Composition for intervertebral disc regeneration

Abstract

This invention provides a composition for promoting nucleus pulposus regeneration of intervertebral discs, comprising a monovalent metal salt of low-endotoxin alginate and mesenchymal stem cells. Specifically, the composition of this invention promotes nucleus pulposus regeneration of the intervertebral disc through activation of nucleus pulposus cells induced by high-purity mesenchymal stem cells derived from human bone marrow and / or differentiation of high-purity mesenchymal stem cells derived from human bone marrow into nucleus pulposus cells.

Description

[Technical Field]

[0001] This invention relates to a composition for intervertebral disc regeneration containing a monovalent metal salt of low-endotoxin alginate and mesenchymal stem cells. This invention relates to a composition for intervertebral disc regeneration containing high-purity mesenchymal stem cells derived from human bone marrow. [Previous Technology]

[0002] The human spine (vertebrae) is composed of 24 bones (vertebrae), and the tissue that cushions the vertebrae is called the intervertebral disc. Intervertebral discs can degenerate and become damaged due to aging, trauma, or disease. Degeneration of the intervertebral disc involves a decrease in the number of cells, water content, and extracellular matrix (type II collagen, aggregatin, etc.). If degeneration occurs, the intervertebral disc loses its function as an shock absorber. Specifically, intervertebral disc degeneration and injury include disc herniation, disc disease, degenerative spondylolisthesis, purulent discitis, degenerative spinal diseases, spinal stenosis, and disc damage caused by trauma. For example, in a herniated disc, the annulus fibrosus covering the nucleus pulposus deforms or cracks, causing it to protrude outward from the disc. The nucleus pulposus that protrudes from the deformed or cracked area compresses the spinal nerves, causing pain, numbness, etc.

[0003] One treatment for herniated discs is discectomy (nucleotomy), which has been confirmed to be effective. However, it is known that during discectomy (nucleotomy), because the surgical site is not treated after the procedure, degenerative changes in the disc sometimes occur. If a portion of the nucleus pulposus is removed during discectomy, a cavity (also referred to as a "defect" in this manual) is created in the nucleus pulposus. Because the nucleus pulposus has very low self-repair and regeneration capabilities, the cavity is easily weakened physically. Furthermore, sometimes fibroblast-like cells accumulate in the cavity, forming tissue with different mechanical properties than the original nucleus pulposus. Therefore, the recurrence rate of herniation after discectomy is high. Although the recurrence rate within 5 years after discectomy is said to be around 4-15%, recent long-term data shows that more than half recur after 10 years. Although recurrence would require further surgery, the spinal cord nerves are buried in the scar tissue formed after the first surgery, making it difficult to pinpoint their location.

[0004] To date, a high-purity hardening gel based on alginate has been developed, and it has been proven that even when the gel is applied alone (acellular), the intervertebral disc tissue can be naturally repaired (Japanese Patent No. 6487110, Tsujimoto et al., EBioMedicine 37 (2018) 521-534). Clinical trials have been conducted on patients aged 20 to 49 years with lumbar disc herniation who have undergone herniation removal surgery and have had the gel implanted.

[0005] Furthermore, mesenchymal stem cells (MSCs) are among the most widely used somatic stem cells in clinical applications after hematopoietic stem cells due to the fewer ethical issues associated with cell harvesting and their diverse differentiation capabilities, including bone, cartilage, and fat. Since MSCs can be isolated using relatively simple techniques, they are primarily induced to differentiate into cartilage or bone in experimental tubes before being transplanted locally, making them widely used as materials for regenerative medicine. Then, in the development of clinical applications, the ability to produce the necessary quantity of cells to maintain certain functions becomes a necessary condition for productization.

[0006] The inventors then established a purification and separation method for isolating LNGFR and Thy-1 co-positive cells from bone marrow by flow cytometry to obtain rapidly expanding clone (REC) cells, which can exclude the poor proliferative properties of MSCs derived from donors (Japanese 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] Japanese Patent No. 6487110 [Patent Document 2] Japanese Patent No. 6463029 [Patent Document 3] International Publication No. 2016 / 017795 (Booklet) [Non-Patent Document]

[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]

[0009] [The problem the invention aims to solve]

[0010] As mentioned above, to date, exploratory physician-led clinical trials have been conducted on the development of high-purity sclerosing gels based on alginate, using these gels after lumbar disc herniation removal surgery. However, the nucleus pulposus, which is said to have almost no self-repair and regeneration capabilities, is even less capable of self-repair in middle-aged and elderly individuals, making gel-only treatment for such patients clearly limited. Therefore, it is necessary to develop further components capable of repairing and regenerating intervertebral disc tissue. [Means of Problem Solving]

[0011] The inventors, in order to address the aforementioned problems, discovered that a composition containing a monovalent metal salt of low-endotoxin alginate and mesenchymal stem cells can solve the aforementioned problems, thus completing the present invention. Furthermore, in other embodiments of the present invention, a composition containing high-purity mesenchymal stem cells derived from human bone marrow was discovered, which can solve the aforementioned problems. That is, the present invention is as follows. [1] A composition for intervertebral disc regeneration, comprising a monovalent metal salt of low-endotoxin alginate and mesenchymal stem cells. [1-1] A composition for intervertebral disc regeneration, comprising a monovalent metal salt of alginate and mesenchymal stem cells. [2] The composition described in [1] or [1-1] promotes the regeneration of the nucleus pulposus of the intervertebral disc through the activation of nucleus pulposus cells caused by mesenchymal stem cells and / or the differentiation of mesenchymal stem cells into nucleus pulposus cells. [3] The composition described in any one of [1] to [2], wherein the aforementioned mesenchymal stem cells are high-purity mesenchymal stem cells derived from human bone marrow. [4] The composition described in [3], wherein the aforementioned high-purity mesenchymal stem cells derived from human bone marrow are cell clusters of rapidly proliferating mesenchymal stem cell lines that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and are cell clusters that satisfy at least one of the following characteristics (a) and (b): (a) the coefficient of variation of forward scattering light in flow cytometry is less than 35%, and (b) the average cell size is less than 20 μm. [4-1] The composition described in [3], wherein the aforementioned high-purity mesenchymal stem cells derived from human bone marrow are cell groups of rapidly proliferating mesenchymal stem cell lines isolated using LNGFR(CD271) as a positive indicator or LNGFR(CD271) and Thy-1(CD90) as co-positive indicators, and are cell groups that meet at least one of the following characteristics (a) and (b): (a) the coefficient of variation of forward scattered light in flow cytometry is less than 35%, (b) the average cell size is less than 20 μm. [4-2] As described in [3], the aforementioned high-purity mesenchymal stem cells derived from human bone marrow are cell clusters derived from rapidly proliferating mesenchymal stem cell lines that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and are cell clusters that satisfy at least one of the following characteristics (a) and (b): (a) the coefficient of variation of forward scattering light in flow cytometry is less than 35%, and (b) the average cell size is less than 20 μm.[4-3] The composition described in [3] wherein the aforementioned high-purity mesenchymal stem cells derived from human bone marrow are cell clusters of rapidly proliferating mesenchymal stem cell lines that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and are cell clusters that satisfy at least one of the following characteristics (a) and (b): (a) the coefficient of variation of forward scattered light in flow cytometry is less than 40%, and (b) the average cell size is less than 20 μm. [4-4] As described in [3], the aforementioned high-purity mesenchymal stem cells derived from human bone marrow are cell groups of rapidly proliferating mesenchymal stem cell lines isolated using LNGFR (CD271) as a positive indicator or LNGFR (CD271) and Thy-1 (CD90) as co-positive indicators, and are cell groups that meet at least one of the following characteristics (a) and (b): (a) the coefficient of variation of forward scattered light in flow cytometry is less than 40%, (b) the average cell size is less than 20 μm. [4-5] The composition described in [3] wherein the aforementioned high-purity mesenchymal stem cells derived from human bone marrow are cell clusters derived from rapidly proliferating mesenchymal stem cell lines that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and are cell clusters that satisfy at least one of the following characteristics (a) and (b): (a) the coefficient of variation of forward scattered light in flow cytometry is less than 40%, (b) the average cell size is less than 20 μm. [5] The composition described in any one of [1] to [4-5] wherein the composition is applied to the intervertebral disc of the subject and is fluid upon application. [5-1] The composition described in any one of [1] to [4] wherein the composition is applied to the nucleus pulposus of the intervertebral disc of the subject in such a manner that the crosslinking agent contacts at least a portion of the surface of the composition after application and is fluid upon application to the nucleus pulposus. [5-2] The composition described in any of [1] to [4] is applied to the nucleus pulposus of the intervertebral disc of the subject in a manner that hardens a portion after application, and is fluid when applied to the nucleus pulposus. [6] The composition described in any of [5] to [5-2] is applied to the aforementioned nucleus pulposus as filling the aforementioned composition into a defective portion of the nucleus pulposus. [7] The composition described in [5-2] or [6] is hardened 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] is a divalent or higher metal ion compound. [8-1] The composition described in any of [1] to [8] is a divalent metal salt of alginate.[9] The composition described in any of [1] to [8-1], wherein the monovalent metal salt of the aforementioned low endotoxin alginate has a weight-average molecular weight (absolute molecular weight) of 80,000 or more as determined by GPC-MALS.

[10] The composition described in any of [1] to [9], wherein the concentration of the monovalent metal salt of the low endotoxin alginate is 0.5 w / w% to 5.0 w / w%.

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

[10] is used for the treatment, prevention or inhibition of recurrence of intervertebral disc degeneration and / or intervertebral disc injury.

[12] As described in

[11] , wherein the aforementioned disc degeneration and / or disc injury is selected from at least one of the groups consisting of disc herniation, disc disease, degenerative spondylolisthesis, purulent discitis, degenerative spinal disease, spinal stenosis, lumbar spinal stenosis, and disc herniation and disc injury associated with lumbar spinal stenosis. [12-1] As described in

[11] or

[12] , wherein the aforementioned disc degeneration and / or disc injury is accompanied by chronic low back pain.

[0012]

[13] A composition for intervertebral disc regeneration, comprising high-purity mesenchymal stem cells derived from human bone marrow applied to the nucleus pulposus of an intervertebral disc.

[14] The composition described in

[13] promotes nucleus pulposus regeneration of the intervertebral disc through activation of nucleus pulposus cells induced by high-purity mesenchymal stem cells derived from human bone marrow and / or differentiation of high-purity mesenchymal stem cells derived from human bone marrow into nucleus pulposus cells.

[15] In the composition described in

[13] or

[14] , the aforementioned high-purity mesenchymal stem cells derived from human bone marrow are applied in an undifferentiated state and / or without induction of differentiation.

[16] The composition described in any of

[13] to

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

[13] to

[15] , wherein the aforementioned high-purity mesenchymal stem cells derived from human bone marrow are cell groups of rapidly proliferating mesenchymal stem cell lines isolated using LNGFR (CD271) as a positive indicator or LNGFR (CD271) and Thy-1 (CD90) as co-positive indicators, and are cell groups that satisfy at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scatter light in flow cytometry is less than 35%, (b) The average cell size is less than 20 μm. [16-2] The composition described in any of

[13] to

[15] , wherein the aforementioned high-purity mesenchymal stem cells derived from human bone marrow are cell groups derived from rapidly proliferating mesenchymal stem cell lines that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and are cell groups that satisfy at least one of the following characteristics (a) and (b): (a) the coefficient of variation of forward scattering light in flow cytometry is less than 35%, and (b) the average cell size is less than 20 μm. [16-3] The composition described in any of

[13] to

[15] , wherein the aforementioned high-purity mesenchymal stem cells derived from human bone marrow are cell groups of rapidly proliferating mesenchymal stem cell lines that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and are cell groups that satisfy at least one of the following characteristics (a) and (b): (a) the coefficient of variation of forward scattering light in flow cytometry is less than 40%, and (b) the average cell size is less than 20 μm.[16-4] The composition described in any of

[13] to

[15] , wherein the aforementioned high-purity mesenchymal stem cells derived from human bone marrow are cell groups of rapidly proliferating mesenchymal stem cell lines isolated using LNGFR (CD271) as a positive indicator or LNGFR (CD271) and Thy-1 (CD90) as co-positive indicators, and are cell groups that satisfy at least one of the following characteristics (a) and (b): (a) the coefficient of variation of forward scattered light in flow cytometry is less than 40%, and (b) the average cell size is less than 20 μm. [16-5] The composition described in any of

[13] to

[15] , wherein the aforementioned high-purity mesenchymal stem cells derived from human bone marrow are a cell mass derived from a rapidly proliferating mesenchymal stem cell line that is positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and is a cell mass that satisfies at least one of the following characteristics (a) and (b): (a) the coefficient of variation of forward scattering light in flow cytometry is less than 40%, (b) the average cell size is less than 20 μm.

[17] The composition described in any of

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

[18] The composition described in

[17] , wherein the aforementioned carrier is selected from the group consisting of alginate, hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratin sulfate, heparin, acetylheparin sulfate, and galactosaminoglycuronglycan sulfate, and pharmaceutically permissible salts thereof.

[19] The composition described in

[17] , wherein the aforementioned carrier is a monovalent metal salt of alginate.

[20] The composition described in any one of

[13] to

[19] is applied to the nucleus pulposus of the intervertebral disc of the subject in such a manner as to allow the crosslinking agent to contact at least a portion of the surface of the composition after application, and is fluid when applied to the nucleus pulposus.

[21] The composition described in any one of

[13] to

[19] is applied to the nucleus pulposus of the intervertebral disc of the subject in such a manner as to partially harden after application, and is fluid when applied to the nucleus pulposus.

[22] The composition described in

[20] or

[21] is applied to the aforementioned nucleus pulposus site to fill the aforementioned composition in the nucleus pulposus defect.

[23] The composition described in any one of

[20] to

[22] , wherein the aforementioned crosslinking agent is a metal ion compound with a valence of 2 or higher.

[24] The composition described in any one of

[19] to

[23] , wherein the aforementioned monovalent metal salt of alginate has a weight average molecular weight (absolute molecular weight) of 80,000 or higher as determined by GPC-MALS.

[25] The composition described in any of

[19] to

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

[26] The composition described in any of

[19] to

[25] , wherein the aforementioned monovalent metal salt of alginate is a low endotoxin monovalent metal salt of alginate.

[27] The composition described in any of

[13] to

[26] is used for the treatment, prevention or inhibition of recurrence of intervertebral disc degeneration and / or intervertebral disc injury.

[28] The composition described in

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

[27] or

[28] , wherein the aforementioned disc degeneration and / or disc injury is accompanied by chronic low back pain.

[29] The composition described in any one of [1] to [28-1] is used to suppress disc-related pain.

[30] The composition described in

[29] , wherein the aforementioned disc-related pain is chronic low back pain.

[0013]

[31] A composition for intervertebral disc regeneration, for application in a fluid state to the nucleus pulposus of an intervertebral disc, comprising a monovalent metal salt of alginate and highly purified mesenchymal stem cells derived from human bone marrow. [31-1] The composition for intervertebral disc regeneration described in

[31] above is intended for use where, after application in a fluid state to the nucleus pulposus of an intervertebral disc, no hardening treatment of the composition is required. [31-2] The composition for intervertebral disc regeneration described in

[31] or [31-1] above is used where, after application in a fluid state to the nucleus pulposus of an intervertebral disc, no crosslinking agent comes into contact with the composition.

[32] The composition described in any of

[31] to [31-2] promotes the regeneration of the nucleus pulposus of the intervertebral disc through activation of nucleus pulposus cells derived from high-purity mesenchymal stem cells from human bone marrow and / or differentiation of high-purity mesenchymal stem cells derived from human bone marrow into nucleus pulposus cells.

[33] The composition described in any of

[31] to

[32] wherein the aforementioned high-purity mesenchymal stem cells derived from human bone marrow are applied in an undifferentiated state and / or without induction of differentiation.

[34] The composition described in any of

[31] to

[33] , wherein the aforementioned high-purity mesenchymal stem cells derived from human bone marrow are cell groups of rapidly proliferating mesenchymal stem cell lines that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and are cell groups that satisfy at least one of the following characteristics (a) and (b): (a) the coefficient of variation of forward scattering light in flow cytometry is less than 40%, and (b) the average cell size is less than 20 μm. [34-1] The composition described in any of

[31] to

[33] , wherein the aforementioned high-purity mesenchymal stem cells derived from human bone marrow are cell groups of rapidly proliferating mesenchymal stem cell lines isolated using LNGFR (CD271) as a positive indicator or LNGFR (CD271) and Thy-1 (CD90) as co-positive indicators, and are cell groups that satisfy at least one of the following characteristics (a) and (b): (a) the coefficient of variation of forward scattered light in flow cytometry is less than 40%, and (b) the average cell size is less than 20 μm. [34-2] The composition described in any of

[31] to

[33] , wherein the aforementioned high-purity mesenchymal stem cells derived from human bone marrow are cell groups derived from rapidly proliferating mesenchymal stem cell lines that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and are cell groups that satisfy at least one of the following characteristics (a) and (b): (a) the coefficient of variation of forward scattered light in flow cytometry is less than 40%, and (b) the average cell size is less than 20 μm.[34-3] The composition described in any of

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

[35] The composition described in any of

[31] to

[34] , wherein the application to the aforementioned nucleus pulposus site is for filling the aforementioned composition into the nucleus pulposus defect.

[36] The composition described in any of

[31] to

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

[37] The composition described in any of

[31] to

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

[38] The composition described in any of

[31] to

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

[39] The composition described in any of

[31] to

[38] is used for the treatment, prevention, or suppression of recurrence of intervertebral disc degeneration and / or intervertebral disc injury.

[40] The composition described in

[39] wherein the aforementioned intervertebral disc degeneration and / or intervertebral disc injury is selected from at least one of the groups consisting of intervertebral disc herniation, intervertebral disc disease, degenerative spondylolisthesis, purulent discitis, chronic low back pain, degenerative spinal disease, spinal stenosis, lumbar spinal stenosis, intervertebral disc herniation (mixed lumbar spinal stenosis) and intervertebral disc injury associated with lumbar spinal stenosis.

[41] The group described in

[39] or

[40] wherein the aforementioned intervertebral disc degeneration and / or intervertebral disc injury is accompanied by chronic low back pain.

[42] The composition described in any of

[31] to

[41] is used to suppress intervertebral disc pain.

[43] As described in

[42] , the aforementioned intervertebral disc pain is chronic low back pain.

[0014]

[44] A composition for application to the nucleus pulposus of a subject in a fluid state, comprising a monovalent metal salt of alginate and a high-purity mesenchymal stem cell derived from human bone marrow for suppressing intervertebral disc pain. [44-1] The intervertebral disc pain suppression composition described in

[44] above is intended for use where, after application to the nucleus pulposus of a subject in a fluid state, no hardening of the composition is required. [44-2] The intervertebral disc pain suppression composition described in

[44] or [44-1] above is used where, after application to the nucleus pulposus of a subject in a fluid state, the crosslinking agent does not come into contact with the composition.

[45] The composition described in any one of

[44] to [44-2], wherein the aforementioned pain is pain accompanied by at least one of the following groups: herniated disc, disc disease, degenerative spondylolisthesis, purulent discitis, degenerative spinal disease, spinal stenosis, lumbar spinal stenosis, herniated disc with lumbar spinal stenosis (mixed lumbar spinal stenosis), and disc injury.

[46] The composition described in

[44] to

[45] , wherein the aforementioned pain is chronic low back pain.

[47] The composition described in any one of

[44] to

[46] , wherein the aforementioned high-purity mesenchymal stem cells derived from human bone marrow are administered in an undifferentiated state and / or without induced differentiation.

[48] ​​The composition described in any of

[44] to

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

[44] to

[47] , wherein the aforementioned high-purity mesenchymal stem cells derived from human bone marrow are cell groups of rapidly proliferating mesenchymal stem cell lines isolated using LNGFR (CD271) as a positive indicator or LNGFR (CD271) and Thy-1 (CD90) as co-positive indicators, and are cell groups that satisfy at least one of the following characteristics (a) and (b): (a) the coefficient of variation of forward scattered light in flow cytometry is less than 40%, and (b) the average cell size is less than 20 μm.[48-2] The composition described in any one of

[44] to

[47] , wherein the aforementioned high-purity mesenchymal stem cells derived from human bone marrow are a cell mass derived from a rapidly proliferating mesenchymal stem cell line that is positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and is a cell mass that satisfies at least one of the following characteristics (a) and (b), the composition described in any one of

[44] to

[47] : (a) the coefficient of variation of forward scattered light in flow cytometry is less than 40%, (b) the average cell size is less than 20 μm. [48-3] The composition described in any one of

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

[44] to [48-3], wherein the application to the aforementioned nucleus pulposus site is a filler for the aforementioned composition in the nucleus pulposus defect.

[49] The composition described in any of

[44] to [48-4], wherein the weight-average molecular weight (absolute molecular weight) of the aforementioned monovalent metal salt of alginate, as determined by GPC-MALS, is 80,000 or more.

[50] The composition described in any of

[44] to

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

[51] The composition described in any of

[44] to

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

[52] The composition described in any of

[31] to

[51] is applied to the nucleus pulposus of the intervertebral disc under X-ray fluoroscopy, and is fluid when applied to the nucleus pulposus without allowing the crosslinking agent to come into contact with the composition.

