JP2005270237A - Material for nerve regeneration and nerve regeneration material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a material for nerve regeneration capable of demonstrating excellent neural cell proliferation accelerating action and nerve axon extending action, urging the proliferation of damaged or defective neural cells and neural tissue, restoring them and regenerating nerves, and its manufacturing method. <P>SOLUTION: The material for the nerve regeneration contains a bioabsorptive polymer and cells. For the material for the nerve regeneration and its manufacturing method, in this case, the cells are composed of (1) the cells of CD29 positive (CD29<SP>+</SP>), CD90 positive (CD90<SP>+</SP>), CD11 negative (CD11<SP>-</SP>) and CD34 negative (CD34<SP>-</SP>) and/or (2) the cells composed by differentiating and inducing the cells of (1). For the material for the nerve regeneration, the bioabsorptive polymer usable together with the cells is composed of a polymer formed from polysaccharides, acid polysaccharide ester, protein, polyglygolic acid, polylactic acid, cyanoacrylate-based copolymer, or the acid polysaccharide ester and a polyamino compound having two or more α-amino groups deriving from natural amino acid. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a nerve regeneration material, a nerve regeneration material, and a production method thereof.

  Treatment of peripheral nerve damage caused by various accidents such as traffic accidents and occupational accidents occupies a large position in the surgical field, particularly in the orthopedic field. In recent years, surgical techniques that connect severed nerves have made significant progress with the introduction of microsurgery. However, when the nerve defect is large enough to be compensated for, the treatment is a challenge for the surgeon. The currently practiced method is autologous nerve transplantation using the sural nerve. The autonomic nerve is the most ideal nerve regeneration material, but its collection is limited due to the burden on the patient and the complexity of the operation. Moreover, since the sensory nerve on the little finger side from the ankle to the back of the foot disappears due to the resection of the sural nerve, the quality of life of the patient is lowered, and it is preferable to avoid autologous nerve transplantation if possible. Under such circumstances, development of a transplant material to replace the autonomic nerve is eagerly desired, and various studies have been conducted. For example, autologous blood vessel transplantation and autologous fascia transplantation have been performed as autotransplantation using tissues other than nerves, but in terms of using the patient's biological tissue, the burden on the patient cannot be eliminated, and at the time of surgery In terms of complexity, it is not much different from autologous nerve transplantation. In addition, although the peripheral nerve is a recoverable organ, when the gap is large, it takes a long time to recover the function. Therefore, a material for nerve regeneration that recovers the nerve function in a shorter time has been demanded.

  Furthermore, the central nervous system represented by the brain and spinal cord of mammals, in particular, is irreversible once it is damaged by traffic injury or sports injury, and is thought to ultimately follow a process of neurodegeneration. Sensory nerves, motor nerves, and autonomic nerves distal to the spinal cord lesion with neurodegeneration are severely damaged, and visceral diseases and excretory dysfunction are also caused, and lifelong functions are not restored. Even in the case of minor injury, you will be forced to live in a wheelchair, but in severe cases you may have to use a ventilator. The social return of these patients is a major social problem.

  The following has been proposed as the reason why the central nerve does not regenerate.

  1) Oligodendrocytes have a strong inhibitory action on nerve regeneration. Specifically, it has been shown that a molecule called Nogo extracted from oligodendrocytes inhibits (Non-patent Documents 1 and 2). In experiments using a culture system, it has been found that when a neurite is extended, contact with an oligodendrocyte-derived myelin sheath not only stops the extension, but also retracts the protrusion (contact inhibition). It is also shown that neurites do not extend where there is a myelin sheath, and that the process advances to avoid them in culture.

  2) Next, it is known that astrocytes that have caused gliosis produce various inhibitors, and proteoglycans such as keratin sulfate and chondroitin sulfate are mentioned (Non-patent Document 3). It is presumed that these substances act as a physical barrier for regenerating axons and inhibit the axons from extending.

  On the other hand, the peripheral nerve is a reproducible organ. Unlike the central nervous system, Schwann cells are the main constituent cells in peripheral nerve tissue. The nerve fibers of peripheral nerves are covered with Schwann cells regardless of whether they are myelinated or unmyelinated. Schwann cells are derived from neural crest cells, and central glia (oligodendrocytes, astrocytes) that inhibit nerve regeneration are originated from neural tubes. It has a different origin.

  Peripheral nerves are thought to regenerate by the following mechanism.

  In peripheral nerves, when damage such as amputation occurs, Wallerian degeneration occurs more peripherally than the damaged site. This means that Schwann cells revert from a differentiated form that formed myelin sheaths to an undifferentiated state, are activated, divide and proliferate, and form a cord. In the peripheral nerve, individual nerve fibers are independently wrapped in Schwann cells, and the outside is covered with a basement membrane. That is, each nerve is in a separate basement membrane sheath. Therefore, even if degeneration occurs, Schwann cells activated in the sheath of the basement membrane proliferate and form a cord-like structure, which can be used as a clue to reconstruct the original neural circuit.

  As mentioned above, the myelin sheath of central glia regenerates a substance that is inhibitory to axon elongation, whereas the same myelin sheath has little inhibitory effect on Schwann cells and seems to be permissive ( Non-patent document 4). It is also known that there is a difference in the composition of the proteins that make up myelin between Schwann cells and oligodendrocytes. In this way, even if the central glia is damaged, it does not divide and proliferate by changing to an undifferentiated form like Schwann cells in peripheral nerves, and it does not change the form greatly, comparing It maintains a phenotype that remains fully differentiated. In that respect, the peripheral nerves are characterized by a quick and flexible response to the damage received.

  Schwann cells in nerve regeneration are thought to have the following roles. Schwann cells produce many factors and release them diffusively. The surface of its cell membrane is covered with a basement membrane, which contains an extracellular matrix component. Furthermore, it is known that cell adhesion molecules are expressed in the Schwann cell membrane, and it is considered that these factors work comprehensively to induce nerve regeneration.

  Schwann cells are known to produce many neurotrophic factors, but the main ones related to regeneration are listed below: 1) Neurotrophin family, such as nerve growth factor, brain- derived neurotrophic factor, neurotrophin-3, neurotrophin-4 / 5; 2) ciliary neurotrophic factor; 3) FGF family, eg acidic and basic fibroblast growth factor; 4) insulin family, eg insulin -like growth factor-I, II; 5) transforming growth factor-β2, β3. The neurotrophin has not only survival to nerve cells but also powerful effects such as process elongation.

  Further, the basement membrane provided by Schwann cells includes extracellular matrix components fibronectin, laminin, type IV collagen, tenascin and the like. In particular, in experiments such as culture systems, fibronectin and laminin are said to play a supporting role in nerve regeneration.

Furthermore, many cell adhesion molecules have been identified. As related to Schwann cells and nerve regeneration, 1) the immunoglobulin superfamily NCAM (neural cell adhesion molecule) and L1 are expressed especially on the Schwann cell membrane, and nerve fibers are in contact with the Schwann cell as a scaffold. When it stretches, it plays a big role as an adhesion molecule. 2) The cadherin superfamily cadherin is a calcium-dependent cell adhesion molecule, and many types have been identified. N-cadherin is particularly involved in nerve regeneration. Like NCAM and L1, which are the immunoglobulin superfamily, this also plays a major role when nerve fibers come in contact with Schwann cells and grow while recognizing. 3) Integrin Superfamily Integrins are receptors on the cell side for the aforementioned extracellular matrix. It is a heterodimeric molecule composed of two subunits, α and β. In addition, it is linked to the cytoskeleton like cadherin, and is thought to work directly in information transmission between cells. In Schwann cells, an α ₆ β ₄ subtype is expressed and plays a role in the process of remyelination during regeneration.

Attempts to regenerate the central nervous system have been attempted by many researchers over the past decades. In summary, 1) regeneration is recognized by excising part of peripheral nerves such as hands and feet and autotransplanting them into the central nerve. The purpose of this is to prepare an environment for promoting the regeneration of the central nerve by utilizing the above-described permissive effect on the axon regeneration of Schwann cells. These studies were started in 1983 by Aguago et al. In Canada (Non-patent Document 5). 2) Further, as described above, it is known that the central nerve itself has a function of suppressing nerve regeneration. It has been reported that myoline-related proteins, such as Nogo factor published by Nature in 2000 and previously known, have an inhibitory effect on axonal regeneration. Therefore, it has been reported that the central nervous system regenerates more or less if these factors are injected to neutralize these factors. 3) Regeneration can be promoted by transplanting cells. There are two types:
-A) Supplying nerve cells that have been degenerated and lost due to damage to the central nervous system, and reconstructing the neural circuit. This approach uses neural stem cells and embryonic neurons.
-B) Instead of supplying the nerve itself, transplant and reconstruct cells (glial cells) that can induce nerve regeneration ability. As such cells, Schwann cells derived from peripheral nerves, glial cells derived from central nerves into which neurotrophic factors have been introduced, ependymocytes derived from central nerves, support cells derived from odor nerves, and the like have been tried. However, each of the above methods has advantages and disadvantages, and an innovative method has not been developed yet.

  Until now, regeneration of the central nerve has been attempted by the method using Schwann cells responsible for the tissue structure of the peripheral nerve described in 3) -b) above (Non-Patent Documents 6, 7, 8 and 9). ). Schwann cells are present in peripheral nerves, but have the ability to induce regeneration, including the central nervous system as well as their own peripheral nerves, and provide effective scaffolds for regenerating fibers by transplantation to the damaged site. It has also been revealed that myelin, which is responsible for nerve jump conduction, which is an essential element for the normal functioning of nerves, can also be reconstructed by Schwann cell transplantation.

  However, there are various difficulties in collecting and culturing Schwann cells, which are relatively easy at the level of animal experiments. For example, Schwann cells are present in peripheral nerves, so hand and foot nerve fragments must be collected and isolated from them, leaving the donor in a disorder after collection. Furthermore, there is a limit to the proliferation ability of adult-derived Schwann cells, and it can be mentioned that mass culture is time consuming and difficult. In addition, neural crest cells, which are thought to differentiate into Schwann cells, can be collected only from fetal peripheral nerves. Therefore, there is a need to provide a natural Schwann cell substitute that can be used in nerve regeneration therapy and can be obtained in large quantities by culture.

  On the other hand, a polysaccharide that has been treated with a polyhydric aldehyde such as glutaraldehyde or glyoxal to form a hemiacetal bond or an acetal bond, and a substrate for nerve regeneration composed of the crosslinked polysaccharide have been proposed (Patent Literature). 1). However, the cross-linked polysaccharide cross-linked with a polyhydric aldehyde is easily hydrolyzed in its hemiacetal bond or acetal bond, and is inferior in long-term stability and hot water resistance. ) Has the disadvantage of decomposing. In addition, the hemiacetal bond or acetal bond in the cross-linked polysaccharide is hydrolyzed by indwelling over a long period of time, so that the polyvalent aldehyde is eluted and exhibits strong cytotoxicity.

  Further, a nerve regeneration material is known in which collagen is applied to and attached to a mesh tube of polyglycolic acid (PGA) (Non-patent Document 10). However, this nerve regeneration material does not show a sufficient nerve regeneration effect.

