Composition for regeneration of intervertebral disc

Abstract

To provide a composition for promoting regeneration of the nucleus pulposus of an intervertebral disc.SOLUTION: There is provided a composition for promoting regeneration of the nucleus pulposus of an intervertebral disc, the composition comprising a low endotoxin monovalent metal salt of an alginic acid and mesenchymal stem cells. In particular, the composition of the present invention promotes the regeneration of the nucleus pulposus of an intervertebral disc via activation of nucleus pulposus cells by human bone marrow-derived high-purity mesenchymal stem cells and / or differentiation of human bone marrow-derived high-purity mesenchymal stem cells into nucleus pulposus cells.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a composition for intervertebral disc regeneration, which contains a low-endotoxin monovalent metal salt of alginic acid and mesenchymal stem cells.The present invention relates to a composition for intervertebral disc regeneration, which contains highly purified mesenchymal stem cells derived from human bone marrow. [Background technology]

[0002] The human spine is made up of 24 bones (vertebrae), and the tissue that acts as a cushion between the vertebrae is called the intervertebral disc. Intervertebral discs can degenerate or become damaged due to aging, trauma, disease, etc. Intervertebral disc degeneration is a state in which the number of cells, water content, and extracellular matrix (type II collagen, aggrecan, etc.) of the intervertebral disc are reduced, and as the condition progresses, the intervertebral disc can no longer function as a shock absorber. Specifically, intervertebral disc degeneration and disc damage include herniated discs, disc disease, degenerative spondylolisthesis, pyogenic discitis, degenerative spondylosis, spinal canal stenosis, and disc damage due to trauma, etc. For example, in herniated discs, the annulus fibrosus that covers the nucleus pulposus forms a herniation due to deformation or cracking, and protrudes outside the intervertebral disc, and the nucleus pulposus that has deviated from the deformation or cracking compresses the spinal nerve, causing pain and paralysis.

[0003] One of the treatments for herniated discs is disc nucleotomy (resection), which has been confirmed to be effective to a certain extent. However, it is known that disc nucleotomy (resection) may cause the degenerative changes of the intervertebral disc to progress because no treatment is given to the surgical site after disc nucleotomy. When a part of the nucleus pulposus is removed by disc nucleotomy, a cavity (also referred to as a "defective part" in this specification) is formed at the nucleus pulposus site. Since the nucleus pulposus has a significantly low self-repairing ability and regenerative ability, the cavity of the nucleus pulposus is likely to become physically weak. In addition, fibroblast-like cells may accumulate in the cavity to form tissue with mechanical properties different from those of the original nucleus pulposus. For this reason, the recurrence rate of herniation is high after disc nucleotomy. The recurrence rate within 5 years after disc nucleotomy is said to be about 4 to 15%, but recent long-term data has revealed that the majority of cases recur after 10 years. If the herniation recurs, a second surgery is necessary, but the spinal nerves are buried in scar tissue that forms after the first surgery, making it difficult to identify their location.

[0004] To date, they have developed a high-purity hardening gel based on alginate and demonstrated that administration of the gel alone (acellular) results in natural repair of intervertebral disc tissue (Patent No. 6487110, Tsujimoto et al., EBioMedicine 37 (2018) 521-534). Clinical trials are currently being conducted in which the gel is implanted in patients aged 20 to 49 with lumbar disc herniation after herniotomy.

[0005] Mesenchymal stem cells (MSCs) are one of the somatic stem cells that have been widely applied clinically after hematopoietic stem cells, because there are few ethical issues associated with cell collection and they have the ability to differentiate into various types of cells such as bone, cartilage, and fat. Since MSCs can be isolated by relatively simple procedures, they are widely used as a material for regenerative medicine, mainly by inducing differentiation into cartilage or bone in vitro and then transplanting them into local areas. When proceeding with clinical applications, it is essential to be able to produce the necessary amount of cells that maintain a certain level of function for commercialization.

[0006] The inventors then isolated LNGFR and Thy-1 co-positive cells from bone marrow fluid by flow cytometry, obtained rapidly expanding clones (RECs), and established a purification and isolation method that can eliminate differences in the proliferation of MSCs derived from donors (Patent No. 6463029, WO2016 / 017795, Mabuchi Y. et al., Stem Cell Reports 1(2): 152-165, 2013). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 6487110 [Patent Document 2] Patent No. 6463029 [Patent Document 3] International Publication No. 2016 / 017795 Brochure [Non-patent literature]

[0008] [Non-Patent Document 1] Tsujimoto T et al., An acellular bioresorbable ultra-purified alginate gel promotes intervertebral disc repair: A preclinical proof-of-concept study, EBioMedicine 37 (2018) 521-534 [Non-Patent Document 2] Mabuchi Y et al., LNGFR+ Thy-1+ Vcam-1hi+ cells reveal functionally distinct subpopulations in mesenchymal stem cells. Stem Cell Reports 1(2): 152-165, 2013 Summary of the Invention [Problem to be solved by the invention]

[0009] As mentioned above, an alginate-based high-purity hardening gel has been developed, and an exploratory investigator-initiated clinical trial has been conducted in which the gel is implanted in patients with lumbar disc herniation after herniotomy. However, the nucleus pulposus, which is said to have almost no self-repair or regeneration ability, is even less able to repair itself in middle-aged and elderly people, and there are clear limitations to gel treatment alone for such patients. For this reason, there is a need to develop further compositions that can repair and regenerate intervertebral disc tissue. [Means for solving the problem]

[0010] As a result of investigations to solve the above problems, the present inventors found that a composition containing a monovalent metal salt of low endotoxin alginic acid and mesenchymal stem cells can solve the above problems, and thus completed the present invention. In another aspect of the present invention, the inventors found that a composition containing highly purified mesenchymal stem cells derived from human bone marrow can solve the above problems. That is, the present invention is as follows. [1] A composition for intervertebral disc regeneration, comprising a low-endotoxin monovalent metal salt of alginic acid and mesenchymal stem cells. [1-1] A composition for intervertebral disc regeneration, comprising a monovalent metal salt of alginic acid and mesenchymal stem cells. [2] The composition described in [1] or [1-1], which promotes regeneration of nucleus pulposus of an intervertebral disc through activation of nucleus pulposus cells by mesenchymal stem cells and / or differentiation of mesenchymal stem cells into nucleus pulposus cells. [3] The composition according to any one of [1] to [2], wherein the mesenchymal stem cells are highly purified mesenchymal stem cells derived from human bone marrow. [4] The composition described in [3], wherein the highly purified human bone marrow-derived mesenchymal stem cells are a cell population of rapidly proliferating mesenchymal stem cell clones that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and which satisfy at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scatter in flow cytometry is 35% or less. (b) the average cell size is 20 μm or less [4-1] The composition described in [3], wherein the highly purified human bone marrow-derived mesenchymal stem cells are a cell population of rapidly proliferating mesenchymal stem cell clones isolated based on the positivity of LNGFR (CD271) or co-positivity of LNGFR (CD271) and Thy-1 (CD90), and the cell population satisfies at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scatter in flow cytometry is 35% or less. (b) the average cell size is 20 μm or less [4-2] The composition described in [3], wherein the highly purified mesenchymal stem cells derived from human bone marrow are a cell population of rapidly proliferating mesenchymal stem cell clones derived from cells that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and which satisfy at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scatter in flow cytometry is 35% or less. (b) the average cell size is 20 μm or less [4-3] The composition described in [3], wherein the highly purified human bone marrow-derived mesenchymal stem cells are a cell population of rapidly proliferating mesenchymal stem cell clones that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and which satisfy at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scatter in flow cytometry is 40% or less. (b) the average cell size is 20 μm or less [4-4] The composition described in [3], wherein the highly purified human bone marrow-derived mesenchymal stem cells are a cell population of rapidly proliferating mesenchymal stem cell clones isolated based on the positivity of LNGFR (CD271) or co-positivity of LNGFR (CD271) and Thy-1 (CD90), and the cell population satisfies at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scatter in flow cytometry is 40% or less. (b) the average cell size is 20 μm or less [4-5] The composition described in [3], wherein the highly purified mesenchymal stem cells derived from human bone marrow are a cell population of rapidly proliferating mesenchymal stem cell clones derived from cells that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and which satisfy at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scatter in flow cytometry is 40% or less. (b) the average cell size is 20 μm or less [5] The composition described in any one of [1] to [4-5], which is applied to a subject's intervertebral disc and has fluidity when applied. [5-1] A composition described in any one of [1] to [4], which is applied to a target intervertebral disc nucleus pulposus site and is used so as to contact at least a portion of the surface of the composition with a crosslinker after application, and which has fluidity when applied to the nucleus pulposus site. [5-2] A composition described in any one of [1] to [4], which is applied to the target intervertebral disc nucleus pulposus site and used to harden a portion of the site after application, and which has fluidity when applied to the nucleus pulposus site. [6] The composition described in any one of [5] to [5-2], wherein the application to the nucleus pulposus site is the filling of the composition into a defective portion of the nucleus pulposus. [7] The composition described in [5-2] or [6], wherein the partial curing is carried out by contacting at least a portion of the surface of the composition with a crosslinking agent. [8] The composition described in [5-1] or [7], wherein the crosslinking agent is a divalent or higher metal ion compound. [8-1] The composition according to any one of [1] to [8], wherein the monovalent metal salt of alginic acid is a low-endotoxin monovalent metal salt of alginic acid. [9] The composition described in any one of [1] to [8-1], wherein the monovalent metal salt of low endotoxin alginic acid has a weight average molecular weight (absolute molecular weight) of 80,000 or more as measured by a GPC-MALS method.

[10] The composition according to any one of [1] to [9], wherein the concentration of the low-endotoxin monovalent metal salt of alginic acid is 0.5 w / w% to 5.0 w / w%.

[11] The composition described in any one of [1] to

[10] , which is used for treating, preventing, or suppressing recurrence of intervertebral disc degeneration and / or intervertebral disc damage.

[12] The composition described in

[11] , wherein the intervertebral disc degeneration and / or intervertebral disc damage is at least one selected from the group consisting of intervertebral disc herniation, intervertebral disc disease, degenerative spinal spondylolisthesis, pyogenic discitis, degenerative spondylosis, spinal stenosis, lumbar spinal stenosis, and intervertebral disc herniation and intervertebral disc damage accompanied by lumbar spinal stenosis. [12-1] The composition described in

[11] or

[12] , wherein the intervertebral disc degeneration and / or intervertebral disc damage is accompanied by chronic lower back pain.

[0011]

[13] A composition for intervertebral disc regeneration, which contains highly purified mesenchymal stem cells derived from human bone marrow and is applied to the site of the nucleus pulposus of a target intervertebral disc.

[14] The composition described in

[13] , which promotes regeneration of nucleus pulposus of an intervertebral disc through activation of nucleus pulposus cells by highly purified mesenchymal stem cells derived from human bone marrow and / or differentiation of highly purified mesenchymal stem cells derived from human bone marrow into nucleus pulposus cells.

[15] The composition described in

[13] or

[14] , wherein the human bone marrow-derived highly purified mesenchymal stem cells are in an undifferentiated state at the time of application and / or are applied without any induced differentiation treatment.

[16] The composition described in any one of

[13] to

[15] , wherein the highly purified human bone marrow-derived mesenchymal stem cells are a cell population of rapidly proliferating mesenchymal stem cell clones that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and which meets at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scatter in flow cytometry is 35% or less. (b) the average cell size is 20 μm or less [16-1] The composition described in any one of

[13] to

[15] , wherein the highly purified human bone marrow-derived mesenchymal stem cells are a cell population of rapidly proliferating mesenchymal stem cell clones isolated based on the positivity of LNGFR (CD271) or co-positivity of LNGFR (CD271) and Thy-1 (CD90), and which satisfies at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scatter in flow cytometry is 35% or less. (b) the average cell size is 20 μm or less [16-2] The composition described in any one of

[13] to

[15] , wherein the highly purified mesenchymal stem cells derived from human bone marrow are a cell population of rapidly proliferating mesenchymal stem cell clones derived from cells that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and which satisfies at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scatter in flow cytometry is 35% or less. (b) the average cell size is 20 μm or less [16-3] The composition described in any one of

[13] to

[15] , wherein the highly purified human bone marrow-derived mesenchymal stem cells are a cell population of rapidly proliferating mesenchymal stem cell clones that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and which satisfies at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scatter in flow cytometry is 40% or less. (b) the average cell size is 20 μm or less [16-4] The composition described in any one of

[13] to

[15] , wherein the highly purified human bone marrow-derived mesenchymal stem cells are a cell population of rapidly proliferating mesenchymal stem cell clones isolated based on the positivity of LNGFR (CD271) or co-positivity of LNGFR (CD271) and Thy-1 (CD90), and which satisfies at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scatter in flow cytometry is 40% or less. (b) the average cell size is 20 μm or less [16-5] The composition described in any one of

[13] to

[15] , wherein the highly purified mesenchymal stem cells derived from human bone marrow are a cell population of rapidly proliferating mesenchymal stem cell clones derived from cells that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and which satisfies at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scatter in flow cytometry is 40% or less. (b) the average cell size is 20 μm or less

[17] The composition described in any one of

[13] to [16-5], further comprising a carrier for embedding cells.

[18] The composition described in

[17] , wherein the carrier is selected from the group consisting of alginic acid, hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparan sulfate, and galactosaminoglycan sulfate, and pharma-ceutically acceptable salts thereof.

[19] The composition described in

[17] , wherein the carrier is a monovalent metal salt of alginic acid.

[20] The composition described in any one of

[13] to

[19] , which is applied to a target intervertebral disc nucleus pulposus site and is used so that after application, at least a portion of the surface of the composition is contacted with a crosslinker, and which has fluidity when applied to the nucleus pulposus site.

[21] The composition described in any one of

[13] to

[19] , which is applied to a target intervertebral disc nucleus pulposus site and used to harden a portion of the site after application, and which has fluidity when applied to the nucleus pulposus site.

[22] The composition described in

[20] or

[21] , wherein the application to the nucleus pulposus site is the filling of the composition into a defective portion of the nucleus pulposus.

[23] The composition according to any one of

[20] to

[22] , wherein the crosslinking agent is a divalent or higher metal ion compound.

[24] The composition according to any one of

[19] to

[23] , wherein the monovalent metal salt of alginic acid has a weight average molecular weight (absolute molecular weight) of 80,000 or more as measured by a GPC-MALS method.

[25] The composition described in any one of

[19] to

[24] , wherein the concentration of the monovalent metal salt of alginic acid is 0.5 w / w% to 5.0 w / w%.

[26] The composition described in any one of

[19] to

[25] , wherein the monovalent metal salt of alginic acid is a low-endotoxin monovalent metal salt of alginic acid.

[27] The composition described in any one of

[13] to

[26] , which is used for treating, preventing, or suppressing recurrence of intervertebral disc degeneration and / or intervertebral disc damage.

[28] The composition described in

[27] , wherein the intervertebral disc degeneration and / or intervertebral disc damage is at least one selected from the group consisting of intervertebral disc herniation, intervertebral disc disease, degenerative spinal spondylolisthesis, pyogenic discitis, degenerative spondylosis, spinal stenosis, lumbar spinal stenosis, intervertebral disc herniation associated with lumbar spinal stenosis (mixed lumbar spinal stenosis), and intervertebral disc damage. [28-1] The composition described in

[27] or

[28] , wherein the intervertebral disc degeneration and / or intervertebral disc damage is accompanied by chronic lower back pain.

[29] The composition described in any one of [1] to [28-1], which is used for suppressing intervertebral disc pain.

[30] The composition described in

[29] , wherein the discogenic pain is chronic lower back pain.

[0012]

[31] A composition for intervertebral disc regeneration comprising a monovalent metal salt of alginic acid and highly purified mesenchymal stem cells derived from human bone marrow, the composition being intended to be applied in a fluid state to the site of the nucleus pulposus of a target intervertebral disc. [31-1] A composition for intervertebral disc regeneration as described in

[31] above, for use without requiring a procedure for hardening the composition after it has been applied in a fluid state to the nucleus pulposus site of a target intervertebral disc. [31-2] A composition for intervertebral disc regeneration according to the above item

[31] or [31-1], which is applied in a fluid state to the target intervertebral disc nucleus pulposus site and then used without contacting the composition with a crosslinking agent.

[32] The composition described in any one of

[31] to [31-2], which promotes regeneration of nucleus pulposus of an intervertebral disc through activation of nucleus pulposus cells by highly purified mesenchymal stem cells derived from human bone marrow and / or differentiation of highly purified mesenchymal stem cells derived from human bone marrow into nucleus pulposus cells.

[33] The composition according to any one of

[31] to

[32] , wherein the human bone marrow-derived highly purified mesenchymal stem cells are in an undifferentiated state at the time of application and / or are applied without any treatment for induced differentiation.

[34] The composition described in any one of

[31] to

[33] , wherein the highly purified human bone marrow-derived mesenchymal stem cells are a cell population of rapidly proliferating mesenchymal stem cell clones that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and which satisfies at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scatter in flow cytometry is 40% or less. (b) the average cell size is 20 μm or less [34-1] The composition described in any one of

[31] to

[33] , wherein the highly purified human bone marrow-derived mesenchymal stem cells are a cell population of rapidly proliferating mesenchymal stem cell clones isolated based on the positivity of LNGFR (CD271) or co-positivity of LNGFR (CD271) and Thy-1 (CD90), and which satisfies at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scatter in flow cytometry is 40% or less. (b) the average cell size is 20 μm or less [34-2] The composition described in any one of

[31] to

[33] , wherein the highly purified mesenchymal stem cells derived from human bone marrow are a cell population of rapidly proliferating mesenchymal stem cell clones derived from cells that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and which satisfies at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scatter in flow cytometry is 40% or less. (b) the average cell size is 20 μm or less [34-3] The composition according to any one of

[34] to [34-2], wherein the coefficient of variation is 35% or less.

[35] The composition described in any one of

[31] to

[34] , wherein the application to the nucleus pulposus site is filling a defective portion of the nucleus pulposus with the composition.

[36] The composition according to any one of

[31] to

[35] , wherein the monovalent metal salt of alginic acid has a weight average molecular weight (absolute molecular weight) of 80,000 or more as measured by a GPC-MALS method.

[37] The composition described in any one of

[31] to

[36] , wherein the concentration of the monovalent metal salt of alginic acid is 0.5 w / w% to 5.0 w / w%.

[38] The composition described in any one of

[31] to

[37] , wherein the monovalent metal salt of alginic acid is a low-endotoxin monovalent metal salt of alginic acid.

[39] The composition described in any one of

[31] to

[38] , which is used for treating, preventing, or suppressing recurrence of intervertebral disc degeneration and / or intervertebral disc damage.

[40] The composition described in

[39] , wherein the intervertebral disc degeneration and / or intervertebral disc damage is at least one selected from the group consisting of intervertebral disc herniation, intervertebral disc disease, degenerative spinal spondylolisthesis, pyogenic discitis, chronic lower back pain, degenerative spondylosis, spinal stenosis, lumbar spinal stenosis, intervertebral disc herniation associated with lumbar spinal stenosis (mixed lumbar spinal stenosis), and intervertebral disc damage.

[41] The composition described in

[39] or

[40] , wherein the intervertebral disc degeneration and / or intervertebral disc damage is accompanied by chronic lower back pain.

[42] The composition according to any one of

[31] to

[41] , which is used to suppress intervertebral disc pain.

[43] The composition described in

[42] , wherein the discogenic pain is chronic lower back pain.

[0013]

[44] A composition for suppressing discogenic pain, comprising a monovalent metal salt of alginic acid and highly purified mesenchymal stem cells derived from human bone marrow, the composition being intended to be applied in a fluid state to the site of the nucleus pulposus of a subject. [44-1] A composition for suppressing discogenic pain described in

[44] above, for use after application in a fluid state to the subject's intervertebral disc nucleus pulposus site without requiring a procedure to harden the composition. [44-2] A composition for suppressing discogenic pain described in

[44] or [44-1] above, which is applied in a fluid state to the target intervertebral disc nucleus pulposus site and then used without contacting the composition with a crosslinking agent.

[45] The composition described in any one of

[44] to [44-2], wherein the pain is pain accompanied by at least one selected from the group consisting of intervertebral disc herniation, intervertebral disc disease, degenerative spondylolisthesis, pyogenic discitis, degenerative spondylosis, spinal stenosis, lumbar spinal stenosis, intervertebral disc herniation associated with lumbar spinal stenosis (mixed lumbar spinal stenosis), and intervertebral disc injury.

[46] The composition described in

[44] to

[45] , wherein the pain is chronic lower back pain.

[47] The composition described in any one of

[44] to

[46] , wherein the human bone marrow-derived highly purified mesenchymal stem cells are in an undifferentiated state at the time of application and / or are applied without any treatment for induced differentiation.

[48] ​​The composition described in any one of

[44] to

[47] , wherein the highly purified human bone marrow-derived mesenchymal stem cells are a cell population of rapidly proliferating mesenchymal stem cell clones that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and which satisfies at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scatter in flow cytometry is 40% or less. (b) the average cell size is 20 μm or less [48-1] The composition described in any one of

[44] to

[47] , wherein the highly purified human bone marrow-derived mesenchymal stem cells are a cell population of rapidly proliferating mesenchymal stem cell clones isolated based on the positivity of LNGFR (CD271) or co-positivity of LNGFR (CD271) and Thy-1 (CD90), and which satisfies at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scatter in flow cytometry is 40% or less. (b) the average cell size is 20 μm or less [48-2] The composition described in any one of

[44] to

[47] , wherein the highly purified human bone marrow-derived mesenchymal stem cells are a cell population of rapidly proliferating mesenchymal stem cell clones derived from cells that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and which satisfies at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scatter in flow cytometry is 40% or less. (b) the average cell size is 20 μm or less [48-3] The composition described in any one of

[48] to [48-2], wherein the coefficient of variation is 35% or less. [48-4] The composition described in any one of

[44] to [48-3], wherein the application to the nucleus pulposus site is filling the composition into a defective portion of the nucleus pulposus.

[49] The composition described in any one of

[44] to [48-4], wherein the monovalent metal salt of alginic acid has a weight average molecular weight (absolute molecular weight) of 80,000 or more as measured by a GPC-MALS method.

[50] The composition described in any one of

[44] to

[49] , wherein the concentration of the monovalent metal salt of alginic acid is 0.5 w / w% to 5.0 w / w%.

[51] The composition according to any one of

[44] to

[50] , wherein the monovalent metal salt of alginic acid is a low-endotoxin monovalent metal salt of alginic acid.

[52] The composition described in any one of

[31] to

[51] , which is applied to the target intervertebral disc nucleus pulposus site under X-ray fluoroscopy, has fluidity when applied to the nucleus pulposus site, and is used without contacting the composition with a crosslinking agent.

