WO2022163872A1 - 椎間板再生用組成物 - Google Patents
椎間板再生用組成物 Download PDFInfo
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- WO2022163872A1 WO2022163872A1 PCT/JP2022/004342 JP2022004342W WO2022163872A1 WO 2022163872 A1 WO2022163872 A1 WO 2022163872A1 JP 2022004342 W JP2022004342 W JP 2022004342W WO 2022163872 A1 WO2022163872 A1 WO 2022163872A1
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- intervertebral disc
- composition
- cells
- nucleus pulposus
- mesenchymal stem
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Definitions
- the present invention relates to a composition for intervertebral disc regeneration containing a low endotoxin monovalent metal salt of alginic acid and mesenchymal stem cells.
- TECHNICAL FIELD The present invention relates to a composition for intervertebral disc regeneration containing high-purity human bone marrow-derived mesenchymal stem cells.
- the human spine (vertebrae) consists of 24 bones (vertebrae), and the tissue that acts as a cushion between the vertebrae is called an intervertebral disc.
- Intervertebral discs are degenerated and damaged due to aging, injury, disease, and the like.
- Intervertebral disc degeneration is a state in which the number of intervertebral disc cells, water content, extracellular matrix (type II collagen, aggrecan, etc.), etc. are reduced, and as it progresses, the intervertebral disc cannot function as a shock absorber.
- intervertebral disc degeneration and intervertebral disc damage include intervertebral disc herniation, intervertebral disc disease, spinal degenerative spondylolisthesis, suppurative discitis, spondylolisthesis, spinal canal stenosis, and intervertebral disc damage due to trauma.
- intervertebral disc herniation the annulus fibrosis that covers the nucleus pulposus is deformed or cracked, forming a herniation and protruding outside the intervertebral disc. cause paralysis, etc.
- intervertebral disc nucleus pulposus removal excision
- a certain effect has been confirmed.
- intervertebral disc nucleus pulpectomy excision
- a cavity also referred to herein as a “defect”
- the cavity of the nucleus pulposus tends to be physically weak.
- fibroblast-like cells may accumulate in the hollow portion to form a tissue having mechanical properties different from those of the original nucleus pulposus.
- the herniation recurrence rate is high after intervertebral disc nucleus pulposus surgery. It is said that the recurrence rate within 5 years after the nucleus pulposus of the intervertebral disc is about 4 to 15%, but recent long-term data have revealed that more than half of the cases will recur after 10 years. If the hernia recurs, a second operation is required, but the spinal nerve is buried in the scar tissue formed after the first operation, making it difficult to confirm the position of the spinal nerve.
- mesenchymal stem cells have few ethical problems associated with cell collection, and have a variety of differentiation potentials such as bone, cartilage, and fat. It is one of the somatic stem cells that have been performed in Japan. Since MSCs can be isolated by a relatively simple procedure, they are widely used as a material for regenerative medicine, mainly by inducing differentiation into cartilage, bone, etc. in vitro and then transplanting them locally. In promoting clinical application, it is essential for commercialization to be able to produce the necessary amount of cells that maintain a certain level of function.
- the present inventors separated LNGFR and Thy-1 co-positive cells from bone marrow fluid by flow cytometry, and obtained clones with rapid expansion (Rapidly Expanding Clones: REC), and the proliferation of donor-derived MSCs Established a purification separation method that can eliminate gender differences (Patent No. 6463029, WO2016/017795, Mabuchi Y. et al., Stem Cell Reports 1(2): 152-165, 2013)
- Patent No. 6487110 Patent No. 6463029 International publication 2016/017795 pamphlet
- the present inventors found that a composition containing a low endotoxin monovalent metal salt of alginic acid and mesenchymal stem cells can solve the above problems, and have completed the present invention. Completed. Moreover, in another aspect of the present invention, it was found that a composition containing human bone marrow-derived high-purity mesenchymal stem cells can solve the above problems. That is, the present invention is as follows. [1] A composition for intervertebral disc regeneration, comprising a monovalent metal salt of low endotoxin alginic acid and mesenchymal stem cells.
- a composition for intervertebral disc regeneration containing a monovalent metal salt of alginic acid and mesenchymal stem cells. [2] promoting nucleus pulposus regeneration of intervertebral discs through activation of nucleus pulposus cells by mesenchymal stem cells and/or differentiation of mesenchymal stem cells into nucleus pulposus cells, [1] or [1-1] The composition according to . [3] The composition according to any one of [1] to [2], wherein the mesenchymal stem cells are high-purity mesenchymal stem cells derived from human bone marrow.
- the human bone marrow-derived high-purity mesenchymal stem cells are LNGFR (CD271)-positive, or LNGFR (CD271) and Thy-1 (CD90) co-positive fast-proliferating mesenchymal stem cell clone cell populations.
- the human bone marrow-derived highly purified mesenchymal stem cells are LNGFR (CD271 ) is positive, or LNGFR (CD271) and Thy-1 (CD90) are co-positive.
- the coefficient of variation of forward scattered light in flow cytometry is 35% or less
- the average cell size is 20 ⁇ m or less [4-2]
- LNGFR CD271
- Thy-1 CD90
- the coefficient of variation of forward scattered light in flow cytometry is 40% or less
- the average cell size is 20 ⁇ m or less [4-5]
- LNGFR LNGFR
- Thy-1 CD90
- co-positive cell population of fast-proliferating mesenchymal stem cell clones derived from cells wherein at least the following (a) and (b)
- the coefficient of variation of forward scattered light in flow cytometry is 40% or less
- the average cell size is 20 ⁇ m or less ] to [4-5].
- [5-2] Any one of [1] to [4], which is applied to the nucleus pulposus region of the target intervertebral disc, used to partially harden after application, and has fluidity when applied to the nucleus pulposus region The composition according to .
- the cross-linking agent is a divalent or higher metal ion compound.
- the monovalent metal salt of alginic acid is a low endotoxin monovalent metal salt of alginic acid.
- the composition according to . [10] The composition according to any one of [1] to [9], wherein the concentration of the low endotoxin monovalent metal salt of alginic acid is 0.5 w/w% to 5.0 w/w%.
- the composition according to any one of [1] to [10] which is used for treatment, prevention or suppression of recurrence of intervertebral disc degeneration and/or intervertebral disc injury.
- intervertebral disc degeneration and/or intervertebral disc injury is intervertebral disc herniation, intervertebral disc disease, spinal degenerative spondylolisthesis, suppurative discitis, spondylolisthesis, spinal canal stenosis, lumbar spinal canal stenosis, and lumbar spinal canal stenosis.
- the composition according to [11] which is at least one selected from the group consisting of intervertebral disc herniation and intervertebral disc injury associated with [12-1]
- the composition of [11] or [12] wherein the intervertebral disc degeneration and/or intervertebral disc injury is associated with chronic low back pain.
- a composition for intervertebral disc regeneration which contains highly purified human bone marrow-derived mesenchymal stem cells and is applied to a target intervertebral disc nucleus pulposus site.
- [14] Promote regeneration of the nucleus pulposus of intervertebral discs through activation of nucleus pulposus cells by highly purified human bone marrow-derived mesenchymal stem cells and/or differentiation of highly purified human bone marrow-derived mesenchymal stem cells into nucleus pulposus cells , the composition according to [13].
- the human bone marrow-derived high-purity mesenchymal stem cells are LNGFR (CD271)-positive or LNGFR (CD271) and Thy-1 (CD90) co-positive fast-proliferating mesenchymal stem cell clone cell populations.
- the coefficient of variation of forward scattered light in flow cytometry is 40% or less
- the average cell size is 20 ⁇ m or less [16-5] ) positive or LNGFR (CD271) and Thy-1 (CD90) co-positive cell population of fast-proliferating mesenchymal stem cell clones derived from cells, wherein at least the following (a) and (b)
- the composition further comprises a carrier for embedding the cells, The composition according to any one of [13] to [16-5].
- the carrier is selected from the group consisting of alginic acid, hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparan sulfate, and galactosaminoglycrine glycan sulfate, and pharmaceutically acceptable salts thereof.
- Any one of [13] to [19], which is applied to the nucleus pulposus region of the target intervertebral disc, is used to partially harden after application, and has fluidity when applied to the nucleus pulposus region composition.
- the cross-linking agent is a divalent or higher metal ion compound.
- the monovalent metal salt of alginic acid has a weight average molecular weight (absolute molecular weight) of 80,000 or more as measured by GPC-MALS. thing.