  Furthermore, attempts have been made to culture neural stem cells for the purpose of regenerating nerve tissue (Patent Document 2, Patent Document 3 and Patent Document 4). However, both relate to in vitro culture methods, and no nerve regeneration effect has been confirmed in vivo.

On the other hand, the present inventors have filed an application earlier, finding that nerve tissue is regenerated when a nerve defect part is filled with a gel of polysaccharide cross-linked with ethylenediamine or a salt thereof (Patent Document 5). In addition, when a nerve deficient material consisting of a polysaccharide gel crosslinked with ethylenediamine or a salt thereof and a nerve stem cell is filled in a nerve defect portion, the nerve tissue was found to regenerate to near normality and was filed earlier ( Patent Document 6). Furthermore, we found that nerve regeneration is promoted by transplantation of bone marrow stromal cell-derived Schwann cells to the optic nerve or sciatic nerve defect, and transplantation of bone marrow stromal cell-derived neurons to the linear body of Parkinson model rats. Filed earlier (Patent Documents 7 and 8). Although it can be said that regeneration of nerve tissue has become possible by these techniques, it is desired to construct a more rapid and more effective means for regeneration of nerve tissue.
JP-A-8-333402 Japanese National Patent Publication No. 8-502652 Japanese National Patent Publication No. 10-505754 Japanese National Patent Publication No. 10-509592 JP 2000-198738 A JP 2002-78792 A JP 2003-61647 A JP 2003-144155 A Schwab, ME et al: Rat CNS myelin and a subtype of oligodendrocytes in culture represent a nonpermissive substrate for neurite growth and fibroblast spreading.J Neurosci 8: 2381-2393, 1988. Chen, MS et al.; Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1.Nature 403: 434-439, 2000. Snow, DM et al: Sulfated proteoglycans in astroglial barriers inhibit neurite out-growth in vitro.Exp Neurobiol 109: 111-130, 1990. Bastmeyer, M et al: Similarities and differences between fish oligodendrocytes and Schwann cells in vitro.Glia 11: 300-314, 1994. Aguago, AJ et al.; A potential for axonal regeneration in neurons of the adult manmalian nervous system.In Nervous System Regeneration, B. Haber, JR Perez-Polo, GA Hashim and AMG Stella eds., Pp.327-340, Alan R. Liss, New York, 1983. Kromer, LF et al: Transplants of Schwann cell cultures promote axonal regeneration in the adult mammalian brain.Proc Natl Acad Sci USA 82: 6330-6334, 1985. Xu, XM et al: Axonal regeneration into Schwann cell-seeded guidance channels grafted into transected adult rat spinal cord.J Comp Neurol 351: 145-160, 1995. Guest, JD et al: The ability of human Schwann cell grafts to promote regeneration in the transected nude rat spinal cord.Exp Neurol 148: 502-522, 1997. Weidner, N et al: Nerve growth factor-hypersecreting Schwann cell grafts augment and guide spinal cord axonal growth and remyelinate central nervous system axons in a phenotypically appropriate manner that correlates with expressin of L1.J Comp Neurol 413: 495-506, 1999. Suzuki, K. et al., Jpn. J. Artif., Organs, 27, 490-494, 1998.

  An object of the present invention is to regenerate nerves by promoting the growth of nerve cells and nerve tissues in which damage or loss has occurred, which can exhibit excellent nerve cell proliferation promoting action and nerve axon extension action, and repairing them. Nerve regeneration material that has no fear of infection and is excellent in safety and compatibility with living body, nerve regeneration material including the material, and material for nerve regeneration capable of producing them efficiently in industrial production process And it is providing the manufacturing method of a nerve regeneration material.

As a result of intensive studies to solve the above problems, the present inventors have determined that a predetermined cell [CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) in a predetermined bioabsorbable polymer. And cells that are CD34 negative (CD34 ⁻ ) and / or cells in which differentiation of these cells is induced) are used as a nerve regeneration material, and the present invention is completed. It was.

That is, the present invention
[1] A nerve regeneration material comprising cells that can be used together with a bioabsorbable polymer, wherein the cells are (1) CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) And CD34 negative (CD34 ⁻ ) cells, and / or (2) a nerve regeneration material comprising cells obtained by inducing differentiation of the cells of (1) above,
[2] The nerve regeneration material according to [1], wherein the cells of (2) are Schwann cells or neurons.
[3] The nerve regeneration material according to [1] or [2], wherein the cell is a mammalian cell.
[4] The nerve regeneration material according to [3], wherein the mammalian cell is a human cell,
[5] The nerve regeneration material according to any one of [1] to [4], wherein the cell is a cell derived from another family or autologous.
[6] The nerve regeneration material according to any one of [1] to [5], wherein some or all of the cells are cells derived from bone marrow.
[7] A nerve regeneration material comprising a bioabsorbable polymer that can be used with cells, wherein the polymer is a polysaccharide and a salt thereof, a crosslinked polysaccharide and a salt thereof, an acidic polysaccharide ester, or a crosslinked acidic polysaccharide Ester, protein, polypeptide, polyglycolic acid, polylactic acid, glycolic acid / lactic acid copolymer, glycol / carbonate copolymer, polydioxanone, polyε-caprolactone, lactic acid / ε-caprolactone copolymer, and cyanoacrylate copolymer A nerve regeneration material comprising at least one selected from the group consisting of polymers;
[8] The nerve regeneration material according to [7] above, wherein the bioabsorbable polymer comprises a polysaccharide and / or a salt thereof,
[9] The nerve regeneration material according to [8], wherein the polysaccharide and / or salt thereof is an acidic polysaccharide and / or salt thereof,
[10] The nerve regeneration material according to [9], wherein the acidic polysaccharide and / or salt thereof is a polysaccharide having a carboxyl group and / or a salt thereof,
[11] The nerve regeneration material according to [10], wherein the polysaccharide having a carboxyl group and / or a salt thereof is at least one selected from the group consisting of alginic acid and a salt thereof, and hyaluronic acid and a salt thereof,
[12] The nerve regeneration material according to [7], wherein the bioabsorbable polymer comprises a crosslinked polysaccharide and / or a salt thereof,
[13] The polysaccharide and / or salt thereof in which the crosslinked polysaccharide and / or salt thereof has a carboxyl group is represented by the following general formula (I):

The material for nerve regeneration according to the above [12], wherein the material is covalently crosslinked with at least one crosslinking reagent selected from the group consisting of a compound represented by the formula:
[14] The nerve regeneration material according to [13], wherein the cross-linking reagent is an N-hydroxysuccinimide salt of the compound represented by the general formula (I),
[15] The N-hydroxysuccinimide salt of the compound represented by the general formula (I) is a 2N-hydroxysuccinimide salt of diaminoethane, a 2N-hydroxysuccinimide salt of diaminohexane, N, N ′. [14] which is at least one selected from the group consisting of 4N-hydroxysuccinimide salt of -di (lysyl) -diaminoethane and 3N-hydroxysuccinimide salt of N- (lysyl) -diaminohexane The nerve regeneration material described in the above,
[16] A nerve regeneration material comprising a bioabsorbable polymer that can be used with cells, wherein the polymer has an acidic polysaccharide ester and two or more α-amino groups derived from natural amino acids A nerve regeneration material comprising a polymer formed from
[17] The nerve regeneration material according to [16], wherein the acidic polysaccharide ester is propylene glycol alginate and / or propylene glycol hyaluronate,
[18] The nerve regeneration material according to [16] or [17], wherein the polyamino compound is ε-polylysine,
[19] A tube in which the bioabsorbable polymer according to any one of [7] to [11] and / or the bioabsorbable polymer according to any one of [12] to [18] is made of a bioabsorbable material. A nerve regeneration material that is filled,
[20] A nerve regeneration material comprising a cell and a bioabsorbable polymer, wherein the cell comprises the cell according to any one of [1] to [6], and the bioabsorbable polymer is the above [7]. ] To [18] a nerve regeneration material comprising the bioabsorbable polymer according to any one of
[21] The nerve regeneration material according to [20], wherein cells and a bioabsorbable polymer are filled in a tube made of a bioabsorbable material.
[22] (1) Step of collecting cells from biological material, and (2) CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ ) from the obtained cells. A step of sorting the cells
A method for producing cells for nerve regeneration material that can be used with a bioabsorbable polymer,
[23] (2 ′) the production method of the above-mentioned [22], further comprising a step of inducing differentiation of the sorted cells,
[24] The method according to [22] or [23], wherein the cells are collected from bone marrow of the living body in step (1),
[25] (1) A step of collecting cells from biological material,
(2) a step of sorting cells that are CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ ) from the obtained cells, and (3) sorting Cells, polysaccharides and salts thereof, crosslinked polysaccharides and salts thereof, acidic polysaccharide esters, crosslinked acidic polysaccharide esters, proteins, polypeptides, polyglycolic acid, polylactic acid, glycolic acid / lactic acid copolymer, glycol A bioabsorbable polymer comprising at least one selected from the group consisting of: / carbonate copolymer, polydioxanone, polyε-caprolactone, lactic acid / ε-caprolactone copolymer, and cyanoacrylate copolymer; A step of combining a saccharide ester and a bioabsorbable polymer formed from a polyamino compound having two or more α-amino groups derived from natural amino acids. The a, a manufacturing method of nerve regeneration material,
[26] The method according to [25] above, further comprising (2 ′) a step of inducing differentiation of the sorted cells between steps (2) and (3), and [27] in step (1) The method according to [25] or [26] above, wherein cells are collected from bone marrow of a living body,
About.

  According to the present invention, it is easy to collect as compared with the conventional one, and there is substantially no damage to the cell collection target (for example, human), and the proliferation rate is high, and the supply is quick in large quantities. Provided is a nerve regeneration material comprising the predetermined cell that can be used. The cells can be autotransplanted, which is a great advantage. Therefore, if the cells are collected from a certain individual, differentiation induction is performed as desired, the material can be produced using the obtained cells, and if the material is transplanted into the impaired nerve of the same individual, no rejection occurs. Therefore, administration of an immunosuppressant or the like is unnecessary, and stable regeneration of nerves can be achieved by using the material together with a predetermined bioabsorbable polymer. Since the raw material cells of the cells can also be obtained from the bone marrow bank, realistic measures can be taken from the supply side.

  The present invention also provides a nerve regeneration material comprising a predetermined bioabsorbable polymer that can be used with the cells. The polymer is excellent in safety and compatibility with a living body, and stable nerve regeneration is possible by using the material in combination with the predetermined cells.

  Furthermore, according to the nerve regeneration material including the material for nerve regeneration of the present invention, nerves can be regenerated safely and satisfactorily. Therefore, the nerve regeneration material is used for diseases of the central nervous system and peripheral nervous system due to damage or defect of nerve cells and nerve tissues, for example, peripheral nervous system diseases such as peripheral neuropathy, peripheral neuropathy and localized neuropathy, Alzheimer's , Parkinsonism, Huntington's disease, amyotrophic lateral sclerosis, treatment of diseases such as Shy-Drager syndrome, traumatic diseases such as cerebrovascular diseases such as spinal diseases, head trauma, and stroke It can be used effectively for the treatment. Further, it can be suitably used as a nerve regeneration material used for surgery for nerve tissue repair.

  Moreover, according to this invention, the nerve regeneration material and nerve regeneration material which have the outstanding performance as above-mentioned can be produced efficiently.

