • Rats were placed under isoflurane anesthetia. Using asceptic precautions, the neck musculature was split midline and the lamina of the fourth or fifth vertebra were partially removed. After opening the dura, the right dorsolateral funiculus of the spinal cord was be cut with a No. 11 scalpel blade in order to transect the rubrospinal tract. Upon hemostasis, microporous alginate implants or microporous alginate implants cultured with primary rat microglia were immediately placed into the lesion/injury site. Control animals did not receive an implant (lesion only). The musculature/wound was washed with sterile saline and
  • mice Prior to euthanasia, animals were either retrogradely tract traced with Fluorogold (FG) or anterogradely tract traced with biotinylated dextran amine (BDA). The animals were fixation perfused and spinal cord and brain removed intact. The collected tissue was post- fixed in 4% (w/v) paraformaldehyde. Spinal cord tissue was cryo-preserved with 30% (w/v) sucrose in phosphate buffered saline. The red nucleus in the rat midbrain was vibratome sectioned (50 microns) and counterstained with 3% (w/v) cresyl violet (CV).
  • FG Fluorogold
  • BDA biotinylated dextran amine
  • Sections were viewed under brightfield optical microscopy and red nucleus neurons were enumerated using stereological counting techniques and the MCID system. FG labeled red nucleus neurons were enumerated in a similar fashion (without CV), except using fluorescence microscopy. Digital images of serial sections were captured for the entire red nucleus.
  • Cryosectioned spinal cord specimens were cut (-25°C, 20 microns) and processed using immunohistochemical staining techniques.
  • Antibodies against neurofilaments, macrophages, microglia, astrocytes, Schwann cells, monocytes, peripheral nerve fibers, proregenerative proteins and nerve cells were employed using biotin/avidin/peroxidase labeling.
  • Diaminobenzidine was used as the chromophore. Sections were imaged using optical microscopy and fluorescence.
  • microporous alginate foam microglia compositions showed immunopositive regenerating/surviving axons growing into the microporous alginate implant.
  • the implant filled the lesion site.
  • Glial scarring was minimal and astrocytes were found throughout the interface of the host tissue/microporous alginate implant.
  • Immunostaining for peripheral nerve fibers (calcitonin gene related peptide (CGRP)) was negative, indicating neurofilament positive fibers were of CNS origin. Alpha-internexin neurofilament reactivity was also found. Cresyl violet counterstaining revealed a dense, cellular infiltrate, that
  • Covalently crosslinked alginate microporous foams were removed from the culture plates using microforceps and placed into sterile 50 ml centrifuge tubes. Microporous foams were autoclaved fully hydrated, suspended in 30 ml nanopure water, on a programmed liquid cycle. These preparations were stored refrigerated until further biological use.
  • Example 23 Primary rat microglia cultures were prepared as detailed in Example 1. In a biological cabinet, sterile microporous alginate foams were sized to approximately 1mm 3 and placed into sterile 500 ⁇ l microfuge tubes. Microglia were harvested as described above. Cell viability was measured by vital staining. A dense microglial cell suspension was micropipetted into the microfuge tube. The tube was placed on an orbital rotator in an incubator for one hour prior to implantation into the injured rat spinal cord. Immediately prior to surgery, tubes were placed on ice.
  • Microporous alginate implants cultured with primary rat microglia were implanted as detailed in Example 1. Euthanasia, tract tracing and immunohistochemical analysis was conducted as previously noted in Example 1. Results of in vivo analysis of these compositions were also as those described in Example 1 and demonstrated that covalently crosslinked alginate/microglia compositions promoted the regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using microglia containing gel compositions.
  • HA hyaluronic acid
  • the plate was loaded into a cold 900 ml Labconco flash freeze flask and lyophilized overnight (-10 ⁇ m Hg, -50 °C).
  • HA microporous foams were removed from the culture plates using microforceps and placed into sterile 50 ml centrifuge tubes. Twenty milliliters of 0.025 M CrK(SO 4 ) 2 crosslinking solution was pipetted into each tube. Microporous HA foams were allowed to incubate in the crosslinking solution for 30 minutes. The tubes were drained and foams repeatedly washed with nanopure water for 2 days. Microporous foams were autoclaved fully hydrated, suspended in 30 ml nanopure water, on a programmed liquid cycle.
  • Example 1 Primary rat microglia cultures were prepared as detailed in Example 1. In a biological cabinet, sterile microporous alginate foams were cut to approximately 1mm 3 and placed into sterile 500 ⁇ l microfuge tubes. Microglia were harvested as noted above. Cell viability was confirmed by vital staining. A dense microglial cell suspension was micropipetted into the microfuge tube. The tube was placed on an orbital rotator in an incubator for one hour prior to implantation into the injured rat spinal cord. Immediately prior to surgery, tubes were placed on ice.
  • Microporous HA implants cultured with primary rat microglia were implanted as detailed in Example 1. Euthanasia, tract tracing and immunohistochemical analysis was conducted as previously noted in Example 1. Results of in vivo analysis of these compositions were also as those described in Example 1 and demonstrate that HA/microglia compositions promoted regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using microglia containing gel compositions.
  • HYALURONIC ACD3 COMPOSITIONS CONTAINING PRIMARY MICROGLIA
  • HA hyaluronic acid
  • Covalently crosslinked HA microporous foams were removed from the culture plates using microforceps and placed into sterile 50 ml centrifuge tubes. Microporous foams were autoclaved fully hydrated, suspended in 30 ml nanopure water, on a programmed liquid cycle.
  • rat microglia cultures were prepared as detailed in Examplel .
  • sterile microporous alginate foams were cut to approximately 1mm 3 and placed into sterile 500 ⁇ l microfuge tubes.
  • Microglia were harvested as described above. Cell viability was confirmed by vital staining.
  • a dense microglial cell suspension was micropipetted into the microfuge tube. The tube was placed on an orbital rotator in an incubator for one hour prior to implantation into the injured rat spinal cord. Immediately prior to surgery, tubes were placed on ice.
  • Example 26 Microporous HA implants cultured with primary rat microglia were implanted as detailed in Example 1. Euthanasia, tract tracing and immunohistochemical analysis was conducted as previously noted in Example 1. Results of in vivo analysis of these compositions were also as those described in Example 1 and demonstrated that HA microglia compositions promoted regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using microglia containing gel compositions.
  • CMC CELLULOSE COMPOSITIONS CONTAINING PRIMARY MICROGLIA
  • CMC carboxymethyl cellulose
  • CMC microporous foams were removed from the culture plates using microforceps and placed into sterile 50 ml centrifuge tubes. Twenty milliliters of 0.025 M CrK(SO 4 ) 2 crosslinking solution was pipetted into each tube. Microporous CMC foams were allowed to incubate in the crosslinking solution for 30 minutes. The tubes were drained and foams repeatedly washed with nanopure water for 2 days. Microporous foams were autoclaved fully hydrated, suspended in 30 ml nanopure water, on a programmed liquid cycle.
