JP2009513269A - A cell-free, bioabsorbable tissue regeneration matrix produced by incubating cell-free blood products - Google Patents

Abstract

The present invention provides methods and compositions useful for the regeneration of damaged, defective and / or degenerated tissues in humans and animals. The present invention provides a cell-free, bioabsorbable tissue regeneration matrix that is produced by incubating for a period of time sufficient for the matrix to form a product derived from cell-free blood. Also provided are methods of making such matrices and methods of using such matrices for regeneration of damaged, defective and / or degenerated tissue. In certain embodiments, the methods and compositions of the invention are useful for the treatment of damaged, defective and / or degenerated neural tissue.

Description

(Citation of related application)
This application is a co-pending application with US Provisional Patent Application No. 60 / 730,614 (filed October 26, 2005), and shares a common inventor with this US application, and this US Application Claim priority. The contents of this US application are hereby incorporated by reference in their entirety.

(background)
The present invention relates to the formation of a bioabsorbable proteinaceous matrix derived from animal tissue. The invention also relates to the use in vivo of this matrix that functions to induce and promote tissue growth and production / regeneration.

All other body tissues except blood consist of cells arranged in an integrated structure that requires an extracellular support scaffold or matrix to maintain proper growth differentiation and function. is there. In the field of tissue engineering, either artificial or natural polymers with properties that allow cell attachment, proliferation and patterning to provide replacement tissue for tissue lost during injury or disease It aims to provide a way to achieve complex structures using. An ideal scaffold material is non-immunogenic and will mimic as closely as possible the natural scaffold support structure found in the body. One of these attributes is that the skeleton is biodegradable so that regenerated or repaired tissue can reach the ultimate level of homeostasis and function. It is known in the prior art to build a bioabsorbable scaffold that can provide support structures for cell adhesion and growth as well as delivery of biologically active chemicals, proteins and peptides. The most widely used are hydrogels composed of various polymers including polysaccharides. Biodegradable hydrogels produced from biodegradable polysaccharides alone or in combination with natural extracellular matrix proteins such as collagen have been used as vehicles for drug delivery (Non-patent Document 1). Non-patent document 2; Non-patent document 3; Non-patent document 4; Non-patent document 5) and providing structural support for the regenerative tissue (Non-patent document 6; Non-patent document 7; Non-patent document 8) Non-patent document 9; Non-patent document 10; Non-patent document 11; Non-patent document 12; Non-patent document 13; Non-patent document 14; Recently, it has been reported that the hydrogel skeleton is used in the treatment of spinal cord injury (“SCI”) as a bridging structure that can provide an inductive channel for the regeneration of neural tissue (Non-patent Document 16). The generation of extracellular matrix or skeletal material from blood or other tissues has been described by US Pat. No. 5,057,097 (Vacanti and Vacanti, “Biological Scaffolding Material”, filed Oct. 17, 2003). This material is described as consisting essentially of cells, cell debris and cell debris. However, this material has not been reported to have biological functionality that promotes wound repair or regeneration, and further requires live cells or other structures with similar properties to cells (non-patent literature). 17). Despite these findings, non-immunogenic (or low immunogenic) that further has cell adhesion, growth promoting properties and supports or stimulates the regeneration / repair properties of the intact tissue surrounding the wound site There is still a need for skeleton materials.
US Patent Publication No. 2004/0137613 Cascone, M .; G et al., “Bioartificial polymer materials based on polysaccharides”, J Biomaterial. Sci. Polym. Ed. 12th edition, 267-281, 2001 Jeong, B.D. Et al., “Drug release from biogradable injectable thermosensitive hydrogen of PEG-PLGA-PEG triblock copolymer”, J Control Release, 63, pp. 155-163. Kopecek, J. et al. "Smart and genetically engineered biomaterials and drug delivery systems", Eur. J. et al. Pharm. Sci. 20, 20-16, 2003 Peppas, N.M. A. Et al., “Hydrogels in pharmaceutical formulation”, Eur. J. et al. Pharm. Biopharm. 50, 27-46, 2000 Zhang, Y. et al. And Chu, C.I. C. , "In vitro release behavior of insulin from biogradable hybrid hydro networks of polysaccharide and synthetic biodegradables." Biometer. Appl. 16, 305-325, 2002 Arevalo-Silva, C.I. A. Et al., “Internal support of tissue-engineered cartridge”, Arch. Otalyngol. Head Neck Surg. 126, 1448-1452, 2000 Desgrandchamps, F.M. , “Biomaterials in functional restructuring”, Curr. Opin. Urol. 10, pp. 201-206, 2000 Kim, T .; K. Et al., “Experimental model for cartridge tissue engineering to regenerative the zone organization of artificial cartridge,” page 11, 653-466. Kojima, K .; Et al., “A composite tissue-engineered trachea using sheep nasal chondrocycle and epithelial cells,” Faceb J. et al. 17, 823-828, 2003 Mailer, J. et al. J. et al. Et al., “Soft-tissue augmentation with injectable aggregate and syngeneic fiblasts”, Plast. Reconstr. Surg. 105, 2049-2058, 2000 Saim, A .; B. Et al., “Engineering autogenous cartridge in the shape of a helix using an injectable hydroscaffold”, Laryngoscope, 110, 1694-1697, 2000. Thompson, C.I. A. "Percutaneous transversal cellular cardiomyopathy: A novel non-surgical approach for myocardial cell plantation", J. et al. Am. Coll. Cardiol. 41, 1964-1971, 2003 Wake, M.M. C. Et al., “Dynamics of fibrous tissue tissue in hydrogen foams”, Cell Transplant. 4, 275-279, 1995 Weng, Y .; Et al., “Tissue-engineered compositions of bones and cartridges for convenient constitutive construction”, J. et al. Oral Maxillofac. Surg. 59, 185-190, 2001 Zimmermann, U. "Hydrogel-based non-autologous cell and tissue therapy", Biotechnique, 29, 564-572, 574, 576, 2000 Tsai, E .; C. Et al., “Synthetic hydrologic guidance channels facillate regeneration of adult rat brain motor after complete spin cord transection”, J. et al. Neurotrauma. 21 789-804, 2004 Vacanti, M.M. P. Et al., “Identification and initial charac- terization of spore-like cells in adult mammals,” J. et al. Cell Biochem. 80, 455-460, 2001

(Summary of the Invention)
In certain embodiments, the present invention provides a method for generating a bioabsorbable structure (hereinafter “regeneration matrix”) having biological regeneration properties. Such a regeneration matrix can be from any animal tissue including, but not limited to, blood, liver, kidney, muscle, heart, pancreas, olfactory mucosa, bone marrow, cerebrospinal fluid, lymph fluid and combinations thereof. Can be generated. In certain embodiments, the regeneration matrix is generated by processing either blood clots or blood collected using anticoagulants. In certain embodiments, the regeneration matrix is generated from a tissue sample from which cells were first removed. The regeneration matrix can be derived from the subject's own tissue, thus allowing application to autologous transplantation. Alternatively, the tissue types of the regeneration matrix can be matched, and application between allogeneic species is possible. In certain embodiments, the regeneration matrix is derived from dissimilar tissues. Cross-species application has utility in a research setting, but is usually not a therapeutic form applied to humans or animals. In certain embodiments, the regeneration matrix is generated from blood, eg, the subject's own blood.

  The regeneration matrix produced by certain methods of the present invention is therapeutic in that it can initiate, increase, support and / or guide tissue regeneration at sites of tissue damage, defects and / or degeneration. It has properties that allow the surrounding tissue to regenerate into normal, functional tissue. In certain embodiments, the present invention provides a method of administering a regeneration matrix to a subject, wherein the regeneration matrix initiates and / or increases tissue regeneration. In certain embodiments, damaged tissue regenerated by the present invention includes, but is not limited to, neural tissue, muscle tissue, liver tissue, heart tissue, lung tissue and / or skin tissue. For example, certain embodiments provide a method for taking advantage of the natural regenerative components of blood and / or clots and manipulating them into a regeneration matrix, delivering the regeneration matrix to the wound site at the appropriate time. The regenerative response can be maximized while minimizing the occurrence of non-functional scar formation.

  In certain embodiments, the regeneration matrix has nerve regeneration properties that are useful for treating conditions that lead to damage, loss and / or degeneration of nerve tissue. In certain embodiments, the neural tissue that is regenerated includes neural tissue from the central nervous system (“CNS”). In certain embodiments, as a result of a disease and / or injury such as, for example, spinal cord injury, spinal cord cancer, amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, stroke and / or multiple sclerosis, Missing. By adding one or more therapeutic agents, including but not limited to proteins, peptides, drugs, cytokines, extracellular matrix molecules and / or growth factors, enhance or supplement the regeneration activity of the regeneration matrix. be able to. In certain embodiments, administration of a therapeutic agent into the regeneration matrix increases one or more therapeutic effects of the therapeutic agent.

  In certain embodiments, the present invention provides a regeneration matrix comprising a bioabsorbable structure with tissue regeneration properties. In certain embodiments, such a regeneration matrix comprises one or more proteins. For example, such a regeneration matrix may comprise transferrin, serum albumin, serum albumin precursor, complement component 3, AD chain hemoglobin, IgM, IgG1, medalacin inhibitor 2, carbonic anhydrase and / or CA1 protein. One or more can be included. In certain embodiments, the regeneration matrix lacks substantial metabolic activity.

  In certain embodiments, one or more therapeutic agents, including but not limited to proteins, peptides, drugs, cytokines, extracellular matrix molecules and growth factors, are used to treat the regeneration matrix of the present invention. Complement. In certain embodiments, administration of a therapeutic agent into the regeneration matrix increases one or more therapeutic effects of the therapeutic agent. In certain embodiments, the regeneration matrix may be seeded with or mixed with cells, including but not limited to stem cells, progenitor cells, neural cells and / or glial cells.

  In certain embodiments, the present invention provides a bioabsorbable tissue regeneration matrix for inducing functional tissue regeneration in a subject at an application site. In certain embodiments, the regeneration matrix promotes tissue self-renewal at the site of application. In certain embodiments, the regeneration matrix increases the biological activity of the surrounding tissue. In certain embodiments, the regeneration matrix is in solid or semi-solid form. For example, the regeneration matrix can be in the form of a three-dimensional matrix. In certain embodiments, the regeneration matrix can be in the form of a suspension.

(Detailed description of specific embodiments)
Definitions “Cell-free sample”: As used herein, the term “cell-free sample” refers to a biological sample obtained by removing cells from an isolated tissue sample. In certain embodiments, a cell-free sample is used to obtain a cell-free bioabsorbable tissue regeneration matrix. The cell-free sample can be obtained from any of a variety of tissue sample types. For example, a cell-free sample can be obtained from any of the four basic animal tissue types (muscle tissue, connective tissue, epithelial tissue and nerve tissue), and three basic plant tissue types (basic tissue, Epidermal tissue and vascular tissue), any of a variety of other specific tissue types (eg, blood or liver tissue), and / or any combination of these or other tissue types Can be obtained from In certain embodiments, the cell-free sample is from placental tissue, from umbilical cord tissue, and / or cardiovascular, digestive, endocrine, excretion, immunity, jacket, lymph, muscle, nerve, reproduction, breathing and / or skeletal From one or more tissues of the organ system. One skilled in the art will recognize other tissue types from which cell-free samples can be obtained. In certain embodiments, the cell-free sample is obtained from a tissue sample comprising two or more different tissue types. In certain embodiments, a cell-free sample is obtained by passing a tissue sample through a filter, and the filter has a filtration diameter that is small enough to pass such a filter and exclude cells. In certain embodiments, the cell-free sample is obtained by centrifuging the tissue sample and using the supernatant. One skilled in the art will recognize other methods useful for removing cells from a tissue sample. When a cell-free sample is obtained from a tissue sample, it is understood that some or all of the cells may be lysed. In certain embodiments, the cell-free sample comprises one or more components from such lysed cells. In certain embodiments, the cell-free sample does not exhibit substantial metabolic activity (see definition of “substantial metabolic activity” below).

  “Bioabsorbable”: As used herein, the term “bioabsorbable” refers to the characteristic of being present for a limited period of time in a biological environment. In certain embodiments, the regeneration matrix of the present invention and / or the regeneration matrix produced by one or more methods of the present invention is bioabsorbable. When it is “bioabsorbable” in the context of a bioabsorbable regeneration matrix, the regeneration matrix is present in its initial form when it is observed at a given time after placement in the biological environment. That means it is no longer allowed. In certain embodiments, the bioabsorbable regeneration matrix can exist for days, weeks or months when placed in a biological environment. For example, when placed in a biological environment, the bioabsorbable regeneration matrix is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110 , 120, 130, 140, 150, 160, 170 or 180 days or more. A bioabsorbable regeneration matrix can be absorbed into the living body by any of a variety of mechanisms. In certain embodiments, the bioabsorbable regeneration matrix is capable of being absorbed into the living body through the action of cellular activity. For example, a bioabsorbable regeneration matrix is absorbed by the living body through the action of macrophages that disassemble the bioabsorbable regeneration matrix. In certain embodiments, the bioabsorbable regeneration matrix is absorbed into the body after being disassembled via mechanical, chemical, metabolic and / or enzymatic degradation. It will be appreciated by those skilled in the art that the exact mechanism of bioabsorbability is not critical as long as the regenerated matrix disassembly products can be absorbed and / or excreted from the body.

  “Incubate”: As used herein, the term “incubate” in the context of generating a regeneration matrix refers to a process that allows the regeneration matrix to take shape over a given time. In certain embodiments, the regeneration matrix is generated by incubating a cell-free sample in an incubation chamber (see definition of “incubation chamber” below). In certain embodiments, the cell-free sample is suspended and / or solubilized in the processing medium during the incubation. In certain embodiments, the regeneration matrix is generated by incubating the cell-free sample at any of a variety of temperatures, but only if the cell-free sample remains in a liquid state. For example, such incubation can be performed with -2, -1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 or 42 ° C This can be done at these temperatures, but only if the cell-free sample remains in a liquid state at these temperatures. In certain embodiments, the temperature can be increased and / or decreased during the incubation period, and thus incubation occurs over a given range of temperatures. In certain embodiments, the regeneration matrix is generated by incubating a cell-free sample at any of various atmospheric pressures. For example, a cell-free sample can be incubated at standard atmospheric pressure (“ATM”) or at pressures below or above ATM. In certain embodiments, the air pressure is changed while incubating the cell-free sample. For example, the atmospheric pressure can be increased and / or decreased during the incubation. In certain embodiments, the regeneration matrix is generated by incubating a cell-free sample at any of a variety of ambient oxygen concentrations. In certain embodiments, the ambient oxygen concentration is lower than the oxygen concentration at standard atmospheric pressure. For example, the ambient oxygen concentration during incubation is about 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 2 or 1% or less. In certain embodiments, the ambient oxygen concentration during the incubation is approximately or exactly 0%. In certain embodiments, the regeneration matrix is generated by incubating a cell-free sample at any of a variety of ambient humidity. In certain embodiments, ambient humidity is kept low during incubation, resulting in increased evaporation of the liquid in the cell-free sample. In certain embodiments, a liquid containing a new solute (such as, but not limited to, a “treatment medium” or “physiological solution”; see definition below) may be used to prevent evaporation from occurring. Add continuously or periodically to cell samples. It will be appreciated that in such embodiments, the osmolarity of the cell-free sample increases over time as a result of evaporation and addition of a liquid containing new solutes. In certain embodiments, the regeneration matrix is generated by incubating a cell-free sample at any of a variety of effective gravitational field strengths. In certain embodiments, the regeneration matrix, the cell-free sample, and the effective gravity field strength approximately equal to the gravity field strength of the earth's gravity (eg, at sea level and / or at one or more separate sea levels above the sea level). Produced by incubation in In certain embodiments, the regeneration matrix is generated by incubating a cell-free sample at an effective gravity field strength that is greater than the gravity field strength of Earth's gravity at sea level. For example, an effective gravity field strength higher than that of the Earth's gravity can be obtained by incubating below sea level or in a centrifuge, but only if such a method does not cause any shaking. It is done. In certain embodiments, the regeneration matrix is generated by incubating a cell-free sample at an effective gravity field strength that is lower than the gravity field strength of Earth's gravity at sea level. For example, an effective gravity field strength that is lower than the gravity field strength of the Earth's gravity can be obtained by incubating at high sea level or in space.

  “Incubation chamber”: As used herein, the term “incubation chamber” is used to incubate a cell-free sample during the formation of a regeneration matrix (see definition of “incubate” above). Refers to any of a variety of containers that can be made. In certain embodiments, the incubation chamber includes standard laboratory vessels such as flasks, culture dishes, beakers and others. Those skilled in the art will recognize other suitable and useful standard laboratory containers. In certain embodiments, the incubation chamber comprises a closed or semi-closed container. In certain embodiments, the incubation chamber is a closed or semi-closed container that is permeable or semi-permeable to one or more substances. For example, in certain embodiments, the incubation chamber includes a container that is permeable to air and / or gaseous molecules. In certain embodiments, the incubation chamber comprises an Opticell® cassette (BioCrystal Ltd., Westerville, Ohio). In certain embodiments, the sealed or semi-closed incubation chamber is designed such that the material can be added to and / or removed from the container after initiating the incubation step. For example, an incubation chamber that is permeable or semi-permeable to air and / or gaseous molecules, and liquid and / or solid material added to the chamber at one or more time points during and / or after incubation And / or can be designed to be removable from the chamber. In certain embodiments, a sealed or semi-closed incubation chamber is designed such that material can be added and / or removed by use of a needle or other tool that can penetrate the incubation chamber. In certain embodiments, a sealed or semi-sealed incubation chamber that is pierced with a needle or other device can itself “reseal” (and / or reseal), so that the incubation chamber It is desirable to maintain a hermetic or semi-hermetic state.

  “Matrix”: As used herein, the term “matrix” refers to any physical structure and / or suspension, including but not limited to solid or semi-solid structures. Point to. In certain embodiments, the present invention provides a matrix having regenerative characteristics (see definition of “regenerative” below). Such a reproducible matrix is referred to herein as a “regenerative matrix”.

  “Processing medium”: As used herein, the term “processing medium” refers to a liquid solution that can be added to a tissue sample, a cell-free sample, or both during the process of obtaining a regeneration matrix. Point to. In certain embodiments, the treatment medium comprises a physiological solution (see definition of “physiological solution” below). In certain embodiments, the treatment medium includes a minimal salt solution. For example, the processing medium can be PBS. One skilled in the art will recognize other minimal salt solutions that can be used in accordance with the present invention. Further, those skilled in the art may include, but are not limited to, any of pH, osmolarity, buffer capacity, concentration of one or more specific ions and / or a variety of other solution properties. It will be appreciated that compositions and methods useful for modifying a minimal salt solution to achieve one or more desirable solution properties. In certain embodiments, the treatment medium includes one or more therapeutic agents (see definition of “therapeutic agent” below). In certain embodiments, the treatment medium is added to the tissue sample before, during and / or after isolation. In certain embodiments, the treatment medium is added to the cell-free sample before, during and / or after cell removal. In certain embodiments, the treatment medium is added during the step of incubating the cell-free sample. In certain embodiments, the treatment medium is added at multiple time points during the step of incubating. In certain embodiments, one or more components of the treatment medium that are added during the regeneration matrix generation process become part of the generated regeneration matrix.

  “Physiological solution”: As used herein, the term “physiological solution” refers to a solution that resembles or is identical to one or more physiological conditions, or the physiological of a particular physiological environment. Refers to a solution that can change state. A “physiological solution” as used herein also refers to a solution that can support the growth of cells (including but not limited to, mammals, vertebrates and / or other cells). . In certain embodiments, the physiological solution includes a defined medium, where the concentration of each of the media components is known and / or controlled. Typically, defined media are nutrients necessary to support cell growth, including but not limited to energy sources such as salts, amino acids, vitamins, lipids, trace elements, and carbohydrates. Contains all. Non-limiting examples of defined media include: DMEM, Eagle basal medium (BME), 199 medium; F-12 (Ham) Nutrient Mixture; F-10 (Ham) Nutrient Mixture; Basal medium (MEM), Williams E Medium, and RPMI 1640. One skilled in the art will recognize other defined media that can be used in accordance with the present invention. In certain embodiments, the physiological solution comprises a mixture of one or more defined media. In certain embodiments, the physiological solution is a composite medium, in which at least one of the medium components is unknown and uncontrolled. Physiological solutions are useful for generating a regeneration matrix by the method of the present invention, but as will become clear from this detailed description, the cell-free sample that is incubated to generate the regeneration matrix is Contains no cells (see definition of “cell-free sample” above). Thus, even though the treatment medium in such a case can support cell growth, it is understood that cell growth is not a necessary component of regeneration matrix generation. However, in certain embodiments, the cells can be added to the incubation chamber at one or more specific times during the regeneration matrix formation process. In these cases, it is understood that the physiological solution can support the viability of the cells added to the incubation chamber by itself or in combination with the regeneration matrix solution.

  “Regeneration”, “regenerate”, “regenerative”: As used herein, these terms refer to the growth, regrowth, repair of weak, damaged, defective, and / or degenerated tissue. Any process or property that initiates, increases, regulates, promotes, supports and / or guides the natural process of functionality, patterning, binding, strengthening, vitality and / or wound healing Point to. These terms also initiate, increase, promote, and support weak, fatigued and / or normal tissue growth, strengthening, functionality, vitality, toughness, efficacy and / or health, It also refers to any process or property that is and / or leads. In certain embodiments, the present invention provides compositions and methods useful for the regeneration of damaged, defective and / or degenerated tissue. For example, the methods and compositions of the present invention can be used to initiate, increase, regulate, support, promote and / or guide regeneration of damaged, defective and / or degenerated tissue. it can. In certain embodiments, the invention provides a regeneration matrix (see definition of “regeneration matrix” above) that exhibits one or more regeneration properties or activities. In certain embodiments, regeneration includes initiation, increase, regulation, promotion, support and / or guidance of one or more of the following processes: natural wound healing, tissue growth, tissue functionality, pattern Cellular genes involved in formation, connectivity, angiogenesis, proliferation and / or progenitor cell activation, cell growth and / or proliferation, cell special differentiation and / or elongation, cell dedifferentiation and / or differentiation, regeneration Up-regulation and / or suppression of scar formation. However, regeneration is not limited to these processes, properties and activities, and those skilled in the art will recognize other “regeneration”, “regenerate” or “renewable” in the art. You will understand the process, nature or activity.