  In the present specification, the term “material for nerve regeneration” is used to mean a material for healing, adhesion, reinforcement and / or regeneration of a nerve that has been damaged such as damage or defect (material for nerve regeneration material). The nerve regeneration material of the present invention includes a cell-containing material and a bioabsorbable polymer. Such materials are not used individually for nerve regeneration, but are used simultaneously or sequentially. Used. The form to be used at the same time includes the use of the term “nerve regeneration material” in which cells and a bioabsorbable polymer are combined and integrated.

The term “natural Schwann cell” is used to mean Schwann cells collected from peripheral nerves present in the body of an organism. The cells are S-100 ⁺ (calcium binding protein positive).

The term “natural nerve cell” is used to mean a nerve cell collected from a peripheral nerve or a central nerve existing in the body of an organism. The cell is neurofilament positive ( ⁺ ).

The term “cell-derived Schwann cells that are CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ )” is (i) morphologically very similar to natural Schwann cells. (Ii) P75 (nerve growth factor (NGF) receptor, nerve growth factor receptor, low affinity), S-100, GFAP by immunostaining. (Gial fibrillary acidic protein, a type of intermediate filament), Nestin (a type of intermediate filament), and O4 (a marker of cells that produce myelin such as Schwann cells and oligodendrocytes), similar to natural Schwann cells And (iii) have the same traits as natural Schwann cells with respect to their ability to regenerate nerves, but can be distinguished from native Schwann cells due to differences in differentiation induction history. It is used in the sense of emission cells.

The term “cell-derived neurons that are CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ )” refers to (i) morphologically cell bodies and neurons. (Ii) biochemically positive for MAP-2, neurofilament, beta3-tubulin, and Tuj1, exhibiting similar responses to natural neurons, and (iii) Although it has the same traits as neurons in terms of the ability to regenerate nerves, it is used in the sense of neurons that can be distinguished from natural neurons due to differences in differentiation induction history.

In the present specification, “cells that are CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ )” may be collectively referred to as the cells of the present invention. is there. Further, accompanying this, the “cell-derived Schwann cells that are CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ )” and the cell-derived Schwann cells of the present invention, “Cell-derived nerve cells that are CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ )” may be referred to as the cell-derived nerve cells of the present invention.

  The nerve regeneration material of the present invention is composed of a bioabsorbable polymer, or a cell of the present invention and / or a cell obtained by inducing differentiation of the cell of the present invention, and these are used in combination for nerve regeneration. In the material for nerve regeneration of the present invention, the bioabsorbable polymer and the cell of the present invention and / or the cell in which differentiation of the cell of the present invention is induced do not necessarily have to be combined from the beginning. In the nerve regeneration material of the present invention, the bioabsorbable polymer and the cell of the present invention and / or the cell obtained by inducing differentiation of the cell of the present invention may be combined or separated. In using the nerve regeneration material of the present invention, the bioabsorbable polymer and the cells of the present invention and / or the cells induced to differentiate the cells of the present invention are combined and applied to a treatment site where nerve regeneration is required. Or the bioabsorbable polymer and the cell of the present invention and / or the cell of which differentiation of the cell of the present invention is induced to be induced separately, and / or a treatment site where nerve regeneration is required and / or It may be applied to other locations (for example, cerebrospinal fluid) simultaneously or sequentially. When applied sequentially, the bioabsorbable polymer is first applied to the treatment site where nerve regeneration is required, and then the cell of the present invention and / or the cell in which differentiation of the cell of the present invention is induced, or The cell of the present invention and / or the cell obtained by inducing differentiation of the cell of the present invention may be applied first, then the bioabsorbable polymer may be applied, and the time interval for applying both may be short, or A certain period (for example, about 7 to 30 days) may be provided.

Here, the combination of the bioabsorbable polymer and the cell of the present invention and / or the cell of which differentiation of the cell of the present invention is induced means that the cell of the present invention and / or the cell of the present invention is induced to differentiate. When the cells and the bioabsorbable polymer are combined, for example, when the cells and the bioabsorbable polymer are mixed, when the cell is supported by the bioabsorbable polymer, the cell is in the bioabsorbable polymer. The bioabsorbable polymer and the main body, such as when the cell is contained in a fabric or other shape made of the bioabsorbable polymer, or when the cell is impregnated with the bioabsorbable polymer. The cells of the invention and / or the cells obtained by inducing differentiation of the cells of the present invention are in contact with each other. In addition, the state in which the bioabsorbable polymer and the cell of the present invention and / or the cell of which the cell of the present invention has been induced to separate are in a state of being separated from each other and not in contact with each other. Say.

  The present invention provides a nerve regeneration material (hereinafter sometimes referred to as a cell material) comprising cells that can be used with a bioabsorbable polymer, and the cells are cells of the present invention and / or cells of the present invention. It consists of cells induced to differentiate.

  The method for obtaining and obtaining the cells of the present invention is not particularly limited, and such cells can be collected from biological materials such as bone marrow, peripheral blood, umbilical cord blood, muscle tissue, etc. in vivo, but mammals including humans. In order to obtain the cells from the above, a method of collecting from the bone marrow is preferably used. Although it can be collected from anywhere within the bone marrow of the body, it is more preferably collected from the bone marrow and / or periosteum of the pelvis (iliac bone) and long bones of the limbs (femur, tibia). In addition, it may be collected from the bone marrow of the alveolar bone, the palate or the periosteum of the alveolar bone.

  When the cells of the present invention are collected from bone marrow, bone marrow stromal cells are first separated from the bone marrow according to a known method. When cells are collected from a site other than the bone marrow, a group of cells to be used for sorting the cells of the present invention described later is separated according to the case of separating bone marrow stromal cells.

  Thus, the cell of this invention is fractionated from the cell group derived from the tissue etc. used as the origin. This is due to the following reason. That is, it has been conventionally known that nerve regeneration is caused by bone marrow stromal cell-derived Schwann cells and the like. However, in nerve regeneration by cells derived from bone marrow stromal cells, the balance of the cells present is important, whether the nerve regeneration ability is concentrated on specific cells, or there are cells / components to be removed The key to nerve regeneration, such as whether to do so, was unclear. The present inventors have found for the first time that it is important for nerve regeneration to substantially remove cells other than the predetermined cells of the present invention. Further, the present inventors have found that cells derived from any tissue or the like can function effectively as long as they correspond to the cells of the present invention. Therefore, it is necessary to sort cells as described above.

The cell surface marker of the present invention is CD29 ⁺ , CD90 ⁺ , CD11 ⁻ , CD34 ⁻ from a cell group isolated from a tissue or the like as described above, for example, by the following cell sorting method. Obtained by sorting cells.

  The cell sorting method is not particularly limited, but is preferably performed by flow cytometry, and most preferably sorted by a fluorescence-activated cell sorter (FACS) manufactured by Becton-Dickinson with the name FACScan or FACScalibur. By this technique, for example, cells having a CD29 marker on a cell are labeled with a specific fluorescent dye by an anti-CD29 antibody bound to the specific fluorescent dye or the like. Similarly, cellular CD90 markers are labeled with different fluorescent dyes by anti-CD90 antibodies conjugated to another dye. The stained cells are applied to an analyzer and irradiated with an argon laser beam that excites fluorescent dyes in the flowing cells and emits light. This luminescence is detected by a photomultiplier tube (PMT) of a fluorescent dye specific to the emission wavelength by a set of optical filters. The signal detected by the PMT is amplified by its own channel and is represented by the computer in a variety of different forms, such as a columnar graph, a stippled display or a contour display. That is, fluorescent cells emitting at one wavelength react with a specific fluorescent dye-labeling reagent, but non-fluorescent cells or fluorescent cells emitting at different wavelengths do not express this molecule and fluoresce at other wavelengths. Expresses a molecule that reacts to the fluorescent dye-labeling reagent that emits. This flow cytometry is also semi-quantitative in that it shows the amount of fluorescence (fluorescence intensity) displayed by the cells. This is relative to the number of molecules expressed by the cell.

  Flow cytometry also has the ability to measure non-fluorescent parameters such as molecular size and light scattered by cells upon irradiation with a laser beam. Cell size is usually measured directly. Light scattering PMTs detect light scattering by cells in either forward (forward scatter; FSC) or right angle (side scatter; SSC). Both parameters are affected by other factors. Note that FSC is an indicator of cell size, while SSC is an indicator of cell complexity.

  The flow cytometry preferably comprises one or more types of PMT emission detectors. The additional PMT can detect other emission wavelengths and simultaneously detect one or more fluorescent dyes in separate channels. The computer allows the analysis of each channel or the correlation between each parameter and another parameter. Fluorescent dyes typically used in FACS include fluorescein isocyanate (FITC) having an emission peak at 525 nm (green), R-phycoerythrin (PE) having an emission peak at 575 nm (orange red), and 620 nm (red). Propidium iodide (PI) having an emission peak at 660, 7-aminoacthiomycin D (7-AAD) having an emission peak at 660 nm (green), and R-phycoerythrin Cy5 having an emission peak at 670 nm (red) PE-Cy5), allophycocyanin (APC) having an emission peak at 655-750 nm (dark red) is included.

Among the cell materials of the present invention, the cells of the present invention consist of cells that are CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ ). It is not excluded that cells other than those cells, that is, cells other than those having surface markers CD29 and CD90 and not having CD11 and CD34 are included. For example, the ratio of the number of cells of the present invention contained in all cells in the cell material can be selected depending on the type and mode of the neurological disease. The ratio is preferably 95% or more, more preferably 99% or more, and particularly preferably 99.5% or more from the viewpoint of obtaining an excellent nerve regeneration effect.

  Further, as the cells of the present invention, those obtained by dedifferentiating stem cells derived from other organs (hematopoietic system, liver, intestinal tract, skeletal muscle, mammary gland, skin) and differentiated into the cells of the present invention can also be used. it can. For example, those obtained by differentiating totipotent human-derived ES cells (embryonic stem cells) can be used. Such dedifferentiation and differentiation induction can be performed according to a known method.

  As a cell in which differentiation of the cell of the present invention is induced (hereinafter sometimes referred to as differentiation-inducing cell), Schwann cell or nerve cell is preferable. The differentiation-inducing cell can be suitably obtained by inducing differentiation after the proliferation of the cell of the present invention having the self-replicating ability capable of proliferating and repeating passage.

  The method for inducing differentiation is not particularly limited. For example, as a method for obtaining the cell-derived Schwann cell of the present invention, the cell of the present invention is cultured in a medium obtained by adding serum to a standard basal medium; A differentiation-inducing agent is added to the medium and further cultured; and a cyclic AMP-elevating agent or cyclic AMP analog, and / or glial cell differentiation survival agent A differentiation induction method (see Japanese Patent Application Laid-Open No. 2003-61647) is preferably used in which the above-mentioned medium is added and further cultured to obtain the cell-derived Schwann cells of the present invention. For example, as a method for obtaining the cell-derived nerve cell of the present invention, the cell of the present invention is cultured in a medium obtained by adding serum to a standard basal medium; a Notch gene and / or a Notch signaling-related gene is introduced. And further culturing; and adding a cyclic AMP-elevating drug or cyclic AMP analog and / or a cell differentiation survival agent to the medium, and further culturing the differentiation-inducing method for obtaining nerve cells (specifically, No. 2003-144155) is preferably used.