[53] The composition described in

[52] , which is applied to the target intervertebral disc nucleus pulposus site using a pressure gauge syringe under X-ray fluoroscopy, which is fluid when applied to the nucleus pulposus site, and which is used without contacting the composition with a crosslinking agent.

[0014]

[54] A method for evaluating the regenerative ability of a composition applied to an intervertebral disc of a sheep severe intervertebral disc degeneration model using the model, the method comprising the step of administering a cell-containing composition to an intervertebral disc of a severe intervertebral disc degeneration model prepared by a method comprising the following steps (a1) or (a2) and (b1) or (b2): (a1) A step of removing nucleus pulposus tissue from a sheep intervertebral disc in an amount equivalent to 0.00004% to 0.00005% of the sheep's body weight to prepare a degenerated intervertebral disc. (a2) Removing 20 mg of nucleus pulposus tissue from a sheep intervertebral disc to prepare a degenerated intervertebral disc. (b1) a step of further removing nucleus pulposus tissue in an amount equivalent to 0.00014% to 0.000175% of the sheep's body weight, 4 weeks after the step (a1) or (a2). (b2) removing an additional 70 mg of nucleus pulposus tissue 4 weeks after step (a1) or (a2);

[55] The method according to

[54] , wherein the step of administering the composition is carried out after step (b1) or (b2).

[56] The method according to

[54] or

[55] , wherein the evaluation is performed on the vertebral bodies and intervertebral discs collected from the degeneration model after administration of the composition using at least one evaluation method selected from the group consisting of MRI, tissue staining, and immunohistochemical staining (IHC).

[0015] The present invention also includes the following aspects.

[57] A method for treating, preventing, or inhibiting recurrence of intervertebral disc degeneration and / or intervertebral disc injury, comprising: The method comprises applying a composition containing a monovalent metal salt of alginic acid and mesenchymal stem cells to the nucleus pulposus site of the intervertebral disc of a subject in need of the treatment, prevention, or suppression of recurrence.

[58] A method for treating, preventing, or inhibiting recurrence of intervertebral disc degeneration and / or intervertebral disc injury, comprising: The method comprises applying highly purified mesenchymal stem cells derived from human bone marrow to the nucleus pulposus site of the intervertebral disc of a subject in need of the treatment, prevention, or suppression of recurrence.

[59] A method for treating, preventing, or inhibiting recurrence of intervertebral disc degeneration and / or intervertebral disc injury, comprising: The method comprises applying a composition comprising highly purified mesenchymal stem cells derived from human bone marrow and a monovalent metal salt of alginic acid to the nucleus pulposus site of the intervertebral disc of a subject in need of said treatment, prevention or recurrence suppression, wherein said composition is fluid when applied to the nucleus pulposus site.

[60] A method for treating, preventing, or inhibiting the recurrence of discogenic pain, comprising: The method comprises applying a composition comprising highly purified mesenchymal stem cells derived from human bone marrow and a monovalent metal salt of alginic acid to the nucleus pulposus site of the intervertebral disc of a subject in need of said treatment, prevention or recurrence suppression, wherein said composition is fluid when applied to the nucleus pulposus site.

[61] A monovalent metal salt of alginic acid and mesenchymal stem cells for use in the treatment, prevention, or suppression of recurrence of intervertebral disc degeneration and / or intervertebral disc injury, comprising applying a composition containing a monovalent metal salt of alginic acid and mesenchymal stem cells to the nucleus pulposus site of the intervertebral disc of a subject in need of such treatment, prevention, or suppression of recurrence of intervertebral disc degeneration and / or intervertebral disc injury.

[62] A composition comprising a monovalent metal salt of alginic acid and mesenchymal stem cells for use in treating, preventing, or inhibiting recurrence of intervertebral disc degeneration and / or intervertebral disc damage.

[63] Highly purified human bone marrow derived mesenchymal stem cells for use in the treatment, prevention or inhibition of recurrence of intervertebral disc degeneration and / or disc injury.

[64] A composition comprising highly purified human bone marrow-derived mesenchymal stem cells and a monovalent metal salt of alginic acid for use in the treatment, prevention, or suppression of recurrence of intervertebral disc degeneration and / or intervertebral disc injury, the composition being applied to the nucleus pulposus site of an intervertebral disc of a subject in need of said treatment, prevention, or suppression of recurrence, wherein the composition has flowability when applied to the nucleus pulposus site.

[65] A composition comprising highly purified human bone marrow-derived mesenchymal stem cells and a monovalent metal salt of alginic acid for use in the treatment, prevention, or suppression of recurrence of discogenic pain, the composition being applied to the nucleus pulposus site of an intervertebral disc of a subject in need of said treatment, prevention, or suppression of recurrence, the composition having flowability when applied to the nucleus pulposus site. Effect of the Invention

[0016] The present invention provides a composition for filling nucleus pulposus capable of promoting the regeneration of nucleus pulposus of intervertebral disc. The composition of the present invention can suppress degenerative changes in the entire intervertebral disc tissue including not only the nucleus pulposus of intervertebral disc but also the annulus fibrosus. The composition of the present invention also has the effect of increasing the proportion of type II collagen-positive hyaline cartilage-like cells in the nucleus pulposus. The composition can also promote regeneration in cases where the self-repair function of middle-aged and elderly people is weak, such as mixed lumbar spinal canal stenosis (combination of spinal canal stenosis and herniated disc), which is often seen in middle-aged and elderly people over 50 years old.

[0017] Moreover, in one aspect of the present invention, taking into consideration the ease of administering the composition and reduced invasiveness to the body, a carrier for embedding cells is used without a treatment for hardening, and pain associated with disc disease, particularly lower back pain, and more preferably chronic lower back pain, can be suppressed. [Brief description of the drawings]

[0018] [Figure 1]Figure 1 shows cell sorting data for the separation of carboxyfluorescein diacetate succinimidyl ester (CFDA-SE)-labeled bone marrow-derived mesenchymal stem cells (BMSCs) and unlabeled nucleus pulposus cells (NPCs) after 7 days of co-culture using sodium alginate solution (UPAL, low endotoxin ultra-purified alginate gel). (a): Two-dimensional (2D) dot plot of co-cultured cells. P1 gate was placed around a single live cell. (b): 2D dot plot of sorted unlabeled NPCs in P2 gate and CFDA-SE-labeled BMSCs in P3 gate. FSC-A: forward scatter area; FSC-W: forward scatter width; SSC-A: side scatter area; CFDA-SE-A: CFDA-SE-area. Gene expression of each cell was plotted on a logarithmic scale (y-axis) relative to the housekeeping gene GAPDH. Data are averages of four different rabbit NPC lines. (c): HIF-1α, (d): GLUT-1, (e): Brachyury, (f): CDMP-1, (g): TGF-β, (h): IGF-1, (i): type II collagen, and (j): aggrecan. Data are expressed as mean ± SEM, and p values ​​were determined by one-way ANOVA with Tukey-Kramer post-hoc test. [Diagram 2] Figure 1. Bone marrow-derived mesenchymal stem cells (BMSCs) survive in the intervertebral disc (IVD). Implanted bone-derived mesenchymal stem cells (BMSCs) labeled with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) survive in the intervertebral disc (IVD) at 4 and 12 weeks after surgery. Frozen sections of IVDs were stained with 4',6-diamino-2-phenylindole (DAPI). Images are representative of 4 replicates (intact control and BMSC+Gel, n = 4; 4 and 12 weeks). Scale bar = 50 μm. [Diagram 3]Figure 1: Gel combined with bone marrow-derived mesenchymal stem cells (BMSCs) preserves water content in degenerated intervertebral discs (IVDs) after discectomy. (a): T2-weighted, midsagittal images of degenerated IVDs 4 and 12 weeks after surgery. Images are representative of 8 scans. (b): Pfirrmann classification of IVD degeneration. Data are means ± SEM (intact control, puncture, discectomy, gel, and BMSCs + gel, n=8; 4 and 12 weeks). (c) Magnetic resonance imaging (MRI) index for degenerative changes in NP (nucleus pulposus (NP) area × mean signal intensity). Values ​​are expressed as percentages compared to intact control IVDs. Data are means ± SEM, p values ​​were determined by one-way ANOVA with post-hoc analysis using Tukey-Kramer test. [Figure 4] Figure 1 shows that gel combined with bone marrow-derived mesenchymal stem cells (BMSCs) prevents intervertebral disc (IVD) degeneration after discectomy. (a, b): Images of mid-sagittal sections of IVD stained with hematoxylin and eosin (H&E) or Safranin O (representative of images from eight experiments) (intact control, puncture, discectomy, gel, BMSCs+gel, n = 8; 4 and 12 weeks). AF: annulus fibrosus, NP: nucleus pulposus. Scale bars = (A): 500 μm (first and third sections from the top), 50 mm (second and fourth sections from the top), and (B): 500 μm. (c): Histological degeneration at 4 and 12 weeks. Data are means ± SEM, p values ​​were determined by one-way ANOVA with Tukey-Kramer post-hoc test. [Diagram 5]Figure showing type II collagen positive cells in rabbit nucleus pulposus (NP). (a): Midsagittal sections of rabbit intervertebral discs (IVDs) were stained for type II collagen. Images are representative of eight replicates (intact control, puncture, discectomy, gel, BMSC+gel, n= 8; 4 and 12 weeks). Arrows indicate cells positive for type II collagen. Scale bars = 500 μm (first and third sections from top) and 20 μm (second and fourth sections from top). (b): Percentage of type II collagen positive cells. Data are means ± SEM, p values ​​determined by one-way ANOVA with Tukey-Kramer post-hoc test. [Figure 6] Figure showing nucleus pulposus (NP) marker positive cells in rabbit NP. (ac): Horizontal sections of rabbit intervertebral discs (IVD) stained for HIF-1α, GLUT-1, and Brachyury on days 1, 7, and 28. Images are representative of four replicate experiments (intact control, discectomy, and BMSC+gel, n=4; day 1, 7, 28). Scale bar=50 μm. (df): Percentage of NP marker positive cells relative to total cells. (gi): Percentage of NP marker positive cells relative to CFDA-SE positive cells (representative of transplanted BMSCs). Data are mean ± SEM, p-values ​​were determined using paired t-test. [Figure 7] Diagram showing the mechanism of intervertebral disc (IVD) regeneration. Transplanted bone marrow-derived mesenchymal stem cells (BMSCs) produce growth factors and extracellular matrix (ECM) that lead to activation of pre-existing nucleus pulposus cells (NPCs). Pre-existing NPC activation also increases the production of growth factors and ECM. Transplanted BMSCs differentiate into NPCs. UPAL: Ultra-purified alginate (also known as low-endotoxin high-purity alginate). [Figure 8] FIG. 1 shows the results of comprehensive analysis of REC clones. [Figure 9] FIG. 1 shows the expression levels of various genes in healthy human intervertebral disc nucleus pulposus cells (NPCs) and highly purified mesenchymal cells (RECs). [Figure 10] FIG. 1 shows the results of MRI of the intervertebral disc in a sheep model 4 weeks after implantation. [Figure 11] FIG. 1 shows the results of histological examination of the intervertebral disc in a sheep model 4 weeks after transplantation. [Figure 12] FIG. 1 shows the results of histological examination of the intervertebral disc in a sheep model 4 weeks after transplantation. [Figure 13] Figure 1. Expression profiles of human NPCs and RECs after 7 days of 3D co-culture. (A) Schematic showing RECs isolation. Flow cytometry profile of human bone marrow cells stained for CD271 (LNGFR) and CD90 (THY-1). Cells collected from one well are seeded into 35 mm culture dishes and expanded up to 14 days to obtain a homogenous cell population with high differentiation and proliferation potential. (B and C) Cell sorting data is used to distinguish CFDA-SE labeled RECs from unlabeled NPCs. (B) P1 gate excludes dead and debris live cells. (C) 2D dot plot showing unlabeled NPCs in P2 gate and CFDA-SE labeled RECs in P3 gate. (D-K) Gene expression levels of each cell were normalized to the expression level of the housekeeping gene GAPDH and plotted on a logarithmic scale (y-axis). Data obtained from four different human NPC lines were averaged. (D) HIF-1α, (E) GLUT-1, (F) brachyury, (G) CDMP-1, (H) TGF-β, (I) IGF-1, (J) type II collagen, (K) aggrecan. Data are presented as mean ± SD (n = 4). Significance was assessed by one-way analysis of variance with post hoc Tukey-Kramer test. NPC: nucleus pulposus cells REC: rapidly proliferating clones CFDA-SE: 5,6-carboxyfluorescein diactosuccinimidyl ester ANOVA: analysis of variance FSC-A: forward scatter area FSC-W: forward scatter width SSC-A: side scatter area CFDA-SE-A: CFDA-SE area [Figure 14]Figure showing the elasticity ratio of the two types of gels. (A) Formation of disk-shaped UPAL and REC + UPAL gels after CaCl2-induced gelation (diameter, 4.5 mm; thickness, 2 mm). (B and C) Tensile compression mechanical testing apparatus. Samples were compressed at a constant rate of 0.5 mm / min. (D) Stress-strain curves. Young's modulus was calculated according to the slope of the tangent line between 10 and 20% compression values. Four replicates were performed and representative images are shown. (E) Young's modulus of the two types of gels. Data are shown as mean ± SD values ​​(n = 4). Significant differences were evaluated by Student's t-test after Welch's test. NS: no significant difference UPAL: ultra-purified alginate (also known as low-endotoxin high-purity alginate) REC: fast-growing clones SD: standard deviation [Figure 15] Diagram showing the experimental schedule and treatment details for each group. (A and B) In the first surgery, 20 mg of fresh NP tissue was removed from the treated IVD to establish a severely degenerated IVD model. (C) Four weeks after the first surgery, 70 mg of degenerated NP tissue was further removed from the degenerated IVD. (D) After removal of the degenerated NP, UPAL or REC + UPAL solution was implanted into the intradiscal space. NP: nucleus pulposus UPAL: ultra-purified alginate (also called low endotoxin high purity alginate) IVD: intervertebral disc [Figure 16]Figure 1: MRI evaluation of treated IVDs at 4 and 24 weeks after implantation. (A) T2-weighted, midsagittal images of IVDs at 4 and 24 weeks after surgery in sheep. Images are representative of 6 or 8 replicates. (B) Pfirrmann grade of IVD degeneration. (C) MRI index (NP area x mean signal intensity) values ​​for degenerative changes in NP. Values ​​are expressed as percentages of values ​​for untreated control IVDs. (D) Disc height index for IVD treatment. Values ​​are expressed as percentages of values ​​for untreated control IVDs. Data are expressed as mean ± SD values ​​(untreated control, n = 6; discectomy, n = 6; gel, n = 8; REC + gel, n = 8). Significant differences were assessed by one-way ANOVA with post hoc analysis using the Tukey-Kramer test. NP: nucleus pulposus MRI: magnetic resonance imaging IVD: intervertebral disc ANOVA: analysis of variance SD: standard deviation [Figure 17] Figure 1 shows histological evaluation of treated IVDs 4 and 24 weeks after implantation. (A and B) Representative midsagittal sections of treated IVDs stained with H&E or Safranin-O staining (untreated control, n=6; discectomy, n=6; gel, n=8; REC+gel, n=8). Scale bars (A); 50 μm (second and fourth sections from the top) or 1 mm (first and third sections from the top) (B); 1 mm. (C) Histological grade was determined via modified Boos classification. Data are presented as mean ± standard deviation. Significant differences were assessed by one-way analysis of variance with post hoc Tukey-Kramer test. IVD: intervertebral disc H&E: hematoxylin and eosin SD: standard deviation AF: annulus fibrosus NP: nucleus pulposus [Figure 18]Figure showing type II or type I collagen positive cells in treated IVDs 4 and 24 weeks after implantation. (A and B) Representative mid-sagittal sections of treated IVDs stained for type II or type I collagen (untreated control, n=6; discectomy, n=6; gel, n=8; REC+gel, n=8). Scale bars, 1 mm (1st and 3rd lines) or 50 μm (2nd and 4th lines). (C and D) Percentage of type II or type I collagen positive cells relative to total cells in treated IVDs. Data are presented as mean ± standard deviation. Significant differences were assessed by one-way analysis of variance with post hoc Tukey-Kramer test. IVD: disc REC: fast-growing clones SD: standard deviation ANOVA: analysis of variance [Figure 19] Figure 1. Histological assessment 4 weeks after NP tissue removal. (A) Midsagittal sections (stained with H&E or Safranin-O) of NP tissue removed from the IVD (untreated control or 20, 50, 100, 200 mg removed). In the sheep model, IVD degeneration was associated with removal of ≥20 mg of NP tissue after 4 weeks. Scale bar; 1 mm (all). (B) Histological grade was determined via modified Boos classification. Data are mean ± standard deviation. NP: nucleus pulposus IVD: intervertebral disc H&E: hematoxylin and eosin SD: standard deviation [Figure 20] Figure 1. Measurement of disc height relative to adjacent vertebral disc height using T2-weighted mid-sagittal images. BC and EF are anterior and posterior disc heights, respectively, and AB and DE are cephalad adjacent vertebral body heights. DHI values ​​were calculated as the ratio of disc height (BC + EF) to vertebral body height (AB + DE), and relative DHI values ​​were calculated as the ratio of DHI of treated IVD to DHI of untreated control IVD. DHI: Disc Height Index IVD: Disc DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] 1. Overview The present invention relates to a composition for regenerating an intervertebral disc, comprising a monovalent metal salt of alginic acid and mesenchymal stem cells (e.g., high-purity mesenchymal stem cells derived from human bone marrow). In one aspect of the present invention, the present invention relates to a composition for regenerating an intervertebral disc, comprising a monovalent metal salt of low-endotoxin alginic acid and mesenchymal stem cells (e.g., high-purity mesenchymal stem cells derived from human bone marrow). The composition of the present invention promotes regeneration of the nucleus pulposus of an intervertebral disc through activation of nucleus pulposus cells by mesenchymal stem cells and / or differentiation of mesenchymal stem cells into nucleus pulposus cells. The composition of the present invention is used to apply to the nucleus pulposus site of a subject and harden a portion thereof after application. In some aspects of the present invention, the composition of the present invention is used to apply to the nucleus pulposus site of a subject and contact at least a portion of the surface with a crosslinking agent.

[0020] In this invention, it was demonstrated in an in vivo test using rabbit-derived mesenchymal stem cells that the combined use of bone marrow-derived mesenchymal stem cells (BMSCs) and gel significantly promoted the tissue repair effect compared to gel alone, and the mechanism behind this was revealed to be that co-culture of gel-embedded BMSCs and nucleus pulposus cells led to 1) mutual activation of both cells through the production of growth factors, 2) differentiation of BMSCs into nucleus pulposus cells, and 3) improved ability to produce extracellular matrix from both cells.

[0021] The alginic acid contained in the composition of the present invention is a polysaccharide extracted from brown algae such as Ecklonia cava, Eisenia bifida, and Laminaria kelp, and has the property of crosslinking and hardening when divalent metal ions such as calcium are added. By utilizing this property, it is possible to harden the surface and retain it at the affected area by contacting it with metal ions at the affected area. In the present invention, it was found that when a composition containing low endotoxin sodium alginate in a sol state is injected into the intervertebral disc nucleus pulposus site and a crosslinking agent is contacted with the injection port to harden a part of the composition, the degeneration of the nucleus pulposus is inhibited, the proportion of type II collagen positive cells favorable for nucleus pulposus regeneration is increased, and the regeneration of the intervertebral disc nucleus pulposus is promoted.

[0022] In the present invention, by embedding mesenchymal stem cells in a monovalent metal salt of alginic acid (e.g., low-endotoxin alginic acid), the embedded mesenchymal stem cells remain in the affected area and exert their effects for a long time. That is, the embedded high-purity mesenchymal stem cells produce growth factors and extracellular matrix, and activate the nucleus pulposus cells. The activated nucleus pulposus cells then produce growth factors and extracellular matrix. As a result, the embedded mesenchymal stem cells differentiate into nucleus pulposus cells, and regeneration of the damaged area is promoted through the interaction between the nucleus pulposus cells and the mesenchymal stem cells. In another embodiment of the present invention, the cells can be embedded in a biocompatible material such as hyaluronic acid.

[0023] Furthermore, one embodiment of the mesenchymal stem cells contained in the composition of the present invention, namely human bone marrow-derived highly purified mesenchymal stem cells (REC), are ultra-high purity human mesenchymal stem cells produced by a technique of direct isolation from bone marrow using two types of antibodies and a cell sorter. As a homogeneous cell population is obtained by isolation using a cell sorter, quality control is easy, and as the proliferation rate is extremely high, it is possible to produce pharmaceutical preparations by mass culture.

[0024] The present invention also relates to a composition for regenerating intervertebral discs, comprising human bone marrow-derived highly purified mesenchymal stem cells. In one aspect of the present invention, the composition of the present invention is applied to the intervertebral disc nucleus pulposus in an undifferentiated state and / or without differentiation induction treatment. The composition of the present invention promotes regeneration of the nucleus pulposus of an intervertebral disc through activation of nucleus pulposus cells by human bone marrow-derived highly purified mesenchymal stem cells and / or differentiation of mesenchymal stem cells into nucleus pulposus cells. The composition of the present invention can be used to apply to the nucleus pulposus site of a subject and contact at least a portion of the surface with a crosslinking agent. In one aspect of the present invention, the composition of the present invention can be used to apply to the nucleus pulposus site of a subject and harden the portion after application.

[0025] 2.Definition "Low endotoxin" and "monovalent metal salt of alginic acid" will be described later. An "intervertebral disc" is a cylindrical tissue located between the vertebrae of the spine. An intervertebral disc is a disk-shaped avascular tissue, and is structured with a nucleus pulposus surrounded by an annulus fibrosus and end plates arranged above and below it. The "nucleus pulposus" is a gel-like tissue that exists in the center of the intervertebral disc, and contains mainly nucleus pulposus cells, an extracellular matrix mainly composed of proteoglycan and type II collagen, and water. It is believed that the nucleus pulposus has extremely low self-repair and regenerative abilities.

[0026] "Nucleus pulposus filling" refers to filling the degenerated, reduced or removed portion of the nucleus pulposus that has been degenerated, reduced or removed due to aging, trauma, infection, or surgical procedures for those reasons (e.g., intervertebral disc nucleotomy (resection)). Note that in this specification, the term "nucleus pulposus filling" is used to mean the same thing as "nucleus pulposus filling," and the "composition for filling nucleus pulposus" of the present invention is synonymous with the "composition for filling nucleus pulposus." The "nucleus pulposus site" refers to the site where the nucleus pulposus is present, the site where degeneration or shrinkage of the nucleus pulposus has occurred, or a defective portion of the nucleus pulposus formed by removing at least a portion of the nucleus pulposus, and also includes the surrounding area of ​​the site where the nucleus pulposus is present.