  “Substantial metabolic activity”: As used herein, the term “substantial metabolic activity” refers to the metabolic activity that an intact cell typically exhibits in vitro or in vivo. In certain embodiments, the cell-free sample and / or the regeneration matrix of the present invention is devoid of cells and thus exhibits no substantial metabolic activity. A variety of techniques are known to those skilled in the art, whereby metabolic activity can be detected and / or measured. For example, intact cells produce significant amounts of ATP by aerobic and / or anaerobic disassembly of macromolecules (eg, sugars, amino acids, lipids and others). Thus, typically a significant level of production of ATP indicates that there are unaltered cells in the sample. Those skilled in the art will appreciate that certain phenomena indicative of the presence of unchanged cells may be detected in a cell-free sample. However, such a phenomenon pointing to a cell is typically observed at a lower or reduced level or scale as compared to the level or magnitude of such a phenomenon that occurs as a result of the presence of the cell. Thus, for example, a cell-free sample and / or regeneration matrix may exhibit low levels of ATP production, and this level is produced when there are intact cells in the cell-free sample and / or regeneration matrix. Well below the level that would be done.

  “Therapeutic agent”: As used herein, the term “therapeutic agent” indicates one or more beneficial therapeutic effects when used in conjunction with the methods and / or regeneration matrices of the present invention. Refers to any of a variety of substances. Examples of therapeutic agents that can be used with the contemplated regeneration matrices and methods include, but are not limited to, proteins, peptides, drugs, cytokines, extracellular matrix molecules and / or growth factors. One skilled in the art will recognize other suitable and / or advantageous therapeutic agents that may be used in accordance with the present invention. In certain embodiments, administering a therapeutic agent into the regeneration matrix increases the magnitude of one or more beneficial therapeutic effects of the therapeutic agent. In certain embodiments, administration of a therapeutic agent into the regeneration matrix prolongs the activity of one or more beneficial therapeutic effects of the therapeutic agent. In certain embodiments, when the therapeutic agent is administered into the regeneration matrix, one or more beneficial therapeutic effects of the therapeutic agent are released over time. In certain embodiments, when the therapeutic agent is administered in the regeneration matrix, one or more beneficial therapeutic effects of the therapeutic agent are protected from substantial reduction over time. In certain embodiments, two or more therapeutic agents are administered in the regeneration matrix.

  “Tissue sample”: As used herein, the term “tissue sample” refers to a biological sample containing cells and / or extracellular material. For example, the tissue sample may be one of four basic animal tissue types (muscle tissue, connective tissue, epithelial tissue and nerve tissue), or three basic plant tissue types (basic tissue, epidermis). Tissue and vascular tissue), any of a variety of other specific tissue types (eg, blood or liver tissue), and / or any combination of these or other tissue types Can be included. In certain embodiments, the tissue sample is from placental tissue, from umbilical cord tissue, and / or cardiovascular, digestive, endocrine, excretion, immunity, jacket, lymph, muscle, nerve, reproduction, breathing and / or skeletal. Includes tissue from one or more tissues of the organ system. One skilled in the art will recognize other tissue types that can be used in accordance with the present invention. In certain embodiments, the tissue sample comprises two or more different tissue types. Tissue samples can be isolated from any of a variety of organisms. As a non-limiting example, tissue samples can be isolated from humans, pigs, rats, salamanders, cows, dogs, cats, mice and / or rabbits. One skilled in the art will recognize other suitable organisms from which tissue samples can be isolated. In certain embodiments, the tissue sample is used as a starting material for generating a regeneration matrix. In certain embodiments, the tissue sample used to generate the regeneration matrix is isolated from the type of organism for which the regeneration matrix is to be used. For example, a regeneration matrix intended for use in humans can be generated from a starting tissue sample isolated from humans. In certain embodiments, the tissue sample used to generate the regeneration matrix is isolated from individual organisms that intend to use the regeneration matrix. For example, a regeneration matrix intended for use in an individual can be generated from a starting tissue sample isolated from that individual. In certain embodiments, the regeneration matrix is generated from the blood of an individual who intends to use the regeneration matrix.

SUMMARY In certain embodiments, the present invention provides a regeneration matrix that can initiate, increase, support and / or guide tissue regeneration at the site of tissue damage. For example, the regeneration matrix according to the present invention can be used to regenerate nerve tissue, muscle tissue, liver tissue, heart tissue, lung tissue and / or skin tissue.

  The ability of the regeneration matrix to repair degenerated tissue, including but not limited to, tissue of the central nervous system (CNS), is based on the science of wound healing and tissue development. Although the process of wound healing has not been fully elucidated, the present invention finds that the regeneration matrix can initiate, increase, support and / or guide regeneration of damaged and / or degenerated tissue. Is included.

  One advantage of the regeneration matrix of the present invention is that it can inhibit certain processes of wound healing that do not enhance tissue regeneration while enhancing other processes that allow the patient to regenerate tissue. It is. It is worth noting that the body can quickly repair itself. However, certain aspects of the wound healing process do not allow the original regenerative functionality found in lower animal species such as salamanders. By suppressing undesirable features of the wound healing and tissue development processes and enhancing or reproducing the desired features, the regeneration matrix can achieve superior results. Unlike lower animal species such as salamanders, the natural process of wound healing in humans is programmed to recover most quickly from injury by providing scar tissue and isolating the damaged site from the rest of the body. This is because, in the case of spinal cord or other CNS injuries, the recovery of function is due to the limited or lack of effect of other parallel wound healing processes. Will be lacking. The same is true for damage and degeneration of other parts of the central nervous system and other tissues of the body.

  In certain embodiments, the methods and / or regeneration matrices of the present invention are useful for the treatment of damaged, defective and / or degenerated neural tissue, eg, central nervous system tissue. Historically, there has been no significant progress and no treatment available for central nervous system damage, including spinal cord damage. The main reason for the lack of treatments to regenerate tissues, particularly central nervous system tissues, is due to the nature of these tissues being life-threatening and highly complex.

  The present invention facilitates the repair of this complex biological and structural essence and the correct patterning and recombination of the spinal cord in SCI patients with a highly complex biological and structural essence Including the finding that it can be repaired using products with related activities that promote The same is true for other areas of the central nervous system. The regeneration matrix of the present invention is designed to have these and other advantageous features. Furthermore, additional functionality can be added to the regeneration matrix of the present invention in a relatively simple and direct manner. To date, developed to treat CNS injury and SCI patients, developed using an all-inclusive approach aimed at repairing complex functionalities of the central nervous system, such as the spinal cord in SCI patients There is no other.

Wound healing In the process of wound healing, several types of cells are involved in different stages of the wound healing process, each independently contributing to regenerating damaged tissue and restoring its functionality . In general, first line cells arise from the bloodstream, including macrophages. The task of macrophages is to clean up the site of injury, creating room for new cell growth, and releasing growth factors that activate the cells that are needed in the next stage of the wound healing process. Macrophages alone have a relatively small effect on spinal cord regeneration, but should have a slight effect rather than no effect at all. However, when combined with other mechanisms of action, activated macrophages have a more pronounced effect on the regeneration process. In certain embodiments, we have observed that the regeneration matrix of the present invention increases the rate of multiple activated macrophages entering the site of spinal cord injury within a few days after transplantation, thus There is no need to inject at the site of tissue damage. However, even though the regenerative matrix of the invention promotes macrophage tissue invasion into the damaged site, the macrophage is still administered at or near the site of injury along with or in addition to the regenerative matrix of the present invention. Those skilled in the art will understand that

  The next step in the natural process of wound healing is the recruitment of new blood vessels, and in the case of traumatic injury, the recruitment of fibroblasts that create the scar that surrounds the traumatic injury site, thereby While the damage is slowly repaired, the surrounding undamaged tissue continues to function and can compensate for the missing functionality of the damaged tissue. Growth factors released by activated macrophages promote the recruitment of these new blood vessels and fibroblasts. Unfortunately, the central nervous system is surrounded by the blood-brain barrier, which limits the invasion of macrophages and the promotion of new blood vessel recruitment. Inhibitors surrounding the blood-brain barrier, especially in the case of CNS damage or degeneration, limit the entry of new blood vessels into the CNS damage site, resulting in scarring by fibroblasts and glial cells. However, new blood vessels are not formed at the same time. The formed scar becomes very thick and cannot penetrate, and lack of new blood vessels means that the damaged site cannot be restored. The net result is the formation of a lesion (a hollow structure surrounded by a thick, non-penetrating scar), leading to long-term (chronic) CNS damage, and in the case of spinal cord injury, chronic paralysis. In certain embodiments, the regeneration matrix of the present invention has wound healing properties that simultaneously promote the formation of new blood vessels while slowing and inhibiting the pace of scar formation. Absence of blood vessels will result in lesions (cavities filled with bodily fluids, in which none of the cells are present), so new blood vessels will be in and around these lesions and / or areas where the lesions are about to form Conveniently formed. Without intending to be bound by theory, the regeneration matrix of the present invention transforms activated macrophages into a unique pathway, ie, von Willebrand factor positive endothelial-like cells that spontaneously form new blood vessels (transformation). It is assumed that new blood vessels can be created (see FIGS. 1A, 1B and 1C). Thus, according to this hypothesis, after activated macrophages attracted to the damaged site of the CNS by such a regeneration matrix have finished the work of purifying the damaged site and released growth factors, the macrophages become new blood vessels. Are transformed into new, specially differentiated types of cells. The net result is a new vascular network that provides nutrients and oxygen to new cells and new CNS tissue. In animal models of CNS injury, the transplanted regeneration matrix is already new within one week of transplantation (this vascular network does not resemble the type of vascular proliferation seen in tumors, but instead, (Similar to the type of large vascular network that exists in development) and has been found to be limited in the presence of any inhibitory scars (eg, FIGS. 2A, 2B, See 2C and 2D).

  After the formation of a new vascular network, typically the next step in the process of wound healing will move surrounding cells to the site of injury. These cells move to the site of injury to freshly replenish the nutrients and oxygen provided by the newly formed blood vessels. Unfortunately, in the case of nerve tissue damage, neurons are the only long-term cells in the body that do not migrate and / or divide (proliferate) once differentiated (transformed into adult cells) − If something happens to move, every time these cells move or divide, their own memory will be lost. In short, they will “forget” their memory (whether somatic or autonomic) involving specific neurons. However, as is known to those skilled in the art, neurons can newly make memory or connections through the formation and / or elongation of axons and dendrites. In certain embodiments, the regeneration matrix promotes the expression of one or more genes that result in the formation of such new bonds and correct patterning and connectivity in the nerve cells surrounding the injury site, such that the expression is Doubled (see, eg, FIGS. 18-19). In certain embodiments, the regeneration matrix extends nerve cells into the site of injury and creates effective and reasonable new bonds to the cells, such as bonds controlled by GAP-43 and Netrin family genes, for example. Make it. These genes are upregulated by the regeneration matrix. Such new connections can be used to obtain new CNS functionality that can be exploited by training and / or education, which would be a convenient functionality for new CNS organizations. In certain embodiments, such new functionality is achieved within a short time frame, eg, about two months. As a non-limiting example, in the case of spinal cord injury, this may require subsequent training / education of the nerves to coordinate and move the muscles to allow movement. As another non-limiting example, in the case of a broker region of the brain (language center), this would require subsequent training / education of the nerves to produce language in an orderly manner.

  In certain embodiments, the regeneration matrix of the present invention further enhances the formation of new CNS tissue by mobilizing and increasing the proliferation of triphasic neural progenitor cells. Triphasic neural progenitor cells are a unique and highly mobile young cell type that, according to neuroscientists, is implicated in the cause of spontaneous growth of new CNS tissue. Spontaneous growth of new CNS tissue was observed in some cases in humans, particularly in the brain, where human damaged CNS tissue was itself at least partially repaired. Triphasic neural progenitor cells are difficult to study in vitro (in dishes). This is because the three-phase neural progenitor cell recognizes that its environment lacks one type of nerve cell or glial cell (nerve support cell: astrocyte and oligodendrocyte) due to its nature. This is because they immediately differentiate (transform) into these cells (a triphasic neural progenitor cell appears to change to a cell type lacking its environment). However, in the present invention, in the presence of the regeneration matrix, the triphasic neural progenitor cells continue to proliferate until the regeneration matrix is removed or biodegraded (eg, in the body), and the regeneration matrix is removed or biodegraded. At this point, the triphasic neural progenitor cells contain the finding that they transform and produce a broad neural network. While not intending to be bound by theory, one hypothesis is that as a final step in the process of generating new CNS tissue, a triphasic neural progenitor cell is said to partially fill the missing portion. This missing part is the part that other surrounding neurons and glial cells did not satisfy during those phases of the regeneration process of the regeneration matrix, in which new CNS tissue was created. Become. Regardless of the exact mechanism, the end result is that the triphasic neural progenitor cells that are stimulated and proliferated by the inventive regeneration matrix produce new neural tissue.

With respect to other approaches in the field of wound healing, all of the conventional products and approaches have gained significant improvement in the experimental animals to a slight to bare extent compared to untreated control animals. Furthermore, clinical trials to date have not demonstrated any significant improvement over placebo with these single approach products. Moreover, in some cases, the technical problems associated with adverse immune responses in human transplantation have not been overcome. Another difficulty is that the product is washed away by cerebrospinal fluid (which typically moves at a rate of about 5 mm / min at 120 mm H ₂ O pressure in humans) and / or during implantation The product cannot be delivered to a human patient without causing any additional damage. The regeneration matrix of the present invention overcomes these and other difficulties. In certain embodiments, the regeneration matrix of the present invention provides a significant improvement in experimental animals compared to untreated control animals, with no toxicological effects. Furthermore, the regeneration matrix of the present invention is readily available in a form that can be delivered to human patients without long-term immunological problems.

Generation of Regeneration Matrix The present invention encompasses the finding that a regeneration matrix can be generated by any of a variety of methods. In certain embodiments, regeneration matrices that exhibit different physical characteristics, biological properties and / or therapeutic activity are obtained from different production methods. Furthermore, in certain embodiments, the physical characteristics, biological properties and / or therapeutic activity of the regeneration matrix produced by the methods of the present invention provides one or more exogenous factors during the production process. Change by things.

  In certain embodiments, the regeneration matrix of the present invention is generated by incubating a cell-free sample for a given period of time. In certain embodiments, the cell-free sample is isolated from the tissue sample. A cell-free sample is taken from any of the four basic tissue types (muscle tissue, connective tissue, epithelial tissue and neural tissue), from any of a variety of other non-basic tissue types, or It can be obtained from any combination of these or other tissue types. In certain embodiments, the cell-free sample is one of four basic animal tissue types (muscle tissue, connective tissue, epithelial tissue and neural tissue), and three basic plant tissue types. (Basic tissue, epidermal tissue and vascular tissue), any of a variety of other specific tissue types (eg, blood or liver tissue), and / or of these or other tissue types It can be obtained from any combination. In certain embodiments, the cell-free sample is from placental tissue, from umbilical cord tissue, and / or cardiovascular, digestive, endocrine, excretion, immunity, jacket, lymph, muscle, nerve, reproduction, breathing and / or skeletal From one or more tissues of the organ system.

  The regeneration matrix can be formed from any animal within the animal kingdom, and / or any plant within the plant kingdom, especially all tissues and body fluids. However, in certain embodiments, the regeneration matrix is generated from blood. Without intending to be bound by theory, it is assumed that the regeneration matrix is formed, at least in part, by tissue components, particularly cellular components that have suffered or are in the vicinity of extensive tissue damage. Yes. By such a mechanism, the cells seem to induce surrounding cells to regenerate damaged tissue. A similar but different and poorly understood phenomenon was observed by Becker et al. In 1974 (Becker, RO et al., “Regeneration of the ventricular myocardium in amphibians” while studying newt hearts. ”Nature, 248, 145-147, 1974). According to Becker's observation, when 30% to 50% of a newt heart is excised, the surrounding red blood cells are lysed and the nuclei they release agglutinate to form new myocardial tissue, and the newt heart is about 4 hours old. It could be regenerated later and therefore many newts could survive. As will become clear from the following description, the regeneration matrix of the present invention operates via a mechanism different from that observed by Becker et al. This is because the regeneration matrix of the present invention is generated from a cell-free sample and as such is not a cell-based viable tissue. Nevertheless, in certain embodiments, the regeneration matrix of the present invention is seeded with or mixed with cells during or after formation of the regeneration matrix to produce one or more advantageous features or on the regeneration matrix. Give function. However, as will be appreciated, such cells are not required to form the regeneration matrix of the present invention.

  It is understood that a functional regeneration matrix can be generated from any of a variety of tissues, but the exact composition of the regeneration matrix is influenced, among other things, by the tissue type from which the cell-free sample is obtained. I will. As a non-limiting example, the regeneration matrix can be generated from liver tissue, whole blood and / or one or more blood fractions. When the regeneration matrix is generated from whole blood, the main protein components of the regeneration matrix are typically albumin and hemoglobin. When the regeneration matrix is generated from the plasma-platelet-buffy coat fraction of blood, the main protein component of the regeneration matrix is typically albumin. Nonetheless, both the regeneration matrix generated from whole blood and the regeneration matrix generated from the plasma-platelet-buffy coat fraction function in tissue damage, defect and / or degenerated sites to regenerate tissue. Start, increase, support and / or lead. In certain embodiments, the regeneration matrix is generated from a cell-free sample obtained from whole blood. In certain embodiments, the regeneration matrix is cell-free from one or more blood fractions including, but not limited to, the red blood cell fraction, buffy coat fraction, platelet fraction and / or plasma fraction. Generate from a sample. Such a fraction can be obtained, for example, by sedimenting and separating whole blood by gravity. In certain embodiments, the blood fraction is obtained by centrifugation. One skilled in the art will recognize other techniques for separating blood into fractions that can be used to generate the regeneration matrix of the present invention. In certain embodiments, the regeneration matrix is generated from frozen blood or other tissue. In certain embodiments, such tissues are frozen for a period of days, weeks, months or years. In certain embodiments, the yield and growth of the regeneration matrix is increased when the blood or other tissue is subjected to multiple freeze-thaw cycles in the temperature range near the freezing to thawing point of the sample. In certain embodiments, the regeneration matrix is generated from blood or other tissue isolated from cadaver (eg, cadaver after several weeks). In certain embodiments, the regeneration matrix is generated from placental or umbilical cord blood. One skilled in the art will recognize other sources that can be used to generate a regeneration matrix of blood, body fluids (eg, cerebrospinal fluid and lymph) and tissues.

  Furthermore, in the case of whole blood, as shown in Examples 1 and 2, the presence of an anticoagulant in the blood sample results in a regenerated matrix having different adhesive properties. Any of a variety of anticoagulants can be used, including but not limited to heparin, EDTA and / or citric acid. A person skilled in the art has other known anticoagulants as well as anticoagulants that can be used to generate a regeneration matrix with different physical characteristics, biological properties and / or therapeutic activity. You will understand.

  In certain embodiments, the cell-free sample is obtained by removing cells from the tissue sample. Any of a variety of techniques can be used to remove such cells. For example, tissue cells can be centrifuged to separate the tissue sample into a cell-containing fraction and a cell-free fraction, and then the cell-free fraction can be isolated. In certain embodiments, the cells are removed from the tissue sample by passing the tissue sample through a filter having a pore size that is sufficiently small to exclude the cells. For example, cells can be removed from a tissue sample by passing the tissue sample through a filter having a pore size of 5 μm, 1.2 μm and / or 0.8 μm. In certain embodiments, the pore size of the filter changes the physical characteristics, biological properties and / or therapeutic activity of the generated regeneration matrix. If you are an expert, the exact pore size depends on the experimental and / or laboratory constraints, the characteristics you want for the regeneration matrix, and / or a variety of other factors that the expert considers important. Can be determined. In certain embodiments, the tissue sample is suspended, solubilized and / or diluted prior to removal of the cells. For example, the tissue sample can be suspended, solubilized and / or diluted in a processing medium and / or physiological solution prior to removing the cells from the tissue sample.

  A regeneration matrix can be generated by incubating a cell-free sample for any of a variety of incubation periods. For example, a cell-free sample may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24. , 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55 or 60 days or more. In an in vitro assay, a functional regeneration matrix has been detected in the generated regeneration matrix after 6, 12, 15, 21, and 28 days and is functional over a longer incubation period of 6 weeks or more. Is maintained. By modifying the environmental parameters and / or processing media used in the production process, a functional regeneration matrix can be produced in a shorter period of time. For example, by reducing the oxygen concentration to about 0% and / or increasing the agglomeration characteristics or effects of the treatment medium, eg by increasing the salt concentration, the functional regeneration matrix can be produced in minutes or hours. Or can be generated within a few days. In certain embodiments, the activity of the regeneration matrix produced by incubating the cell-free sample increases as the incubation period increases. In certain embodiments, the regeneration matrix produced by incubating a cell-free sample reaches maximum or near maximum activity after incubation for a given period of time. In such embodiments, even further incubation, the activity of the regeneration matrix is only additive, if at all. In such embodiments, upon further incubation, the regeneration matrix may lose activity.

  Applicants have found that shaking or otherwise perturbing a cell-free sample during the early stages of formation of the regeneration matrix results in an incomplete and / or defective regeneration matrix and / or no regeneration matrix is produced at all. It has been discovered that, in addition, the biological activity of such imperfect and / or defective regeneration matrices is correspondingly reduced. In certain embodiments, the cell-free sample is incubated for a period of time during which the regeneration matrix is formed such that the sample is not shaken or otherwise disturbed. For example, a cell-free sample can be obtained without shaking or other disturbance, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days or more. In certain embodiments, the cell-free sample is incubated so that the sample is not shaken or otherwise disturbed over the entire period of forming the regeneration matrix.

  By adding specific incubation conditions and / or one or more exogenous factors during the production process, the incubation period required for the regeneration matrix to achieve the desired level of activity can be varied. As a non-limiting example, reducing the ambient oxygen concentration also reduces the amount of time required to achieve the desired level of regeneration matrix activity. In certain embodiments, the ambient oxygen concentration during incubation is about 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% or less. In certain embodiments, the ambient oxygen concentration during the incubation is approximately or exactly 0%. In certain embodiments, when incubated in a hypoxic environment, a regeneration matrix with the desired level of activity is obtained after only 3 days. In certain embodiments, when incubated in a hypoxic environment, a regeneration matrix with the desired level of activity is obtained after only 7 days. In certain embodiments, the ambient oxygen concentration during incubation is changed (eg, increased / decreased) during the incubation step. The person skilled in the art will consider the time constraints when forming the regeneration matrix, the type of tissue damage using the regeneration matrix, the experimental and / or laboratory constraints, and / or the variety that the skilled worker considers important. Depending on any of the other factors, one or more suitable ambient oxygen concentrations could be chosen.