  As the differentiation-inducing cells of the present invention, it is not necessary that all cells exhibit a single differentiated cell reaction or trait. For example, the ratio of the number of cells showing the response or trait of a single differentiated cell in the differentiation-inducing cell of the present invention can be arbitrarily selected depending on the type and mode of the neurological disease. The ratio is preferably 40% or more, more preferably 60% or more, and particularly preferably 80% or more from the viewpoint of obtaining an excellent nerve regeneration effect.

  As the cell of the present invention and / or the cell obtained by inducing differentiation of the cell of the present invention, any cell can be used as long as it can proliferate and differentiate in a nerve defect part. Those derived from mammals (mammals such as humans, monkeys, chimpanzees, and sheep) are preferred, and those derived from humans are more preferred. The cell may be a cell derived from another family (living donor) or an autologous cell, preferably derived from another family, and more preferably derived from the family. The other house is an individual different from the individual to which the nerve regeneration material of the present invention is applied, and the own family is the same as the individual to which the nerve regeneration material of the present invention is applied. It means that it is an individual.

  In addition, the cells of the present invention and / or the cells obtained by differentiating the cells of the present invention are preferably derived from bone marrow, and some or all of the cells may be cells derived from bone marrow. Note that some of the cells of the present invention and / or the cells in which differentiation of the cells of the present invention is induced are not derived from bone marrow, and partially include cells derived from bone marrow. Means that.

The concentration of the cell of the present invention and / or the differentiation-inducing cell of the present invention in the cell material of the present invention is not particularly limited, but is preferably 10 ³ to 10 ¹⁰ cells / ml, more preferably 10 ⁵ to 10. ⁹ cells / ml. The mixing ratio of both cells when using both the cell of the present invention and the differentiation-inducing cell of the present invention is not particularly limited. In addition to the cells of the present invention and / or the differentiation-inducing cells of the present invention, the cell material of the present invention includes other components such as water, salts, growth factors, antibiotics, polysaccharides, proteins, peptides, lipids, Amino acids, vitamins, (heavy) metal ions, and the like may be included. The cell material of the present invention may be the cell of the present invention and / or the differentiation-inducing cell itself obtained as described above. However, when the other component is further contained, the cell of the present invention and / or Alternatively, it can be prepared by obtaining differentiation-inducing cells and appropriately mixing them with other desired components.

As described above, the cell material of the present invention can be obtained.
(1) Step of collecting cells from biological material, and (2) CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ ) from the obtained cells. A process of sorting cells,
There is also provided a method for producing a cell for nerve regeneration material that can be used with a bioabsorbable polymer. The production method may further include (2 ′) a step of inducing differentiation of the sorted cells. The collection of cells in step (1) is preferably performed from bone marrow of the living body. In the present specification, the biological material may be derived from a living body or a non-living body. In addition to the above-described materials, for example, subcutaneous tissue, hair follicle tissue, liver, pancreas, Small intestine, large intestine, (soft) bone tissue, adipose tissue and the like may be used.

  The present invention also provides a nerve regeneration material comprising a bioabsorbable polymer that can be used with cells, the polymer comprising (a) a polysaccharide and its salt, a cross-linked polysaccharide and its salt, and an acidic polysaccharide. Ester, cross-linked acidic polysaccharide ester, protein, polypeptide, polyglycolic acid, polylactic acid, glycolic acid / lactic acid copolymer, glycol / carbonate copolymer, polydioxanone, polyε-caprolactone, lactic acid / ε-caprolactone copolymer , And at least one selected from the group consisting of cyanoacrylate copolymers, or (b) a polymer formed from an acidic polysaccharide ester and a polyamino compound having two or more α-amino groups derived from natural amino acids Consists of. Hereinafter, the nerve regeneration material comprising the bioabsorbable polymer (a) is referred to as polymer material a, and the nerve regeneration material comprising (b) is referred to as polymer material b. In addition, the polymer of (a) and the polymer of (b) can also be mixed and used.

  The bioabsorbable polymer constituting the material for nerve regeneration of the present invention is composed of the above-mentioned polymers, but these polymers are safe for the living body and can be decomposed and absorbed in the living body. It can be decomposed and absorbed in the body along with the regeneration of nerve tissue and disappear when nerve regeneration is completed. In addition, it has high biological safety and is safe to be implanted in the body.

  Examples of polysaccharides and salts thereof, crosslinked polysaccharides and salts thereof, and acidic polysaccharide esters that are bioabsorbable polymers constituting the polymer material a include alginic acid, crosslinked alginic acid, chitin, hyaluronic acid, crosslinked hyaluronic acid, cellulose, Examples thereof include starch, cross-linked starch, pectin, carboxymethylcellulose, carboxymethyldextran, and derivatives (eg, esters) thereof. Examples of proteins include gelatin, cross-linked gelatin, collagen, casein, fibrin, and albumin. Polypeptide usually refers to a peptide in which 10 to 100 amino acids are linked by peptide bonds, and examples thereof include polyaspartic acid, polyglutamic acid, and polylysine. The molecular weight of the polysaccharide is usually about 50,000 to 5,000,000 Da.

  The bioabsorbable polymer constituting the polymer material a is preferably a polysaccharide and / or a salt thereof, more preferably an acidic polysaccharide and / or a salt thereof, alginic acid, hyaluronic acid, pectin, carboxymethylcellulose. Polysaccharides having a carboxyl group such as carboxymethyl dextran and / or salts thereof are more preferably used, and at least one selected from the group consisting of alginic acid, hyaluronic acid and salts thereof is more preferably used. Among these, a water-soluble salt of alginic acid and / or a water-soluble salt of hyaluronic acid is particularly preferably used from the viewpoint of high safety for living bodies. As a water-soluble salt of a polysaccharide having a carboxyl group, for example, a water-soluble salt of alginic acid or a water-soluble salt of hyaluronic acid, sodium salt, potassium salt, ammonium salt and the like are preferably used, and sodium salt is more preferably used. . As a water-soluble salt of a polysaccharide having a carboxyl group, the viscosity at 20 ° C. of the aqueous solution is 100 centipoise when the aqueous solution has a concentration of 1% by weight from the viewpoint of ensuring good strength of the resulting polymer material a. Those having (cp) or more are preferably used, and those having 300 cp or more are more preferably used. The viscosity can be measured with a BL type viscometer.

  In addition, as the bioabsorbable polymer constituting the polymer material a, a polysaccharide and / or a salt thereof obtained by crosslinking a polysaccharide and / or a salt thereof is also preferably used. And / or a cross-linked polysaccharide obtained by covalently cross-linking a salt thereof with at least one cross-linkable reagent selected from the group consisting of the compound represented by the above general formula (I) and a salt thereof and / or The salt is more preferably used.

  The crosslinking reagent may be any amine compound and / or salt thereof included in the compound represented by the above general formula (I) (hereinafter referred to as amine compound (I)). As amine compound (I) and / or its salt, the compound and / or its salt whose n is 2-8 in said general formula (I) are used preferably.

  Specific examples of the amine compound (I) include diaminoethanes such as diaminoethane, diaminopropane, diaminobutane, diaminopentane, diaminohexane, diaminoheptane, diaminooctane, diaminononane, diaminodecane, diaminododecane, and diaminooctadecane. Or N- (lysyl) -diaminoethane, N, N′-di (lysyl) -diaminoethane, N- (lysyl) -diaminohexane, N, N′-di (lysyl) -diaminohexane or the like Examples thereof include di (lysyl) diaminoalkanes and salts thereof.

  When the crosslinkable reagent is composed of a salt of amine compound (I), N-hydroxysuccinimide is preferably used as a component for forming the salt. Particularly suitable N-hydroxysuccinimide salts of amine compound (I) include 2N-hydroxysuccinimide salt of diaminoethane, 2N-hydroxysuccinimide salt of diaminohexane, N, N′-di (lysyl). ) -Diaminoethane 4N-hydroxysuccinimide salt and at least one selected from the group consisting of N- (lysyl) -diaminohexane 3N-hydroxysuccinimide salt. These compounds have higher safety, biocompatibility, and the like, and a crosslinked polysaccharide obtained by using these compounds as a crosslinkable reagent can exhibit a more excellent nerve regeneration action.

  A polysaccharide in which a polysaccharide having a carboxyl group and / or a salt thereof is covalently crosslinked with a crosslinkable reagent comprising an amine compound (I) and / or a salt thereof generally exhibits a gel-like shape (from this point, a covalent bond). Cross-linked polysaccharides may be referred to as “cross-linked polysaccharide gels”). The covalent crosslinking rate of the polysaccharide having a carboxyl group and / or its salt by the crosslinking reagent (reaction rate of the crosslinking agent to the polysaccharide having a carboxyl group and / or its salt) is Or it can control by the use molar ratio of the crosslinkable reagent with respect to the salt. Lowering the molar ratio of the crosslinkable reagent yields a soft and highly water-crosslinked cross-linked polysaccharide gel, while increasing the crosslinkable reagent molar ratio results in a strong and low moisture content. A crosslinked crosslinked polysaccharide gel is obtained.

  The covalent crosslinking rate is appropriately selected as desired. However, if the covalent crosslinking rate is too low, the strength of the crosslinked polysaccharide gel is lowered, and if a large amount of crosslinking reagent is used to increase the covalent crosslinking rate, the crosslinking reagent is not used. Since there is a possibility that it remains in the crosslinked polysaccharide gel as it is reacted, as a crosslinking rate, 1 to 50 mol% of the carboxyl groups possessed by the polysaccharide having a carboxyl group and / or its salt are crosslinkable. It is preferable to react with a reagent, and it is more preferable that 5-30 mol% of carboxyl groups are reacting with a crosslinkable reagent. The crosslinking rate can be determined by analysis (NMR method) using a nuclear magnetic resonance apparatus.

  The covalent cross-linking reaction between the polysaccharide having a carboxyl group and / or a salt thereof and a cross-linking reagent comprising the amine compound (I) and / or a salt thereof may be performed using a dehydration condensing agent such as a water-soluble carbodiimide. it can.

  The covalent cross-linking reaction of a polysaccharide having a carboxyl group and / or a salt thereof with a crosslinkable reagent comprising an amine compound (I) and / or a salt thereof is generally carried out by using a polysaccharide having a carboxyl group and / or a salt thereof in water. Dissolve to prepare an aqueous solution having a concentration of about 0.1 to 2% by weight and a viscosity (20 ° C.) of about 100 to 1200 cp, to which a crosslinkable reagent comprising amine compound (I) and / or a salt thereof and dehydration are prepared. The condensing agent is preferably mixed uniformly in such an amount that a predetermined covalent bond crosslinking rate as described above can be obtained, and reacted at a temperature of 4 to 50 ° C. for about 1 to 100 hours.

  The cross-linked polysaccharide gel obtained by the above shows practical strength and stability by itself, but if desired, ion-bonded cross-links and hydrophobic bond cross-links together with covalent cross-links with amine compounds (I) and / or their salts. Other crosslinks such as may be applied.