[53] The composition described in

[52] is applied to the nucleus pulposus of the intervertebral disc of the subject under X-ray fluoroscopy using a pressure gauge syringe. It is fluid when applied to the nucleus pulposus and does not allow the crosslinking agent to come into contact with the composition.

[0015]

[54] A method for assessing the regenerative capacity of an intervertebral disc, which uses a sheep severe intervertebral disc degeneration model to assess the regenerative capacity of the intervertebral disc in the composition of the intervertebral disc applied to the model, comprising the steps of (a1) or (a2) and (b1) or (b2) of the following steps, administering a cellular composition to the intervertebral disc of the severe intervertebral disc degeneration model, which is prepared by the following steps: (a1) removing 0.00004% to 0.00005% of the sheep's body weight of nucleus pulposus tissue from the sheep intervertebral disc to prepare a degenerated intervertebral disc; (a2) removing 20 mg of nucleus pulposus tissue from the sheep intervertebral disc to prepare a degenerated intervertebral disc; (b1) further removing 0.00014% to 0.000175% of the sheep's body weight of nucleus pulposus tissue 4 weeks after the aforementioned steps (a1) or (a2); (b2) Four weeks after the aforementioned step (a1) or (a2), a further step of removing 70 mg of nucleus pulposus tissue.

[55] The method described in

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

[56] The method described in

[54] or

[55] wherein the assessment is performed after the administration of the composition on the vertebral body and intervertebral disc of the self-deformation pattern by at least one assessment method selected from the group consisting of MRI, tissue staining, and immunohistochemical staining (IHC).

[0016] Furthermore, the present invention includes the following aspects.

[57] A method for treating, preventing and / or inhibiting recurrence of intervertebral disc degeneration and / or intervertebral disc injury, comprising: applying a composition containing a monovalent metal salt of alginate and mesenchymal stem cells to the nucleus pulposus of the intervertebral disc of a subject requiring the aforementioned treatment, prevention or inhibition of recurrence.

[58] A method for treating, preventing and / or inhibiting recurrence of intervertebral disc degeneration and / or intervertebral disc injury, comprising: applying high-purity mesenchymal stem cells derived from human bone marrow to the nucleus pulposus of the intervertebral disc of a subject requiring the aforementioned treatment, prevention or inhibition of recurrence.

[59] A method for treating, preventing, and / or inhibiting recurrence of intervertebral disc degeneration and / or intervertebral disc injury, comprising: applying a composition comprising highly purified mesenchymal stem cells derived from human bone marrow and a monovalent metal salt of alginate to the nucleus pulposus of an intervertebral disc in a subject requiring the aforementioned treatment, prevention, or inhibition of recurrence, wherein the composition is fluid when applied to the nucleus pulposus.

[60] A method for treating, preventing, and / or inhibiting recurrence of intervertebral disc pain, comprising: applying a composition comprising highly purified mesenchymal stem cells derived from human bone marrow and a monovalent metal salt of alginate to the nucleus pulposus of an intervertebral disc in a subject requiring the aforementioned treatment, prevention, or inhibition of recurrence, wherein the composition is fluid when applied to the nucleus pulposus.

[61] A monovalent metal salt of alginate and mesenchymal stem cells for the treatment, prevention or inhibition of recurrence of intervertebral disc degeneration and / or intervertebral disc injury, wherein the composition comprising a monovalent metal salt of alginate and mesenchymal stem cells is applied to the nucleus pulposus of the intervertebral disc in a patient requiring treatment, prevention or inhibition of recurrence of intervertebral disc degeneration and / or intervertebral disc injury.

[62] A composition comprising a monovalent metal salt of alginate and mesenchymal stem cells, for the treatment, prevention or inhibition of recurrence of intervertebral disc degeneration and / or intervertebral disc injury.

[63] A high-purity mesenchymal stem cell derived from human bone marrow, for the treatment, prevention or inhibition of recurrence of intervertebral disc degeneration and / or intervertebral disc injury.

[64] A composition comprising highly purified mesenchymal stem cells derived from human bone marrow and a monovalent metal salt of alginate, for the treatment, prevention, or suppression of recurrence of intervertebral disc degeneration and / or intervertebral disc injury, the composition comprising: application to the nucleus pulposus of the intervertebral disc of the subject requiring the aforementioned treatment, prevention, or suppression of recurrence, and the composition being fluid when applied to the nucleus pulposus.

[65] A composition comprising highly purified mesenchymal stem cells derived from human bone marrow and a monovalent metal salt of alginate, for the treatment, prevention, or suppression of recurrence of intervertebral disc pain, the composition comprising: application to the nucleus pulposus of the intervertebral disc of the subject requiring the aforementioned treatment, prevention, or suppression of recurrence, and the composition being fluid when applied to the nucleus pulposus. [Effects of the Invention]

[0017] According to the present invention, a nucleus pulposus filling composition is provided that can promote the regeneration of the nucleus pulposus of the intervertebral disc. The composition according to the present invention not only inhibits the degenerative changes of the intervertebral disc nucleus pulposus but also the entire intervertebral disc tissue including the annulus fibrosus. Furthermore, the composition of the present invention has the effect of increasing the proportion of hyaline chondrocytes positive for type II collagen in the nucleus pulposus. Therefore, even in cases where the self-repair function is weak in middle-aged and elderly individuals, such as mixed lumbar spinal stenosis (combined spinal stenosis and disc herniation) commonly seen in middle-aged and elderly individuals over 50 years of age, a regeneration-promoting effect can be obtained. Furthermore, in one embodiment of the present invention, considering the ease of application and reduced invasiveness to the body, it is used without the treatment of hardening the carrier used to embed the cells, and can suppress pain associated with intervertebral disc diseases, especially low back pain, and preferably chronic low back pain.

Implementation Method

[0019] 1. Summary

[0020] This invention relates to a composition for intervertebral disc regeneration containing a monovalent metal salt of alginate and mesenchymal stem cells (e.g., high-purity mesenchymal stem cells derived from human bone marrow). In one embodiment of the invention, the composition relates to an intervertebral disc regeneration composition containing a monovalent metal salt of low-endotoxin alginate and mesenchymal stem cells (e.g., high-purity mesenchymal stem cells derived from human bone marrow). The composition of this invention promotes the regeneration of the nucleus pulposus of the intervertebral disc through activation of nucleus pulposus cells induced by mesenchymal stem cells and / or differentiation of mesenchymal stem cells into nucleus pulposus cells. The composition of this invention is applied to the nucleus pulposus of the object and used in a manner that partially hardens it after application. In several embodiments of the invention, the composition of this invention is applied to the nucleus pulposus of the object in a manner that brings a crosslinking agent into contact with at least a portion of the surface.

[0021] In this invention, an in vivo experiment using interlobular stem cells derived from rabbits demonstrated that the combined use of bone marrow interlobular stem cells (BMSCs) and gel significantly promoted tissue repair compared to gel alone. The mechanism is understood to be that co-culturing gel-embedded BMSCs and nucleus pulposus cells leads to: 1) mutual activation of the two cells due to the production of growth factors; 2) differentiation of BMSCs into nucleus pulposus cells; and 3) an enhanced ability to produce extracellular matrix from both cells.

[0022] The alginate contained in the composition of this invention is a polysaccharide extracted from brown algae such as Ecklonia cava, Eisenia bicyclis, and kelp. It has the property of cross-linking and hardening upon the addition of divalent metal ions such as calcium. Utilizing this property, by contacting it with metal ions at the affected area, its surface hardens, allowing it to remain at the affected area. In this invention, it was found that injecting a composition containing low-endotoxin sodium alginate in a sol state into the nucleus pulposus of the intervertebral disc, and contacting a cross-linking agent at the injection port to harden a portion of the composition, inhibits nucleus pulposus degeneration, increases the proportion of type II collagen-positive cells which are better for nucleus pulposus regeneration, and promotes the regeneration of the intervertebral disc nucleus pulposus.

[0023] In this invention, mesenchymal stem cells are embedded in a monovalent metal salt of alginate (e.g., low-endotoxin alginate), allowing the implanted mesenchymal stem cells to exert a long-term effect at the affected area. Specifically, the implanted high-purity mesenchymal stem cells produce growth factors or extracellular matrix, activating nucleus pulposus cells. Then, the activated nucleus pulposus cells produce growth factors or extracellular matrix. As a result, the implanted mesenchymal stem cells differentiate into nucleus pulposus cells, promoting regeneration of the affected area through the interaction between nucleus pulposus cells and mesenchymal stem cells. In other embodiments of this invention, cells can be embedded in biocompatible materials such as hyaluronic acid.

[0024] Furthermore, among the mesenchymal stem cells contained in the components of the present invention, one type of high-purity mesenchymal stem cells (REC) derived from human bone marrow are ultra-high-purity human mesenchymal stem cells produced by direct separation from bone marrow using two antibodies and a cell sorting instrument. Since uniform cell clusters can be obtained through separation using a cell sorting instrument, quality management is easy, and due to their extremely high proliferative capacity, large-scale formulation through culture is possible.

[0025] This invention also relates to a composition for intervertebral disc regeneration containing high-purity mesenchymal stem cells derived from human bone marrow. In one embodiment of the invention, the composition is applied to the nucleus pulposus of the intervertebral disc in an undifferentiated state and / or under non-differentiation-inducing treatment. The composition promotes the regeneration of the nucleus pulposus of the intervertebral disc through activation of nucleus pulposus cells induced by high-purity mesenchymal stem cells derived from human bone marrow and / or differentiation of mesenchymal stem cells into nucleus pulposus cells. The composition can be applied to the nucleus pulposus of the object in such a manner that at least a portion of the crosslinking agent contacts the surface. In another embodiment of the invention, the composition can be applied to the nucleus pulposus of the object in such a manner that a portion of it hardens after application.

[0026] 2. Definitions of "low endotoxin" and "monovalent metal salts of alginate" are described below. "Intervertebral disc" is a cylindrical tissue connecting the vertebrae of the spine. An intervertebral disc is a plate-shaped, avascular tissue with a nucleus pulposus at its center, surrounded by an annulus fibrosus, and further arranged with endplates above and below. The "nucleus pulposus" is a gel-like tissue located in the center of the intervertebral disc, mainly containing nucleus pulposus cells, an extracellular matrix mainly composed of proteoglycans and type II collagen, and water. It is believed that the self-repair and regeneration capabilities of the nucleus pulposus are significantly low.

[0027] The term "nucleus pulposus filling" refers to filling the degenerated, shrunken, or removed portion of the nucleus pulposus due to aging, trauma, infection, or surgical procedures such as discectomy (nucleotomy). Furthermore, the term "nucleus pulposus filling" in this specification is used to mean the same thing as "nucleus pulposus filling," and the "composition for nucleus pulposus filling" in this invention is synonymous with "composition for nucleus pulposus filling." The term "nucleus pulposus location" refers to the location where the nucleus pulposus is present, the location where degeneration or shrinkage of the nucleus pulposus occurs, or the defect in the nucleus pulposus formed by the removal of at least a portion of the nucleus pulposus, and also includes the periphery of the location where the nucleus pulposus is present.

[0028] "Object" refers to humans or other organisms, such as birds and non-human mammals (e.g., cattle, monkeys, cats, mice, rats, guinea pigs, hamsters, pigs, dogs, rabbits, sheep, and horses). "Application" means filling the nucleus pulposus of the present invention into the nucleus pulposus in an amount sufficient to address the degenerated, shrunken, removed, or defective portions of the intervertebral disc. "Embedding" refers to suspending or mixing cells in a biocompatible material, preferably a solution of a monovalent metal salt of alginate. "Partial hardening" is as described below.

[0029] The term "containing a monovalent metal salt of low-endotoxin alginate" means that the nucleus pulposus portion of the composition of the present invention contains a monovalent metal salt of low-endotoxin alginate in an amount sufficient for nucleus pulposus regeneration. The term "having fluidity" is as described below. The terms "intervertebral disc degeneration and / or intervertebral disc injury," and "treatment, prevention, or inhibition of recurrence" are as described below. The term "discal pain" means pain originating from the intervertebral disc. The term "low back pain" refers to pain occurring around the intervertebral disc, including back pain and buttock pain. The term "chronic low back pain" means continuous low back pain for more than 12 weeks since the onset of the low back pain.

[0030] The components of the present invention can be provided in a solution state using a solvent, or in a dried state such as freeze-dried material (especially freeze-dried powder). When provided in a dried state, the components of the present invention are used in a solvent-based, fluid state such as a solution. While there is no particular limitation on the solvent, any solvent suitable for use in living organisms may be used, examples include water for injection, purified water, distilled water, ion-exchanged water (or deionized water), Milli-Q water, physiological saline, phosphate-buffered saline (PBS), etc. Preferably, water for injection, distilled water, physiological saline, etc., suitable for human and animal treatment are preferred.

[0031] 3. Monovalent Metal Salt of Alginic Acid In this invention, the "monovalent metal salt of alginic acid" used as a carrier is a soluble salt produced by ion exchange between the hydrogen atom of the carboxylic acid at the 6-position of alginic acid and a monovalent metal ion such as Na+ or K+. Specifically, examples of monovalent metal salts of alginic acid include sodium alginate and potassium alginate, but commercially available sodium alginate is particularly preferred. When the solution of the monovalent metal salt of alginic acid is mixed with a crosslinking agent, a gel is formed.

[0032] The "alginic acid" used in this invention is a biodegradable high-molecular-weight polysaccharide, a linear polymer of two uric acid compounds, D-mannuronic acid (M) and L-guluronic acid (G). More specifically, it is a block copolymer formed by arbitrary bonding of the homopolymer portion of D-mannuronic acid (MM portion), the homopolymer portion of L-guluronic acid (GG portion), and the randomly arranged portion of D-mannuronic acid and L-guluronic acid (MG portion). The composition ratio of D-mannuronic acid to L-guluronic acid in alginic acid (M / G ratio) varies mainly depending on the type of source organism such as seaweed, and is also affected by the habitat or season of the organism, ranging 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.

[0033] Alginic acid, a monovalent metal salt, is a high-molecular-weight polysaccharide. While accurately determining its molecular weight is difficult, too low a molecular weight results in lower viscosity, potentially weakening its adhesion to surrounding tissues at the application site. Conversely, excessively high molecular weights are difficult to manufacture, leading to reduced solubility, excessively high viscosity when formulated as a solution resulting in poor handling, and difficulty in maintaining physical properties during long-term storage. Generally, the weight-average molecular weight is between 10,000 and 10,000,000, preferably between 20,000 and 8,000,000, and even more preferably between 50,000 and 5,000,000. In this specification, the numerical range indicated by "~" represents the minimum and maximum values, respectively, for the values ​​before and after "~".

[0034] On the other hand, it is known that in the determination of molecular weight of polymers derived from natural substances, the values ​​may vary depending on the determination method. For example, the weight-average molecular weight determined by gel permeation chromatography (GPC) or gel filtration chromatography (these are also collectively referred to as size exclusion chromatography) is preferably 100,000 or more, more preferably 500,000 or more, more preferably 5,000,000 or less, and more preferably 3,000,000 or less, according to the effects shown in the embodiments of the present invention. The preferred range is 100,000 to 5,000,000, more preferably 500,000 to 3,500,000.

[0035] Furthermore, for example, the absolute weight-average molecular weight can be determined using the GPC-MALS method, which combines gel permeation chromatography (GPC) with multi-angle light scattering (MALS). The weight-average molecular weight (absolute molecular weight) determined by the GPC-MALS method, based on the effects shown in the embodiments of the present invention, is preferably 10,000 or more, more preferably 80,000 or more, further preferably 90,000 or more, even more preferably 1,000,000 or less, more preferably 800,000 or less, further preferably 700,000 or less, and particularly preferably 500,000 or less. Its preferred range is 10,000 to 1,000,000, more preferably 80,000 to 800,000, even more preferably 90,000 to 700,000, and particularly preferably 90,000 to 500,000.

[0036] Typically, when calculating the molecular weight of high-molecular-weight polysaccharides using the method described above, a measurement error of 10% to 20% or more may occur. For example, if the value is 400,000, the value may vary between 320,000 and 480,000; if the value is 500,000, the value may vary between 400,000 and 600,000; and if the value is 1,000,000, the value may vary between approximately 800,000 and 1,200,000.

[0037] The molecular weight of monovalent metal salts of alginate can be determined using conventional methods. Representative conditions for molecular weight determination using gel permeation chromatography are as described in the examples in this specification. For example, a column such as GMPW-XL×2+G2500PW-XL (7.8mm ID×300mm) can be used, and the elution buffer can be, for example, a 200mM sodium nitrate aqueous solution. Polytriglucose can be used as the molecular weight standard.

[0038] Representative conditions for using GPC-MALS in molecular weight determination are as described in the examples in this specification. As detectors, for example, RI detectors and light scattering detectors (MALS) can be used.

[0039] Although monovalent metal salts of alginic acid have large molecular weights and high viscosity when initially extracted from brown algae, their molecular weights decrease and viscosity decreases through processes such as heat drying and purification. By managing conditions such as temperature during manufacturing, selecting the appropriate brown algae as raw material, and differentiating molecular weights during manufacturing, monovalent metal salts of alginic acid with varying molecular weights can be produced. Furthermore, by mixing with another batch of monovalent metal salts of alginic acid with different molecular weights or viscosities, monovalent metal salts of alginic acid with the target molecular weight can be created.

[0040] The monovalent metal salt of alginate used in this invention is preferably prepared by dissolving the monovalent metal salt of alginate in MilliQ water to make a 1 w / w% concentration solution. The apparent viscosity measured using a cone-plate viscometer at 20°C is preferably 40 mPa·s to 800 mPa·s, more preferably 50 mPa·s to 600 mPa·s. The conditions for measuring apparent viscosity are as described below. Furthermore, in this specification, "apparent viscosity" is sometimes simply referred to as "viscosity".

[0041] The alginic acid used in this invention can be either naturally derived or synthetic, but naturally derived alginic acid is preferred. Examples of naturally derived alginic acid include those extracted from brown algae. While brown algae containing alginic acid are abundant in coastal areas worldwide, the number of algae actually usable as raw materials for alginic acid is limited, with examples including giant kelp from South America, kelp from North America, kelp or ascophyllum from Europe, and ascophyllum from Australia. Examples of brown algae used as raw materials for alginic acid include genera such as *Lessonia*, *Macrocystis*, *Laminaria* (kelp genus), *Ascophyllum*, *Durvillea*, *Eisenia*, and *Ecklonia*.

[0042] In other embodiments of the present invention, other biocompatible materials may also 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, acetylheparin sulfate, and galactosaminoglycan (GGGS), containing pharmaceutically permissible salts (physiological salts), may be used as carriers for embedding cells. In other embodiments of the present invention, while there are no particular limitations on the carriers for embedding cells, any one or more of the following may be used: polysaccharides such as cellulose, cellulose derivatives, agarose gel, chitin, chitosan, starch, pectin, proteins such as gelatin, collagen, and polypeptides, amino acid derivatives, copolymers of these, and derivatives thereof. In other embodiments of the present invention, hydrogels composed of collagen or gelatin can also be used as carriers.

[0043] 4. Low Endotoxin Treatment The monovalent metal salt of alginate used in this invention is not particularly limited, but may be, for example, a monovalent metal salt of alginate with low endotoxin. In one embodiment of this invention, the carrier used for cell encapsulation is preferably low in endotoxin. That is, the monovalent metal salt of alginate used in this invention is preferably a monovalent metal salt of alginate with low endotoxin. Low endotoxin refers to an endotoxin level so low that it does not substantially cause inflammation or fever. More preferably, it is a monovalent metal salt of alginate treated with low endotoxin.

[0044] Low endotoxin treatment can be carried out according to known methods or methods based thereon. For example, it can be carried out by the methods of Suga et al. for purifying sodium hyaluronate (e.g., see Japanese Patent Application Publication No. 9-324001, etc.), the methods of Yoshida et al. for purifying β1,3-glucan (e.g., see Japanese Patent Application Publication No. 8-269102, etc.), the methods of William et al. for purifying alginate, galvanic acid, and other biopolymer salts (e.g., see Japanese Patent Application Publication No. 2002-530440, etc.), the methods of James et al. for purifying polysaccharides (e.g., see International Publication No. 93 / 13136, etc.), the methods of Lewis et al. (e.g., see US Patent No. 5589591, etc.), the methods of Hermann Frank et al. for purifying alginate (e.g., see Appl Microbiol Biotechnol (1994) 40:638-643, etc.), or based on these methods. The low endotoxin treatment of this invention is not limited to these methods. It can be implemented using known methods such as washing, filtration using filters (endotoxin removal filters or charged filters, etc.), ultrafiltration, purification using columns (endotoxin adsorption affinity columns, gel filtration columns, ion exchange resin columns, etc.), adsorption on hydrophobic substances, resins, or activated carbon, organic solvent treatment (extraction using organic solvents, precipitation using added organic solvents, sedimentation, etc.), and surfactant treatment (for example, see Japanese Patent Application Laid-Open No. 2005-036036, etc.), or appropriate combinations thereof. Known methods such as centrifugation can also be appropriately combined with these treatment steps. Appropriate selection of the type of alginic acid is recommended.