- the monovalent metal salt of alginic acid is a low endotoxin monovalent metal salt of alginic acid.
- intervertebral disc degeneration and/or intervertebral disc injury is intervertebral disc herniation, intervertebral disc disease, spinal degenerative spondylolisthesis, suppurative discitis, spondylolisthesis, spinal canal stenosis, lumbar spinal canal stenosis, and lumbar spinal canal stenosis.
- the composition of [27] which is at least one selected from the group consisting of intervertebral disc herniation (mixed lumbar spinal canal stenosis) and intervertebral disc injury associated with spinal cord stenosis.
- [28-1] The composition of [27] or [28], wherein the intervertebral disc degeneration and/or intervertebral disc injury is associated with chronic low back pain.
- [30] The composition of [29], wherein the disc pain is chronic low back pain.
- composition for intervertebral disc regeneration comprising a monovalent metal salt of alginic acid and high-purity human bone marrow-derived mesenchymal stem cells, which is to be applied to the nucleus pulposus of the intervertebral disc in a fluid state.
- composition for regeneration [31-1] The composition for intervertebral disc regeneration according to [31] above, for use that does not require a treatment of hardening the composition after being applied to the target intervertebral disc nucleus pulposus in a fluid state.
- intervertebral disc according to [31] or [31-1] above which is used without contacting the composition with a cross-linking agent after being applied to the target intervertebral disc nucleus pulposus in a fluid state.
- composition for regeneration [32] Promote regeneration of the nucleus pulposus of intervertebral discs through activation of nucleus pulposus cells by highly purified human bone marrow-derived mesenchymal stem cells and/or differentiation of highly purified human bone marrow-derived mesenchymal stem cells into nucleus pulposus cells , the composition according to any one of [31] to [31-2].
- the human bone marrow-derived high-purity mesenchymal stem cells are LNGFR (CD271)-positive or LNGFR (CD271) and Thy-1 (CD90) co-positive fast-proliferating mesenchymal stem cell clone cell populations.
- the coefficient of variation of forward scattered light in flow cytometry is 40% or less
- the average cell size is 20 ⁇ m or less
- the human bone marrow-derived high-purity mesenchymal stem cells are ) is positive, or LNGFR (CD271) and Thy-1 (CD90) are co-positive.
- the human bone marrow-derived high-purity mesenchymal stem cells are ) positive or LNGFR (CD271) and Thy-1 (CD90) co-positive cell population of fast-proliferating mesenchymal stem cell clones derived from cells, wherein at least the following (a) and (b) The composition according to any one of [31] to [33], which is a cell population that satisfies one characteristic.
- intervertebral disc degeneration and/or intervertebral disc injury is intervertebral disc herniation, intervertebral disc disease, spondylolisthesis, suppurative discitis, chronic low back pain, spondylosis osteoarthritis, spinal canal stenosis, lumbar spinal canal stenosis, lumbar spinal column
- the composition of [39] which is at least one selected from the group consisting of intervertebral disc herniation associated with canal stenosis (mixed lumbar spinal stenosis) and intervertebral disc injury.
- [41] The composition of [39] or [40], wherein the disc degeneration and/or disc injury is associated with chronic low back pain.
- [42] The composition of any one of [31] to [41], which is used for suppressing intervertebral disc pain.
- [43] The composition of [42], wherein the disc pain is chronic low back pain.
- a composition for suppressing intervertebral disc pain containing a monovalent metal salt of alginic acid and high-purity human bone marrow-derived mesenchymal stem cells, which is applied in a fluid state to the nucleus pulposus region of the subject composition.
- the pain is associated with intervertebral disc herniation, intervertebral disc disease, spinal degenerative spondylolisthesis, suppurative discitis, spondylolisthesis, spinal canal stenosis, lumbar spinal canal stenosis, intervertebral disc herniation associated with lumbar spinal canal stenosis (mixed The composition according to any one of [44] to [44-2], which is pain associated with at least one selected from the group consisting of lumbar spinal canal stenosis and intervertebral disc injury. [46] The composition of [44] to [45], wherein the pain is chronic low back pain.
- the human bone marrow-derived high-purity mesenchymal stem cells are LNGFR (CD271)-positive or LNGFR (CD271) and Thy-1 (CD90) co-positive fast-proliferating mesenchymal stem cell clone cell populations.
- the composition according to any one of [44] to [47] which is a cell population that satisfies at least one of the following characteristics (a) and (b).
- the coefficient of variation of forward scattered light in flow cytometry is 40% or less
- the average cell size is 20 ⁇ m or less [48-2]
- LNGFR LNGFR
- Thy-1 CD90
- co-positive cell population of fast-proliferating mesenchymal stem cell clones derived from cells wherein at least the following (a) and (b) The composition according to any one of [44] to [47], which is a cell population that satisfies one characteristic.
- the coefficient of variation of forward scattered light in flow cytometry is 40% or less
- the average cell size is 20 ⁇ m or less [48-3] the coefficient of variation is 35% or less, [48]-[ 48-2].
- the monovalent metal salt of alginic acid has a concentration of 0.5 w/w% to 5.0 w/w%.
- composition according to any one of [44] to [50], wherein the monovalent metal salt of alginic acid is a low endotoxin monovalent metal salt of alginic acid.
- the composition is applied to the nucleus pulposus region of the target intervertebral disc under X-ray fluoroscopy, has fluidity when applied to the nucleus pulposus region, and is used without contacting the composition with a cross-linking agent [31]-[ 51].
- a method of evaluating the regenerative ability of an intervertebral disc in a composition applied to an intervertebral disc of a sheep model of severe intervertebral disc degeneration comprising the steps of (a1) or (a2) below, and (b1) or (b2).
- Step (a1) Step of removing nucleus pulposus tissue in an amount corresponding to 0.00004% to 0.00005% of the body weight of sheep from the intervertebral disc of sheep to prepare a degenerated intervertebral disc
- Step (b1) of preparing a degenerated intervertebral disc Four weeks after the step (a1) or (a2), a step of removing an amount of nucleus pulposus tissue corresponding to 0.00014% to 0.000175% of the body weight of the sheep ( b2) 4 weeks after step (a1) or (a2), removing an additional 70 mg of nucleus pulposus tissue [55] administering the composition after step (b1) or (b2), The method of [54].
- the vertebral bodies and intervertebral discs collected from the degeneration model are evaluated by at least one evaluation method selected from the group consisting of MRI, tissue staining, and immunohistochemical staining (IHC). , [54] or [55].
- a method for treating, preventing, or inhibiting recurrence of intervertebral disc degeneration and/or injury comprising: The above method, comprising applying a composition containing a monovalent metal salt of alginic acid and mesenchymal stem cells to the nucleus pulposus region of the intervertebral disc of a subject in need of the above treatment, prevention or suppression of recurrence.
- a method for treating, preventing, or inhibiting recurrence of intervertebral disc degeneration and/or injury comprising: The above method, comprising applying high-purity human bone marrow-derived mesenchymal stem cells to the nucleus pulposus region of the intervertebral disc of the subject in need of the above treatment, prevention, or suppression of recurrence.
- a method for treating, preventing or inhibiting recurrence of intervertebral disc degeneration and/or injury comprising: applying a composition containing high-purity human bone marrow-derived mesenchymal stem cells and a monovalent metal salt of alginic acid to the nucleus pulposus region of the intervertebral disc of the subject in need of the treatment, prevention, or suppression of recurrence; is flowable when applied to the nucleus pulposus site.
- a method for treating, preventing, or suppressing recurrence of discogenic pain comprising: applying a composition containing high-purity human bone marrow-derived mesenchymal stem cells and a monovalent metal salt of alginic acid to the nucleus pulposus region of the intervertebral disc of the subject in need of the treatment, prevention, or suppression of recurrence; is flowable when applied to the nucleus pulposus site.
- Human bone marrow-derived high-purity mesenchymal stem cells for use in treating, preventing, or suppressing recurrence of intervertebral disc degeneration and/or intervertebral disc injury.
- a composition comprising high-purity human bone marrow-derived mesenchymal stem cells and a monovalent metal salt of alginic acid for use in the treatment, prevention, or suppression of recurrence of intervertebral disc degeneration and/or intervertebral disc injury, said composition comprises applying to the nucleus pulposus region of the intervertebral disc of a subject in need of said treatment, prevention or suppression of recurrence, said composition having flowability when applied to said nucleus pulposus region.