  Further, as the acidic polysaccharide ester used for the bioabsorbable polymer constituting the polymer material b, a polysaccharide having a carboxyl group in which two or more carboxyl groups are esterified with alcohols is preferably used. It is done. Specific examples of the above-mentioned acidic polysaccharide having a carboxyl group that forms the base of the acidic polysaccharide ester include carboxyl group-containing polysaccharides such as alginic acid, xanthan gum, gellan gum, and hyaluronic acid, and those artificially acceptable physiologically. Carboxyl groups were artificially introduced into polysaccharides that did not originally contain carboxyl groups, such as derivatives, carboxymethylated cellulose, carboxymethylated dextran, carboxymethylated pullulan, partially succinylated chitosan, carboxymethylated chitosan, and carboxymethylated chitin. The thing etc. can be mentioned.

  Suitable examples of the alcohols in which acidic groups, particularly carboxyl groups in the acidic polysaccharide are esterified include aliphatic alcohols, aromatic alcohols, cycloaliphatic alcohols, and heterocyclic alcohols. Among these, aliphatic monovalent or polyhydric alcohols having 1 to 16 carbon atoms are preferable, and specific examples include methanol, ethanol, propanol, ethylene glycol, propylene glycol, glycerin and the like. In the case of a polyhydric alcohol, it is preferable that only one of the plurality of hydroxyl groups is ester-linked (—COO—) to the carboxyl group in the carboxyl group-containing acidic polysaccharide.

  The type of the acidic polysaccharide ester is not particularly limited, and any acidic polysaccharide ester that can be formed between the above acidic polysaccharide and the above alcohol may be used. For example, alginic acid or hyaluronic acid may be used as the acidic polysaccharide. For example, propylene glycol ester of alginic acid, ethylene glycol ester, trimethylene glycol ester, butylene glycol ester, pentylene glycol ester, propylene glycol ester of hyaluronic acid, ethylene glycol ester, trimethylene glycol ester, butylene glycol ester, Examples include pentylene glycol ester. These can be used alone or in admixture of two or more. Among them, propylene glycol ester of alginic acid and / or propylene glycol ester of hyaluronic acid is preferably used because of high biological safety.

  The production method of the acidic polysaccharide ester is not particularly limited. For example, the method described in JP-A-52-36177, edited by the Chemical Society of Japan, “Experimental Chemistry Course 22 Organic Synthesis IV-Acid / Amino Acid / Peptide”, No. 4th edition, pages 24 to 83 (1992, issued by Maruzen). Yalpani, “Tetrahedron” (M. Yalpani, Tetrahedron), Vol. 41, page 2957 (1985). Among them, as described in JP-A-52-36177, 1,2-epoxides such as ethylene oxide and propylene oxide, and 1,3-epoxides such as trimethylene oxide have an acidic polysaccharide having a carboxyl group. The method of reacting with is preferably employed.

  On the other hand, the “polyamino compound having two or more α-amino groups derived from natural amino acids” used for the bioabsorbable polymer constituting the polymer material b is an amino acid found naturally (alanine, glycine, phenylalanine, serine). , Valine, lysine, glutamic acid, etc.) and a compound having two or more primary amino groups derived from the α-amino group. The polyamino compound is preferably a water-soluble polymer having two or more α-amino groups. In addition, the molecular weight of a polyamino compound is about 500-10000 Da normally. Further, the upper limit of the number of α-amino groups in the polyamino compound is usually about 100.

  The polyamino compound may be any compound as long as it has two or more α-amino groups derived from natural amino acids. Among them, polyamino acids that are polymers of natural amino acids, polyamino acids modified with amino acids having α-amino groups, and the like. Saccharides and the like are preferably used, and these polyamino compounds can be produced by introducing natural amino acid derivatives, whose functional groups are optimally protected, into polysaccharides, polyamino acids and the like by chemical modification and then deprotecting them. In the present invention, ε-polylysine is particularly preferably used as the polyamino compound. As shown in the following chemical formula, ε-polylysine is a water-soluble polymer in which an ε-position amino group and an α-position carboxyl group are condensed by an amide bond, and the side chain has α-amino of lysine. A group is present.

  The bioabsorbable polymer constituting the polymer material b of the present invention can be produced, for example, according to the method described in JP-A-2001-278984 using the acidic polysaccharide ester and the polyamino compound.

  The bioabsorbable polymer used in the nerve regeneration material of the present invention may be in a state swollen with water (hydrous gel), in a dry state before being swollen with water (hydrous gel), or with water. However, it may not be completely swollen but may contain some water.

  In addition, the form of the bioabsorbable polymer is not particularly limited, for example, sponge-like, film-like, sheet-like, mat-like, non-woven-like, woven-like, knitted-like, net-like, fibrous, pellet-like, small lump-like, It can be in any form such as large block, powder, particle, tubular, and linear.

The content of the bioabsorbable polymer in each of the polymer materials a and b of the present invention is not particularly limited, but is preferably 1 to 50% by weight, more preferably 5 to 20% by weight. In addition to the bioabsorbable polymer, the polymer materials a and b of the present invention include, as other components, for example, moisture content control, elastic modulus / strength adjustment, degradation rate control, cell adhesion, Hydrophilic properties such as inorganic ions such as Na ⁺ , Ca ⁺⁺ , Mg ⁺⁺ , ethylene glycol, propylene glycol, glycerin, polyethylene glycol, polyvinyl alcohol, polyacrylic acid, etc. for the purpose of maintaining or changing the differentiation state of It is possible to add an additive such as a polymer compound. Further, any pharmacologically acceptable drug can be added alone or in a plurality of types. Moreover, it is also possible to fix | immobilize these chemical | medical agents on a bioabsorbable polymer individually or in multiple types by chemical reaction. Examples of drugs that can be used include disinfectants, antibiotics, antibacterial agents, actinosin, blood circulation improvers such as PEG1, TGFβ, PDGF, b-FGF, platelet-derived growth factor (PDNF-AA), ciliary neurotrophic Anti-factors such as growth factors such as factor (CNTF), glial-derived neurotrophic factor (GDNF), nerve growth factor (NGF), heregulin, enzyme inhibitors such as urinastatin and TIMP, steroids and non-steroidal anti-inflammatory agents Inflammatory agents, structural proteins such as fibrin and collagen, medium such as Eagle's alpha-modified minimum essential medium, serum such as fetal bovine serum, SH reagent such as β-mercaptoethanol, cyclic AMP elevating activity such as forskolin And a demethylating agent such as an agent (or cyclic AMP analog) and 5-azacytidine.

  The above-described bioabsorbable polymer can be used as it is as the polymer materials a and b of the present invention. However, when the other components are further included, for example, a bioabsorbable polymer is obtained and the desired one is obtained. It can be prepared by appropriately mixing other components.

  Further, as one aspect of the present invention, a bioabsorbable polymer constituting the polymer material a of the present invention and / or a bioabsorbable polymer constituting the polymer material b is filled in a tube made of a bioabsorbable material. A nerve regeneration material excellent in surgical operability is provided. Such a nerve regeneration material can be produced in accordance with a nerve regeneration material containing a bioabsorbable polymer and a cell of the present invention and / or a cell in which differentiation of the cell of the present invention is induced, which will be described later. The mixing ratio of the bioabsorbable polymer constituting the polymer material a and the bioabsorbable polymer constituting the polymer material b is not particularly limited.

  The present invention is also a nerve regeneration material comprising a cell and a bioabsorbable polymer, wherein the cell comprises the cell of the present invention and / or a cell obtained by inducing differentiation of the cell of the present invention. Provided is a nerve regeneration material in which a polymer is composed of a bioabsorbable polymer constituting the polymer material a of the present invention and / or a bioabsorbable polymer constituting the polymer material b of the present invention. Such a nerve regeneration material is a combination of cells and bioabsorbable polymer used in the above-described nerve regeneration material, and can be directly transplanted to a portion requiring regeneration of nerve tissue, etc. It can be conveniently used with good operability in surgery and the like. Furthermore, in consideration of handling properties and storage stability, a nerve regeneration material in which cells and a bioabsorbable polymer are filled in a tube made of a bioabsorbable material is preferable.

The mixing ratio of the bioabsorbable polymer in the nerve regeneration material of the present invention and the cell of the present invention and / or the cell obtained by inducing differentiation of the cell of the present invention is not particularly limited, and the situation of the biologically damaged part to which the nerve regeneration material is applied Can be adjusted according to the type of damaged nerve, etc. Usually, preferably 1 × 10 ³ to 1 × 10 ⁹ cells, more preferably 1 ×, of the cells of the present invention and / or the cells induced to differentiate the cells of the present invention with respect to 1 mg of the dry weight of the bioabsorbable polymer. It is desirable to mix them at a ratio of 10 ⁴ to 1 × 10 ⁸ from the viewpoint of exerting good nerve regeneration performance.

  As the tube for forming the nerve regeneration material of the present invention, a tube made of a bioabsorbable material and a physiologically acceptable material is preferably used. Specific examples of materials that can be used to form the tube include alginic acid, crosslinked alginic acid, chitin, chitosan, hyaluronic acid, crosslinked hyaluronic acid, cellulose, starch, crosslinked starch, and derivatives thereof; gelatin, crosslinked gelatin, Proteins such as collagen, casein, fibrin and albumin; polypeptides such as polyaspartic acid, polyglutamic acid and polylysine; polyglycolic acid, polylactic acid, glycolic acid / lactic acid copolymer, glycolic acid / carbonate copolymer, polyε- Examples include synthetic polymer materials such as caprolactone, lactic acid / ε-caprolactone copolymer, polydioxanone, and cyanoacrylate polymers; inorganic materials such as hydroxyapatite, tricalcium phosphate, and calcium carbonate. Among them, a tube made of a synthetic polymer material such as polyglycolic acid, polylactic acid, glycolic acid / lactic acid copolymer, polyε-caprolactone, lactic acid / ε-caprolactone copolymer has stability, flexibility, transparency, It is preferably used because it is excellent in terms of heat resistance, moist heat resistance and strength.

  Such a tube is preferably open at both ends. Further, the form of the tube is not particularly limited, and for example, a non-woven fabric, a woven fabric, a knitted fabric, a felt, a net, a film, a sheet, a mat, a sponge, etc. made of the above-described substances that are bioabsorbable and physiologically acceptable. And the like, and those obtained by directly forming a porous or non-porous tube by hollow spinning or tubular extrusion, etc. it can. The inner diameter, thickness, length, etc. of the tube are not particularly limited, and the use of the nerve regeneration material comprising the tube filled with the bioabsorbable polymer and cells used in the present invention, the state of the affected part where it is used Various adjustments can be made according to the method of surgery. The inner diameter of the tube is usually in the range of 0.5 to 20 mm, preferably in the range of 1 to 10 mm from the viewpoint of ease of use in surgery and the like, and preferably 2 to 5 mm. More preferably within the range. The thickness of the tube is preferably within a range of 0.1 to 1 mm, and more preferably within a range of 0.1 to 0.5 mm.

  The nerve regeneration material of the present invention can be preferably produced using the nerve regeneration material of the present invention. Filling the tube with cells and a bioabsorbable polymer can be appropriately performed by a known method.

Although the nerve regeneration material of the present invention is obtained as described above, the present invention includes, as one aspect thereof,
(1) a step of collecting cells from biological material;
(2) a step of sorting cells that are CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ ) from the obtained cells, and (3) sorting There is also provided a method for producing a nerve regeneration material, comprising the step of combining the prepared cells and the bioabsorbable polymer used in the present invention. The production method may further include (2 ′) a step of inducing differentiation of the sorted cells between steps (2) and (3). The collection of cells in step (1) is preferably performed from bone marrow of the living body.