  In certain embodiments, ambient humidity is adjusted or controlled during incubation. For example, a regeneration matrix is generated by incubating a cell-free sample at any of a variety of ambient humidity. In certain embodiments, the ambient humidity is kept low during the incubation, thereby increasing the evaporation of the liquid in the cell-free sample. In certain embodiments, a liquid containing new solutes (eg, “treatment medium” and / or “physiological solution”) is added continuously or periodically to a cell-free sample in which evaporation occurs. It will be appreciated that in such embodiments, the osmolarity of the cell-free sample increases over time as a result of evaporation and addition of a liquid containing new solutes. In certain embodiments, such an increase in osmolarity results in a change in the physical characteristics, biological properties and / or therapeutic activity of the resulting regeneration matrix. As one non-limiting example, increasing the osmolarity of a solution and / or suspension containing a cell-free sample during the step of incubating increases the osmolarity, or It has been shown to obtain a regeneration matrix with a higher physical hardness compared to a regeneration matrix produced by a process increased by a smaller amount. If it is desired that the regeneration matrix maintain a particular physical shape for an extended period of time, such a regeneration matrix can be used as an implantable composition. For example, if it is desired to inject a regeneration matrix into a subject, a regeneration matrix with a lower physical hardness (eg, by maintaining the osmolarity constant or increasing it by a smaller amount). The generated reproduction matrix or the like is useful. In certain embodiments, the ambient humidity is changed (eg, increased and / or decreased) during the step of incubating to control the rate at which the osmolarity changes. The person skilled in the art will consider the time constraints when forming the regeneration matrix, the type of tissue damage using the regeneration matrix, the experimental and / or laboratory constraints, and / or the variety that the skilled worker considers important. Depending on any of the other factors, one or more suitable ambient humidity could be chosen. Furthermore, the density, porosity and / or level of hydration of the formed regeneration matrix can be changed by centrifuging or lyophilizing the formed regeneration matrix. For example, the formed regeneration matrix could be centrifuged at 5000 × g to increase the density and hardness for embedding in bone defects in order to support bone regeneration.

  In certain embodiments, the regeneration matrix of the present invention is generated by incubating a cell-free sample for a given period in an incubation chamber. Any of a variety of incubation chambers can be used. In certain embodiments, the incubation chamber comprises a closed or semi-closed container that is permeable or semi-permeable to one or more substances, such as air and / or gaseous molecules. For example, an Opticell® cassette (BioCrystal Ltd., Westerville, Ohio) can be used as an incubation chamber to generate a regeneration matrix from a cell-free sample. In certain embodiments, the sealed or semi-closed incubation chamber is designed such that the material can be added to and / or removed from the container after initiating the incubation step. For example, an incubation chamber that is permeable or semi-permeable to air and / or gaseous molecules, and liquid and / or solid material added to the chamber at one or more time points during and / or after incubation And / or can be designed to be removable from the chamber. Such an incubation chamber is advantageous when it is desired to increase the osmolarity of the cell-free sample during the step of incubating. For example, the osmolarity of the cell-free sample is determined during the step of incubating the liquid solution or suspension solvent containing the cell-free sample (this solution or solvent, eg, treatment medium or physiological solution). Can be obtained by adding to a cell-free sample and / or tissue sample to obtain a cell-free sample) over time and allowing the liquid containing the new solute to be continuously or periodically added to the cell-free sample. Can be increased. Liquids containing such solutes can include processing media and / or physiological solutions. If the cell-free sample is solubilized or suspended in the processing medium and / or physiological solution, the cell-free sample is incubated during the step of incubating the same processing medium and / or physiological solution. Can be added. In addition or alternatively, different processing media and / or physiological solutions can be added to the cell-free sample during the incubation step. In addition or alternatively, the liquid containing the solute added to the cell-free sample is neither a processing medium nor a physiological solution.

  In certain embodiments, the regeneration matrix exhibits different physical characteristics, biological properties and / or therapeutic activity, depending on the processing medium and / or physiological solution used during the production process. For example, as shown in Example 11, the regeneration matrix formed using TR-10 medium has increased biological activity compared to the regeneration matrix produced using DMEM / F-12 medium. Show. Those skilled in the art will be able to use a suitable processing medium to achieve a regeneration matrix that exhibits one or more desired physical characteristics, biological properties and / or therapeutic activity without undue experimentation. A physiological solution could be determined.

  At any of various points during the regeneration matrix generation process, the treatment medium and / or physiological solution can be added to the tissue sample and / or the cell-free sample. In certain embodiments, during the isolation of the tissue sample, before removing the cells from the tissue sample, before incubating the cell-free sample, and / or during the step of incubating, the treatment medium may be the tissue sample and / or Add to cell-free sample.

  In certain embodiments, a regeneration matrix is generated by incubating a cell-free sample in the presence of one or more exogenous factors. For example, a cell-free sample can be incubated in the presence of one or more therapeutic agents, one or more cell types, or both. In certain embodiments, such exogenous factors alter the physical characteristics of the regeneration matrix and / or enhance biological and / or therapeutic activity. For example, as shown in Example 9, a regeneration matrix supplemented by the addition of ITS, EGF and bFGF during the generation of the regeneration matrix results in an increased effect on the neurite outgrowth activity of the regeneration matrix.

  Any of a variety of therapeutic agents can be used to increase and / or supplement the activity of the regeneration matrix of the present invention. Examples of therapeutic agents that can be used with the inventive regeneration matrix and methods include, but are not limited to, proteins, peptides, drugs, cytokines, extracellular matrix molecules and / or growth factors. . One skilled in the art will recognize other suitable and / or advantageous therapeutic agents that may be used in accordance with the present invention. In certain embodiments, administering a therapeutic agent into the regeneration matrix increases the magnitude of one or more beneficial therapeutic effects of the therapeutic agent. In certain embodiments, administration of a therapeutic agent into the regeneration matrix prolongs the activity of one or more beneficial therapeutic effects of the therapeutic agent. In certain embodiments, when the therapeutic agent is administered into the regeneration matrix, one or more beneficial therapeutic effects of the therapeutic agent are released over time. In certain embodiments, when the therapeutic agent is administered in the regeneration matrix, one or more beneficial therapeutic effects of the therapeutic agent are protected from substantial reduction over time. In certain embodiments, two or more therapeutic agents are administered in the regeneration matrix. In certain embodiments, the therapeutic agent is added during the process of generating the regeneration matrix. For example, the therapeutic agent may include, but is not limited to, during isolation of a tissue sample, before removing cells from a tissue sample, before incubating a cell-free sample, and / or during an incubation step. Can be added in one or more steps of the production process.

  In certain embodiments, the therapeutic agent is evenly distributed throughout the regeneration matrix. In certain embodiments, the therapeutic agent is unevenly distributed throughout the regeneration matrix. For example, the therapeutic agent can be concentrated at or near the core of the regeneration matrix. This can be achieved, for example, by introducing a therapeutic agent during the first few days or the first half of the regeneration matrix formation period and then removing most or all of the solution surrounding the regeneration matrix being formed in the incubation chamber. Achieved by removing and adding the initial cell-free sample or solution to the incubation chamber until the desired level is reached, then further incubating the cell-free sample and the regeneration matrix to continue the regeneration matrix formation process Will be able to. Additional cell-free samples or solutions could also be added along with another therapeutic agent. This other therapeutic agent then becomes non-uniformly concentrated in the vicinity of the outer surface of the regeneration matrix being formed, and therefore, if the regeneration matrix biodegrades or degrades, the first therapeutic agent It will be released to the surrounding environment before the agent. This method could be used in applications where it is desired that therapeutic agents be delivered to specific sites at different times (eg, one after the other). In certain embodiments, such unequal distribution affects the properties and / or function of the therapeutic agent in vivo. For example, such unequal distribution changes the magnitude of one or more beneficial therapeutic effects of the therapeutic agent, prolonging the activity of one or more beneficial therapeutic effects of the therapeutic agent, The sustained release characteristics of one or more beneficial therapeutic effects of the therapeutic agent are altered and / or one or more beneficial therapeutic effects of the therapeutic agent are protected from substantial reduction over time Is done.

  Any of a variety of cell types can be used to increase and / or supplement the activity of the regeneration matrix of the present invention. However, although cells can be used to increase and / or supplement the activity of the regeneration matrix of the present invention, such cells are not required to form the regeneration matrix. You will understand that. In certain embodiments, the cells are added to a cell-free sample from which the cells have been removed. In certain embodiments, a different type of cell is used than the cells present in the tissue sample from which the cell-free sample is obtained. Examples of cell types that can be used according to the present invention include, but are not limited to, stem cells, progenitor cells and / or somatic cells. One skilled in the art will recognize other suitable and / or advantageous cell types that can be used in accordance with the present invention. In certain embodiments, cells are added during the process of generating the regeneration matrix. For example, one of the production steps including, but not limited to, isolating a tissue sample, before removing cells from the tissue sample, before incubating the cell-free sample, and / or during the incubation step. In one or more steps, cells can be added. In certain embodiments, the cells are evenly distributed throughout the regeneration matrix. In certain embodiments, the cells are distributed unevenly throughout the regeneration matrix. For example, the cells can be concentrated at or near the core of the regeneration matrix. In certain embodiments, such unequal distribution affects cellular properties and / or functions in vivo.

  One of the most effective ways to generate a promising regeneration matrix for implantation into a patient without eliciting a significant immune response is to derive the regeneration matrix from the patient's own blood and / or tissue. However, the regeneration matrix of the present invention is not limited to such an autogenous regeneration matrix. In certain embodiments, a ready-made regeneration matrix for immediate implantation is made using blood or other tissue from the same species. In such embodiments, it may be desirable to administer such a regenerative matrix in combination with one or more immunosuppressive agents. Such immunosuppressive agents are known to those skilled in the art. In certain embodiments, a regeneration matrix for use in one species is derived from cells from different species. For example, a regeneration matrix produced using tissue from an animal (eg, a pig) is administered to a human. In certain embodiments, the regeneration matrix of the present invention is useful for animal applications, such as repairing damaged and / or degenerated tissue of livestock and / or pets.

  In certain embodiments, the physical characteristics, biological properties, and / or therapeutic activity of the regeneration matrix are changed after the production process is substantially complete. For example, in many cases it will be desirable to store the regeneration matrix for an extended period of time after fabrication. As is known in the art, hydrolysis is one cause of shortening the shelf life of a variety of biological and non-biological materials. Thus, in certain embodiments, the shelf life of the regeneration matrix of the present invention can be extended by removing some or all of the liquid present in such regeneration matrix. Any of a variety of techniques can be used to remove such liquids. For example, the regeneration matrix can be centrifuged to condense proteinaceous and / or other biological macromolecular components, after which a desired amount of liquid can be removed from the centrifuged sample. In addition or alternatively, a filtration step (e.g., by gravity or low speed centrifugation) that uses a filter having a pore size that is large enough to pass liquid but not regenerated matrix material. Liquid can also be removed from the regeneration matrix by applying it to the regeneration matrix. In addition or alternatively, liquid can be removed from the regeneration matrix by dehydrating the regeneration matrix for a given period of time in the presence or absence of a desiccant. One skilled in the art will recognize other suitable and / or useful methods for removing liquid from the regeneration matrix to improve the shelf life of the regeneration matrix. It will also be appreciated that removal of liquid from the regeneration matrix may change one or more of the biological properties and / or therapeutic activity of the regeneration matrix. In certain embodiments, such biological properties and / or therapeutic activities are enhanced by a liquid removal process.

  Furthermore, the removal of such liquids provides additional benefits by changing the physical characteristics of the regeneration matrix such that the regeneration matrix more closely matches the physical environment in which the regeneration matrix is administered. Can do. For example, reducing the level of hydration of the regeneration matrix can result in a regeneration matrix having a greater density. Such a dense regeneration matrix can be advantageously used, for example, for bone and / or cartilage regeneration. Those skilled in the art will know, based on the present description, other advantageous applications of such a high density regeneration matrix, and in such applications, such regeneration matrices can be used without undue experimentation. Could be used.

Regeneration Matrix Composition and Features The regeneration matrix of the present invention comprises an unequal mixture of proteins, lipids, carbohydrates, salts and nucleic acids. In addition, the regeneration matrix is stored at refrigerated temperatures for up to 3 months without loss of function. Perhaps the regeneration matrix could be stored indefinitely using appropriate conditions. For example, the moisture content of the regeneration matrix can be reduced (eg, by centrifugation, dehydration, lyophilization, and the like) to extend the period during which the regeneration matrix can be stored. In certain embodiments, the primary structure consists mostly of aggregates of spherical structures with a diameter of about 1-4 μm. In certain embodiments, the primary structure consists mostly of aggregates of spherical structures with a diameter of about 100 nm. In certain embodiments, the primary structure consists mostly of aggregates of spherical structures that are at least about 100 nm in diameter. In certain embodiments, the regeneration matrix comprises such a spherical structure interspersed with fibers. As described above, the processing conditions used to form the starting material can affect the ultimate size of the spherical structure. For example, if the starting material is filtered through a 5 μm filter, the diameter of the predominant spherical structure is typically about 2 to 4 μm. When the starting material is filtered through a 1.2 μm filter, the diameter of the predominant spherical structure is typically about 1 to 2 μm.

  In certain embodiments, an antibody against CD56 recognizes the surface of the sphere, indicating the presence of a neural cell adhesion molecule (NCAM). Without intending to be bound by theory, it is postulated that NCAM-mediated stimulation is one mechanism by which the regeneration matrix mobilizes neurons and stimulates regeneration when damage occurs. In certain embodiments, the regeneration matrix acts as an extracellular growth matrix (ECGM) during tissue repair, tissue regeneration and / or tissue formation. When the regeneration matrix is formed from blood, the physical and functional properties will allow the lysate to be filtered prior to the method of blood collection (eg, anticoagulant presence and / or type of anticoagulant) and incubation period. It is affected by the pore size used to do. When blood is collected by adding heparin, an anticoagulant, a structure similar to that formed using blood collected without the anticoagulant is formed. The regeneration matrix formed from this whole blood usually adheres to both the lower and upper parts of the generator (eg Opticell®), and the arrangement of the structures is slightly different on each surface. However, when blood is collected with the addition of anticoagulant EDTA or citrate, the regeneration matrix formed from this whole blood loosely adheres to the generator and has a gel-like appearance and hardness.

  In certain embodiments, the regeneration matrix of the present invention includes water as a major component. In certain embodiments, the non-aqueous composition of the regeneration matrix comprises primarily proteins. In certain embodiments, the protein content of the regeneration matrix ranges from about 5 to 15% by weight. However, this ratio can be manipulated by using different ratios of tissue samples and / or cell-free samples to processing media in the production process. In certain embodiments, by utilizing such different ratios, the protein content of the generated regeneration matrix is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30% or more. This ratio can be further changed by changing the level of hydration of the generated regeneration matrix. In certain embodiments, by utilizing such changes in the level of hydration of the regeneration matrix, the protein content of the regeneration matrix can be less than about 0.1% or greater than about 90%. Let's go. When the regeneration matrix is generated from whole blood, the main protein components of the regeneration matrix are typically albumin and hemoglobin. When the regeneration matrix is generated from the plasma-platelet-buffy coat fraction of blood, the main protein component of the regeneration matrix is typically albumin. However, those skilled in the art will also appreciate that other protein or peptide components of blood are also present in these regeneration matrices. In addition, those skilled in the art will appreciate that other protein or peptide components may also be present when the regeneration matrix is generated from a cell-free sample obtained from a tissue other than blood.

  The regeneration matrix has important properties and possibilities for regeneration and tissue formation. The regeneration matrix can assist and / or serve as a scaffold for cells involved in tissue regeneration and tissue formation. In certain embodiments, the regeneration matrix of the present invention allows cells to adhere, grow and / or differentiate on or around the regeneration matrix. In certain embodiments, the regeneration matrix of the present invention stimulates nerve cells to elongate neurites that adhere to the regeneration matrix. In vitro cell culture assays using neurons, where the regeneration matrix selectively stimulates genes associated with neuronal cell adhesion, neuronal survival, axon proliferation and regeneration, pattern formation, and connectivity Has been demonstrated. In vitro cell culture assays using human fibroblasts demonstrate that the regeneration matrix produced by filtration using a 5 μm filter has an inhibitory effect on the growth of these cells. The magnitude of such fibroblast inhibitory activity can be influenced by the processing conditions used to form the regeneration matrix. A regeneration matrix produced from coagulated blood inhibits fibroblast proliferation more strongly than a regeneration matrix produced from blood collected in the presence of an anticoagulant. In certain embodiments, this difference is advantageous because fibroblasts secrete collagen in the wound tissue, which is needed to some degree to provide support structure, but for most severe tissue damage, Deletions become mainly filled with connective tissue scars. Extensive connective tissue scarring inhibits the regeneration process and leads to permanent loss of function in damaged tissues and organs. Thus, in certain embodiments, the magnitude of the activity of the regeneration matrix is adjusted or controlled to provide optimal wound healing characteristics based on the severity of tissue damage at the wound site.

  Using an in vivo model of spinal cord injury, the regeneration matrix of the present invention is not limited to these, but includes, but is not limited to, angiogenic properties, axonal proliferation, reduced lesion size and number, and astrocyte glial scar formation It has been demonstrated that it exhibits several advantageous effects, including a suppressive function against In vivo, the regeneration matrix typically degrades over time (eg, usually in about 4-8 weeks) primarily by macrophage ingestion, whereas the regeneration matrix of the present invention has such degradation time. In addition, it is not limited to the decomposition mechanism. As will be apparent from this description, such degradation includes, but is not limited to, mechanical, chemical, metabolic and / or enzymatic degradation due to cellular activity (eg, through the action of macrophages). It can occur through any of a variety of mechanisms, including the beginning. In certain embodiments, such degradation of the regeneration matrix stimulates macrophages so that the macrophages themselves exhibit additional regenerative properties (eg, by differential transformation into von Willebrand factor positive endothelial-like cells). become. When the regeneration matrix of the present invention is transplanted into a damaged, deleted or excised part of a tissue or organ, the matrix of the present invention supports the regeneration and formation of new tissue, and the damaged or deleted part of the tissue or organ. Help restore the functionality of the. Non-limiting examples of tissues that can be implanted with a regeneration matrix to initiate, increase, support and / or guide tissue regeneration and tissue formation include: liver, kidney, pancreas, heart, ovary, Examples include damaged, deleted or excised parts of the thyroid, brain, spinal cord and other neural tissues.

  In the case of spinal cord regeneration, the regeneration matrix may be transplanted into a damaged, degenerated, deleted or excised portion of the spinal cord with cells such as neural stem cells, neural progenitor cells and / or differentiated neurons. However, it may be transplanted without cells. Further expand or manipulate the functionality of the regeneration matrix by adding one or more therapeutic agents including, but not limited to, growth factors, cytokines, drugs and / or other ingredients. Can do. In certain embodiments, when the regeneration matrix is generated without the addition of exogenous therapeutic agents, partial functionality with respect to neurite outgrowth and neurogene upregulation is observed. In such embodiments, the magnitude of these functions can be enhanced by incorporating exogenous therapeutic agents during the generation of the regeneration matrix. Non-limiting examples of desirable additional therapeutic agents include growth factors such as bFGF, EGF, BDNF and / or NGF. In certain embodiments, when the regeneration matrix is transplanted into the injured spinal cord, motor function deficits are functionally restored only 4 weeks after transplantation.

  The biological scaffolds encompassed by certain embodiments of the present invention have unique multifaceted regeneration properties, are implantable and degradable, and the formation of these biological scaffolds is completely unprecedented. . Several cell types produce simple extracellular matrices, but such matrices appear to be only structural. The generation of self-assembling matrix or skeletal materials that structurally and biologically promote, support and assist in the regeneration and growth of new tissue has never been reported. Such matrix or framework materials are encompassed by certain embodiments of the present invention.

(Example 1)
Induction of regenerated matrix from coagulated whole blood After venous blood (from Research Blood Components, Brightton, Mass.) From healthy donors was procured in 12 ml of vaccutainer and allowed to clot at RT for at least 30 minutes. Frozen at −20 ° C. until use. Treatment was initiated by subjecting the blood to 5 freeze (−20 ° C.)-Thaw (RT) cycles. The contents of vaccutainer containing 10 ml of blood were put into a sterile mortar. Supplemented with 2 × ITS adjuvant (Gibco / Invitrogen, Gaithersburg, MD), 20 ng / ml recombinant human EGF (R & D Systems, Minneapolis, MN) and 40 ng / ml recombinant human bFGF (R & D Systems) Processing medium consisting of a 1: 1 mixture of DMEM (Mediatech, Herndon, VA) and Ham F12 medium (Mediatech) is added, and the clot is manually ground using a sterile pestle Destroyed by. During the grinding process, the liquid containing the liquefied whole blood clot and processing medium is aspirated from the mortar using a sterile 25 ml serum pipette and transferred to a 40 μm mesh filter from which the contents are transferred. Filtered by gravity into a sterile 50 ml conical centrifuge tube. Fresh processing medium was added to the remaining solid clot and the process was repeated until the clot had all liquefied and reached a final volume of 200 ml. The tube was centrifuged at 500 × g for 30 minutes, and the supernatant was filtered directly through a 5 μm syringe filter (Pall / Gelman) into an Opticell® cassette. The Opticell® cassette was incubated at 37 ° C. and 7.5% CO ₂ . After 6 to 8 days of incubation, 3 ml of fresh processing medium was added to each Opticell® cassette. Thereafter, 1.5 ml of fresh processing medium was added every other day until the regeneration matrix was collected for further analysis. The regeneration matrix formed using this method has adhesive properties because it needs to be collected by removing the upper membrane of the Opticell® cassette and scraping from the lower membrane.

(Example 2)
Induction from Whole Blood Collected Using Regenerative Matrix Anticoagulants From healthy donors, venous blood (Research Blood Components, Brightton, Mass.) From 12 ml of vaccutainer containing Na ₄ -EDTA® ) And frozen at −20 ° C. until use. The process was initiated by subjecting the blood to 5 consecutive freeze (−20 ° C.)-Thaw (RT) cycles. The contents of vaccutainer containing 10 ml of blood were poured into a sterile container and the solution was gently mixed after the treatment medium (see Example 1) was added to a final volume of 200 ml. This starting material was gravity filtered through a 40 μm mesh filter. After collecting the filtrate, it was centrifuged at 500 × g for 30 minutes, and the supernatant was directly filtered into an Opticell (registered trademark) cassette by 10 ml through a 5 μm syringe filter. The Opticell® cassette was incubated at 37 ° C. and 7.5% CO ₂ in air. After 6 to 8 days of incubation, 3 ml of fresh processing medium was added to each Opticell® cassette. Thereafter, 1.5 ml of fresh processing medium was added every other day until the regeneration matrix was collected for further analysis.

In another experiment, venous blood (from Research Blood Components, Brightton, Mass.) Was sourced from healthy donors in 12 ml of vaccutainer® containing Na ₄ -EDTA and kept on ice. The contents of vaccutainer containing 10 ml of blood were poured into a sterile container and the solution was gently mixed after the treatment medium (see Example 1) was added to a final volume of 200 ml. This starting material was filtered through a 5 μm syringe filter. After collecting the filtrate, it was centrifuged at 500 × g for 30 minutes, and the supernatant was directly filtered into an Opticell (registered trademark) cassette by 10 ml through a 5 μm syringe filter. The Opticell® cassette was incubated at 37 ° C. and 7.5% CO ₂ . After 6 to 8 days of incubation, 3 ml of fresh processing medium was added to each Opticell® cassette. Thereafter, 1.5 ml of fresh processing medium was added every other day until the regeneration matrix was collected for further analysis.