[0027] A "subject" is a human or a non-human organism, such as birds and non-human mammals (eg, cows, monkeys, cats, mice, rats, guinea pigs, hamsters, pigs, dogs, rabbits, sheep, and horses). By "application" is meant filling the nucleus pulposus site with the composition of the present invention in an amount sufficient to fill the degenerated, reduced, removed, missing, etc. portion of the nucleus pulposus site of the intervertebral disc. "Embedding" refers to suspending or mixing the cells in a biocompatible material, preferably a solution of a monovalent metal salt of alginic acid. The term "partially hardening" is as described below.

[0028] "Containing a monovalent metal salt of low-endotoxin alginic acid" means that the composition of the present invention contains a sufficient amount of a monovalent metal salt of low-endotoxin alginic acid to regenerate nucleus pulposus at the site of application. The term "having liquidity" is defined as follows. The terms "intervertebral disc degeneration and / or intervertebral disc damage" and "treatment, prevention, or suppression of recurrence" are as defined below. "Discogenic pain" means pain caused by the intervertebral disc. "Low back pain" refers to pain occurring around the intervertebral disc, including back pain and buttock pain. "Chronic low back pain" means low back pain that continues for 12 weeks or more from the onset of the pain.

[0029] The composition of the present invention may be provided in a solution state using a solvent, or in a dry state such as a lyophilized body (particularly, a lyophilized powder). When provided in a dry state, the composition of the present invention is used in a state having fluidity such as a solution state using a solvent at the time of application. The solvent is not particularly limited as long as it is a solvent that can be applied to a living body, and examples of the solvent include water for injection, purified water, distilled water, ion-exchanged water (or deionized water), Milli-Q water, physiological saline, phosphate buffered saline (PBS), etc. Preferred are water for injection, distilled water, physiological saline, etc. that can be used for the treatment of humans and animals.

[0030] 3. Monovalent metal salts of alginic acid In the present invention, the "monovalent metal salt of alginic acid" used as a carrier is a metal salt in which the hydrogen atom of the 6-position of the carboxylate of alginic acid is replaced by Na + Or K + Alginic acid is a water-soluble salt made by ion exchange with monovalent metal ions such as sodium alginate and potassium alginate. Specific examples of monovalent metal salts of alginic acid include sodium alginate and potassium alginate, with sodium alginate being particularly preferred, which is commercially available. A solution of a monovalent metal salt of alginic acid forms a gel when mixed with a crosslinking agent.

[0031] The "alginic acid" used in the present invention is a biodegradable polymeric polysaccharide, which is a polymer formed by linear polymerization of two types of uronic acid, D-mannuronic acid (M) and L-guluronic acid (G). More specifically, it is a block copolymer in which a homopolymer fraction of D-mannuronic acid (MM fraction), a homopolymer fraction of L-guluronic acid (GG fraction), and a fraction in which D-mannuronic acid and L-guluronic acid are randomly arranged (MG fraction) are arbitrarily bonded. The composition ratio (M / G ratio) of D-mannuronic acid and L-guluronic acid in alginic acid varies mainly depending on the type of organism from which seaweed and the like are derived, and is also influenced by the growing place and season of the organism, and ranges widely from a high G type with an M / G ratio of about 0.4 to a high M type with an M / G ratio of about 5.

[0032] Monovalent metal salts of alginic acid are polymeric polysaccharides, and it is difficult to determine the molecular weight accurately, but if the molecular weight is too low, the viscosity will be low and there is a risk of weak adhesion to the surrounding tissues at the application site, and if the molecular weight is too high, it will be difficult to manufacture and will have problems such as reduced solubility, too high viscosity when made into a solution making it difficult to handle, and difficulty in maintaining physical properties during long-term storage. Therefore, the weight-average molecular weight is generally in the range of 10,000 to 10 million, preferably 20,000 to 8 million, and more preferably 50,000 to 5 million. In this specification, the numerical range indicated using "~" indicates a range that includes the numerical values ​​written before and after "~" as the minimum and maximum values, respectively.

[0033] On the other hand, it is known that the molecular weight measurement of polymeric substances derived from natural products may result in different values ​​depending on the measurement method. For example, the weight average molecular weight measured by gel permeation chromatography (GPC) or gel filtration chromatography (collectively referred to as size exclusion chromatography) is preferably 100,000 or more, more preferably 500,000 or more, and preferably 5 million or less, more preferably 3 million or less, according to the effects shown in the examples of the present invention. The preferred range is 100,000 to 5 million, more preferably 500,000 to 3.5 million.

[0034] Also, for example, the absolute weight average molecular weight can be measured by a GPC-MALS method combining gel permeation chromatography (GPC) and a multi-angle light scattering detector (Multi Angle Light Scattering: MALS). According to the effects shown in the examples of the present invention, the weight average molecular weight (absolute molecular weight) measured by the GPC-MALS method is preferably 10,000 or more, more preferably 80,000 or more, even more preferably 90,000 or more, and preferably 1,000,000 or less, more preferably 800,000 or less, even more preferably 700,000 or less, and particularly preferably 500,000 or less. The preferred range is 10,000 to 1,000,000, more preferably 800,000 to 800,000, even more preferably 90,000 to 700,000, and particularly preferably 90,000 to 500,000.

[0035] Usually, when the molecular weight of a polymeric polysaccharide is calculated by the above-mentioned method, a measurement error of 10 to 20% or more may occur. For example, if the molecular weight is 400,000, the value may vary within a range of 320,000 to 480,000, if the molecular weight is 500,000, the value may vary within a range of 400,000 to 600,000, and if the molecular weight is 1,000,000, the value may vary within a range of 800,000 to 1,200,000.

[0036] The molecular weight of the monovalent metal salt of alginic acid can be measured by a conventional method. Representative conditions for using gel permeation chromatography for molecular weight measurement are as described in the Examples of this specification. For example, a GMPW-XL×2+G2500PW-XL (7.8 mm ID×300 mm) column can be used, for example, a 200 mM sodium nitrate aqueous solution can be used as the eluent, and pullulan can be used as the molecular weight standard.

[0037] Representative conditions for using GPC-MALS for molecular weight measurement are as described in the Examples of this specification. As a detector, for example, an RI detector and a light scattering detector (MALS) can be used.

[0038] When monovalent metal salts of alginic acid are first extracted from brown algae, they have a large molecular weight and a high viscosity, but the molecular weight decreases and the viscosity decreases during processes such as drying by heat and refining. Monovalent metal salts of alginic acid with different molecular weights can be produced by techniques such as controlling the conditions of the production process, such as the temperature, selecting the brown algae used as the raw material, and fractionating by molecular weight during the production process. Furthermore, it is possible to produce a monovalent metal salt of alginic acid with the desired molecular weight by mixing it with a different lot of monovalent metal salt of alginic acid with a different molecular weight or viscosity.

[0039] The monovalent metal salt of alginic acid used in the present invention is preferably a solution having a concentration of 1 w / w% dissolved in MilliQ water, and the apparent viscosity measured at 20°C using a cone-plate viscometer is preferably 40 mPa·s to 800 mPa·s, more preferably 50 mPa·s to 600 mPa·s. The apparent viscosity is preferably measured under the conditions described below. In this specification, the "apparent viscosity" may be simply referred to as "viscosity".

[0040] The alginic acid used in the present invention may be either naturally derived or synthetic, but is preferably naturally derived. Examples of naturally derived alginic acid include those extracted from brown algae. Brown algae containing alginic acid are found in abundance in coastal areas around the world, but the seaweed that can actually be used as a raw material for alginic acid is limited, and representative examples include Lessonia in South America, Macrocystis in North America, Laminaria and Ascophyllum in Europe, and Durvillea in Australia. Examples of brown algae that can be used as a raw material for alginic acid include the genera Lessonia, Macrocystis, Laminaria (kelp), Ascophyllum, Durvillea, Eisenia, and Ecklonia.

[0041] In another embodiment of the present invention, other biocompatible materials may be used as carriers for embedding cells. For example, one or more natural or synthetic glycosaminoglycans (GAGs) or mucopolysaccharides, such as hyaluronic acid (HA), chondroitin sulfate, dermatan sulfate, keratin sulfate, heparin, heparan sulfate, galactosaminoglycuronglycan sulfate (GGGS), including pharma- ceutically acceptable salts (physiological salts) thereof, may be used as carriers for embedding cells. In another embodiment of the present invention, the carriers for embedding cells may be any one or more selected from, but not limited to, cellulose, cellulose derivatives, agarose, chitin, chitosan, starch, pectin, and other polysaccharides, gelatin, collagen, polypeptides, and other proteins, amino acid derivatives, copolymers thereof, and derivatives thereof. In another embodiment of the present invention, hydrogels, such as collagen and gelatin-like compositions, may be used as carriers.

[0042] 4. Low endotoxin treatment The monovalent metal salt of alginic acid used in the present invention is not particularly limited, and may be, for example, a monovalent metal salt of alginic acid with low endotoxin. In one embodiment of the present invention, the carrier for embedding cells is preferably low in endotoxin. That is, the monovalent metal salt of alginic acid used in the present invention is preferably a monovalent metal salt of alginic acid with low endotoxin. Low endotoxin means that the endotoxin level is low enough to not substantially cause inflammation or fever. More preferably, it is desirable to use a monovalent metal salt of alginic acid that has been treated to reduce endotoxin.

[0043] The endotoxin reduction treatment can be carried out by a known method or a method similar thereto. For example, the purification of sodium hyaluronate can be carried out by the method of Suga et al. (see, for example, Japanese Patent Application Laid-Open No. 9-324001), the purification of β1,3-glucan by Yoshida et al. (see, for example, Japanese Patent Application Laid-Open No. 8-269102), the purification of biopolymer salts such as alginate and gellan gum by Williams et al. (see, for example, Japanese Translation of PCT International Publication No. 2002-530440), the purification of polysaccharides by James et al. (see, for example, the pamphlet of International Publication No. 93 / 13136), the method of Lewis et al. (see, for example, the specification of U.S. Patent No. 5,589,591), the purification of alginate by Herman Frank et al. (see, for example, Appl Microbiol Biotechnol (1994) 40:638-643), or methods similar thereto. The low endotoxin treatment of the present invention is not limited to these, and may be carried out by known methods such as washing, filtration with a filter (endotoxin removal filter, charged filter, etc.), ultrafiltration, purification using a column (endotoxin adsorption affinity column, gel filtration column, column with ion exchange resin, etc.), adsorption to hydrophobic substances, resins or activated carbon, organic solvent treatment (extraction with organic solvent, precipitation / sedimentation by adding organic solvent, etc.), surfactant treatment (see, for example, JP-A-2005-036036, etc.), or by appropriately combining these. These treatment steps may be appropriately combined with known methods such as centrifugation. It is desirable to appropriately select the method according to the type of alginic acid, etc.

[0044] The endotoxin level can be confirmed by known methods, for example, a method using Limulus Alkaline Lympholysate (LAL) or a method using Entospecy (registered trademark) ES-24S set (Seikagaku Corporation).

[0045] The method for treating endotoxins in the monovalent metal salt of alginic acid contained in the composition of the present invention is not particularly limited, but as a result, when endotoxin is measured using a Limulus reagent (LAL), the endotoxin content of the monovalent metal salt of alginic acid is preferably 500 endotoxin units (EU) / g or less, more preferably 100 EU / g or less, particularly preferably 50 EU / g or less, and particularly preferably 30 EU / g or less. Low-endotoxin treated sodium alginate is available, for example, under the trademark Sea Matrix (Mochida Pharmaceutical Co., Ltd.), PRONOVA TM It is available commercially as a product such as UP LVG (FMC BioPolymer). Such low-endotoxin treated sodium alginate is also referred to herein as "high-purity sodium alginate" (UPAL).

[0046] 5. Preparation of Solutions of Monovalent Metal Salts of Alginic Acid The composition of the present invention may be prepared using a solution of a monovalent metal salt of alginic acid. The solution of a monovalent metal salt of alginic acid can be prepared by a known method or a method similar thereto. That is, the monovalent metal salt of alginic acid used in the present invention can be produced by a known method such as an acid method or a calcium method using the above-mentioned brown algae. Specifically, for example, alginic acid can be obtained by extracting from these brown algae using an alkaline aqueous solution such as an aqueous sodium carbonate solution, and then adding an acid (e.g., hydrochloric acid, sulfuric acid, etc.), and a salt of alginic acid can be obtained by ion exchange of alginic acid. As described above, low endotoxin treatment is performed. The solvent for the monovalent metal salt of alginic acid is not particularly limited as long as it is a solvent that can be applied to a living body, and examples thereof include purified water, distilled water, ion-exchanged water, Milli-Q water, physiological saline, and phosphate-buffered saline (PBS). These are preferably sterilized, and low endotoxin treatment is preferable. For example, Milli-Q water can be used after sterilization by filtration.

[0047] Even when the composition of the present invention is provided in a dry state such as a lyophilized form, it can be prepared into a flowable solution using the above-mentioned solvent. In addition, all operations for obtaining the composition of the present invention are desirably carried out in an environment with low endotoxin and bacterial levels. For example, the operations are preferably carried out on a clean bench using sterilized instruments, and the instruments used may be treated with a commercially available endotoxin remover.

[0048] 6. Apparent Viscosity of the Composition of the Present Invention The composition of some embodiments of the present invention is in a fluid liquid state, i.e., in a solution state. The composition of the present invention has fluidity when applied to the nucleus pulposus site. In one embodiment of the present invention, the composition of the present invention preferably has fluidity that allows the composition to be injected with a 21G injection needle after being left to stand at 20°C for 1 hour. The apparent viscosity of the composition of the present invention in this embodiment is not particularly limited as long as the effect of the present invention can be obtained, but if the viscosity is too low, the adhesion to the surrounding tissues at the application site may be weakened, so it is preferably 10 mPa·s or more, more preferably 100 mPa·s or more, even more preferably 200 mPa·s or more, and particularly preferably 500 mPa·s or more. If the apparent viscosity is too high, the handling may be poor, so it is preferably 50000 mPa·s or less, more preferably 20000 mPa·s or less, and even more preferably 10000 mPa·s or less. When the apparent viscosity is 20000 mPa·s or less, application with a syringe or the like can be more easily performed. However, even if the apparent viscosity is 20000 mPa·s or more, it can be applied using a pressurized or electric filling device or other means. The preferred range of the composition of the present invention is 10 mPa·s to 50000 mPa·s, more preferably 100 mPa·s to 30000 mPa·s, even more preferably 200 mPa·s to 20000 mPa·s, even more preferably 500 mPa·s to 20000 mPa·s, and particularly preferably 700 mPa·s to 20000 mPa·s. In another preferred embodiment, it may be 500 mPa·s to 10000 mPa·s, or 2000 mPa·s to 10000 mPa·s. The composition of some embodiments of the present invention has a viscosity that can be applied to a target using a syringe or the like.

[0049] The apparent viscosity of a composition containing a monovalent metal salt of alginic acid, such as an aqueous solution of alginic acid, can be measured according to a conventional method. For example, the apparent viscosity can be measured using a coaxial double cylinder rotational viscometer, a single cylinder rotational viscometer (Brookfield type viscometer), a cone-plate type rotational viscometer (corn-plate type viscometer), etc., in the rotational viscometer method. It is preferable to follow the viscosity measurement method of the Japanese Pharmacopoeia (16th edition). In the present invention, it is preferable to measure the viscosity at 20°C. When the composition of the present invention contains a substance that does not dissolve in a solvent, such as cells, as described below, in order to accurately measure the viscosity, it is preferable that the apparent viscosity of the composition is the apparent viscosity in a state in which the cells are not contained.

[0050] In the present invention, it is more preferable to use a cone-plate viscometer to measure the apparent viscosity of a composition containing a monovalent metal salt of alginic acid. For example, it is preferable to measure under the following measurement conditions. The sample solution is prepared using MilliQ water. The measurement temperature is 20°C. The rotation speed of the cone-plate viscometer is 1 rpm when measuring a 1% solution of monovalent metal salt of alginic acid, and 0.5 rpm when measuring a 2% solution, and is determined based on this. The reading time is measured for 2 minutes when measuring a 1% solution of monovalent metal salt of alginic acid, and the average value from 1 minute to 2 minutes is taken. When measuring a 2% solution, the measurement is performed for 2.5 minutes, and the average value from 0.5 minutes to 2.5 minutes is taken. The test value is the average value of three measurements.

[0051] The apparent viscosity of the composition of the present invention can be adjusted, for example, by controlling the concentration, molecular weight, or M / G ratio of the monovalent metal salt of alginic acid. The apparent viscosity of a solution of a monovalent metal salt of alginic acid is high when the concentration of the monovalent metal salt of alginic acid in the solution is high, and low when the concentration is low. Also, the viscosity is high when the molecular weight of the monovalent metal salt of alginic acid is high, and low when the molecular weight is low.

[0052] Since the apparent viscosity of a solution of a monovalent metal salt of alginic acid is affected by the M / G ratio, for example, an alginic acid having a preferred M / G ratio can be appropriately selected depending on the viscosity of the solution, etc. The M / G ratio of the alginic acid used in the present invention is about 0.1 to 5.0, preferably about 0.1 to 4.0, and more preferably about 0.2 to 3.5. As mentioned above, since the M / G ratio is mainly determined by the type of seaweed, the type of brown algae used as the raw material affects the viscosity of the solution of the monovalent metal salt of alginic acid. The alginic acid used in the present invention is preferably derived from brown algae of the genus Lessonia, Macrocystis, Laminaria, Ascophyllum, or Durvillea, more preferably from brown algae of the genus Lessonia, and particularly preferably from Lessonia nigrescens.

[0053] 7. Mesenchymal stem cells The mesenchymal stem cells used in the present invention are somatic stem cells derived from mesodermal tissue (mesenchyme), and are expected to be applied to regenerative medicine, such as the reconstruction of bones, blood vessels, and cardiac muscle. Mesenchymal stem cells can be obtained from various tissues such as bone marrow, adipose tissue, placental tissue, dental pulp, or umbilical cord tissue. The purification process is, for example, as follows. A small amount of fat fragments collected from humans or non-human mammals (e.g., cows, monkeys, cats, mice, rats, guinea pigs, hamsters, pigs, dogs, rabbits, sheep, and horses, goats, rabbits, etc.) are enzymatically treated to obtain a mixed population of cell types. A floating fat cell population is separated by centrifugation, and fibroblast-like cells that sink and grow on the bottom surface when left in contact with the ceiling surface of a culture vessel filled with culture medium are then proliferated by subculture. In the present invention, it is also possible to use mesenchymal stem cells derived from iPS cells or commercially available mesenchymal stem cells. In the present invention, the mesenchymal stem cells are preferably applied to the nucleus pulposus in an undifferentiated state and / or without treatment for induced differentiation. An undifferentiated state means that the differentiation-potential stem cells are maintained in an undifferentiated state. "Without treatment for differentiation induction" means, for example, that a treatment such as differentiation of the differentiation-potential stem cells into a specific cell using a differentiation-inducing medium is not performed.

[0054] 8. Highly purified mesenchymal stem cells derived from human bone marrow (1)REC Clone In previous studies, the present inventors succeeded in isolating rapidly expanding cell clones (RECs) from mesenchymal stem cells positive for LNGFR (CD271) (CD271+ cells) or mesenchymal stem cells co-positive for LNGFR (CD271) and Thy-1 (CD90) (CD271+CD90+ cells).

[0055] These RECs can reach confluence in two weeks when seeded individually onto a 96-well plate and cultured, and have over 1,000 times the proliferation, differentiation, and migration capabilities of mesenchymal stem cells obtained by conventional methods. In particular, because they retain their migration capabilities, they can be administered intravenously, and are expected to be useful for treating severe systemic diseases such as osteochondrodysplasia. In the present invention, among the above-mentioned REC clones, cell clones with less variation in differentiation potential and proliferation potential can be used.

[0056] The cell population containing the cell clone of the present invention is a population of mesenchymal stem cell clones that are co-positive for LNGFR (CD271) and Thy-1 (CD90) and rapidly proliferate, and satisfies at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scatter in flow cytometry is 40% or less. (b) The average cell size is less than 20 μm.

[0057] (2) Method for enriching human mesenchymal stem cells In the present invention, mesenchymal stem cells that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90) can be obtained according to the method described in WO2009 / 31678, for example. The method is outlined below.

[0058] First, mesenchymal stem cells are highly enriched by selecting a cell fraction positive for LNGFR (CD271) (CD271+) or co-positive for CD271 and CD90 (CD271+CD90+) from a cell population containing human mesenchymal stem cells. If the cell population containing human mesenchymal stem cells contains blood cells, a step of selecting cells co-negative for CD45 and CD235a (CD45-CD235a-) may be added to select non-blood cells.

[0059] Cell populations containing mesenchymal stem cells can be prepared by flow cytometry or affinity chromatography. The material for obtaining this cell population is not particularly limited, but examples thereof include bone marrow, adipose tissue, umbilical cord blood, peripheral blood (including peripheral blood after administration of G-CSF), etc. In addition, bone marrow from the spine, sternum, ilium, etc. may be used. In addition, examples of cells include ES cells and iPS cells.

[0060] During cell preparation, if the material is in the form of a cell mass involving mesenchymal stem cells, the material can be subjected to physical treatment such as pipetting, or enzyme treatment with trypsin, collagenase, etc. If the material is contaminated with red blood cells, it is preferable to hemolyze the red blood cells in advance. The cell population prepared as described above is used to select CD271+ cells or CD271+CD90+ cells.

[0061] Methods for selecting CD271 + cells or CD271 + CD90 + cells include, for example, methods using antibodies. The antibodies are anti-CD271 and / or anti-CD90 antibodies that can select CD271+ cells or CD271+CD90+ cells. When using flow cytometry for selection, live cells can be selected in a short time by using anti-CD271 antibodies labeled with different fluorescent dyes such as FITC, PE, and APC, or by using an appropriate combination of anti-CD271 and anti-CD90 antibodies. In addition to flow cytometry, CD271+CD90+ cells can be selected by methods using magnetic beads or affinity chromatography. Before using these methods, dead cells may be removed by reacting a cell population with a fluorescent dye (e.g., PI) that stains dead cells and removing the fluorescently stained cells.

[0062] (3) REC cell enrichment Next, the selected LNGFR-positive cells or LNGFR and Thy1 co-positive cells are cultured as single cells (clones), and by selecting the lot with the fastest proliferation, high-purity human mesenchymal stem cells (REC: Rapidly Expanding Clone) with excellent proliferation, differentiation, and migration capabilities are obtained.

[0063] Mononuclear cells are prepared from human bone marrow or adipose tissue / placental chorion, and bone marrow mononuclear cells are stained with anti-LNGFR alone or with anti-LNGFR and anti-Thy1. Next, LNGFR-positive cells or LNGFR-positive and Thy1-positive cells are clonally sorted into a 96-well culture plate using flow cytometry (cell sorter). That is, one cell is seeded per well. After two weeks of single cell culture, the culture plate is photographed under a microscope, wells that have become confluent or semi-confluent are selected, and the cells contained in the wells are designated as REC.