- a composition comprising high-purity human bone marrow-derived mesenchymal stem cells and a monovalent metal salt of alginic acid for use in treating, preventing, or suppressing recurrence of disc pain, wherein the composition comprises , applying to a nucleus pulposus site of an intervertebral disc of a subject in need of prevention or inhibition of recurrence, wherein said composition has flowability when applied to said nucleus pulposus site.
- the present invention provides a nucleus pulposus replacement composition capable of promoting regeneration of the nucleus pulposus of intervertebral discs.
- a nucleus pulposus replacement composition capable of promoting regeneration of the nucleus pulposus of intervertebral discs.
- the composition of the present invention it is possible to suppress degenerative changes in the entire intervertebral disc tissue including not only the nucleus pulposus of the intervertebral disc but also the annulus fibrosus.
- the composition of the present invention also has the effect of increasing the proportion of type II collagen-positive hyaline cartilage-like cells in the nucleus pulposus.
- a carrier for embedding cells is used without treatment for hardening, and the composition is used in patients with intervertebral disc disease. Pain, especially low back pain, more preferably chronic low back pain can be suppressed.
- FIG. 1 Carboxyfluorescein diacetate succinimidyl after 7 days of co-culture with a sodium alginate solution (hereinafter sometimes referred to as "UPAL" (low endotoxin ultra-purified alginate gel))
- UPAL low endotoxin ultra-purified alginate gel
- NPCs non-labeled nucleus pulposus cells
- CFDA-SE Cell sorting data for separation of bone marrow-derived mesenchymal stem cells (BMSCs) and non-labeled nucleus pulposus cells labeled with Lester
- 2D Two-dimensional (2D) dot plot of co-cultured cells. A P1 gate was placed around a single live cell.
- FIG. 2 A diagram showing that bone marrow-derived mesenchymal stem cells (BMSCs) survive in the intervertebral disc (IVD).
- BMSCs bone marrow-derived mesenchymal stem cells
- BMSCs Carboxyfluorescein diacetate succinimidyl ester
- DAPI 4′,6-diamino-2-phenylindole
- (b): Pfirrmann classification of IVD degeneration. Data are mean ⁇ SEM (intact control, puncture, discectomy, gel, and BMSC+gel, n 8; 4 and 12 weeks).
- FIG. 5 A diagram showing type II collagen-positive cells in rabbit nucleus pulposus (NP).
- NP rabbit nucleus pulposus
- IVD rabbit intervertebral disc
- Scale bar 500 ⁇ m (first and third sections from top) and 20 ⁇ m (second and fourth sections from top).
- FIG. 6 A diagram showing nucleus pulposus (NP) marker-positive cells in rabbit NP.
- NP marker-positive cells Percentage of NP marker-positive cells to CFDA-SE-positive cells (representative of engrafted BMSCs). Data are mean ⁇ s.e.m. p-values were determined using a paired t-test.
- FIG. 7 A diagram showing the mechanism of intervertebral disc (IVD) regeneration.
- BMSCs bone marrow-derived mesenchymal stem cells
- ECM extracellular matrix
- Pre-existing NPC activation also increases the production of growth factors and ECM.
- Transplanted BMSCs differentiate into NPCs.
- UPAL ultra-purified alginate (also called low-endotoxin high-purity alginate).
- FIG. 8 A diagram showing the results of comprehensive analysis of REC clones.
- FIG. 9 A diagram showing expression levels of various genes in healthy human intervertebral disc nucleus pulposus cells (NPC) and highly purified mesenchymal cells (REC).
- FIG. 10 A diagram showing the results of MRI of an intervertebral disc in a sheep model 4 weeks after transplantation.
- FIG. 11 A diagram showing histological examination results of an intervertebral disc in a sheep model 4 weeks after transplantation.
- FIG. 12 A diagram showing histological examination results of an intervertebral disc in a sheep model 4 weeks after transplantation.
- FIG. 13 Expression profiles of human NPCs and RECs after 7 days of 3D co-culture.
- A Schematic showing REC isolation. Flow cytometry profile of human bone marrow cells stained for CD271 (LNGFR) and CD90 (THY-1). Cells harvested from one well are seeded into 35 mm culture dishes and grown for up to 14 days to obtain a homogeneous cell population with high differentiation and proliferation potential.
- B and C Cell sorting data are used to distinguish between CFDA-SE labeled RECs and unlabeled NPCs.
- B P1 gate excludes dead cells and debris live cells.
- DK Gene expression levels in each cell were normalized to the expression level of the housekeeping gene GAPDH and plotted on a logarithmic scale (y-axis). Data obtained from four different human NPC lines were averaged.
- D HIF-1 ⁇ , (E) GLUT-1, (F) brachyury, (G) CDMP-1, (H) TGF- ⁇ , (I) IGF-1, (J) type II collagen,
- NPC nucleus pulposus cells REC: rapidly growing clones
- CFDA-SE 5,6-carboxyfluorescein diactatosuccinimidyl ester
- FSC-A forward scatter area
- FSC-W forward scatter width
- SSC-A side scattering area
- CFDA-SE-A CFDA-SE area
- FIG. 14 A diagram showing the elastic ratio of two types of gels.
- A Formation of disk-shaped UPAL and REC+UPAL gels after CaCl 2 -induced gelation (diameter, 4.5 mm; thickness, 2 mm).
- B and C Tension Compression Mechanical Test Apparatus. The sample was compressed at a constant speed of 0.5 mm/min.
- NP nucleus pulposus
- UPAL ultra-purified alginic acid (also called low-endotoxin high-purity alginic acid)
- IVD intervertebral disc [Fig. 16] MRI evaluation of treated IVD at 4 and 24 weeks post-implantation.
- A T2-weighted, mid-sagittal images of IVD at 4 and 24 weeks post-op in sheep. Images are representative of the results of 6 or 8 replicates.
- B Pfirrmann grade IVD modification.
- MRI index NP area x mean signal intensity values for degenerative changes in NPs. Values are expressed as a percentage of the value for the untreated control IVD.
- NP nucleus pulposus MRI: magnetic resonance imaging IVD: intervertebral disc ANOVA: analysis of variance SD: standard deviation .
- Scale bar A; 50 ⁇ m (2nd and 4th parts from top) or 1 mm (1st and 3rd parts from top) (B); 1 mm.
- C Histologic grade was determined via modified Boos classification. Data are expressed as mean ⁇ standard deviation. Significant differences were evaluated by one-way ANOVA with post hoc Tukey-Kramer test.
- IVD intervertebral disc H&E: hematoxylin & eosin SD: standard deviation AF: annulus fibrosus NP: nucleus pulposus
- FIG. 18 Diagram showing type II or type I collagen-positive cells in treated IVD 4 weeks and 24 weeks after implantation.
- C and D Percentage of type II or type I collagen positive cells to total cells in treated IVDs.
- FIG. 20 shows the measurement results of intervertebral disc height relative to the disc height of adjacent vertebrae using T2-weighted mid-sagittal images.
- BC and EF are the anterior and posterior disc heights, respectively, and AB and DE are the cephalad adjacent vertebral body heights.
- DHI values were calculated as the ratio of disc height (BC+EF) to vertebral body height (AB+DE), and relative DHI values were calculated as the ratio of treated IVD DHI to untreated control IVD DHI.
- DHI Intervertebral disc height index IVD: Intervertebral disc
- the present invention relates to a composition for intervertebral disc regeneration containing a monovalent metal salt of alginic acid and mesenchymal stem cells (for example, human bone marrow-derived high-purity mesenchymal stem cells).
- a composition for intervertebral disc regeneration containing a low endotoxin monovalent metal salt of alginic acid and mesenchymal stem cells (for example, human bone marrow-derived high-purity mesenchymal stem cells).
- composition of the present invention promotes regeneration of the nucleus pulposus of intervertebral discs through activation of nucleus pulposus cells by mesenchymal stem cells and/or differentiation of mesenchymal stem cells into nucleus pulposus cells.
- the compositions of the present invention are used to apply to the nucleus pulposus site of a subject and to harden the portion after application.
- a composition of the invention is applied to a nucleus pulposus site of a subject and used to contact at least a portion of the surface with a cross-linking agent.
- BMSCs bone marrow-derived mesenchymal stem cells
- gel significantly promoted the tissue repair effect compared to gel alone.