  • Example 27 Primary rat microglia cultures were prepared as detailed in Example 1. In a biological cabinet, sterile microporous alginate foams were cut to approximately 1mm 3 and placed into sterile 500 ⁇ l microfuge tubes. Microglia were harvested as described above. Cell viability was confirmed by vital staining. A dense microglial cell suspension was micropipetted into the microfuge tube. The tube was placed on an orbital rotator in an incubator for one hour prior to implantation into the injured rat spinal cord. Immediately prior to surgery, tubes were placed on ice.
  • Microporous CMC implants cultured with primary rat microglia were implanted as detailed in Example 1. Euthanasia, tract tracing and immunohistochemical analysis was conducted as previously noted in Example 1. Results of in vivo analysis of these compositions were also as those described in Example 1 and demonstrated that CMC/microglia compositions promote regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using microglia containing gel compositions.
  • Solutions of 1% (w/v) agarose were prepared using high-speed mechanical mixing techniques at 45°C. Phosphate buffer containing 0.025 M calcium phosphorylcholine was used as the solvent. Solutions were stored in sterile 200 ml glass screw top bottles. Prior to plating, the agarose solution was microwaved on high power for thirty seconds to one minute. One milliliter of agarose solution was pressure injected into each well of a 12 well Falcon (Sigma) cell culture plate. The plate was sealed with parafilm, solution allowed to settle and gel, and frozen overnight in a - 20 °C freezer. The plate was loaded into a cold 900 ml
  • Agarose microporous foams were removed from the culture plates using microforceps and stored in sterile 50 ml conicals. Dry agarose foams were ethylene oxide sterilized.
  • Primary rat microglia cultures were prepared as detailed in Example 1. In a biological cabinet, sterile microporous agarsose foams were cut to approximately 1mm 3 and placed into sterile 500 ⁇ l microfuge tubes. Microglia were harvested as described above. Cell viability was confirmed by vital staining. A dense microglial cell suspension was micropipetted into the microfuge tube. The tube was placed on an orbital rotator in an incubator for one hour prior to implantation into the injured rat spinal cord. Immediately prior to surgery, tubes were placed on ice.
  • the agarose/microglial composition is implanted into the injured adult spinal cord as described in Example 1. Euthanasia, tract tracing and immunohistochemical analysis is conducted as previously noted in Example 1. Results of in vivo analysis of these compositions is similar to those described in Example 1 and demonstrated that agarose/microglia compositions promote regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using microglia containing gel compositions.
  • chitosan solutions were prepared using high-speed mechanical mixing techniques in dilute acetic acid at pH 4.5. Phosphate buffer containing 0.025 M calcium chloride was used as the solvent. Chitosan solutions were air pressure filtered (70 micron) into in sterile 200 ml glass screw top bottles. One ml of chitosan solution was pressure
  • Chitosan microporous foams were removed from the culture plates using microforceps and stored in sterile 50 ml conicals. Foams were covalently crosslinked with genipin (40mg/ml) overnight on a clinical rotator. Reaction was allowed to continue until a strong blue chromophore was detected. Foams were washed repeatedly with nanopure water to remove excess genipin. Foams were dried under vacuum at ambient temperature and ethylene oxide sterilized. Prior to implantation, foams were washed repeatedly with sterile filtered cell culture media (without protein or serum).
  • Example 1 Primary rat microglia cultures were prepared as detailed in Example 1. In a biological cabinet, sterile microporous chitosan foams were cut to approximately 1mm 3 and placed into sterile 500 ⁇ l microfuge tubes. Microglia were harvested as described above. Cell viability was confirmed by vital staining. A dense microglial cell suspension was micropipetted into the microfuge tube. The tube was placed on an orbital rotator in an incubator for one hour prior to implantation into the injured rat spinal cord. Immediately prior to surgery, tubes were placed on ice.
  • the chitosan/microglial composition was implanted into the injured adult spinal cord as described in Example 1. Euthanasia, tract tracing and immunohistochemical analysis was conducted as noted in Example 1. Results of in vivo analysis of these compositions was similar to those described in Example 1 and demonstrated that chitosan/microglia compositions promote regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using microglia containing gel compositions.
  • Dextran microporous foams were removed from the culture plates using microforceps and stored in sterile 50 ml conicals. Foams were covalently crosslinked with genipin (40mg/ml) overnight on a clinical rotator. Reaction was allowed to continue until a strong blue chromophore was detected. Foams were washed repeatedly with nanopure water to remove excess genipin. Foams were dried under vacuum at ambient temperature and ethylene oxide sterilized. Prior to implantation, foams were washed repeatedly with sterile filtered cell culture media (without protein or serum).
  • Example 1 Primary rat microglia cultures were prepared as detailed in Example 1. In a biological cabinet, sterile microporous chitosan foams were cut to approximately 1mm 3 and placed into sterile 500 ⁇ l microfuge tubes. Microglia were harvested as described above. Cell viability was confirmed by vital staining. A dense microglial cell suspension was micropipetted into the microfuge tube. The tube was placed on an orbital rotator in an incubator for one hour prior to implantation into the injured rat spinal cord. Immediately prior to surgery, tubes were placed on ice.
  • Example 31 The dextran/microglial composition is implanted into the injured adult spinal cord as described in Example 1. Euthanasia, tract tracing and immunohistochemical analysis is conducted as previously noted in Example 1. Results of in vivo analysis of these compositions is similar to those described in Example 1 and demonstrated that dextran/microglia compositions promote regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using microglia containing gel compositions.
  • Viscous solutions of 10% (w/v) gelatin were prepared at 45°C. Tris buffer was used as the solvent. Solutions were stored in sterile 200 ml glass screw top bottles. Prior to plating, the gelatin solution was gently heated in a vacuum oven to 45 °C. One milliliter of gelatin solution was pressure injected into each well of a 12 well Falcon (Sigma) cell culture plate. The plate was sealed with parafilm, solution allowed to settle, cool to ambient temperature and gel, and frozen overnight in a - 20 °C freezer. The plate was loaded into a cold 900 ml Labconco flash freeze flask and lyophilized overnight (-10 ⁇ m Hg, -50 °C). Microporous gelatin foams were crosslinked and further processed as detailed in Example 5.
  • Example 1 Primary rat microglia cultures were prepared as detailed in Example 1. In a biological cabinet, sterile microporous chitosan foams were sized to approximately 1mm 3 and placed into sterile 500 ⁇ l microfuge tubes. Microglia were harvested as described above. Cell viability was confirmed by vital staining. A dense microglial cell suspension was micropipetted into the microfuge tube. The tube was placed on an orbital rotator in an
  • the gelatin composition is implanted into the injured adult spinal cord as described in Example 1. Euthanasia, tract tracing and immunohistochemical analysis is conducted as previously noted in Example 1. Results of in vivo analysis of these compositions is similar to those described in Example 1 and demonstrate that microporous gelatin implant compositions promote the regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using microglia containing gel compositions.