[0064] Here, "fast proliferation" and "rapid proliferation" mean that when one cell is seeded into each well of a 96-well culture plate and cultured, the cell has a proliferation rate such that the culture plate becomes confluent or semi-confluent 2 weeks or earlier after the start of culture (doubling time is 26±1 hours).

[0065] Confluence is a state in which 90% or more of the culture vessel surface (culture surface) is covered with cultured cells. Semi-confluence is a state in which 70-90% of the culture vessel surface (culture surface) is covered with cultured cells. The size and type of culture equipment used can be changed appropriately depending on the cell growth rate. Moderately / Slowly Expanding Cells, that is, cells that do not become semi-confluent or confluent even after 2 weeks of single cell culture, are discarded. The RECs collected from each well selected as RECs are transferred to a culture flask for each well and further cultured until they become confluent (expansion culture). The expanded cells are then collected separately. RECs from one well are counted as one lot.

[0066] The RECs used in the present invention are obtained by clonal sorting in which one cell is seeded per well, and therefore the genetic traits of the expanded cells are all identical. Therefore, in the present invention, the entire cell population may be referred to as a "clone," or each individual cell constituting the cell population may be referred to as a "clone."

[0067] In the present invention, the REC to be used for selection can be evaluated in advance using a REC marker (anti-Ror2). For example, after the expansion culture, the attached and proliferated cells are collected from all the lots, and a portion of each lot (1 to 3 × 10 5 A total of about 100 cells (approximately 1000 cells) are selected and single stained with an anti-Ror2 monoclonal antibody. A method for single staining with an anti-Ror2 monoclonal antibody is known (WO2016 / 17795). In summary, the ratio of REC marker-positive cells in the collected cells is determined by flow cytometry analysis using the REC marker. The ratio may be determined by quantifying the mRNA expression of Ror2 using quantitative PCR, or the ratio may be determined manually using a microscope. A lot (cell population) with the positive ratio above a certain value (e.g., 65%) is considered to be acceptable, and can be used for the selection described below.

[0068] (4) Selection of stem cell populations In the present invention, by examining the cell proliferation ability, fat differentiation ability, expression level of REC-specific markers, and uniformity of cell size for each lot of REC clone and analyzing the correlation between each, it has become possible to select RECs with high purity and higher cell performance. In the present invention, the coefficient of variation (CV value) of forward scattered light and the average size of cells are used as indices for selection. Forward scatter is light that is scattered at a small angle in the forward direction relative to the axis of the laser light. Forward scatter is made up of scattered light, diffracted light, and refracted light of the laser light generated on the cell surface, and provides information about the size of the sample.

[0069] The coefficient of variation (CV) is the standard deviation divided by the mean, and is used to relatively evaluate the variation in data with different units, or the relationship between the data and the variation in relation to the mean. In the present invention, the above CV value is selected to be 40% or less, preferably 35% or less. A cell population with a CV value of 40% or less, more preferably 35% or less, is a cell population composed of cells of uniform size. Preferably, the CV value is 30% or less, 25% or less, or 20% or less. Furthermore, the average size of cells in the cell population selected by the present invention is 20 μm or less. Preferably, it is 18 μm or less, and is in the range of 14 μm to 18 μm.

[0070] The present invention also provides a method for evaluating the quality of a cell population of LNGFR-positive cells or a fast-growing mesenchymal stem cell clone that is co-positive for LNGFR (CD271) and Thy-1 (CD90). In the present invention, a cell population that satisfies at least one of the following characteristics (a) and (b), preferably both characteristics, is determined to be of high quality. (a) The coefficient of variation of forward scatter in flow cytometry is 40% or less. (b) The average cell size is less than 20 μm. The cell population thus evaluated and selected is not limited in the number of cell clones constituting the population. For example, 0.8×10 7 ~1.2X10 7 It has about 10 cells.

[0071] In a preferred embodiment of the present invention, a cell population that satisfies at least one of the following characteristics (a) and (b), preferably both of these characteristics, is determined to be of high quality. (a) The coefficient of variation of forward scatter in flow cytometry is 35% or less. (b) The average cell size is less than 20 μm. The cell population thus evaluated and selected is not limited in the number of cell clones constituting the population. For example, 0.8×10 7 ~1.2X10 7 It has about 10 cells.

[0072] 9. Preparation of the Compositions of the Invention The composition of the present invention is characterized by containing, for example, a monovalent metal salt of low endotoxin alginic acid and the above-mentioned mesenchymal stem cells as active ingredients. The present inventors have found for the first time that when the composition of the present invention is filled into the nucleus pulposus site of a living body, the monovalent metal salt of alginic acid itself exerts a regenerative or therapeutic effect on nucleus pulposus tissue. The term "containing as an active ingredient" means that the monovalent metal salt of low endotoxin alginic acid is contained in an amount that can exert a regenerative or therapeutic effect on nucleus pulposus tissue when applied to the affected area, and is preferably at least 0.1 w / v% or more of the entire composition, more preferably 0.5 w / v% or more, and even more preferably 1 w / v%. The preferred concentration of the monovalent metal salt of alginic acid in the composition of the present invention is preferably 0.5 w / v% to 5 w / v%, more preferably 1 w / v% to 5 w / v%, even more preferably 1 w / v% to 3 w / v%, and particularly preferably 1.5 w / v% to 2.5 w / v%. In another embodiment, the concentration of the monovalent metal salt of alginic acid in the composition of the present invention may be preferably 0.5 w / w% to 5 w / w%, more preferably 1 w / w% to 5 w / w%, even more preferably 1 w / w% to 3 w / w%, and particularly preferably 1.5 w / w% to 2.5 w / w%.

[0073] When a composition is prepared as described above using a monovalent metal salt of alginic acid that has been purified to exhibit a preferred endotoxin level, the endotoxin content of the composition is typically 500 EU / g or less, more preferably 300 EU / g or less, even more preferably 150 EU / g or less, and particularly preferably 100 EU / g or less.

[0074] The number of cells (cell concentration) contained in the composition of the present invention is, for example, 1 × 10 4 / ml or more, or 1×10 5 / ml or more, preferably 1 x 10 4 pieces / ml~1×10 7 The cells are counted per ml.

[0075] The composition of the present invention may also contain a factor that promotes cell growth. Examples of such factors include BMP, FGF, VEGF, HGF, TGF-β, IGF-1, PDGF, CDMP (cartilage-derived-morphogenetic protein), CSF, EPO, IL, PRP (platelet rich plasma), SOX, and IF. These factors may be produced by recombinant methods or purified from protein compositions. Some embodiments of the composition of the present invention do not contain these growth factors. Even if growth factors are not contained, the regeneration of the nucleus pulposus is sufficiently good, and is safer than the case where cell growth is actively promoted.

[0076] The composition of the present invention may also contain a factor that inhibits cell death. Factors that cause cell death include, for example, caspase and TNFα, and factors that inhibit them include antibodies and siRNA. These factors that inhibit cell death may be produced by recombinant methods or may be purified from protein compositions. Note that the composition of some embodiments of the present invention does not contain these factors that inhibit cell death. Even if the composition does not contain a factor that inhibits cell death, the regeneration of the nucleus pulposus is sufficiently good, and is safer than when cell death is actively inhibited.

[0077] In one embodiment of the present invention, the composition of the present invention does not contain any component that exerts a pharmacological effect on the nucleus pulposus tissue of the intervertebral disc other than the low endotoxin monovalent metal salt of alginic acid. Even a composition containing only the low endotoxin monovalent metal salt of alginic acid as an active ingredient can exert a sufficient regenerative or therapeutic effect on the nucleus pulposus. In some embodiments of the present invention, the composition of the present invention may contain, as necessary, other medicament active ingredients, or ingredients normally used in pharmaceuticals, such as conventional stabilizers, emulsifiers, osmotic pressure adjusting agents, buffers, isotonicity adjusting agents, preservatives, soothing agents, coloring agents, etc.

[0078] 10. Curing of the composition of the present invention The compositions of the present invention are used to harden the area following application to the nucleus pulposus site. "Partial hardening" refers to contacting a part of the composition of the present invention having fluidity with a crosslinking agent, and gelling and hardening a part of the composition that has come into contact with the crosslinking agent, but not the whole of the composition. Preferably, the composition of the present invention is partially hardened by contacting at least a part of the surface of the composition of the present invention having fluidity with the crosslinking agent. In the present invention, a crosslinking agent and hardening means suitable for the carrier to be used can be selected.

[0079] In some embodiments of the present invention, "partially hardening the composition after application to the nucleus pulposus site" may be demonstrated by filling a 6 mm diameter test tube in vitro with 500 μL of low-endotoxin sodium alginate and a crosslinking agent using the same method and ratio of crosslinking agent as used for filling the nucleus pulposus site, and leaving it for 1 hour, whereby at least 50% of the volume of the composition in the test tube has not gelled, and the ungelled portion can be aspirated using a syringe equipped with a 21 G needle.

[0080] Since the composition shows such properties even after being filled into the nucleus pulposus site, it is considered that the composition will not escape even if a compressive force is applied from the cranial and caudal sides of the intervertebral disc after filling. "At least a part of the surface of the composition" is, for example, an opening on the surface of the intervertebral disc that leads to the nucleus pulposus, and is preferably an opening on the surface of the intervertebral disc that is used to apply the composition to the nucleus pulposus site, that is, a filling port of the composition. By gelling and solidifying at least a part of the surface of the composition, it is possible to effectively prevent the composition from leaking out of the intervertebral disc. The filling port of the composition on the surface of the intervertebral disc is, for example, an opening used for filling the composition on the surface of the intervertebral disc with a syringe needle or scalpel, or an opening on the surface of the intervertebral disc made with a scalpel or the like when removing a herniated disc. In this embodiment, the intervertebral disc is preferably an annulus fibrosus.

[0081] The composition of the present invention preferably does not contain a crosslinking agent in an amount that hardens the composition before application to the nucleus pulposus site of the subject. Therefore, the composition of the present invention may contain a crosslinking agent in an amount that does not harden the composition even after a certain period of time has elapsed. The certain period of time here is not particularly limited, but is preferably about 30 minutes to 12 hours. The fact that the composition "does not contain a crosslinking agent in an amount that hardens the composition" may be shown, for example, by the fact that the composition can be left to stand at 20°C for 1 hour and then injected with a syringe equipped with a 21G injection needle. The composition of some embodiments of the present invention does not contain a crosslinking agent.

[0082] The crosslinking agent is not particularly limited as long as it can crosslink the solution of the monovalent metal salt of alginic acid to fix the surface. 2+ , Mg 2+ , B.A. 2+ , Sr 2+ Examples of the divalent or higher metal ion compounds include CaCl2, MgCl2, CaSO4, BaCl2, etc., and examples of the crosslinking reagent having 2 to 4 amino groups in the molecule include diaminoalkanes that may have a lysyl group (-COCH(NH2)-(CH2)4-NH2) on the nitrogen atom, that is, diaminoalkanes and derivatives in which the amino group is substituted with a lysyl group to form a lysylamino group, and specific examples include diaminoethane, diaminopropane, N-(lysyl)-diaminoethane, etc., but it is particularly preferable to use a CaCl2 solution for reasons such as easy availability and gel strength.

[0083] In some embodiments of the present invention, the timing of contacting the surface of the composition of the present invention with the crosslinking agent is preferably after the composition of the present invention is applied to the nucleus pulposus site. The method of contacting a part of the composition of the present invention with the crosslinking agent (e.g., divalent or higher metal ion) is not particularly limited, but examples include a method of spraying a solution of divalent or higher metal ion onto the surface of the composition using a syringe, injector (spray), etc. For example, the crosslinking agent may be slowly sprayed onto the filling port of the composition formed on the intervertebral disc for several seconds to 10-odd seconds. After that, if necessary, a process of removing the crosslinking agent remaining near the filling port may be added. The crosslinking agent may be removed by, for example, washing the application site with physiological saline solution or the like.

[0084] The amount of the crosslinking agent used is preferably adjusted appropriately in consideration of the amount of the composition of the present invention applied, the size of the composition filling port on the surface of the intervertebral disc, the size of the application site of the nucleus pulposus of the intervertebral disc, etc. In order to prevent the crosslinking agent from strongly affecting the tissue surrounding the filling port of the composition, the amount of the crosslinking agent used is adjusted so as not to be excessive. The amount of the divalent or higher metal ion used is not particularly limited as long as it is an amount that can solidify the surface of the composition containing a monovalent metal salt of alginic acid. However, for example, when using a 100 mM CaCl2 solution, if the filling port on the surface of the intervertebral disc has a diameter of about 1 mm, the amount of the CaCl2 solution used is preferably about 0.3 ml to 5.0 ml, more preferably about 0.5 ml to 3.0 ml. If the filling port on the surface of the intervertebral disc is made with a scalpel or the like during removal of the herniated disc and has a margin of about 5 mm x 10 mm, the amount of the 100 mM CaCl2 solution used is preferably about 0.3 ml to 10 ml, more preferably about 0.5 ml to 6.0 ml. The amount can be increased or decreased as appropriate while observing the condition of the composition of the present invention at the application site.

[0085] It is known that when calcium is contained in the crosslinking agent, the higher the calcium concentration, the faster the gelation and the harder the gel can be formed. However, since calcium is cytotoxic, if the concentration is too high, it may have a negative effect on the intervertebral disc nucleus pulposus regeneration effect of the composition of the present invention. Therefore, when using, for example, a CaCl2 solution to harden the surface of the composition containing a monovalent metal salt of alginic acid, the concentration is preferably 25 mM to 200 mM, more preferably 50 mM to 150 mM.

[0086] In the present invention, it is preferable to add the crosslinking agent to the composition, leave it for a certain period of time, and then remove the crosslinking agent remaining at the site of addition by washing or the like. The certain period of time for leaving it is not particularly limited, but it is preferable to leave it for about 1 minute or more, more preferably for about 4 minutes or more to gel the surface of the composition. Alternatively, it is preferable to leave it for about 1 minute to about 10 minutes, more preferably for about 4 minutes to about 10 minutes, about 4 minutes to about 7 minutes, and even more preferably for about 5 minutes. During this certain period of time, it is preferable to keep the composition in contact with the crosslinking agent, and the crosslinking agent may be appropriately added so that the liquid surface of the composition does not dry.

[0087] For example, alginate beads are made by dropping a sodium alginate solution into a CaCl2 solution and gelling it. However, alginate beads need to be applied by pressing them against the application site, and they need to be made to fit the size of the application site, which is technically difficult to use in actual clinical practice. In addition, when a CaCl2 solution is used as a crosslinking agent, the Ca ions on the surface of the beads come into contact with the surrounding tissue, which causes problems with calcium cytotoxicity. In contrast, the composition of the present invention is in the form of a solution, so it can be easily applied to any shape of application site, and the entire application site can be covered with the composition, and it has good adhesion to the surrounding tissue. The part of the composition of the present invention that comes into contact with the surrounding tissue can maintain a low calcium concentration, and there is little problem with calcium cytotoxicity. The part of the composition of the present invention that comes into contact with the surrounding tissue is less affected by the crosslinking agent, so the composition of the present invention can easily contact the cells and tissues at the application site. Preferably, the composition of the present invention fuses with the tissue of the living body so that it cannot be distinguished at the application site after about 4 weeks have passed since it is applied to the nucleus pulposus site, and it has high affinity with the living body.

[0088] When the composition of the present invention is applied to the nucleus pulposus site, if a part of the composition is gelled by a crosslinking agent, the composition of the present invention partially hardens at the affected site and is localized in a state of being in close contact with the surrounding tissue, thereby preventing leakage from the nucleus pulposus site. In addition, the composition of the present invention is in close contact with the surrounding tissue, so that the nucleus pulposus regeneration effect of the composition of the present invention is more potently exerted.

[0089] When the entire filling material filled into the nucleus pulposus area is gelled and hardened, the hardened gel escapes from the filling port on the surface of the intervertebral disc when compressive force is applied from the cranial and caudal sides, whereas when the solution-like composition of the present invention is filled into the nucleus pulposus area, there is no escape from the filling port on the surface of the intervertebral disc even when compressive force is applied from the cranial and caudal sides. In other words, when the nucleus pulposus is actually filled with the composition of the present invention, there is little risk of the filled composition leaking out even when pressure is applied from above and below the intervertebral disc.

[0090] Furthermore, when filling the nucleus pulposus site with a hardened gel, there is a risk that the hardened gel will protrude into the spinal canal and cause serious nerve damage, whereas the solution-like composition of the present invention has a low risk of such a problem and a low risk of developing complications.

[0091] In one embodiment of the present invention, the carrier in which the cells are embedded may be used without hardening. Depending on the clinical symptoms and the size and shape of the damaged area, it may also be applied without using a crosslinking agent.

[0092] 11. Application of the composition of the present invention The compositions of the present invention are applied to the nucleus pulposus site of the intervertebral disc of humans or non-human organisms, such as birds and non-human mammals (e.g., cows, monkeys, cats, mice, rats, guinea pigs, hamsters, pigs, dogs, rabbits, sheep, and horses), and are used to promote regeneration of the nucleus pulposus.

[0093] The form of the composition of the present invention is preferably a fluid liquid, that is, a solution. In the present invention, "having fluidity" means that the composition has a property of changing its form into an indefinite shape, and does not necessarily have a property of always flowing like a solution. Preferably, the composition has such fluidity that it can be sealed in a syringe or the like and injected into the nucleus pulposus of the intervertebral disc. In one of several aspects of the present invention, the composition is desirably fluid enough to be injected into the nucleus pulposus of the intervertebral disc with a syringe equipped with a 14G to 26G injection needle after leaving the composition at 20°C for 1 hour, and more preferably with a 21G injection needle. When the composition of the present invention is provided in a dry state such as a freeze-dried body, it can be made into a fluid composition as described above by using a solvent or the like at the time of application.

[0094] The composition of the present invention in the form of a solution can be easily applied to the nucleus pulposus site of the intervertebral disc using a syringe, a gel pipette, a special injection device, a special injector, a filling device, or the like. When the viscosity of the composition of the present invention is high, it is difficult to apply it with a syringe, so a pressurized or electric syringe may be used. Even if a syringe is not used, the composition may be applied to the defective part of the nucleus pulposus with, for example, a spatula or a rod. When injecting with a syringe, it is preferable to use, for example, a needle of 14G to 27G or 14G to 26G.

[0095] The method of applying the composition of the present invention to the nucleus pulposus site is not particularly limited, but preferably, the composition of the present invention can be applied to the nucleus pulposus site after exposing the affected area under direct vision by a known surgical technique, or under a microscope or endoscope using a syringe, a filling instrument, etc. In one preferred embodiment, for example, the needle of a filling instrument may be inserted from the surface of the annulus fibrosus toward the nucleus pulposus site, and the composition of the present invention may be applied.

[0096] Since the composition of the present invention is in the form of a solution, it can be adapted to any shape of nucleus pulposus site, such as a reduced nucleus pulposus, a cavity or defect in the nucleus pulposus site, and can fill the entire reduced nucleus pulposus, cavity or defect. The reduced nucleus pulposus, a cavity or defect in the nucleus pulposus site may be caused by degeneration or damage of the intervertebral disc, or may be caused by removing or aspirating at least a portion of the nucleus pulposus in a surgical operation. It is preferable to apply the composition of the present invention to a nucleus pulposus defect formed by removing at least a portion of the nucleus pulposus.

[0097] The removal of at least a part of the nucleus pulposus is not particularly limited, and may be, for example, a discectomy performed under direct vision, percutaneously, microscopically, or endoscopically. Alternatively, for example, a method (Love method) may be used in which a 2-10 cm incision is made in the back, muscles are detached from the posterior surface of the posterior element of the spine called the vertebral arch, the ligament between the vertebral arch is excised, the nerve and the herniated disc are identified, and the herniated disc compressing the nerve is removed. Alternatively, a method in which a laser is irradiated to the nucleus pulposus to reduce the volume of the nucleus pulposus may be used.

[0098] After application of the composition of the present invention to the nucleus pulposus site, a portion of the composition can be hardened with a crosslinking agent as described above. The amount of the composition of the present invention to be applied may be determined according to the volume of the application site of the nucleus pulposus to be applied, and is not particularly limited, but is, for example, 0.01 ml to 10 ml, more preferably 0.1 ml to 5 ml, and even more preferably 0.2 ml to 3 ml. When the composition of the present invention is applied to a defective site of the nucleus pulposus, it is desirable to inject it so as to sufficiently fill the volume of the defective site of the nucleus pulposus site.

[0099] The number and frequency of application of the composition of the present invention can be increased or decreased depending on the symptoms and effects. For example, it may be applied only once, or it may be applied continuously once a month to once a year. Alginic acid is a substance that does not naturally exist in the bodies of animals, so animals do not possess the enzymes that specifically degrade alginic acid. Although alginic acid is gradually degraded in the animal body by normal hydrolysis, it degrades more slowly than polymers such as hyaluronic acid. In addition, since there are no blood vessels in the nucleus pulposus, when it is filled into the nucleus pulposus, it is expected to have a long-lasting effect.

[0100] Even if the composition of the present invention is not provided together with the cells or growth factors described above, the composition of the present invention may be used in combination with the cells or growth factors described above, cell death inhibitors, other drugs described below, etc. when applied to the nucleus pulposus site. The composition of the present invention, when applied to the nucleus pulposus site, exhibits the effects of inhibiting degenerative changes in the entire intervertebral disc tissue and the nucleus pulposus and promoting regeneration, and is therefore preferably used as a composition for filling the nucleus pulposus of an intervertebral disc.

[0101] A preferred embodiment of the composition of the present invention is a composition for inhibiting degeneration of intervertebral discs, more preferably a composition for inhibiting degeneration of intervertebral disc nucleus pulposus. "Degeneration of intervertebral disc or nucleus pulposus" refers to a state in which the number of cells, water content, extracellular matrix (type II collagen, aggrecan, etc.) of the intervertebral disc are reduced due to aging, resulting in morphological changes and functional decline, and as the condition progresses, the intervertebral disc is no longer able to function as a shock absorber. In this specification, "inhibition of degeneration" refers to the inhibition of degenerative changes compared to the untreated case, and does not necessarily mean that the condition is free of degeneration.

[0102] One embodiment of the composition of the present invention is a composition for regenerating nucleus pulposus. Nucleus pulposus regeneration aims to prevent the accumulation of fibroblast-like cells and regenerate a nucleus pulposus with a high ratio of nucleus pulposus cells, and is intended to regenerate nucleus pulposus tissue rich in type II collagen and proteoglycan. The term "nucleus pulposus regeneration" also includes the inhibition of degeneration of nucleus pulposus. In one preferred embodiment of the present invention, it is desirable that the composition of the nucleus pulposus regenerated by applying the composition of the present invention is close to the composition of a natural normal nucleus pulposus.