  The regeneration matrix formed using these methods has weak adhesive properties and is collected by aspiration through an 18 gauge needle using one of the access ports of the Opticell® cassette. be able to.

(Example 3)
Derivation from whole blood fractions obtained using continuous filtration of the regeneration matrix. The activity of the regeneration matrix was determined using the supernatant from the centrifugation step (described in Examples 1 and 2) and 5 μm and 1.2 μm filters. Further improvements were made by passing the combined sets together. This step removes large particles from the starting material, resulting in a change in functional properties. An example of a reproduction matrix structure in an Opticell® cassette is shown (FIG. 3). FIG. 4 shows that the regeneration matrix generated from whole blood does not show any signs of unchanged cells or nuclei. Hematoxylin staining was completely negative in all sections analyzed, but the material stained strongly with eosin. Using a high numerical aperture lens (high resolution), the bead-like structure can be clearly resolved. In addition to the bead-like structure that makes up most of the regeneration matrix, a small area of a flat sheet that appears to be of a denser structure can be observed.

(Example 4.1)
Scanning electron microscope observation (SEM) of the regeneration matrix
For electron microscopy, the regeneration matrix produced by the method described in Examples 1, 2 and 3 was used with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (from Electron Microscience Sciences) Fix for 16 hours at 4 ° C. Immobilization was performed directly in Opticell® by replacing the medium with 10 ml fixative. The next day, the fixative was removed and PBS (10 ml) was added to Opticell®. The Opticell (registered trademark) was opened with a scalpel, and the Opticell (registered trademark) membrane to which the regeneration matrix was adhered was cut into 0.5 x 1 cm pieces. Small pieces were transferred to 1.5 ml microcentrifuge tubes and post-fixed with 1% osmium tetroxide in water for 2 hours. The sample was then immediately kept in a 50% ethanol solution for 2 minutes before being continuously dehydrated in 70, 80, 90, 95 and 100% ethanol solutions. After the last ethanol step, the samples were kept at −20 ° C. until shipped to the Central Microscopy Core Facility at the University of Massachusetts Amherst. The sample was dried to the critical point and sputter coated. The sample was photographed at a magnification of 100 to 7500 using a JEOL JSM-5400 scanning electron microscope. Images were imported into a computer via a digital interface and exported as a TIFF format with a resolution of 640 x 480 pixels.

  Regeneration matrices with different microstructures were obtained from each of the preparation methods (FIGS. 5, 6 and 7). From the lower porosity filtration, a similar overall morphology was obtained, but the size of the sphere for the 1.2 μm treatment (1-2 μm diameter, FIG. 6) is the same as that of the sphere for the 5.0 μm treatment. It was smaller than the size (diameter 2-3 μm, FIG. 5). The regeneration matrix produced using coagulated blood experienced a mature phase after approximately 2-3 weeks of incubation, in which a wavy structure was observed on the upper surface of the matrix. The regeneration matrix produced using non-coagulated blood does not have such an appearance, but the spherical structures are aggregated together to form a continuous structure in which fibers of about 100 nm are scattered all over ( FIG. 7).

  For histological examination, the regeneration matrix was fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (from Electron Microscience Sciences) at 4 ° C. for 16 hours. Immobilization was performed directly in Opticell® by replacing the medium with 10 ml fixative. The next day, the fixative was removed and PBS (10 ml) was added to Opticell®. The Opticell® is opened with a scalpel and the Opticell® membrane with the regeneration matrix attached is cut into 2 × 2 cm pieces and mounted between sponges in a tissue cassette for histological examination. . Samples were transferred to 70% ethanol and sent for standard histological examination by Mass History (Worcester, Mass.). Samples were dehydrated continuously in ethanol and embedded in paraffin, then positioned and cut. Sections were stained with hematoxylin-eosin using normal immobilization conditions. Images were acquired with dark field optics using a modified Leica Microscope optimized by Richardson technologies for contrast enhancement and noise reduction. Images were collected at 20, 40 and 100 times using a video camera's 640 × 480 pixel resolution. An image of the sample collected at 100x magnification is shown (Figure 8). The regeneration matrix is dyed with eosin but not with hematoxylin.

(Example 4.2)
Transmission electron microscope observation (TEM) of regenerated matrix
The regeneration matrix prepared from tissue samples and passed through a 5 μm filter after 21 days was cut and scanned by TEM (FIGS. 9-11). It is clear from the TEM analysis that the spherical particles are part of a continuous material since the spherical particles do not show boundaries between them under TEM. The spheres are hard and not hollow inside and do not have a membrane and any other type of structure around them. The interior of this material is homogeneous. The very dark spot in FIG. 9 is the staining background.

(Example 5)
Biochemical characterization of regeneration matrix Regeneration matrices made from whole blood were analyzed for protein, lipid, nucleic acid and carbohydrate content and metabolic activity as a sensitive indicator of the presence of living cells. Biochemical data was normalized with respect to mass in grams of the wet matrix analyzed. Using standard methods for protein, DNA, RNA and lipid quantification, the composition of several different lots of the regeneration matrix was determined at multiple points in the production process of the regeneration matrix (detailed methods are , Indicated in individual terms).

5.1. Total protein content of the regeneration matrix The most abundant biological component of the regeneration matrix is protein, representing 8.8 ± 1.2% by weight, with the remaining mass being the liquid bound to the regeneration matrix . A standardized protein content in the regeneration matrix was pre-weighed and a certain amount of regeneration matrix was added to SDS lysis buffer (150 mM NaCl, 50 mM Tris-HCI (pH 7.5), 10 mM EDTA, 1% SDS) and Complete (trademark). ) Determined by solubilization in a miniprotease inhibitor cocktail (Catalog No. 11-836-153, Roche). The protein concentration in the lysate was determined according to the manufacturer's instructions using a protein assay kit based on the Raleigh method (catalog number 500-0112, manufactured by Bio-Rad). A standard curve was generated using a BSA standard (catalog number 500-0007, manufactured by Bio-Rad). The optical density (750 nm) was measured using a Spectramax Plus spectrophotometer (Model 384, manufactured by Molecular Devices), and the protein concentration of the sample was measured in the linear range of a standard curve using Softmax Pro software (produced by Molecular Devices). Determined by interpolation.

  Total protein content (per regeneration matrix) is determined for samples collected at day 0, day 15 and day 21 after initial seeding of OptiCell® cassette with respect to regeneration matrix starting material (0). did.

Table 1. Total Protein Content of Regeneration Matrix in Opticell® Cassette After removing the regeneration matrix generating liquid and solubilizing the regeneration matrix in SDS buffer, the total protein content was determined. At each time point, 4 Opticell® cassettes were sampled and analyzed. Results from 4 independent batches of regeneration matrix generation are reported as mean ± standard deviation.

５．２．再生マトリックスのＳＤＳ−Ｐａｇｅ分析
再生マトリックス中に存在するタンパク質の主な種を、還元ＳＤＳ−ＰＡＧＥゲルから切り取ったすべての目に見えるクーマシーバンドに対する質量分析法によって決定した。上記に準じて、ＳＤＳ−ＰＡＧＥのために、試料を調製し、定量化した後、あらかじめ成型したポリアクリルアミド勾配ゲル（ＰＡＧＥ ４〜２０％勾配、カタログ番号３４５−００３３、Ｂｉｏ−Ｒａｄ製）に、ウェルあたり１０μｇで積んだ。主なタンパク質のバンドを、Ｓｉｍｐｌｙ Ｂｌｕｅ（登録商標）ゲル染色液（カタログ番号ＬＣ６０６０、Ｉｎｖｉｔｒｏｇｅｎ製）を用いて染色することによって可視化した。

5.2. SDS-Page Analysis of Regeneration Matrix The main species of proteins present in the regeneration matrix were determined by mass spectrometry on all visible Coomassie bands excised from reducing SDS-PAGE gels. In accordance with the above, after preparing and quantifying samples for SDS-PAGE, pre-molded polyacrylamide gradient gel (PAGE 4-20% gradient, catalog number 345-0033, manufactured by Bio-Rad) Loaded at 10 μg per well. Major protein bands were visualized by staining with Simply Blue® gel stain (Cat. No. LC6060, Invitrogen).

  The excised protein bands were sent to Midwest Bio Services (Overland Park, KS (www.midwestbioservices.com)) for identification. Briefly, peptide extraction and separation on a microcapillary reversed-phase column after reduction, alkylation and in-gel trypsinization. Peptides were eluted and electrosprayed directly onto an LCQ Deca XP Plus ion trap mass meter. Full MS spectra and MS / MS spectra were obtained and data were analyzed by TurboSEQUEST software. In this type of analysis, the mass of the peptide is used to dereference the known molecular weight of peptide fragments of known proteins so that the presence of partially degraded protein in the gel band will also be detected. Let's go. The main protein species identified in the regeneration matrix are listed below: transferrin, serum albumin, serum albumin precursor, complement component 3, AD chain hemoglobin, IgM, IgG1, medalacin inhibitor 2, carbonic anhydride Enzymes and CA1 protein.

  A consistent protein banding pattern was observed in samples from all batches (51-56) on both day 15 and day 21. Most proteins degraded to a low molecular weight (about 67% <10 KDa), about 15% to 33 KDa, and the remaining about 19% to a molecular weight between 34 and 103 KDa. A photograph of the SDS gel is shown in FIG.

5.3. Nucleic acid content of regeneration matrix According to the case of protein, DNA was purified from the regeneration matrix (M) and quantified, and then compared with the starting material (O) at time points 0, 15, 21 Weighed samples were prepared in 2 × SDS / Proteinase K lysis buffer (40 mM Tris-HCl [pH 8.0], 50 mM EDTA, 200 mM NaCl, 2% SDS and 6 μl Proteinase K stock (20 mg / ml, catalog number 25530-049). , Manufactured by Invitrogen) and incubated overnight at 50 ° C. The lysate was aliquoted with an equal volume of Tris-HCl saturated phenol-chloroform-isoamyl alcohol (PCI, 25: 24: 1, pH 8.0), Extracted with 1 drop of Phase Lock Gel (PLG, catalog number 955 15 403-7, manufactured by Eppendorf) Samples were centrifuged at 14,000 g for 10 minutes at 4 ° C. to separate the phases. , PLG added, new 2.0ml Transfer to a microcentrifuge tube and repeat extraction and centrifugation Transfer the aqueous phase to a new 2.0 ml microcentrifuge tube with PLG, extract with an equal volume of chloroform, 12,000 × g, 4 ° C. The aqueous phase was transferred to a fresh 1.5 ml microcentrifuge tube and the DNA was transferred to 0.7 volumes of isopropanol (Catalog No. 3032-06, from Mallinckrodt) and 10 μg of glycogen (Catalog No. 10910393001). From Roche) and left at RT for 15 minutes Samples were centrifuged at 14,000 × g for 20 minutes at 4 ° C. The pellet was washed with 0.5 ml of 70% ethanol (catalog number). EX0289-1, manufactured by EM Science) twice, air-dried, and 30 μl TE Resuspended in impulse, pH 8.0 The concentration of DNA was determined by optical spectrophotometric measurement (Spectra Max Plus, Model 384, Molecular Devices) at 260 nm optical density (OD). The OD ratio of / 280 was also determined to indicate the level of protein contamination in the DNA isolation, and the total DNA content (μg) of the regeneration matrix at day 15 and day 12 was calculated as Comparison with total DNA content.

Table 2. Total DNA content of regeneration matrix in Opticell® cassette After removing the regeneration matrix generating liquid and solubilizing the R regeneration matrix in SDS buffer, the total DNA content was determined. At each time point, 4 Opticell® cassettes were sampled and analyzed. Results from 4 independent batches of regeneration matrix generation are reported as mean ± standard deviation.

再生マトリックス（Ｍ）からＲＮＡを単離し、Ｔｒｉｚｏｌ試薬（カタログ番号１５５９６−０１８、Ｉｎｖｉｔｒｏｇｅｎ製）を使用して、０、１５および２１の時点で、出発材料（Ｏ）のＲＮＡと比較した。１．０ｍｌのＴｒｉｚｏｌを、あらかじめ決定した量の質量に添加し、可溶化するまでＲＴで放置した（１０〜２０分）。試料を、１２，０００×ｇ、４℃で１０分間遠心分離し、水相を、新しい１．５ｍｌの微量遠心チューブに移した。溶液を、２００μｌのクロロホルム（カタログ番号４４４０−０４、Ｍａｌｌｉｎｃｋｒｏｄｔ製）で抽出し、１２，０００×ｇ、４℃で２０分間遠心分離した。水相を、新しい１．５ｍｌの微量遠心チューブに移し、ＲＮＡを、５００μｌのイソプロピルアルコール（カタログ番号３０３２−０６、Ｍａｌｌｉｎｃｋｒｏｄｔ製）を添加することによって沈殿させ、１２，０００×ｇ、４℃で３０分間遠心分離した。ＲＮＡペレットを、７５％エタノール中で洗浄し、７，５００×ｇ、４℃で５分間遠心分離し、風乾した後、試料を６５℃で１５分間加熱することによって、ＲＮａｓｅを含まない２５μｌの水中に再懸濁した。ＲＮＡを、分光光度計（Ｓｐｅｃｔｒａｍａｘ、Ｍｏｄｅｌ ３８４、Ｍｏｌｅｃｕｌａｒ Ｄｅｖｉｃｅｓ製）を使用して、２６０ｎｍのＯＤによって定量化した。第１５日および第１２日における再生マトリックスの全ＲＮＡ含有量（μｇ）を、０時の再生マトリックス出発材料中の全ＲＮＡ含有量と比較した。

RNA was isolated from the regeneration matrix (M) and compared to the starting material (O) RNA at 0, 15 and 21 using Trizol reagent (Cat. No. 15596-018, Invitrogen). 1.0 ml Trizol was added to a predetermined amount of mass and left at RT until solubilized (10-20 minutes). Samples were centrifuged at 12,000 × g for 10 minutes at 4 ° C. and the aqueous phase was transferred to a new 1.5 ml microcentrifuge tube. The solution was extracted with 200 μl of chloroform (Catalog No. 4440-04, Mallinckrodt) and centrifuged at 12,000 × g at 4 ° C. for 20 minutes. The aqueous phase is transferred to a new 1.5 ml microcentrifuge tube and the RNA is precipitated by adding 500 μl of isopropyl alcohol (Catalog No. 3032-06, Mallinckrodt), 12,000 × g, 30 ° C. at 4 ° C. Centrifuged for minutes. The RNA pellet is washed in 75% ethanol, centrifuged at 7,500 × g, 4 ° C. for 5 minutes, air dried, and then the sample is heated at 65 ° C. for 15 minutes to give 25 μl water without RNase. Resuspended. RNA was quantified by OD of 260 nm using a spectrophotometer (Spectramax, Model 384, Molecular Devices). The total RNA content (μg) of the regeneration matrix on day 15 and day 12 was compared to the total RNA content in the regeneration matrix starting material at 0:00.

Table 3. Total RNA content of regeneration matrix in Opticell® cassette After removing the regeneration matrix generating liquid and solubilizing the regeneration matrix in SDS buffer, the total RNA content was determined. At each time point, 4 Opticell® cassettes were sampled and analyzed. Results from 4 independent batches of regeneration matrix generation are reported as mean ± standard deviation.

５．４．再生マトリックスの脂質含有量。

5.4. The lipid content of the regeneration matrix.

The total lipid content in the regeneration matrix (M) was determined by the method of Bligh and Dyer (Bright, EG and Dyer, WJ, “A rapid method of total extraction and purification”, Can. J. Biochem. Physiol., 37, 911-917, 1959) and compared to the total lipid content of the starting material (O) at 0, 15 and 21 time points. Samples were stored frozen at -85 ° C until analysis and thawed at RT during analysis. For the regeneration matrix, 375 μl of methanol: chloroform (2: 1 v / v) was added to the pre-weighed sample and the medium speed, RT in the vortex mixer (Digital mini vortexer, catalog number 14005-824, VWR). Incubated for 15 minutes. Then, 475 μl of a methanol: chloroform: water mixture (catalog number IB05174, manufactured by IBI Shelton) was added and incubated for 15 minutes at RT in a vortex mixer as above. Samples were centrifuged at 250 xg for 10 minutes at RT. The liquid phase (both chloroform and water phases) was transferred to a fresh 1.5 ml microcentrifuge tube, leaving only the regeneration matrix pellet in the tube. The regeneration matrix pellet was resuspended in 475 μl of a 2: 1: 0.8 mixture of methanol: chloroform: water and extracted by vortexing again at medium speed for 15 minutes. 125 μml of chloroform was added, the sample was vortexed for 2 minutes, 125 μml of water was added, the sample was vortexed for an additional 2 minutes and then centrifuged at 250 × g, RT for 10 minutes. The liquid phase was pooled to the sample from the initial extraction and centrifuged at 1,000 × g, RT for 10 minutes. The chloroform phase was transferred to a fresh 1.5 ml microcentrifuge tube and dried in a vacuum oven for 45 minutes or until the chloroform was completely evaporated. 50 μl of chloroform was added to the dried lipid pellet and the sample was vortexed for 15 minutes to completely solubilize the lipid. Samples were stored at -85 ° C until final analysis. By making a dilution series of standard lipids (L4646, Sigma), adding 10 μl of extracted lipid sample to 500 μl of concentrated sulfuric acid (Catalog No. SX1244-6, EMD Chemicals) and boiling the tube for 10 minutes, The sample was hydrolyzed. Samples were cooled in a water bath for 2 minutes at RT, and 40 μl of sample was diluted with 600 μl of phosphate-vanillin reagent (0.6 g vanillin (Cat # VX0045-1, from EM Science), 10 ml 100% ethanol (catalog No. EX0289-1, EM Science), 90 ml dH ₂ O and 400 ml phosphoric acid (Catalog No. PX099-6, EMD Chemicals)), mixed, then protected from light and incubated for 45 minutes at RT . Lipid concentration was determined by measuring OD at 525 nm (Spectra Max 384, Molecular Devices) and interpolating with a standard curve using SoftMax provided in the spectrophotometer. The total lipid content (μg) of the regeneration matrix on day 15 and day 12 was compared to the total lipid content in the starting material at 0:00.

Table 4. Total lipid content of regeneration matrix in Opticell® cassette The regeneration matrix production liquid was removed and the regeneration matrix total lipid content was determined as described above. At each time point, 4 Opticell® cassettes were sampled and analyzed. Results from 4 independent batches of regeneration matrix generation are reported as mean ± standard deviation.

核酸および脂質の分析結果は、これらの材料が出発材料中に存在し、時間の経過と共に分解することを示唆している。ゼロ時における出発材料中に検出されたＤＮＡおよびＲＮＡの量の変動の程度が大きく、それに伴って、初期の分解速度も変動するようである。異なる試料間でのこの変動は、第１５日および第２１日までには、時間と共に減少した。これらの成分のうちのいずれかもが増加する場合はなく、これは、細胞の活性が存在しないことのさらなる証拠となる。

Nucleic acid and lipid analysis results suggest that these materials are present in the starting material and degrade over time. It appears that the amount of variation in the amount of DNA and RNA detected in the starting material at time zero is large, with the initial degradation rate varying accordingly. This variation between the different samples decreased with time by day 15 and day 21. There is no increase in any of these components, which provides further evidence that there is no cellular activity.

(Example 5.5)
Protein and nucleic acid synthesis during the formation of the regeneration matrix using radiolabeled substrates Establish whether the formation of the regeneration matrix is actively involved in the basic cellular processes (DNA, RNA, protein synthesis) For this purpose, the regeneration matrix starting material was incubated with tritium labeled substrates for the synthesis of DNA, RNA and proteins. Five Opticell® containing the starting material were incubated with the addition of radioisotope labeled substrate. One Opticell® was used for each substrate. The substrates used were ³ H-L-amino acid mixture (1 mCi / ml, catalog number 20063, manufactured by MP Biomedicals), ³ H-dTTP (10-20 Ci / mmol, catalog number 24044, manufactured by MP Biomedicals), ³ H-thymidine. (60-90 Ci / mmol, catalog number 24060, manufactured by MP Biomedicals), ³ H-dUTP (35-50 Ci / mmol, catalog number 24061, manufactured by MP Biomedicals) and ³ H-uridine (35-50 Ci / mmol, catalog number 24046) MP Biomedicals). For introduction into an Opticell® cassette, 25 μl of the isotope is diluted in 0.5 ml of DMEM: F-12 medium, then injected into each Opticell®, swirled to mix. Promoted. Immediately after injection and mixing, 1.0 ml of each was removed from Opticell® (total volume 10 ml) and treated as a zero time control (see below). Opticell® was incubated at 37 ° C. in an atmosphere of 5.0% CO _{2 in} air. At the time of day 1, day 3 and day 7, 1.0 ml each was removed for analysis. The sample was kept on ice in a 1.5 ml microcentrifuge tube, 100 μl (0.1 vol) 100% trichloroacetic acid was added, the tube was inverted and the contents mixed. Samples were incubated on ice for 20 minutes to precipitate nucleic acids and proteins. The precipitate was added to a GF / C filter (Whatman) in 0.1% TCA (ice cold) using a Pasteur pipette. The contents of the microcentrifuge tube were added to the filter and rinsed 5 times using ice-cold 0.1% TCA. The filter was transferred onto the foil and air dried for 1 hour. The filter was transferred into a 4 ml Scintillation Cocktail (ScintiSafe, Fisher Scientific) and added to a 5 ml plastic scintillation vial. Vials were counted for 1 minute on a tritium energy channel using a Beckman liquid scintillation counter. The TCA-precipitating material results (counts per minute) are reported in the table below. Cultured human fibroblasts were used as a positive control for the assay.

  Table 5. Total counts per minute for each radiolabeled substrate at time zero, day 1, day 3 and week 1 after initial seeding of regenerative matrix-forming material

これらの結果は、再生マトリックス形成工程の間には検出可能な細胞の活性がないことを示している。

These results indicate that there is no detectable cellular activity during the regeneration matrix formation process.

(Example 5.6)
Metabolic Inhibition Studies In the first set of experiments (see FIGS. 13 and 14), aphidicolin (dissolved in DMSO, 10 μg / ml final concentration), α-amanitin (dissolved in water, 10 μg / ml final concentration) and Cycloheximide (dissolved in ethanol, final concentration of 10 μg / ml) was used to inhibit the activity of DNA polymerase, RNAII polymerase and protein synthesis, respectively, in the regeneration matrix generated from whole blood. Inhibitors were added to Opticell® on day 0, ie the first feeding day (5th or 6th day). Thereafter, a normal medium was supplied to the culture. Samples from each treatment and control were taken on day 21 and analyzed.

  The results of these inhibition studies are shown in FIGS. Control fibroblast cultures containing 0, 10, 30 and 100 nM α-amanitin were cultured for 1 week. Fibroblasts without added α-amanitin became confluent by day 7. Cultures containing 10, 30, and 100 nM α-amanitin exhibited mortality of about 60-70%, 80-90%, and 100% by day 7, respectively.