  • HSA human serum albumin
  • Phosphate buffer was used as the solvent. Solutions were microfiltered (70 micron) into sterile 200 ml glass screw top bottles. Solutions were stored refrigerated. One ml of HSA solution was pressure injected into each well of a 12 well Falcon (Sigma) cell culture plate. The plate was sealed with parafilm, solution allowed to settle and frozen overnight in a - 20 °C freezer. The plate was loaded into a cold 900 ml Labconco flash freeze flask and lyophilized overnight (-10 ⁇ m Hg, -50 °C). Microporous HSA foams were crosslinked and further processed as detailed in Example 5.
  • Phosphate buffer was used as the solvent. Solutions were microfiltered (70 micron) into sterile 200 ml glass screw top bottles. Solutions were stored refrigerated. One ml of HSA solution was pressure injected into each well of a 12 well Falcon (Sigma) cell culture plate. The plate was sealed with parafilm, solution allowed to settle and frozen overnight in
  • Example 1 Primary rat microglia cultures were prepared as detailed in Example 1. In a biological cabinet, sterile microporous HSA foams were sized to approximately 1mm 3 and placed into sterile 500 ⁇ l microfuge tubes. Microglia were harvested as described above. Cell viability was confirmed by vital staining. A dense microglial cell suspension was micropipetted into
  • the tube was placed on an orbital rotator in an incubator for one hour prior to implantation into the injured rat spinal cord. Immediately prior to surgery, tubes were placed on ice.
  • the HSA composition is implanted into the injured adult spinal cord as described in Example 1. Euthanasia, tract tracing and immunohistochemical analysis is conducted as previously mentioned in Example 1. Results of in vivo analysis of these compositions is similar to those described in Example 1 and demonstrate that microporous HSA implant compositions promote the regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using microglia containing gel compositions.
  • Microporous alginate foams were synthesized as detailed in Example 1. Sterile foams are incubated in recombinant human BDNF (0.75 mg/ml) in an incubator (37°C, 8% CO 2 ) for 6 to 12 hours.
  • Example 1 In a biological cabinet, sterile microporous alginate foams were sized to approximately 1mm 3 and placed into sterile 500 ⁇ l microfuge tubes.
  • the microporous alginate implant adsorbed with BDNF is implanted as detailed in Example 1. Euthanasia, tract tracing and immunohistochemical analysis is conducted as previously mentioned in Example 1. Results of in vivo analysis of these compositions is similar to those noted in Example 1 and demonstrate that alginate compositions for the localized, controlled and sustained release of BDNF promote the regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using gel compositions.
  • Microporous alginate foams were synthesized as detailed in Example 1. Sterile foams are incubated in recombinant human NT-3 (0.75 mg/ml) in an incubator (37°C, 8%
  • Example 1 In a biological cabinet, sterile microporous alginate foams were sized to approximately 1mm 3 and placed into sterile 500 ⁇ l microfuge tubes. The microporous alginate implant adsorbed with NT-3 is implanted as detailed in Example 1. Euthanasia, tract tracing and immunohistochemical analysis is conducted as previously noted in Example 1. Results of in vivo analysis of these compositions is similar to those described in Example 1 and demonstrate that alginate compositions for the localized, controlled and sustained release of NT-3 promote the regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using gel compositions.
  • Microporous alginate foams were synthesized as detailed in Example 1. Sterile foams are incubated in recombinant human NGF (0.1 mg/ml) in an incubator (37°C, 8% CO 2 ) for 6 to 12 hours.
  • microporous alginate foams were sized to approximately 1mm 3 and placed into sterile 500 ⁇ l microfuge tubes.
  • Example 35 alginate implant adsorbed with NGF is implanted as detailed in Example 1. Euthanasia, tract tracing and immunohistochemical analysis is conducted as previously noted in Example 1. Results of in vivo analysis of these compositions is similar to those described in Example 1 and demonstrate that alginate compositions for the localized, controlled and sustained release of NGF promote the regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using gel compositions.
  • Microporous alginate foams were synthesized as detailed in Example 1. Sterile foams are incubated in recombinant human M-CSF (0.1 mg/ml) in an incubator (37°C, 8% CO 2 ) for 6 to 12 hours.
  • Example 1 In a biological cabinet, sterile microporous alginate foams were sized to approximately 1mm 3 and placed into sterile 500 ⁇ l microfuge tubes.
  • the microporous alginate implant adsorbed with M-CSF is implanted as detailed in Example 1. Euthanasia, tract tracing and immunohistochemical analysis is conducted as previously noted in Example 1. Results of in vivo analysis of these compositions is similar to those described in Example 1 and demonstrate that alginate compositions for the localized, controlled and sustained release of M-CSF promote the regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using gel compositions.
  • Microporous alginate foams were synthesized as detailed in Example 1. Sterile foams are incubated in recombinant human NT-3 (0.75 mg/ml) and BDNF (0.75 mg/ml) in an incubator (37°C, 8% CO 2 ) for 6 to 12 hours.
  • sterile microporous alginate foams were sized to approximately 1mm 3 and placed into sterile 500 ⁇ l microfuge tubes.
  • the microporous alginate implant adsorbed with NT-3 and BDNF is implanted as detailed in Example 1. Euthanasia, tract tracing and immunohistochemical analysis is conducted as previously noted in Example 1. Results of in vivo analysis of these compositions is similar to those described in Example 1 and demonstrate that alginate compositions for the localized, controlled and sustained release of the combination of NT-3 and BDNF promote the regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using gel compositions.
  • Microporous alginate foams were synthesized as detailed in Example 1. Sterile foams are incubated in fibronectin (10 mg/ml) in an incubator (37°C, 8% CO 2 ) for 6 to 12 hours. Following the incubation period, foams were washed three times with sterile minimal essential media (MEM).
  • fibronectin 10 mg/ml
  • MEM sterile minimal essential media
  • Example 37 In a biological cabinet, sterile microporous alginate foams were sized to approximately 1mm 3 and placed into sterile 500 ⁇ l microfuge tubes.
  • the microporous alginate implant adsorbed with fibronection is implanted as detailed in Example 1. Euthanasia, tract tracing and immunohistochemical analysis is conducted as previously noted in Example 1. Results of in vivo analysis of these compositions is similar to those described in Example 1 and demonstrate that alginate compositions adsorbed with the ECM molecule fibronectin promote the regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using gel compositions.
  • Microporous alginate foams were synthesized as detailed in Example 1. Sterile foams are incubated in the fibronectin-like engineered protein polymer RGD (0.7 mg/ml) in an incubator (37°C, 8% CO 2 ) for 6 to 12 hours. Following the incubation period, foams were washed three times with sterile minimal essential media (MEM).