[0103] In addition, the composition of the present invention in a preferred embodiment is used for the treatment, prevention, or suppression of recurrence of intervertebral disc degeneration and / or intervertebral disc damage. In this specification, "treatment, prevention, or suppression of recurrence" includes treatment, prevention, suppression of recurrence, reduction, suppression, improvement, elimination, reduction in incidence, delay in onset, suppression of progression, reduction in severity, reduction in recurrence rate, delay in recurrence, alleviation of clinical symptoms, etc. In this specification, "treatment, prevention, or suppression of recurrence" includes relief of (chronic) pain. In a preferred embodiment of the composition of the present invention, particularly when the carrier in which the cells are embedded is used without hardening, it is used to suppress (chronic) pain (particularly lower back pain) associated with intervertebral disc degeneration and / or intervertebral disc damage.

[0104] The preferred embodiments of the composition of the present invention and the method of using the composition are as described above. The degeneration and / or injury of the intervertebral disc is at least one condition or disease selected from the group consisting of, for example, herniated disc, disc disease, degenerative spondylolisthesis, pyogenic discitis, degenerative spondylosis, spinal canal stenosis, lumbar spinal canal stenosis, injury of the intervertebral disc, herniated disc associated with lumbar spinal canal stenosis (also called mixed lumbar spinal canal stenosis). The degeneration and / or injury of the intervertebral disc may be accompanied by lower back pain. The composition of the present invention may be used without hardening the carrier in which the cells are embedded, depending on the clinical symptoms and the size and shape of the damaged area. In such an embodiment, the composition is used to suppress pain associated with intervertebral disc degeneration and / or disc injury, particularly chronic lower back pain.

[0105] 12.Treatment method The present invention provides a method for treating, preventing or suppressing recurrence of intervertebral disc degeneration and / or intervertebral disc injury using the composition of the present invention.Preferably, the treatment method of the present invention is a method for treating, preventing or suppressing recurrence of intervertebral disc degeneration and / or intervertebral disc injury, which comprises applying a flowable composition containing a monovalent metal salt of low endotoxin alginic acid to the nucleus pulposus site of the intervertebral disc of a subject who needs the treatment, prevention or suppression of recurrence, and hardening a part of the applied composition.

[0106] The treatment methods of the present invention may include the step of removing at least a portion of the nucleus pulposus prior to applying the composition of the present invention to the site of the nucleus pulposus. The disc degeneration and / or disc damage is at least one condition or disease selected from the group consisting of disc herniation, discopathy, degenerative spondylolisthesis, pyogenic discitis, degenerative spondylosis, spinal canal stenosis, and disc damage. In some embodiments of the treatment method of the present invention, the disc degeneration and / or disc damage is disc herniation, particularly lumbar disc herniation. In some embodiments of the treatment method of the present invention, the disc degeneration and / or disc damage is disc herniation associated with lumbar spinal canal stenosis (also called mixed lumbar spinal canal stenosis). The disc degeneration and / or disc damage may be chronic lower back pain. The disc degeneration and / or disc damage may be a combination of these conditions or diseases.

[0107] In some embodiments of the present invention, a method for inhibiting degenerative changes in an intervertebral disc is provided using the composition of the present invention. In a preferred embodiment of the present invention, a method for regenerating nucleus pulposus of an intervertebral disc is provided using the composition of the present invention. These methods include applying a flowable composition containing mesenchymal stem cells and a monovalent metal salt of low endotoxin alginic acid to the nucleus pulposus site of a subject's intervertebral disc in need of inhibition of disc degeneration or regeneration of nucleus pulposus, and hardening a portion of the applied composition. The method may include a step of removing at least a portion of the nucleus pulposus before applying the composition of the present invention to the nucleus pulposus site.

[0108] The preferred embodiment of the composition of the present invention, the specific method of applying the composition to the nucleus pulposus site of the intervertebral disc, the method of hardening the composition, the meaning of terms, etc. are as described above. The treatment method of the present invention may be appropriately combined with other treatment methods or therapeutic agents for the intervertebral disc.

[0109] In addition, before, simultaneously, or after applying the composition of the present invention to the nucleus pulposus site, antibiotics such as streptomycin, penicillin, tobramycin, amikacin, gentamicin, neomycin, and amphotericin B, aspirin, nonsteroidal antipyretic analgesics (NSAIDs), anti-inflammatory drugs such as acetaminophen, protease, corticosteroids, and HMG-CoA reductase inhibitors such as simvastatin and lovastatin may be filled. These drugs may be mixed into the composition of the present invention. Or they may be administered orally or parenterally in combination. In addition, muscle relaxants, opioid analgesics, neuropathic pain relievers, etc. may be administered orally or parenterally in combination as necessary.

[0110] The present invention also relates to the use of a low-endotoxin monovalent metal salt of alginic acid for the preparation of a composition of the present invention. The use of the present invention is the use of a monovalent metal salt of low-endotoxin alginic acid to manufacture a composition for treating, preventing or suppressing recurrence of intervertebral disc degeneration and / or intervertebral disc damage, the composition being applied to the nucleus pulposus site of a subject, partially hardening after application, and having flowability when applied to the nucleus pulposus site.

[0111] The present invention further provides a low-endotoxin monovalent metal salt of alginic acid for use in the treatment, prevention, or suppression of recurrence of intervertebral disc degeneration and / or intervertebral disc injury, comprising applying a flowable composition containing mesenchymal stem cells and a low-endotoxin monovalent metal salt of alginic acid to the nucleus pulposus site of the intervertebral disc of a subject in need of treatment, prevention, or suppression of recurrence of intervertebral disc degeneration and / or intervertebral disc injury, and hardening a portion of the applied composition.

[0112] 13. Evaluation of the Composition of the Present Invention The composition of the present invention can be evaluated using a sheep severe intervertebral disc degeneration model newly established by the inventors. The severe intervertebral disc degeneration model can be prepared by (a) removing nucleus pulposus tissue in an amount equivalent to 0.00004% to 0.00005% of the sheep's body weight from the sheep intervertebral disc in the first operation to prepare a degenerated intervertebral disc, and (b) further removing nucleus pulposus tissue in an amount equivalent to 0.00014% to 0.000175% of the sheep's body weight from the degenerated intervertebral disc prepared in (a) four weeks after the first operation.

[0113] In a preferred embodiment, a sheep model of severe intervertebral disc degeneration can be prepared by: (a) removing 20 mg of nucleus pulposus tissue from a sheep intervertebral disc weighing 40 to 50 kg in a first surgery to prepare a degenerated intervertebral disc; and (b) four weeks after the first surgery, removing an additional 70 mg of nucleus pulposus tissue from the degenerated intervertebral disc prepared in (a). The composition of the present invention can be evaluated by the following procedures (a) to (d): (a) in a first operation, 20 mg of nucleus pulposus tissue is removed from a sheep intervertebral disc to prepare a degenerated intervertebral disc, (b) in a second operation, 4 weeks after the first operation, an additional 70 mg of nucleus pulposus tissue is removed from the degenerated intervertebral disc prepared in (a) to prepare a severe intervertebral disc degeneration sheep model, (c) after the second operation, a target composition is administered to the resulting space, and (d) after administration of the composition, the regeneration of the intervertebral disc is evaluated by at least one evaluation method selected from the group consisting of MRI, tissue staining, and immunohistochemical staining (IHC) for the vertebral body and intervertebral disc taken from the degenerated model.

[0114] In the conventional large animal model, a degenerated intervertebral disc model was prepared by partially removing a normal intervertebral disc, which may have led to spontaneous healing of the intervertebral disc. However, this animal model, in which an already degenerated intervertebral disc is partially removed, allows for a more accurate evaluation of the effect of the present embodiment on a degenerated human intervertebral disc.

[0115] Working Example The present invention will be described in more detail below with reference to examples, although the scope of the present invention is not limited to these examples. EXAMPLES

[0116] Treatment of intervertebral disc disorder in rabbit model In this example, a composition comprising bone marrow-derived mesenchymal stem cells (BMSCs) and bioabsorbable ultra-purified alginate (also known as low-endotoxin highly purified alginate, UPAL) gel was used to test the regenerative effect of transplanting BMSCs into a degenerated intervertebral disc (IVD) after IVD resection.

[0117] 1. Materials and Methods 1.1.Animal testing All animal procedures were approved by the Hokkaido University Animal Care and Use Committee (approval number: 13-0051) and were performed in accordance with the approved guidelines. Male Japanese white rabbits (20 weeks old, 3.2–3.5 kg) were obtained from Sankyo Lab Service Co., Ltd. (Tokyo, Japan).

[0118] 1.2. Preparation of rabbit nucleus pulposus cells (NPCs) Nucleus pulposus (NP) samples were obtained from lumbar IVDs (L1 / 2 to L5 / 6; 20 IVDs in total) of four rabbits after euthanasia via intravenous pentobarbital overdose. NPCs were isolated from NP tissue and cultured as previously described [2,5,7,8]. Specifically, under sterile conditions, gelatinous NP tissue was separated from the annulus fibrosus (AF) using microforceps. Tissue specimens were placed in culture medium containing 10% fetal bovine serum (Nichirei Bioscience, Tokyo, Japan), 1% penicillin / streptomycin, and 1.25 mg / ml fungizone (Life Technologies, Waltham, MA, USA) in Dulbecco's modified Eagle's medium (Sigma-Aldrich, St. Louis, MO, USA). No exogenous growth factors were used. Samples were resuspended in medium supplemented with 0.25% collagenase (Wako Pure Chemical Industries, Osaka, Japan), incubated for 4 h at 37°C, 20% O2 and 5% CO2 in a shaking incubator, and isolated by enzymatic digestion. Cells isolated from NP tissues were grown in culture dishes and cultured in the above medium at 37°C with 20% O2 and 5% CO2 in a humidified atmosphere. The medium was changed twice a week, and NPCs were used at passage 2.

[0119] 1.3. Preparation of rabbit allogeneic BMSCs Rabbit allogeneic BMSCs were purchased from Cyagen (Santa Clara, CA, USA; Catalog No.: RBXMX-01,001, Lot No.: 151114I31). TM Rabbit mesenchymal stem cells were used. These cells were tested for characteristics, post-thaw viability, cell cycle, verification of undifferentiated state, and multipotent differentiation potential along osteogenic, chondrogenic, and adipogenic lineages. BMSCs were cultured according to the manufacturer's instructions, medium was changed twice a week, and BMSCs were used at passage 2.

[0120] 1.4. Preparation of UPAL Gels and Three-Dimensional (3D) Cultures In this example, UPAL gel (Mochida Pharmaceutical Co. Ltd., Tokyo, Japan) was used as an alginate scaffold for 3D culture [2]. The purification process of UPAL gel was as previously reported [2]. Specifically, alginate in seaweed was extracted by converting it to water-soluble sodium alginate through a clarification procedure [2]. This alginate solution was highly viscous and was diluted with a large amount of water [2]. The extract was then filtered to separate the sodium alginate solution from the fibrous residue [2]. Acid was added to the solution to isolate high-quality alginate [2].

[0121] A 2% (w / v) UPAL solution in phosphate-buffered saline (Wako Pure Chemical Industries, Ltd.) and 102 mM CaCl2 was prepared for gelation. Prior to 3D culture, BMSCs were fluorescently labeled with 5,6-carboxyfluorescein diactate succinimidyl ester (CFDA-SE; CFDA-SE Cell Proliferation Assay Kit; BIO RAD, Hercules, CA, USA) at a final concentration of 20 mM according to the manufacturer's instructions [9]. Subsequently, labeled BMSCs and unlabeled NPCs were mixed at a 1:1 ratio (1 × 10 6 The cells were embedded in UPAL solution at a ratio of 2 × 10 6 A final cell concentration of 1 × 10 cells / ml was obtained [9,10]. The UPAL / cell mixture was passed through a 22-gauge needle into 102 mM CaCl2 for gelation. The gel beads were cultured in the above medium for 7 days under hypoxic conditions (5% O2 and 5% CO2)

[10] . Furthermore, NPCs and BMSCs were cultured at 1 × 10 6 The gel beads were embedded separately in UPAL solution at a cell concentration of 1000 cells / ml. The cell concentration was set based on a previous study

[11] in which different total cell numbers were used in each group to compare the effects. After gelation, the gel beads were cultured under hypoxic conditions as described above. The experimental groups were as follows: (a) NPC monoculture; (b) BMSC monoculture; and (c) NPC + BMSC coculture.

[0122] After 0 and 7 days of culture, all gel beads were dissolved in 55 mM sodium citrate until the gel and cells were separated according to a previously reported method [8]. For NPC + BMSC cocultures, the harvested cells were sorted using a BD FACSAria III High speed cell sorter with Diva software version 7.0 (BD Biosciences, San Jose, CA, USA) [1,12]. Dead cells and debris were removed, and cells fluorescing at 530 nm were identified as BMSCs, and non-fluorescent cells were identified as NPCs.

[0123] 1.5. RNA extraction and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) The collected NPCs and BMSCs were placed in 1 ml of TRIzol. (TM) (Invitrogen, Carlsbad, CA, USA) and total RNA was extracted from the samples using the RNeasy Mini kit (Qiagen, Valencia, CA, USA). TM Gene Expression Assays and Custom TaqMan TM Real-time qRT-PCR analysis was performed using the Gene Expression Assay (Table 1) (Applied Biosystems, Waltham, MA, USA). Cycle threshold values ​​(Ct) were obtained for each sample and were then compared by 2 -ΔCt The relative mRNA expression of each target gene was calculated based on the Ct value of the housekeeping gene GAPDH using the method [1].

[0124] [Table 1]

[0125] 1.6. In vivo study using rabbit degenerative IVD model A total of 48 rabbits were used for the in vivo study. The sample size was determined based on previous reports [2,7,8] for each of the two time points employed. Thirty-two of the 48 rabbits were randomly selected for qualitative analysis of IVD degeneration (magnetic resonance imaging (MRI), histology, and immunohistochemistry (IHC)), and a total of 80 IVDs were randomly assigned (8 IVDs in each group) to the intact control group, the puncture group (only puncture to create degeneration), the discectomy group (partial discectomy to create a cavity for degenerated IVDs), the gel group (partial discectomy and UPAL gel implantation for degenerated IVDs), and the BMSC+gel group (partial discectomy and BMSC and UPAL gel implantation for degenerated IVDs).

[0126] Frozen sections of NP tissue from four rabbits were used to assess the survival of transplanted BMSCs, and a total of eight IVDs were randomly assigned to intact control and BMSC+gel groups (four IVDs per group). The remaining 12 rabbits were used to assess the expression of HIF-1a, GLUT-1, and Brachyury, which have been proposed as key markers expressed in healthy human NPCs

[13] (using IHC on paraffin sections of rabbit NP tissue). A total of 36 IVDs were randomly assigned to intact control, discectomy, and BMSC+gel groups (four IVDs per group).

[0127] BMSC labeling and embedding in UPAL solution In the in vivo experiment, the same OriCell cells as in the in vitro experiment were used as transplant cells. TM Rabbit mesenchymal stem cells were used at passage 2. BMSCs were labeled with CFDA-SE before transplantation, embedded in 2% UPAL solution, and 1 × 10 6 The final cell concentration was adjusted to 100 cells / ml

[14] .

[0128] 1.8. Creation of IVD degeneration and cell transplantation Twenty-week-old rabbits are not aged sufficiently to mimic the state of the elderly human population, and it is difficult to obtain aged rabbits with uniform aging. Therefore, in this example, we used a rabbit intervertebral disc annulus fibrosus (AF) puncture model to obtain degenerated IVDs [8,15,16]. General anesthesia was achieved by intravenous injection of ketamine (10 mg / kg) and xylazine (3 mg / kg), and anesthesia was maintained with O2 and air (3.0 l / min) mixed with sevoflurane (2–3%) under spontaneous ventilation. IVD degeneration was created by AF puncture using an 18-gauge needle at the L2 / 3 and L4 / 5 IVDs. The L3 / 4 IVD was left untreated as a control.

[0129] Four weeks after IVD puncture, UPAL solution containing BMSCs was transplanted into the sites of degenerated IVDs at L2 / 3 and L4 / 5 [8]. Under general anesthesia, the spine was exposed via an anterolateral retroperitoneal approach. In the discectomy group, gel group, and BMSC+gel group, an IVD cavity was created by puncturing the spine with an 18-gauge needle, and then the degenerated NP tissue was aspirated using a 10-ml syringe, and the remaining NP tissue was removed using microforceps (Nagashima Medical Instruments Co., Ltd., Tokyo, Japan) in the L2 / 3 and L4 / 5 IVDs (approximately 10–12 mg wet weight per IVD). The L3 / 4 IVD was left untreated as a control.

[0130] In the gel group, the IVD defect was filled with 20 μl of 2% UPAL solution using a microsyringe equipped with a 27-gauge needle (Hamilton Medical, Bonaduz, Switzerland), and in the BMSC+gel group, the IVD defect was filled with UPAL solution containing BMSCs. Notably, a 27-gauge needle was used because it has been shown that a 26-gauge needle does not affect cell viability or tissue degeneration [2,7,8,17]. Next, 1 mL of 102 mM CaCl2 was sprinkled on top of the UPAL solution to induce gelation. After 5 min, the surgical wound was washed with saline and closed. A sham operation was performed in the puncture group. The rabbits were sacrificed by intravenous pentobarbital overdose at 4 and 12 weeks after surgery for analysis of IVD degeneration, or at 1, 7, and 28 days after surgery for evaluation of NPC marker expression. To evaluate the effect of human BMSCs on rabbit IVD, similar implantation experiments were also performed using bone marrow-derived human mesenchymal stem cells (hMSC-BM; PromoCell, Heidelberg, Germany; Catalog number: C-12974, Lot number: 412Z022.4).

[0131] 1.9. Assessment of transplanted BMSC survival At weeks 4 and 12 after implantation, the survival of implanted BMSCs was confirmed based on CFDA-SE fluorescent labeling [18,19]. IVDs (intact control and BMSC+gel groups) were cut horizontally in half, frozen in liquid nitrogen, sectioned into 5 mm slices, and then stained with 4',6-diamidino-2-phenylindole (DAPI; Invitrogen; P36935) as a counterstain. Notably, the survival rate of transplanted BMSCs could not be quantified because DAPI-positive cells cannot identify live versus dead NPCs and BMSCs.

[0132] 1.10.MRI analysis T2-weighted midsagittal images of the IVD were obtained at 4 and 12 weeks after surgery using a 7.0-T MR scanner (Varian Unity Inova; Varian Medical Systems, Palo Alto, CA, USA) [2,8,16]. IVD degeneration was graded using the Pfirrmann classification (grade 5 was classified as severe degeneration)

[20] . Quantitative analysis was also performed using analysis software version 12.0 (AnalyzeDirect, Overland Park, KS, USA) to calculate the MRI index. The MRI index (product of NP area and mean signal intensity) was applied to quantify the degeneration of NPs, and quantitative data were expressed as a percentage of the MRI index obtained in untreated control IVDs (relative MRI index) [2,8,16].

[0133] 1.11.Histological analysis After MRI analysis, each IVD was processed for histological staining. Midsagittal sections (5 mm thick) were stained with hematoxylin and eosin (H&E) and Safranin O-Fast Green to evaluate proteoglycan expression [8]. Semiquantitative analysis of IVDs was performed and graded from 0 (normal) to 5 (severely degenerated) [21,22]. Specifically, this histological scale focuses on the morphological changes of AF structures.

[0134] 1.12.IHC analysis Immunohistochemistry (IHC) staining was performed to detect type I and type II collagen at 4 and 12 weeks after surgery [8], and to detect HIF-1α, GLUT-1, and Brachyury at 1, 7, and 28 days after surgery. For type I and type II collagen staining, mouse monoclonal antibodies against type I collagen (Sigma-Aldrich; C2456, RRID: AB_476836) and type II collagen (Kyowa Pharma Chemical, Toyama, Japan; F-57) were applied. Staining was developed with 3,3'-diaminobenzidine hydrochloride (Dako) and Mayer's hematoxylin (Merck, Darmstadt, Germany) as counterstain. For HIF-1α, GLUT-1, and Brachyury staining, DyLight 550-conjugated rabbit polyclonal antibody against HIF-1α (Novus Biologicals, Centennial, CO, USA; NB100-479R, RRID: AB_1642267), PE-conjugated rabbit polyclonal antibody against GLUT-1 (LS Bio, Seattle, WA, USA; LS-A109342-100), and unconjugated rabbit polyclonal antibody against Brachyury (LS Bio; LS-C31179-100, RRID: AB_911118) were applied. Additionally, Alexa Fluor 594-conjugated goat anti-rabbit polyclonal antibody (Invitrogen; A32740) was used as the secondary antibody for Brachyury. Staining was developed with DAPI as a counterstain.

[0135] Cells positive for collagen I and II, HIF-1α, GLUT-1 and Brachyury were counted separately in 5 independent randomly selected fields [2,8]. Fields spanned the width of the NP, including both deep and superficial regions. Values ​​are expressed as percentage of positive cells relative to total cell counts for all endpoints and percentage of CFDA-SE positive cells for NPC marker assessment. All experiments were performed at each time point in 8 IVDs for collagen I and II assessment and 4 IVDs for NPC marker assessment from each treatment group.

[0136] 1.13.Statistical analysis All data are presented as mean ± standard error (SE). One-way analysis of variance (ANOVA) and Tukey-Kramer post-hoc test were performed for multiple group comparisons. Paired t-test was performed for two-group comparisons. All statistical calculations were performed using JMP Pro-version 14.0 statistical software (SAS Institute, Cary, NC, USA) with a significance threshold of p < 0.05.

[0137] 2.Results 2.1. NPC and BMSC co-culture promotes differentiation of BMSC into NPC and production of growth factors and ECM To investigate the effect of NPC and BMSC coculture on each cell type, unlabeled NPC and CFDA-SE labeled BMSC were embedded in UPAL gel for 3D culture. We collected both cell types in the coculture group using a cell sorter. Phosphate buffered saline / cell suspension analysis was performed using forward and side scatter. A P1 gate was drawn in a 2D dot plot (Fig. 1a) to exclude dead cells and debris. Unlabeled NPC and CFDA-SE labeled BMSC were sorted using different gates in a fluorescence vs. side scatter dot plot (Fig. 1b). Thus, a P2 gate was set above the unlabeled cells and a P3 gate was set above the CFDA-SE labeled cells, with a gap between the two gates to avoid cross contamination [1]. Following gel lysis and cell sorting, six types of cells were obtained: (a) NPC control (day 0), (b) NPC monoculture, (c) NPC coculture, (d) BMSC control (day 0), (e) BMSC monoculture, and (f) BMSC coculture. To analyze BMSC differentiation, gene expression of NPC markers HIF-1α, GLUT-1, and Brachyury, growth factors CDMP-1, TGF-β, and IGF-1, and extracellular matrix (ECM) markers type II collagen and aggrecan were evaluated in the six types of cells using qRT-PCR.