[0045] Endotoxin levels can be confirmed by known methods, such as by using the Limulus Amebocyte Lysate (LAL) reagent or by using the Endospecy (registered trademark) ES-24S kit (Biochemical Industrial Co., Ltd.).

[0046] While there is no particular limitation on the method for treating the endotoxin content of the monovalent metal salt of alginate contained in the composition of the present invention, when determining the endotoxin content of the monovalent metal salt of alginate using the horseshoe crab reagent (LAL), a result of 500 endotoxin units (EU) / g or less is preferred, more preferably 100 EU / g or less, particularly preferably 50 EU / g or less, and especially preferably 30 EU / g or less. Low-endotoxin treated sodium alginate can be obtained from commercially available products such as Sea Matrix (registered trademark) (Mochida Pharmaceutical Co., Ltd.) and PRONOVATMUP LVG (FMC BioPolymer). Such low-endotoxin treated sodium alginate is also referred to as "high-purity sodium alginate" (UPAL) in this specification.

[0047] 5. Preparation of a Solution of a Monovalent Metal Salt of Alginate The components of this invention can also be prepared using a solution of a monovalent metal salt of alginate. The solution of a monovalent metal salt of alginate can be prepared using known methods or methods based thereon. That is, the monovalent metal salt of alginate used in this invention can be manufactured using the aforementioned brown algae using known methods such as acid methods or calcium methods. Specifically, for example, after extraction from such brown algae using an alkaline aqueous solution such as sodium carbonate solution, an acid (e.g., hydrochloric acid, sulfuric acid, etc.) is added to obtain alginate, and alginate salts can be obtained through ion exchange of alginate. As mentioned above, low endotoxin treatment is performed. The solvent for the monovalent metal salt of alginate is not particularly limited as long as it is a solvent applicable to living organisms, but examples include purified water, distilled water, ion-exchanged water, Milli-Q water, physiological saline, phosphate-buffered saline (PBS), etc. These are preferred if they have been treated with bacteria or have low endotoxin levels. For example, Milli-Q water can be filtered and sterilized before use.

[0048] When the composition of the present invention is provided in a dried state such as a freeze-dried product, it can be prepared into a free-flowing solution using the aforementioned solvent. Furthermore, it is preferable that all operations to obtain the composition of the present invention be carried out in an environment with low endotoxin and bacterial levels. For example, it is better to operate on a clean bench using sterilization equipment, or the equipment can be treated with commercially available endotoxin removers.

[0049] 6. Apparent Viscosity of the Composition of the Invention The composition of several embodiments of the present invention is a fluid liquid, i.e., a solution. The composition of the present invention has fluidity when applied to the nucleus pulposus. Preferably, one embodiment of the present invention has fluidity such that it can be injected with a 21G injection needle after standing at 20°C for 1 hour. While the apparent viscosity of this embodiment of the composition is not particularly limited as long as the effects of the present invention are achieved, excessively low viscosity may weaken the adhesion to the surrounding tissues at the application site. Therefore, 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. Excessively high apparent viscosity may lead to poor operability. Therefore, it is preferably 50,000 mPa·s or less, more preferably 20,000 mPa·s or less, and even more preferably 10,000 mPa·s or less. When the apparent viscosity is below 20,000 mPa·s, application with a syringe or the like is relatively easy. However, even when the apparent viscosity is above 20,000 mPa·s, it can be applied using pressurized or electrically operated filling devices or other means. The preferred range of the composition of the present invention is 10 mPa·s to 50,000 mPa·s, more preferably 100 mPa·s to 30,000 mPa·s, further preferably 200 mPa·s to 20,000 mPa·s, even more preferably 500 mPa·s to 20,000 mPa·s, and particularly preferably 700 mPa·s to 20,000 mPa·s. Other preferred embodiments may have a viscosity of 500 mPa·s to 10,000 mPa·s or 2,000 mPa·s to 10,000 mPa·s. The compositions of several embodiments of the present invention have viscosities that can also be applied to the target object using a syringe or the like.

[0050] The apparent viscosity of compositions containing monovalent metal salts of alginic acid, such as aqueous solutions of alginic acid, can be determined using conventional methods. For example, a coaxial double-cylinder rotational viscometer, a single-cylinder rotational viscometer (Bruch's viscometer), or a cone-plate rotational viscometer (cone-plate viscometer) can be used. It is preferable to follow the viscosity determination method in the Japanese Pharmacopoeia (16th edition). In this invention, viscosity determination is preferably performed at 20°C. As will be described later, when the composition of this invention contains cells or other substances insoluble in the solvent, it is preferable to set the apparent viscosity of the composition to the apparent viscosity of the state without cells or other substances in order to accurately perform viscosity determination.

[0051] In this invention, the determination of the apparent viscosity of a composition containing a monovalent metal salt of alginate is particularly preferably performed using a cone-plate viscometer. For example, the following measurement conditions are preferred: The sample solution is prepared using MilliQ water. The measurement temperature is set at 20°C. The rotational speed of the cone-plate viscometer is set to 1 rpm for a 1% solution of the monovalent metal salt of alginate and 0.5 rpm for a 2% solution. The reading time is set to 2 minutes for a 1% solution of the monovalent metal salt of alginate, and the average value from the first 1 to 2 minutes is taken. For a 2% solution, the reading time is set to 2.5 minutes, and the average value from the first 0.5 to 2.5 minutes is taken. The test value is the average of three measurements.

[0052] 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 alginate. The apparent viscosity of the solution of the monovalent metal salt of alginate increases when the concentration of the monovalent metal salt of alginate in the solution is high, and decreases when the concentration is low. Furthermore, the viscosity increases when the molecular weight of the monovalent metal salt of alginate is large, and decreases when the molecular weight is small.

[0053] The apparent viscosity of a solution of a monovalent metal salt of alginate is affected by the M / G ratio. Therefore, alginate with a better M / G ratio can be appropriately selected based on factors such as the viscosity of the solution. The M / G ratio of the alginate used in this 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, the M / G ratio is mainly determined by the type of algae. Therefore, the type of brown algae used as a raw material affects the viscosity of the solution of a monovalent metal salt of alginate. The alginate used in this invention is preferably derived from brown algae of the genera *Klebsiella*, *Kelp*, *Laminaria*, *Syngonium*, and *Syngonium*, more preferably from brown algae of the genus *Klebsiella*, and especially preferably from *Lessonia nigrescens*.

[0054] 7. Mesenchymal Stem Cells The present invention uses mesenchymal stem cells, which are somatic stem cells derived from mesodermal tissue (mesenchyma), and are expected to be used in regenerative medicine applications such as bone or blood vessel, myocardial remodeling, etc. 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 as follows: A small amount of fat flakes taken from humans or non-human mammals (e.g., cattle, monkeys, cats, mice, rats, guinea pigs, hamsters, pigs, dogs, rabbits, sheep, and goats, rabbits, etc.) are enzyme-treated to obtain a mixed aggregate of cell types. Free adipocytes are separated from this mixed aggregate by centrifugation. Fibroblast-like cells that settle to the bottom and proliferate when placed in contact with the top surface of a culture vessel filled with culture medium are then proliferated through subculture. Furthermore, in the present invention, mesenchymal stem cells derived from iPS cells or commercially available mesenchymal stem cells can also be used. In this invention, mesenchymal stem cells are preferably applied to the nucleus pulposus in an undifferentiated state and / or without any induction of differentiation. An undifferentiated state refers to stem cells with differentiation capacity remaining undifferentiated. "Without induction of differentiation" means that no treatment, such as using a differentiation-inducing medium to differentiate the stem cells into specific cells, has been performed.

[0055] 8. High-purity mesenchymal stem cell (1) REC strain derived from human bone marrow. Based on previous research, the inventors have successfully isolated a rapidly proliferating cell line (Rapidly Expanding Clone: ​​REC) from mesenchymal stem cells (CD271+ cells) that are positive for LNGFR (CD271) or mesenchymal stem cells (CD271+CD90+ cells) that are co-positive for LNGFR (CD271) and Thy-1 (CD90).

[0056] This REC, obtained by seeding cells one by one in a 96-well plate and culturing them, reaches cell confluence after two weeks. Compared with mesenchymal stem cells obtained by conventional methods, it has a proliferation capacity, differentiation capacity, and migration capacity that are all more than 1000 times greater. Furthermore, it maintains its migration capacity, making it suitable for intravenous administration and potentially applicable to serious systemic diseases such as osteochondral dysplasia. In this invention, cell lines with minimal deviations in differentiation or proliferation capacity from the aforementioned REC strains can be used.

[0057] A cell cluster comprising the cell line of the present invention, which is co-positive for LNGFR (CD271) and Thy-1 (CD90) and is a rapidly proliferating intercalary stem cell line cluster, satisfies at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scattered light in flow cytometry is less than 40%. (b) The average cell size is less than 20 μm.

[0058] (2) Method for concentrating human mesenchymal stem cells In this invention, to obtain mesenchymal stem cells that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), the method described, for example, in WO2009 / 31678 can be followed. The outline of the method is as follows.

[0059] First, from a cell mass containing human mesenchymal stem cells, mesenchymal stem cells are highly concentrated by sorting cells that are positive for LNGFR (CD271) (CD271+) or co-positive for CD271 and CD90 (CD271+CD90+). Furthermore, when the cell mass containing human mesenchymal stem cells includes hemocytocytes, a step can be added to sort non-hemocytocyte cells by sorting cells that are co-negative for CD45 and CD235a (CD45-CD235a-).

[0060] Cell clusters containing mesenchymal stem cells can be modulated using flow cytometry or affinity chromatography. While there are no particular limitations on the materials used to obtain these cell clusters, examples include bone marrow, adipose tissue, umbilical cord blood, and peripheral blood (including peripheral blood after G-CSF administration). Furthermore, bone marrow from the spine, sternum, or intestine can be used. Additionally, examples of cells include ES cells and iPS cells.

[0061] During cell preparation, when the material is composed of mesenchymal stem cell blocks, the material may be subjected to physical treatments such as aspiration or transfer, or enzymatic treatments such as trypsin or collagenase, as needed. Furthermore, if red blood cells are mixed in the material, it is preferable to pre-hemolyze the red blood cells. Using the cell groups prepared as described above, CD271+ cells or CD271+CD90+ cells are sorted.

[0062] As a method for sorting CD271+ cells or CD271+CD90+ cells, an example is the use of antibodies. The antibodies are anti-CD271 antibodies and / or anti-CD90 antibodies that can sort CD271+ cells or CD271+CD90+ cells. When using flow cytometry for sorting, by appropriately combining anti-CD271 antibodies labeled with different fluorescent dyes such as FITC, PE, and APC, or by using anti-CD271 antibodies and anti-CD90 antibodies, live cells can be sorted in a short time. Furthermore, besides flow cytometry, CD271+CD90+ cells can also be sorted using magnetic beads or affinity chromatography. In addition, before using these methods, the fluorescent dye (e.g., PI) used to stain dead cells can be reacted with the cell clusters to remove the fluorescently stained cells, thereby removing dead cells.

[0063] (3) REC cell concentration Next, the sorted LNGFR positive cells, or LNGFR and Thy1 co-positive cells are cultured as single cells (lineages) and the batches with rapid proliferation are selected to obtain high-purity human mesenchymal stem cells (REC: Rapidly Expanding Clone) with excellent proliferation, differentiation and migration capabilities.

[0064] Mononuclear cells were prepared from human bone marrow or adipose tissue, or placental chorionic villus. Bone marrow mononuclear cells were stained with anti-LNGFR alone, or with both anti-LNGFR and anti-Thy1. Then, using flow cytometry (cell sorting system), LNGFR-positive cells, or LNGFR-positive and Thy1-positive cell lines, were sorted into 96-well culture dishes. That is, one cell was seeded in each well. After two weeks of single-cell culture, the culture dishes were photographed under a microscope, and wells containing confluence or half-confluence of cells were sorted out. The cells in these wells were designated as RECs.

[0065] Here, the terms "rapid proliferation" and "high-speed proliferation" refer to the proliferation rate at which the culture dish reaches the degree of cell confluence or half-cell confluence after two weeks of culture or before the start of culture when one cell is seeded in each well of a 96-well culture dish for culture (doubling time is 26±1 hours).

[0066] Cell confluence refers to a state where cultured cells cover more than 90% of the surface (culture surface) of the culture container. Half-cell confluence refers to a state where cultured cells cover 70-90% of the surface (culture surface) of the culture container. The size and type of culture equipment used can be appropriately changed according to the cell proliferation rate. Slowly proliferating cells (Moderately / Slowly Expanding Cells), i.e., cells that have not reached half-cell confluence or cell confluence after two weeks of single-cell culture, are discarded. RECs recovered from each well (sorted as RECs) are transferred to a culture flask and cultured until cell confluence is achieved (expansion culture). Afterwards, the expanded cultured cells are individually recovered. RECs originating from one well are defined as one batch.

[0067] Since the REC used in this invention is obtained by sorting a lineage by seeding one cell per well, the genetic characteristics of the proliferated cells are all identical. Therefore, in this invention, the entire cell group can be referred to as a "lineage", or each individual cell constituting the cell group can be referred to as a "lineage".

[0068] Furthermore, in this invention, the RECs used for sorting can also be evaluated beforehand using REC labeling (anti-Ror2). For example, after the aforementioned expanded culture, attached and proliferating cells are recovered from the entire batch, and a portion (approximately 1 to 3 × 10⁵ cells) of each batch is separated for single staining with a monoclonal antibody against Ror2. The method for single staining with a monoclonal antibody against Ror2 is well known (WO2016 / 17795). In summary, the percentage of REC-labeled positive cells in the recovered cells is determined by analyzing the REC-labeled flow cytometry. The percentage can be obtained by quantifying Ror2 mRNA expression using quantitative PCR, or manually by microscopy. Batches (cell groups) with a positive percentage of a certain value (e.g., 65%) or higher are considered acceptable, and this can also be used for sorting as described later.

[0069] (4) Sorting of Stem Cell Groups In this invention, for each batch of REC strains, the cell proliferation capacity, adipogenesis capacity, REC-specific marker expression level, and cell size uniformity are investigated, and their correlations are analyzed, thereby enabling the sorting of RECs with higher cell performance with high purity. In this invention, the coefficient of variation (CV value) of forward scattering light and the average cell size are set as sorting indicators. Forward scattering light is light scattered at a small angle relative to the frontal direction of the laser light. Forward scattering light is composed of scattered light, diffracted light, and refracted light from the laser light generated on the cell surface, and information about the sample size can be obtained.

[0070] The coefficient of variation (CV) is the value obtained by dividing the standard deviation by the mean. It is a value used when evaluating the deviation of data with different units of measurement, or the relationship between data and deviation relative to the mean. In this invention, cells with a CV value of 40% or less, preferably 35% or less, are sorted. Cell clusters with a CV value of 40% or less, more preferably 35% or less, are cell clusters 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 the cells in the cell clusters sorted according to this invention is 20 μm or less. Preferably, it is 18 μm or less, within the range of 14 μm to 18 μm.

[0071] This invention also provides a method for evaluating the quality of cell clusters of LNGFR-positive cells, or high-proliferation mesenchymal stem cell lines co-positive for LNGFR (CD271) and Thy-1 (CD90). In this invention, cell clusters that satisfy at least one of the following characteristics (a) and (b), preferably both, are considered to be of high quality. (a) The coefficient of variation of forward scattered light in flow cytometry is less than 40%. (b) The average cell size is less than 20 μm. The number of cell lines constituting the cell clusters evaluated and sorted in this way is not limited, for example, approximately 0.8 x 10⁷ to 1.2 x 10⁷ cells per 1 ml of solution.

[0072] Furthermore, in a preferred embodiment of the present invention, a cell cluster satisfying at least one of the following characteristics (a) and (b), preferably satisfying both characteristics, is determined to be of high quality. (a) The coefficient of variation of the forward scattered light in the flow cytometer is 35% or less. (b) The average cell size is 20 μm or less. The number of cell lines constituting the cell cluster evaluated and sorted in this manner is not limited, for example, approximately 0.8 x 10⁷ to 1.2 x 10⁷ cells per 1 ml of solution.

[0073] 9. Preparation of the Composition of the Invention The composition of the present invention is characterized by containing, for example, a monovalent metal salt of low-endotoxin alginate and the aforementioned mesenchymal stem cells as active ingredients. The inventors, upon filling the composition of the present invention into the nucleus pulposus of a living organism, first discovered that the monovalent metal salt of alginate itself exerts a regenerative or therapeutic effect on the nucleus pulposus tissue. When the monovalent metal salt of low-endotoxin alginate is applied to the affected area as an active ingredient, it is sufficient to contain an amount capable of exerting a regenerative or therapeutic effect on the nucleus pulposus tissue, preferably at least 0.1 w / v% of the total 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 alginate 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. Furthermore, in other samples, the concentration of monovalent metal salt of alginate in the composition of the present invention is 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 most preferably 1.5 w / w% to 2.5 w / w.

[0074] When using a monovalent metal salt of alginate purified to show a better endotoxin level, as described above, the endotoxin content of the composition is usually less than 500 EU / g, more preferably less than 300 EU / g, even more preferably less than 150 EU / g, and especially preferably less than 100 EU / g.

[0075] The number of cells (cell concentration) contained in the composition of the present invention is, for example, 1×104 cells / ml or more, or 1×105 cells / ml or more, preferably 1×104 cells / ml to 1×107 cells / ml.

[0076] The components of the present invention may also contain factors that promote 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 can be manufactured by recombinant methods or purified from protein components. Furthermore, several forms of the components of the present invention do not contain these growth factors. Even without growth factors, nucleus pulposus regeneration is excellent, and the safety profile is higher compared to cases where cell growth is actively promoted.

[0077] The components of the present invention may also contain factors that inhibit cell death. Examples of factors that cause cell death include caspase, TNFα, etc., and examples of factors that inhibit these include antibodies or siRNA. These factors that inhibit cell death can be manufactured by recombinant methods or purified from protein components. Furthermore, several forms of the components of the present invention do not contain factors that inhibit cell death. Even in the case where factors that inhibit cell death are not contained, nucleus pulposus regeneration is very good, and the safety is also higher compared to the case where cell death is actively inhibited.

[0078] Furthermore, in one embodiment of the present invention, the composition of the present invention, apart from the monovalent metal salt of low-endotoxin alginate, does not contain any components that exert pharmacological effects on the nucleus pulposus tissue of the intervertebral disc. Even in compositions containing only the monovalent metal salt of low-endotoxin alginate as an active ingredient, the regeneration or therapeutic effects on the nucleus pulposus can be fully exerted. In several embodiments of the present invention, the composition of the present invention may also contain other pharmaceutically active ingredients, or conventionally used pharmaceutical ingredients such as stabilizers, emulsifiers, osmotic pressure adjusters, buffers, isotonic agents, preservatives, soothing agents, and colorants, as needed.

[0079] 10. Hardening of the Composition of the Invention The composition of the present invention can be used in a manner that hardens a portion after application to the nucleus pulposus. "Hardening a portion" means that a portion of the composition of the present invention, which is in a flowable state, is brought into contact with a crosslinking agent, not the entire composition in contact with the crosslinking agent, but only a portion thereof, is gelled and solidified. Preferably, a portion of the composition of the present invention is hardened by bringing the crosslinking agent into contact with at least a portion of the surface of the flowable composition of the present invention. In the present invention, a crosslinking agent or hardening means suitable for use can be selected.

[0080] In several embodiments of the present invention, the term "hardening a portion after applying the composition to the nucleus pulposus" can also be expressed as follows: using the same method and ratio of application of the crosslinking agent as for filling the nucleus pulposus, in vitro, after filling a 6 mm diameter test tube with 500 μL of low endotoxin sodium alginate and the crosslinking agent and allowing it to stand for 1 hour, at least 50% of the volume of the composition in the test tube is not gelled, and at least 50% of the volume of the ungelled portion of the composition in the test tube can be aspirated by a syringe equipped with a 21 G injection needle.

[0081] It is believed that the composition exhibits the same properties after filling the nucleus pulposus, and that the composition will not dislodge even when compressive forces are applied from the head and tail sides of the intervertebral disc after filling. "At least a portion of the surface of the composition" is, for example, an opening on the surface of the intervertebral disc connecting the nucleus pulposus, preferably an opening on the surface of the intervertebral disc used for applying the composition to the nucleus pulposus, i.e., a filling port of the composition. By gelling and solidifying at least a portion of the surface of the composition, leakage of the composition from the intervertebral disc can be effectively prevented. The filling port of the composition on the surface of the intervertebral disc is preferably, for example, an opening on the surface of the intervertebral disc made with a syringe needle or scalpel for filling the composition, or an opening on the surface of the intervertebral disc made with a scalpel during disc herniation removal. In this embodiment, the intervertebral disc is preferably the annulus fibrosus.