  • RGD fibronectin-like engineered protein polymer
  • sterile microporous alginate foams were sized to approximately 1mm 3 and placed into sterile 500 ⁇ l microfuge tubes.
  • the microporous alginate implant adsorbed with RGD is implanted as detailed in Example 1. Euthanasia, tract tracing and immunohistochemical analysis is conducted as previously noted in Example 1. Results of in vivo analysis of these compositions is similar to those described in Example 1 and demonstrate that alginate compositions adsorbed with the ECM-like molecule RGD promote the regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using gel compositions.
  • Microporous alginate foams were synthesized as detailed in Example 1. Sterile foams are incubated in the ECM gel matrix derived from the Swarm mouse sarcoma (diluted to 0.5 mg/ml) in an incubator (37°C, 8% CO 2 ) for 6 to 12 hours. Following the incubation period, foams were washed three times with sterile minimal essential media (MEM).
  • ECM sterile minimal essential media
  • sterile microporous alginate foams were sized to approximately 1mm 3 and placed into sterile 500 ⁇ l microfuge tubes.
  • the microporous alginate implant adsorbed with ECM is implanted as detailed in Example 1. Euthanasia, tract tracing and immunohistochemical analysis is conducted as previously noted in Example 1. Results of in vivo analysis of these compositions is similar to those described in Example 1 and demonstrate that alginate compositions adsorbed with ECM promotes the regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using gel compositions.
  • Microporous alginate foams were synthesized as detailed in Example 1. Sterile foams are incubated in laminin from human placenta (ECM) (diluted to 0.5 mg/ml) in an incubator (37°C, 8% CO 2 ) for 6 to 12 hours. Following the incubation period, foams were washed three times with sterile minimal essential media (MEM).
  • ECM human placenta
  • microporous alginate foams were sized to approximately 1mm 3 and placed into sterile 500 ⁇ l microfuge tubes.
  • alginate implant adsorbed with ECM is implanted as detailed in Example 1. Euthanasia, tract tracing and immunohistochemical analysis is conducted as previously noted in Example 1. Results of in vivo analysis of these compositions is similar to those described in Example 1 and demonstrate that alginate compositions adsorbed with ECM promotes the regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using gel compositions.
  • Microporous alginate foams were synthesized as detailed in Example 1. Sterile foams are incubated in the IKVAV polymer (diluted to 0.3 mg/ml) in an incubator (37°C, 8% CO 2 ) for 6 to 12 hours. Following the incubation period, foams were washed three times with sterile minimal essential media (MEM).
  • MEM sterile minimal essential media
  • sterile microporous alginate foams were sized to approximately 1mm 3 and placed into sterile 500 ⁇ l microfuge tubes.
  • the microporous alginate implant adsorbed with the laminin-like IKVAV polymer is implanted as detailed in Example 1. Euthanasia, tract tracing and immunohistochemical analysis is conducted as previously noted in Example 1. Results of in vivo analysis of these compositions is similar to those described in Example 1 and demonstrate that alginate compositions adsorbed with IKVAV laminin promotes the regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using gel compositions.
  • DNA was extracted from yellow onion following standard isolation protocol and precipitated in a non-solvent (isopropanol). Phosphate buffer was used as the solvent. Solutions were repeatedly washed in 95% ethanol and vacuum dried. The DNA precipitate was loaded into 12 well cell culture plates, frozen overnight (- 20°C freezer) and lyophilized overnight (-10 ⁇ m Hg, -50°C). Viscous solutions of 1.5% (w/v) DNA (Kelco HV) were prepared using high-speed mechanical mixing techniques. One milliliter of DNA solution was pressure injected into each well of a 12 well Falcon (Sigma) cell culture plate. The plate was sealed with parafilm, solution allowed to settle and frozen overnight in a - 20°C freezer. The plate was loaded into a cold 900 ml Labconco flash freeze flask and lyophilized overnight (-10 ⁇ m Hg, -50°C).
  • DNA microporous foams were removed from the culture plates using microforceps and placed into sterile 50 ml centrifuge tubes. Twenty milliliters of 0.025 M CrK(SO 4 ) 2 and 50mM glutathione crosslinking solution was pipetted into each tube. Ionically crosslinked microporous DNA foams were allowed to incubate in the crosslinking solution for 30 minutes. The tubes were drained and foams repeatedly washed with nanopure water for 2 days and dried under vacuum. Microporous foams were ethylene oxide sterilized and aerated for 24 hours. These preparations were stored refrigerated until further biological use.
  • rat microglia were cultured and seeded on DNA foams as detailed in Example 1.
  • sterile DNA foams were sized to approximately 1mm 3 and placed into sterile 500 ⁇ l microfuge tubes containing culture media (no serum or protein). Microglia were harvested as described above. Cell viability was confirmed by vital staining.
  • the media was aspirated from the microfuge tube and a dense microglial cell suspension was micropipetted into the tube.
  • the tube was placed on an orbital rotator in an incubator for one hour prior to implantation into the injured rat spinal cord. Immediately prior to surgery, tubes were placed on ice.
  • microporous DNA implant cultured with primary microglia was implanted as detailed in Example 1. Euthanasia, tract tracing and immunohistochemical analysis was conducted as previously noted in Example 1. Results of in vivo analysis of these compositions was similar to those described in Example 1 and demonstrated that DNA compositions promoted the regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using microglia containing gel compositions.
  • DNA was extracted from yellow onion following standard isolation protocol and precipitated in a non-solvent (isopropanol). Phosphate buffer was used as the solvent. Solutions were repeatedly washed in 95% ethanol and vacuum dried. The DNA precipitate was loaded into 12 well cell culture plates, frozen overnight (- 20°C freezer) and lyophilized overnight (-10 ⁇ m Hg, -50°C). Viscous solutions of 1.5% (w/v) DNA (Kelco HV) were prepared using high-speed mechanical mixing techniques. One milliliter of DNA solution was pressure injected into each well of a 12 well Falcon (Sigma) cell culture plate. The plate was sealed with parafilm, solution allowed to settle and frozen overnight in a - 20°C freezer. The plate was loaded into a cold 900 ml Labconco flash freeze flask and lyophilized overnight (-10 ⁇ m Hg, -50°C).
  • DNA microporous foams were removed from the culture plates using microforceps and placed into sterile 15 ml borosilicate test tubes. Twenty mis of genipin (40 mg/ml)in buffer (pH 12.0) crosslinking solution was pipetted into each tube. Test tubes were placed in a convection oven at 95°C for 1 hour or until the reaction medium turned dark brown/blue. Covalently crosslinked foams were cooled to ambient temperature and tumbled on a clinical rotator for 12 hours. DNA foams were repeatedly washed with nanopure water and vacuum dried. Foams were ethylene oxide sterilized and aerated for 24 hours. These preparations were stored refrigerated until further biological use.