[0138] Gene expression of HIF-1α was significantly increased in NPC cocultures compared to NPC controls (p = 0.0218, Tukey-Kramer test) and in BMSC cocultures compared to BMSC controls (p = 0.0041, Tukey-Kramer test) and monocultures (p = 0.0041, 0.0116, Tukey-Kramer test) (Figure 1c). The expression of GLUT-1 in NPC cocultures showed a significant increase compared to NPC controls (p < 0.0001, Tukey-Kramer test) and monocultures (p < 0.0001, Tukey-Kramer test), and in NPC monocultures showed a significant increase compared to NPC controls (p < 0.0001, Tukey-Kramer test). BMSC cocultures showed a significant increase in GLUT-1 expression compared to BMSC controls (p = 0.0189, Tukey-Kramer test) (Fig. 1d). In contrast, gene expression of Brachyury showed no statistically significant difference among the three types of NPC. Furthermore, gene expression of Brachyury was only observed in BMSC cocultures, but not in either BMSC controls or monocultures (Fig. 1e).

[0139] Gene expression of CDMP-1, TGF-β and IGF-1 in NPC cocultures was significantly increased compared to NPC controls (p<0.0001, p<0.0001, p=0.0002, Tukey-Kramer test) and monocultures (p<0.0001, p<0.0001, p=0.0082, Tukey-Kramer test), and expression in NPC monocultures was significantly increased compared to NPC controls (p=0.0179, p<0.0001, p=0.0079, Tukey-Kramer test). Expression in BMSC cocultures was significantly increased compared to BMSC controls (p = 0.0011, p < 0.0001, p < 0.0001, Tukey-Kramer test) and monocultures (p = 0.0015, p = 0.0386, p < 0.0001, Tukey-Kramer test) (Figure 1f-h).

[0140] Expression of type II collagen in NPC cocultures and monocultures was significantly increased compared to NPC controls (p = 0.0036, p = 0.0035, Tukey-Kramer test), and expression in BMSC cocultures was significantly increased compared to BMSC controls (p = 0.004, Tukey-Kramer test) and monocultures (p = 0.0226, Tukey-Kramer test) (Fig. 1i). For aggrecan, gene expression in NPC cocultures was significantly increased compared to NPC controls (p = 0.0047, Tukey-Kramer test) and monocultures (p = 0.0392, Tukey-Kramer test), and expression in monocultures was significantly increased compared to NPC controls (p = 0.0274, Tukey-Kramer test). Notably, aggrecan was expressed only in BMSC cocultures, but not in BMSC controls or monocultures (Fig. 1j).

[0141] 2.2. Survival of transplanted BMSCs In the in vivo study, CFDA-SE-labeled BMSCs were observed in the BMSC + gel group, but not in the intact control group at 4 and 12 weeks after surgery (Figure 2). The human BMSC group also showed similar findings to the BMSC + gel group (rabbit BMSCs), confirming that the transplanted BMSCs survived in the IVD at 12 weeks after transplantation. Upon incision, no gel extrusion was observed.

[0142] 2.3. Combination of BMSCs and UPAL gel promotes IVD regeneration after discectomy in degenerative IVD Degenerative changes in the treated IVDs were qualitatively analyzed by MRI, capturing T2-weighted midsagittal images (Fig. 3a). The Pfirrmann grade in the BMSC+gel group was significantly lower than that in the discectomy group at week 4 (p=0.003, Tukey-Kramer test), and significantly lower in the puncture and discectomy groups at week 12 (p=0.0009, p<0.0001, Tukey-Kramer test). Furthermore, the degree of degeneration in the gel group was also significantly lower than that in the discectomy group at week 12 (p=0.0028, Tukey-Kramer test) (Fig. 3b). No significant differences were observed between the gel and BMSC+gel groups. The MRI index of the BMSC+gel group was significantly higher than that of the discectomy group at 4 weeks (p=0.0085, Tukey-Kramer test) and the puncture, discectomy and gel groups at 12 weeks (p=0.0002, p<0.0001, p=0~0248, Tukey-Kramer test). Also, the index of the gel group was significantly higher than that of the discectomy group at 12 weeks (p=0.0092, Tukey-Kramer test) (Fig. 3c). Notably, no significant differences were observed in either the Pfirrmann grade or the MRI index between the rabbit BMSC group and the human BMSC group.

[0143] Prior to histological classification, the overall structure of the IVD, including the NP and AF, was evaluated to observe any gross differences between the groups. Histological evaluation of the IVD revealed that the intact control specimens showed a typical oval shape of the NP tissue without structural collapse of the internal AF (Figure 4a and b). In the discectomy group, the medial AF was collapsed and fibrotic changes of the NP tissue were observed at both 4 and 12 weeks. However, the medial AF in the BMSC + gel group appeared to be well preserved at both 4 and 12 weeks with minimal fibrotic changes of the NP tissue, and the medial AF in the gel group also appeared to be relatively well preserved (Figure 4a and b). The histological degeneration scores of the BMSC+gel group were significantly lower than those of the puncture group (p=0.0006, p<0.0001, Tukey-Kramer test), discectomy group (p<0.0001, p<0.0001, Tukey-Kramer test), and gel group (p=0.00,134, p<0.0001, Tukey-Kramer test) at both 4 and 12 weeks, and the degeneration score of the gel group was significantly lower than that of the discectomy group at 12 weeks (p=0.0006, Tukey-Kramer). No significant difference was observed between the rabbit BMSC group and the human BMSC group. No osteophyte formation was observed in any group.

[0144] 2.4. Combination of BMSCs and UPAL gel promotes ECM production in degenerated IVDs Next, we evaluated ECM production in the IVD. In particular, type II collagen is an essential component required for IVD function, whereas increased synthesis of type I collagen is observed during IVD degeneration

[23] . The percentage of type II collagen positive cells was significantly higher in the BMSC+gel group than in the puncture and discectomy groups at 4 weeks (p=0.0001, p<0.0001, Tukey-Kramer test), and significantly higher than the puncture, discectomy, and gel groups at 12 weeks (p<0.0001, p<0.0001, p<0.0001, Tukey-Kramer test). Furthermore, the gel group showed a significantly higher rate than the discectomy group at 4 weeks (p<0.0001, Tukey-Kramer test), and at 12 weeks was significantly higher than the puncture group and discectomy group (p=0.0231, p<0.0001, Tukey-Kramer test) (Figure 5).

[0145] On the other hand, the percentage of type I collagen positive cells was significantly lower in the BMSC+gel group than in the puncture group (p<0.0001, p<0.0001, Tukey-Kramer test), discectomy group (p<0.0001, p<0.0001, Tukey-Kramer test), and gel group (p<0.0001, p<0.0001, Tukey-Kramer test) at both 4 and 12 weeks. The percentage was also significantly lower in the gel group than in the discectomy group at 4 weeks (p<0.0001, Tukey-Kramer test), and significantly lower than in the puncture and discectomy groups at 12 weeks (p=0.0007, p<0.0001, Tukey-Kramer test). No significant differences were observed between the rabbit BMSC group and the human BMSC group in both evaluations by IHC analysis.

[0146] 2.5. Differentiation of transplanted BMSCs into NPCs in degenerated IVDs Finally, we investigated the mechanism by which transplanted BMSCs differentiate into NPCs in vivo by observing HIF-1α, GLUT-1, and Brachyury-positive cells over time (Fig. 6a-c). In the intact control group, almost all cells were positive for the three NPC markers at all time points. In the BMSC+gel group, HIF-1α, GLUT-1, and Brachyury-positive cells were observed at low levels on day 1, but the number of positive cells increased over time. The proportion of cells positive for the three NPC markers relative to the total number of cells was significantly higher on day 28 compared with days 1 and 7 (p<0.0001, p<0.0001, Student's t-test). In contrast, in the discectomy group, approximately 20% of cells were positive at all time points (Fig. 6d-f). Similarly, the ratio of cells positive for the three NPC markers to the number of CFDA-SE positive cells (representing transplanted BMSCs) was significantly higher on day 28 compared to days 1 and 7 (p<0.0001, p<0.0001, Student's t-test) (Figure 6g-i).

[0147] 3. Discussion Since the regenerative capacity of IVD is significantly low, the defect caused by discectomy may result in insufficient tissue repair, leading to further IVD degeneration [2]. In this study, UPAL gel alone inhibited IVD degeneration compared to after discectomy, but the combination of BMSCs and UPAL gel exerted a stronger effect of inducing IVD regeneration. Furthermore, in human BMSCs and rabbit BMSCs, IVD regeneration was observed equally between the two groups. Several previous studies have also demonstrated the regenerative ability of various BMSCs in degenerated IVD [18, 24, 25]. Furthermore, UPAL gel exhibits high biocompatibility and sufficient biomechanical properties to support endogenous repair treatment strategies [2], providing an optimal environment for IVD regeneration and prevention of further degeneration after transplantation of BMSCs into degenerated IVD sites.

[0148] In this example, gene expression of NPC markers, growth factors, and ECM was significantly increased in 3D cocultures of NPCs and BMSCs compared to each 3D monoculture in vitro. These results indicate that coculture of NPCs with BMSCs leads to BMSC differentiation into NPCs, which in turn leads to mutual activity between NPCs and BMSCs, resulting in enhanced ECM production in both cell types. This is consistent with the findings of several other in vitro studies showing that coculture of NPCs with BMSCs stimulates BMSCs to differentiate into an NPC-like phenotype [1,12,26] and supports the mutual activation between NPCs and BMSCs via upregulation of growth factor production [1,11,27] and ECM synthesis [1,5,10,26].

[0149] Furthermore, similar results were observed in vivo, showing increased expression of NPC markers in implanted BMSCs over time and enhanced ECM production compared to results after discectomy alone. To our knowledge, no reports have examined the mechanism by which BMSCs differentiate into NPCs when transplanted with alginate and BMSCs in vivo, although one study demonstrated that transplanted MSCs embedded in atelocollagen could differentiate into NPCs

[28] . Specifically, autologous BMSCs labeled with green fluorescent protein were transplanted into adult rabbit IVDs, and differentiation of the transplanted cells was measured by IHC analysis. At 48 weeks after transplantation, the transplanted BMSCs were shown to be positive for HIF-1α, GLUT-1, and MMP-2, meaning that the BMSCs had differentiated into cells expressing some of the representative phenotypic characteristics of NPCs

[28] .

[0150] In vivo studies, histological analysis further demonstrated that implantation of gel alone prevented IVD degeneration compared to after discectomy, and the combination of BMSCs + gel exerted a stronger effect on preventing degeneration. This is consistent with the MRI findings suggesting that gel alone preserves IVD water content compared to discectomy, but the combination of BMSCs + gel has a stronger effect on water content preservation. IHC results further suggested that the combination of BMSCs + gel promoted ECM synthesis in degenerated NP tissue, which is important for proper IVD function and leads to the prevention of degeneration progression via reduced production of type I collagen. Furthermore, the histological results of BMSCs + gel compared to the puncture group (degenerated IVD without discectomy) suggested that BMSC transplantation led to IVD regeneration. Together, these results indicate that transplanted BMSCs differentiate into NPCs over time, resulting in IVD regeneration in vivo.

[0151] Regarding AF regeneration, the gel group and BMSC+gel group significantly reduced collagen I production in the NP, but the puncture-induced AF defect was repaired by filling with gel and BMSC+gel. Because IVD degeneration is characterized by the degradation of the NP extracellular matrix [2], in this study we mainly focused on the preservation / regeneration of NP.

[0152] In intervertebral disc cell therapy, there are reports of the risk of transplanted cells leaking from the injection site without engraftment in the intervertebral disc, resulting in the formation of osteophytes [5,29,30]. Therefore, in the case of BMSC transplantation after discectomy, a carrier material is necessary to prevent cell leakage. In the present invention, osteophyte formation was not observed in the BMSC+gel group. The combination of UPAL gel and BMSC has the important characteristic of immediate hardening, and as a result, it is clinically useful in terms of preventing cell leakage.

[0153] Based on our results and previous reports [1,11,12,26-28], we hypothesized that the mechanism of IVD regeneration is as follows. 1) The implanted BMSCs can be localized within the IVD cavity via embedding in UPAL gel without leaking outside the intervertebral disc. 2) The implanted BMSCs produce growth factors and ECM, leading to the activation of existing NPCs. 3) Activated NPCs also increase production of growth factors and ECM. 4) As a result, the implanted BMSCs differentiate into NPCs. 5) Furthermore, BMSCs and pre-existing NPCs activate each other, resulting in IVD regeneration (Figure 7).

[0154] In the present in vitro experiments, if BMSCs and NPCs had fused with each other, cell separation would have been impossible, so we believe that cell binding is unlikely to constitute the main mechanism of action of IVD degeneration. In addition, BMSCs and NPCs were not in direct contact in vivo. Therefore, the underlying mechanism of action may be due to growth factors and / or some important humoral factors that regulate the interaction between BMSCs and NPCs.

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[21] Sakai D, Mochida J, Iwashina T, et al. Regenerative effects of transplanting mesenchymal stem cells embedded in atelocollagen to the degenerated intervertebral disc. Biomaterials 2006;27(3):335-45.

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[23] Le Maitre CL, Pockert A, Buttle DJ, Freemont AJ, Hoyland JA. Matrix synthesis and degradation in human intervertebral disc degeneration. Biochem Soc Trans 2007;35(Pt 4):652-5.

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[29] Li YY, Diao HJ, Chik TK, et al. Delivering mesenchymal stem cells in collagen microsphere carriers to rabbit degenerative disc: reduced risk of osteophyte formation. Tissue Eng Part A 2014;20(9 10):1379-91.

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[31] Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006;8(4):315-7.

[32] Raynaud CM, Maleki M, Lis R, et al. Comprehensive characterization of mesenchymal stem cells from human placenta and fetal membrane and their response to osteoactivin stimulation. Stem Cells Int 2012;2012:658356.

[33] Serigano K, Sakai D, Hiyama A, Tamura F, Tanaka M, Mochida J. Effect of cell number on mesenchymal stem cell transplantation in a canine disc degeneration model. J Orthop Res 2010;28:1267-75. EXAMPLES

[0156] Changes in gene expression by co-culture (in vitro) of nucleus pulposus cells (NPC) and highly purified mesenchymal stem cells (REC) 1. Materials and Methods 1.1. Healthy human intervertebral disc nucleus pulposus cells (NPCs) Healthy human intervertebral disc nucleus pulposus cells (NPCs) were obtained during anterior spinal fusion surgery in a young patient (15.3±3.3 years old) with adolescent idiopathic scoliosis at Hokkaido University Hospital.

[0157] 1.2. Preparation of highly homogeneous and highly pure mesenchymal stem cells (1) Measurement of forward scatter (FSC) CV value by flow cytometry Multiple REC clones that had been prepared in advance by a known method (WO2016 / 17795) were used for measuring the CV value of forward scattered light by flow cytometry. In flow cytometry, FSC is proportional to the surface area or size of a cell. In this example, the CV value of FSC was used as an index to evaluate the variation in cell size. (a) Individual REC clones were stained with PI, and a gate was set on the PI-negative live cell population to exclude dead cells from the analysis. (b) The PI-negative live cell population was expanded on an FSC / SSC cytogram, and a gate (P1) was set on the main cell population to exclude debris and noise from the analysis. (c) The cell population within the P1 gate was expanded on an FSC histogram, a marker (M1) was set, and the CV value was measured.

[0158] (2) Evaluation of cell proliferation and adipogenesis For cell proliferation evaluation, 1x10 5 The REC cells were seeded onto a 100 mm culture dish and cultured at 37°C in a 5% CO2 environment for 5 days, after which the cell count and average cell size were measured using a cell counter. The culture medium used was DMEM medium (FUJIFILM Wako Pure Chemical Industries, Ltd.) supplemented with FBS, basic FGF, Hepes, and Penicillin-Streptomycin. In addition, for the evaluation of fat differentiation potential, 5x10 4 REC cells were seeded in a 24-well plate and cultured at 37°C and 5% CO2 for 2 days, then replaced with fat differentiation induction medium and cultured for another 14 days. After 14 days of culture, Oil Red 0 staining was performed and the lipid droplet area was calculated by image analysis. The fat differentiation induction medium used was the above culture medium supplemented with Dexamethasone, Indomethacin, and IBMX.

[0159] (3) Comprehensive analysis When the FSC CV value, cell proliferation rate (proliferation potential), average cell size, and fat differentiation potential of multiple REC clones were examined, it was found that REC clones with low FSC CV values ​​(CV = 35% or less) had both high proliferation and differentiation potential and a small average cell size (average cell size: 20 μm or less) (Figure 8).

[0160] On the other hand, REC clones with high FSC CV values ​​(CV = 35% or higher) were found to have low proliferation and differentiation abilities and large average cell sizes. The average proliferation rate of clones with a CV value of 30% to 35% was 6.4, that of clones with a CV value of 25% to 30% was 9.2, and that of clones with a CV value of less than 25% was 15.1. The average proliferation rate of clones with an average cell size of 18 μm to 20 μm was 7.0, that of clones with an average cell size of 16 μm to 18 μm was 12.7, and that of clones with an average cell size of less than 16 μm was 21.3. Hereinafter, in this Example, REC clones with low FSC CV values ​​(CV=25% or less) were used.

[0161] 1.3. Preparation of UPAL Compositions Containing REC NPCs and fluorescently labeled high-purity mesenchymal cells (RECs) were divided into individual and mixed groups and co-cultured or cultured alone in a 3D gel of ultra-high-purity alginate (UPAL: provided by Mochida Pharmaceutical). Before culture (day 0) and after 7 days of culture, the 3D gel was dissolved and the cells were separated into the following types using a cell sorter. (a) NPC control group (day 0) (b) NPC single culture group (c) NPC co-culture group (d) REC control group (day 0) (e) REC single culture group (f)REC co-culture group

[0162] The expression of the following genes was measured for the above six groups of cells by quantitative PCR analysis (qRT-PCR). Nucleus pulposus cell markers HIF-1α GLUT-1 Brachyury growth factors CDMP-1 TGF-β IGF-1 extracellular matrix Type II collagen aggrecan The gene expression level was measured in accordance with Example 1.

[0163] 2.Results When RECs were co-cultured with nucleus pulposus cells, differentiation indicators (HIF-1α, GLUT-1, Brachrury) increased in both nucleus pulposus cells and mesenchymal stem cells compared to when they were cultured alone (Figure 9). The same was true for growth factors (CDMP-1, TGFβ, IGF-1) and extracellular matrices (Type II collagen, Aggrecan). It is suggested that nucleus pulposus cells and highly purified mesenchymal stem cells interact with each other and become activated, and that the mesenchymal stem cells differentiate into nucleus pulposus cells. EXAMPLES

[0164] Regenerative effects (MRI analysis) and histological analysis in a sheep intervertebral disc degeneration model 1. Materials and Methods Seven sheep (40-50 kg) were treated according to GLP. The L1 / L2 (disc between the first and second lumbar vertebrae), L2 / L3 (disc between the second and third lumbar vertebrae), L3 / L4 (disc between the third and fourth lumbar vertebrae), and L4 / L5 (disc between the fourth and fifth lumbar vertebrae) were divided into the following four groups. Control group (n=6): No treatment Discectomy group (n=6): only removal of nucleus pulposus tissue Gel group (n=8): After the nucleus pulposus tissue was removed, alginate gel was implanted. REC+gel group (n=8): After the removal of nucleus pulposus tissue, REC and alginate gel were implanted.

[0165] Except for the control group, 20 mg of nucleus pulposus tissue was extracted from the intervertebral disc of each group under anesthesia to induce severe disc degeneration. Four weeks after the initial extraction of nucleus pulposus tissue, an additional 70 mg of nucleus pulposus tissue was extracted under anesthesia and injected into the intradiscal space at a final cell concentration of 1 × 10 6 REC (REC prepared in Example 2, section 1.2) suspended in UPAL at cell / ml (100 μl) was injected. Four weeks after implantation, the sheep were euthanized. The treated discs were analyzed using 3 Tesla magnetic resonance imaging (MRI) to quantitatively evaluate degeneration, and the discs were then histologically evaluated by H&E and Safranin-O staining, and immunohistochemically evaluated for Type II collagen expression. All data are presented as mean ± standard error, and differences between groups were tested by one-way analysis of variance (ANOVA) followed by Tukey-Kramer post hoc test.

[0166] 2.Results After implantation of the gel and REC+Gel into the affected areas of sheep intervertebral discs, the degeneration of the intervertebral disc was evaluated by MRI 4 weeks later. The Pfirrmann classification was significantly lower in the gel and REC+Gel groups than in the untreated group (Discectomy group), and the MRI index was significantly higher in the REC+Gel group than in the untreated group (Discectomy group) (Fig. 10). The histological degeneration score (Boos classification) by tissue staining was significantly lower in the gel and REC+Gel groups than in the untreated group (Discectomy group), and the REC+Gel group was significantly lower when comparing the gel group and the REC+Gel group (Fig. 11). Immunohistochemical analysis showed that the percentage of type II collagen-positive cells was significantly higher in the REC+Gel group than in the untreated group (Discectomy group) and the gel group (Fig. 12).

[0167] Implantation of UPAL gel containing REC inhibited IVD degeneration after discectomy in severely degenerated IVDs, and type II collagen-positive cells were significantly higher in the REC+gel group. Thus, REC combined with UPAL gel was shown to promote spontaneous IVD regeneration. Furthermore, the inventors demonstrated that the combination of REC and UPAL gel promoted endogenous NPC activation along with growth factor and ECM production, promoted differentiation into NPCs, and enhanced IVD regeneration. EXAMPLES

[0168] In this example, we demonstrated that the combination of bone marrow-derived highly purified mesenchymal stem cells (RECs) with UPAL gel enhanced IVD regeneration in situ after discectomy (up to 24 weeks) in a sheep model exhibiting severe IVD degeneration. Furthermore, we co-cultured healthy human NPCs and RECs in a 3D system in UPAL gel to evaluate the mechanisms underlying IVD regeneration. We also performed co-cultures of NPCs and commercial BMSCs, and co-cultures of NPCs and REC cells to compare the effects of RECs and commercial BMSCs on NPCs. Our findings suggest the clinical applicability of the combination of RECs with an in situ hardening gel for the treatment of herniation in degenerative human IVDs.

[0169] 1. Materials and Methods Study Design The study design in this example was as follows. (i) To investigate the mechanism of IVD regeneration via 3D co-culture of human NPCs and RECs in vitro (ii) Comparison of the effects of REC and commercially available human BMSCs on NPCs through 3D co-culture of REC and commercially available human BMSCs with NPCs (iii) Measurement of the mechanical properties of UPAL and REC + UPAL gels (iv) Assessment of the regenerative potential of severely degenerated IVDs after discectomy in vivo (v) Assessment of tumor formation in transplanted IVDs

[0170] We hypothesized that transplantation of RECs embedded in UPAL gel after discectomy would promote spontaneous IVD regeneration. The ethical committee of the Hokkaido University Graduate School of Medicine approved the use of healthy human IVDs. Animal experimental procedures were approved by the Institutional Animal Care and Use Committees of Hokkaido University and Hamri Co., Ltd. (Ibaraki Prefecture) and were performed in accordance with the guidelines recommended by these committees.