[0082] Preferably, the composition of the present invention does not contain an amount of crosslinking agent that would harden the composition before being applied to the nucleus pulposus of the target. Therefore, the composition of the present invention may contain an amount of crosslinking agent that does not harden the composition even after a certain period of time. Although there is no particular limitation on the certain period of time, it is preferably about 30 minutes to 12 hours. "Does not contain an amount of crosslinking agent that would harden the composition" can also be expressed, for example, by the following: after the composition has been left to stand at 20°C for 1 hour, it can be injected into a syringe with a 21G injection needle. Several embodiments of the composition of the present invention do not contain crosslinking agents.

[0083] As a crosslinking agent, there are no particular limitations as long as it can be used to immobilize the surface of a monovalent metal salt of alginate by crosslinking a solution of the metal salt of alginate. Examples of crosslinking agents include divalent or higher metal ion compounds such as Ca2+, Mg2+, Ba2+, and Sr2+, as well as crosslinking reagents having 2 to 4 amino groups in their molecules. More specifically, examples of divalent or higher metal ionic compounds include CaCl2, MgCl2, CaSO4, and BaCl2. Examples of crosslinking reagents with 2 to 4 amino groups in the molecule include diaminoalkanes with a lysyl group (-COCH(NH2)-(CH2)4-NH2) on the nitrogen atom, including diaminoalkanes and their derivatives with lysyl amino groups formed by the substitution of amino groups with lysyl groups. Specifically, examples include diaminoethane, diaminopropane, and N-(lysyl)-diaminoethane, but CaCl2 solution is preferred in particular due to its ease of acquisition and gel strength.

[0084] In one of the several embodiments of the present invention, the preferred time point for contacting the crosslinking agent with the surface of the composition of the present invention is after applying the composition of the present invention to the nucleus pulposus. While there is no particular limitation on the method for contacting a portion of the composition of the present invention with the crosslinking agent (e.g., a divalent or higher metal ion), examples include applying a solution of divalent or higher metal ions to the surface of the composition using a syringe, sprayer, or similar device. For example, the crosslinking agent can be slowly and continuously applied to the filling opening of the composition formed in the intervertebral disc for several seconds to ten seconds. Afterwards, if necessary, the residual crosslinking agent near the filling opening can be removed. Removal of the crosslinking agent can be achieved, for example, by washing the application site with physiological saline solution.

[0085] The amount of crosslinking agent used should be appropriately adjusted taking into account the application amount of the composition of the present invention, the size of the filling orifice of the composition on the intervertebral disc surface, and the size of the application site of the nucleus pulposus of the intervertebral disc. In order to prevent the crosslinking agent from strongly affecting the tissue surrounding the filling orifice of the composition, the amount of crosslinking agent used should be adjusted in a way that does not exceed the limit. As for the amount of divalent or higher metal ions used, there is no particular limitation as long as it is enough to solidify the surface of the composition containing a monovalent metal salt of alginate. However, for example, when using a 100mM CaCl2 solution, if the filling orifice on the surface of the intervertebral disc is about 1mm in diameter, the amount of CaCl2 solution used is preferably about 0.3ml to 5.0ml, and more preferably about 0.5ml to 3.0ml. The filling opening on the surface of the intervertebral disc is made using a scalpel or similar tool during disc herniation removal, with an edge approximately 5mm × 10mm. The amount of 100mM CaCl2 solution used is preferably 0.3ml to 10ml, more preferably 0.5ml to 6.0ml. The amount can be adjusted appropriately by observing the state of the components of this invention at the application site.

[0086] When the crosslinking agent contains calcium, it is known that higher calcium concentrations result in faster gelation and the formation of a harder gel. However, since calcium is cytotoxic, excessively high concentrations may negatively impact the nucleus pulposus regeneration of the intervertebral disc in the composition of this invention. Therefore, when solidifying the composition containing a monovalent metal salt of alginate, for example using a CaCl2 solution, a concentration of 25 mM to 200 mM is preferred, and more preferably 50 mM to 150 mM is also suitable.

[0087] In this invention, it is preferable to add the crosslinking agent to the composition, allow it to stand for a certain period of time, and then remove the remaining crosslinking agent by washing or other means. While there is no particular limitation on the standing time, it is preferable to stand for about 1 minute or more, more preferably about 4 minutes or more, to allow the surface of the composition to gel. Alternatively, it can stand for about 1 minute to about 10 minutes, more preferably about 4 minutes to about 10 minutes, about 4 minutes to about 7 minutes, and even more preferably about 5 minutes. This certain time is appropriate to ensure that the composition is in contact with the crosslinking agent, and an appropriate amount of crosslinking agent can be added to prevent the liquid surface of the composition from drying out.

[0088] For example, alginate beads are made by gelling sodium alginate solution dropped into a CaCl2 solution. However, alginate beads must be squeezed onto the application site for application, and they must be made to fit the size of the application site, which presents technical difficulties in actual clinical use. Furthermore, when using CaCl2 solution as a crosslinking agent, calcium cytotoxicity is a problem because the Ca ions on the bead surface come into contact with the surrounding tissue. In contrast, since the composition of the present invention is in solution form, it can be easily applied to application sites of any shape, and the composition can cover the entire application site, exhibiting good adhesion to surrounding tissues. The portion of the composition of the present invention in contact with surrounding tissue maintains a low calcium concentration, resulting in fewer calcium cytotoxicity problems. Because the crosslinking agent has minimal influence on the portion of the composition of the present invention in contact with surrounding tissue, the composition of the present invention can easily contact the cells or tissues at the application site. Preferably, after the composition of the present invention is applied to the nucleus pulposus, it fuses with living tissue at the application site to an undetectable degree after about 4 weeks, and also has high affinity for living tissue.

[0089] When the composition of the present invention is applied to the nucleus pulposus, if a portion is gelled by a cross-linking agent, the composition of the present invention hardens in a portion of the affected area and exists locally in close contact with the surrounding tissue, thus preventing leakage from the nucleus pulposus. Furthermore, by adhering closely to the surrounding tissue, the nucleus pulposus regeneration effect of the composition of the present invention is more strongly exerted.

[0090] It has been found that when the filling material used to fill the nucleus pulposus is gelled and hardened, if a compressive force is applied to the intervertebral disc from the head-to-tail side, the hardened gel will detach from the filling port on the surface of the intervertebral disc. In contrast, when the solution-like composition of the present invention is filled into the nucleus pulposus, it will not detach from the filling port on the surface of the intervertebral disc when a compressive force is applied from the head-to-tail side. That is, in practice, when the nucleus pulposus is filled with the composition of the present invention, even under pressure from above and below the intervertebral disc, the risk of leakage of the filled composition can be said to be low.

[0091] Furthermore, when the hardened gel is filled into the nucleus pulposus, the hardened gel may protrude into the spinal canal, posing a risk of causing significant neurological disorders. On the other hand, the solution-like composition of the present invention has a low risk of such problems and a low risk of developing complications.

[0092] In one embodiment of the present invention, the carrier for embedding cells can also be used without hardening. Depending on the clinical symptoms or the size and shape of the lesion, it can also be applied without using a cross-linking agent.

[0093] 11. Application of the composition of the present invention The composition of the present invention is applied to the nucleus pulposus of the intervertebral disc of humans or other organisms, such as birds and non-human mammals (e.g., cattle, monkeys, cats, mice, rats, guinea pigs, hamsters, pigs, dogs, rabbits, sheep and horses) to promote the regeneration of the nucleus pulposus.

[0094] The composition of the present invention is preferably in the form of a fluid liquid, i.e., a solution. In the present invention, "fluidity" means having the property of changing its shape amorphously, and does not need to have the property of continuous flow like a solution. Preferably, it has the fluidity to allow the composition to be sealed in a syringe or similar device and injected into the nucleus pulposus of the intervertebral disc. Furthermore, in one of the several embodiments of the present invention, it is preferable to have the fluidity to allow injection into the nucleus pulposus of the intervertebral disc using a syringe with a 14G to 26G needle after the composition has been left to stand at 20°C for 1 hour, and more preferably, to allow injection using a 21G needle. When the composition of the present invention is provided in a dried state such as a freeze-dried product, it can be prepared into a fluid composition as described above using a solvent or similar agent during application.

[0095] The solution-like composition of the present invention can be easily applied to the nucleus pulposus of the intervertebral disc using a syringe, gel pipette, special syringe, special injector, or filling device. When the viscosity of the composition of the present invention is high, application with a syringe is difficult; therefore, a pressure-type or electric syringe can also be used. Alternatively, it can be applied to the defect in the nucleus pulposus without a syringe, for example, using a scraper or rod. When injecting with a syringe, a 14G-27G or 14G-26G needle is preferred.

[0096] While there are no particular limitations on the method of applying the composition of the present invention to the nucleus pulposus, it is preferable that the affected area be exposed under direct vision using known surgical techniques, or that the composition of the present invention be applied to the nucleus pulposus using a syringe, filling device, etc., under a microscope or endoscope. In one preferred embodiment, the composition of the present invention may also be applied, for example, by inserting a needle of a filling device into the nucleus pulposus from the surface of the annulus fibrosus.

[0097] Since the composition of the present invention is in solution form, it is suitable for any shape of nucleus pulposus, such as shrinkage, cavities, or defects in the nucleus pulposus, and can also fill the entire shrinkage, cavity, or defect of the nucleus pulposus. Shrinkage, cavities, or defects in the nucleus pulposus may be caused by degeneration or injury of the intervertebral disc, or may be caused by surgical removal or aspiration of at least a portion of the nucleus pulposus. It is preferable to apply the composition of the present invention to nucleus pulposus defects formed by removing at least a portion of the nucleus pulposus.

[0098] There are no particular limitations on the removal of at least a portion of the nucleus pulposus. For example, it can be performed under direct vision, percutaneously, under microscopic observation, or endoscopically. Another example is the LOVE method, which involves making a 2-10 cm incision in the back, dissecting the muscles behind the posterior element of the vertebral arch (the vertebral arch), removing the intervertebral ligaments, identifying the nerve and disc herniation, and removing the herniation compressing the nerve. Alternatively, it can be a method of reducing the volume of the nucleus pulposus by irradiating it with a laser.

[0099] After the composition of the present invention is applied to the nucleus pulposus, as described above, a portion of the composition can be hardened by a cross-linking agent. The amount of the composition of the present invention applied is determined by the volume of the application site in the nucleus pulposus of the subject to which it is applied. Although there is no particular limitation, it 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 applying the composition of the present invention to a nucleus pulposus defect, it is preferable to inject it in a manner that fully fills the volume of the defect in the nucleus pulposus.

[0100] The frequency and number of applications of the components of this invention can be adjusted according to the symptoms and effects. For example, it can be applied only once, or it can be applied continuously once a month to once a year. Since alginic acid is a substance that does not naturally exist in the animal body, animals do not possess the enzymes that specifically break down alginic acid. In the animal body, alginic acid is slowly decomposed through normal hydrolysis, but its decomposition is slower than that of polymers such as hyaluronic acid. Furthermore, since there are no blood vessels in the nucleus pulposus, the filling of the nucleus pulposus can be expected to provide long-term effects.

[0101] Even if the composition of the present invention is not provided together with the cells or growth factors as described above, when the composition of the present invention is applied to the nucleus pulposus, it can be used in conjunction with the aforementioned cells or growth factors, cell death inhibitors, and other agents described later. The composition of the present invention, by being applied to the nucleus pulposus, exerts the effect of inhibiting degenerative changes in the entire intervertebral disc tissue and the nucleus pulposus, and promoting regeneration. Therefore, the composition of the present invention is preferably used as a composition for filling the nucleus pulposus of the intervertebral disc.

[0102] One preferred embodiment of the composition of the present invention is a composition for inhibiting intervertebral disc degeneration, more preferably a composition for inhibiting intervertebral disc nucleus pulposus degeneration. "Intervertebral disc or nucleus pulposus degeneration" refers to morphological changes caused by aging, such as a decrease in cell number, water content, and extracellular matrix (type II collagen, proteoglycans, etc.), resulting in reduced function. If this continues, the disc will no longer function as an shock absorber. In this specification, "inhibiting degeneration" means simply inhibiting degenerative changes compared to the untreated state, not necessarily achieving a state without degeneration.

[0103] One aspect of the composition of the present invention is a composition for inhibiting nucleus pulposus regeneration. Nucleus pulposus regeneration refers to the regeneration of the nucleus pulposus with the aim of preventing the aggregation of fibroblast-like cells and achieving a high proportion of nucleus pulposus cells, thereby regenerating nucleus pulposus tissue rich in type II collagen or proteoglycans. This terminology also includes inhibiting nucleus pulposus degeneration. One preferred aspect of the present invention is that the composition of the regenerated nucleus pulposus after applying the composition of the present invention is close to the composition of a naturally occurring normal nucleus pulposus.

[0104] Furthermore, the preferred form of the composition of the present invention is used for the treatment, prevention, or suppression of recurrence of intervertebral disc degeneration and / or intervertebral disc injury. In this specification, "treatment, prevention, or suppression of recurrence" includes treatment, prevention, suppression of recurrence, reduction, inhibition, improvement, removal, reduction of incidence, delay of onset, inhibition of progression, reduction of severity, reduction of recurrence rate, delay of recurrence time, and alleviation of clinical symptoms. In this specification, "treatment, prevention, or suppression of recurrence" includes the alleviation of (chronic) pain. The preferred form of the composition of the present invention, particularly when used without hardening the carrier of embedded cells, is used to suppress (chronic) pain (especially low back pain) associated with intervertebral disc degeneration and / or intervertebral disc injury.

[0105] The preferred embodiments of the components of this invention, the methods of using the components, etc., follow the description above. Intervertebral disc degeneration and / or intervertebral disc injury refers to at least one condition or disease selected from the group consisting of intervertebral disc herniation, intervertebral disc disease, degenerative spondylolisthesis, purulent discitis, degenerative spinal disease, spinal stenosis, lumbar spinal stenosis, intervertebral disc injury, and intervertebral disc herniation associated with lumbar spinal stenosis (also known as mixed lumbar spinal stenosis). Intervertebral disc degeneration and / or intervertebral disc injury may be accompanied by low back pain. The components of this invention, depending on the clinical symptoms or the size and shape of the injured area, can also be used without hardening the carrier of the embedded cells. In such embodiments, they are used to suppress pain associated with intervertebral disc degeneration and / or intervertebral disc injury, especially chronic low back pain.

[0106] 12. Treatment Method The present invention provides a method for treating, preventing, or inhibiting recurrence of intervertebral disc degeneration and / or intervertebral disc injury using the aforementioned composition of the present invention. Preferably, the treatment method of the present invention is a method for treating, preventing, and / or inhibiting recurrence of intervertebral disc degeneration and / or intervertebral disc injury, comprising: applying a composition containing a monovalent metal salt of low endotoxin alginate and having fluidity to the nucleus pulposus of the intervertebral disc of the subject requiring the aforementioned treatment, prevention, or inhibition of recurrence, and sclerotherapy a portion of the applied composition.

[0107] The treatment method of the present invention may also 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. The aforementioned disc degeneration and / or disc injury is, for example, a condition or disease selected from at least one of the group consisting of disc herniation, disc disease, degenerative spondylolisthesis, purulent discitis, degenerative spinal disease, spinal stenosis, and disc injury. In several embodiments of the treatment method of the present invention, the aforementioned disc degeneration and / or disc injury is disc herniation, particularly lumbar disc herniation. In several embodiments of the treatment method of the present invention, the aforementioned disc degeneration and / or disc injury is disc herniation accompanying lumbar spinal stenosis (also known as mixed lumbar spinal stenosis). Disc degeneration and / or disc injury may be chronic low back pain. Disc degeneration and / or disc injury may also be a combination of these conditions or diseases.

[0108] Furthermore, in one of the several embodiments of the present invention, a method for inhibiting intervertebral disc degeneration using the aforementioned composition of the present invention is provided. Also, in one preferred embodiment of the present invention, a method for regenerating the nucleus pulposus of an intervertebral disc using the aforementioned composition of the present invention is provided. These methods include: applying a composition containing mesenchymal stem cells and a low-endotoxin alginate monovalent metal salt, and having fluidity, to the nucleus pulposus region of the intervertebral disc of a subject requiring inhibition of intervertebral disc degeneration or nucleus pulposus regeneration, thereby hardening a portion of the applied composition. The aforementioned methods also 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 region.

[0109] The preferred form of the composition of the present invention, the specific method of application to the nucleus pulposus of the intervertebral disc, the method of sclerosis of the composition, and the meaning of the terms used are as described above. Other intervertebral disc treatment methods or medications may also be appropriately combined to perform the treatment method of the present invention.

[0110] Furthermore, before, simultaneously with, or after applying the composition of the present invention to the nucleus pulposus, antibiotics such as streptomycin, penicillin, tobramycin, amikacin, gentamicin, neomycin, and amphotericin B, aspirin, nonsteroidal anti-inflammatory drugs (NSAIDs), anti-inflammatory drugs such as acetaminophen, proteolytic enzymes, corticosteroids, and HMG-CoA reductase inhibitors such as simvastatin and lovastatin can be added. These agents can be mixed into the composition of the present invention. They can also be administered orally or in combination. In addition, muscle relaxants, opioid analgesics, and neuropathic pain relievers can be administered orally or in combination as needed.

[0111] This invention also relates to the use of a low-endotoxin monovalent metal salt of alginate for manufacturing the composition of this invention. The use of this invention is for a low-endotoxin monovalent metal salt of alginate, which is used to manufacture a composition for the treatment, prevention, or inhibition of recurrence of intervertebral disc degeneration and / or intervertebral disc injury, wherein the aforementioned composition is applied to the nucleus pulposus of the subject to partially harden after application, and is fluid when applied to the nucleus pulposus.

[0112] The present invention further provides a monovalent metal salt of low endotoxin alginate for the treatment, prevention or inhibition of recurrence of intervertebral disc degeneration and / or intervertebral disc injury. The composition, which contains mesenchymal stem cells and a monovalent metal salt of low endotoxin alginate, is applied to the nucleus pulposus of the intervertebral disc of a subject who requires the treatment, prevention or inhibition of recurrence of intervertebral disc degeneration and / or intervertebral disc injury, thereby hardening a portion of the applied composition.

[0113] 13. Evaluation of the Components of the Invention The components of the present invention can be evaluated using a newly established sheep severe intervertebral disc degeneration model by the inventors. The severe intervertebral disc degeneration model can be created by the following (a) and (b): (a) during the first surgery, removing 0.00004% to 0.00005% of the sheep's body weight of nucleus pulposus tissue from the sheep intervertebral disc to create a degenerated intervertebral disc, and (b) 4 weeks after the first surgery, further removing 0.00014% to 0.000175% of the sheep's body weight of nucleus pulposus tissue from the degenerated intervertebral disc created in (a).

[0114] In a preferred embodiment, a sheep model of severe intervertebral disc degeneration can be created from sheep weighing 40-50 kg by the following (a) and (b): (a) during the first surgery, 20 mg of nucleus pulposus tissue is removed from the sheep intervertebral disc to create a degenerated intervertebral disc; and (b) four weeks after the first surgery, 70 mg of nucleus pulposus tissue is further removed from the degenerated intervertebral disc created in (a). The components of the present invention can be evaluated by the following procedures (a) to (d). (a) In the first surgery, 20 mg of nucleus pulposus tissue was removed from the sheep intervertebral disc to create a degenerated intervertebral disc; (b) As a second surgery, four weeks after the first surgery, a severely degenerated sheep intervertebral disc model was created by removing 70 mg of nucleus pulposus tissue from the degenerated intervertebral disc created in (a); (c) After the second surgery, the composition of the subject was administered into the resulting space; and (d) After the administration of the composition, the regeneration of the intervertebral disc was assessed on the vertebral body and intervertebral disc of the degenerated model using at least one assessment method selected from the group consisting of MRI, tissue staining, and immunohistochemical staining (IHC).

[0115] In previous large animal models, since the disc degeneration model was created by partially removing the normal intervertebral disc, the intervertebral disc had the potential for natural healing. However, in this animal model, by partially removing the already degenerated intervertebral disc, the effect of this embodiment on degenerated intervertebral discs in humans can be evaluated more accurately.

[0116] Examples The present invention will be further described in detail below by way of examples. However, the scope of the present invention is not limited by these examples. [Example 1]

[0117] Intervertebral Disc Disorder Treatment Experiment in Rabbit Model In this embodiment, a composition comprising bone marrow mesenchymal stem cells (BMSCs) and in vivo absorbable ultrapure alginate (low endotoxin high purity alginate, also known as UPAL) gel was used to test the regenerative effect of transplanted BMSCs on degenerated IVDs after intervertebral disc (IVD) resection. 1. Materials and Methods 1.1. Animal Experiments All animals were handled in accordance with the guidelines approved by the Animal Management and Use Committee of Hokkaido University (Approval No.: 13-0051). Male Japanese white rabbits (20 weeks old, 3.2–3.5 kg) were obtained from Sankyo Laboratory Services Co., Ltd. (Tokyo, Japan).