  Another set of experiments showed that addition of ATPase / ADPase apyrase (dissolved in water, final concentration of 10 μg / ml) can partially inhibit matrix formation in 5 μm filtered samples. . Apyrase has two functions. That is, apyrase has ATPase / ADPase on the one hand and anticoagulant activity on the other hand. It is possible that components present in samples filtered at 5 μm are sensitive to the anticoagulant activity of apyrase, resulting in the “precipitation” observed in their cultures. The reason for the absence of “sedimentation” in the sample filtered at 1 μm is probably because these components, which remain when the filter pore size is small, have been removed.

(Example 5.7)
Fatty Acid Analysis Analysis by mass spectrometry was performed from whole blood and filtered through a 5 μm or 1 μm filter to determine the fatty acid content in the regenerated matrix culture after 3 weeks. As shown in Table 6, many of the detected fatty acid species were not present in the supernatant, but were exclusively present in the matrix. In the case of the matrix, the ratio of fatty acids per 1 mg of lipid was higher in the sample filtered at 1 μm than in the sample filtered at 5 μm. This ratio was reversed in the case of the supernatant. Since the data presented is based on only one sample and one analysis, no conclusions can be drawn at this stage.

  (Table 6.3: Regeneration matrix after 3 weeks (prepared from whole blood), results of fatty acid analysis by Folch partitioning of supernatant, shown as μg / mg lipid)

（実施例５．８）
浸透圧モル濃度
２つの異なる培養物の上清からの凍結試料の浸透圧モル濃度を、異なる期間にわたって分析した。培養が進むにつれて、浸透圧モル濃度が劇的に増加することが、下記の表７から明らかである。

(Example 5.8)
Osmolarity The osmolarity of frozen samples from the supernatants of two different cultures was analyzed over different time periods. It is clear from Table 7 below that the osmolarity increases dramatically as the culture progresses.

  (Table 7. Measured Osmolarity of Regenerated Matrix Culture)

（実施例５．９）
ＡＴＰ促進性代謝活性に関する再生マトリックスの分析
再生マトリックスおよびそれに使用した生成用液体を、アデノシン三リン酸（ＡＴＰ）のレベルについて、２、３、４、６および８週が経過した時点でアッセイし、これらのレベルを、出発材料中のＡＴＰのレベルと比較した。ＥＮＬＩＴＥＮ ＡＴＰ Ａｓｓａｙシステムを使用して、ＡＴＰを、発光によって迅速かつ定量的に検出した。メーカーの指示に従って、材料を、ＳＤＳで抽出した後、ＴＣＡを用いてあらかじめ沈殿させた。その上、いくつかの代謝酵素の存在を、同一の試料中で、ウェスタンブロット法を使用して試験した。ＡＴＰレベルについては、検出反応を、三つ組で行い、３つの異なる日にアッセイを行った。３バッチの再生マトリックスの出発材料は、ピコモル量（１×１０^{−１２ｍｏｌ}）のＡＴＰを含有したが、ＡＴＰは、培養第６日で検出不可能となり、その後の培養時点のすべてで検出不可能な状態を維持した。

(Example 5.9)
Analysis of regeneration matrix for ATP-stimulated metabolic activity The regeneration matrix and the production fluid used therein were assayed for adenosine triphosphate (ATP) levels at 2, 3, 4, 6 and 8 weeks, These levels were compared to the level of ATP in the starting material. ATP was detected rapidly and quantitatively by luminescence using the ENLITEN ATP Assay system. Following the manufacturer's instructions, the material was extracted with SDS and then pre-precipitated with TCA. In addition, the presence of several metabolic enzymes was tested in the same sample using Western blotting. For ATP levels, detection reactions were performed in triplicate and assayed on three different days. The starting material for the three batches of regeneration matrix contained picomolar amounts (1 × 10 ^{−12 mol} ) of ATP, but ATP became undetectable at day 6 of culture and undetectable at all subsequent time points of culture. The state was maintained.

  The presence of the metabolic enzymes glucose-6-phosphate dehydrogenase, aldolase, pyruvate dehydrogenase and cytochrome reductase was assessed using Western blotting. Aldolase is an enzyme that catalyzes the degradation of glucose via glycolysis and is present in the starting material, which is detected at reduced levels between day 21 and day 28 of incubation, It was impossible to detect. Glucose-6-phosphate dehydrogenase (pentose phosphate pathway) was detected in the starting material and over time was detected predominantly in the production liquid used in the regeneration matrix. Its level in the regeneration matrix decreased by day 28 and remained low for the remainder of the culture. However, the band on the gel of this enzyme has a lower molecular weight, suggesting that only the degraded form was detected. Pyruvate dehydrogenase (Krebs cycle) was detected in the starting material and its level did not appear to change until day 28. Again, the band has a lower molecular weight than expected, suggesting that degradation products have been detected. This enzyme could not be detected at any time thereafter. Cytochrome reductase (oxidative phosphorylation) is detected in the starting material, and faster migratory proteins (not present in the starting material) are observed at all subsequent time points, which This suggests that it was detected in the regeneration matrix.

  The absence of detectable levels of ATP in the regeneration matrix, just 6 days after the initial sowing, supports the conclusion that these enzymes are not functional. Both pyruvate dehydrogenase and cytochrome reductase catalyze reactions that produce large amounts of ATP (36 ATP per reaction).

(Example 5.10)
Other analysis Matrix composition analysis indicates that there is no evidence of cellular metabolic activity in the regeneration matrix. The pH of the medium remained constant throughout the culture, thus this indicates very little or no metabolic activity. Furthermore, the osmolarity of the medium increases dramatically during the culture period, making it unacceptable for cell growth.

  Comparison of Coomassie stained SDS-PAGE gels revealed a slight difference in band pattern between samples filtered at 5 μm and samples filtered at 1 μm. An image of the gel is shown in FIG. The blue band designation indicates the band previously identified by mass spectrometry. New bands (M7 and M8) indicated by red circles are bands observed only with the matrix filtered at 1 μm. The appearance of these bands may be due to the enrichment of certain bands as a result of removal of other proteins. The sensitivity of the regeneration matrix to different proteases and nucleases was tested.

  Nucleases such as DNase and RNase had no effect on the regeneration matrix. Proteolytic enzymes such as pepsin, trypsin, papain, proteinase K were able to digest the matrix.

(Example 6)
Activation of neurotrophic genes in vitro using a regeneration matrix Human neuroblastoma cells (cell line SH-SY5Y, catalog number CRL-2266, manufactured by American Type Tissue Culture Collection, Manassas, VA) The neurotrophic activity of the regeneration matrix was characterized. Cells were cultured using the following basal medium (BM): supplemented with 10% FCS (Catalog No. 26140-079, Invitrogen) and 5 μg / ml antibiotic gentamicin (Catalog No. 15710064, Invitrogen). DMEM: F12 (catalog number 30-2006, ATCC). This was amplified and maintained periodically by passage at confluence using T125 tissue culture flasks.

For gene induction analysis, cells were trypsinized and seeded at 10 ⁵ cells / ml in T75 flasks and starting material was added after 3 days. The materials tested were basal medium (BM), matrix medium (R), OptiCell® starting material (O) and regeneration matrix (M). To inactivate the liquid test material, OptiCell® starting material (O) was placed in a 15 ml polystyrene conical culture tube and heated at 60 ° C. for 30 minutes. In order to inactivate the regeneration matrix, the liquid used for the regeneration matrix was removed from the OptiCell® and the regeneration matrix was washed 3 times with PBS (Catalog Number 21-030-CM, Mediatech) (10 ml each). Washed twice. 10 ml of formalin (3.7% formaldehyde in PBS, catalog number 2106-01, from JT Baker) was added to OptiCell® containing the regeneration matrix and incubated for 30 minutes. The regeneration matrix was washed 3 times with PBS and stored in PBS until collected for experiments.

For regeneration matrix treatment (active or fixed), the entire contents of OptiCell® were added to a T75 culture flask containing 70% confluent SH-SY5Y neuroblastoma cells. After 3 hours, the cells were collected by washing with PBS, scraping the cells and pipetting them into a 2.0 ml microcentrifuge tube. Cells were centrifuged at 12,000 RPM and the supernatant was discarded. Total cellular RNA was isolated using the Atlas Total RNA Isolation Kit (Cat. No. K1036-1, BD Biosciences, originally from Clontech) according to the manufacturer's instructions. The RNA pellet was dissolved in RNase free H ₂ O and quantified by OD ₂₆₀ . RT-PCR was performed according to the manufacturer's instructions using an Ambion Retroscript Kit (Catalog No. 1710, Ambion, Austin, TX) using 2 μg RNA template. For RT-PCR, human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), growth-related protein 43 (GAP-43), Netrin-1, neural cell adhesion molecule (NCAM-1), neutrophin -3 (NT-3), Neutrophin-6 (NT-6), glial-derived neurotrophic factor (GDNF) and fibroblast growth factor-9 (FGF-9) specific primers were used. GAPDH was used as a control since GAPDH transcription should not be affected by the type of stimulus used in this assay. The primers used are shown below.

ＰＣＲのために、５μｌのＲＴ反応産物を、４５μｌのＰＣＲ反応カクテル（５μｌの１０×ＰＣＲ緩衝液、１００ｍＭ Ｔｒｉｓ−ＨＣｌ、ｐＨ８．３、５００ｍＭ ＫＣｌ、１５ｍＭ ＭｇＣｌ_２、２．５μｌの２．５ｍＭ ｄＮＴＰミックス、３２．１μｌのｄＨ_２Ｏ（ヌクレアーゼを含まない）、各２．５μｌのセンスおよびアンチセンス１０μＭプライマーストック、０．４μｌの５Ｕ／μｌ Ｔａｑ Ｐｏｌｙｍｅｒａｓｅ（カタログ番号２０５２、Ａｍｂｉｏｎ製））に添加した。ＰＣＲパラメーターは、９４℃、２分（最初の変性）、次いで、９４℃、３０秒、５５℃、３０秒、および７２℃、４０秒の３０サイクル、さらに、７２℃、５分の仕上げの最終ステップであった。ＰＣＲ産物を、ウェルあたり１０μｌのＰＣＲ産物を使用して、２．５％アガロースゲルを５８Ｖで２時間流して分解し、０．５μｇ／ｍｌ臭化エチジウムで染色した。ゲルのデジタル画像を、上方制御の倍増について、Ｓｃｉｏｎ画像分析ソフトウェア（Ｓｃｉｏｎ製、Ｆｒｅｄｅｒｉｃｋ、メリーランド州）を使用して分析した。添加する媒体単独から産生したＲＮＡのＲＴ−ＰＣＲ産物を含有するレーンのバンド強度を使用して、その他の処置からのシグナルを規準化した（データを示さず）。

For PCR, 5 μl RT reaction product was added to 45 μl PCR reaction cocktail (5 μl 10 × PCR buffer, 100 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl ₂ , 2.5 μl 2.5 mM dNTP). Mix, 32.1 μl dH ₂ O (without nuclease), 2.5 μl each of sense and antisense 10 μM primer stock, 0.4 μl of 5 U / μl Taq Polymerase (Cat # 2052, Ambion)) . PCR parameters are 94 ° C., 2 minutes (initial denaturation), then 30 cycles of 94 ° C., 30 seconds, 55 ° C., 30 seconds, and 72 ° C., 40 seconds, plus a final finish of 72 ° C., 5 minutes. It was a step. PCR products were digested by running 2.5% agarose gels at 58V for 2 hours using 10 μl of PCR product per well and stained with 0.5 μg / ml ethidium bromide. A digital image of the gel was analyzed for up-regulation doubling using Scion image analysis software (Scion, Frederick, MD). The band intensity of the lane containing the RT-PCR product of RNA produced from the added vehicle alone was used to normalize the signal from the other treatments (data not shown).

  RT-PCR analysis showed that FGF-9, Netrin-1, NT-3, NCAM-1 and GAP-43 genes were activated in response to incubation with the regeneration matrix. Gene activation by the regeneration matrix appears to be selective. This is because transcription of the genes GAPDH, NT-6 and GDNF was not stimulated in these experiments. These data indicate that the human neuroblastoma cell line SH-SY5Y selectively responds to the regeneration matrix and activates genes related to function and differentiation. Production media supplemented with growth factors alone showed a slight increase in gene upregulation activity for FGF-9 and Netrin alone compared to vehicle alone. These results suggest that the upregulation activity of the gene in the regeneration matrix is due to the inherent properties of the regeneration matrix and not simply due to the presence of growth factors.

  Activation of gene expression of NT-3, NCAM-1, Netrin-1, GAP-43 and FGF-9 by regeneration matrix in SH-SY5Y cells is inactivated by treating the matrix at 60 ° C. for 30 minutes (FIG. 19). Finally, treatment of the regeneration matrix with formalin or high energy gamma irradiation (30 kGy, 30 minutes) also inactivates the regeneration matrix (data not shown).

  For periodic analysis, the effect of regeneration matrix on the upregulation of GAPDH (control), GAP-43, NCAM-1 and NT-3 was determined for four consecutive production batches. The regeneration matrix from each batch was sampled on days 15 and 21 and compared to the gene induction activity of the starting material (Table 8). Up-regulation doubling was determined by RT-PCR from four production runs (two OptiCell® cassettes at each time point). Results were reported as mean ± standard deviation.

  (Table 8. Generation of GAPDH, NT-3, NCAM-1 and GAP- in SH-SY5Y neuroblastoma cells after incubation with regeneration matrix on day 0 (starting material), day 15 and day 21) 43 doubling of up-regulation)

（実施例６．１）
ヒト神経芽腫細胞における、増殖因子が補足されていない再生マトリックスが遺伝子の誘導に及ぼす効果
再生マトリックスを、いずれの増殖因子も補足せず、ＤＭＥＭ：Ｆ１２培地単独を使用して生成した。ＧＡＰＤＨ、ＮＴ−３、ＮＣＡＭ−１およびＧＡＰ−４３について、ＳＨ−ＳＹ５Ｙ神経芽腫細胞における遺伝子の上方制御応答を、ＩＴＳ（２×）、ＥＧＦ（２０ｎｇ／ｍｌ）およびｂＦＧＦ（４０ｎｇ／ｍｌ）を補足して生成した再生マトリックスと比較した。増殖因子なしで生成した再生マトリックスは、再生マトリックスを増殖因子を補足した生成媒体中で生成する場合と変わらない遺伝子の上方制御活性を有する（データを示さず）。

(Example 6.1)
Effect of regeneration matrix not supplemented with growth factors on gene induction in human neuroblastoma cells Regeneration matrices were generated using DMEM: F12 medium alone without supplementing any growth factors. For GAPDH, NT-3, NCAM-1 and GAP-43, the gene upregulation response in SH-SY5Y neuroblastoma cells was expressed as ITS (2 ×), EGF (20 ng / ml) and bFGF (40 ng / ml). It was compared with the regeneration matrix generated supplementarily. The regeneration matrix produced without growth factors has gene upregulation activity that is the same as when the regeneration matrix is produced in a production medium supplemented with growth factors (data not shown).

(Example 6.2)
Regeneration matrix neuronal gene upregulation activity The regeneration matrix formed for 12 days has neuronal cell upregulation activity, the surrounding solution (CM) and the first (day 0) solution that will form the regeneration matrix (Rep) has limited neuronal upregulation activity. 18A-18C show RT-PCR results on human SHSY neuroblastoma cells treated with regeneration matrix for 3 hours. The value of doubling up-regulation compared to the standard control is shown. A regeneration matrix was generated simultaneously from non-coagulated (EDTA-treated) whole blood from the same human donor. A = DMEM / F12 basal medium with supplement (standard regeneration matrix production medium). ITS = DMEM / F12 basal medium supplemented with insulin + transferrin + selenium supplement. EGF = DMEM / F12 basal medium supplemented with epidermal growth factor supplement. FGF = DMEM / F12 basal medium supplemented with fibroblast growth factor 2 supplement. Total = DMEM / F12 basal medium supplemented with ITS + EGF + FGF supplement. None = DMEM / F12 basal medium without supplements. No feed = no additional medium was added after the start of regeneration matrix formation period (day 0).

  The rows A to S in FIGS. 18A to 18C indicate the following reproduction matrices.

A = complete medium for generating the regeneration matrix;
B = Day 12, all, RM-1;
C = Day 12, all, CM-1;
D = Day 12, ITS, RM-1;
E = Day 12, ITS, CM-1;
F = day 12, EGF-ITS, RM-1;
G = day 12, EGF-ITS, CM-1;
H = day 12, FGF-ITS, RM-1;
I = Day 12, FGF-ITS, CM-1;
J = day 12, none, RM-1;
K = day 12, none, CM-1;
L = day 12, no supply, RM-1;
M = day 12, no supply, CM-1;
N = day 0, EGF-ITS, Rep-1;
O = Day 0, EGF-ITS, Rep-2;
P = Day 0, FGF-ITS, Rep-1;
Q = Day 0, FGF-ITS, Rep-2;
R = day 0, none, Rep-1;
S = Day 0, none, Rep-2.

  As can be seen in FIGS. 18A-18C, in each case the regeneration matrix showed increased neuronal gene upregulation activity at day 21 compared to its starting solution (day 0). . The ambient solution on day 21 continued to show limited activity observed in the starting solution (day 0). Thus, only the matrix formed showed increased activity, and the formation of the regeneration matrix did not reduce the activity of the surrounding solution. Thus, the overall neuronal gene upregulation activity of the culture increased dramatically with time as the regeneration matrix was formed.

(Example 7)
Effect of regeneration matrix on rat Neuroscreen-1® cells (induction of neurite outgrowth)
Neuroscreen® cells, an enhanced subclone from rat PC-12 pheochromocytoma cells (Tsuji, M., et al., “Induction of neural outgrowth in PC12 cells by alpha-phenyl-N-tert-butylthyrotrophic) activation of protein kinase C and the Ras-extracellular signal-regulated kinase pathway, J. Biol. Chem., 276, 32779-32785, 2001; Wu, Y. Y. a. and Brad. Synergistic induction of neural out-by-by n rve growth factor or epidemic growth factor and interleukin-6 in PC 12 cells ", J Biol Chem, 271, 13033-13039, 1996) from Cellomics (Catalog No. R04-0001-C) Incubated. Neuroscreen® cells responded by extending neurites. In addition, in the presence of the regeneration matrix, the Neuroscreen® cell morphology changes, becomes flattened and becomes elongated (FIG. 20).

  For Neuroscreen® cell-regeneration matrix interaction studies, cell and regeneration matrix sample materials were prepared using the following method. Prior to the interaction assay, Neuroscreen® cells were maintained by regular culture on plastic coated with collagen, RPMI (Catalog No. 10-040-CV, Mediatech), 4.0 mM glutamine ( Consisting of catalog number 25-005-CI, manufactured by Mediatech) supplemented with 20% horse serum (catalog number 35-030-CV, manufactured by Mediatech) and 10% fetal calf serum (catalog number 35-010, manufactured by Mediatech) Culture medium was used to pass at confluence. The day before establishing the regeneration matrix interaction, the cells were trypsinized and then counted and plated at 2,000 cells per well in 96-well cell culture plates (BD Biosciences, catalog number 47743-953, Axygen). Open the Opticell® cassette containing the regeneration matrix, transfer the regeneration matrix to a 100 μm nylon cell strainer (Catalog No. 352360, BD Falcon), sieve and then 1 ml PBS (Catalog No. MT21-030). -CM, manufactured by Mediatech). 10 μl of the suspension containing the regeneration matrix was added to each well containing Neuroscreen® cells seeded the day before.

  For each production batch, samples were taken on day 0, day 15 and day 21 for the above analysis. Neuroscreen® cells were assayed for neurite outgrowth using the following β-tubulin immunostaining. After 4 days in culture, cells were fixed by adding 10% v / v 37% formaldehyde (Catalog No. 2106-01, JT Baker) directly to the medium in the wells. Plates were incubated at 37 ° C. for 30 minutes, washed twice with PBS, permeabilized, and blocked (10% Normal goat serum: catalog number S26-100M, Chemicon, 5% BSA: catalog number 2910, Anti-β-tubulin antibody (mouse monoclonal, cell culture) blocked for 2.5 hours with Omnipure, 0.1% Saponin: catalog number 102855, MP-Biochemicals and diluted 1: 100 in blocking buffer Using a supernatant, catalog number E7, Developmental Studies Hybridoma Bank), staining was performed at 4 ° C. for 16 hours. Cells were washed twice in PBS and labeled with alexa488 goat anti-mouse secondary antibody (Cat. No. A11029, Invitrogen) diluted 1: 200 in blocking buffer. Cells were counterstained with Hoechst 33342 (Cat. No. H1399, Invitrogen) at 2 μg / ml in PBS for 30 minutes and then washed once with PBS. The plates were scanned using a neurite Outgrowth Bio-application V2.0 using a KineticScan microscope (Cellomics, V2.2.0.0 Build 19).

  The selected output parameters included total cells, the neurite outgrowth index (NOI), which is the percentage of cells in wells with neurite length over 10 μm, and the average neurite length (ANL) μm in each cell.

  As a positive control for this assay, nerve growth factor (NGF, catalog number 13257-019, manufactured by Invitrogen) was added at 100 ng / ml. In summary, the treatment used is shown below. For each, NGF added or not added: regeneration matrix, Neuroscreen-basal medium, Neuroscreen-basal medium + 100 ng / ml NGF, regeneration matrix made without growth factors.

  Only wells with a cell count greater than 100 per field were analyzed. The average NOI per well was divided by the average NOI value from positive control wells containing Neuroscreen® basal medium and 100 ng / ml NGF. This value is reported as a percentage of NGF response. Data from four independent experiments that tested neurite outgrowth stimulation from Neuroscreen® cells are shown in FIG. FIG. 22 shows the neurite outgrowth stimulation by the regeneration matrix prepared in the absence of the growth factor adjuvant compared to the neurite outgrowth stimulation by the regeneration matrix prepared in the presence of the growth factor adjuvant.

  These results show that the regeneration matrix has significant neurite inducing activity when tested with Neuroscreen® cells. However, unlike the results from studies of gene upregulation using neuroblastoma cells, the addition of ITS, EGF and bFGF adjuvants during regeneration matrix generation significantly increased the neurite outgrowth activity of the regeneration matrix. Can be increased.