[0171] Healthy human NPCs and RECs were co-cultured in a 3D system in UPAL gel to evaluate the mechanisms underlying IVD regeneration. After 0 and 7 days of culture, the expression of NPC markers (including HIF-1α, GLUT-1, and brachyury), growth factors (including CDMP-1, TGF-β, and IGF-1), and ECM components (including type II collagen and aggrecan) were analyzed in all cell types using qRT-PCR (n = 4) (2,9,30,31). Furthermore, NPCs and commercially available BMSCs were co-cultured to compare the effects of RECs and commercially available BMSCs on NPCs.

[0172] Next, the mechanical properties of UPAL and REC + UPAL gels were evaluated using unconfined compression tests. Gel disks were compressed at a constant rate of 0.5 mm / min, and Young's modulus was calculated (n = 4) (3, 15, 32, 33). In the animal study, 14 sheep (total of 56 IVDs) were divided into 4 groups: Untreated control group (4 weeks, n = 6; 24 weeks, n = 6): Untreated group Discectomy group (4 weeks, n = 6; 24 weeks, n = 6): only removal of nucleus pulposus tissue, also known as discectomy group Gel group (4 weeks, n = 8; 24 weeks, n = 8): After the nucleus pulposus tissue was removed, alginate gel was implanted. REC + gel group (4 weeks, n = 8; 24 weeks, n = 8): After the extraction of nucleus pulposus tissue, REC and alginate gel were implanted.

[0173] To generate severely degenerated IVDs, we removed 20 mg of NP tissue from the treated IVDs. We further removed 70 mg of NP tissue from the degenerated IVDs 4 weeks after the first surgery. In preliminary experiments, removal of NP tissue of 20 mg or more in a sheep model caused IVD degeneration after 4 weeks, so the initial amount of tissue to be removed was set to 20 mg (FIG. 19). The amount of tissue removed in the second round was set to 70 mg based on the ratio of the amount removed in normal and degenerated IVDs from previous rabbit model experiments (2, 3). After the second discectomy, a solution containing UPAL or a combination of REC and UPAL was implanted into the intradiscal space. The sheep were euthanized 4 weeks (Example 3) and 24 weeks after implantation. To evaluate IVD degeneration, IVDs were qualitatively analyzed using 3.0-T MRI (2, 3, 15, 37, 38). The IVDs were then stained with H&E and Safranin-O for histological analysis, and for analysis of ECM components, the levels of collagen types II and I were assessed by IHC. Finally, tumorigenesis analysis was performed using tissue specimens (2,3,15).

[0174] 1.2.Statistical analysis Sample size for quantitative data was determined by power analysis using the Tukey-Kramer test with an alpha level of 0.05 and a power of 0.8. Statistical analysis was performed using JMP Pro version 14.0 software (SAS Institute, Cary, NC, USA), and values ​​were considered significant when p<0.05. All data are presented as mean ± SD values. One-way analysis of variance (ANOVA) with Tukey-Kramer post-hoc test was performed for multiple group comparisons. Student's t test or Mann-Whitney U test and Welch's test were used for comparison between two groups. The inventors randomized the samples and were blinded to the samples being tested.

[0175] 1.3. In vitro REC and 3D co-culture of commercially available human BMSCs and human NPCs Preparation of REC In this study, we used frozen GMP-compliant RECs provided by Japan Tissue Engineering Co., Ltd. For comparative analysis of RECs and commercially available human BMSCs, three clones of RECs, namely, REC-02 prototype #003-P6-191220, REC-02 HLWF-1-P6-210114, and REC-02 QRKF-1-P6-210210, were prepared at passage 6. Cells were thawed in a 37°C water bath before 3D culture.

[0176] Preparation of commercially available human BMSCs Commercially available human BMSCs (hMSC-BM; PromoCell, Heidelberg, Germany; C-12974, lot number: 412Z022.4) were obtained as previously described. (2) The characteristics of these cells belonging to osteogenic, chondrogenic, and adipogenic lineages, their post-thaw viability, cell cycle, undifferentiated state, and multipotency were examined. BMSCs were cultured in complete culture medium, i.e., Dulbecco's modified Eagle's medium (low glucose level; 2 mg / mL) containing L-glutamine and phenol red (DMEM; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 20% HyClone fetal bovine serum (FBS; Cytiva, Tokyo, Japan), 1% penicillin / streptomycin, 1.25 mg / mL fungizone (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA), 1% HEPES (Life Technologies, Thermo Fisher Scientific), and 0.1% bFGF (Kaken Pharmaceutical Co., Ltd., Tokyo, Japan), according to the manufacturer's instructions. The medium was changed twice a week, and BMSCs from the fourth passage process were used (a total of six passages was the same as for REC).

[0177] 2. Preparation of UPAL Gels and 3D Cultures UPAL gel (Mochida Pharmaceutical Co. Ltd., Tokyo, Japan) was used as an alginate scaffold for 3D culture ( 2 , 3 ). A 2% (w / v) UPAL solution was prepared in phosphate-buffered saline (PBS; FUJIFILM Wako Pure Chemical Industries) and gelled with CaCl2 solution (102 mM). RECs or commercially available BMSCs were added at a concentration of 1 × 10 6 The 3D cells were mixed with UPAL solution at a final cell concentration of 10 ...

[0178] Analysis of cell proliferation ability After 7 days of 2D or 3D culture, the four types of cells obtained were re-seeded for secondary culture. The cells were harvested at confluence and cell proliferation was evaluated based on doubling time (Td), defined as the time required for the cell number to double. Td was calculated using the following formula: Td =(t2-t1)× ln(2) / ln(N2 / N1) (where N2 and N1 are the numbers of cells at times t2 and t1.)

[0179] Flow cytometry analysis PBS (containing 2% FBS) was added to each of the four cell types to obtain 1 × 10 6A cell suspension was prepared at a final density of 1000 cells / mL. Cells were then stained with anti-CD90 antibody (PE anti-human CD90 (Thy1), BioLegend, San Diego, CA, USA) and anti-CD45 antibody (FITC anti-human CD45, Beckman Coulter, Brea, CA, USA). Staining was also performed with mouse IgG1 antibody (mouse IgG1-PE, BioLegend; mouse IgG1-FITC, Beckman Coulter) as a negative control. Propidium iodide (PI) was used to detect dead cells. Flow cytometry analysis was performed using a CytoFLEX System (Beckman Coulter) to evaluate cell viability and the uniformity and positivity of each cell surface antigen. Data were analyzed using FlowJo software (Becton Dickinson, Franklin Lakes, NJ, USA).

[0180] Preparation of healthy human NPCs Human NP samples were obtained from nine patients (mean age ± SD, 15.3 ± 3.3 years) who underwent anterior spinal fusion surgery for adolescent idiopathic scoliosis at Hokkaido University Hospital. Informed consent was obtained from the participants who participated in this study, and consent forms were written and stored. Samples were obtained at the time of surgical procedures. All IVDs were analyzed preoperatively via MRI and assessed for degenerative changes using the Pfirrmann classification (36). All IVDs were classified as grade 1, suggesting that all samples were non-degenerative IVDs.

[0181] Cells were isolated from human NP samples and cultured as previously described (2, 3). First, each gel-like NP was separated from the AF under a dissecting microscope, and the tissue specimen was placed in culture medium. The preparation was washed twice by centrifugation (1,000 rpm, 3 min) and resuspended in culture medium supplemented with 0.25% collagenase. For cell isolation, the preparation was incubated in a shaking incubator (37°C, 4 h) and then centrifuged twice (1,000 rpm, 3 min). Cells separated from the matrix were placed in 10 cm tissue culture dishes and incubated as described above. The medium was changed twice a week, and NPCs from the fourth passage were used.

[0182] Preparation of RECs and commercially available human BMSCs The above REC (REC-02_prototype #003-P6-191220) and commercially available human BMSCs were also used for 3D co-culture. The two types of cells were cultured in the same manner as the above 2D culture, following the manufacturer's instructions. The medium was changed twice a week. Both cells from the second passage (REC, 8 passages in total; BMSC, 4 passages in total) were used.

[0183] 3D co-culture and monoculture We prepared 2% UPAL solution and used CaCl2 solution (102 mM) for gelation as previously reported (2, 3). Prior to 3D culture, RECs and BMSCs were fluorescently labeled with 20 mM CFDA-SE (CFDA-SE Cell Proliferation Assay Kit; BIO RAD, Hercules, CA, USA) according to the manufacturer's manual (2, 15, 29). Then, labeled cells and unlabeled NPCs were added to the UPAL solution in the same ratio (1 × 10 cells each). 6 cells / mL (2, 31, 49) to a final cell concentration of 2 × 10 6 The cell-UPAL mixture was gelled by inserting it into 102 mM CaCl2 solution using a 22-gauge needle. The resulting gel was cultured for 7 days under hypoxic conditions (5% O2 and 5% CO2) (2, 49). In addition, the three types of cells were cultured at 1 × 10 6The gel beads were mixed separately in UPAL solution at a concentration of 1000000000000000 cells / mL. The cell concentration was selected based on our previously published results (2). After gelation, the gel beads were cultured under hypoxic conditions in the same manner as above, and the following experimental groups were used (n = 4 per group): (a) Monocultured NPCs (b) Monocultured BMSCs (c) Monoculture REC (d) Co-culture NPC+BMSC (e) Co-culture NPC+REC.

[0184] On the first and seventh day of culture, all gel beads were dissolved in 55 mM sodium citrate to separate the gel from the cells. In the coculture group, the collected cells were sorted using a BD FACSAria III High speed cell sorter with Diva software version 7.0 (BD Biosciences, San Jose, CA, USA) (2, 31). After removing debris and dead cells, 530 nm fluorescent cells were selected as RECs and BMSCs, and non-fluorescent cells were selected as NPCs.

[0185] RNA extraction and qRT-PCR The collected 10 cell types (control NPCs, monocultured NPCs, NPCs cocultured with commercial BMSCs, NPCs cocultured with RECs, control BMSCs, monocultured BMSCs, cocultured BMSCs, control RECs, monocultured RECs, and cocultured RECs) were dissolved in 1 mL of TRIzol® (Invitrogen, Thermo Fisher Scientific), and total RNA was extracted from the samples using an RNeasy Mini kit (Qiagen, Valencia, CA, USA). Real-time qRT-PCR was performed using TaqMan® gene expression assays (Applied Biosystems, Thermo Fisher Scientific) (Table 3). Cycle threshold (Ct) values ​​were obtained for each sample. Furthermore, the relative mRNA expression levels of each target gene, NPC marker, growth factor, and ECM component were calculated via the 2‐ΔCt method (2). Expression levels were normalized by the expression level of the housekeeping gene GAPDH (2,31).

[0186] 1.4. Elastic analysis of gels using unconfined compression tests The mechanical properties of UPAL and REC-UPAL gels were evaluated using unconfined compression tests. Two types of disk-shaped gels with a diameter of 4.5 mm and a thickness of 2 mm were prepared (Figure 14(a)). The samples were placed in a tensile-compression mechanical tester (Autograph AG-X; Shimadzu Corporation, Kyoto, Japan) and compressed at a constant rate of 0.5 mm / min using a 100 N load cell until the gels were crushed (Figure 14, B and C) (3, 15). Young's modulus was calculated based on the obtained stress-strain curve (Figure 14D) using an approximation line between compression values ​​of 10 and 20% (n = 4 gels / group) (Figure 14E) (3, 15, 32, 33).

[0187] 1.5. In Vivo Testing in a Sheep Model of Degenerative IVDs In this example, all procedures involving the sheep model were performed in a pharmaceutical GLP-compliant laboratory (Hamri Co., Ltd.) (3). Fourteen male, Suffolk sheep (2 years old, weighing 40-60 kg) were used to perform a qualitative analysis of IVD degeneration (3). A total of 56 IVDs were randomly assigned to untreated control (4 weeks, n = 6; 24 weeks, n = 6), discectomy (4 weeks, n = 6; 24 weeks, n = 6), gel (4 weeks, n = 8; 24 weeks, n = 8), and REC + gel (4 weeks, n = 8; 24 weeks, n = 8) groups. First, NP tissue removal surgery was performed to create a severely degenerated IVD model. Anesthesia was induced by intramuscular injection of a 4:1 mixture of ketamine (0.2 mg / kg) and xylazine (20 mg / kg) at a rate of 0.5 mL / kg, and maintenance of anesthesia was achieved by inhalation anesthesia (isoflurane). Surgery was performed via a right lateral retroperitoneal approach to expose the vertebral bodies and IVDs from L1 to L5. A solid cancellous screw (ZIMMER BIOMET, Warsaw, IN, USA) was inserted into the L2 vertebral body as a vertebral landmark. In the discectomy group, gel group, and REC + gel group, 20 mg of NP tissue was extracted after AF incision (5 × 3 mm) to induce IVD degeneration (Figure 15, A,B) (3,8).

[0188] Removal and transplantation of degenerated NP tissue Four weeks after the initial surgery, the vertebral bodies and IVDs were exposed using the same approach. We further extracted 70 mg of NP tissue from the degenerated IVDs obtained from the three treatment groups to create IVD cavities after AF incision, as described above (Figure 15C). After discectomy, the gel group was implanted with 110-120 μL of 2% UPAL solution into the intradiscal space, while the REC + gel group was implanted with a mixture of REC and UPAL solution (final concentration, 1 × 10 6 The sheep were then euthanized with pentobarbital at 4 and 24 weeks after implantation, and the lumbar spinal column was excised en bloc (3).

[0189] MRI T2-weighted midsagittal section images were obtained using a 3.0-T MR scanner (MAGNETOM Prisma; Siemens, Munich, Germany). To evaluate the signal changes of the treated IVDs, we scored the degree of IVD degeneration using the Pfirrmann classification (36), which includes five grades (1: normal to 5: severely degenerated). Furthermore, we measured the MRI index value (the product of the mean signal intensity of the NPs and the NP area) and quantified the brightness of the NP tissue using Analyze 14.0 software (AnalyzeDirect, Overland Park, KS, USA). Furthermore, we evaluated the relative MRI index, which is the ratio of the MRI index in the untreated control group, in all three treatment groups (2, 3, 15, 37, 38). We also measured the DHI, which is the ratio of the disc height to the height of the adjacent vertebral body on the cranial side (4, 39). We determined the relative DHI, which is the percentage value of the DHI in the untreated control group.

[0190] Histological analysis After MRI, samples were fixed in 10% formaldehyde, desalted in 10% EDTA (pH 7.5), and embedded in paraffin. Sagittal 5-micrometer-thick paraffin sections were deparaffinized in xylene, treated with alcohol, rinsed in water, and stained with H&E and Safranin-O. The degree of IVD degeneration was scored from 0 (normal) to 36 (severely degenerated) using a modified Boos' classification (3, 40, 41).

[0191] IHC The expression of collagen types II and I in the IVD was determined by IHC. Sections were deparaffinized in xylene and treated with 0.1% trypsin for 30 min for antigen activation. Sections were then treated with 3% H2O2 in methanol for 10 min, followed by protein blocking for 30 min using protein block serum-free solution (DAKO, Agilent, Santa Clara, CA, USA). Goat anti-collagen type I (1:40; Southern Biotech, Birmingham, AL, USA) was used with anti-collagen type I antibody, and anti-hCL(II) and purified IgG (1:400; Kyowa Pharma Chemical Co., Ltd., Toyama, Japan) were used as primary antibodies with anti-collagen type II antibody. After washing the cells with PBS, EnVision+ System-HRP-labeled polymer anti-mouse (DAKO) for collagen type II and Histofine® Simple Stain Max PO(G) (Nichirei Biosciences, Tokyo, Japan) for collagen type I were used as secondary antibodies. Finally, the sections were stained with DAB (DAKO) and hematoxylin. The number of collagen type II and collagen type I positive cells was determined in five randomly selected fields, and the positive cell rate in the total cells was calculated (2, 3, 15).

[0192] Tumorigenesis analysis To assess tumorigenesis of transplanted and pre-existing cells in the transplanted IVD, evaluation of (i) invasive growth, (ii) karyocytes, (iii) binucleated cells, and (iv) nucleoli was performed. During the 24-week evaluation period, the total number of positive cells was counted using H&E stained specimens in the untreated control and REC + gel groups in 15 randomly selected visualization fields (15 visualization fields at 400x magnification; 26.5 visualization fields, totaling 5 mm 2 ) and the numbers per square millimeter were calculated.

[0193] 2.Results 2.1. Results of in vitro REC and 3D co-culture of commercially available human BMSCs and human NPCs Comparison of RECs with commercially available human BMSCs The details of the stability confirmation results for RECs and commercially available human BMSCs before and after incorporation into the gel are shown in Tables 2 and 3.

[0194] The ratio of PI-unstained cells to the total number of cells was defined as cell viability. There was no significant difference in the ratio between RECs and BMSCs (p = 0.6087, Table 2). The coefficient of variation (CV) of forward scatter, which is proportional to the cell diameter, was calculated to evaluate the uniformity of cell size. The %CV value was significantly lower in RECs than in BMSCs (p = 0.0366, Table 2). Cell proliferation ability was evaluated by calculating Td. Td was significantly shorter in RECs than in BMSCs (p = 0.0074, Table 2). Cell surface antigen confirmation test showed that the percentage of CD90-positive cells was significantly higher in RECs than in BMSCs (p = 0.0004). No significant difference was observed in CD45-positive cells between the groups (p = 0.0907, Table 2).

[0195] We further compared REC and commercial BMSCs after 1 week of culture (Table 3). There was no significant difference between REC and BMSCs in terms of Td after 2D and 3D culture (p = 0.3339, p = 0.6996, respectively). However, the CD90 positive cell rate was significantly higher in REC than in BMSCs in both 2D and 3D culture (p = 0.0039, p = 0.0007, respectively). As for CD45 positive cells, the rate was significantly lower in REC than in BMSCs after 2D culture (p = 0.0038). There was no significant difference between REC and BMSCs after 3D culture (p = 0.3221). In a 3D culture environment with alginate, which mimics the cellular environment after transplantation into the intervertebral disc in vitro, RECs maintained the expression of cell surface markers characteristic of mesenchymal stem cells compared to BMSCs, indicating that RECs stably maintain the characteristics of mesenchymal stem cells even in the alginate environment.

[0196] [Table 2]

[0197] [Table 3]

[0198] Co-culture of REC with human NP cells (NPCs) We investigated the effect of co-culture of REC with human NPCs and that of co-culture of human NPCs with commercially available human BMSCs, which are different cell types, and compared the two. RECs or BMSCs labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), and unlabeled NPCs were embedded in UPAL gel and cultured in three dimensions (3D) (2,15,27). After 7 days of culture, the gel was dissolved, and debris and dead cells were removed using a cell sorter. The cells were then classified into CFDA-SE positive cells, RECs, BMSCs, unlabeled cells, and NPCs. After cell sorting, the following 10 types of cells were obtained: (a) Control NPCs (uncultured) (b) Monocultured NPCs (c) NPCs co-cultured with commercially available BMSCs (d) NPCs cocultured with RECs (e) Control BMSCs (uncultured). (f) Monocultured BMSCs (g) Co-cultured BMSCs (h) Control REC (non-cultured). (i) Monoculture REC (j) Co-culture REC

[0199] To analyze the differentiation, cell-cell interactions, and ECM production of BMSCs and RECs, NPC markers (including HIF-1α, GLUT-1, and brachyury), growth factors (including CDMP-1, TGF-β, and IGF-1), and ECM components (including type II collagen and aggrecan) expression were measured by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) (Table 4) (2, 9, 30, 31).

[0200] [Table 4]

[0201] In NPCs cocultured with RECs, the expression levels of three NPC markers, HIF-1α, GLUT-1, and brachyury, were significantly higher than those in control NPCs, monocultured NPCs, and NPCs cocultured with commercially available BMSCs. The GLUT-1 expression level was significantly higher in NPCs cocultured with commercially available BMSCs than in control NPCs (Figure 13, D-F). The expression levels of CDMP-1 and IGF-1 were significantly increased in NPCs cocultured with RECs compared with those in control NPCs, monocultured NPCs, and NPCs cocultured with commercially available BMSCs. The expression level of TGF-β was significantly increased in NPCs cocultured with RECs compared with those in control and monocultured NPCs. NPCs cocultured with commercially available BMSCs showed a significant increase in CDMP-1 and IGF-1 expression compared with those in control NPCs and monocultured NPCs (Figure 13, G-I). The expression levels of type II collagen and aggrecan were significantly higher in NPCs cocultured with RECs than in control and monocultured NPCs, and the expression levels in NPCs cocultured with commercially available BMSCs were also significantly higher than the expression levels in control and monocultured NPCs ( Fig. 13 , J and K).

[0202] The expression levels of all three NPC markers in cocultured RECs were significantly higher than those in control and monocultured RECs (Fig. S13, D–F). The expression levels of CDMP-1, TGF-β, and IGF-1 were also significantly increased in cocultured RECs compared with those in control and monocultured RECs (Fig. S13, G–I). Similarly, the expression levels of type II collagen and aggrecan were significantly higher in cocultured RECs than those in control and monocultured RECs (Fig. S13, J and K).

[0203] 2.2. Results of elastic analysis of gel using unconfined compression test Effect of REC embedded in UPAL gel To evaluate and compare the mechanical properties of REC-UPAL and UPAL gels, unconfined compression tests were performed (3, 15, 32, 33). Two types of cylindrical gel samples (4.5 mm in diameter and 2 mm in thickness) were prepared, and the axial compressive stress-to-strain ratios (Figure 14, A–C) were calculated. At compression levels of 10–20%, the Young's moduli were 18.0 ± 3.5 kPa and 18.8 ± 2.1 kPa for UPAL and REC-UPAL gels, respectively, and there was no significant difference in the mechanical properties between the two groups (p = 0.6942) (Figure 14, D and E). Moreover, these two values ​​were comparable to those of normal human NP tissues (3, 32).

[0204] 2.3. Results of in vivo studies in a sheep model of degenerative IVDs Combination of REC and UPAL gel enhances IVD regeneration The regenerative potential of IVDs was evaluated in a sheep model after implantation of gel or REC+gel (Figure 15). Sheep have been widely used to study IVDs after discectomy, including biomaterials (3, 34, 35). Fifty-six IVDs from 14 sheep were divided into the following groups: Untreated control group (n = 6 IVD) Discectomy group (n = 6 IVD) Gel group (n = 8 IVD) REC + Gel group (n = 8 IVD)

[0205] To create the IVD degeneration model, 20 mg of NP tissue was removed during the first surgery (FIG. 15, A and B), and the amount to be removed was set to 20 mg based on the results of a preliminary experiment (FIG. 19). Four weeks after the first surgery (after observing degenerative changes), the NP tissue was removed again (FIG. 15C). The IVD defect was then implanted with UPAL or REC + UPAL solution, and gelled by exposure to 102 mM CaCl2 after the second discectomy (FIG. 15D). The implanted lumbar spine was harvested for various analyses 4 and 24 weeks after implantation.