- co-culture of gel-embedded BMSCs and nucleus pulposus cells caused 1) mutual activation of both cells by growth factor production, 2) differentiation of BMSCs into nucleus pulposus cells, 3) from both cells clarified the improvement of the extracellular matrix production ability of
- the alginic acid contained in the composition of the present invention is a polysaccharide extracted from brown algae such as Ecklonia cava, Arame, and Kelp, and has the property of cross-linking and hardening when divalent metal ions such as calcium are added. By utilizing this property, contacting the affected area with metal ions makes it possible to harden the surface and fix it to the affected area.
- a composition containing low-endotoxin sodium alginate is injected in a sol state into the nucleus pulposus of an intervertebral disc, and the injection port is brought into contact with a cross-linking agent to partially harden the composition, thereby suppressing degeneration of the nucleus pulposus. , increased the ratio of type II collagen-positive cells, which are favorable for nucleus pulposus regeneration, and promoted regeneration of the intervertebral disc nucleus pulposus.
- the implanted mesenchymal stem cells remain in the affected area and exhibit long-term effects. That is, the implanted high-purity mesenchymal stem cells produce growth factors and extracellular matrix, and activate nucleus pulposus cells. The activated nucleus pulposus cells then produce growth factors and extracellular matrix. As a result, the implanted mesenchymal stem cells are differentiated into nucleus pulposus cells, and regeneration of the lesion is promoted through interaction between the nucleus pulposus cells and the mesenchymal stem cells.
- cells may be embedded in a biocompatible material such as hyaluronic acid.
- human bone marrow-derived high-purity mesenchymal stem cells which is one aspect thereof, are produced by a technique of directly separating from bone marrow using two types of antibodies and a cell sorter. It is an ultra-pure human mesenchymal stem cell. A homogeneous cell population can be obtained by separation using a cell sorter, so quality control is easy.
- the present invention also relates to a composition for intervertebral disc regeneration containing high-purity mesenchymal stem cells derived from human bone marrow.
- the compositions of the invention are applied to the intervertebral disc nucleus pulposus in an undifferentiated state and/or without treatment to induce differentiation.
- the composition of the present invention promotes regeneration of the nucleus pulposus of intervertebral discs through activation of nucleus pulposus cells by human bone marrow-derived high-purity mesenchymal stem cells and/or differentiation of mesenchymal stem cells into nucleus pulposus cells.
- compositions of the present invention can be applied to a nucleus pulposus site of a subject and used to contact at least a portion of the surface with a cross-linking agent.
- the compositions of the invention can be applied to a subject's nucleus pulposus site and used to harden the portion after application.
- An "intervertebral disc” is a columnar tissue between the vertebrae that connect the spine.
- An intervertebral disc is a disk-shaped avascular tissue, and has a structure in which an annulus fibrosus surrounds the nucleus pulposus at the center, and end plates are arranged above and below.
- the “nucleus pulposus” is a gel-like tissue present in the center of the intervertebral disc, and mainly contains nucleus pulposus cells, an extracellular matrix mainly composed of proteoglycan and type II collagen, and water. The nucleus pulposus is considered to have remarkably low self-repairing ability and regeneration ability.
- Nucleus pulposus replacement refers to degeneration, shrinkage, or degeneration or shrinkage of the nucleus pulposus that has been degenerated, reduced, or removed due to aging, trauma, infection, or surgical operations (e.g., intervertebral disc nucleus pulposus removal (excision)). It means to make up for the minute or the removed portion.
- the term "nucleus pulposus filling” is used in the same sense as “nucleus pulposus filling”
- the "nucleus pulposus filling composition" of the present invention is synonymous with “nucleus pulposus filling composition”. be.
- Nucleus pulposus site means a site where the nucleus pulposus exists, a site where the nucleus pulposus is degenerated or shrunk, or a nucleus pulposus defect formed by removing at least a part of the nucleus pulposus. , including the periphery of the site where the nucleus pulposus is present.
- a “subject” is a human or non-human organism, such as avian and non-human mammals (e.g., cows, monkeys, cats, mice, rats, guinea pigs, hamsters, pigs, dogs, rabbits, sheep, and horses). be.
- applying is meant filling the nucleus pulposus region of the intervertebral disc with the composition of the present invention in an amount sufficient to fill degeneration, reduction, removal, defect, etc. of the nucleus pulposus region.
- Embedding refers to suspending or mixing cells in a biocompatible material, preferably a solution of a monovalent metal salt of alginic acid. The phrase “partially cured” will be described later.
- Constant a monovalent metal salt of low endotoxin alginic acid means that the composition of the present invention contains a sufficient amount of monovalent metal salt of low endotoxin alginic acid to regenerate the nucleus pulposus at the nucleus pulposus site to which the composition is applied. means to contain "Having fluidity” is as described later.
- Intervertebral disc degeneration and/or intervertebral disc injury” and “treatment, prevention or suppression of recurrence” are as described below.
- Intervertebral disc pain means pain caused by an intervertebral disc.
- Low back pain is pain that occurs around intervertebral discs, and includes back pain and buttock pain. By “chronic back pain” is meant back pain lasting 12 weeks or more from the onset of back pain.
- the composition of the present invention may be provided in a solution state using a solvent, or may be provided in a dry state such as a freeze-dried product (especially a freeze-dried powder).
- a dry state the composition of the present invention is used in a fluid state such as a solution by using a solvent at the time of application.
- the solvent is not particularly limited as long as it is applicable to living organisms.
- a saline solution (PBS) and the like are included. Preferred are water for injection, distilled water, physiological saline, etc., which can be used for treatment of humans and animals.
- the "monovalent metal salt of alginic acid" used as a carrier is a monovalent metal ion such as Na + or K + and a hydrogen atom of the carboxylic acid at the 6-position of alginic acid. It is a water-soluble salt made by exchange. Specific examples of the monovalent metal salt of alginic acid include sodium alginate and potassium alginate, and sodium alginate, which is commercially available, is particularly preferred. A solution of a monovalent metal salt of alginic acid forms a gel when mixed with a crosslinker.
- Alginic acid used in the present invention is a biodegradable high-molecular-weight polysaccharide, and is a polymer in which two types of uronic acid, D-mannuronic acid (M) and L-guluronic acid (G), are linearly polymerized. is. More specifically, D-mannuronic acid homopolymer fraction (MM fraction), L-guluronic acid homopolymer fraction (GG fraction), and D-mannuronic acid and L-guluronic acid are randomly arranged. The fraction (MG fraction) is an optionally conjugated block copolymer.
- composition ratio (M/G ratio) of D-mannuronic acid and L-guluronic acid in alginic acid varies depending on the type of organisms from which it is derived, such as seaweed, and is also affected by the habitat and season of the organisms. It ranges widely from high G type with M/G ratio of about 0.4 to high M type with M/G ratio of about 5.
- the monovalent metal salt of alginic acid is a high-molecular polysaccharide, and it is difficult to determine its molecular weight accurately.
- the weight average molecular weight is generally in the range of 10,000 to 10,000,000, preferably 20,000 to 8,000,000, more preferably 50,000 to 5,000,000.
- a numerical range indicated using " ⁇ " indicates a range including the numerical values before and after " ⁇ " as the minimum and maximum values, respectively.
- the value may differ depending on the measurement method.
- the weight average molecular weight measured by gel permeation chromatography (GPC) or gel filtration chromatography (also collectively referred to as size exclusion chromatography) is preferably It is 100,000 or more, more preferably 500,000 or more, and preferably 5,000,000 or less, more preferably 3,000,000 or less.
- the preferred range is 100,000 to 5,000,000, more preferably 500,000 to 3,500,000.
- the absolute weight average molecular weight can be measured by a GPC-MALS method that combines gel permeation chromatography (GPC) and multi-angle light scattering (MALS).
- the weight average molecular weight (absolute molecular weight) measured by the GPC-MALS method is preferably 10,000 or more, more preferably 80,000 or more, and still more preferably 90,000 or more, according to the effects shown in the examples of the present invention. It is preferably 1,000,000 or less, more preferably 800,000 or less, still more preferably 700,000 or less, and particularly preferably 500,000 or less.
- the preferred range is 10,000 to 1,000,000, more preferably 80,000 to 800,000, even more preferably 90,000 to 700,000, and particularly preferably 90,000 to 500,000.
- a measurement error of 10 to 20% or more can occur.