(Example 8)
Protection and Enhancement of Growth Factor Activity by Regeneration Matrix Human peripheral blood is collected in 10 8 ml Vacutainer ™ tubes containing K2EDTA, shipped overnight on ice, and then separated by gravity In a + 4 ° C. refrigerator for 6 hours. The Vactainer ™ tube containing gravity separated blood was then carefully placed vertically in a −20 ° C. freezer for storage. Two days later, four tubes were removed from the freezer, placed vertically in a tube stand, and melted at 20 ° C. When thawed, the plasma-platelet-buffy coat fractions of the two tubes were removed and placed in a 50 ml conical tube, and the entire contents of the remaining two tubes were placed in another 50 ml conical tube. After thorough mixing of the contents of each conical tube, 5 ml of each conical tube was placed in another 50 ml conical tube along with 10 ml TR-10 medium containing 100 ng / ml nerve growth factor (NGF). . Table 9 shows the composition of TR-10 medium. After thorough mixing of the contents of each conical tube, the solution was filtered through a 5 μm syringe filter followed by a 1.2 μm syringe filter and then placed into a new 50 ml conical tube. Sufficient TR-10 medium containing 100 ng / ml NGF was added to make the contents of each conical tube equally 50 ml. After thorough mixing of the contents of each conical tube, a 10 ml volume of solution is injected into a separate set of 5 Opticell®, then 37 ° C., 20% CO 2, 2% O 2 non-humidified Placed horizontally in the incubator. Therefore, each Opticell® contained 1 μg NGF. Samples were left for 6 days, after which 3 ml of TR-10 medium was added to each Opticell®. On days 8, 10 and 12, an additional 1 ml of TR-10 medium was added to each Opticell®. On day 13, the contents of each Opticell® were placed in a separate 15 ml conical tube and spun at 500 × g. The supernatant was removed and saved for subsequent analysis. The contents of each 15 ml conical tube were then washed with PBS, spun at 500 × g, the supernatant removed and discarded. This was repeated two more times to remove all the original solution that each of the regeneration matrices was surrounded (when the regeneration matrix was in Opticell®). Each regeneration matrix (mass about 1 g and volume about 1 ml) was then saved for subsequent analysis. Each regeneration matrix contained approximately 5 mg of protein. The regeneration matrix was centrifuged at 5,000 × g for 30 minutes (with removal of the resulting liquid supernatant), and the level of hydration of the regeneration matrix could be halved.

  (Table 9. TR-10 composition)

２週間後、Ｎｅｕｒｏｓｃｒｅｅｎ（登録商標）アッセイを、実施例９に従って準備した。神経増殖因子（ＮＧＦ）の応答曲線を作成した（図２３を参照）。Ｎｅｕｒｏｓｃｒｅｅｎ（登録商標）アッセイの９６ウェルプレートの各ウェルは、２００μｌの溶液を含むので、１００ｎｇ／ｍｌの濃度のＮＧＦを有するウェルは、総計２０ｎｇのＮＧＦを含んだ。

Two weeks later, the Neuroscreen® assay was prepared according to Example 9. A nerve growth factor (NGF) response curve was generated (see FIG. 23). Each well of the Neuroscreen® assay 96-well plate contained 200 μl of solution, so wells with a concentration of 100 ng / ml NGF contained a total of 20 ng NGF.

  Early studies showed that addition of 10 mg regeneration matrix doubled the average neurite length (of neurites> 10 μm in length) for Neuroscreen® cells exposed to 100 ng / ml NGF (see Table 10). In this assay, an experiment was performed to determine the effect of adding nGF to the starting material solution.

  (Table 10. Mean neurite length of Neuroscreen ™ cells exposed to RMx and 100 ng / ml NGF. Values are expressed as a percentage of the positive control (100 ng / ml NGF))

各群から、５つの再生マトリックスのうちの３つおよびそれらの対応する溶液（保存した上清）を使用して、Ｎｅｕｒｏｓｃｒｅｅｎ（登録商標）アッセイにおける応答曲線を決定した（下記を参照）。Ｎｅｕｒｏｓｃｒｅｅｎ（登録商標）細胞を、ＮＧＦ含有再生マトリックスに曝露すると、Ｎｅｕｒｏｓｃｒｅｅｎ（登録商標）細胞は、（長さ１０μｍ超の神経突起の）平均神経突起長が倍増するという非常に類似した結果を得た（下記を参照）。しかし、この倍増効果は、全血から作製した０．２ｍｇの再生マトリックス、および血漿−血小板−バフィーコート画分（赤血球画分を有しない、図２４を参照）から作製した０．０３ｍｇの再生マトリックスの場合のみで得られた。仮に出発材料溶液に添加したＮＧＦが、形成されつつある再生マトリックスと周囲の溶液との間で等しく分散しているとすると、Ｎｅｕｒｏｓｃｒｅｅｎ（登録商標）アッセイのウェルに添加した再生マトリックスの全量は、全血および血漿−血小板−バフィーコート画分から作製した再生マトリックスに関して、それぞれ、わずか０．０２ｎｇおよび０．００３ｎｇのＮＧＦを含有するはずである。仮にすべてのＮＧＦが再生マトリックスに集まるとすると、その場合には、ウェルあたりのＮＧＦの全量は、それぞれ、０．２ｎｇおよび０．０３ｎｇとなるであろう。しかし、（これらの実験と平行して、同時に同一の（新鮮な）ＮＧＦを用いて行った）ＮＧＦ応答の標準曲線の点は、ウェルあたり１０ｎｇ未満の量では、新鮮なＮＧＦがＮｅｕｒｏｓｃｒｅｅｎ（登録商標）細胞に及ぼす効果には制限があるか、効果がなく、最大の応答がウェルあたり１００ｎｇのＮＧＦで生ずることを示している。血漿−血小板−バフィーコート画分から作製した再生マトリックスは、この最大の応答の倍増を、そのようなＮＧＦの量（または濃度）の１／３０００から１／３００００（またはそれ未満）で示すことから、再生マトリックスには、Ｎｅｕｒｏｓｃｒｅｅｎ（登録商標）細胞に対して増殖因子感作（またはそれに類似する）効果が存在するに違いない。さらに、平均神経突起長が、ＮＧＦ単独で達成することができる最大閾値を上回って倍増したことから、再生マトリックスは、その他の神経突起増殖経路も刺激するに違いない。再生マトリックス単独（いずれのＮＧＦもなし）では、Ｎｅｕｒｏｓｃｒｅｅｎ（登録商標）細胞の平均神経突起長は、陽性対照（１００ｎｇ／ｍｌのＮＧＦ）の約６０％となることが決定されている。

From each group, 3 of 5 regeneration matrices and their corresponding solutions (preserved supernatants) were used to determine response curves in the Neuroscreen® assay (see below). When Neuroscreen® cells were exposed to an NGF-containing regeneration matrix, Neuroscreen® cells gave very similar results that the average neurite length (of neurites> 10 μm in length) doubled. (See below). However, this doubling effect is due to the 0.2 mg regeneration matrix made from whole blood and the 0.03 mg regeneration matrix made from the plasma-platelet-buffy coat fraction (without the red blood cell fraction, see FIG. 24). Only in the case of If the NGF added to the starting material solution is evenly distributed between the regeneration matrix being formed and the surrounding solution, the total amount of regeneration matrix added to the wells of the Neuroscreen® assay is For regeneration matrices made from blood and plasma-platelet-buffy coat fractions, they should contain only 0.02 ng and 0.003 ng NGF, respectively. If all NGF gathers in the regeneration matrix, then the total amount of NGF per well would be 0.2 ng and 0.03 ng, respectively. However, the standard curve point of NGF response (performed in parallel with these experiments, simultaneously with the same (fresh) NGF) is that the amount of fresh NGF is less than 10 ng per well. ) The effect on the cells is limited or ineffective, indicating that the maximum response occurs with 100 ng NGF per well. The regeneration matrix made from the plasma-platelet-buffy coat fraction shows this doubling of maximum response as 1/3000 to 1 / 30,000 (or less) of the amount (or concentration) of such NGF, The regeneration matrix must have a growth factor sensitization (or similar) effect on Neuroscreen® cells. Furthermore, the regeneration matrix must also stimulate other neurite growth pathways, as the average neurite length doubled above the maximum threshold that can be achieved with NGF alone. Regeneration matrix alone (without any NGF) has been determined that the average neurite length of Neuroscreen® cells is approximately 60% of the positive control (100 ng / ml NGF).

  Surprisingly, the supernatant (or the solution surrounding the regeneration matrix in each Opticell® at the end of the 13-day regeneration matrix formation period) also produced the greatest response of Neuroscreen® cells to NGF. Doubled. If the NGF added to the starting material solution is evenly distributed between the regeneration matrix being formed and the surrounding solution (supernatant), the doubling effect (or maximal response) is only slightly per well. Occurs at 5 ng NGF, or 1/20 of the amount of NGF required for a standard maximal response (which is only ½ of the response obtained with the supernatant). Thus, although much less potent, an effect similar to that in the regeneration matrix is seen in the supernatant. This is believed to be due to the properties that are present and formed during the process that would also form small particles of the regeneration matrix prior to agglomeration into the regeneration matrix and regeneration matrix composite.

  Furthermore, since NGF activity decreases with time at 37 ° C., the regeneration matrix appears to also have a growth factor protective effect. The NGF activity assay of the regenerated matrix that has been stored for an additional 2 weeks at 4 ° C. after 13 days shows much higher NGF activity than fresh NGF from the same lot. In early experiments, the same effect was observed with EGF, bFGF and BDNF.

Example 9
Dose response of regeneration matrix prepared with DMEM / F12 basal medium and TR-10 basal medium Table 11 shows neurite outgrowth of regeneration matrix prepared with either DMEM / f12 basal medium or TR-10 basal medium A sex dose response comparison is shown (assay was prepared according to Example 7). Values are expressed as percent of positive control (NGF).

  (Table 11)

（実施例１０）
再生マトリックスのラット胎仔脊髄からの初代細胞培養に及ぼす効果
健常なラットの胎仔（Ｅ１８）および成体から脊髄を得た。使用した神経細胞培養プロトコールは、Ｂｒｅｗｅｒら（「Ｓｅｒｕｍ−ｆｒｅｅ Ｂ２７／ｎｅｕｒｏｂａｓａｌ ｍｅｄｉｕｍ ｓｕｐｐｏｒｔｓ ｄｉｆｆｅｒｅｎｔｉａｔｅｄ ｇｒｏｗｔｈ ｏｆ ｎｅｕｒｏｎｓ ｆｒｏｍ ｔｈｅ ｓｔｒｉａｔｕｍ，ｓｕｂｓｔａｎｔｉａ ｎｉｇｒａ，ｓｅｐｔｕｍ，ｃｅｒｅｂｒａｌ ｃｏｒｔｅｘ，ｃｅｒｅｂｅｌｌｕｍ，ａｎｄ ｄｅｎｔａｔｅ ｇｙｒｕｓ」、Ｊ．Ｎｅｕｒｏｓｃｉ．Ｒｅｓ．、４２巻、６７４〜６８３頁、１９９５年）を改作した。脊髄を、ラットから、４℃の３５ｍｍペトリ皿中のＢ２７（カタログ番号１７５０４−０４４、Ｉｎｖｉｔｒｏｇｅｎ製）および０．５ｍＭ Ｌ−グルタミン（カタログ番号２５−００５−ＣＩ、Ｍｅｄｉａｔｅｃｈ製）で補足した２ｍｌのＨｉｂｅｒｎａｔｅ−Ａ（Ｂｒａｉｎｂｉｔｓ製、Ｓｐｒｉｎｇｆｉｅｌｄ、イリノイ州）中に解体した。髄膜および過剰の白質を同一の培地中で、４℃の第２の皿に取り出した。脊髄を、同一の培地であらかじめ湿らせた無菌の紙に移し、脊髄の長軸に垂直な約０．５ｍｍ厚さの切片を作製し、同一の培地の４℃のチューブに移した。３０℃で８分間振とう後、切片を、広い口径のピペットを用いて、パパインを含有する３０℃の別のチューブに移した。パパイン（１５〜２３単位／タンパク質ｍｇ、システインで活性化されていない）は、１２ｍｇを３７℃で５分間加温した６ｍｌのＨｉｂｅｒｎａｔｅ Ａに溶解させることによって調製した。溶液を、ろ過滅菌し、４℃で保存した。切片を、３０℃の水浴中で３０分間インキュベートし、この際、切片を懸濁させるのに十分なスピード（１７０ｒｐｍ）でプラットフォームを回転させた。切片を、２ｍｌのＨｉｂｅｒｎａｔｅ−Ａ／Ｂ２７を含有する３０℃の１５ｍｌのコニカルチューブに移し、ＲＴで５分間放置した。切片を、シリコン処理パスツールピペット（火炎研磨した先端）を用いて（約３０秒かけて）１０回粉砕した。２分間放置して小片を沈降させ、上清を別のチューブに移した。第１のチューブからの沈降物を、２ｍｌのＨｉｂｅｒｎａｔｅ Ａ／Ｂ２７中に再懸濁させ、粉砕および沈降の工程をさらに２回繰り返した。各粉砕からの上清を組み合わせて、６ｍｌの懸濁液を得た。

(Example 10)
Effect of regeneration matrix on primary cell cultures from rat fetal spinal cord Spinal cords were obtained from healthy rat fetuses (E18) and adults. The neural cell culture protocol used is Brewer et al. 42, 674-683, 1995). Spinal cords were supplemented from rats with B27 (catalog number 17504-044, Invitrogen) and 0.5 mM L-glutamine (catalog number 25-005-CI, Mediatech) in 35 mm Petri dishes at 4 ° C. Dismantled in -A (Brainbits, Springfield, Ill.). Meninges and excess white matter were removed in a second dish at 4 ° C. in the same medium. The spinal cord was transferred to sterile paper pre-wetted with the same medium, and approximately 0.5 mm thick sections perpendicular to the long axis of the spinal cord were made and transferred to 4 ° C. tubes of the same medium. After shaking for 8 minutes at 30 ° C., the sections were transferred to another 30 ° C. tube containing papain using a wide bore pipette. Papain (15-23 units / mg protein, not activated with cysteine) was prepared by dissolving 12 mg in 6 ml of Hibernate A warmed at 37 ° C. for 5 minutes. The solution was sterilized by filtration and stored at 4 ° C. The sections were incubated in a 30 ° C. water bath for 30 minutes, with the platform rotated at a speed sufficient to suspend the sections (170 rpm). The sections were transferred to a 15 ml conical tube at 30 ° C. containing 2 ml of Hibernate-A / B27 and left at RT for 5 minutes. The sections were ground 10 times using a siliconized Pasteur pipette (flame-polished tip) (over about 30 seconds). The pieces were allowed to settle for 2 minutes and the supernatant transferred to another tube. The sediment from the first tube was resuspended in 2 ml of Hibernate A / B27 and the grinding and sedimentation steps were repeated two more times. The supernatant from each grinding was combined to give 6 ml suspension.

Cells are plated on autoclaved glass, pre-coated with 50 μg / ml lysine-arginine copolymer (LAS, BrainBits) at 90-320 / mm ² in 60-150 μl Neurobasal-A / B27, Distributed in 100 mm dishes. After 1 hour from sprinkling and incubating (5.0% O ₂ , 5.0% CO ₂ , the rest N ₂ ), the coverslips are quickly removed, rinsed, and 0.4 ml in a 24-well plate at 37 ° C. Transferred into Neurobasal-A / B27. The medium was aspirated and the coverslips were rinsed once with warm Hibernate-A, and the cells were re-fed with Neurobasal-A / B27, 0.5 mM glutamine, 10 μg / ml gentamicin and 5 ng / ml bFGF. On day 4, the cells were fed by removing half of the medium and replacing it with an equal volume of fresh medium containing 5 ng / ml bFGF. Neurons were allowed to attach and proliferate for 6 days before being used for interaction experiments. These cells showed a healthy appearance, grew steadily and were confluent between 40-60% at the start of the interaction.

  Three different batches of matrix were evaluated on day 16, day 9 and day 7, respectively. All three batches were derived from non-coagulated blood collected using EDTA as an anticoagulant. Two batches contained glucose, glutamine and pyruvate, and one batch did not contain them. A single cell suspension of rat fetus (E18) whole spinal cord was prepared according to the protocol described above. Cell monolayers were grown on glass slides and incubated with either matrix or control medium. On the 7th day after the start of the interaction culture, coverslips were processed for ICC. A control that was a spinal cord primary cell without matrix was used as a positive control for the functionality of the culture system.

  Photomicrographs showing the results of immunocytochemical analysis are shown in FIGS. Controls stained positive for mature neurons and all of these stained negative for glial cells (Figure 25). In the control culture, the number of Tuj1 positive cells is 4.2% and GFAP positive cells is 0.4%. Cells that stained positive for both Tuj1 and GFAP were not detected.

  Nerve cells incubated with the matrix showed a lower proportion of prominent, long axons. However, the percentage of Tuj1 positive cells in the culture increased to 55% with the regeneration matrix (most of the staining was limited to cell bodies and short processes), compared to 4. 2%. The percentage of GFAP positive cells in the culture increased to 92% with the regeneration matrix compared to 0.4% in the control. In cultures treated with regeneration matrix, about 50% of the cells are positive for both Tuj1 and GFAP, indicating that these cells are triphasic neural progenitor cells, and in the controls Such cells were not observed.

  This observation indicates that the regeneration matrix promotes the growth of young and new neurons. An increase in the presence of astrocytes during the same period can explain the phenomenon of tissue neurogenesis and / or the initial response of “damage”, which is required in the healing cascade and precedes it is there.

  As shown in FIG. 26, the cells treated with the regeneration matrix contained cells that were positive for both Tuj1 and GFAP, indicating that these cells are triphasic neural progenitor cells and control. Some such cells were not observed.

Example 11
In Vitro Effect of Regeneration Matrix on Human Fibroblasts Prior to the interaction assay, human neonatal foreskin fibroblasts (cell line number CCD 1079 Sk, catalog number CRL 2097, ATCC) were added to basal fibroblast medium (10 Maintained by periodic culture using DMEM supplemented with% FBS and pen-strep) and passage at confluence. Cells are trypsinized into an empty Opticell® membrane (Opticell®) or an Opticell® membrane (regeneration matrix) to which a regeneration matrix formed using coagulated blood is attached. I whispered on one. The membrane was cut into 1 cm ² . The membrane was pre-attached to the bottom of the 6-well plate using a sterile Vaseline strip. The Vaseline point was placed at the bottom of the well before the medium was added, then the medium was added and finally the membrane was placed by pushing its center from the top. Each well contained two Opticell® treatments and two regeneration matrix treatments. Trypsinized cells from confluent plates were counted and seeded at a density of 20,000 cells per well in 3 ml of medium. Plates were placed in an incubator and after 2 hours, some representative samples were fixed before staining nuclei and estimating cell numbers. The remaining membrane was gently kept for another 2 days. BrdU (Catalog number 203806, EMD Biosciences) was added by diluting a 10 mM stock to 20 μM in growth medium. After the incubation period, the membranes were soaked in 15 mM glycine (catalog number G-8790, Sigma) in 1 ml ice-cold 70% ethanol (catalog number UNI170, EM Science) in a 24-well plate. Fixed cells were stored at −20 ° C. for at least 24 hours. For detection of BrdU, the cells were washed once in PBS (Cat. No. MT21-030-CM, Mediatech) and 1 of Kit II monoclonal anti-BrdU antibody-nuclease reagent (Catalog No. 1299964, Roche). : Treated with 10 dilutions and incubated at 37 ° C. for 1 hour. Cells were washed twice in PBS and blocking buffer (10% normal goat serum: catalog number S26-100M, Chemicon, 5% BSA: catalog number 2910, Omnipure, 0.1% saponin: catalog number 102855, A 1: 200 dilution of Alexa-488 labeled goat anti-mouse antibody (Cat. No. A11029, Invitrogen) in MP-Biochemicals, final solution was sterile filtered through a 0.2 μm filter and stored at 4 ° C. And incubated at room temperature for 1 hour. Samples were counterstained with 2 μg / ml Hoechst 33342 in PBS (Cat. No. H1399, Invitrogen, 10 mg / ml stock kept at −20 ° C.) for 30 minutes and washed once in PBS. The membrane was then inverted and mounted again in 50 μl of glycerol on a 24-well plate. Plates were stored at 4 ° C. and scanned within one week.

  Images were analyzed using a KineticScan (Cellomics, V2.2.0.0 Build 19) using Bio-application software Target Activation V2.0. Images were stored on the hard drive in the proprietary format of Cellomics and analyzed while scanning the plate. A setting for the positive cell threshold was obtained by plotting a histogram of negative and positive cells for each staining session. Plates that had detached cells from all wells during fixation and plates where no signal was observed were not included in the final analysis.

  Cells grown on Opticell® membranes are a total of 21.8 ± 2.6% positive for BrdU and only 13.4 ± 2 when grown on the regeneration matrix. Only 0% positive. Early adhesion differences were excluded by counting cells immediately after attachment (ie, the total number of adherent cells was the same in these two samples).

  The number of fibroblasts that adhered to the regeneration matrix at 2 hours was the same as that of the control Opticell®, but the size of the nuclei was reduced (possibly indicating the first stage of apoptosis) and 24 hours. In the case of fibroblasts grown on the regeneration matrix over time, the number of BrdU positive cells is decreasing (see FIGS. 28A-C). Fibroblast cultures grown longer on these regeneration matrices have a dramatic decrease in fibroblast density compared to control cultures (some of these fibroblasts are Undergoing apoptosis). FIG. 27 shows this difference in the proliferation density of fibroblasts grown for 5 days on a regeneration matrix made from 5 μm filtered whole blood.

Example 12
In vitro effect of regeneration matrix on human astrocytes The same treatment and culture conditions as used for fibroblasts, except that the morphological change assay was performed using staining for glial fibrillary acidic protein (GFAP), Used for astrocytes. Human primary astrocytes (Catalog No. 1800, from Sciencell, San Diego, Calif.) Are cultured in basal medium (Astrocyte Medium, Cat. No. 1801, from ScienCell) on a plate coated with poly-D-lysine, and confluence. Passed on. The day before the interaction experiment, cells were trypsinized and then counted and plated for BrdU at 2000 cells per well in a 96-well plate coated with poly-D-lysine (Cat. No. 354461, BD Falcon). It was.