  In the material for nerve regeneration of the present invention, the bioabsorbable polymer and the cell of the present invention and / or the storage form of the cell in which differentiation of the cell of the present invention is induced and the form when applied to a living body are not particularly limited. As described above, the cells of the present invention and / or the cells obtained by inducing differentiation of the cells of the present invention are mixed with the bioabsorbable polymer, or the cells are supported, embedded, included, impregnated in the bioabsorbable polymer, etc. Then, they may be stored together or applied to the affected area, or those that existed in a separated state before being applied to the treatment site of the living body may be used together immediately before application. In addition, the bioabsorbable polymer and the cells of the present invention and / or cells that have undergone differentiation induction of the cells of the present invention are not combined, and the affected area and / or other places (for example, cerebrospinal fluid) that need to be individually regenerated. Or a bioabsorbable polymer and one of the cells of the present invention and / or a cell in which differentiation of the cell of the present invention is induced to be applied to the affected area, and then the other after the predetermined period. A sequential method applied to the same affected area and / or other places may be adopted. In the case of adopting the sequential method, the bioabsorbable polymer is first applied to the affected area where nerve regeneration is required, and then the cell of the present invention and / or the cell in which the cell of the present invention is induced to be differentiated, Alternatively, the cell of the present invention and / or the cell in which differentiation of the cell of the present invention is induced may be first applied to the affected area and then the bioabsorbable polymer may be applied, but the bioabsorbable polymer is preferably applied first. . In addition, the time interval for performing both may be short or may be applied after a certain period (for example, about 7 to 30 days), and an appropriate method should be adopted depending on the type and situation of the affected area. Can do. The amount of the cell and the bioabsorbable polymer applied to the affected area where nerve regeneration is required is not particularly limited, but the ratio of the cell and the bioabsorbable polymer as described for the nerve regeneration material is preferable. The amount of bioabsorbable polymer applied can be determined according to the state of the affected area.

  When the bioabsorbable polymer and the cell of the present invention and / or the cell of which the cell of the present invention is induced to be differentiated are applied to the affected area, the cell of the present invention and / or the present invention are used depending on the form of the bioabsorbable polymer. The cells obtained by inducing differentiation of the cells of the invention may be combined with the bioabsorbable polymer by mixing, carrying, embedding, inclusion, impregnation or the like.

  After transplantation of the nerve regeneration material or nerve regeneration material of the present invention, any pharmacologically acceptable drug can be applied to the patient at any time and at any location and in any manner. Applicable drugs include disinfectants, antibiotics, antibacterials, blood flow improving drugs such as actosine, PEG1, TGFβ, PDGF, b-FGF, platelet-derived growth factor (PDNF-AA), ciliary neurotrophic factor ( CNTF), glial-derived neurotrophic factor (GDNF), nerve growth factor (NGF), growth factors such as heregulin, enzyme inhibitors such as urinastatin and TIMP, anti-inflammatory agents such as steroids and nonsteroidal anti-inflammatory agents , Structural proteins such as fibrin and collagen, medium such as Eagle's alpha-modified minimum essential medium, serum such as fetal bovine serum, SH reagent such as β-mercaptoethanol, cyclic AMP elevating agent such as forskolin (Forskolin) Alternatively, a demethylating agent such as cyclic AMP analog) or 5-azacytidine can be used alone or in combination with a plurality thereof.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to only this Example at all.

<< Examples 1-9 and Comparative Examples 1-2 >>
(1) Production of covalently bonded alginic acid crosslinked gel (i) 2.3 g (20 mmol) of N-hydroxysuccinimide (manufactured by Peptide Institute, Inc.) was dissolved in 150 ml of ethyl acetate, and 10 ml of ethyl acetate was dissolved in this solution. 0.6 g (10 mmol) of ethylenediamine (diaminoethane) (manufactured by Wako Pure Chemical Industries, Ltd.) was added dropwise at room temperature while stirring. After completion of the dropwise addition, stirring was continued for another hour. The precipitated crystals were collected by filtration and dried under reduced pressure to obtain 2.9 g (yield 100%) of ethylenediamine 2N-hydroxysuccinimide salt.

(Ii) Ethylenediamine 2N-hydroxysuccinimide salt obtained in (i) above in 550 ml (carboxyl group; 275 mmol) of a 1% by weight aqueous solution (viscosity 500-600 cp) of sodium alginate (manufactured by Wako Pure Chemical Industries, Ltd.) 2.42 g (8.5 mmol) and 17.6 g (92 mmol) of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (manufactured by Peptide Institute, Inc.) were added and dissolved, thereby obtaining The obtained solution was cast on a Teflon-coated aluminum tray (15 cm × 25 cm) and allowed to stand at room temperature. After about 51 hours, a hydrogel (crosslinking rate: 15 mol%) was obtained.

(Iii) The water-containing gel obtained in the above (ii) is prepared by using calcium chloride and chloride so that the concentration of calcium ions and sodium ions is the same as that in the cell interstitial fluid (calcium ions 5 meq, sodium ions 143 meq). After thoroughly washing with an aqueous solution in which sodium was dissolved, it was thoroughly washed with pure water and then freeze-dried to obtain about 5 g of an alginate crosslinked gel sponge composed of a covalently crosslinked alginate gel.

(Iv) The alginate crosslinked gel sponge obtained in the above (iii) was trimmed to obtain an alginate crosslinked gel (about 10 mg) having a size of about 10 × 5 × 2 mm (Example 1).

(2) Obtaining bone marrow stromal cells and cells that are CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ ), and differentiation induction (i) GFP ( Stromal cells are collected from the bone marrow of adult rat (Wistar species) -positive (green fluorescent protein) and cultured. The medium used was modified Eagle's Minimum Essential Eagle Alpha Modification with 20 (v / v)% fetus carf serum added. Subcultured up to 4 generations.

(Ii) The obtained cells were sorted with a FACScan (fluorescence-activated cell sorter) manufactured by Becton-Dickinson, and CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative ( CD34 ⁻ ) cells were obtained.

(Iii) The cells obtained in (ii) were cultured again, and when 50% confluent was reached, β-mercaptoethanol was added at a concentration of 1 mM and cultured for 24 hours. . Thereafter, the medium was switched to a medium supplemented with 35 ng / ml retinoic acid. The medium used was modified Eagle's minimum essential medium alpha supplemented with 20 (v / v)% fetal calf serum. After 3 days, Forskolin 5 μM, Platelet-derived growth factor-AA 5 ng / ml, basic-Fibroblast growth factor 10 ng / ml, Heregulin (registered trademark) 200 ng / ml was further switched to the medium. Cells were immunostained after 7 days. When reacted with P75, O4, S-100, GFAP, and Nestin antibodies, the reaction was similar to that of natural Schwann cells. The bone marrow stromal cells were induced to a morphology morphologically similar to natural Schwann cells.

(Iv) The cells obtained in (ii) were cultured again, and the Notch intracellular domain gene was introduced when the cells reached 80-90% confluence. This was recombined by inserting the EcoRI-XbaI fragment of the 3.1 kb Notch intracellular domain into the EcoRI-XbaI multicloning site of Promega, pCI-neo mammalian expression vector (# E1841). For introduction, a system of LipofectAMINE2000 (GibcoBRL, 11668-027) was used. On the next day after introduction, G418sulfate (GibcoBRL, 83-5027) was added at a concentration of 200 ng / ml, and selection of the introduced cells was performed for 10 days. After the cell number has recovered to 90% confluence, Forskolin 5 μM (Calbiochem, 344273), basic-Fibroblast growth factor 10 ng / ml (Peprotech EC, LTD, 100-18B), ciliary neurotroph / 55 NT) was added. As a result of analyzing the cells after about 10 days, a characteristic morphology of the nerve cells was observed. Induced cells showed a positive reaction to MAP-2 (Chemicon MAB364), neurofilament (Boehringer Mannheim, 814342), beta3-tubulin, Tuj1. Since MAP-2, neurofilament, beta3-tubulin and Tuj1 are markers of nerve cells, it can be determined that the induced cells have the properties of nerve cells.

(V) The cells obtained in (i) above were collected by Trypsin treatment, and purified and isolated bone marrow stromal cells were obtained by repeating pipetting, standing, and supernatant aspiration (Comparative Example 1) .

(Vi) The cells obtained in (ii) above are repeatedly pipetted, allowed to stand, and the supernatant is aspirated, and purified and isolated, CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ^-) and CD34-negative (CD34 ^-) cells were obtained is (example 2).

(Vii) The cells obtained in (iii) above were collected by Trypsin treatment, and the operations of pipetting, standing, and supernatant aspiration were repeated, and purified and isolated CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), Cell-derived Schwann cells that were CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ ) were obtained (Example 3).

(Viii) The cells obtained in (iv) above were collected by Trypsin treatment, and the operations of pipetting, standing, and supernatant aspiration were repeated, and purified and isolated CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), Cell-derived neurons that were CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ ) were obtained (Example 4).

(3) (i) Preparation of nerve regeneration material: bone marrow obtained in (2) (v) above on alginate cross-linked gel (about 10 mg) of about 10 × 5 × 2 mm obtained in (1) above by to drop-impregnated with a medium containing a stromal cell ^106, nerve regeneration material was prepared (Comparative example 2). All operations were performed under sterile conditions.

(Ii) Preparation of nerve regeneration material: On about 10 mg of the alginate crosslinked gel obtained in (1) above, CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ) obtained in (2) (vi) above, CD11-negative (CD11 ^-) and CD34-negative (CD34 ^-) a is to dropping-impregnated with a culture medium containing ¹⁰⁶ cells and neural regeneration material was prepared (example 5). All operations were performed under sterile conditions.

(Iii) Preparation of nerve regeneration material: On about 10 mg of the alginate crosslinked gel obtained in (1) above, CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ) obtained in (2) (vii) above, CD11-negative (CD11 ^-) and CD34-negative (CD34 ^-) a is cell-derived Schwann cells 10 ⁶ or be dropped, impregnated with a medium containing, nerve regeneration material was prepared (example 6). All operations were performed under sterile conditions.

(Iv) Preparation of nerve regeneration material: On about 10 mg of the alginate crosslinked gel obtained in (1) above, CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ) obtained in (2) (viii) above, CD11-negative (CD11 ^-) and CD34-negative (CD34 ^-) a is cell-derived neurons 10 ⁶ or be dropped, impregnated with a medium containing, nerve regeneration material was prepared (example 7). All operations were performed under sterile conditions.

(4) Obtaining CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ ) cells derived from peripheral blood, and induction of differentiation
(i) Blood was collected from the heart of a GFP-positive adult rat (Wistar species) and fractionated with a FACScan (fluorescence-activated cell sorter) manufactured by Becton-Dickinson, and CD29-positive (CD29 ⁺ ), CD90-positive (CD90 ⁺ ), Cells that were CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ ) were obtained (Example 8).