- the value may fluctuate in the range of 320,000 to 480,000 for 400,000, 400,000 to 600,000 for 500,000, and 800,000 to 1,200,000 for 1 million.
- the molecular weight of the monovalent metal salt of alginic acid can be measured according to a conventional method. Typical conditions for using gel permeation chromatography for molecular weight determination are as described in the Examples herein.
- Typical conditions for using gel permeation chromatography for molecular weight determination are as described in the Examples herein.
- For the column for example, GMPW-XL ⁇ 2+G2500PW-XL (7.8 mm ID ⁇ 300 mm) can be used, and for the eluent, for example, a 200 mM sodium nitrate aqueous solution can be used, and pullulan is used as a molecular weight standard. can be used.
- GPC-MALS for molecular weight measurement
- detectors for example, RI detectors and light scattering detectors (MALS) can be used.
- the monovalent metal salt of alginic acid has a large molecular weight and high viscosity when it is first extracted from brown algae, but the molecular weight becomes smaller and the viscosity becomes lower during the process of drying and refining with heat.
- Monovalent metal salts of alginic acid with different molecular weights can be produced by controlling conditions such as temperature in the production process, selecting brown algae as a raw material, and molecular weight fractionation in the production process. Furthermore, it is possible to obtain a monovalent metal salt of alginic acid having a desired molecular weight by mixing with another lot of monovalent metal salt of alginic acid having a different molecular weight or viscosity.
- the monovalent metal salt of alginic acid used in the present invention is preferably prepared by dissolving the monovalent metal salt of alginic acid in MilliQ water to form a solution having a concentration of 1 w/w%, and using a cone-plate type viscometer to obtain a solution at 20°C. It is preferable that the apparent viscosity is 40 mPa ⁇ s to 800 mPa ⁇ s, more preferably 50 mPa ⁇ s to 600 mPa ⁇ s when the viscosity is measured under the conditions. It is desirable that the conditions for measuring the apparent viscosity conform to the conditions described later. In this specification, "apparent viscosity" may be simply referred to as "viscosity".
- the alginic acid used in the present invention may be naturally derived or synthetic, but is preferably naturally derived.
- naturally occurring alginic acid include those extracted from brown algae.
- Alginic acid-containing brown algae grow abundantly in coastal areas around the world, but the seaweeds that can actually be used as alginate raw materials are limited. A typical example is the Durvillea.
- brown algae that are raw materials for alginic acid include, for example, the genus Lessonia, the genus Macrocystis, the genus Laminaria (the genus Laminaria), the genus Ascophyllum, the genus Durvillea, the genus Arame ( Eisenia) genus, Ecklonia genus, and the like.
- biocompatible materials may be used as carriers for embedding cells.
- hyaluronic acid HA
- chondroitin sulfate dermatan sulfate, keratin sulfate, heparin, heparan sulfate, galactosaminoglycuronglycan sulfate (GGGS), including pharmaceutically acceptable salts thereof (physiological salts)
- GAGs glycosaminoglycans
- mucopolysaccharides such as, may be used as carriers for embedding the cells.
- the carrier for embedding cells is not particularly limited, but may be cellulose, cellulose derivatives, agarose, chitin, chitosan, starch, polysaccharides such as pectin, gelatin, collagen, polypeptides, or the like. It is selected from proteins, amino acid derivatives, copolymers thereof, and derivatives thereof, and one or more of them may be used.
- hydrogels such as collagen or gelatin-like compositions may be used as carriers.
- the monovalent metal salt of alginic acid used in the present invention is not particularly limited, and is, for example, a low endotoxin monovalent metal salt of alginic acid.
- the carrier for embedding the cells is preferably low endotoxin. That is, the monovalent metal salt of alginic acid used in the present invention is preferably a monovalent metal salt of alginic acid with low endotoxin.
- a low endotoxin level means that the endotoxin level is so low that it does not substantially cause inflammation or fever. More preferably, it is a monovalent metal salt of alginic acid treated with low endotoxin.
- Low endotoxin treatment can be performed by a known method or a method based thereon.
- the method of Suga et al. for purifying sodium hyaluronate see, for example, JP-A-9-324001
- the method by Yoshida et al. for purifying ⁇ 1,3-glucan see, for example, JP-A-8-269102 etc.
- the method of William et al. for purifying biopolymer salts such as alginate and gellan gum see, for example, JP-A-2002-530440
- the method for purifying polysaccharides by James et al. 93/13136 see, e.g., U.S.
- the low endotoxin treatment of the present invention is not limited to them, but washing, filtration with a filter (endotoxin removal filter, charged filter, etc.), ultrafiltration, column (endotoxin adsorption affinity column, gel filtration column, ion exchange resin column, etc.) ), adsorption to hydrophobic substances, resins or activated carbon, organic solvent treatment (extraction with organic solvent, precipitation / sedimentation by addition of organic solvent, etc.), surfactant treatment (for example, JP 2005-036036 See publications, etc.) or a combination thereof.
- a known method such as centrifugation may be appropriately combined with these treatment steps. It is desirable to appropriately select it according to the type of alginic acid.
- the endotoxin level can be confirmed by a known method, and can be measured, for example, by a method using Limulus reagent (LAL), a method using Entospecie (registered trademark) ES-24S set (Seikagaku Corporation), or the like. .
- LAL Limulus reagent
- Entospecie registered trademark
- ES-24S set Seikagaku Corporation
- the method for treating the endotoxin of the monovalent metal salt of alginic acid contained in the composition of the present invention is not particularly limited. is preferably 500 endotoxin units (EU)/g or less, more preferably 100 EU/g or less, particularly preferably 50 EU/g or less, particularly preferably 30 EU/g or less.
- Low endotoxin-treated sodium alginate is commercially available, for example, Sea Matrix (registered trademark) (Mochida Pharmaceutical Co., Ltd.), PRONOVA TM UP LVG (FMC BioPolymer), and the like.
- Such endotoxin-reduced sodium alginate is also referred to herein as "pure sodium alginate" (UPAL).
- compositions of the present invention may be prepared using solutions of monovalent metal salts of alginic acid.
- a solution of a monovalent metal salt of alginic acid can be prepared by a known method or a method analogous thereto. That is, the monovalent metal salt of alginic acid used in the present invention can be produced by a known method such as an acid method or a calcium method using the aforementioned brown algae.
- alginic acid can be obtained by extracting these brown algae with an alkaline aqueous solution such as an aqueous sodium carbonate solution, and then adding an acid (eg, hydrochloric acid, sulfuric acid, etc.).
- a salt of alginic acid can be obtained by ion exchange. Low endotoxin treatment is performed as previously described.
- the solvent for the monovalent metal salt of alginic acid is not particularly limited as long as it is a solvent that is applicable to living organisms. (PBS) and the like. They are preferably sterilized and preferably treated with low endotoxin. For example, Milli-Q water can be used after being filter sterilized.
- composition of the present invention when the composition of the present invention is provided in a dry state such as a lyophilized product, it can be prepared into a fluid solution using the above solvents. Moreover, it is desirable that all the operations for obtaining the composition of the present invention be performed in an environment with low endotoxin levels and low bacterial levels. For example, the operation is preferably performed on a clean bench using sterilized instruments, and the instruments used may be treated with a commercially available endotoxin remover.
- compositions of some embodiments of the invention are in the form of flowable liquids, ie, solutions.
- the compositions of the present invention are flowable when applied to the nucleus pulposus site.
- the composition of the present invention preferably has a fluidity that permits injection with a 21G needle after allowing the composition to stand at 20° C. for 1 hour.
- the apparent viscosity of the composition of the present invention in this aspect is not particularly limited as long as the effects of the present invention can be obtained.
- It is preferably 10 mPa ⁇ s or more, more preferably 100 mPa ⁇ s or more, still more preferably 200 mPa ⁇ s or more, and particularly preferably 500 mPa ⁇ s or more. If the apparent viscosity is too high, the handleability may deteriorate. When the apparent viscosity is 20,000 mPa ⁇ s or less, application with a syringe or the like can be performed more easily. However, even if the apparent viscosity is 20000 mPa ⁇ s or more, it can be applied by using a pressurized or electric filling device or other means.