[0206] Degenerative changes in the treated IVDs were evaluated using T2-weighted midsagittal images obtained via MRI (Figure 16A). For morphological analysis of the IVDs, the Pfirrmann score and MRI indices were evaluated to evaluate signal changes in the implanted IVDs (2, 3, 15, 36-38), and the disc height index (DHI). Based on the Pfirrmann score, the degenerative scores in the Gel group were significantly lower than those in the Discectomy group at 24 weeks, and the degenerative scores in the REC + Gel group were significantly lower than those in the Discectomy group and Gel group at both 4 and 24 weeks (Figure 16B).

[0207] The MRI index was significantly higher in the REC + Gel group than in the Discectomy group at week 4. At week 24, the MRI index was significantly higher in the Gel group than in the Discectomy group, and was significantly higher in the REC + Gel group than in the Discectomy and Gel groups (Figure 16C). In addition, the disc height of the treated IVD was measured using MRI images. The ratio of disc height to the height of the upper adjacent vertebral body, i.e., DHI, was measured anteriorly and posteriorly of the IVD. The relative DHI, i.e., the ratio of the DHI values ​​of the three treatment groups to the DHI value of the untreated control group, was then determined (Figure 20) (4, 39). The DHI values ​​of the three treatment groups were significantly lower than that of the untreated control group at both 4 and 24 weeks. There was no significant difference among the three groups at 4 weeks after implantation, but at 24 weeks after implantation, the DHI of the gel group was significantly higher than that of the Discectomy group, and the REC + Gel group was significantly higher than that of the Discectomy group and the Gel group (Figure 16D).

[0208] Histological analysis was performed using hematoxylin and eosin (H&E) and Safranin-O staining. At both 4 and 24 weeks in the untreated control group, the IVD showed a spindle shape, there was no fibrotic change in the NP tissue, and the annulus fibrosus (AF) had a concentric structure and was stained uniformly with Safranin-O. In the discectomy group, significant scar tissue, fibrotic change, tissue loss, and endplate collapse / destruction were observed (Figure 17, A, B). The histological degree of degeneration in the gel group, as determined based on the modified Boos' classification (3, 40, 41), was significantly lower than that in the discectomy group at both 4 and 24 weeks, and the REC + Gel group was significantly lower than that in the discectomy and gel groups at both 4 and 24 weeks (Figure 17C).

[0209] The expression levels of collagen types II and I in the treated IVDs were evaluated by immunohistochemistry (IHC) (Figure 18, A and B). The percentage of collagen types II and I positive cells in the total number of cells in five randomly selected visual fields was evaluated in the NP tissue (2, 3, 15). Type II collagen is an essential ECM component in the NP tissue, and as degeneration progresses, it is replaced by collagen type I. Analysis of collagen type II showed that the NP tissue was stained uniformly in the untreated control group, but was slightly reduced in the REC + gel group. Scattered non-stained areas were observed in the gel group, and extensive non-stained areas were observed in the discectomy group. The percentage of collagen type II positive cells was significantly higher in the REC + gel group than in the gel and discectomy groups at both 4 and 24 weeks, and significantly higher in the gel group than the discectomy group at 24 weeks (Figure 18C). Conversely, the percentage of type I collagen positive cells was significantly lower in the REC + Gel group than in the Discectomy group at the 4-week evaluation period, and significantly lower than in the Discectomy and Gel groups at the 24-week evaluation period. The percentage of these cells was also significantly lower in the Gel group than in the Discectomy group at 24 weeks (Figure 18D).

[0210] Effect of REC+gel injection on the IVD During the 24-week evaluation period, tumor formation was analyzed in histological specimens from the untreated control and REC + gel groups. In all specimens from both groups, invasive growth was evaluated along with mitosis, and the number of binucleated cells and nucleoli per square millimeter were measured. No invasive growth, mitotic figures, or nucleoli were observed in either group. The number of binucleated cells in the untreated control and REC + gel groups was 0.03 ± 0.07 and 0.23 ± 0.25, respectively; i.e., values ​​were <1 / mm2, and no significant differences were observed between the groups (p = 0.3311) (Table 5).

[0211] [Table 5]

[0212] 3. Discussion This example demonstrated the superior properties of REC compared to commercially available human BMSCs in terms of cell proliferation capacity, cell size uniformity, and expression of cell surface antigens. Furthermore, this example demonstrated that the expression levels of NPC markers, growth factors, and ECM components were significantly increased in 3D cocultures of human NPCs and RECs compared to those observed in 3D cocultures of NPCs with commercially available BMSCs. The efficacy of the combination of REC with UPAL gel was observed at the site of IVD degeneration in a sheep lumbar spine model. UPAL gel alone inhibited IVD degeneration compared to the discectomy group, but the combination of REC with the gel enhanced IVD regeneration more effectively.

[0213] In this example, it was suggested that co-culture of RECs with NPCs led to the differentiation of RECs into NPCs, thereby improving the production of ECM components in both cell types. Similar results were observed in rabbits (Example 1), where it was revealed that the expression of NPC markers was increased after transplantation of BMSCs embedded in UPAL gel, and that the production of ECM was increased in the BMSC+UPAL gel group compared to the discectomy group (2).

[0214] Based on the above results and those of previous studies ( 2 , 31 , 47 , 48 , 50 - 52 ), the following possible mechanisms underlying IVD regeneration by transplantation of RECs embedded in UPAL gel were suggested. 1) RECs can be embedded in a gel and transplanted into the IVD defect. 2) RECs produce growth factors and ECM components, thereby activating pre-existing NPCs (paracrine mechanism). 3) NPCs produce more ECM and growth factors. 4) As a result, RECs directly differentiate into NPCs. 5) Finally, RECs and NPCs have a positive intercellular feedback loop that leads to IVD regeneration.

[0215] Biomaterials / hydrogels used for IVD repair need to be biologically and mechanically suitable. In this example, we selected the sheep lumbar model to implant candidate hydrogels in a preclinical animal model because the biomechanics and geometry of the lumbar IVD are comparable to those of humans (3,4,34). We have previously shown that UPAL gel implanted in sheep lumbar IVDs after discectomy exhibits suitable biomechanical properties, does not result in material extrusion, and does not require suturing of the AF after discectomy (3). In this example, unconstrained compression tests revealed no significant difference in Young's modulus between the UPAL and REC-UPAL groups, indicating that REC embedded in the UPAL gel did not change the mechanical properties of the gel. Due to its crucial ability to produce rapid healing, the combination of UPAL gel with REC offers clinical advantages in terms of preventing cell leakage without suturing the AF.

[0216] MRI and histological evaluation in animals implanted with (and without) UPAL gel indicate that IVD degeneration was inhibited after implantation of UPAL gel alone. Furthermore, the combination of REC and UPAL gel prevented degeneration even more effectively compared with UPAL gel alone. There was also no significant difference in the number of binucleated cells between the untreated group and the REC + gel group, suggesting that the combination of REC and gel does not induce tumor formation. Furthermore, the IHC results suggest that the combination of REC and gel inhibited the progression of IVD degeneration by enhancing ECM synthesis and inhibiting the production of type I collagen, which are essential for normal IVD function. These results are consistent with the results obtained using a rabbit model of IVD degeneration (Example 1), which demonstrated that BMSCs embedded in UPAL gel inhibited degeneration more effectively (2). Example 1 also showed that the histological grade score of degeneration after BMSC + UPAL gel implantation was lower compared with animals that did not undergo discectomy, which created a degenerated IVD via AF needle puncture. This suggests that BMSC transplantation led to IVD regeneration (2). Taken together, our findings indicate that transplantation of RECs embedded in UPAL gel enhances IVD regeneration in vivo (2).

[0217] References in Example 4 1. JN Katz, Lumbar disc disorders and low-back pain: Socioeconomic factors and consequences. J. Bone Joint Surg. Am. 88, 21-24 (2006). 2. D. Ukeba, H. Sudo, T. Tsujimoto, K. Ura, K. Yamada, N. Iwasaki, Bone marrow mesenchymal stem cells combined with ultra-purified alginate gel as a regenerative therapeutic strategy after discectomy for degenerated intervertebral discs. EBioMedicine 53, 102698 (2020). 3. T. Tsujimoto, H. Sudo, M. Todoh, K. Yamada, K. Iwasaki, T. Ohnishi, N. Hirohama, T. Nonoyama, D. Ukeba, K. Ura, YM Ito, N. Iwasaki, An acellular bioresorbable ultra-purified alginate gel promotes intervertebral disc repair: A preclinical proof-of-concept study. EBioMedicine 37, 521–534 (2018). 4. SR Sloan Jr, C Wipplinger, S Kirnaz, R Navarro-Ramirez, F Schmidt, D McCloskey, T Pannellini, A Schiavinato, R Hartl, l. J. 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Tryfonidou, CL Maitre, K. Ito, Characterization of biomaterials intended for use in the nucleus pulposus of degenerated intervertebral discs. Acta Biomater. 114, 1-15 (2020). 573 EXAMPLES

[0218] Suppression of inflammation and pain by sodium alginate and mesenchymal stem cells in a rat intervertebral disc nucleus pulposus defect model 1. Creation of a rat intervertebral disc puncture degeneration model A rat disc puncture degeneration model (Mohd Isa et al. Sci Adv 2018) will be used for the experiments involving the administration of test substances (IHC analysis, histological analysis, pain-related behavior analysis). A total of 60 12-week-old female SD rats (260-300 g) will be randomly assigned to the following groups (n=3 per time point per group): skin incision only (sham group), disc puncture only (punch group), disc puncture followed by implantation of UPAL (UPAL group), and disc puncture followed by implantation of UPAL and REC (REC group). Anesthesia will be induced by inhalation of 5% isoflurane, followed by intraperitoneal injection of a mixture of ketamine and medetomidine (ketamine:medetomidine=75 mg / kg : 0.5 mg / kg). After confirming the depth of anesthesia by a tail pinch test using forceps, incise the dorsal skin of Co4 / 5 -5 / 6. Remove the connective tissue to expose Co4 / 5 -5 / 6, and injure the intervertebral disc of Co4 / 5 -5 / 6 using a 19 G needle (diameter 1 mm, depth 2 mm).

[0219] In the Gel and REC groups, dry fibrous UPAL (400-600 mPa / s, Mochida Pharma Co. Ltd., Tokyo, Japan) was dissolved in physiological saline (0.9%, Otsuka) to prepare a 2% (w / v) solution, and 4 μl of the UPAL solution was injected into the center of the intervertebral disc using a microsyringe (Hamilton) equipped with a 26 G needle via a route separate from the puncture site. In the REC group, the concentration was 1x10 6 The surgical site is irrigated with saline and the fascia, connective tissue, and skin are sutured closed.

[0220] 2. Immunohistological Evaluation Immunohistological evaluation of rat intervertebral discs was performed to detect TNF-α, IL-6, and TrkA positive cells 1, 4, 7, and 28 days after surgery. Rats (n=3 per group per time point) were deeply anesthetized with isoflurane inhalation and euthanized by cervical dislocation.

[0221] The entire tail (Co 4 / 5-Co 5 / 6) was surgically removed and the soft tissue was removed under sterile conditions to obtain only the caudal vertebrae and intervertebral discs. The discs were fixed in 4% (w / v) paraformaldehyde (48 h at room temperature) and embedded in paraffin. The specimens were transected at the center of the disc to obtain mid-coronal transverse sections (5 μm thick). The sections were deparaffinized with xylene and then incubated in proteinase K (Dako, Agilent Technologies, Santa Clara, CA, USA) (37°C, 15 min). They were then blocked with 1% hydrogen peroxide in methanol (w / v) (37°C, 30 min), incubated in 2% (w / v) bovine serum albumin (room temperature, 30 min), and incubated with primary antibodies overnight at 4°C. Anti-TNF-α mouse monoclonal antibody (ab220210, Abcam), anti-IL-6 mouse monoclonal antibody (ab9324, Abcam), and anti-TrkA rabbit monoclonal antibody (ab86474, Abcam) will be used.

[0222] For color development, Histofine® Fast Red II (Nichirei Bioscience) was used for TNF-α analysis, HistoGreen Substrate kit for Peroxidase (Cosmo Bio Co., Ltd., Tokyo, Japan) for IL-6 analysis, and Histofine® DAB (Nichirei Bioscience) was used for TrkA analysis. To improve visibility, cell nuclei were counterstained with hematoxylin for TNF-α or TrkA staining and fast red for IL-6 staining, respectively. TNF-α, IL-6, or TrkA positive cells were counted individually in five randomly selected fields using a light microscope (Olympus, Tokyo, Japan), and the number of positive nucleus pulposus or annulus fibrosus cells for each staining was calculated as a percentage of the total number of nucleus pulposus or annulus fibrosus cells in the field. All evaluations were performed by two independent blinded observers. Each observer performed three evaluations for each specimen, and the average value for each specimen was calculated and compared between groups.

[0223] 3. Histological Evaluation Intervertebral disc tissue degeneration was evaluated 28 days after surgery. Rats (n=3) were euthanized in the same manner as for immunohistochemical evaluation, and the intervertebral discs were harvested. The harvested intervertebral discs were fixed in 4% (w / v) paraformaldehyde for 48 hours, decalcified in Kristensen's decalcification solution for 2 weeks, washed in tap water for 24 hours, and embedded in paraffin (Mohd Isa et al. Sci Adv 2018). Sagittal midline specimens (5 μm thick) of rat intervertebral discs were stained with hematoxylin and eosin, safranin O, or Alcian blue (AB) to evaluate the histological score (Rutges et al. Osteoarth Carti 2013). AB staining is not directly related to the score, but is performed for auxiliary purposes because it is a suitable stain for evaluating the extracellular matrix. All evaluations were performed by two blinded independent observers. Each observer will perform three evaluations for each specimen, calculate the individual mean values, and then compare between groups.

[0224] 4. Pain-related behavior analysis Twelve rats (n=3 per group) survived until 28 days after surgery for immunohistological evaluation, and 12 rats (n=3 per group) were used for histological analysis, for a total of 24 rats (n=6 per group) for pain-related behavior analysis. All rats were subjected to Hargreaves, von Frey, and tail flick tests. 24 hours and immediately before each test, each rat was allowed to acclimate to the test environment for 20 min. All tests were performed by the same blinded examiner. Multiple measurements were performed on each rat, and the average values ​​were calculated, and the results obtained were compared between groups.

[0225] 4.1 Hargreaves test The Hargreaves test is performed on the second day (day-2) before surgery and on the second, seventh, fourteenth, and twenty-seventh days after surgery (Mohd Isa et al. Sci Adv 2018) using the Hargreaves test apparatus (Ugo Basile Biological Instruments, Gemonio, Italy). Rats are placed in individual compartments (with an air hole at the top) on a glass plate (Ugo Basile Biological Instruments). An infrared beam is applied to the ventral side of the skin incision as a thermal stimulus. The latency to show escape behavior in response to the thermal stimulus is recorded. The intensity of the beam is set to 50% of the maximum output. A cut-off time of 20 seconds is set to prevent tissue damage. Four measurements are performed at each time point on the same rat, with at least a 1-minute break between each measurement.

[0226] 4.2 von Frey test The von Frey test is performed on the second day before surgery (Day-2) and on the 2nd, 7th, 14th, and 27th days after surgery using a dynamic plantar aesthesiometer (Ugo Basile Biological Instruments). The rat is placed in a small chamber similar to that used for the Hargreaves test, which is set on a wire mesh. A 0.5 mm diameter filament is placed on the ventral side of the skin incision and a linearly increasing force is applied starting from 0 g to 5 g over 10 seconds, after which a constant force of 5 g is applied until 30 seconds after the start of the test. The latency until the rat shows any escape behavior is recorded. Five measurements are performed on the same rat at each time point, with at least a 10 second break between each measurement.

[0227] 4.3 Tail flick test The tail flick test is performed using a heat flux radiometer (Ugo Basile Biological Instruments). To avoid tissue damage due to excessive heat stimulation caused by the same schedule as the Hargreaves test, the test is performed on the first day before surgery (Day-1) and on the 3rd, 8th, 15th, and 28th days after surgery (Mohd Isa et al. Sci Adv 2018). After wrapping each rat in a towel and allowing it to settle for 10 minutes, the tail is placed on the device while the body is covered with a towel, and an infrared beam is irradiated 5 cm proximal to the ventral side of the tail from the distal end. The latency to the tail flick response to the heat stimulation is recorded. The cutoff time is set to 20 seconds to prevent tissue damage. Four measurements are performed at each time point on the same rat, with at least a 15-second break between each measurement.

[0228] 5. Statistical analysis All data are expressed as mean ± standard error (SE). One-way ANOVA was used for multiple group comparisons. Unpaired Student-t test was used for two-group comparisons. All ANOVA results were further evaluated using Tukey-Kramer post-hoc test or Kruskal-Wallis test. Differences were considered statistically significant at the 5% significance level (P < 0.05).

[0229] 6.Results A pain suppression effect was confirmed in the group implanted with UPAL and REC (REC group).

Claims

1. A composition for regenerating an intervertebral disc, comprising a monovalent metal salt of alginic acid and mesenchymal stem cells.

2. The composition according to claim 1, which promotes regeneration of nucleus pulposus of an intervertebral disc through activation of nucleus pulposus cells by mesenchymal stem cells and / or differentiation of mesenchymal stem cells into nucleus pulposus cells.

3. The composition according to claim 1 or 2, wherein the mesenchymal stem cells are highly purified mesenchymal stem cells derived from human bone marrow.

4. The composition of claim 3, wherein the highly purified human bone marrow-derived mesenchymal stem cells are a cell population of rapidly proliferating mesenchymal stem cell clones that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and satisfy at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scattered light in flow cytometry is 40% or less. (b) the average cell size is 20 μm or less;

5. The composition according to claim 3 or 4, wherein the highly purified human bone marrow-derived mesenchymal stem cells are a cell population of rapidly proliferating mesenchymal stem cell clones isolated based on the positivity of LNGFR (CD271) or co-positivity of LNGFR (CD271) and Thy-1 (CD90), and the cell population satisfies at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scattered light in flow cytometry is 40% or less. (b) the average cell size is 20 μm or less;

6. The composition according to any one of claims 1 to 5, which is used to apply to a subject's intervertebral disc and to bring a crosslinking agent into contact with at least a portion of the surface of the composition after application, and which has flowability when applied.

7. The composition according to claim 6 for application to the nucleus pulposus site of an intervertebral disc.

8. The composition according to claim 7 , wherein the application to the nucleus pulposus site is the filling of a defective portion of the nucleus pulposus with the composition.

9. The composition according to any one of claims 6 to 8, wherein the crosslinking agent is a divalent or higher metal ion compound.

10. The composition according to any one of claims 1 to 9, wherein the monovalent metal salt of alginic acid is a low-endotoxin monovalent metal salt of alginic acid.

11. The composition according to any one of claims 1 to 10, wherein the monovalent metal salt of alginic acid has a weight average molecular weight (absolute molecular weight) of 80,000 or more as measured by a GPC-MALS method.

12. The composition according to any one of claims 1 to 11, wherein the concentration of the monovalent metal salt of alginic acid is 0.5 w / w% to 5.0 w / w%.

13. The composition according to any one of claims 1 to 12, which is used for treating, preventing, or suppressing the recurrence of intervertebral disc degeneration and / or intervertebral disc damage.

14. The composition according to claim 13, wherein the intervertebral disc degeneration and / or intervertebral disc damage is at least one selected from the group consisting of intervertebral disc herniation, intervertebral disc disease, degenerative spondylolisthesis, pyogenic discitis, degenerative spondylosis, spinal stenosis, and intervertebral disc damage.

15. A composition for intervertebral disc regeneration comprising a monovalent metal salt of alginic acid and highly purified mesenchymal stem cells derived from human bone marrow, the composition being intended to be applied in a fluid state to the nucleus pulposus site of a subject's intervertebral disc.

16. The composition for intervertebral disc regeneration according to claim 15, which is applied in a fluid state to the site of the intervertebral disc nucleus pulposus of a subject and then used without contacting the composition with a crosslinking agent.

17. The composition according to claim 15 or 16, which promotes regeneration of the nucleus pulposus of an intervertebral disc through activation of nucleus pulposus cells by the human bone marrow-derived highly purified mesenchymal stem cells and / or differentiation of the human bone marrow-derived highly purified mesenchymal stem cells into nucleus pulposus cells.

18. The composition according to any one of claims 15 to 17, wherein the human bone marrow-derived highly purified mesenchymal stem cells are in an undifferentiated state at the time of application and / or are applied without any treatment for induced differentiation.

19. The composition according to any one of claims 15 to 18, wherein the highly purified human bone marrow-derived mesenchymal stem cells are a cell population of fast-proliferating mesenchymal stem cell clones that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and which satisfy at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scattered light in flow cytometry is 40% or less. (b) the average cell size is 20 μm or less;

20. The composition according to any one of claims 15 to 19, wherein the highly purified human bone marrow-derived mesenchymal stem cells are a cell population of rapidly proliferating mesenchymal stem cell clones isolated based on an indicator that they are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and the cell population satisfies at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scattered light in flow cytometry is 40% or less. (b) the average cell size is 20 μm or less;

21. The composition according to any one of claims 15 to 20, wherein the monovalent metal salt of alginic acid is a low-endotoxin monovalent metal salt of alginic acid.

22. The composition according to any one of claims 15 to 21, wherein the monovalent metal salt of alginic acid has a weight average molecular weight (absolute molecular weight) of 80,000 or more as measured by a GPC-MALS method.

23. The composition according to any one of claims 15 to 22, wherein the concentration of the monovalent metal salt of alginic acid is 0.5 w / w% to 5.0 w / w%.

24. The composition according to any one of claims 15 to 23, which is used for treating, preventing, or suppressing the recurrence of intervertebral disc degeneration and / or intervertebral disc damage.

25. The composition according to claim 24, wherein the intervertebral disc degeneration and / or intervertebral disc damage is at least one selected from the group consisting of intervertebral disc herniation, intervertebral disc disease, degenerative spondylolisthesis, pyogenic discitis, chronic lower back pain, degenerative spondylosis, spinal stenosis, lumbar spinal stenosis, intervertebral disc herniation associated with lumbar spinal stenosis (mixed lumbar spinal stenosis), and intervertebral disc damage.

26. 26. The composition of claim 24 or 25, wherein the disc degeneration and / or disc damage is accompanied by chronic lower back pain.

27. The composition according to any one of claims 15 to 23, which is used for suppressing intervertebral disc pain.

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