(Ii) When the cells obtained in (i) were cultured and reached 50% confluent, β-mercaptoethanol was added at a concentration of 1 mM and cultured for 24 hours. . Thereafter, the medium was switched to a medium supplemented with 35 ng / ml retinoic acid. The medium used was modified Eagle's minimum essential medium alpha supplemented with 20 (v / v)% fetal calf serum. After 3 days, Forskolin 5 μM, Platelet-derived growth factor-AA 5 ng / ml, basic-Fibroblast growth factor 10 ng / ml, and Heregulin (registered trademark) 200 ng / ml were further switched to the medium. Seven days later, cells were immunostained. When reacted with P75, O4, S-100, GFAP and Nestin antibodies, the reaction was similar to that of natural Schwann cells. Cells derived from the peripheral blood derived from CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ ) are induced to a morphology morphologically similar to natural Schwann cells. It was done.

(Iii) The cells obtained in (ii) above are collected by Trypsin treatment, and the operations of pipetting, standing, and supernatant aspiration are repeated, and purified and isolated from peripheral blood CD29 positive (CD29 ⁺ ), CD90 positive Schwann cells derived from (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ ) cells were obtained (Example 9).

<< Test Example 1 >>
Central nerve regeneration surgery: Ten 30-day-old rats were used for the study. Each rat was anesthetized with ether, and then laminectomy of the thoracic spine was performed under a microscope to expose the spinal cord. Using a sharp scalpel, a gap of 2 mm was formed in the spinal cord at the 8th to 10th thoracic vertebrae level, and the nerve regeneration material of Example 6 obtained in the above (3) and (iii) (CD29 positive (CD29 to the alginate crosslinked gel) ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ ) impregnated with cell-derived Schwann cells) immediately trimmed to 2 × 2 × 2 mm size Filled. After the transplantation, the bone was returned, and the surgery was completed by suturing several muscles and several skins. Post-operative rats were given cyclosporine daily as an immunosuppressant.

<< Test Example 2 >>
(I) Ten 30-day-old rats were used in the test, and a 2 mm gap was formed in the spinal cord by the same method as in Test Example 1, and the bioabsorbable polymer of Example 1 obtained in (1) above was formed there. After filling an alginic acid cross-linked gel trimmed to a size of 2 × 2 × 2 mm (about 1.5 mg), the bone was returned, and several muscles and several skins were sutured.

(Ii) On the 10th day after the operation of (i) above, the spinal cord was exposed again, and the CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ) of Example 3 obtained in (2) (vii) above were exposed there. ), CD 11 negative (CD 11 ^-) and CD34-negative (CD34 ^{-) after six} cell-derived Schwann cells 10 were injected with a microsyringe is likewise returned to the bone, several places muscle, several points sutured skin And completed the surgery. Rats were given cyclosporine daily after the operation (i).

<< Test Example 3 >>
Ten 30-day-old rats were used for the test, a 2 mm gap was formed in the spinal cord by the same method as in Test Example 1, and the nerve regeneration material of Example 5 (alginate crosslinked gel) obtained in (3) (ii) above. CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ ) cells impregnated with 2 × 2 × 2 mm Was immediately filled. After the transplantation, the bone was returned, and the surgery was completed by suturing several muscles and several skins. Post-operative rats were given cyclosporine daily.

<< Test Example 4 >>
Ten 30-day-old rats were used in the test, a 2 mm gap was formed in the spinal cord by the same method as in Test Example 1, and the nerve regeneration material of Example 7 (alginate crosslinked gel) obtained in (3) and (iv) above. CD29-positive (CD29 ⁺ ), CD90-positive (CD90 ⁺ ), CD11-negative (CD11 ⁻ ) and CD34-negative (CD34 ⁻ ) cells impregnated with cell-derived neurons have a size of 2 × 2 × 2 mm The trimmed one was immediately filled. After the transplantation, the bone was returned, and the surgery was completed by suturing several muscles and several skins. Post-operative rats were given cyclosporine daily.

<< Test Example 5 >>
Peripheral nerve regeneration surgery: Ten 6-week-old rats were used for the study. Each rat was given general anesthesia by intraperitoneal injection of ketalal, and then a 7 mm gap was formed in the sciatic nerve at the center of the right lower leg using a sharp scalpel, obtained in (3) (ii) above. Further, 2 of the nerve regeneration material of Example 5 (in which alginate gel was impregnated with CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ ) cells) I transplanted them. Each stump was sandwiched between two alginate gels. After transplantation, several muscles and several skins were sutured, and nerve regeneration material was transplanted and wounds were closed. Post-operative rats were given cyclosporine daily.

<< Test Example 6 >>
Ten 6-week-old rats were used in the test, and a 7 mm gap was formed in the sciatic nerve at the center of the right lower leg by the same method as in Test Example 5. The nerve of Example 6 obtained in (3) (iii) above. Two regenerated materials (alginate gel impregnated with cell-derived Schwann cells impregnated with CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ )) . Each stump was sandwiched between two alginate gels. After transplantation, several muscles and several skins were sutured, and nerve regeneration material was transplanted and wounds were closed. Post-operative rats were given cyclosporine daily.

<< Test Example 7 >>
(i) Ten 30-day-old rats were used in the test, and a 2 mm gap was formed in the spinal cord by the same method as in Test Example 1, and the bioabsorbable polymer of Example 1 obtained in (1) above was formed there. After filling an alginic acid cross-linked gel trimmed to a size of 2 × 2 × 2 mm (about 1.5 mg), the bone was returned, and several muscles and several skins were sutured.

(Ii) On the 10th day after the operation of (i) above, the spinal cord was exposed again, and the CD29 positive (CD29 ⁺ ) and CD90 positive of Example 9 obtained in (4) and (iii) of Example 1 there (CD90 ^+), CD11-negative (CD 11 ^-) and CD34-negative (CD34 ^-) Schwann cells 10 ⁶ derived cells is a, after injecting using a microsyringe, likewise returns the bone, several points muscles, The operation was completed by suturing several skins. Rats were continuously administered cyclosporine after the operation (i).

<< Comparative Test Example 1 >>
Ten 30-day-old rats were used in the test, a 2 mm gap was formed in the spinal cord by the same method as in Test Example 1, and the nerve regeneration material of Example 1 obtained in (1) (iv) above was used. After the alginic acid cross-linked gel was trimmed to a size of 2 × 2 × 2 mm, the bone was returned, and several muscles and several skins were sutured to complete the operation. Post-operative rats were treated with cyclosporine every day for the same experimental conditions.

<< Comparative Test Example 2 >>
Ten 30-day-old rats were used in the test, a 2 mm gap was formed in the spinal cord by the same method as in Test Example 1, and the nerve regeneration material of Comparative Example 2 (alginate crosslinked gel) obtained in (3) (i) above. Implanted with bone marrow stromal cells) and immediately trimmed to 2 × 2 × 2 mm in size. After the transplantation, the bone was returned, and the surgery was completed by suturing several muscles and several skins. Post-operative rats were given cyclosporine daily.

<< Comparative Test Example 3 >>
Ten 30-day-old rats were used in the test, a 2 mm gap was formed in the spinal cord by the same method as in Test Example 1, and the nerve regeneration material of Example 1 obtained in (1) (iv) above was used. after filling those trimmed alginate crosslinked gel in advance other nerve regeneration material which collected from a Wistar rat cultured neural stem ¹⁰⁶ cells was impregnated with the size of 2 × 2 × 2 mm, back bones, muscles The surgical operation was completed by suturing several places and several places on the skin. Post-operative rats were given cyclosporine daily.

<< Comparative Test Example 4 >>
Ten 30-day-old rats were used in the test, a 2 mm gap was formed in the spinal cord by the same method as in Test Example 1, and the nerve regeneration material of Example 1 obtained in (1) (iv) above was used. after filling those trimmed alginate crosslinked gel formed by previously taken-cultured natural Schwann cells 10 impregnated ^six from autologous peripheral nerve nerve regeneration material to a size of 2 × 2 × 2 mm, back bones, muscles The surgical operation was completed by suturing several places and several places on the skin. Post-operative rats were treated with cyclosporine every day for the same experimental conditions.

<< Comparative Test Example 5 >>
Ten 30-day-old rats were used in the test, and a 2 mm gap was formed in the spinal cord by the same method as in Test Example 1, and the CD29 positive of Example 3 obtained in (2) (vii) above (CD29 ⁺ ), CD90-positive (CD90 ^+), CD11-negative (CD 11 ^-) and CD34-negative (CD34 ^{-) after six} cell-derived Schwann cells 10 were injected with a microsyringe a back bone, several points muscles, The operation was completed by suturing the skin several places. Post-operative rats were given cyclosporine daily.

<< Comparative Test Example 6 >>
Ten 6-week-old rats were used for the test, and a 7 mm gap was formed in the sciatic nerve at the center of the right lower leg by the same method as in Test Example 5. The nerve of Example 1 obtained in (1) (iv) above was used. Two regeneration materials (alginate cross-linked gel) were transplanted. Each stump was sandwiched between two alginate cross-linked gels. After transplantation, several muscles and several skins were sutured, and nerve regeneration material was transplanted and wounds were closed. Post-operative rats were treated with cyclosporine every day for the same experimental conditions.

"Test results"
I. Postoperative initial morphological observation (1) Central nerve regeneration test: In Test Examples 1 and 2 and Comparative Test Example 5 above, CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 Morphological observation was performed using immunohistochemical techniques on the 6th and 14th days after transplantation of the negative (CD34 ⁻ ) cell-derived Schwann cells.

(I) 6 days after the operation: the gaps in Test Examples 1 and 2 are positive for a number of GFPs (markers indicating that they are transplanted cells), and S100 antibody, Nestin antibody, P0 antibody, P75 antibody, and O4 antibody positive CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ ) cell-derived Schwann cells were observed. The CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ ) cell-derived Schwann cells adhered well to the remaining alginate gel. In addition, GAP43 (growth associated protein-43, a protein expressed when regenerating axons grow and extend) positive neurons were observed near the cranial stump and caudal stump. Regenerating axons are observed in the same position as cell-derived Schwann cells that are CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ ), and these Schwann cells are regenerated It was confirmed that adhesion to axons and myelin formation occurred. In Test Examples 1 and 2, the number and morphology of cell-derived Schwann cells that are CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ ) are large. No difference was observed. On the other hand, in Comparative Test Example 5, transplanted GFP-positive CD29-positive (CD29 ⁺ ), CD90-positive (CD90 ⁺ ), CD11-negative (CD11 ⁻ ) and CD34-negative (CD34 ⁻ ) cell-derived Schwann cells from the transplant site The cells were flowing out, and the cells were not observed in the gap.

(Ii) 14 days after surgery: From the gaps in Test Examples 1 and 2, many CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ ) Cell-derived Schwann cells were observed, and more cells were observed in Test Example 2. CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ ) cell-derived Schwann cells adhered well to the residual alginate gel. A regenerating axon surrounded by GAP43-positive thick myelin was observed in the center of the gap. There was no significant difference in the number of observed axons between Test Examples 1 and 2. In Comparative Test Example 5, GFP-positive CD29-positive (CD29 ⁺ ), CD90-positive (CD90 ⁺ ), CD11-negative (CD11 ⁻ ) and CD34-negative (CD34 ⁻ ) cell-derived Schwann cells and regenerative axons in the gap Observed.