[0118] 1.2. Modified nucleus pulposus (NP) samples from rabbit nucleus pulposus cells (NPCs) were obtained from IVDs (from L1 / 2 to L5 / 6; a total of 20 IVDs) in the lumbar region after euthanizing four rabbits by intravenous overdose of pentobarbital. NPCs were isolated from the nucleus pulposus (NP) tissue and cultured using previously reported methods [2,5,7,8]. Specifically, gelatinous NP tissue was separated from the annulus fibrosus (AF) under aseptic conditions using microforceps. Tissue specimens were placed in Dulbecco modified Eagle medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (Nichirei Bioscience, Tokyo, Japan), 1% penicillin / streptomycin, and 1.25 mg / ml Fungizone (Life Technologies, Waltham, MA, USA). No exogenous growth factors were used. The sample was resuspended in medium supplemented with 0.25% collagenase (Wako Pure Chemical Industries, Osaka, Japan) and incubated in a shaking incubator at 37°C, 20% O2, and 5% CO2 for 4 hours, followed by enzymatic digestion for cell isolation. Cells isolated from NP tissue were then multiplied in culture dishes and cultured in the above medium at 37°C under humidified atmospheric conditions containing 20% ​​O2 and 5% CO2. The medium was changed twice a week, and NPC was used for subculture 2.

[0119] 1.3. Modulation of Rabbit Allogeneic BMSCs Rabbit allogeneic BMSCs were prepared using OriCell™ rabbit mesenchymal stem cells purchased from Cyagen (Santa Clara, CA, USA; Model: RBXMX-01,001, Batch No.: 151114I31). These cells were used to test their characteristics, post-thaw viability, cell cycle, undifferentiated state, and pluripotent differentiation capacity along the osteogenic, chondrogenic, and adipogenic systems. BMSCs were cultured according to the manufacturer's instructions, with the culture medium changed twice weekly for each passage.

[0120] 1.4. Preparation of UPAL Gel and Three-Dimensional (3D) Culture In this embodiment, UPAL gel (Mochida Pharmaceutical Co. Ltd., Tokyo, Japan) was used as an alginate scaffold for 3D culture [2]. The purification process of UPAL gel is as previously reported [2]. Specifically, alginate in seaweed was converted into water-soluble sodium alginate by a clarification process [2] for extraction. This alginate solution was diluted with a large amount of water because it was highly viscous [2]. Then, the extract was filtered to separate the sodium alginate solution from the fibrous residue [2]. In order to isolate high-quality alginic acid, acid was added to this solution [2].

[0121] A 2% (w / v) UPAL solution was prepared for gelation by dissolving in phosphate-buffered saline (Wako Pure Chemical Industries) and 102 mM CaCl2. Before 3D culture, BMSCs were fluorescently labeled with 20 mM 5,6-carboxyluciferin diacetate succinimide (CFDA-SE; CFDA-SE Cell Proliferation Assay Kit; BIO RAD, Hercules, CA, USA) according to the manufacturer's instructions [9]. Then, labeled BMSCs and unlabeled NPCs were embedded in UPAL solution at a ratio of 1:1 (1×106 cells / ml, respectively) [1,10] to obtain a final cell concentration of 2×106 cells / ml [9,10]. The UPAL / cell mixture was then placed in 102 mM CaCl2 through a 22 gauge needle for gelation. The gel beads were cultured for 7 days in the above-mentioned medium under hypoxic conditions (5% O2 and 5% CO2)

[10] . Then, NPC and BMSC were embedded in UPAL solution at a cell concentration of 1×106 cells / ml. The cell concentration was set based on previous reports comparing the effects of different total cell numbers in each group

[11] . After gelation, the gel beads were cultured under the same hypoxic conditions as described above. The experimental groups were as follows: (a) NPC monoculture; (b) BMSC monoculture; and (c) NPC + BMSC coculture.

[0122] After culture for 0 and 7 days, all gel beads were dissolved in 55 mM sodium citrate according to previously reported methods [8] until the gel and cells were separated. In the NPC+BMSC co-culture group, the recovered cells were sorted using a BD FACSAria III high-speed cell sorter equipped with Diva software version 7.0 (BD Biosciences, San Jose, CA, USA) [1,12]. Dead cells and debris were removed, and cells emitting 530 nm fluorescence were identified as BMSCs, while non-fluorescent cells were identified as NPCs.

[0123] 1.5. RNA extraction and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) The recovered NPC and BMSC were dissolved in 1 ml of TRIzol™ (Invitrogen, Carlsbad, CA, USA), and total RNA was extracted from the samples using the RNeasy Mini kit (Qiagen, Valencia, CA, USA). Real-time qRT-PCR analysis was performed using TaqMan™ gene expression assay and custom TaqMan™ gene expression assay (Table 1) (Applied Biosystems, Waltham, MA, USA). Regarding the cycle threshold (Ct) obtained for each sample, the relative mRNA expression of each target gene was calculated based on the Ct value of the housekeeping gene GAPDH using the 2-ΔCt method [1].

[0124]

[0125] 1.6. In vivo trial using rabbit degenerative IVD model. A total of 48 rabbits were used in the in vivo trial. The sample size was determined based on previous reports [2,7,8] for each of the two time points used. 32 rabbits were randomly selected from the 48 rabbits for qualitative analysis of IVD degeneration (magnetic resonance imaging (MRI), histology, immunohistochemistry (IHC)). A total of 80 IVDs were randomly assigned to the following groups: Intact control group, Puncture group (puncture only for creating degeneration), Discectomy group (partial discectomy for creating cavities in degenerative IVDs), Gel group (partial discectomy and UPAL gel implantation for degenerative IVDs), and BMSC+Gel group (partial discectomy and implantation of BMSCs and UPAL gel for degenerative IVDs) (8 IVDs in each group).

[0126] The survival of transplanted BMSCs was assessed using frozen sections of NP tissue from 4 rabbits. A total of 8 IVDs were randomly assigned to a complete control group and a BMSC+gel group (4 IVDs in each group). The remaining 12 rabbits were used to assess the expression of HIF-1a, GLUT-1, and Brachyury. These markers are advocated as major markers of expression in healthy human NPCs

[13] (using IHC of paraffin sections of rabbit NP tissue). A total of 36 IVDs were randomly assigned to a complete control group, a discectomy group, and a BMSC+gel group (4 IVDs in each group).

[0127] 1.7. BMSC labeling and embedding in UPAL solution In vivo experiments, the same OriCell™ rabbit mesenchymal stem cells as those used in in vitro experiments were used as transplanted cells in passage 2. Before transplantation, BMSCs were labeled with CFDA-SE and embedded in 2% UPAL solution to achieve a final cell concentration of 1×106 cells / ml

[14] .

[0128] 1.8. IVD Degeneration and Cell Transplantation 20-week-old rabbits are not sufficiently aged to mimic the state of an aged human population, making it difficult to obtain rabbits with uniform aging. Therefore, in this embodiment, a rabbit annulus fibrosus (AF) puncture pattern was used to obtain degenerated IVDs [8,15,16]. General anesthesia was administered via intravenous injection of ketamine (10 mg / kg) and xylazine hydrochloride (3 mg / kg), and anesthesia was maintained by spontaneous ventilation using O2 mixed with sevoflurane (2-3%) and air (3.0 L / min). IVD degeneration was achieved by AF puncture of L2 / 3 and L4 / 5 IVDs using an 18-gauge needle. L3 / 4 IVD served as a control without treatment.

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

[0130] In the gel group, a microinjector (Hamilton Medical, Bonaduz, Switzerland) with a 27-gauge needle was used to fill the IVD defect with 20 μl of 2% UPAL solution. In the BMSC+ gel group, the IVD defect was filled with UPAL solution containing BMSCs. In particular, a 27-gauge needle was used because a 26-gauge needle showed no effect on cell viability or tissue degeneration [2,7,8,17]. Next, 1 mL of 102 mM CaCl2 was spread on the UPAL solution to induce gelation. After 5 minutes, the surgical wound was washed with physiological saline and closed. A sham operation was performed in the puncture group. Rabbits were euthanized by intravenous overdose of pentobarbital 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 performance. To evaluate the effect of human BMSCs on rabbit IVD imaging, the same implantation experiment was also conducted using human mesenchymal stem cells derived from bone marrow (hMSC-BM; PromoCell, Heidelberg, Germany; model: C-12974, batch number: 412Z022.4).

[0131] 1.9. Assessment of transplanted BMSC survival: At weeks 4 and 12 post-implantation, survival of implanted BMSCs was confirmed based on CFDA-SE fluorescent labeling [18,19]. IVDs (intact control group and BMSC + gel group) were halved horizontally, frozen in liquid nitrogen, sectioned into 5 mm sections, and stained for contrast with 4',6-diamidinyl-2-phenylindole (DAPI; Invitrogen; P36935). In particular, because DAPI-positive cells could not define the survival of surviving NPCs and BMSCs, the survival rate of transplanted BMSCs could not be quantified.

[0132] 1.10. MRI analysis was performed at 4 and 12 weeks postoperatively, using a 7.0-T MR scanner (Varian Unity Inova; Varian Medical Systems, Palo Alto, CA, USA) to obtain T2-weighted midsagittal images of IVD [2,8,16]. IVD degeneration was graded using the Pfirrmann classification (grade 5 for 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 NP degeneration, and the quantitative data were expressed as a percentage of the MRI index obtained relative to the untreated control IVD (relative MRI index) [2,8,16].

[0133] 1.11. Histological Analysis After MRI analysis, each IVD was processed for histological staining. To assess proteoglycan representation, midsagittal sections (5 mm thick) were stained with hematoxylin and eosin (H&E) and safranin O-fast green [8]. Semi-quantitative analysis of the IVD was performed, graded from 0 (normal) to 5 (highly degenerated) [21,22]. Specifically, this histological scale focused on morphological changes in the AF structure.

[0134] 1.12. IHC analysis Immunohistochemical staining (IHC) was performed at 4 and 12 weeks postoperatively to detect type I and type II collagen [8]. HIF-1α, GLUT-1 and Brachyury were detected on postoperative day 1, 7 and 28. 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 used. 3,3'-diaminobenzidine hydrochloride (Dako) and Mayer's hematoxylin (Merck, Darmstadt, Germany) were used for contrast staining. For HIF-1α, GLUT-1, and Brachyury staining, DyLight 550 conjugated rabbit multiclonal antibody against HIF-1α (Novus Biologicals, Centennial, CO, USA; NB100-479R, RRID: AB_1642267), PE conjugated rabbit multiclonal antibody against GLUT-1 (LS Bio, Seattle, WA, USA; LS-A109342-100), and unconjugated rabbit multiclonal antibody against Brachyury (LS Bio; LS-C31179-100, RRID: AB_911118) were used. Furthermore, Alexa Fluor 594 conjugated goat anti-rabbit multiclonal antibody (Invitrogen; A32740) was used as a secondary antibody against Brachyury. DAPI was used for contrast staining.

[0135] Positive cells for type I and type II collagen, HIF-1α, GLUT-1, and Brachyury were measured using independently and randomly selected 5 fields of view [2,8]. The field of view extends to the width of the NP, encompassing both deep and surface regions. Values ​​are expressed as the proportion of positive cells relative to the total number of cells in all assessment items, and also as the proportion of CFDA-SE positive cells used for NPC marker assessment. All experiments, with respect to the 8 IVDs used for type I and type II collagen assessment, and the 4 IVDs used for NPC marker assessment from each treatment group, were performed at separate time points.

[0136] 1.13. Statistical analysis of all data, expressed as mean ± standard error (SE). One-way analysis of variance (ANOVA) and Tukey-Kramer post-hoc tests were performed for multiple group comparisons. Paired t-tests were 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. Co-culture of NPC and BMSC promotes the differentiation of BMSC into NPC and the production of growth factors and ECM. In order to investigate the effect of co-culture of NPC and BMSC for each cell type, unlabeled NPC and CFDA-SE labeled BMSC were embedded in UPAL gel for 3D culture. In the co-culture group, the cell types of both were collected using a cell sorter. Phosphate-buffered saline / cell suspension analysis was performed using forward scattering and side scattering. P1 gate was plotted in a 2D scatter plot (Fig. 1a), excluding dead cells and debris. Unlabeled NPC and CFDA-SE labeled BMSC were sorted in a fluorescence side scatter plot using different gates (Fig. 1b). Therefore, P2 gate was set on unlabeled cells and P3 gate was set on CFDA-SE labeled cells, and a gap was set between the two gates to avoid cross-contamination [1]. After gel dissolution and cell sorting, six cell types 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. Regarding the analysis of BMSC differentiation, qRT-PCR was used in the six cell types to assess the gene expression of HIF-1α, GLUT-1, and Brachyury as NPC markers, the gene expression of CDMP-1, TGF-β, and IGF-1 as growth factors, and the gene expression of type II collagen and proteoglycans as extracellular matrix (ECM).

[0138] The expression of HIF-1α in NPC co-culture was significantly increased compared with the NPC control (p=0.0218, Tukey-Kramer test), and in BMSC co-culture it was significantly increased compared with the BMSC control (p=0.0041, Tukey-Kramer test) and in single culture (p=0.0041, 0.0116, Tukey-Kramer test) (Figure 1c). The expression of GLUT-1 in NPC co-culture was significantly increased compared with the NPC control (p<0.0001, Tukey-Kramer test) and in single culture (p<0.0001, Tukey-Kramer test), and in NPC single culture it was significantly increased compared with the NPC control (p<0.0001, Tukey-Kramer test). In BMSC co-culture, a significant increase in GLUT-1 expression was observed compared to the BMSC control (p=0.0189, Tukey-Kramer test) (Figure 1d). In contrast, Brachyury gene expression did not show statistically significant differences among the three NPCs. Furthermore, Brachyury gene expression was observed only in BMSC co-culture and not in either the BMSC control or monoculture (Figure 1e).

[0139] The gene expression of CDMP-1, TGF-β and IGF-1 in NPC co-culture was significantly increased compared with NPC control (p<0.0001, p<0.0001, p=0.0002, Tukey-Kramer test) and single culture (p<0.0001, p<0.0001, p=0.0082, Tukey-Kramer test). The expression in NPC single culture was significantly increased compared with NPC control (p=0.0179, p<0.0001, p=0.0079, Tukey-Kramer test). The performance of BMSCs in co-culture was significantly improved compared with that of BMSC control (p=0.0011, p<0.0001, p<0.0001, Tukey-Kramer test) and single culture (p=0.0015, p=0.0386, p<0.0001, Tukey-Kramer test) (Figure 1f-h).

[0140] The expression of type II collagen in NPC co-culture and monoculture was significantly increased compared with the NPC control (p=0.0036, p=0.0035, Tukey-Kramer test). The expression in BMSC co-culture was significantly increased compared with the BMSC control (p=0.004, Tukey-Kramer test) and monoculture (p=0.0226, Tukey-Kramer test) (Fig. 1i). Regarding agglutinin, gene expression in NPC co-culture was significantly increased compared with the NPC control (p=0.0047, Tukey-Kramer test) and monoculture (p=0.0392, Tukey-Kramer test). The expression in monoculture was significantly increased compared with the NPC control (p=0.0274, Tukey-Kramer test). In particular, agglutinin was only expressed in BMSC co-culture, but not in BMSC control or monoculture (Fig. 1j).

[0141] 2.2. Survival of transplanted BMSCs In vivo trials, CFDA-SE labeled BMSCs were observed in the BMSC+gel group, but not in the intact control group at weeks 4 and 12 post-transplantation (Figure 2). The human BMSC group also showed the same observations as the BMSC+gel group (rabbit BMSCs), confirming that the transplanted BMSCs survived in IVD at week 12 post-transplantation. No gel extrusion was observed upon incision.

[0142] 2.3. The combined use of BMSC and UPAL gel promotes IVD regeneration after discectomy in degenerative IVDs. Degenerative changes in the treated IVDs were qualitatively analyzed using MRI, capturing T2-weighted midsagittal images (Figure 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 than that in the puncture group and discectomy group 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) (Figure 3b). No significant difference was observed between the gel group and the BMSC + gel group. 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 also significantly higher than that of the puncture, discectomy, and gel groups at 12 weeks (p=0.0002, p<0.0001, p=0–0248, Tukey-Kramer test). Furthermore, the index of the gel group was also significantly higher than that of the discectomy group after 12 weeks (p=0.0092, Tukey-Kramer test) (Figure 3c). In particular, no significant differences were observed between the rabbit BMSC group and the human BMSC group in either the Pfirrmann grade or the MRI index.

[0143] Prior to histological classification of degeneration, the overall structure of the IVD, including NP and AF, was evaluated, and macroscopic differences between groups were observed. The results of histological evaluation of the IVD showed that intact control specimens showed no structural collapse of the internal AF, and the NP tissue exhibited a typical elliptical shape (Figs. 4a and b). In the discectomy group, medial AF collapse was observed, with fibrous changes in the NP tissue observed at both weeks 4 and 12. However, in the BMSC_+ gel group, fibrous changes in the medial AF and NP tissue were minimal, appearing well-preserved at both weeks 4 and 12. The medial AF in the gel group also appeared relatively well-preserved (Figs. 4a and b). The histological degeneration scores in the BMSC + gel group were significantly lower than those in the puncture group (p=0.0006, p<0.0001, Tukey-Kramer test), the discectomy group (p<0.0001, p<0.0001, Tukey-Kramer test), and the gel group (p=0.00, 134, p<0.0001, Tukey-Kramer test) at both weeks 4 and 12. At week 12, the degeneration score in the gel group was also significantly lower than that in the discectomy group (p=0.0006, Tukey-Kramer test). Furthermore, no significant differences were observed between the rabbit BMSC group and the human BMSC group. No osteophyte formation was observed in any group.

[0144] 2.4. Combined use of BMSC and UPAL gel to promote ECM production in denatured IVDs. The inventors then evaluated ECM production in IVDs. In particular, type II collagen is an essential component for IVD function, but an increase in type I collagen synthesis was observed during IVD denaturation

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

[0145] On the other hand, the proportion 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), the discectomy group (p < 0.0001, p < 0.0001, Tukey-Kramer test), and the gel group (p < 0.0001, p < 0.0001, Tukey-Kramer test) at both weeks 4 and 12. This proportion was also significantly lower in the gel group than in the discectomy group at week 4 (p < 0.0001, Tukey-Kramer test), and significantly lower in the gel group than in the puncture group and the discectomy group at week 12 (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 the two assessments performed by IHC analysis.

[0146] 2.5. Differentiation of transplanted BMSCs into NPCs in degenerative IVD Finally, the mechanism of differentiation of transplanted BMSCs into NPCs in vivo was investigated by observing HIF-1α, GLUT-1, and Brachyury positive cells over time (Figs. 6a-c). In the complete control group, almost all cells were positive for all three NPC markers at all time points. In the BMSC + gel group, although HIF-1α, GLUT-1, and Brachyury positive cells were observed at low levels on day 1, 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 day 1 and day 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 (Figs. 6d-f). Similarly, the proportion of NPC marker-positive cells relative to CFDA-SE positive cells (representing transplanted BMSCs) was significantly higher on day 28 compared to day 1 and day 7 (p < 0.0001, p < 0.0001, Student's t-test) (Figure 6 g-i).

[0147] 3. Due to the significantly low regenerative capacity of IVD, the repair of the defective tissue generated by discectomy is insufficient, which may lead to IVD degeneration[2]. In this embodiment, even UPAL gel alone inhibited IVD degeneration more than after discectomy, but when BMSC and UPAL gel were used together, a strong effect was achieved by inducing IVD regeneration. Furthermore, IVD regeneration was observed to be equal in human BMSC and rabbit BMSC between the two groups. The regenerative capacity of various BMSCs in degenerated IVD has also been demonstrated in several previous studies[18, 24, 25]. Furthermore, UPAL gel, due to its high biocompatibility and adequate biomechanical properties, supports the endogenous repair treatment strategy[2], thus providing the optimal environment for IVD regeneration and further degeneration prevention after transplantation of BMSCs at the site of degenerated IVD.

[0148] In this embodiment, the gene expression of NPC markers, growth factors, and ECM was significantly increased in 3D co-culture of NPC and BMSCs compared with each 3D single culture in vitro. These results indicate that co-culture of NPC and BMSCs guides the differentiation of BMSCs into NPCs, which in turn leads to the mutual activation between NPCs and BMSCs, resulting in enhanced ECM production in both cell types. This is also consistent with the insights of several other in vitro studies showing that co-culture of NPC and BMSCs stimulates the differentiation of BMSCs into NPC-like phenotypes [1,12,26], supporting the mutual activation between NPCs and BMSCs through the production of growth factors [1,11,27] and the up-regulation of ECM synthesis [1,5,10,26].

[0149] Furthermore, in in vivo experiments, the expression of NPC markers in implanted BMSCs increased over time, showing the same result as the enhancement of ECM production compared to the results after discectomy alone. To our knowledge, there are no reports on the mechanism of BMSC differentiation into NPCs during in vivo transplantation of alginate and BMSCs, but one study demonstrated that transplanted MSCs implanted in atelocollagen can differentiate into NPCs

[28] . Specifically, autologous BMSCs labeled with green fluorescent protein were transplanted into mature rabbit IVD, and the differentiation of transplanted cells was determined by IHC analysis. At 48 weeks post-transplantation, transplanted BMSCs showed positivity for HIF-1α, GLUT-1, and MMP-2, which means that BMSCs differentiated into cells exhibiting several representative phenotypes of NPCs

[28] .