- composition of the present invention is 10 mPa ⁇ s to 50000 mPa ⁇ s, more preferably 100 mPa ⁇ s to 30000 mPa ⁇ s, still more preferably 200 mPa ⁇ s to 20000 mPa ⁇ s, and even more preferably 500 mPa ⁇ s to 20000 mPa ⁇ s. ⁇ s, particularly preferably 700 mPa ⁇ s to 20000 mPa ⁇ s. In another preferred embodiment, it may be 500 mPa ⁇ s to 10000 mPa ⁇ s, or 2000 mPa ⁇ s to 10000 mPa ⁇ s.
- Compositions of some embodiments of the present invention are of a viscosity such that they can be applied to a subject, such as with a syringe.
- the apparent viscosity of a composition containing a monovalent metal salt of alginic acid, such as an aqueous solution of alginic acid can be measured according to a conventional method.
- a coaxial double cylindrical rotational viscometer, a single cylindrical rotational viscometer (Brookfield viscometer), a cone-plate rotational viscometer (cone plate viscometer), etc. can be measured by Preferably, it is desirable to follow the viscosity measurement method of the Japanese Pharmacopoeia (16th edition). In the present invention, it is desirable to measure the viscosity at 20°C.
- the apparent viscosity of the composition is the apparent viscosity of the composition without cells or the like in order to accurately measure the viscosity. is preferred.
- the apparent viscosity of the composition containing the monovalent metal salt of alginic acid is particularly preferably measured using a cone-plate viscometer.
- a cone-plate viscometer For example, it is desirable to measure under the following measurement conditions.
- a sample solution is prepared using MilliQ water.
- the measurement temperature shall be 20°C.
- the number of rotations of the cone-plate viscometer is set to 1 rpm when measuring a 1% solution of monovalent metal alginate and 0.5 rpm when measuring a 2% solution of alginic acid monovalent metal salt, and the determination is made using this as a guideline.
- the reading time is measured for 2 minutes in the case of measurement of a 1% solution of monovalent metal salt of alginic acid, and the average value from 1 minute to 2 minutes is taken as the reading time. In the case of 2% solution measurement, measure for 2.5 minutes and take the average value from 0.5 minutes to 2.5 minutes. The test value is the average of three measurements.
- the apparent viscosity of the composition of the present invention can be adjusted, for example, by controlling the concentration, molecular weight, M/G ratio, etc. of the monovalent metal salt of alginic acid.
- the apparent viscosity of a solution of a monovalent metal salt of alginic acid is high when the concentration of the monovalent metal alginate in the solution is high, and is low when the concentration is low. Also, when the molecular weight of the monovalent metal salt of alginic acid is large, the viscosity is high, and when the molecular weight is small, the viscosity is low.
- alginic acid having a preferred M/G ratio can be appropriately selected according to, for example, the viscosity of the solution.
- the M/G ratio of alginic acid used in the present invention is about 0.1-5.0, preferably about 0.1-4.0, more preferably about 0.2-3.5.
- the type of brown alga used as a raw material affects the viscosity of the solution of monovalent metal salt of alginic acid.
- the alginic acid used in the present invention is preferably derived from brown algae of the genus Lessonia, macrocystis, laminaria, genus Ascophyllum, and genus Durvillea, more preferably brown algae of the genus Lessonia, and particularly preferably from the genus Lessonia. It is from Lessonia nigrescens.
- the mesenchymal stem cells used in the present invention are somatic stem cells derived from mesodermal tissue (mesenchyme), and are expected to be applied to regenerative medicine such as reconstruction of bones, blood vessels, and myocardium. It is Mesenchymal stem cells can be obtained from various tissues such as bone marrow, adipose tissue, placental tissue, dental pulp or umbilical cord tissue. The purification process is, for example, as follows.
- a floating adipocyte population is separated by centrifugation from the mixed population of cell types obtained, and when left to stand in contact with the ceiling surface of a culture vessel filled with culture medium, it settles on the lower floor surface and proliferates. Fibroblast-like cells are grown by subculture.
- iPS cell-derived mesenchymal stem cells and commercially available mesenchymal stem cells can also be used.
- mesenchymal stem cells are preferably applied to the nucleus pulposus in an undifferentiated state and/or without treatment for inducing differentiation.
- undifferentiated state refers to the maintenance of a state in which stem cells with differentiation potential have not differentiated.
- without differentiation-inducing treatment means that, for example, stem cells with differentiation potential are not treated to differentiate into specific cells using a differentiation-inducing medium.
- This REC is a cell that can reach confluence in two weeks when cells are seeded one by one in a 96-well plate and cultured. All of the differentiation ability and migration ability have 1000-fold or more ability. In particular, since it retains migration ability, it can be administered intravenously, and application to serious systemic diseases such as osteogenesis imperfecta and achondroplasia can be expected. In the present invention, among the above REC clones, cell clones with little variation in differentiation and proliferation ability can be used.
- the cell population containing the cell clones of the present invention is a population of mesenchymal stem cell clones that are co-positive for LNGFR (CD271) and Thy-1 (CD90) and proliferate rapidly, and the following (a) and ( At least one characteristic of b) is satisfied.
- the coefficient of variation of forward scattered light in flow cytometry is 40% or less.
- the average cell size is 20 ⁇ m or less;
- LNGFR (CD271)-positive (CD271+) or CD271 and CD90 co-positive (CD271+CD90+) cell fractions were selected to extract highly mesenchymal stem cells.
- LNGFR CD271-positive (CD271+) or CD271 and CD90 co-positive (CD271+CD90+) cell fractions were selected to extract highly mesenchymal stem cells.
- CD45-CD235a- cells that are co-negative for CD45 and CD235a (CD45-CD235a-) are selected in order to select non-hematopoietic cells.
- a process may be added.
- a cell population containing mesenchymal stem cells can be prepared by flow cytometry or affinity chromatography.
- Materials for obtaining this cell population are not particularly limited, but examples thereof include bone marrow, adipose tissue, umbilical cord blood, peripheral blood (including peripheral blood after administration of G-CSF), and the like.
- the bone marrow the bone marrow of the spine, sternum, ilium, or the like may be used.
- Cells can also include ES cells and iPS cells.
- the material is a cell mass involving mesenchymal stem cells
- physical treatment such as pipetting, or enzymatic treatment with trypsin, collagenase, etc.
- trypsin a cell mass involving mesenchymal stem cells
- red blood cells a cell mass involving mesenchymal stem cells
- CD271+ cells or CD271+CD90+ cells are sorted.
- Methods for selecting CD271+ cells or CD271+CD90+ cells include, for example, methods using antibodies.
- the antibody is an anti-CD271 antibody and/or an anti-CD90 antibody capable of sorting CD271+ cells or CD271+CD90+ cells.
- viable cells can be isolated by using anti-CD271 antibodies labeled with different fluorescent dyes such as FITC, PE, APC, or anti-CD271 antibodies and anti-CD90 antibodies in appropriate combinations. It becomes possible to sort by time.
- CD271 + CD90 + cells can be sorted by a method using magnetic beads or a method using affinity chromatography. Before using these methods, dead cells may be removed by reacting a cell population with a fluorescent dye (e.g., PI) that stains dead cells and removing fluorescently-stained cells. good.
- a fluorescent dye e.g., PI
- Mononuclear cells are prepared from human bone marrow or adipose/placental chorion, and the bone marrow mononuclear cells are stained with anti-LNGFR alone or anti-LNGFR and anti-Thy1.
- LNGFR-positive cells or LNGFR-positive and Thy1-positive cells are clonally sorted in a 96-well culture plate. That is, one cell is seeded per well. Two weeks after the single cell culture, the culture plate is photographed under a microscope, confluent or semi-confluent wells are selected, and the cells contained in the wells are designated as REC.
- rapid growth and “high-speed growth” refer to two weeks after the start of culture or before the culture plate becomes confluent when cells are seeded one by one in each well of a 96-well culture plate and cultured. Or it means having a growth rate (doubling time (doubling time) of 26 ⁇ 1 hour) to the extent of becoming semi-confluent.
- Confluent is a state in which 90% or more of the culture vessel surface (culture surface) is covered with cultured cells.
- semi-confluent refers to a state in which 70 to 90% of the culture vessel surface (culture surface) is covered with cultured cells.
- the size and type of culture device to be used can be appropriately changed according to the growth rate of cells.
- Moderately/Slowly Expanding Cells ie cells that have not become semi-confluent or confluent after 2 weeks of single-cell culture, are discarded.
- REC collected from each well selected as REC is transferred to each well into a culture flask and cultured until confluent (expansion culture). The expanded cells are then harvested separately. REC derived from one well is taken as one lot.