(2) Peripheral nerve regeneration test: In Test Examples 5 and 6 and Comparative Test Example 6 above, morphology observation was performed using an immunohistochemical technique on the 6th and 14th days after nerve regeneration material or nerve regeneration material transplantation Went. In Test Examples 5 and 6, CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), and CD11 negative negative on GFP positive and S100 antibody, Nestin antibody, P0 antibody, P75 antibody, and O4 antibody positive on the sixth day after surgery Cell-derived Schwann cells that were (CD11 ⁻ ) and CD34 negative (CD34 ⁻ ) were observed, and these Schwann cells adhered well to the remaining alginate gel. In addition, many neurofilament antibody-positive regenerative axons myelinated in these Schwann cells were observed, and in both test cases, a large number of axons were observed in the center of the gap. On the 14th day after the operation, the regenerating axon had reached the distal stage end beyond the gap. No morphologically significant difference was observed between the two test cases. On the other hand, in Comparative Test Example 6, a small number of regenerating axons surrounded by GFP-negative Schwann cells were observed near the proximal stump on the sixth day after the operation. On the 14th day after surgery, the tip of the regenerating axon reached only near the center of the gap.

II. Central nerve regeneration in the postoperative chronic phase (1) Evaluation method of central nerve regeneration: Using the electromyograph ("The Nicolet Viking" manufactured by Nicolet Biomedical Instruments) at 5 weeks after the above surgery Somatomotor evoked potentials (MEP) and somatosensory evoked potentials (SEP) were recorded. At that time, MEP applied electrical stimulation to the cerebral motor cortex and recorded the evoked potentials generated by the peripheral gastrocnemius muscle. In addition, SEP applied electrical stimulation to the lower limbs and recorded the evoked potentials generated by postcruciate sensory cortex. Furthermore, the morphological evaluation of the tissue was performed at 12 weeks using an optical microscope and an electron microscope.

(2) Evaluation results of central nerve regeneration: (i) In Test Examples 1, 2, 3, 4 and 7, and Comparative Test Examples 2 and 3, MEP and SEP were already recorded 5 weeks after the operation, and nerve regeneration It was evaluated as being. Among them, in Test Examples 1, 2, 3, 4 and 7, both MEP and SEP had a short latency and a high potential, and an evaluation closer to normal was obtained. Further, in Test Examples 2 and 7, the latency was shorter and the potential was higher, and good functional recovery was achieved.

(Ii) On the other hand, in Comparative Test Examples 1, 4 and 5, MEP and SEP were not recorded 5 weeks after the operation, and nerve regeneration was not performed smoothly.

(Iii) In the optical microscope specimen at 12 weeks, in Test Examples 1, 2, 3, 4 and 7, and Comparative Test Examples 2 and 3, many myelinated and unmyelinated axons were observed in the center of the gap. . Among them, in Test Examples 1, 2, 3, 4 and 7, more myelinated axons were observed, and the axons had a one-to-one relationship with Schwann-like cells and had thick myelin myelin sheaths. Further, in Test Examples 2 and 7, more myelinated axons were observed and had thick myelin myelin sheaths.

(Iv) On the other hand, almost no myelinated axons were observed at the center of the gap in Comparative Test Examples 1, 4 and 5 in the 12-week specimen.

(V) Further, in the transmission electron microscope observation at 12 weeks, in Test Examples 1 to 7 and Comparative Test Examples 1 to 4 and 6, macrophages and lymphocytes are embedded in a portion for implanting a nerve regeneration material or a nerve regeneration material. Was hardly observed and almost no foreign body reaction and chronic inflammatory reaction occurred.

(Vi) From the above results, the bioresorbable polymer covalently cross-linked alginate cross-linked gel and especially CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ It can be seen that a high level of neurological function regeneration can be obtained by the combined use with the cell-derived Schwann cells. This is because of the excellent biocompatibility of covalently cross-linked alginate cross-linked gel and cell-derived Schwann that is CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ ). It is presumed to be due to a synergistic effect of nerve cell proliferation promoting action and nerve axon extension action by cells. In addition, cell-derived Schwann cells that are bioabsorbable polymers (covalently crosslinked alginate crosslinked gel) and CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ ) Rather than using it as a nerve regeneration material as a whole, as in Test Examples 2 and 7, after applying the covalently crosslinked alginate crosslinked gel to the affected area first, CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), It can be seen that more advanced nerve regeneration can be achieved by applying cell-derived Schwann cells that are CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ ) and sequentially using them together.

  Nerve regeneration material or nerve regeneration material of the present invention comprising a predetermined bioabsorbable polymer and predetermined cells and / or cells in which the cells have been induced to differentiate, in particular, a polysaccharide having a carboxyl group as a bioabsorbable polymer and / or Alternatively, a bioabsorbable polymer comprising a cross-linked polysaccharide obtained by covalently cross-linking a salt thereof with an amine compound (I) and / or a cross-linking reagent comprising the salt, and a predetermined cell and / or differentiation of the cell. A nerve regeneration material or nerve regeneration material composed of induced cells can exert nerve cell proliferation promoting action and nerve axon extension action, and is excellent in terms of safety, biocompatibility, etc. Effectively used for the treatment of diseases of the central nervous system and peripheral nervous system due to damage or loss of nerve tissue, and for the treatment of traumatic diseases such as cerebrovascular diseases such as spinal diseases, head trauma, and stroke. It can be.

  The material for nerve regeneration and the material for nerve regeneration of the present invention are free from the occurrence of infectious diseases, are excellent in safety and compatibility with living bodies, and can be easily produced industrially with high productivity. Further, the nerve regeneration material of the present invention in which the cells constituting the nerve regeneration material of the present invention and the bioabsorbable polymer are filled in a tube made of a bioabsorbable material is excellent in handleability and is suitable for nerve tissue repair. It can be used conveniently and with good operability in surgical operations.

Claims (27)

A nerve regeneration material comprising cells that can be used with a bioabsorbable polymer, wherein the cells are (1) CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 A material for nerve regeneration, comprising negative (CD34 ⁻ ) cells and / or (2) cells obtained by inducing differentiation of the cells of (1).   The nerve regeneration material according to claim 1, wherein the cell (2) is a Schwann cell or a nerve cell.   The nerve regeneration material according to claim 1 or 2, wherein the cell is a mammal-derived cell.   The material for nerve regeneration according to claim 3, wherein the mammalian cell is a human cell.   The nerve regeneration material according to any one of claims 1 to 4, wherein the cell is an allogeneic or autologous cell.   The nerve regeneration material according to any one of claims 1 to 5, wherein some or all of the cells are cells derived from bone marrow.   A nerve regeneration material comprising a bioabsorbable polymer that can be used with cells, wherein the polymer is a polysaccharide and a salt thereof, a crosslinked polysaccharide and a salt thereof, an acidic polysaccharide ester, a crosslinked acidic polysaccharide ester, a protein , Polypeptides, polyglycolic acid, polylactic acid, glycolic acid / lactic acid copolymer, glycol / carbonate copolymer, polydioxanone, polyε-caprolactone, lactic acid / ε-caprolactone copolymer, and cyanoacrylate copolymer A nerve regeneration material comprising at least one selected from the group consisting of:   The material for nerve regeneration according to claim 7, wherein the bioabsorbable polymer comprises a polysaccharide and / or a salt thereof.   The nerve regeneration material according to claim 8, wherein the polysaccharide and / or salt thereof is an acidic polysaccharide and / or salt thereof.   The nerve regeneration material according to claim 9, wherein the acidic polysaccharide and / or salt thereof is a polysaccharide having a carboxyl group and / or a salt thereof.   The nerve regeneration material according to claim 10, wherein the polysaccharide having a carboxyl group and / or a salt thereof is at least one selected from the group consisting of alginic acid and a salt thereof, and hyaluronic acid and a salt thereof.   The material for nerve regeneration according to claim 7, wherein the bioabsorbable polymer comprises a crosslinked polysaccharide and / or a salt thereof. The crosslinked polysaccharide and / or salt thereof is a polysaccharide having a carboxyl group and / or a salt thereof represented by the following general formula (I):

The nerve regeneration material according to claim 12, which is formed by covalently crosslinking with at least one crosslinking reagent selected from the group consisting of a compound represented by the formula:   The material for nerve regeneration according to claim 13, wherein the crosslinking reagent is an N-hydroxysuccinimide salt of the compound represented by the general formula (I).   The N-hydroxysuccinimide salt of the compound represented by the general formula (I) is diaminoethane 2N-hydroxysuccinimide salt, diaminohexane 2N-hydroxysuccinimide salt, N, N′-di ( The nerve regeneration according to claim 14, which is at least one selected from the group consisting of 4N-hydroxysuccinimide salt of lysyl) -diaminoethane and 3N-hydroxysuccinimide salt of N- (lysyl) -diaminohexane. Materials.   A nerve regeneration material comprising a bioabsorbable polymer that can be used with cells, wherein the polymer is formed from an acidic polysaccharide ester and a polyamino compound having two or more α-amino groups derived from natural amino acids. A material for nerve regeneration, which is made of a polymer that has been prepared.   17. The nerve regeneration material according to claim 16, wherein the acidic polysaccharide ester is alginate propylene glycol ester and / or hyaluronic acid propylene glycol ester.   The nerve regeneration material according to claim 16 or 17, wherein the polyamino compound is ε-polylysine.   Nerve regeneration in which the bioabsorbable polymer according to any one of claims 7 to 11 and / or the bioabsorbable polymer according to any one of claims 12 to 18 is filled in a tube made of a bioabsorbable material. Materials.   A nerve regeneration material comprising a cell and a bioabsorbable polymer, wherein the cell comprises the cell according to any one of claims 1 to 6, and the bioabsorbable polymer according to any one of claims 7 to 18. A nerve regeneration material comprising a bioabsorbable polymer.   21. The nerve regeneration material according to claim 20, wherein cells and a bioabsorbable polymer are filled in a tube made of a bioabsorbable material. (1) Step of collecting cells from biological material, and (2) CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ ) from the obtained cells. A process of sorting cells,
A method for producing cells for nerve regeneration material that can be used with a bioabsorbable polymer.   The production method according to claim 22, further comprising the step of (2 ') inducing differentiation of the sorted cells.   The production method according to claim 22 or 23, wherein the cells are collected from bone marrow of the living body in the step (1). (1) a step of collecting cells from biological material;
(2) a step of sorting cells that are CD29 positive (CD29 ⁺ ), CD90 positive (CD90 ⁺ ), CD11 negative (CD11 ⁻ ) and CD34 negative (CD34 ⁻ ) from the obtained cells, and (3) sorting Cells, polysaccharides and salts thereof, crosslinked polysaccharides and salts thereof, acidic polysaccharide esters, crosslinked acidic polysaccharide esters, proteins, polypeptides, polyglycolic acid, polylactic acid, glycolic acid / lactic acid copolymer, glycol A bioabsorbable polymer comprising at least one selected from the group consisting of: / carbonate copolymer, polydioxanone, polyε-caprolactone, lactic acid / ε-caprolactone copolymer, and cyanoacrylate copolymer; A step of combining a saccharide ester and a bioabsorbable polymer formed from a polyamino compound having two or more α-amino groups derived from natural amino acids. The a method for producing a nerve regeneration material.   The method according to claim 25, further comprising (2 ') a step of inducing differentiation of the sorted cells between the steps (2) and (3).   27. The production method according to claim 25 or 26, wherein the cells are collected from bone marrow of the living body in step (1).