[0150] In the in vivo trial, histological analysis further demonstrated that gel transplantation alone prevented IVD degeneration better than discectomy, and that the combination of BMSCs and gel exerted a stronger effect in preventing degeneration. This is consistent with MRI results, which showed that gel alone preserved IVD water content better than discectomy, but the combination of BMSCs and gel resulted in a stronger effect on water content preservation. IHC results further showed that the combination of BMSCs and gel promoted ECM synthesis in degenerated NP tissue, which is important for proper IVD function, leading to prevention of degeneration by reducing type I collagen production. Furthermore, histological results of BMSCs and gel compared with the puncture group (degenerated IVD without discectomy) showed that BMSC transplantation led to IVD regeneration. In summary, these results show that transplanted BMSCs differentiate into NPCs over time, resulting in in vivo IVD regeneration.

[0151] Regarding AF regeneration, although the production of collagen I in NP was significantly reduced in the gel group and BMSC+ gel group, the AF defects were repaired by puncture in the filling of gel and BMSC+ gel. Since the characteristic of IVD denaturation is the decomposition of NP extracellular matrix[2], this embodiment mainly focuses on the preservation / regeneration of NP.

[0152] Reports indicate that in intervertebral disc cell therapy, transplanted cells may fail to attach within the intervertebral disc, leading to leakage from the injection site and, consequently, osteophyte formation [5,29,30]. Therefore, in cases of BMSC transplantation after discectomy, a carrier material is needed to prevent cell leakage. In this invention, no osteophyte formation was observed in the BMSC + gel group. The combination of UPAL gel and BMSC exhibits the important characteristic of immediate sclerosis, which is clinically useful in preventing cell leakage.

[0153] Based on the results of this invention and previous reports [1,11,12,26-28], the inventors believe that the mechanism of IVD regeneration is as follows: 1) Implanted BMSCs, through encapsulation in UPAL gel, can exist locally within the cavities of the IVD without leakage outside the intervertebral disc. 2) Implanted BMSCs produce growth factors and ECM, leading to the activation of existing NPCs. 3) Activated NPCs further increase the production of growth factors and ECM. 4) As a result, implanted BMSCs differentiate into NPCs. 5) Furthermore, BMSCs and existing NPCs mutually activate each other, resulting in IVD regeneration (Figure 7).

[0154] In this in vitro experiment, it was considered that if BMSCs and NPCs fused, cell separation would be impossible. Therefore, it was deemed unlikely that cell binding constituted the main mechanism of IVD degeneration. Furthermore, no direct contact between BMSCs and NPCs was confirmed in vivo. Therefore, the underlying mechanism is likely due to growth factors and / or several important humoral factors regulating the interaction between BMSCs and NPCs.

[0155] [Example 2]

[0156] Changes in gene expression induced by co-culture (in vitro) of nucleus pulposus cells (NPC) and high-purity mesenchymal stem cells (REC) 1. Materials and Methods 1.1. Human healthy intervertebral disc nucleus pulposus cells (NPC) Human healthy intervertebral disc nucleus pulposus cells (NPC) were obtained from young patients (15.3±3.3 years old) with adolescent idiopathic scoliosis at Hokkaido University Hospital during anterior spinal fixation surgery.

[0157] 1.2. Modulation of high-purity mesenchymal stem cells with high homogeneity (1) CV value determination by forward scattering (FSC) of flow cytometer Multiple REC lines pre-modulated by a known method (WO2016 / 17795) are used for CV value determination by forward scattering of flow cytometer. The FSC value in flow cytometer is proportional to the surface area or size of the cells. In this embodiment, the CV value of FSC is used as an indicator to assess the deviation of cell size. (a) Each REC line is PI stained, and a gate is set for PI-negative live cell clusters, excluding dead cells. (b) PI-negative live cell clusters are expanded with an FSC / SSC cell histogram, and a gate (P1) is set for the main cell clusters, excluding junk or noise. (c) Expand the cell clusters within the P1 ring using an FSC histogram, set a marker (M1), and measure the CV value.

[0158] (2) Assessment of cell proliferation and adipogenesis capacity To assess cell proliferation capacity, 1 x 10⁵ REC cells were seeded in 100 mm culture dishes and cultured at 37°C and 5% CO₂ for 5 days. Cell count and average cell size were then measured using a cell counter. The culture medium used was DMEM (Fujifilm and Hikari Pure Chemicals Co., Ltd.) supplemented with FBS, basic FGF, Hepes, and penicillin-streptomycin. To assess adipogenesis capacity, 5 x 10⁴ REC cells were seeded in 24-well plates and cultured at 37°C and 5% CO₂ for 2 days. The culture medium was then replaced with adipogenesis induction medium and cultured for 14 days. Oil Red O staining was performed after 14 days, and the fat droplet area was calculated using image analysis. The adipogenesis induction medium used was a medium supplemented with dexamethasone, indomethacin, and IBMX.

[0159] (3) Comprehensive analysis of multiple REC strains: After investigating the CV value of FSC, cell proliferation rate (proliferation capacity), average cell size, and adipogenic differentiation capacity, it was found that REC strains with low CV value of FSC (CV=35% or less) have high proliferation capacity and differentiation capacity, and small average cell size (average cell size: less than 20μm) (Figure 8).

[0160] On the other hand, it was found that REC strains with high CV values ​​(CV = 35% or higher) from FSC had low proliferation and differentiation abilities, and large average cell size. The average proliferation rate of strains with CV values ​​of 30% to 35% was 6.4, the average proliferation rate of strains with CV values ​​of 25% to 30% was 9.2, and the average proliferation rate of strains with CV values ​​below 25% was 15.1. Furthermore, the average proliferation rate of strains with an average cell size of 18 μm to 20 μm was 7.0, the average proliferation rate of strains with an average cell size of 16 μm to 18 μm was 12.7, and the average proliferation rate of strains with an average cell size below 16 μm was 21.3. Hereinafter, REC strains with low CV values ​​from FSC (CV = 25% or lower) were used in this embodiment.

[0161] 1.3. Modulation of UPAL composition containing REC: NPC and fluorescently labeled high-purity mesenchymal cells (REC) were divided into individual groups and mixed groups, and co-cultured or cultured alone in a 3D gel containing 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 groups using a cell sorter. (a) NPC control group (day 0) (b) NPC culture alone group (c) NPC co-culture group (d) REC control group (day 0) (e) REC culture alone group (f) REC co-culture group

[0162] The expression of the following genes in the above 6 groups of cells was determined by quantitative PCR (qRT-PCR). The expression levels of HIF-1α, GLUT-1, Brachyury, CDMP-1, TGF-β, IGF-1, and extracellular matrix type II collagen agglutinin genes were determined according to Example 1.

[0163] 2. Results: When RECs were co-cultured with nucleus pulposus cells, differentiation markers (HIF-1α, GLUT-1, Brachrury) increased in both nucleus pulposus cells and mesenchymal stem cells compared to cultured alone (Figure 9). The same results were observed with growth factors (CDMP-1, TGFβ, IGF-1) and extracellular matrix (type II collagen, proteoglycans). Nucleus pulposus cells were shown to interact with and be activated by high-purity mesenchymal stem cells, leading to differentiation of mesenchymal stem cells into nucleus pulposus cells. [Example 3]

[0164] Regeneration-promoting effect (MRI analysis) and histological analysis in sheep intervertebral disc degeneration patterns 1. Materials and methods Seven sheep (40-50kg) underwent GLP surgery. Intervertebral discs L1 / L2 (between the first and second lumbar vertebrae), L2 / L3 (between the second and third lumbar vertebrae), L3 / L4 (between the third and fourth lumbar vertebrae), and L4 / L5 (between the fourth and fifth lumbar vertebrae) were divided into the following four groups: Control group (n=6): No treatment group; Intervertebral disc resection group (n=6): Removal of nucleus pulposus tissue only; Gel group (n=8): Removal of nucleus pulposus tissue followed by implantation of alginate gel REC+ gel group (n=8): Removal of nucleus pulposus tissue followed by implantation of REC and alginate gel.

[0165] Except for the untreated group, 20 mg of nucleus pulposus tissue was removed from the intervertebral discs of each group under anesthesia to induce severe disc degeneration. Four weeks after the initial nucleus pulposus tissue removal, 70 mg of nucleus pulposus tissue was removed under anesthesia, and REC (prepared in Example 2, 1.2) suspended in UPAL was injected into the intervertebral disc space at a final cell concentration of 1 × 10⁶ cells / ml (100 μl). Four weeks after the implantation surgery, the sheep were euthanized. For the purpose of quantifying the degeneration of the treated intervertebral discs, analysis was performed using 3 Tesla magnetic resonance imaging (MRI). Subsequently, the intervertebral discs were histologically evaluated using H&E staining and Safranin-O staining, and the expression of type II collagen was evaluated using immunohistochemistry. All data are expressed as mean ± standard error, and the differences between groups were determined by one-way ANOVA and Tukey-Kramer post-hoc test.

[0166] 2. Results: Four weeks after implantation of gel and REC+ gel into the affected area of ​​the sheep intervertebral disc, MRI was used to assess the disc degeneration. In the Pfirrmann classification, the gel and REC+ gel groups were significantly lower than the untreated group (discotomy group), and in the MRI index, the REC+ gel group was significantly higher than the untreated group (discotomy group) (Figure 10). Using tissue staining for histological degeneration scores (Boos classification), the gel and REC+ gel groups were significantly lower than the untreated group (discotomy group), and the REC+ gel group was significantly lower than the gel group (Figure 11). In immunohistochemical analysis, the proportion of type II collagen-positive cells was significantly higher in the REC+ gel group than in the untreated group (discotomy group) and the gel group (Figure 12).

[0167] When UPAL gel containing REC is implanted, post-disc resection IVD degeneration is inhibited in highly degenerated IVDs, and type II collagen-positive cells are significantly higher in the REC + gel group. Therefore, it is shown that the combined use of REC with UPAL gel promotes spontaneous IVD regeneration. Furthermore, the inventors have demonstrated that the combined use of REC and UPAL gel promotes the production of growth factors and ECM and the activation of intrinsic NPCs, promotes differentiation into NPCs, and enhances IVD regeneration. [Example 4]

[0168] This embodiment empirically demonstrates that, in a sheep model exhibiting severe IVD degeneration, the combination of highly purified mesenchymal stem cells (RECs) derived from bone marrow and UPAL gel enhances in-situ IVD regeneration after discectomy (up to 24 weeks). Furthermore, in this embodiment, healthy human NPCs and RECs were co-cultured in a 3D system within UPAL gel to evaluate the mechanism underlying IVD regeneration. Additionally, co-cultures of NPCs and commercially available BMSCs, as well as co-cultures of NPCs and RECs, were performed, comparing the effects of RECs and commercially available BMSCs on NPCs. The inventors' findings demonstrate the outstanding clinical applicability of the combination of RECs and in-situ sclerosing gel for the treatment of degenerated human IVD.

[0169] 1. Materials and Methods 1.1. Experimental Design The experimental design in this embodiment was carried out as follows: (i) To explore the mechanism of IVD regeneration through 3D co-culture of human NPC and REC in vitro; (ii) To compare the effects of REC and commercially available human BMSCs co-cultured with NPC in 3D on NPC; (iii) To determine the mechanical properties of UPAL and REC+UPAL gels; (iv) To evaluate the regenerative capacity of severely degenerated IVDs after in vivo discectomy; (v) To evaluate tumor formation in transplanted IVDs.

[0170] The inventors hypothesize that spontaneous IVD regeneration can be promoted by transplanting REC embedded in UPAL gel after discectomy. The use of IVD in healthy humans is approved by the Ethics Committee of the Graduate School of Medicine, Hokkaido University. Animal testing procedures were conducted in accordance with the guidelines approved by the Institutional Animal Care and Use Committee of Hokkaido University and Hamri Co., Ltd. (Ibaraki Prefecture).

[0171] Healthy human NPCs and RECs were co-cultured in a UPAL gel 3D system to evaluate the mechanisms underlying IVD regeneration. At day 0 and day 7 of culture, qRT-PCR (n=4) was used to analyze the expression of NPC markers (including HIF-1α, GLUT-1, and brachyyury), growth factors (including CDMP-1, TGF-β, and IGF-1), and ECM components (including type II collagen and proteoglycans) in all cell types (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 an unrestrained compression test. The circular gels were compressed at a constant speed of 0.5 mm / min, and Young's modulus (n=4) (3, 15, 32, 33) was calculated. In the animal experiment, 14 sheep (56 IVDs) were divided into the following 4 groups. The results over 4 weeks are also described in Example 3. Untreated control group (4 weeks, n=6; 24 weeks, n=6): Untreated discectomy group (4 weeks, n=6; 24 weeks, n=6): Only the nucleus pulposus was removed, also known as the discectomy group. Gel group (4 weeks, n=8; 24 weeks, n=8): After removal of the nucleus pulposus, alginate gel was implanted. REC+gel group (4 weeks, n=8; 24 weeks, n=8): After removal of the nucleus pulposus, REC and alginate gel were implanted.

[0173] To create a severely degenerated IVD, the inventors removed 20 mg of NP tissue from the treated IVD. Furthermore, 4 weeks after the initial surgery, the inventors removed 70 mg of NP tissue from the degenerated IVD. Since in the preliminary experiments, removing more than 20 mg of NP tissue in the sheep model resulted in IVD degeneration after 4 weeks, the initial amount of tissue removed was set at 20 mg (Fig. 19). The second removal amount was set at 70 mg, based on the ratio of normal to degenerated IVD removal amounts obtained in previous rabbit experiments (2, 3). After the second discectomy, a solution containing UPAL or a combination of REC and UPAL was transplanted into the intervertebral disc space. The sheep were euthanized 4 weeks after transplantation (Example 3) and 24 weeks after transplantation. To assess IVD degeneration, 3.0-T MRI (2, 3, 15, 37, 38) was used for qualitative analysis of the IVD. Subsequently, for histological analysis, IVD was stained with H&E and safranin-O. For ECM component analysis, the levels of type II and type I collagen were assessed by IHC. Finally, tissue specimens (2, 3, 15) were used for tumorigenesis analysis.

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

[0175] 1.3. Modification of RECs from in vitro culture and commercially available human BMSCs and human NPCs in 3D co-culture. In this experiment, RECs conforming to freezing GMP were used, provided by Japan Tissue Engineering Co., Ltd. In the comparative analysis of RECs and commercially available human BMSCs, three cell lines with RECs modified by passage 6 were used: REC-02 prototype #003-P6-191220, REC-02 HLWF-1-P6-210114, and REC-02 QRKF-1-P6-210210. Cells were thawed in a 37°C bath before 3D culture.

[0176] Modification of commercially available human BMSCs Commercially available human BMSCs (hMSC-BM; PromoCell, Heidelberg, Germany; C-12974, batch number: 412Z022.4) were obtained according to previous reports (2). The characteristics of these cells belonging to the bone formation, chondrogenesis and adipogenesis systems, their viability after thawing, cell cycle, undifferentiated state and multidifferentiation capacity were tested. BMSCs were cultured using complete culture medium, namely, Dulbecco modified Eagle medium (low glucose level; 2 mg / mL) 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) containing L-glutamine and phenol red (DMEM; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), following the manufacturer's instructions. The medium was changed twice a week, using BMSCs after the 4th subculture (a total of 6 subcultures, the same as REC).

[0177] Preparation of UPAL Gel 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 by dissolving in phosphate-buffered saline (PBS; FUJIFILM Wako Pure Chemical Industries) and gelled using CaCl2 solution (102 mM). REC or commercially available BMSCs were mixed with the UPAL solution at a final cell concentration of 1×106 cells / mL (2, 31, 49). For gelation, the cell-UPAL mixture was pipetted into 102 mM CaCl2 solution using a 22-gauge needle. The two gels obtained as beads were cultured together with the culture medium in a humidified environment (20% O2, 5% CO2, 37°C) for 7 days. The culture medium was changed every 3 days. Seven days after 3D culture, to recover the cells, as previously reported (2, 3, 35), they were dissolved in 55 mM sodium citrate, centrifuged (110 × g for 10 minutes at 4°C), and then recovered from the gel beads. Furthermore, as mentioned above, under normal conditions for 7 days of planar culture (2D culture), the prepared RECs and commercially available BMSCs were used as control groups (i.e., the following four groups were set as experimental groups: 1) 2D cultured RECs, 2) 3D cultured RECs, 3) 2D cultured BMSCs, and 4) 3D cultured BMSCs).

[0178] Analysis of Cell Proliferation Capacity: After 7 days of 2D or 3D culture, cells were seeded again for the four cell types obtained from the secondary culture. Cells were recovered at the stage of dense cell production, and cell proliferation capacity was assessed based on the 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 cell numbers at times t2 and t1, respectively).

[0179] Flow cytometry was used to analyze four cell types, each supplemented with PBS (containing 2% FBS) to prepare cell suspensions at a final density of 1 × 10⁶ cells / mL. Cells were then stained with anti-CD90 antibodies (PE anti-human CD90 (Thy1), BioLegend, San Diego, CA, USA) and anti-CD45 antibodies (FITC anti-human CD45, Beckman Coulter, Brea, CA, USA). Mouse IgG1 antibodies (mouse IgG1-PE, BioLegend; mouse IgG1-FITC, Beckman Coulter) were used as negative controls. Dead cells were detected using propidium iodide (PI). Flow cytometry analysis was performed using the CytoFLEX System (Beckman Coulter) to assess cell viability and the homogeneity and positivity of various cell surface antigens. Data analysis was performed using FlowJo software (Becton Dickinson, Franklin Lakes, NJ, USA).

[0180] Human NP samples were obtained from nine patients (mean age ± SD, 15.3 ± 3.3 years) who underwent anterior spinal fixation for adolescent idiopathic scoliosis at Hokkaido University Hospital. Informed consent was obtained from the participants, and consent forms were prepared and retained. Samples were obtained during surgery. All IVDs were analyzed preoperatively by MRI, and degenerative changes were assessed using the Pfirrmann classification (36). All IVDs were classified as grade 1, indicating that all samples were non-degenerative IVDs.

[0181] Cells were isolated from human NP samples and cultured as previously reported (2, 3). First, gel-like NPs were isolated from AF under a dissecting microscope, and the tissue specimens were placed in culture medium. The preparation was washed twice by centrifugation (1,000 rpm, 3 min) and then resuspended in culture medium supplemented with 0.25% collagenase. For cell isolation, the preparation was cultured in a shaking incubator (37°C, 4 h), followed by two centrifugations (1,000 rpm, 3 min). Cells isolated from the matrix were placed in 10 cm tissue culture dishes and cultured in the same manner as above. The culture medium was changed twice a week using NPCs after the 4th passage.

[0182] Preparation of REC and Commercially Available Human BMSCs The aforementioned REC (REC-02_Prototype #003-P6-191220) and commercially available human BMSCs were also used in 3D co-culture. Both cell types were cultured according to the manufacturer's instructions using the same method as described in 2D culture. The culture medium was changed twice a week. Both were derived from second-passage cells (REC, a total of 8 passages; BMSCs, a total of 4 passages).

[0183] 3D Co-culture and Single Culture The inventors prepared a 2% UPAL solution, as previously reported (2, 3), and used a CaCl2 solution (102 mM) for gelation. Before 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 mixed at the same ratio as the UPAL solution (1 × 10⁶ cells / mL each) (2, 31, 49), with a final cell concentration of 2 × 10⁶ cells / mL. The cell-UPAL mixture was gelled using a 22-gauge needle by adding 102 mM CaCl2 solution. The resulting gel was cultured for 7 days under hypoxic conditions (5% O₂ and 5% CO₂) (2, 49). Subsequently, the three cell types were mixed in UPAL solution at a concentration of 1×10⁶ cells / mL. The cell concentration was selected based on previously reported results (2). After gelation, the gel beads were cultured under hypoxic conditions using the same method as described above and used in the following experimental groups (n=4 per group). (a) Monoculture of NPC (b) Monoculture of BMSC (c) Monoculture of REC (d) Co-culture of NPC + BMSC (e) Co-culture of NPC + REC.

[0184] On the first day of culture and 7 days after, all gel beads were dissolved in 55 mM sodium citrate to separate the gel from the cells. In the co-culture group, the recovered cells were sorted using a high-speed cell sorter. The high-speed cell sorter used Diva software version 7.0 (BD Biosciences, San Jose, CA, USA) with BD FACSAria III (2, 31). After removing debris and dead cells, fluorescent cells at 530 nm were selected as RECs and BMSCs, and non-fluorescent cells were selected as NPCs.

[0185] RNA Extraction and qRT-PCR Ten cell types (control NPC, monoculture NPC, NPC co-cultured with commercially available BMSCs, NPC co-cultured with RECs, control BMSCs, monoculture BMSCs, co-cultured BMSCs, control RECs, monoculture RECs, and co-cultured RECs) were recovered and dissolved in 1 mL of TRIzol (registered trademark) (Invitrogen, Thermo Fisher Scientific). Total RNA was extracted using the RNeasy Mini kit (Qiagen, Valencia, CA, USA). Real-time qRT-PCR was performed using the TaqMan gene expression assay (Applied Biosystems, Thermo Fisher Scientific) (Table 3). Cyclic threshold (Ct) values ​​were obtained for each sample. The relative mRNA expression levels of each target gene, NPC marker, proliferation factor, and ECM component were calculated using the 2-ΔCt method (2). Expression levels were standardized by the expression level of the housekeeping gene GAPDH (2,31).