  • rat microglia were cultured and seeded on DNA foams as detailed in Example 1.
  • sterile DNA foams were sized to approximately 1mm 3 and placed into sterile 500 ⁇ l microfuge tubes containing culture media (no serum or protein). Microglia were harvested as described above. Cell viability was confirmed by vital staining.
  • the media was aspirated from the microfuge tube and a dense microglial cell suspension was micropipetted into the tube.
  • the tube was placed on an orbital rotator in an incubator for one hour prior to implantation into the injured rat spinal cord. Immediately prior to surgery, tubes were placed on ice.
  • microporous DNA implant cultured with primary microglia is implanted as detailed in Example 1. Euthanasia, tract tracing and immunohistochemical analysis is conducted as previously noted in Example 1. Results of in vivo analysis of these compositions is similar to those described in Example 1 and demonstrate that DNA compositions promotes the regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using microglia containing gel compositions.
  • Chitosan and DNA solutions were prepared as detailed in Examples, 4 and 23, respectively.
  • the chitosan-DNA solutions (10:2) were mechanically mixed to form a coacervate.
  • One ml the mixture was pressure injected into each well of a 12 well Falcon (Sigma) cell culture plate.
  • the plate was sealed with parafilm, solution allowed to settle and frozen overnight in a - 20°C freezer.
  • the plate was loaded into a cold 900 ml Labconco flash freeze flask and lyophilized overnight (-10 ⁇ m Hg, -50°C).
  • Chitosan-DNA microporous foams were removed from the culture plates using microforceps and placed into sterile 50 ml centrifuge tubes. Twenty milliliters of genipin (40 mg/ml) in phosphate buffer was pipetted into each tube. Foams were tumbled on a clinical rotator for 12 hours at ambient temperature. Covalently crosslinked chitosan-DNA foams were repeatedly washed with nanopure water. Foams were autoclaved on a preprogrammed liquid cycle. These preparations were stored refrigerated until further biological use.
  • rat microglia were cultured and seeded on chitosan-DNA foams as detailed in Example 1.
  • sterile chitosan-DNA foams were sized to approximately 1mm 3 and placed into sterile 500 ⁇ l microfuge tubes containing culture media (no serum or protein). Microglia were harvested as described above. Cell viability was confirmed by vital staining.
  • the media was aspirated from the microfuge tube and a dense microglial cell suspension was micropipetted into the tube.
  • the tube was placed on an orbital rotator in an incubator for one hour prior to implantation into the injured rat spinal cord. Immediately prior to surgery, tubes were placed on ice.
  • microporous chitosan-DNA implant cultured with primary microglia is implanted
  • Example 44 Euthanasia, tract tracing and immunohistochemical analysis is similar to that previously noted in Example 1. Results of in vivo analysis of these compositions is similar to those described in Example 1 and demonstrate that chitosan-DNA compositions promotes the regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using microglia containing gel compositions.
  • Viscous solutions of 2% (w/v) alginate were prepared using high-speed mechanical mixing and air pressure filtration techniques as detailed in Example 1. Solutions were autoclaved on a liquid cycle and stored refrigerated. Primary microglia were cultured as described in Example 1. One ml of a concentrated suspension (35 x 10 6 cells/ml) of microglia was gently mixed with 10 ml of alginate solution. The solution was triturated repeatedly, loaded into a 20 ml syringe and placed in an incubator (37°C, 8% CO 2 ). The cell containing solution was mechanically pressure injected (60 ml/min) into minimal essential media containing 0.075 M CaCl 2 . The water insoluble alginate gel tube containing primary microglia was sized and implanted into the injured adult rat rubrospinal tract as detailed in Example 1.
  • Solutions of 2% (w/v) alginate and 1% (w/v) DNA, (10:2) were prepared using highspeed mechanical mixing and air pressure filtration techniques as detailed in Examples 1 and 24, respectively. Solutions were autoclaved on a liquid cycle and stored refrigerated. Primary microglia were cultured as described in Example 1. One ml of a concentrated suspension (35 x 10 6 cells/ml) of microglia was gently mixed with 10 ml of alginate-DNA solution. The solution was triturated repeatedly, loaded into a 20 ml syringe and placed in an incubator (37°C, 8% CO 2 ). The cell containing solution was mechanically pressure injected (60 ml/min) into minimal essential media containing 0.075 M CaCl 2 . The water insoluble alginate gel tube containing primary microglia was cut and implanted into the injured adult rat rubrospinal tract as detailed in Example 1.
  • Euthanasia, tract tracing and immunohistochemical analysis is similar to that previously noted in Example 1. Results of in vivo analysis of these compositions is similar to those described in Example 1 and demonstrate that DNA-alginate microglia compositions promotes the regeneration of injured nervous tissue in the adult rat spinal cord.
  • Euthanasia, tract tracing and immunohistochemical analysis is similar to that previously noted in Example 1.
  • Results of in vivo analysis of these compositions is similar to those described in Example 1 and demonstrate that alginate-Schwann cell-microglial compositions promote the regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using stem cell and stem cell/microglia containing gel compositions.
  • Ionically crosslinked microporous alginate foams were prepared as detailed in Example 1. Microporous alginate foams were placed in clean borosilicate test tubes. A 10% (w/w) MPC-N- vinyl pyrrolidone (NVP)(3:1) monomer solution was prepared in a dry box under an argon atmosphere. Five milliliters of MPC/NVP monomer solution was pipetted into each test tube under a laminar flow hood. Following an overnight pre-soak, tubes were purged with argon to remove dissolved oxygen, sealed and polymerized using radiation initiation polymerization (0.05 Mrad, 1180 rads/min) in a 60 Co gamma source. MPC/PVP modified foams were further processed, seeded with microglia and is surgically implanted and evaluated as detailed in Example 1.
  • MPC-N- vinyl pyrrolidone (NVP)(3:1) monomer solution was prepared in a dry box under an argon atmosphere. Five milliliter
  • results of in vivo analysis of these compositions is similar to those described in Example 1 and demonstrate that MPC copolymer/alginate/microglia compositions promote the regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using gel compositions.
  • Ionically crosslinked microporous alginate foams were prepared as detailed in Example 1. Microporous alginate foams were placed in clean borosilicate test tubes. A 10% (w/w) MPC-2-hydroxyethyl methacrylate (HEMA)(3:1) monomer solution was prepared in a dry box under an argon atmosphere. Five milliliters of MPC/HEMA monomer solution was pipetted into each test tube under a laminar flow hood. Following an overnight pre-soak,
  • Example 1 48 tubes were purged with argon to remove dissolved oxygen, sealed and polymerized using radiation initiation polymerization (0.05 Mrad, 1180 rads/min) in a 60 Co gamma source. MPC/PHEMA modified foams were further processed, seeded with microglia and is surgically implanted and evaluated as detailed in Example 1.