- the RECs used in the present invention were obtained by clonal sorting in which one cell was seeded per well, so the genetic traits of the proliferated cells were all the same. Therefore, in the present invention, the cell population as a whole may be referred to as a "clone", and individual cells constituting the cell population may be referred to as a "clone”.
- RECs to be used for selection can be evaluated in advance using REC markers (anti-Ror2).
- REC markers anti-Ror2
- adherent and proliferated cells are collected from all lots, and a portion (approximately 1 to 3 ⁇ 10 5 cells) of each lot is selected and single-stained with a monoclonal antibody against Ror2.
- a technique for single staining with a monoclonal antibody to anti-Ror2 is known (WO2016/17795). Briefly, flow cytometry analysis using REC markers determines the percentage of REC marker positive cells in recovered cells. The ratio may be obtained by quantifying Ror2 mRNA expression using quantitative PCR, or by manually determining the same ratio using a microscope. A lot (cell population) with a certain value (for example, 65%) or more of the above-mentioned positive ratio can be accepted and used for the later-described selection.
- REC clones of individual lots are examined for cell proliferation ability, adipogenic ability, REC-specific marker expression level, and cell size uniformity, and their correlations are analyzed. By doing so, it became possible to select RECs with high purity and high cell performance.
- the coefficient of variation (CV value) of forward scattered light and the average cell size are used as indicators for sorting.
- Forward scatter is light that scatters at a small forward angle with respect to the axis of the laser beam.
- Forward scattered light consists of scattered light, diffracted light, and refracted light of laser light generated on the cell surface, and provides information about the size of the sample.
- Coefficient of Variation is the value obtained by dividing the standard deviation by the average value. It is a numerical value used to relatively evaluate the variation of data with different units and the relationship between the data and the variation with respect to the average value. is.
- CV Coefficient of Variation
- those with a CV value of 40% or less, preferably 35% or less are selected.
- a cell population with a CV value of 40% or less, more preferably a cell population with a CV value of 35% or less is a cell population composed of cells of uniform size.
- the CV value is 30% or less, 25% or less, or 20% or less.
- the average size of cells in the cell population sorted by the present invention is 20 ⁇ m or less. The size is preferably 18 ⁇ m or less, and is in the range of 14 ⁇ m to 18 ⁇ m.
- the present invention also provides a method for evaluating the quality of a cell population of LNGFR-positive cells or fast-growing mesenchymal stem cell clones co-positive for LNGFR (CD271) and Thy-1 (CD90).
- a cell population that satisfies at least one of the following characteristics (a) and (b), preferably both characteristics, is determined to be of high quality.
- the coefficient of variation of forward scattered light in flow cytometry is 40% or less.
- the average cell size is 20 ⁇ m or less;
- the cell population evaluated and selected in this way is not limited in the number of cell clones that make up the population, and has about 0.8 ⁇ 10 7 to 1.2 ⁇ 10 7 cells in 1 ml of solution, for example. .
- a cell population that satisfies at least one of the following characteristics (a) and (b), preferably both characteristics, is determined to be of high quality.
- the coefficient of variation of forward scattered light in flow cytometry is 35% or less.
- the average cell size is 20 ⁇ m or less; The cell population evaluated and selected in this way is not limited in the number of cell clones that make up the population, and has about 0.8 ⁇ 10 7 to 1.2 ⁇ 10 7 cells in 1 ml of solution, for example. .
- composition of the present invention is characterized by containing, for example, a low endotoxin monovalent metal salt of alginic acid and the mesenchymal stem cells as active ingredients.
- a low endotoxin monovalent metal salt of alginic acid and the mesenchymal stem cells as active ingredients.
- the present inventors have found for the first time that when the composition of the present invention is applied to the nucleus pulposus of a living body, the monovalent metal salt of alginic acid itself exerts a regenerating or therapeutic effect on the nucleus pulposus tissue.
- Containing as an active ingredient means that when the low endotoxin monovalent metal salt of alginic acid is applied to the affected area, it is contained in an amount that can exhibit the regeneration or therapeutic effect of the nucleus pulposus tissue.
- the preferred monovalent metal salt concentration of alginic acid in the composition of the present invention is preferably 0.5 w/v% to 5 w/v%, more preferably 1 w/v% to 5 w/v%, and more preferably 1 w/v % to 3 w/v %, particularly preferably 1.5 w/v % to 2.5 w/v %.
- the monovalent metal salt concentration of alginic acid 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%. , more preferably 1 w/w% to 3 w/w%, and particularly preferably 1.5 w/w% to 2.5 w/w%.
- the endotoxin content of the composition is usually 500 EU/g or less, more preferably. is 300 EU/g or less, more preferably 150 EU/g or less, and particularly preferably 100 EU/g or less.
- the number of cells (cell concentration) contained in the composition of the present invention is, for example, 1 ⁇ 10 4 cells/ml or more, or 1 ⁇ 10 5 cells/ml or more, preferably 1 ⁇ 10 4 cells/ml to 1 cell/ml. ⁇ 10 7 cells/ml.
- compositions of the present invention can also contain factors that promote cell growth.
- factors that promote cell growth include BMP, FGF, VEGF, HGF, TGF- ⁇ , IGF-1, PDGF, CDMP (cartilage-derived-morphogenic protein), CSF, EPO, IL, PRP (Platelet Rich Plasma), SOX, IF, and the like.
- BMP BMP
- FGF vascular endothelial growth factor
- VEGF vascular endothelial growth factor
- HGF vascular endothelial growth factor
- TGF- ⁇ fibroblast growth factor
- IGF-1 interleukin-1
- PDGF interleukin-1
- CDMP cartilage-derived-morphogenic protein
- CSF CSF
- EPO EPO
- IL IL
- PRP Platinum Rich Plasma
- SOX IF
- the composition of the present invention can also contain a factor that suppresses cell death.
- Factors that cause cell death include, for example, caspase and TNF ⁇ , and factors that suppress these include antibodies, siRNA, and the like. These factors that suppress cell death may be produced by recombinant methods or purified from protein compositions.
- the compositions of some embodiments of the present invention do not contain factors that suppress cell death. Even without a factor that suppresses cell death, the regeneration of the nucleus pulposus is sufficiently good, and the safety is higher than in the case of actively suppressing cell death.
- the composition of the present invention does not contain a component that exerts a pharmacological action on the nucleus pulposus tissue of intervertebral discs, other than the monovalent metal salt of low endotoxin alginic acid. Even a composition containing only a monovalent metal salt of low endotoxin alginic acid as an active ingredient can exhibit sufficient nucleus pulposus regeneration or therapeutic effects.
- other pharmaceutically active ingredients and conventional stabilizing agents, emulsifying agents, tonicity adjusting agents, buffering agents, tonicity agents, preservatives, soothing agents, coloring agents are optionally added.
- the composition of the present invention can also contain components that are commonly used in pharmaceuticals, such as pharmaceutical agents.
- compositions of the invention are used to cure a portion after application to the nucleus pulposus site.
- Partially curing refers to contacting a portion of the flowable composition of the present invention with a cross-linking agent to gel and solidify a portion, but not all, of the composition in contact with the cross-linking agent.
- a portion of the composition of the present invention is cured by contacting at least a portion of the surface of the fluid composition of the present invention with a cross-linking agent.
- a cross-linking agent and curing means suitable for the carrier to be used can be selected.
- curing a portion after applying the composition to the nucleus pulposus site means using the same cross-linking agent usage method and usage ratio as filling the nucleus pulposus site, in In vitro, a test tube with a diameter of 6 mm is filled with 500 ⁇ L of low endotoxin sodium alginate and a cross-linking agent, and after standing for 1 hour, at least 50% of the volume of the composition in the test tube is not gelled, and the non-gelled portion is may be indicated by at least 50% of the volume of the composition in the test tube being able to be aspirated with a syringe fitted with a 21G needle.
- composition exhibits such properties even after being filled into the nucleus pulposus region, it is thought that the composition will not deviate even when compressive force is applied from the craniocaudal side of the intervertebral disc after filling.
- At least a portion of the surface of the composition is, for example, the opening in the surface of the intervertebral disc leading to the nucleus pulposus, preferably the opening in the surface of the intervertebral disc used to apply the composition to the nucleus pulposus site, i.e. Composition fill port.