[0186] 1.4. Elasticity Analysis of Gels Using Unrestrained Compression Testing The mechanical properties of UPAL and REC-UPAL gels were evaluated using unrestrained compression testing. Two types of disc-shaped gels with a diameter of 4.5 mm and a thickness of 2 mm were prepared (Fig. 14(a)). The samples were placed in a tensile-compression mechanical testing machine (Autograph AG-X; Shimadzu Corporation, Kyoto, Japan) until the gel broke, and compressed at a constant speed of 0.5 mm / min using a 100 N force gauge (Fig. 14, B and C) (3, 15). Young's modulus, based on the obtained stress-skew curve (Fig. 14D), was calculated using an approximate straight line between 10% and 20% compression (n=4 gels / group) (Fig. 14E) (3, 15, 32, 33).

[0187] 1.5. In Vivo Trial in Sheep Model of Degenerated IVD In this embodiment, the complete procedure of the sheep model was performed in a pharmaceutical GLP-compliant laboratory (Hamri Co., Ltd.) (3). Qualitative analysis of IVD degeneration was performed using 14 male Suffolk sheep (2 years old, weighing 40–60 kg) (3). All 56 IVDs were randomly assigned to no-treatment 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 hydrochloride (20 mg / kg), and maintained by inhalation anesthesia (isoflurane). The surgery was performed using a right-sided retroperitoneal approach, exposing the vertebral bodies and IVD from L1 to L5. A solid sponge screw (ZIMMER BIOMET, Warsaw, IN, USA) was inserted into the L2 vertebral body as a vertebral marker. In the discectomy group, gel group, and REC+gel group, 20 mg of NP tissue was removed after an AF incision (5×3 mm), inducing IVD degeneration (Fig. 15, A, B) (3, 8).

[0188] Removal and transplantation of degenerated NP tissue: Four weeks after the initial surgery, the vertebral body and IVD were exposed using the same approach. As described above, in order to create an IVD cavity after AF incision, the inventors extracted 70 mg of NP tissue from the degenerated IVD obtained from the three treatment groups (Fig. 15C). After discectomy, 110–120 μL of 2% UPAL solution was transplanted into the intervertebral disc cavity in the gel group, and 110–120 μL of a mixture of REC and UPAL solution (final concentration, 1 × 10⁶ cells / mL) was transplanted into the REC+gel group (Fig. 15D) (2). Immediately, 102 mM CaCl₂ solution was injected onto the surface of the mixture, and gelation was confirmed after 5 minutes. Four and 24 weeks after implantation, sheep were euthanized with pentobarbital, and the lumbar spine was removed in one piece (3).

[0189] MRI was performed using a 3.0-T MR scanner (MAGNETOM Prisma; Siemens, Munich, Germany) to obtain T2-weighted midsagittal slice images. To assess the signal changes in treated IVD, the inventors used a Pfirrmann classification (36) comprising five grades (1: normal to 5: highly degenerated) to fractionate the degree of IVD degeneration. Furthermore, using Analyze 14.0 software (AnalyzeDirect, Overland Park, KS, USA), the MRI index value (the product of the average signal intensity of the NP and the NP area) was measured to quantify the brightness of the NP tissue. Furthermore, the ratio of the MRI index in the untreated control group to the ratio of the MRI index in all three treatment groups (2, 3, 15, 37, 38) was evaluated as the relative MRI index. The ratio of intervertebral disc height to the vertebral body height on the adjacent thorax side was also measured as DHI (4, 39). The percentage value of DHI in the untreated control group was determined as the relative DHI.

[0190] Histological Analysis: Following MRI imaging, samples were fixed in 10% formaldehyde, desalted with 10% EDTA (pH 7.5), and embedded in paraffin. Sagittal 5-micron thick paraffin sections were dewaxed with xylene, treated with alcohol, washed with water, and stained with H&E and safranin-O. The degree of IVD degeneration was fractionated using a modified Boos' classification (3, 40, 41) from 0 (normal) to 36 (highly degenerated).

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

[0192] Tumor Formation Analysis To assess tumor formation in transplanted cells and existing cells in the transplanted IVD, (i) invasive proliferation, (ii) mitotic figures, (iii) binucleated cells, and (iv) nucleoli were evaluated. During the 24-week evaluation period, the total number of positive cells in H&E-stained specimens from the untreated control and REC+ gel group was determined in 15 randomly selected visualization fields (15 visualization fields at 400x magnification; 26.5 visualization fields, totaling 5 mm²), and the number per square millimeter was calculated.

[0193] 2. Results 2.1. Results of 3D co-culture of REC and commercially available human BMSCs with human NPC in vitro Comparison of REC and commercially available human BMSCs Details of the results of confirming the stability of REC and commercially available human BMSCs before and after gel incorporation are presented in Tables 2 and 3.

[0194] Cell viability was defined as the ratio of PI-unstained cells to the total number of cells. There was no significant difference in the ratio between REC and BMSC (p = 0.6087, Table 2). The coefficient of variation (CV) of forward scattered light, which is proportional to cell diameter, was calculated to assess cell size uniformity. The %CV value was significantly lower in REC than in BMSC (p = 0.0366, Table 2). Cell proliferation capacity was assessed by calculating Td. Td was significantly shorter in REC than in BMSC (p = 0.0074, Table 2). Cell surface antigen confirmation assays showed that the proportion of CD90-positive cells was significantly higher in REC than in BMSC (p = 0.0004). No significant differences were observed in CD45-positive cells between groups (p = 0.0907, Table 2).

[0195] The inventors further compared REC and commercially available BMSCs after one week of culture (Table 3). Regarding Td after 2D and 3D culture, there was no significant difference between REC and BMSC (p=0.3339 and p=0.6996, respectively). However, the CD90 positive cell rate was significantly higher in REC than in BMSC in both 2D and 3D cultures (p=0.0039 and p=0.0007, respectively). Regarding CD45 positive cells, the proportion was significantly lower in REC than in BMSC after 2D culture (p=0.0038). There was no significant difference between REC and BMSC after 3D culture (p=0.3221). In the in vitro simulation of the cell environment after intervertebral disc transplantation, i.e., the 3D culture environment with alginate, REC better maintained the characteristic cell surface markers of mesenchymal stem cells compared to BMSC, indicating that REC stably maintained the morphology of mesenchymal stem cells even in the alginate environment.

[0196]

[0197]

[0198] Co-culture of REC and Human NP Cells (NPC) The inventors investigated the effects of co-culturing REC and human NPC, and the effects of co-culturing different cell types, namely commercially available human BMSCs and human NPC, and compared the two. REC or BMSCs labeled with 5,6-carboxyfluorescein diacetate succinimide (CFDA-SE), and unlabeled NPCs were embedded in UPAL gels and cultured in 3D (2,15,27). After 7 days of culture, the gel was dissolved, and residues and dead cells were removed using a cell sorting machine. The cells were then sorted into CFDA-SE positive cells, REC, BMSCs, unlabeled cells, and NPCs. The following 10 cell types were obtained after cell sorting. (a) Control NPC (uncultured) (b) NPC cultured alone (c) NPC co-cultured with commercially available BMSCs (d) NPC co-cultured with RECs (e) Control BMSCs (uncultured) (f) BMSC cultured alone (g) BMSC co-cultured (h) REC control (uncultured) (i) REC cultured alone (j) REC co-cultured

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

[0200]

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

[0202] The expression levels of all three NPC markers in co-cultured RECs were significantly higher than those in control and single-cultured RECs (Fig. 13, D-F). The expression levels of CDMP-1, TGF-β, and IGF-1 were also significantly increased in co-cultured RECs compared to control and single-cultured RECs (Fig. 13, G-I). Similarly, the expression levels of type II collagen and proteoglycans were significantly higher in co-cultured RECs than in control and single-cultured RECs (Fig. 13, J and K).

[0203] 2.2. Results of Elastic Analysis of Gels Using Unrestrained Compression Tests on REC Implantation in UPAL Gel To evaluate and compare the mechanical properties of REC-UPAL and UPAL gels, unrestrained compression tests (3, 15, 32, 33) were performed. Two cylindrical gel samples (4.5 mm in diameter and 2 mm in thickness) were prepared, and the ratio of axial compressive stress to strain was calculated (Fig. 14, A-C). At compression levels of 10-20%, the Young's moduli in UPAL and REC-UPAL gels were 18.0 ± 3.5 kPa and 18.8 ± 2.1 kPa, respectively, with no significant difference in mechanical properties between the two groups (p = 0.6942) (Fig. 14, D and E). Furthermore, these two values ​​are equivalent to those of normal human NP tissue (3, 32).

[0204] 2.3. Results of the in Vivo trial in sheep models of regenerated IVDs: The combined use of REC and UPAL gels improved IVD regeneration. The regenerative capacity of IVDs was evaluated in sheep models after gel or REC+gel transplantation (Figure 15). Sheep are widely used in IVD studies following discectomy containing living material (3, 34, 35). 56 IVDs from 14 sheep were divided into the following groups: No treatment control group (n=6 IVDs), Discectomy group (n=6 IVDs), Gel group (n=8 IVDs), REC+gel group (n=8 IVDs).

[0205] To create an IVD degeneration model, 20 mg of NP tissue was removed during the initial surgery (Fig. 15, A and B). Based on the results of preliminary experiments, the amount to be removed was set at 20 mg (Fig. 19). Four weeks after the initial surgery (after observing degeneration changes), NP tissue was removed again (Fig. 15C). Next, UPAL or REC+UPAL solution was transplanted into the IVD defect, and the second intervertebral disc was excised and exposed to 102 mM CaCl2 to gel (Fig. 15D). The transplanted lumbar vertebrae were harvested 4 weeks and 24 weeks after transplantation for various analyses.

[0206] Degenerative changes in the treated IVD were assessed using T2-weighted central sagittal sectional images obtained via MRI (Fig. 16A). For morphological analysis of the IVD, Pfirrmann scores and MRI indices were evaluated to assess signal changes in the implanted IVDs (2, 3, 15, 36–38) and the disc height index (DHI). Based on the Pfirrmann scores, the degeneration score in the gel group was significantly lower than that in the discectomy group at week 24, and the degeneration score in the REC+gel group was significantly lower than that in both the discectomy group and the gel group at weeks 4 and 24 (Fig. 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 of the gel group was significantly higher than that of the discectomy group, and the REC+ gel group was significantly higher than that of both the discectomy group and the gel group (Fig. 16C). Furthermore, the disc height of the treated IVD was measured using MRI images. The ratio of the disc height to the height of the adjacent superior vertebral body was measured anteriorly and posteriorly to the IVD; this is known as the DHI. The relative DHI, i.e., the ratio of the DHI value of the three treatment groups to the DHI value of the treatment control group, was then determined (Fig. 20) (4, 39). The DHI values ​​of the three treatment groups were significantly lower than those of the untreated control group at both weeks 4 and 24. Although there were no significant differences among the three groups 4 weeks after implantation, the DHI of the gel group was significantly higher than that of the discectomy surgery group 24 weeks after implantation, and the REC+ gel group was significantly higher than that of the discectomy surgery group and the gel group (Figure 16D).

[0208] Histological analysis was performed using hematoxylin & eosin (H&E) and safranin-O staining. In both the untreated control group and at 4 and 24 weeks, IVD showed a spindle-shaped structure without fibrous changes in the NP tissue. The annulus fibrosus (AF) was a concentric structure, uniformly stained with safranin-O. In the discectomy group, significant scar tissue, fibrous changes, tissue defects, and endplate collapse and destruction were observed (Fig. 17, A, B). The histological degeneration degree determined by the modified Boos' classification (3, 40, 41) in the gel group was significantly lower than that in the discectomy group at both 4 and 24 weeks. At both 4 and 24 weeks, the REC+ gel group was significantly lower than both the discectomy group and the gel group (Fig. 17C).

[0209] The expression levels of type II and type I collagen in treated IVD were assessed by immunohistochemistry (IHC) (Fig. 18, A and B). The percentage of type II and type I collagen-positive cells in the total number of cells in five randomly selected visualization fields in NP tissue was assessed (2, 3, 15). Type II collagen is an essential ECM component in NP tissue and is replaced by type I collagen as denaturation progresses. In the analysis of type II collagen, NP tissue was uniformly stained in the untreated control group, but slightly reduced in the REC+ gel group. Unstained areas were observed scattered in the gel group, while extensive unstained areas were observed in the discectomy group. The proportion of type II collagen-positive cells was significantly higher in the REC+ gel group at 4 weeks and 24 weeks than in the gel group, and significantly higher in the gel group than in the discectomy group at 24 weeks (Fig. 18C). Conversely, the proportion of type I collagen-positive cells was significantly lower in the REC+ gel group than in the discectomy group during the 4-week assessment period, and significantly lower in both the discectomy group and the gel group during the 24-week assessment period. Furthermore, the proportion of these cells was significantly lower in the gel group than in the discectomy group at 24 weeks (Figure 18D).

[0210] During the 24-week evaluation period of the effect of REC+ gel injection on IVD, tumor formation was analyzed using histological specimens from the untreated control and the REC+ gel group. In both groups, invasive proliferation was assessed along with mitotic figures, and the number of binucleated cells and nucleoli per square millimeter were measured. No invasive proliferation, mitotic figures, or nucleoli were observed in either group. The number of binucleated cells in the untreated control group and the REC+ gel group were 0.03±0.07 and 0.23±0.25, respectively. That is, the value was <1 / mm2, and no significant difference was observed between the groups (p=0.3311) (Table 5).

[0211]

[0212] 3. In this embodiment, REC demonstrated superior characteristics compared to commercially available human BMSCs in terms of cell proliferation capacity, cell size uniformity, and expression of cell surface antigens. Furthermore, this embodiment demonstrated that the expression levels of NPC markers, growth factors, and ECM components were significantly increased in 3D co-culture of human NPCs and RECs compared to the expression levels observed in 3D co-culture of NPCs with commercially available BMSCs. The effectiveness of the combined use of REC and UPAL gel was observed in IVD degeneration sites in sheep lumbar spine models. While UPAL gel alone inhibited IVD degeneration more effectively than the discectomy group, the combined use of REC and gel more effectively enhanced IVD regeneration.

[0213] This embodiment teaches that co-culturing RECs with NPCs leads to the differentiation of RECs into NPCs, thereby improving the production of ECM components in both cell types. The same results were observed in rabbits (Example 1). That is, it was found that the expression of NPC markers in BMSCs embedded in UPAL gel increased after transplantation in Example 1, and the production of ECM was increased in the BMSC+UPAL gel group compared with the discectomy group (2).

[0214] Based on the above results and previous studies (2, 31, 47, 48, 50-52), the following possibilities are shown as a mechanism by which the transplantation of RECs embedded in UPAL gel becomes the basis for IVD regeneration: 1) RECs embedded in gel can be transplanted to IVD defects. 2) RECs produce growth factors and ECM components, thereby activating existing NPCs (paracrine mechanisms). 3) NPCs produce more ECM and growth factors. 4) As a result, RECs directly differentiate into NPCs. 5) Finally, RECs and NPCs possess a positive intercellular feedback loop leading to IVD regeneration.

[0215] The biomaterial / hydrogel used in IVD repair must be biologically and mechanically suitable. In this embodiment, since the biomechanical and geometric configuration of the lumbar IVD is equivalent to that of humans (3,4,34), a sheep lumbar spine model was chosen to transplant the candidate hydrogel into the preclinical animal model. To date, the inventors have found that UPAL gel implanted in sheep lumbar IVDs after discectomy has shown appropriate biomechanical properties, indicating no material protrusion and no need for suture of the AF after discectomy (3). In this embodiment, unrestrained compression tests showed no significant difference in Young's modulus between the UPAL group and the REC-UPAL group, and showed that the REC implanted with UPAL gel did not alter the mechanical properties of the gel. The combination of UPAL gel and REC offers clinical advantages in preventing cell leakage without suturing the AF due to its decisive ability to provide rapid healing.

[0216] MRI and histological evaluation of animals transplanted with UPAL gel (and those not transplanted) showed that UPAL gel alone inhibited IVD degeneration after transplantation. Furthermore, the combination of REC and UPAL gel was more effective in preventing degeneration than UPAL gel alone. Also, there was no significant difference in the number of binucleated cells between the untreated group and the REC+gel group, indicating that the combination of REC and gel does not induce tumor formation. Furthermore, the IHC results showed that the combined use of REC and gel enhances the ECM synthesis required for the function of normal IVD, thereby inhibiting the progression of IVD degeneration by inhibiting the production of type I collagen. These results are consistent with the results obtained using the rabbit model of IVD degeneration (Example 1), demonstrating that BMSCs embedded in UPAL gel are more effective in inhibiting degeneration (2). Example 1 showed that the histological malignancy score of degeneration after BMSC+UPAL gel transplantation was lower than that of animals that underwent discectomy to create degenerated IVDs via AF needle puncture. This demonstrates that BMSC transplantation leads to IVD regeneration (2). In summary, the inventors' findings show that transplantation of RECs embedded in UPAL gel enhances IVD regeneration in vivo (2).

[0217] Reference to Example 4 [Example 5]

[0218] Inflammation-suppressing and pain-suppressing effects of sodium alginate and mesenchymal stem cell filling in a rat intervertebral disc nucleus pulposus defect model. 1. Construction of a rat intervertebral disc puncture degeneration model. The rat intervertebral disc puncture degeneration model (Mohd Isa et al. Sci Adv 2018) was used in experiments involving the administration of test substances (IHC analysis, histological analysis, pain-related behavioral analysis). A total of 60 12-week-old female SD rats (260-300 g) were randomly assigned to a group with only skin incision (sham group), a group with only intervertebral disc puncture (punch group), a group with UPAL implanted after intervertebral disc puncture (UPAL group), and a group with UPAL and REC implanted after intervertebral disc puncture (REC group) (n=3, at each time point and in each group). Anesthesia was administered via 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 using a forceps pinch test, the dorsal skin of Co4 / 5–5 / 6 was incised. Connective tissue was dissected to expose Co4 / 5–5 / 6, and the intervertebral discs of Co4 / 5–5 / 6 were ruptured using a 19G needle (1 mm in diameter, 2 mm in depth).

[0219] In the Gel and REC groups, dried 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. Using a microinjector (Hamilton) with a 26G needle, 4 μl of the UPAL solution was injected into the center of the intervertebral disc via a different route than the puncture site. In the REC group, the concentration was set at 1 x 10⁶ / ml. The surgical site was cleaned with physiological saline, and the fascia, connective tissue, and skin were sutured closed.

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

[0221] The entire tail (Co4 / 5 to 5 / 6) was surgically removed, and under aseptic conditions, soft tissue was removed, leaving only the caudal vertebrae and intervertebral discs. The harvested intervertebral discs were fixed in 4% (w / v) polyoxymethylene (at room temperature for 48 hours) and embedded in paraffin. The specimen was transversely sectioned at the center of the intervertebral disc to obtain a central coronal section (5 μm thick). After dewaxing with xylene, the sections were cultured in proteinase K (Dako, Agilent Technologies, Santa Clara, CA, USA) (37°C, 15 min). Subsequently, the sections were blocked with 1% hydrogen peroxide methanol (w / v) (37°C, 30 min), cultured in 2% (w / v) bovine serum albumin (at room temperature, 30 min), and then cultured overnight at 4°C with a single antibody. Anti-TNF-α mouse monoclonal antibody (ab220210, Abcam), anti-IL-6 mouse monoclonal antibody (ab9324, Abcam), and anti-TrkA rabbit monoclonal antibody (ab86474, Abcam) were used.

[0222] Regarding staining, Histofine Fast Red II (Nichirei Bioscience) was used for TNF-α analysis, HistoGreen Substrate kit for Peroxidase (Cosmo Bio Co., Ltd., Tokyo, Japan) was used for IL-6 analysis, and Histofine DAB (Nichirei Bioscience) was used for TrkA analysis. Contrast staining of cell nuclei was performed to improve visibility; hematoxylin was used for TNF-α and TrkA staining, and Fast Red was used for IL-6 staining. Using an optical microscope (Olympus, Tokyo, Japan), the number of TNF-α, IL-6, or TrkA positive cells was counted in five randomly selected fields of view for each staining. The number of positive nucleus pulposus or annulus fibrosus cells in each staining was calculated as a percentage of the total number of nucleus pulposus or annulus fibrosus cells in the field of view. Full evaluation was performed by two independent, blinded observers. Each observer performed three assessments on one specimen, calculated the average value for each specimen, and made comparisons between groups.

[0223] 3. Histological evaluation of intervertebral disc degeneration 28 days post-surgery. Rats (n=3) were euthanized using the same method as for immunohistochemical evaluation, and intervertebral discs were harvested. The harvested discs were fixed in 4% (w / v) paraformaldehyde for 48 hours, decalcified with Kristensen decalcification solution for 2 weeks, washed with tap water for 24 hours, and then embedded in paraffin (Mohd Isa et al. Sci Adv 2018). To assess histological fraction using sagittal midline specimens (5 μm thick) of rat intervertebral discs (Rutges et al. Osteoarth Carti 2013), staining was performed with hematoxylin & eosin, safranin O, or Arcian blue (AB). Although AB staining is not directly related to the fraction, it was performed as an auxiliary staining method suitable for extracellular matrix assessment. All evaluations were performed by two independent, blinded observers. Each observer performed three assessments on one specimen, calculated the average value for each individual, and then compared the results between the groups.