For each batch, samples were taken on day 0, day 6, day 12, day 15, day 18 and day 21 for the above analysis. Each treatment was repeated 4 times for each measurement (eg, 4 wells per sample). For growth, after 2 days in the presence of the regeneration matrix, BrdU (Catalog No. 203806, EMD Biosciences) is diluted to 20 μM by diluting a 400 μM stock 1: 200 in 200 μl medium in growth medium. Added. The cells were incubated for an additional 1.5 hours at 37 ° C., 5% O ₂ , 5% CO ₂ and the rest N ₂ . The medium was removed by decantation and the cells were fixed in 15 mM glycine (Catalog No. G-8790, Sigma) using ice-cold 70% ethanol (Catalog No. UNI170, EM Science). Fixed cells were stored at −20 ° C. for at least 24 hours. For detection of BrdU, the cells were washed once in 200 μl of PBS (Cat. No. MT21-030-CM, Mediatech) and monoclonal anti-BrdU antibody-nuclease reagent for KitII (Catalog No. 1299964, Roche). And incubated at 37 ° C. for 1 hour. Cells were washed twice in PBS and blocking buffer (10% normal goat serum: catalog number S26-100M, Chemicon, 5% BSA: catalog number 2910, Omnipure, 0.1% saponin: catalog number 102855, Add a 1: 200 dilution of Alexa-488 labeled goat anti-mouse antibody (Cat. No. A11029, Invitrogen) in MP-Biochemicals, final solution sterile filtered through 0.2 μm and stored at 4 ° C. Incubated for 1 hour at room temperature. Samples were counterstained with 2 μg / ml Hoechst 33342 (catalog number H1399, Invitrogen, 10 mg / ml stock kept at −20 ° C.) in PBS for 30 minutes, washed once in PBS and stored at 4 ° C. Usually scanned within a week. After ethanol fixation and storage, cells were treated with 2N HCl (Catalog No. 2612-14, Mallinckrodt chemicals) for 30 minutes and neutralized with 100 mM Tris, HCl pH 8.5 (Catalog No. 9230, OmniPure). Then, it was washed twice with PBS. Cells were incubated for 16 hours at 4 ° C. in 50 μl of a 1: 200 dilution of Alexa-488 labeled monoclonal anti-BrdU antibody (Cat # MD5420, Caltag laboratories) in blocking buffer. Cells were washed twice in PBS, Alexa546-labeled goat anti-mouse (Cat. No. A11030, Invitrogen) in blocking buffer was added and incubated for 3 hours. Cells were counterstained in 200 μl of 2 μg / ml Hoechst in PBS and stored in PBS. Images were analyzed using a KineticScan (Cellomics, V2.2.0.0 Build 19) using Bio-application software Target Activation V2.0. Images were stored on the hard drive in the proprietary format of Cellomics and analyzed while scanning the plate. A setting for the positive cell threshold was obtained by plotting a histogram of negative and positive cells for each staining session. Plates that had detached cells from all wells during fixation and plates where no signal was observed were not included in the final analysis. In general, astrocytes cultured in the presence of regeneration matrix (13.9 ± 2.3%) have fewer BrdU positive nuclei than astrocytes cultured without regeneration matrix (18.5 ± 2.4%). This probably explains that less glial scarring was observed in spinal cord injuries treated with regeneration matrix compared to controls.

(Example 13)
In vivo effects of regeneration matrix on injured rat spinal cord To assess the in vivo activity of regeneration matrix, three rat models of spinal cord injury model were used.

-Complete crossing (5mm defect)
-One side cut (5mm defect, right side)
-Contusion (50mm weight drop)
All animal experiments were performed using an IACUC approved protocol that includes surgery and standard animal care procedures after surgery. For these experiments, nude rats (Biomedical Research Models, Inc., Worcester, Mass.) Were used to minimize the immunological response to the human regeneration matrix. To access the spinal cord, a midline incision was made on the spine from T8 to T11, and the skin and dorsolateral lumbar ligament were retracted. Both sides of the spine were exposed by blunt dissection. A dorsal laminectomy was performed at the T8-T9 level. After the dura was cut, it was retracted to expose the spinal cord. In the cross-sectional study, the full-thickness area of the spinal cord, cut between T8 and T9, was cut to 5 mm length and carefully removed. In the unilaterally cut SCI model, the spinal cord was exposed as described above and a 5 mm defect on the right side was surgically created in the spinal cord. Regeneration matrix implants were used to fill the defects. The dura mater, upper ligament, muscle and skin were closed with sutures. In the contusion model, rats were allowed to recover for 2 weeks after injury after the exposed dura mater of the spinal cord was struck by a 50 mm weight drop using NYU IMPACTOR. After 2 weeks of recovery, the rats were surgically implanted with regeneration matrix into the spinal cord adjacent to the injured area. All rats were given 3 days of subcutaneous injection of antibiotics to recover in the cage to which they belong and to prevent infection. Sensory and motor functions of all rats are described in a modified version of the BBB neurological scale (Basso, DM, et al., “A sensitive and reliable locomotor for open field testing in rats”, J. Neurotraum Vol. 1 to 21 (1995)). BBB scores were recorded approximately once every two weeks using the criteria listed in Tables 12 and 13.
Table 12. Basso, Beattie and Bresnahan gait assessment scales

（表１３．ＢＢＢの定義）

(Table 13. Definition of BBB)

１５ａ．完全横切脊髄損傷ラットモデル
図２９は、ラットにおける完全横切研究からの結果を示し、ここでは、横切損傷を再生マトリックスで満たし、対照としては、すき間を血液で満たした。一般的に、脊髄損傷の重症度のために、この種の研究に関連する死亡率のレベルが高く、ここでの結果は、少なくとも第６週で生存したラットにおける神経機能の状態を描写する。ＢＢＢスコアを、２週間隔で、移植後１２から１４週間までモニターした。再生マトリックスを移植し、損傷／移植後少なくとも第６週で生存した７匹の全ラットは、改善されたＢＢＢスコアを示した（図２９）。最大の増加が、移植後第６〜８週で見られた。少なくとも第６週で生存した３匹の対照ラットは、１２週間の評価期間にわたり、実質的な改善を示さなかった。

15a. Complete Transverse Spinal Cord Injury Rat Model FIG. 29 shows the results from a complete transversal study in rats, where the transcutaneous injury was filled with a regeneration matrix and, as a control, the gap was filled with blood. In general, because of the severity of spinal cord injury, the level of mortality associated with this type of study is high, and the results here depict the state of neurological function in rats that survived at least 6 weeks. BBB scores were monitored at 2-week intervals from 12 to 14 weeks after transplantation. All seven rats transplanted with regeneration matrix and survived at least 6 weeks after injury / transplantation showed improved BBB scores (FIG. 29). The greatest increase was seen at 6-8 weeks after transplantation. Three control rats that survived at least 6 weeks showed no substantial improvement over the 12 week evaluation period.

  Furthermore, in rats implanted with regeneration matrix, the number of lesions (cysts) and the mean volume of each lesion above and below the spinal cord injury (surgery) site were significantly reduced compared to control rats (FIG. 30). This means that the regeneration matrix exhibits a neuroprotective effect in these animal models, resulting in increased protection of neural tissue above and below the injury site.

15b. Unilaterally Cut Spinal Cord Injury Rat Model FIG. 31 shows an overview of the results of transplanting the regeneration matrix into the spinal cord damaged by unilateral cut. In the unilaterally cut SCI model, a 5 mm defect in the spinal cord was surgically created on the right side after exposure of the spinal cord as described above. This model is not as harsh as the crossing model, and the rat can function normally to some degree. For example, even with surgically induced defects, the bladder can be controlled, but the motor function of the right hind limb is impaired. Transplanting regeneration matrix (n = 4) increased survival and restored function (n = 3) compared to non-transplanted controls (n = 2). Non-transplanted control rats died before the first BBB assessment period in the first week.

15c. Contusion Spinal Cord Injury Rat Model In the contusion model, the dura mater was exposed with the lower spinal cord, and the SCI was measured using the MASCIS IMPACTOR (formerly NYU IMPACTOR, WM Keck Center for Collaborative Neuroscience, Rutgers University, New York, USA). ) Using a 50 mm weight drop. Rats were allowed to recover for 2 weeks, then a second operation was performed, the spinal cord was exposed, and the regeneration matrix was placed adjacent to the injured area. FIG. 32 shows that regeneration matrix implantation also improves the BBB score in rats with spinal cord injured by contusion. In this study, 25 rats had contusion injury on the spinal cord and 19 rats survived the second week after the contusion. Of the surviving rats, 11 rats were transplanted with the regeneration matrix and 8 were controls, which either performed a biologically inactivated transplant or did not undergo a second surgery (thus the transplant None). At 6 weeks after injury, 9 matrix-grafted rats, 4 inactivated matrix-grafted rats, and 3 non-grafted control rats were alive. BBB scores for these surviving rats were evaluated at 2-week intervals (FIG. 32). Regenerated matrix transplanted rats recovered as well as in the complete transverse model and unilaterally cut model. Biologically inactivated matrix-implanted rats partially recover, indicating that the structural properties of the regeneration matrix alone have a significant regeneration potential over controls that did not receive the matrix. ing. One of the control rats clearly showed considerable spontaneous recovery, which was likely due to incomplete damage. However, the implantation matrix overcame the mild but measurable debilitating effect of the second surgery, resulting in a significant improvement in rats.

(Example 16)
Human regeneration matrix is safe, well tolerated in pigs with experimental spinal cord injury and supports functional recovery. In an experimental pilot study, pigs, males, weighing 20 to 45 kg, different types of spinal cord injury ( Impact, penetration, laceration, surgical unilateral cut) and a plug of human regeneration matrix or porcine fibrin was placed at the injury site. Test different models to generate views on the consequences of each type of injury on gait and sensory functionality in pigs, and in which injury model the regeneration matrix seems to work best did. The human regeneration matrix had a clear tendency to promote functional recovery in pigs with SCI, with the most prominent effect seen in severe impact damage resulting in laceration and spinal cord tissue loss. These experiments also provided an opportunity to evaluate the tolerability and expected safety of the regenerative matrix implant. The porcine spinal cord is more than 98% identical to the human spinal cord in terms of the microenvironment from T2 to L2, and thus determines any possible safety concerns that the regeneration matrix may exhibit in the human spinal cord. A good model for. Safety concerns in any pig transplanted with human regeneration matrix, including 3 pigs infused with regeneration matrix twice into the spinal cord in two separate operations at intervals of 1-3 months Was not detected. No significant differences in body weight and behavior were found between control pigs and pigs transplanted with regeneration matrix. Behavior includes general observations made during animal management regarding feeding, drinking, social interactions, and urination and defecation behavior. This indicates that the regeneration matrix is well tolerated and relatively non-toxic when implanted in pigs.

  Tissue samples collected one month after transplantation in several pigs were subjected to Thj-1 staining, indicating that the transplanted regeneration matrix induced the formation of new neurons at the site of spinal cord injury. . FIG. 37 shows that the transplanted regeneration matrix induces the formation of new Tuj-1 positive neurons within one month of transplantation (shown at 200 × magnification).

  These preliminary pilot studies suggested that human regeneration matrix promotes recovery of locomotor activity in pigs with SCI. FIG. 33 shows the results of these preliminary studies, in which a modified ASIA scoring system was used to assess neurological recovery in these pigs. Some pigs started with a very low ASIA score due to the severity of the surgical injury, while other pigs (including all of the control pigs) were less severe and thus more severe Starting with a high ASIA score.

  After the completion of these pilot studies, a 7-week full-blind safety study demonstrated that human regeneration matrix is safe and well tolerated in pigs that received experimental spinal cord injury. In these double-blind studies, pigs were surgically-induced SCI localized to the right side of the T8-T9 spinal cord—after removal of a 5 mm apart double unilateral cut, a 5 mm long piece of spinal cord was removed— Given, either a human regeneration matrix or an equivalent amount of homogenized human clot was transplanted. The left spinal cord served as a control for nonspecific toxicity related to experimental surgery and transplantation. All pigs were observed regularly for 7 weeks after SCI and transplantation for changes in overall health, weight and locomotor response.

  Initially, 16 age- and weight-matched pigs were randomly assigned to 8 groups of 2 animals each, which were implanted with either human regeneration matrix or human clot. Four pigs—two in the regeneration matrix treatment group and two in the clot treatment group—died shortly after severe spinal cord injury. This indicates that the human regeneration matrix has nothing to do with increased mortality compared to the clot transplant group.

  Regenerative matrix transplantation in pigs appeared to be well tolerated and usually safe compared to control pigs transplanted with blood clots. FIG. 34 shows a summary of weekly pig weight gain in the two groups and compares them. In the first 5 weeks, no difference was detected in the average total body weight gain of the pigs, but in the last 2 weeks of the study period, the weight gain of the regenerated matrix transplanted pigs exceeded the subject pigs. These data suggest that the regeneration matrix is well tolerated and not systemic in pigs. Furthermore, the data suggest that in the last two weeks of the study period, regenerated matrix transplanted pigs began to gain more muscle mass than control pigs.

  In all pigs, locomotor function was measured initially at 72 hours and then weekly after SCI using a modified ASIA Sensory and Motor Skil scoring system for the hind legs of the animals. . FIG. 35 shows the locomotor scores of regenerated matrix transplanted pigs or homogenized clot transplanted pigs.

  In these studies, the Sensory and Motor Skil baseline score was measured 72 hours after surgical SCI and transplantation and used as an initial value, and then a 7-week score was determined every week. Pigs from both study groups began to recover locomotor and sensory functions (from spinal shock) within the first week of surgical SCI, and these values remained constant at 2-4 weeks after transplantation. Reached the level. During this 2-4 week period, no significant difference was observed in the locomotor function and sensory function between the regeneration matrix transplantation group and the clot transplantation group, indicating that the regeneration matrix It does not interfere with functional recovery and does not further impair spinal cord function.

  A further increase in sensory and locomotor recovery was observed in most pigs approximately 4-6 weeks after SCI. At these later time points, the level of recovery observed in regenerated matrix transplanted pigs was higher than the level of recovery observed in clot transplanted pigs. A detailed analysis of the difference between these two groups at week 7 shows that there is a significant improvement (p = 0.03) in the function of the right side (damaged side) of the pigs that received the regeneration matrix. It is shown (FIG. 36). At this seventh week, there is a much greater variation within the regeneration matrix transplant group than the control group, indicating that the biological activity of the regeneration matrix is at its endpoint (final exercise score) value. Indicates that it has not yet been reached. The endpoint was estimated to be about 16 weeks.

  Taken together, these studies show that in pigs, the regeneration matrix promotes recovery of sensory and locomotor activity after safe and well tolerated surgically induced SCI.

(Example 17)
Rat GLP Toxicology Studies To evaluate the potential for systemic toxicity associated with transplantation of regeneration matrix, preclinical studies in accordance with GLP, ISO 10993-11 were developed and conducted at NAMSA (Northwood, Ohio). In this study, the regeneration matrix was implanted into the subcutaneous tissue of 14 rats (7 males and 7 females). A control substance (sterile 0.9% saline) was similarly transplanted into another group of 14 rats (7 males and 7 females). Rats were observed daily for overt signs of mortality and toxicity. Detailed examinations for clinical signs of disease and abnormalities were performed weekly. Rat weights were determined prior to transplantation and weekly throughout the study.

  At week 4, rats were euthanized and blood samples were collected for hematology and clinical chemistry analysis (all analyzes were performed by NAMSA). Autopsy was performed and selected organs were excised, weighed and histologically processed. From each rat, the subcutaneous tissue surrounding each implantation site was also excised and examined microscopically. Microscopic evaluation of the transplant site and selected organs was performed by a regular pathologist. Body weight, organ weight, organ weight / body weight ratio, hematology values and clinical chemistry values were analyzed statistically.

  From the data, there was no evidence of systemic toxicity from test substances (regeneration matrix) after subcutaneous implantation in rats. Daily clinical observations, body weights, autopsy findings, rat organ weights and organ weight / body weight ratios were within acceptable limits and were similar between test and control treatment groups and within each group. There were no changes in hematology and clinical chemistry values that were considered to be biologically significant or related to treatment with the test substance in either male or female rats. Microscopic evaluation of selected tissues gave no evidence of systemic toxicity in the group treated with the regeneration matrix. Based on histological criteria, the regeneration matrix in male and female rats was considered to be a moderate irritant when compared to a reference control material that was saline. This latter finding is within the experience of this laboratory for the evaluation of bioabsorbable materials and as a cross-species transplant (in this case, a regeneration matrix derived from human whole blood was transplanted into a rat) Belongs to the lower range. In conclusion, local injection of the regeneration matrix across species does not cause any significant toxicity, and transplantation of the regeneration matrix to human SCI patients does not cause the regeneration matrix to cause local pathology. It is suggested that it will be.

(Example 18)
Microscopic evaluation of sections from spinal cord regeneration studies. Regeneration matrices were implanted in experimental rats from which the 10 mm long spinal cord had been surgically removed. Ten days later, the spinal cord was examined histologically. Microscopically, the center of the spinal cord defect consists of severe Wallerian degeneration of the central axon and neutrophil necrosis, which is about 4.5 mm from the center caused by surgical removal of the 1 cm long spinal cord. It stretched with a radius (full width of 1 cm). Around the regenerative matrix implant are sheets of reactive glial cells and lattice cells, some of which have hemosiderin, and in addition, clearly primitive mesenchymal / reticular type There was also a sheet of cells (see FIGS. 38A and 38B), which appeared to be spreading into the spinal cord defect (filling the spinal cord defect). These cells appeared to be involved in the development of new neural tissue and regeneration of the spinal cord. Around the defect there was a buffy coat thickening with reactive fiber growth. Fibroproliferation appears to spread along the buffy coat as a framework to support these apparently primitive mesenchymal / reticular cells, and appears to support the regeneration of the damaged spinal cord. It was.

  The foregoing description is intended to be illustrative in nature and is not intended to limit the invention or the details relating to the application or use of the invention. Those skilled in the art will appreciate that certain modifications can be made to one or more of the embodiments of the invention described herein. However, such modifications are within the scope of the present invention. Alternative methods and materials for practicing the invention, as well as additional applications, will be apparent to those skilled in the art. They are intended to be included within the scope of the accompanying claims.