  • results of in vivo analysis of these compositions is similar to those described in Example 1 and demonstrate that MPC copolymer/alginate/microglia compositions promote the regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using gel compositions.
  • Solutions of 4% (w/v) alginate were prepared using high-speed mechanical mixing and air pressure filtration techniques as detailed in Example 1.
  • Minimal essential media MEM was used as solvent. Solutions were autoclaved on a liquid cycle and stored refrigerated. Primary microglia were cultured as described in Example 1. Cells were harvested as described in Example 1 and the microglial- conditioned supernatant was loaded into sterile 50 ml centrifuge tubes. Five mis of microglial-conditioned media were gently mixed with 5 ml of alginate solution. The solution was triturated repeatedly, loaded into a 20 ml syringe and placed in an incubator (37°C, 8% CO 2 ).
  • the cell containing solution was mechanically pressure injected (60 ml/min) into sterile filtered (0.2 micron) minimal essential media containing 0.075 M CaCl 2 .
  • the water insoluble alginate gel tube containing primary microglial conditioned media was sterilely cut and implanted into the injured adult rat rubrospinal tract as detailed in Example 1.
  • Solutions of 2% (w/v) alginate were prepared using high-speed mechanical mixing and air pressure filtration techniques. Tris buffer was used as the solvent. Solutions were pressure filtered through 70 micron Spectra filters into sterile 500 ml glass screw top bottles. One ml of alginate solution was pressure injected into each well of a 12 well Falcon (Sigma) cell culture plate. The plate was sealed with parafilm, solution allowed to settle and frozen overnight in a - 20 °C freezer. The plate was loaded into a cold 900 ml Labconco flash freeze flask and lyophilized overnight (-10 ⁇ m Hg, -50 °C).
  • Alginate microporous foams were removed from the culture plates using microforceps and placed into sterile 50 ml centrifuge tubes. Twenty milliliters of 0.1 M CaCl 2 crosslinking solution was pipetted into each tube. Microporous alginate foams were allowed to incubate in the crosslinking solution for 30 minutes. The tubes were drained and foams repeatedly washed with nanopure water for 2 days.
  • Microporous alginate foams were placed in clean borosilicate test tubes.
  • a 10% (w/v) MPC monomer solution was prepared in a dry box under an argon atmosphere.
  • Five milliliters of MPC monomer solution was pipetted into each test tube under a laminar flow hood. Following an overnight pre-soak, tubes were purged with argon to remove dissolved
  • MPC modified alginate foams were repeatedly washed with nanopure water for approximately one week. Characterization of MPC modified foams included Fourier Transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), contact angle goniometry, atomic force microscopy (AFM) and in vitro cell culture diagnostics. Foams were autoclaved fully hydrated on a preprogrammed liquid cycle and stored refrigerated.
  • FTIR Fourier Transform infrared spectroscopy
  • XPS X-ray photoelectron spectroscopy
  • AFM atomic force microscopy
  • Foams were autoclaved fully hydrated on a preprogrammed liquid cycle and stored refrigerated.
  • rat microglia were cultured from perinatal rat brains and isolated as follows. Briefly, rat whole brains were stripped of meninges while immersed in dissociation solution (D). Clean fragments were mechanically minced, transferred to a 50 ml conical tube, and incubated under bi-directional rotation in 0.05% trypsin in solution "D" for 20 minutes at 37°C. An equal volume of minimal essential medium (MEM) containing 10% (v/v) fetal bovine serum (FBS) was added to the suspension to quench the reaction and the tissue was triturated again and passed through a 130 mm Nitex filter before being centrifuged (400 x g, 10 minutes).
  • MEM minimal essential medium
  • FBS fetal bovine serum
  • the resulting pellet was resuspended in 10 ml of MEM with FBS and passed through a 40 mm Nitex filter before being plated.
  • the plating density was 2 brains/150 cm 2 flask. Cultures were incubated (37°C, 8% CO 2 ) for 3 days. Fresh complete medium was added, and the cultures incubated for an additional 7 days after which microglia were harvested every third day. Flasks were placed on an orbital shaker in an incubator (37°C, 200 rpm) for 1 hour. After shaking, the medium was collected and centrifuged (400 x g, 10 minutes), yielding a high concentration of microglia; typically 35 million cells per preparation.
  • sterile microporous alginate foams were cut to approximately lmm 3 and placed into sterile 500 ⁇ l microfuge tubes. Microglia were harvested as described
  • mice Prior to euthanasia, animals were either retrogradely tract traced with Fluorogold (FG) or anterogradely tract traced with biotinylated dextran amine (BDA). The animals were fixation perfused and spinal cord and brain removed intact. The collected tissue was post- fixed in 4% (w/v) paraformaldehyde. Spinal cord tissue was cryo-preserved with 30% (w/v) sucrose in phosphate buffered saline. The red nucleus in the rat midbrain was vibratome sectioned (50 microns) and counterstained with 3% (w/v) cresyl violet (CV).
  • FG Fluorogold
  • BDA biotinylated dextran amine
  • Sections were viewed under brightfield optical microscopy and red nucleus neurons were enumerated using stereological counting techniques and the MCID system. FG labeled red nucleus neurons were enumerated in a similar fashion (without CV), except using fluorescence microscopy. Digital images of serial sections were captured for the entire red nucleus.
  • Cryosectioned spinal cord specimens were cut (-25°C, 20 microns) and processed using immunohistochemical staining techniques. Antibodies against neurofilaments,
  • Cresyl violet counterstaining revealed a dense, cellular infiltrate, that consisted of Schwann cells, macrophages, microglia, fibroblasts, ependymal cells and other non-identified cells. This staining also showed a smooth interface between damaged and healthy tissue.
  • Microporous HA foams were modified with MPC in the same manner as described in Example 30. Primary microglial cultures were prepared as detailed in Example 30. In a biological cabinet, sterile MPC modified microporous HA foams were sized to approximately lmm 3 and placed into sterile 500 ⁇ l microfuge tubes. Microglia were harvested as described above. Cell viability was confirmed by vital staining. A dense microglial cell suspension was micropipetted into the microfuge tube. The tube was placed on an orbital rotator in an incubator for one hour prior to implantation into the injured rat spinal cord. Immediately prior to surgery, tubes were placed on ice.
  • the MPC modified HA/microglial composition is implanted into the injured adult spinal cord as described in Example 30. Euthanasia, tract tracing and immunohistochemical analysis is conducted as previously noted in Example 30. Results of in vivo analysis of these compositions is similar to those described in Example 30 and demonstrated that MPC/HA/microglia compositions promoted regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using microglia containing gel compositions.