[0224] 4. Pain-related behavioral analysis: A total of 24 rats (n=6 per group) were used for pain-related behavioral analysis, including 12 rats that survived up to day 28 post-surgery during immunohistochemical evaluation (n=3 per group) and 12 rats used in histological analysis (n=3 per group). Hargreaves, von Frey, and tail flick tests were performed on all rats. Each rat was individually acclimatized to the test environment for 20 minutes 24 hours prior to the test and again before the start of the test. All tests were conducted blinded by the same examiner. Multiple measurements were performed on each rat, and the average values ​​were calculated. Results were compared between groups.

[0225] 4.1 Hargreaves Test The Hargreaves test was performed using the Hargreaves test apparatus (Ugo Basile Biological Instruments, Gemonio, Italy) on Day 2 before surgery and on Days 2, 7, 14, and 27 after surgery (Mohd Isa et al. Sci Adv 2018). Rats were placed in individual small chambers enclosed on all four sides and above by a glass plate (Ugo Basile Biological Instruments) (with air holes at the top). Infrared beams were irradiated ventrally at the skin incision site as a thermal stimulus. The latency to escape behavior in response to the thermal stimulus was recorded. The beam intensity was set to 50% of the maximum output. To prevent tissue damage, the cutoff time was set to 20 seconds. Although four measurements were performed at each time point in the same rat, there was at least a 1-minute rest period between each measurement.

[0226] 4.2 von Frey Test The von Frey test was performed using a dynamic plantar aesthesiometer (Ugo Basile Biological Instruments) on Day 2 before surgery and on Days 2, 7, 14, and 27 after surgery. Rats were placed in the same small chamber used in the Hargreaves test, set up on a metal mesh. A 0.5 mm diameter fiber was placed on the ventral side of the skin incision, and a linearly increasing force from 0 g to 5 g was applied over 10 seconds, followed by a constant force of 5 g until 30 seconds after the start of the test. The latency until the rat exhibited any escape behavior was recorded. Although five measurements were performed on the same rat at each time point, there was at least a 10-second rest period between each measurement.

[0227] 4.3 Tail Flick Pain Test The tail flick pain test was performed using a heat flux radiometer (Ugo Basile Biological Instruments). To avoid tissue damage due to excessive heat stimulation caused by performing the test on the same schedule as the Hargreaves test, it was performed on Day 1 before surgery and on Days 3, 8, 15, and 28 after surgery (Mohd Isa et al. Sci Adv 2018). After the rats were calmed for 10 minutes with their bodies wrapped in towels, only their tails were placed on the device with their bodies covered by towels. Infrared beams were irradiated onto the ventral side of the tail, about 5 cm from the distal end. The latency of the tail flick response to heat stimulation was recorded. To prevent tissue damage, the cutoff time was set to 20 seconds. Although four measurements were performed at each time point for the same rat, there was a rest period of at least 15 seconds between each measurement.

[0228] 5. Statistical Analysis: All data are expressed as mean ± standard error (SE). One-way ANOVA was used for comparisons among multiple groups. Unpaired Student's t-test was used for comparisons between two groups. All ANOVA results were further evaluated using the Tukey-Kramer post-hoc test or the Kruskal-Wallis test. Differences were considered statistically significant at the 5% significance level (P < 0.05).

[0229] 6. Results: Pain suppression effect was confirmed in the group with UPAL implantation and REC implantation (REC group). [Simplified Explanation of the Diagram]

[0018] [Figure 1] shows cell sorting data for separating bone marrow mesenchymal stem cells (BMSCs) and unlabeled nucleus pulposus cells (NPCs) after 7 days of co-culture with sodium alginate solution (hereinafter, sometimes referred to as "UPAL" (Ultra-purified alginate gel)). (a): Two-dimensional (2D) scatter plot of co-cultured cells. P1 gates are positioned around single live cells. (b): 2D scatter plot of unlabeled NPCs in P2 gates and CFDA-SE labeled BMSCs in P3 gates after sorting. FSC-A: Forward scattering region; FSC-W: Forward scattering width; SSC-A: Lateral scattering region; CFDA-SE-A: CFDA-SE region. Gene expression of each cell was plotted against a logarithmic scale (y-axis) using the housekeeping gene GAPDH as a baseline. Data represent the average values ​​from four different rabbit NPC systems. (c): HIF-1α, (d): GLUT-1, (e): Brachyury, (f): CDMP-1, (g): TGF-β, (h): IGF-1, (i): type II collagen, and (j): agglutinin. Data are expressed as mean ± standard error, and p-values ​​were obtained using a one-way variance analysis with a post-hoc Tukey-Kramer test. [Figure 2] shows the survival of bone marrow intervertebral stem cells (BMSCs) in the intervertebral disc (IVD). Bone marrow mesenchymal stem cells (BMSCs) labeled with carboxyfluorescein diacetate succinimide (CFDA-SE) survived in the intervertebral disc (IVD) at weeks 4 and 12 post-surgery. Frozen sections of the IVD were stained with 4',6-diamino-2-phenylindole (DAPI). Images are representative of four replicates (intact control and BMSCs + gel, n=4; 4 weeks and 12 weeks). Scale bar = 50 μm. [Fig. 3] shows the water content of the degenerated intervertebral disc (IVD) after discectomy in gel preservation combined with bone marrow mesenchymal stem cells (BMSCs). (a): T2-weighted imaging (T2WI), midsagittal plane images of the degenerated IVD at weeks 4 and 12 post-surgery. Images are representative of eight imaging studies. (b): Pfirrmann classification of IVD degeneration. Data are mean ± standard error (complete control, puncture, discectomy, gel, and BMSC+gel, n=8; 4 weeks and 12 weeks). (c) MRI index of NP degeneration (nucleus pulposus (NP) area × mean signal intensity). Values ​​are expressed as a percentage (%) compared to complete control IVD.Data are presented as mean ± standard error. The p-value was determined using the Tukey-Kramer test with one-way ANOVA following post-hoc analysis. [Figure 4] Gels combined with bone marrow-derived intervertebral stem cells (BMSCs) show a pattern of intervertebral disc (IVD) degeneration prevention after discectomy. (a, b): Midsagittal section images of IVD stained with hematoxylin and eosin (H&E) or safranin O (representative examples of images from 8 experiments) (Intact control, Puncture, Discectomy, Gel, BMSC + Gel, n=8; 4 and 12 weeks). AF: Annulus fibrosus, NP: Nucleus pulposus. Scale bar = (A): 500 μm (segments 1 and 3 from above), 50 mm (segments 2 and 4 from above), and (B): 500 μm. (c): Histological degeneration at weeks 4 and 12. Data are mean ± standard error, and p-values ​​were obtained by one-way ANOVA using the Tukey-Kramer post-hoc test. [Fig. 5] Image showing type II collagen-positive cells in rabbit nucleus pulposus (NP). (a): Midsagittal section of rabbit intervertebral disc (IVD) stained with type II collagen. Images are representative of 8 replicates (complete control, puncture, discectomy, gel, BMSC+gel, n=8; 4 and 12 weeks). Arrows indicate type II collagen-positive cells. Scale bar = 500 μm (first and third segments from the top) and 20 μm (second and fourth segments from the top). (b): Proportion of type II collagen-positive cells. Data are presented as mean ± standard error. p-values ​​were determined using a one-way ANOVA with a post-hoc Tukey-Kramer test. [Figure 6] Graph showing nucleus pulposus (NP)-labeled positive cells in rabbit NPs. (ac): Horizontal sections of rabbit intervertebral discs (IVDs) stained with HIF-1α, GLUT-1, and Brachyury on days 1, 7, and 28. Images are representative of four replicates (complete control, discectomy, and BMSCs + gel, n=4; day 1, 7, 28). Scale bar = 50 μm. (df): Proportion of NP-labeled positive cells relative to whole cells. (gi): Proportion of NP-labeled positive cells relative to CFDA-SE positive cells (representing transplanted BMSCs). Data are presented as mean ± standard error. p-values ​​were determined using a paired t-test. [Figure 7] Graph showing the mechanism of intervertebral disc (IVD) regeneration. Transplanted bone marrow mesenchymal stem cells (BMSCs) produce growth factors and extracellular matrix (ECM) that activate existing nucleus pulposus cells (NPCs). The activation of existing NPCs further increases the production of growth factors and ECM. The transplanted BMSCs differentiate into NPCs.UPAL: Ultrapure alginate (also known as low-endotoxin high-purity alginate). [Figure 8] A diagram showing the comprehensive analysis results of the REC strain. [Figure 9] A diagram showing the expression levels of various genes in healthy human intervertebral disc nucleus pulposus cells (NPCs) and high-purity mesenchymal cells (RECs). [Figure 10] A diagram showing the MRI results of intervertebral discs in a sheep model at week 4 post-transplantation. [Figure 11] A diagram showing the histological examination results of intervertebral discs in a sheep model at week 4 post-transplantation. [Figure 12] A diagram showing the histological examination results of intervertebral discs in a sheep model at week 4 post-transplantation. [Figure 13] A diagram showing the phenotypic profile of human NPCs and RECs after 7 days of 3D co-culture. (A) A schematic diagram showing isolated RECs. Flow cytometry profile of human bone marrow cells stained with CD271 (LNGFR) and CD90 (THY-1). Cells collected from one well were seeded into 35 mm culture dishes and allowed to proliferate for 14 days to obtain homogeneous cell clusters with high differentiation and proliferation capabilities. (B and C) Cell sorting data were used to distinguish CFDA-SE labeled RECs from unlabeled NPCs. (B) Dead cells and residual live cells were excluded from the P1 gate. (C) A 2D scatter plot showing unlabeled NPCs in the P2 gate and CFDA-SE labeled RECs in the P3 gate is displayed. (D-K) Gene expression levels of each cell were normalized relative to the expression level of the housekeeping gene GAPDH and plotted on a logarithmic scale (y-axis). Data from four different human NPC strains were averaged. (D) HIF-1α, (E) GLUT-1, (F) brachyury, (G) CDMP-1, (H) TGF-β, (I) IGF-1, (J) type II collagen, (K) agglutinin data are expressed as mean ± SD (n=4). Significant differences were assessed using one-way ANOVA with post-hoc Tukey-Kramer test. NPC: Nucleus pulposus cells; REC: Rapidly proliferating cell line; CFDA-SE: 5,6-carboxyfluorescein diacetate succinimide ester; ANOVA: ANOVA; FSC-A: Forward scattering area; FSC-W: Forward scattering width; SSC-A: Lateral scattering area; CFDA-SE-A: CFDA-SE area [Figure 14] shows the elasticity ratio of the two gels. (A) Formation of disc-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. The samples were compressed at a constant speed of 0.5 mm / min. (D) Stress-strain curves. Young's modulus was calculated based on the slope of the tangent between 10% and 20% of the compression value. Four replicate tests were performed, and representative images are shown. (E) Young's modulus of the two gels.Data show mean ± SD values ​​(n=4). Significant differences were assessed by Student's t-test after Welch's test. NS: No significant difference UPAL: Ultrapure alginate (also known as low endotoxin high purity alginate) REC: Rapidly proliferating cell line SD: Standard deviation [Figure 15] shows the experimental schedule and detailed treatment diagrams for each group. (A and B) 20 mg of fresh NP tissue was removed by autologous IVD at the initial surgery to establish a heavily degenerated IVD pattern. (C) 70 mg of degenerated NP tissue was removed by autologous IVD 4 weeks after the initial surgery. (D) After removal of degenerated NP, UPAL or REC+UPAL solution was transplanted into the intervertebral disc space. NP: Nucleus pulposus UPAL: Ultrapure alginate (also known as low endotoxin high purity alginate) IVD: Intervertebral disc [Figure 16] shows MRI assessments of the treated IVD at 4 and 24 weeks post-implantation. (A) T2-weighted midsagittal images of IVD in sheep at 4 and 24 weeks post-surgery. Images are representative of 6 or 8 replicates. (B) Pfirrmann grade IVD degeneration. (C) MRI index (NP area × mean signal intensity) of degenerative change relative to NP. Values ​​are expressed as a percentage (%) of the value relative to the untreated control IVD. (D) Disc height index used in IVD treatment. Values ​​are expressed as a percentage (%) of the value relative to the untreated control IVD. Data are expressed as mean ± SD (untreated control, n=6; discectomy, n=6; gel, n=8; REC+ gel, n=8). Significance was assessed by a one-dimensional ANOVA using the Tukey-Kramer post-hoc analysis. NP: Nucleus pulposus MRI: Magnetic resonance imaging IVD: Intervertebral disc ANOVA: Analysis of variance SD: Standard deviation [Fig. 17] shows the results of 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 (no treatment control, n=6; discectomy, n=6; gel, n=8; REC+ gel, n=8). Scale bar (A): 50 μm (from the 2nd and 4th segments above) or 1 mm (from the 1st and 3rd segments above) (B): 1 mm. (C) Histological grading was determined by modified Boos classification. Data are expressed as mean ± standard deviation. Significance was assessed by one-way variance analysis using the post-hoc Tukey-Kramer test. IVD: Intervertebral disc H&E: Hematoxylin & Eosin SD: Standard deviation AF: Annulus fibrosus NP: Nucleus pulposus [Fig. 18] shows type II or type I collagen-positive cells in the treated IVD 4 weeks and 24 weeks after implantation.(A and B) Representative midsagittal sections of treated IVDs stained for type II or type I collagen (no treatment control, n=6; discectomy, n=6; gel, n=8; REC+ gel, n=8). Scale bar, 1 mm (lines 1 and 3) or 50 μm (lines 2 and 4). (C and D) Percentage of type II or type I collagen-positive cells relative to total cells in treated IVDs. Data are expressed as mean ± standard deviation. Significance was assessed by one-way ANOVA using the post-hoc Tukey-Kramer test. IVD: disc REC: rapidly proliferating strains SD: standard deviation ANOVA: analysis of variance [Figure 19] shows the histological assessment at 4 weeks after NP tissue removal. (A) Midsagittal portion of NP tissue removed from IVD (no treatment control, or removal of 20, 50, 100, or 200 mg) (stained with H&E or safranin-O). In sheep models, IVD degeneration occurs after 4 weeks due to removal of more than 20 mg of NP tissue. Scale bar: 1 mm (total). (B) Histological grading was determined using a modified Boos classification. Data are mean ± standard deviation. NP: nucleus pulposus; IVD: intervertebral disc; H&E: hematoxylin and eosin; SD: standard deviation. [Fig. 20] shows the results of intervertebral disc height measurements relative to adjacent vertebrae using midsagittal images of T2-weighted imaging. BC and EF represent anterior and posterior disc heights, respectively, and AB and DE represent cephalad adjacent vertebral body heights. The DHI value is calculated as the ratio of intervertebral disc height (BC+EF) to vertebral body height (AB+DE). The relative DHI value is calculated as the ratio of the DHI of the treated IVD to the DHI of the untreated control IVD. DHI: Intervertebral Disc Height Index; IVD: Intervertebral Disc.

Claims

1. A composition for intervertebral disc regeneration, comprising 0.5 w / w% to 5.0 w / w% of a monovalent metal salt of alginate and mesenchymal stem cells derived from human bone marrow, wherein the aforementioned mesenchymal stem cells derived from human bone marrow are cell clusters of rapidly proliferating mesenchymal stem cell lines that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and are cell clusters that satisfy the following characteristics (a), or a combination of (a) and (b): (a) the coefficient of variation of forward scattered light in flow cytometry is less than 40%, and (b) the average cell size is less than 20 μm.

2. A composition for intervertebral disc regeneration, comprising 0.5 w / w% to 5.0 w / w% of a monovalent metal salt of alginate and mesenchymal stem cells derived from human bone marrow, wherein the aforementioned mesenchymal stem cells derived from human bone marrow are cell groups of rapidly proliferating mesenchymal stem cell lines isolated using LNGFR (CD271) as a positive indicator or LNGFR (CD271) and Thy-1 (CD90) as co-positive indicators, and are cell groups that meet the following characteristics (a), or a combination of (a) and (b): (a) the coefficient of variation of forward scattered light in flow cytometry is less than 40%, and (b) the average cell size is less than 20 μm.

3. The components of claim 1 or 2, which promote the regeneration of the nucleus pulposus of the intervertebral disc through activation of nucleus pulposus cells induced by mesenchymal stem cells and / or differentiation of mesenchymal stem cells into nucleus pulposus cells.

4. The composition of claim 1 or 2, applied to the intervertebral disc of the object in such a manner as to allow the crosslinking agent to contact at least a portion of the surface of the composition after application, and having fluidity upon application.

5. The components of claim 4 are intended to be applied to the nucleus pulposus of the intervertebral disc.

6. The composition of claim 5, wherein the application to the aforementioned nucleus pulposus site is for filling the aforementioned composition into the nucleus pulposus defect.

7. The composition of claim 4, wherein the aforementioned crosslinking agent is a metal ion compound with a valence of 2 or higher.

8. The composition of claim 1 or 2, wherein the aforementioned monovalent metal salt of alginate is a low-endotoxin monovalent metal salt of alginate.

9. The composition of the aforementioned alginate monovalent metal salt, as determined by GPC-MALS, has a weight-average molecular weight (absolute molecular weight) of 80,000 or more.

10. The components of claim 1 or 2 are used for the treatment, prevention or inhibition of recurrence of intervertebral disc degeneration and / or intervertebral disc injury.

11. As in claim 10, wherein the aforementioned intervertebral disc degeneration and / or intervertebral disc injury is selected from at least one of the groups consisting of intervertebral disc herniation, intervertebral disc disease, degenerative spondylolisthesis, purulent discitis, degenerative spinal disease, spinal stenosis, and intervertebral disc injury.

12. The use of a composition comprising 0.5 w / w% to 5.0 w / w% of a monovalent metal salt of alginate and a mesenchymal stem cell derived from human bone marrow, for preparing a composition for intervertebral disc regeneration applied in a fluid state to the nucleus pulposus of an intervertebral disc, wherein the aforementioned mesenchymal stem cells derived from human bone marrow are cell clusters of rapidly proliferating mesenchymal stem cell lines that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and are cell clusters that satisfy the following characteristics (a), or a combination of (a) and (b): (a) the coefficient of variation of forward scattered light in flow cytometry is less than 40%, and (b) the average cell size is less than 20 μm.

13. The use of a composition comprising 0.5 w / w% to 5.0 w / w% of a monovalent metal salt of alginate and a human bone marrow-derived mesenchymal stem cell, for use in preparing a composition for intervertebral disc regeneration applied in a fluid state to the nucleus pulposus of an intervertebral disc, wherein the aforementioned human bone marrow-derived mesenchymal stem cells are cell clusters of rapidly proliferating mesenchymal stem cell lines isolated using LNGFR (CD271) as a positive indicator, or LNGFR (CD271) and Thy-1 (CD90) as co-positive indicators, and are cell clusters that satisfy the following characteristics (a), or a combination of (a) and (b): (a) the coefficient of variation of forward scattered light in flow cytometry is less than 40%, and (b) the average cell size is less than 20 μm.

14. As claimed in claim 12 or 13, it is used so that the crosslinking agent does not come into contact with the composition after being applied to the nucleus pulposus of the intervertebral disc in a fluid state.

15. As claimed in claim 12 or 13, it promotes the regeneration of the nucleus pulposus of the intervertebral disc through the activation of nucleus pulposus cells derived from the aforementioned intervertebral bone marrow interlobular stem cells and / or the differentiation of the aforementioned intervertebral bone marrow interlobular stem cells into nucleus pulposus cells.

16. As claimed in claim 12 or 13, wherein the aforementioned mesenchymal stem cells derived from human bone marrow are administered in an undifferentiated state and / or without induced differentiation.

17. As claimed in claim 12 or 13, wherein the aforementioned monovalent metal salt of alginate is a low-endotoxin monovalent metal salt of alginate.

18. As requested in paragraphs 12 or 13, wherein the aforementioned monovalent metal salt of alginate has a weight-average molecular weight (absolute molecular weight) of 80,000 or more as determined by GPC-MALS.

19. As requested in paragraphs 12 or 13, it is for the treatment, prevention or suppression of recurrence of intervertebral disc degeneration and / or intervertebral disc injury.

20. As claimed in claim 19, wherein the aforementioned intervertebral disc degeneration and / or intervertebral disc injury is selected from at least one of the following groups: intervertebral disc herniation, intervertebral disc disease, degenerative spondylolisthesis, purulent discitis, chronic low back pain, degenerative spinal disease, spinal stenosis, lumbar spinal stenosis, intervertebral disc herniation (mixed lumbar spinal stenosis) associated with lumbar spinal stenosis, and intervertebral disc injury.

21. As requested in claim 19, wherein the aforementioned intervertebral disc degeneration and / or intervertebral disc injury is accompanied by chronic low back pain.

22. As requested in items 12 or 13, it is used to suppress intervertebral disc pain.

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