The foregoing features of the invention will be more readily understood by reference to the accompanying drawings and the following detailed description. In the figure, “RMx” indicates a reproduction matrix.
FIGS. 1A, 1B and 1C show the transformation of macrophages into von Willebrand factor positive endothelial-like cells. 2A: Macrophages (light staining) and von Willebrand factor (+) (dark staining) cells at the site of spinal cord injury and regeneration matrix transplantation 2 weeks after transplantation in pigs. The double stain is black. Shown at 200X magnification. 2B: Black staining = macrophages expressing von Willebrand factor (bright staining). Shown at 400X magnification. 2C: Macrophages expressing von Willebrand factor are forming blood vessels. Shown at 400X magnification. Figures 2A, 2B, 2C and 2D show the therapeutic effect of the regeneration matrix on rat spinal cord tissue. A shows healthy rat spinal cord with healthy blood vessels in the absence of regeneration matrix. B shows damaged rat spinal cord with little CNS tissue visible and no healthy blood vessels. C shows a view of the spinal cord of an injured rat treated with a regeneration matrix, where a regenerated healthy CNS tissue and blood vessel cross section is visible. D is another view of C injured spinal cord tissue, showing regenerated CNS tissue and longitudinal sections of healthy blood vessels. FIG. 3 is a photograph of a regeneration matrix produced using non-coagulated blood and 1.2 μm final filtration, contained in an Opticell® production cassette. FIG. 4 shows a paraffin section of a regeneration matrix made from human whole blood after 3 weeks. The regeneration matrix does not show any signs of unchanged cells or nuclei. Hematoxylin staining was completely negative in all sections analyzed, but the material stained strongly with eosin. Using a high numerical aperture lens (high resolution), the bead-like structure can be clearly resolved. In addition to the bead-like structure that makes up most of the RMx, a small region of a flat sheet that appears to be of a denser structure can be observed. FIGS. 5A, 5B, 5C and 5D show scanning electron micrographs of the regeneration matrix produced using coagulated blood and 5 μm final filtration. A-200 times, B-1000 times, C-7500 times, D-7500 times. FIGS. 6A, 6B, 6C and 6D show scanning electron micrographs of the regeneration matrix (day 21) generated using coagulated blood and 1.2 μm final filtration. A-200 times, B-1000 times, C-7500 times, D-7500 times. FIGS. 7A, 7B, 7C and 7D show scanning electron micrographs of the regeneration matrix (day 21) generated using non-coagulated blood (using EDTA) and a final filtration of 1.2 μm. A-200 times, B-1500 times, C-5000 times, D-10000 times. FIG. 8 shows an optical micrograph (100 × magnification) of a regeneration matrix formed using non-coagulated blood and a final filtration of 1.2 μm. FIG. 9 shows transmission electron microscopy (TEM) of a regeneration matrix prepared from a tissue sample and passed through a 5 μm filter after 21 days. In this TME, the rounded spheres seen in the scanning electron micrographs (Figures 5-7) are part of a much larger, relatively homogeneous structure or aggregate of these spheres. Is clear. There is no nuclear evidence. FIG. 10 shows a transmission electron microscope observation of a regeneration matrix prepared from human whole blood after 8 weeks. The regeneration matrix stains homogeneously and the outer surface of each aggregate is larger in protruding spheres and has a somewhat honeycomb shape (particles of particles) than the regeneration matrix after 3 weeks (see FIG. 9). The most effective arrangement method). Many new aggregates of the regeneration matrix formed along the long threads of the regeneration matrix material. FIG. 11 shows a transmission electron microscope observation of a regeneration matrix produced from human whole blood after 8 weeks. The regeneration matrix stains homogeneously and the outer surface of each aggregate is larger in protruding spheres and has a somewhat honeycomb shape (particles of particles) than the regeneration matrix after 3 weeks (see FIG. 9). The most effective arrangement method). Figures 12A and 12B show SDS-PAGE analysis of the regeneration matrix from five different batches. All proteins in the regeneration matrix on day 15 (A) and day 21 (B) were stained with Coomassie blue. The main protein species are numbered 1-6 and are shown in their respective densitometric graphs. Molecular weight standards (KDa) are shown on the left side of the gel. FIG. 13 shows the regeneration matrix formed in the presence of α-amanitin (RNAII polymerase inhibitor), aphidicolin (DNA polymerase inhibitor) or cycloheximide (protein synthesis inhibitor) and culture on day 21 compared to the control. The total RNA content (μg) in the product supernatant (filtered at 5 μm) is shown. FIG. 14 shows the regeneration matrix formed in the presence of α-amanitin (RNAII polymerase inhibitor), aphidicolin (DNA polymerase inhibitor) or cyclohexamide (protein synthesis inhibitor) and day 21 compared to the control. The total protein content in the culture supernatant (filtered at 5 μm) is shown. FIG. 15 shows the total protein content in the regeneration matrix formed in the presence of α-amanitin (RNAII polymerase inhibitor) and the supernatant of the culture on day 21 (filtered at 1 μm) compared to the control. . FIG. 16 shows the total lipid content in the regeneration matrix formed in the presence of [alpha] -amanitin (RNAII polymerase inhibitor)) and the supernatant of the 21st day culture (filtered at 1 [mu] m) compared to the control. . FIG. 17 shows a comparison of Coomassie-stained protein bands for matrix and media from 1 μm filtered matrix to 5 μm filtered matrix on PAGE. FIG. 18 shows the effect of the regeneration matrix (and the supernatant used for each) produced in the treatment medium with different adjuvants on upregulation of neuronal genes. A: NT-3, B: NCAM-1, C: GAP-43. FIG. 19 shows the doubling of gene upregulation and heat inactivated regeneration matrix produced by the regeneration matrix on SH-SY5Y cells. SH-SY5Y cells were incubated with the regeneration matrix for 3 hours and then GAPDH, NT-3, NT-6, NCAM-1, FGF-9, GDNF, Netrin-1 and GAP- using RT-PCR. 43 gene expressions were analyzed. The regeneration matrix washed 3 times in PBS had activity similar to the unwashed regeneration matrix. The regeneration matrix incubated for 30 minutes at 60 ° C. did not have gene upregulation activity. FIG. 20 shows neurite outgrowth by Neuroscreen® cells treated with a regeneration matrix. The figure on the left shows a representative area of Neuroscreen® cells grown in basal medium. The right figure shows a representative area of cells treated with the regeneration matrix. Cell nuclei were stained blue and tubulin was stained green. FIG. 21 shows neurite outgrowth of Neuroscreen® cells cultured and compared in the presence and absence of regeneration matrix. Results are expressed as a percentage of the neurite outgrowth index of NGF treated cells. Results are the average of 4 independent experiments performed in quadruplicate. Error bars indicate the average standard deviation. The values of regenerated matrix treated cells and regenerated matrix untreated cells differ with a P value of less than 0.01 (t test). FIG. 22 compares the neurite outgrowth of Neuroscreen® cells generated with regeneration matrix in the presence and absence of EGF and bFGF growth factor supplements and cultured in the presence of these regeneration matrices. Shows the case. Results are expressed as a percentage of the neurite outgrowth index of NGF treated cells. The result is an average of at least six samples. Error bars indicate the average standard deviation. All values are different from each other with a difference P value of less than 0.01 (t-test). FIG. 23 shows NGF dose response curves for neurite outgrowth of Neuroscreen® cells with different concentrations of NGF. FIG. 24 is a doubling (attenuation) of the average total neurite outgrowth compared to the NGF positive control (FIG. 23; 100 ng / ml NGF) when applying a regeneration matrix generated in TR-10 treated media. Indicates. The regeneration matrix was generated from either whole blood or plasma-platelet-buffy coat fractions. FIG. 25 shows control rat fetal spinal cord primary cells, labeled neuronal tubulin (Tuj1) and glia (GFAP) after 6 days of single cell suspension culture (2 frames are all The dyed components are shown). On average, 4.2% of the cells were Tuj1 positive and 0.4% of the cells were GFAP positive. 100x magnification. FIG. 26 shows the effect of regeneration matrix on primary cells of rat fetal spinal cord, labeling tubulin (Tuj1) and glia (GFAP) of neurons after 6 days of single-cell suspension culture (2 frames are , Shows all stained components). On average, 55% of the cells were Tuj1 positive and 92% of the cells were GFAP positive. Double positive (Tuj-1 and GFAP positive) cells indicate triphasic neural progenitor cells. 100x magnification. FIGS. 27A, 27B, 27C and 27D show on the regeneration matrix (C and D) for immunocytochemical staining for β-tubulin (A) and F-actin (B) of human fibroblasts cultured for 5 days. A comparison with the control (A and B) is shown. The medium used in the control culture was the treatment medium used to generate the regeneration matrix. Human fibroblasts cultured in contact with the regeneration matrix had significantly lower levels of confluence compared to control cultures (it also appeared to have undergone apoptosis). FIG. 28 shows a comparison of a 2-day culture and a control in terms of regeneration matrix fibroblast adhesion, fibroblast nuclear area, and percent BrdU positive fibroblasts. FIG. 29 shows that transplantation of regenerative matrix into a fully crossed rat model of SCI (5 mm) induces recovery of motor function. Recovery of locomotor function as measured by the BBB score was recorded once every two weeks after injury and regeneration matrix transplantation. BBB score for individual animals (Regeneration Matrix-M and Control-C). FIG. 30 shows a decrease in the incidence of cyst formation in a transverse rat model of spinal cord injury that received a regeneration matrix transplant. In regenerated matrix transplanted rats (n = 19), the incidence of cyst formation at and around the SCI site is 15.8%, and in control rats (n = 5), the occurrence of cyst formation around the SCI site. The rate was 60%. FIG. 31 shows that transplantation of regeneration matrix into SCI unilaterally cut rat model (5 mm) induces recovery of motor function. Recovery of locomotor function as measured by the BBB score was recorded once every two weeks after injury and regeneration matrix transplantation. BBB score for individual animals that received a regeneration matrix transplant. Non-transplanted controls did not survive until the first evaluation time point. FIG. 32A: Functional recovery after regeneration matrix transplantation into rat spinal cord with 5 mm complete crossing. FIG. 32B: Functional recovery after regeneration matrix transplantation into rat spinal cord 2 weeks after injury due to contusion (50 mm). FIG. 32C: Combined (full crossing and contusion) model showing the efficacy of the regeneration matrix in the treatment of spinal cord injury. Control rats include empty controls and biological inactivated regeneration matrix controls from both injury models. For each of FIGS. 32A, 32B and 32C, the top line represents data from a regeneration matrix transplanted rat. The lower line shows data from control rats. FIG. 32A: Functional recovery after regeneration matrix transplantation into rat spinal cord with 5 mm complete crossing. FIG. 32B: Functional recovery after regeneration matrix transplantation into rat spinal cord 2 weeks after injury due to contusion (50 mm). FIG. 32C: Combined (full crossing and contusion) model showing the efficacy of the regeneration matrix in the treatment of spinal cord injury. Control rats include empty controls and biological inactivated regeneration matrix controls from both injury models. For each of FIGS. 32A, 32B and 32C, the top line represents data from a regeneration matrix transplanted rat. The lower line shows data from control rats. FIG. 32A: Functional recovery after regeneration matrix transplantation into rat spinal cord with 5 mm complete crossing. FIG. 32B: Functional recovery after regeneration matrix transplantation into rat spinal cord 2 weeks after injury due to contusion (50 mm). FIG. 32C: Combined (full crossing and contusion) model showing the efficacy of the regeneration matrix in the treatment of spinal cord injury. Control rats include empty controls and biological inactivated regeneration matrix controls from both injury models. For each of FIGS. 32A, 32B and 32C, the top line represents data from a regeneration matrix transplanted rat. The lower line shows data from control rats. FIG. 33 shows that regenerative matrix implantation induces motor function recovery in porcine spinal cord injury caused by impact and / or surgical unilateral cutting (including removal of 5 mm spinal cord). Pigs 00-6-5 and 113-1 were damaged by severe impact resulting in tears and loss of spinal cord tissue. Pig 80-1 was damaged by penetration with a 14 gauge needle. Pigs 55-1, 53-2, 54-5 and 54-2, as well as all control pigs, received a right surgical unilateral cut (5 mm long right spinal cord was removed). The results of recovery of locomotor function as measured by a modified numerical ASIA score were recorded weekly after injury and regeneration matrix transplantation. FIG. 34 shows a comparison of average body weight and average body weight gain over 7 weeks between regenerated matrix transplanted pigs and homogenized clot transplanted pigs. FIG. 35 shows a comparison of the recovery of right motor skills over 7 weeks between regenerated matrix transplanted pigs and human clot transplanted pigs after spinal cord injury by right side unilateral cut (5 mm long right side spinal cord removed). FIG. 36 shows a comparison of recovery of locomotor function between human regeneration matrix-treated pigs and clot-treated pigs in the seventh week after SCI. The undamaged left side (used as an internal control) was the same in both groups, but the damaged right side recovered more significantly with the regeneration matrix than with the clot. (P = 0.03). FIG. 37 shows nerve cells (brown) being newly formed at the site of spinal cord injury (and spinal cord removal) in pigs in the first month after regeneration matrix transplantation. Light color = cell nucleus. Dark color = Tuj-1 positive cells. Black = double staining. The magnification is 200 times. FIG. 38 shows microscopic evaluation of longitudinal sections from a rat spinal cord regeneration study. 38A: Primitive cells associated with the regeneration matrix are filling the spinal cord defect. Taken at 10x magnification. 38B: Primitive cells at the site of formation of new neural tissue 10 days after transplantation of regeneration matrix into a 10 mm spinal cord defect. Taken at 40x magnification.

Claims (94)

A method for producing a cell-free, bioabsorbable tissue regeneration matrix comprising:
a) providing an isolated tissue sample;
b) removing cells from the tissue sample to obtain a cell-free sample;
c) incubating the cell-free sample in an incubation chamber for a first time period sufficient to form the cell-free, bioabsorbable tissue regeneration matrix, the cell-free, bioabsorbable tissue At least one regenerative activity of the regenerative matrix changes over the first period, and the change in regenerative activity is selected from the group consisting of appearance of regenerative activity, generation of regenerative activity, increased regenerative activity, and combinations thereof Steps to be performed;
d) isolating the cell-free, bioabsorbable tissue regeneration matrix.   The method of claim 1, wherein the isolated tissue sample is selected from the group consisting of muscle tissue, connective tissue, epithelial tissue, neural tissue, and combinations thereof.   The method of claim 1, wherein the isolated tissue sample is selected from the group consisting of basic tissue, epidermal tissue and vascular tissue, and combinations thereof. The isolated tissue sample is
Organ system tissue selected from the group consisting of cardiovascular, digestive, endocrine, excretion, immunity, envelope, lymph, muscle, nerve, reproduction, breathing, skeletal, and combinations thereof;
Placental tissue;
Umbilical cord tissue;
And the method of claim 1 or 2 selected from the group consisting of combinations thereof.   The method of claim 1, 2, or 4, wherein the tissue sample comprises blood.   6. The method of claim 1, 2, 4 or 5, wherein the tissue sample comprises a blood fraction.   The method of claim 6, wherein the blood fraction comprises a buffy coat layer.   The method of claim 6, wherein the blood fraction comprises a buffy coat / platelet / plasma layer.   10. A method according to any of claims 1-2 or claims 4-9, wherein the tissue sample comprises an anticoagulant.   10. The method according to any of claims 1-2 or claim 4-9, wherein the blood comprises one or more clots and further comprising solubilizing the clot.   8. A method according to any preceding claim, wherein the removing step comprises passing the tissue sample through a size selection filter.   The method of claim 11, wherein the size selection filter excludes particles greater than about 5 μM.   The method of claim 11, wherein the size selection filter excludes particles greater than about 1.2 μM.   The method of claim 11, wherein the size selection filter excludes particles greater than about 0.8 μM.   A method according to any preceding claim, wherein the incubation chamber is permeable to solids or liquids.   The method of claim 15, wherein the incubation chamber is permeable to a gas generated by evaporation of a liquid inside the incubation chamber.   17. A method according to claim 15 or 16, wherein the incubation chamber is incubated in a low humidity environment, thereby increasing the evaporation rate of the liquid inside the incubation chamber.   A method according to any preceding claim, further comprising the step of adding a treatment medium to the cell-free sample.   The step of adding the treatment medium was selected from the group consisting of isolation of the tissue sample, prior to the removing step, prior to the incubating step, during the incubating step, and combinations thereof 19. The method of claim 18, comprising adding at one or more times.   20. The method of claim 18 or 19, wherein the cell-free sample is in the form of a liquid solution or suspension and at least a portion of the solute evaporates from the incubation chamber over the first period.   21. The method of claim 18, 19 or 20, wherein the step of adding the treatment medium results in an increase in osmolarity of the liquid solution or suspension over the first period.   The method according to any of claims 18 to 21, wherein the treatment medium comprises a physiological solution.   The method according to any of the preceding claims, wherein the first period is at least 5 days.   A method according to any preceding claim, wherein the first period is at least 12 days.   The method according to any of the preceding claims, wherein the incubating step comprises incubating the cell-free sample in a hypoxic environment.   26. The method of claim 25, wherein the hypoxic environment comprises an environment having less than about 5% oxygen.   26. The method of claim 25, wherein the hypoxic environment comprises an environment that is substantially devoid of oxygen.   28. The method of claim 25, 26 or 27, wherein the first period is at least 3 days.   28. The method of claim 25, 26 or 27, wherein the first period is at least 7 days.   A method according to any preceding claim, further comprising providing a therapeutic agent.   32. The method of claim 30, wherein the therapeutic agent is selected from the group consisting of proteins, peptides, drugs, cytokines, extracellular matrix molecules, growth factors, and combinations thereof.   Providing the therapeutic agent is selected from the group consisting of isolation of the tissue sample, prior to the removing step, prior to the incubating step, during the incubating step, and combinations thereof 32. A method according to claim 30 or 31, comprising providing at one or more designated times.   The osmolarity of the cell-free sample is controlled so that the cell-free, bioabsorbable tissue regeneration matrix formed in the first period is in the form of a three-dimensional matrix. The method according to any one.   Any of the preceding claims, wherein the osmolarity of the cell-free sample is controlled such that the cell-free, bioabsorbable tissue regeneration matrix formed during the first period is in the form of a suspension. The method of crab.   8. The method according to any of the preceding claims, further comprising the step of seeding or mixing the cell-free, bioabsorbable tissue regeneration matrix with cells after it has been generated.   36. The method of claim 35, wherein the cell is a cell selected from the group consisting of stem cells, progenitor cells, somatic cells, and combinations thereof.   37. The method of claim 35 or 36, wherein the cell is a cell selected from the group consisting of embryonic stem cells, neural stem cells, neural progenitor cells, neural cells, glial cells, and combinations thereof.   8. The method of any preceding claim, further comprising seeding or mixing the cell-free, bioabsorbable tissue regeneration matrix with the cells while it is being produced.   40. The method of claim 38, wherein the cell is a cell selected from the group consisting of stem cells, progenitor cells, somatic cells, and combinations thereof.   40. The method of claim 38 or 39, wherein the cell is a cell selected from the group consisting of embryonic stem cells, neural stem cells, neural progenitor cells, neural cells, glial cells, and combinations thereof.   The cell-free, bioabsorbable tissue regeneration matrix exhibits activity during the first period, the activity being tissue growth, cell growth, gene upregulation, cell inhibition, cell proliferation, cell proliferation Selected from the group consisting of dedifferentiation, cell differentiation, cell differentiation transformation, neurite outgrowth, gene upregulation, or one or more natural wound healing processes, and combinations thereof The method according to any one.   42. The method of claim 41, wherein the activity is determined by measuring the effect of the generated cell-free, bioabsorbable tissue regeneration matrix on one or more cell types.   43. The method of claim 41 or 42, wherein the activity is determined by measuring the effect of the generated cell-free, bioabsorbable tissue regeneration matrix on one or more cell types.   44. A cell-free, bioabsorbable tissue regeneration matrix obtained according to the method of any one of claims 1 to 43. A method of regenerating a target organization,
a) providing a subject that has undergone tissue damage, loss or degeneration;
b) administering a cell-free, bioabsorbable tissue regeneration matrix to a subject at or near the site of tissue damage, loss or degeneration, and
A method wherein the cell-free, bioabsorbable tissue regeneration matrix initiates, increases, supports and / or guides tissue regeneration at sites of tissue damage, loss or degeneration.   46. The method of claim 45, wherein the subject is a human.   46. The method of claim 45, wherein the subject is an animal.   48. The method of claim 45, 46 or 47, wherein the cell-free, bioabsorbable tissue regeneration matrix comprises a therapeutic agent.   49. The method of claim 48, wherein the therapeutic agent is unevenly distributed within the cell-free, bioabsorbable tissue regeneration matrix.   50. The method of claim 48 or 49, wherein the therapeutic agent is evenly distributed within the cell-free, bioabsorbable tissue regeneration matrix.   51. A method according to any of claims 45 to 50, wherein the cell-free, bioabsorbable tissue regeneration matrix comprises two or more therapeutic agents.   52. The method of any one of claims 45 to 51, wherein the therapeutic agent is selected from the group consisting of a protein, peptide, drug, cytokine, extracellular matrix molecule, growth factor, and combinations thereof.   53. A method according to any of claims 45 to 52, wherein the cell-free, bioabsorbable tissue regeneration matrix increases the magnitude of one or more beneficial effects of the therapeutic agent.   The cell-free, bioabsorbable tissue regeneration matrix prolongs one or more beneficial effects of the therapeutic agent by slowly releasing the therapeutic agent in the subject over time; 54. A method according to any one of claims 45 to 53.   55. A cell according to any of claims 45 to 54, wherein the cell-free, bioabsorbable tissue regeneration matrix protects one or more beneficial effects of the therapeutic agent from a substantial decrease over time. the method of.   56. The method of any one of claims 45 to 55, wherein the damaged or degenerated tissue is selected from the group consisting of muscle tissue, connective tissue, epithelial tissue, neural tissue, and combinations thereof.   57. The method of claim 56, wherein the neural tissue is central nervous system tissue. The damaged or degenerated tissue is
Organ system tissue selected from the group consisting of cardiovascular, digestive, endocrine, excretion, immunity, envelope, lymph, muscle, nerve, reproduction, breathing, skeletal, and combinations thereof;
Placental tissue;
Umbilical cord tissue;
58. The method according to any of claims 45 to 57, selected from the group consisting of: and combinations thereof.   59. The method of claim 58, wherein the bioabsorbable tissue regeneration matrix stimulates the recruitment of neural progenitor cells to the damaged, defective or degenerated site.   60. The method of claim 58 or 59, wherein the bioabsorbable tissue regeneration matrix stimulates proliferation of neural progenitor cells.   61. The method of claim 58, 59 or 60, wherein the bioabsorbable tissue regeneration matrix stimulates the recruitment of von Willebrand factor positive cells to the damaged, deficient or degenerated site.   In response to administration of the cell-free, bioabsorbable tissue regeneration matrix to or near the site of the damaged or degenerated tissue, the neurotrophic activity of the damaged or degenerated central nervous system tissue is increased. Item 62. The method according to any one of Items 58 to 61.   Expression of a neurotrophic gene wherein the increased neurotrophic activity is selected from the group consisting of FGF-9, Netrin-1, NT-3, NCAM-1, GAP-43, neuregulin, and combinations thereof 63. A method according to any of claims 58 to 62, characterized by an increase in.   64. The method of any of claims 45-63, further comprising administering cells to or near the site of the damaged or degenerated tissue.   65. The method of any of claims 45 to 64, wherein the cells and the cell-free, bioabsorbable tissue regeneration matrix are administered sequentially.   65. A method according to any of claims 45 to 64, wherein the cells and the cell-free, bioabsorbable tissue regeneration matrix are administered simultaneously.   68. The method of any of claims 45 to 67, wherein the cell-free, bioabsorbable tissue regeneration matrix is seeded or mixed with cells.   68. The method of claim 67, wherein the cell-free, bioabsorbable tissue regeneration matrix is seeded or mixed with cells while the cell-free, bioabsorbable tissue regeneration matrix is being formed.   69. The method of claim 67 or 68, wherein the cell-free, bioabsorbable tissue regeneration matrix is seeded or mixed with cells after the cell-free, bioabsorbable tissue regeneration matrix is formed.   70. The method of any of claims 64 to 69, wherein the cell is a cell selected from the group consisting of stem cells, progenitor cells, somatic cells, and combinations thereof.   71. The method of any of claims 64 to 70, wherein the cell is a cell selected from the group consisting of embryonic stem cells, neural stem cells, neural progenitor cells, neural cells, glial cells, and combinations thereof.   72. The method of any one of claims 45 to 71, wherein the administering step comprises an administration route selected from the group consisting of infusion, surgical implantation, delivery through a catheter, and combinations thereof.   74. A method according to any of claims 45 to 73, wherein the bioabsorbable tissue regeneration matrix dedifferentiates cells into cells capable of giving rise to one or more different cell types.   74. The method of claim 73, wherein the dedifferentiated cell is a macrophage.   75. The method of claim 74, wherein the dedifferentiated macrophages exhibit endothelium-like characteristics.   76. The method of claim 75, wherein the endothelium-like feature comprises expressing von Willebrand factor.   77. A method according to any of claims 74 to 76, wherein the dedifferentiated macrophages produce new blood vessels.   78. The method of claim 77, wherein the new blood vessel supports new tissue growth. An isolated cell-free, bioabsorbable tissue regeneration matrix comprising a proteinaceous core having a protein content of at least 1%;
The structural feature of the cell-free, bioabsorbable tissue regeneration matrix is a sphere having a diameter of at least about 100 nm;
Furthermore, the cell-free, bioabsorbable tissue regeneration matrix lacks substantial metabolic activity;
Further, the cell-free, bioabsorbable tissue regeneration matrix may initiate regeneration of more tissue in a subject having damaged tissue, increase tissue regeneration in a subject having damaged tissue, or A tissue regeneration matrix that is both possible.   One selected from the group consisting of transferrin, serum albumin, serum albumin precursor, complement component 3, AD chain hemoglobin, IgM, IgG1, medalacin inhibitor 2, carbonic anhydrase, CA1 protein, or combinations thereof 80. The isolated cell-free, bioabsorbable tissue regeneration matrix of claim 79 further comprising the above protein.   81. The isolated cell-free, bioabsorbable tissue regeneration matrix of claim 79 or 80, wherein the sphere is recognized by a CD56 antibody.   82. The cell-free, bioabsorbable tissue regeneration matrix of claim 79, 80 or 81, wherein the sphere has a diameter of about 1 to 2 [mu] m.   82. The cell-free, bioabsorbable tissue regeneration matrix of claim 79, 80 or 81, wherein the sphere has a diameter of about 2 to 4 [mu] m.   84. The cell-free, bioabsorbable tissue regeneration matrix according to any of claims 79 to 83, further comprising a therapeutic agent.   85. The cell-free, bioabsorbable tissue regeneration matrix of claim 84, wherein the therapeutic agent is unevenly distributed within the cell-free, bioabsorbable tissue regeneration matrix.   85. The cell-free, bioabsorbable tissue regeneration matrix of claim 84, wherein the therapeutic agent is evenly distributed within the cell-free, bioabsorbable tissue regeneration matrix.   87. The cell-free, bioabsorbable tissue regeneration matrix according to any of claims 84 to 86, comprising two or more therapeutic agents.   88. A cell-free, living organism according to any of claims 84 to 87, wherein the therapeutic agent is selected from the group consisting of proteins, peptides, drugs, cytokines, extracellular matrix molecules, growth factors, and combinations thereof. Absorbable tissue regeneration matrix.   89. A cell-free, bioabsorbable tissue regeneration matrix according to any of claims 84 to 88, which increases the magnitude of one or more beneficial effects of the therapeutic agent.   90. A cell-free according to any of claims 84 to 89, which prolongs one or more beneficial effects of a therapeutic agent by slowly releasing the therapeutic agent over time in the subject. Bioresorbable tissue regeneration matrix.   91. A cell-free, bioabsorbable tissue regeneration matrix according to any of claims 84 to 90, which protects one or more beneficial effects of the therapeutic agent from a substantial decrease over time.   92. The cell-free, bioabsorbable tissue regeneration matrix according to any of claims 79 to 91, further comprising cells.   94. The cell-free, bioabsorbable tissue regeneration matrix of claim 92, wherein the cells are selected from the group consisting of stem cells, progenitor cells, somatic cells, and combinations thereof.   94. The cell-free, bioresorbable of claim 92 or 93, wherein the cell is a cell selected from the group consisting of embryonic stem cells, neural stem cells, neural progenitor cells, neural cells, glial cells, and combinations thereof. Tissue regeneration matrix.

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