  • CMC carboxymethyl cellulose
  • Microporous CMC foams were modified with MPC in the same manner as described in Example 1. Primary microglial cultures were prepared as detailed in Example 30. In a biological cabinet, sterile MPC modified microporous CMC foams were sized to approximately lmm 3 and placed into sterile 500 ⁇ l microfuge tubes. Microglia were harvested as described above. Cell viability was confirmed by vital staining. A dense microglial cell suspension was micropipetted into the microfuge tube. The tube is placed on an orbital rotator in an incubator for one hour prior to implantation into the injured rat spinal cord. Immediately prior to surgery, tubes are placed on ice.
  • the MPC modified CMC/microglial composition is implanted into the injured adult spinal cord as described in Example 30. Euthanasia, tract tracing and immunohistochemical analysis is conducted as previously noted in Example 30. Results of in vivo analysis of these compositions is similar to those described in Example 30 and demonstrate that
  • chitosan solutions were prepared using high-speed mechanical mixing techniques in dilute acetic acid at pH 4.5. Phosphate buffer containing 0.025 M calcium chloride was used as the solvent. Chitosan solutions were air pressure filtered (70 micron) into in sterile 200 ml glass screw top bottles. One ml of chitosan solution was pressure injected into each well of a 12 well Falcon (Sigma) cell culture plate. The plate was sealed with parafilm, solution allowed to settle and frozen overnight in a - 20 °C freezer. The plate was loaded into a cold 900 ml Labconco flash freeze flask and lyophilized overnight (-10 ⁇ m Hg, -50 °C). Microporous chitosan foams were crosslinked and further processed as detailed in Example 30.
  • Microporous chitosan foams were modified with MPC in the same manner as described in Example 30. Primary microglial cultures were prepared as detailed in Example 30.
  • sterile MPC modified microporous chitosan foams were cut to approximately lmm 3 and placed into sterile 500 ⁇ l microfuge tubes.
  • Microglia were harvested as described above. Cell viability was confirmed by vital staining.
  • a dense microglial cell suspension was micropipetted into the microfuge tube.
  • the tube was placed on an orbital rotator in an incubator for one hour prior to implantation into the injured rat spinal cord. Immediately prior to surgery, tubes were placed on ice.
  • the MPC modified chitosan/microglial composition is implanted into the injured adult spinal cord as described in Example 30. Euthanasia, tract tracing and immunohistochemical analysis is conducted as previously noted in Example 30. Results of in vivo analysis of these compositions is similar to those described in Example 30 and demonstrate that MPC/chitosan/microglia compositions promote the regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using microglia containing gel compositions.
  • Microporous dextran DEAE foams were modified with MPC in the same manner as described in Example 30. Primary microglial cultures were prepared as detailed in Example 30. In a biological cabinet, sterile MPC modified microporous dextran foams were sized to approximately lmm 3 and placed into sterile 500 ⁇ l microfuge tubes. Microglia were
  • the MPC modified dextran/microglial composition is implanted into the injured adult spinal cord as described in Example 30. Euthanasia, tract tracing and immunohistochemical analysis is conducted as previously noted in Example 30. Results of in vivo analysis of these compositions is similar to those described in Example 30 and demonstrate that MPC/dextran/microglia compositions promote the regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using microglia containing gel compositions.
  • agarose solutions were prepared using high-speed mechanical mixing techniques at 45°C. Phosphate buffer containing 0.025 M calcium phosphorylcholine was used as the solvent. Solutions were stored in sterile 200 ml glass screw top bottles. Prior to plating, the agarose solution was microwaved on high power for thirty seconds to one minute. One ml of agarose solution was pressure injected into each well of a 12 well Falcon (Sigma) cell culture plate. The plate was sealed with parafilm, solution allowed to settle and gel, and frozen overnight in a - 20 °C freezer. The plate was loaded into a cold 900 ml Labconco flash freeze flask and lyophilized overnight (-10 ⁇ m Hg, -50 °C). Microporous foams were crosslinked and further processed as detailed in Example 30.
  • Microporous agarose foams were modified with MPC in the same manner as described in Example 1. Primary microglial cultures were prepared as detailed in Example 30. In a biological cabinet, sterile MPC modified microporous agarose foams were sized to approximately lmm 3 and placed into sterile 500 ⁇ l microfuge tubes. Microglia were harvested as described above. Cell viability was confirmed by vital staining. A dense microglial cell suspension was micropipetted into the microfuge tube. The tube was placed on an orbital rotator in an incubator for one hour prior to implantation into the injured rat spinal cord. Immediately prior to surgery, tubes were placed on ice.
  • the MPC modified agarose/microglial composition is implanted into the injured adult spinal cord as described in Example 30. Euthanasia, tract tracing and immunohistochemical analysis is conducted as previously noted in Example 30. Results of in vivo analysis of these compositions is similar to those described in Example 30 and demonstrate that MPC/agarose/microglia compositions promote the regeneration of injured nervous tissue in the adult rat spinal cord.
  • Ionically crosslinked microporous alginate foams were prepared as detailed in Example 30. Microporous alginate foams were placed in clean borosilicate test tubes. A 10% (w/w) MPC-2-hydroxyethyl methacrylate (HEMA)(3:1) monomer solution was prepared in a dry box under an argon atmosphere. Five milliliters of MPC/HEMA monomer solution was pipetted into each test tube under a laminar flow hood. Following an overnight pre-soak, tubes were purged with argon to remove dissolved oxygen, sealed and polymerized
  • HEMA MPC-2-hydroxyethyl methacrylate
  • Example 30 using radiation initiation polymerization (0.05 Mrad, 1180 rads/min) in a 60 Co gamma source.
  • MPC/PHEMA modified foams were further processed, seeded with microglia and is surgically implanted and evaluated as detailed in Example 30.
  • results of in vivo analysis of these compositions is similar to those described in Example 30 and demonstrate that MPC copolymer/alginate/microglia compositions promote the regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using microglia containing gel compositions.
  • Ionically crosslinked microporous alginate foams were prepared as detailed in Example 30.
  • Microporous alginate foams were placed in clean borosilicate test tubes.
  • a 10% (w/w) MPC-2-hydroxyethyl methacrylamide (HMA)(3:1) monomer solution was prepared in a dry box under an argon atmosphere.
  • Five mis of MPC/HMA monomer solution was pipetted into each test tube under a laminar flow hood. Following an overnight pre-soak, tubes were purged with argon to remove dissolved oxygen, sealed and polymerized using radiation initiation polymerization (0.05 Mrad, 1180 rads/min) in a 60 Co gamma source.
  • MPC/PHMA modified foams were further processed, seeded with microglia and are surgically implanted and evaluated as detailed in Example 30.
  • results of in vivo analysis of these compositions is similar to those described in Example 30 and demonstrate that MPC copolymer/alginate/microglia compositions promote the regeneration of injured nervous tissue in the adult rat spinal cord. Comparable results are obtained using microglia containing gel compositions.

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Abstract

L'invention concerne une composition implantable conçue afin de stimuler la croissance et l'orientation d'un tissu neural sur un site de lésion de tissu nerveux d'un mammifère humain ou non humain. Ladite composition comprend a) un matériau bioérodable de remplissage d'espace cellulairement compatible, permettant la croissance d'un tissu neural dans ladite composition ou sur celle-ci, et b) au moins une cellule biologiquement active, ensemencée sur ou dans le matériau, capable de produire, lors de l'implantation de la composition chez un mammifère, au moins un facteur neurotrophique, un facteur de croissance, une cytokine, une molécule matricielle extracellulaire ou un mélange de ceux-ci efficace pour fournir un support neurotrophique à un tissu nerveux de mammifère. L'invention concerne également un procédé permettant de promouvoir la régénération neuronale d'un tissu nerveux lésé ou endommagé consistant à implanter ladite composition dans le tissu du mammifère à proximité d'une lésion entre les débris d'axones proximaux et les segments distaux respectifs.
PCT/US2002/030900 2001-09-28 2002-09-30 Compositions de biopolymere et de biopolymere-cellule permettant de reparer un tissu nerveux WO2003026489A2 (fr)

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WO2006116717A2 (fr) * 2005-04-28 2006-11-02 The Board Of Regents Of The University Of Texas System Cellules somatiques autologues du sang peripherique et leurs utilisations
WO2008121331A1 (fr) * 2007-03-30 2008-10-09 Pervasis Therapeutics, Inc. Matériels et procédés de traitement de lésions nerveuses et de promotion de la réparation et de la régénération des nerfs
EP2152860A2 (fr) * 2007-06-13 2010-02-17 FMC Corporation Matériau de matrice cellulaire lié à un peptide pour des cellules souches et leur procédé d'utilisation
US9023377B2 (en) 2005-06-21 2015-05-05 Shire Regenerative Medicine, Inc. Methods and compositions for enhancing vascular access
US9040092B2 (en) 2005-04-21 2015-05-26 Massachusetts Institute Of Technology Materials and methods for altering an immune response to exogenous and endogenous immunogens, including syngeneic and non-syngeneic cells, tissues or organs
EP2838607A4 (fr) * 2012-04-12 2015-12-30 Univ Wake Forest Health Sciences Modèle de conduit pour remplacement du nerf périphérique
AU2012225784B2 (en) * 2011-03-04 2016-03-17 The Regents Of The University Of California Locally released growth factors to mediate motor recovery after stroke
AU2012262679B2 (en) * 2011-06-03 2016-05-26 Central Adelaide Local Health Network Incorporated Method of treating the effects of stroke
WO2016160918A1 (fr) * 2015-03-31 2016-10-06 The University Of North Carolina At Chapel Hill Véhicules d'apport pour cellules souches et leurs utilisations
GB2539006A (en) * 2015-06-03 2016-12-07 Ewos Innovation As Functional feed
CN107715178A (zh) * 2017-11-09 2018-02-23 李瑞锋 一种多层含有细胞因子的高强度人工硬脑膜及其制备方法
US10219895B2 (en) 2012-10-26 2019-03-05 Wake Forest University Health Sciences Nanofiber-based graft for heart valve replacement and methods of using the same
US10632235B2 (en) 2007-10-10 2020-04-28 Wake Forest University Health Sciences Devices and methods for treating spinal cord tissue

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CN115531613B (zh) * 2022-10-20 2023-11-17 武汉轻工大学 一种多孔支架材料填充的神经移植物导管及制备方法和应用

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US20030027760A1 (en) * 1993-12-23 2003-02-06 Gluckman Peter David Composition and methods to improve neural outcome
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Cited By (20)

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US9040092B2 (en) 2005-04-21 2015-05-26 Massachusetts Institute Of Technology Materials and methods for altering an immune response to exogenous and endogenous immunogens, including syngeneic and non-syngeneic cells, tissues or organs
WO2006116717A3 (fr) * 2005-04-28 2007-03-29 Univ Texas Cellules somatiques autologues du sang peripherique et leurs utilisations
WO2006116717A2 (fr) * 2005-04-28 2006-11-02 The Board Of Regents Of The University Of Texas System Cellules somatiques autologues du sang peripherique et leurs utilisations
US9023377B2 (en) 2005-06-21 2015-05-05 Shire Regenerative Medicine, Inc. Methods and compositions for enhancing vascular access
WO2008121331A1 (fr) * 2007-03-30 2008-10-09 Pervasis Therapeutics, Inc. Matériels et procédés de traitement de lésions nerveuses et de promotion de la réparation et de la régénération des nerfs
EP2152860A2 (fr) * 2007-06-13 2010-02-17 FMC Corporation Matériau de matrice cellulaire lié à un peptide pour des cellules souches et leur procédé d'utilisation
JP2010529858A (ja) * 2007-06-13 2010-09-02 エフ エム シー コーポレーション 幹細胞のためのペプチド結合細胞マトリックスおよびその使用法
EP2152860A4 (fr) * 2007-06-13 2011-12-07 Fmc Corp Matériau de matrice cellulaire lié à un peptide pour des cellules souches et leur procédé d'utilisation
US10632235B2 (en) 2007-10-10 2020-04-28 Wake Forest University Health Sciences Devices and methods for treating spinal cord tissue
US9700596B2 (en) 2011-03-04 2017-07-11 The Regents Of The University Of California Locally released growth factors to mediate motor recovery after stroke
AU2012225784B2 (en) * 2011-03-04 2016-03-17 The Regents Of The University Of California Locally released growth factors to mediate motor recovery after stroke
AU2012262679B2 (en) * 2011-06-03 2016-05-26 Central Adelaide Local Health Network Incorporated Method of treating the effects of stroke
US9675358B2 (en) 2012-04-12 2017-06-13 Wake Forest University Health Sciences Conduit for peripheral nerve replacement
EP2838607A4 (fr) * 2012-04-12 2015-12-30 Univ Wake Forest Health Sciences Modèle de conduit pour remplacement du nerf périphérique
US10219895B2 (en) 2012-10-26 2019-03-05 Wake Forest University Health Sciences Nanofiber-based graft for heart valve replacement and methods of using the same
WO2016160918A1 (fr) * 2015-03-31 2016-10-06 The University Of North Carolina At Chapel Hill Véhicules d'apport pour cellules souches et leurs utilisations
US11027047B2 (en) 2015-03-31 2021-06-08 The University Of North Carolina At Chapel Hill Delivery vehicles for stem cells and uses thereof
GB2539006A (en) * 2015-06-03 2016-12-07 Ewos Innovation As Functional feed
CN107715178A (zh) * 2017-11-09 2018-02-23 李瑞锋 一种多层含有细胞因子的高强度人工硬脑膜及其制备方法
CN107715178B (zh) * 2017-11-09 2020-10-09 李瑞锋 一种多层含有细胞因子的高强度人工硬脑膜及其制备方法

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