- the opening for filling the composition on the surface of the intervertebral disc is, for example, an opening used for filling the composition on the surface of the intervertebral disc with a needle of a syringe or a scalpel, or an opening on the surface of the intervertebral disc made with a scalpel or the like when removing an intervertebral disc. It is preferably an opening.
- the intervertebral disc in this aspect is preferably the annulus fibrosus.
- the composition of the present invention preferably does not contain an amount of cross-linking agent that causes the composition to harden prior to application to the nucleus pulposus site of the subject.
- the compositions of the present invention may contain an amount of cross-linking agent that does not cure the composition after a period of time.
- the fixed time here is not particularly limited, but is preferably about 30 minutes to 12 hours.
- Does not contain an amount of a cross-linking agent that hardens the composition may be indicated, for example, by allowing the composition to stand at 20° C. for 1 hour and then injecting it with a syringe fitted with a 21 G injection needle.
- the compositions of some embodiments of the invention do not contain a cross-linking agent.
- the cross-linking agent is not particularly limited as long as it can fix the surface by cross-linking a solution of a monovalent metal salt of alginic acid.
- Examples of cross-linking agents include divalent or higher metal ion compounds such as Ca 2+ , Mg 2+ , Ba 2+ and Sr 2+ , and cross-linking reagents having 2 to 4 amino groups in the molecule. More specifically, CaCl 2 , MgCl 2 , CaSO 4 , BaCl 2 and the like, which are divalent or higher metal ion compounds, are used as cross-linking reagents having 2 to 4 amino groups in the molecule on the nitrogen atom.
- a diaminoalkane which may have a lysyl group (-COCH( NH2 )-( CH2 ) 4 - NH2 ), i.e. a diaminoalkane and its amino group is substituted with a lysyl group to form a lysylamino group.
- Specific examples include diaminoethane, diaminopropane, N-(lysyl)-diaminoethane and the like.
- CaCl2 solution is particularly preferred . is preferable.
- the timing of contacting the surface of the composition of the present invention with the cross-linking agent is preferably after applying the composition of the present invention to the nucleus pulposus site.
- the method for contacting a part of the composition of the present invention with a cross-linking agent is not particularly limited, but for example, a syringe, an injector (spray), or the like may be used to contact a divalent or higher metal ion. and a method of applying a solution of the above to the surface of the composition.
- the cross-linking agent may be slowly applied to the filling opening of the composition formed in the intervertebral disc for several seconds to ten-odd seconds. After that, if necessary, a treatment for removing the cross-linking agent remaining in the vicinity of the filling port may be added. Removal of the cross-linking agent may be, for example, washing the application site with saline or the like.
- the amount of the cross-linking agent used is desirably adjusted in consideration of the application amount of the composition of the present invention, the size of the filling opening of the composition on the surface of the intervertebral disc, the size of the application site of the nucleus pulposus of the intervertebral disc, and the like.
- the amount of the cross-linking agent used should be adjusted so as not to be excessive.
- the amount of the divalent or higher metal ion to be used is not particularly limited as long as it is an amount capable of solidifying the surface of the composition containing the monovalent metal salt of alginic acid.
- the amount of CaCl 2 solution used is preferably about 0.3 ml to 5.0 ml when the diameter of the filling opening on the surface of the intervertebral disc is about 1 mm. , more preferably about 0.5 ml to 3.0 ml.
- the amount of 100 mM CaCl2 solution used is preferably about 0.3 ml to 10 ml, and more. Preferably, it is about 0.5 ml to 6.0 ml. It can be increased or decreased as appropriate while observing the state of the composition of the present invention at the application site.
- the concentration of calcium is preferably 25 mM to 200 mM, more preferably 50 mM to 150 mM. desirable.
- the cross-linking agent is added to the composition and allowed to stand for a certain period of time, it is desirable to remove the cross-linking agent remaining at the added site by washing or the like.
- alginate beads are produced by dropping a sodium alginate solution into a CaCl 2 solution and causing gelation.
- alginate beads need to be applied by being pressed against the application site, and it is necessary to prepare beads that match the size of the application site, which is technically difficult to use in actual clinical practice.
- the composition of the present invention is in the form of a solution, it can be easily applied to any shape of the application site, and the entire application site can be covered with the composition. Good adhesion to tissue.
- the portion of the composition of the present invention that contacts the surrounding tissue can be kept at a low calcium concentration and is less susceptible to calcium cytotoxicity. Since the portion of the composition of the present invention that contacts the surrounding tissue is less affected by the cross-linking agent, the composition of the present invention can easily contact cells and tissues at the application site. Preferably, the composition of the present invention fuses with the tissue of the body to such an extent that it becomes indistinguishable at the site of application within about 4 weeks after being applied to the nucleus pulposus site, and has high biocompatibility.
- composition of the present invention When the composition of the present invention is partially gelled by a cross-linking agent when the composition of the present invention is applied to the nucleus pulposus site, the composition of the present invention partially hardens in the affected area and is localized in close contact with the surrounding tissue. Leakage from the nuclear site can be prevented. In addition, since the composition of the present invention adheres to the surrounding tissue, the nucleus pulposus regeneration effect of the composition of the present invention is exhibited more strongly.
- the composition in solution form of the present invention has a low risk of such complications and a low risk of developing complications.
- the cell-embedding carrier may be used without hardening. It can also be applied without using a cross-linking agent, depending on the clinical symptoms and the size and shape of the injured area.
- compositions of the present invention may be used in human or non-human organisms such as birds and non-human mammals (e.g. cows, monkeys, cats, mice, rats, guinea pigs, hamsters, pigs, dogs). , rabbits, sheep, and horses) to the nucleus pulposus site of the intervertebral disc to promote regeneration of the nucleus pulposus.
- non-human mammals e.g. cows, monkeys, cats, mice, rats, guinea pigs, hamsters, pigs, dogs.
- rabbits, sheep, and horses to the nucleus pulposus site of the intervertebral disc to promote regeneration of the nucleus pulposus.
- the form of the composition of the present invention is preferably fluid liquid, ie, solution.
- “having fluidity” means having the property of changing its form into an amorphous form, and does not need to have the property of always flowing like a solution.
- the composition should be fluid such that it can be enclosed in a syringe or the like and injected into the nucleus pulposus region of the intervertebral disc.
- the composition has fluidity such that it can be injected into the nucleus pulposus region of the intervertebral disc with a syringe fitted with a 14G to 26G injection needle after the composition has been allowed to stand at 20°C for 1 hour. and, more preferably, can be injected with a 21G needle.
- the composition of the present invention is provided in a dry state such as a lyophilized product, it can be made into a fluid composition as described above by using a solvent or the like at the time of application.
- the composition of the present invention which is in the form of a solution, can be easily applied to the nucleus pulposus region of the intervertebral disc using a syringe, a gel pipette, a dedicated injector, a dedicated injector, a filling instrument, or the like.
- a syringe a gel pipette, a dedicated injector, a dedicated injector, a filling instrument, or the like.
- a pressurized or electric syringe may be used.
- a spatula, stick, or the like may be used to apply to the defect in the nucleus pulposus region.
- a needle of, for example, 14G to 27G or 14G to 26G.
- the method of applying the composition of the present invention to the nucleus pulposus site is not particularly limited. , filling instruments and the like can be used to apply the compositions of the present invention to the nucleus pulposus site.
- the composition of the present invention may be applied by inserting a needle of a filling device or the like from the surface of the annulus fibrosus toward the nucleus pulposus site.
- the composition of the present invention is in the form of a solution, it can be adapted to any shape of the nucleus pulposus site, such as a reduced nucleus pulposus, a cavity or defect in the nucleus pulposus site, and a reduced nucleus pulposus, cavity or defect. It is also possible to fill the entire part.
- a reduction in the nucleus pulposus, a cavity or defect at the nucleus pulposus site may result from degeneration or injury of the intervertebral disc, or may result from surgical removal or aspiration of at least a portion of the nucleus pulposus. It can be anything.
- the composition of the present invention is applied to a nucleus pulposus defect formed by removing at least part of the nucleus pulposus.
- the removal of at least part of the nucleus pulposus is not particularly limited, and may be, for example, an intervertebral disc nucleus pulposus that is performed under direct, percutaneous, microscopic, or endoscopic observation. Also, for example, an incision of 2 cm to 10 cm is made in the back, the muscle is separated from the posterior surface of the posterior element of the spine called the vertebral arch, the ligament between the vertebral arches is excised, the nerve and intervertebral disc herniation are confirmed, and the nerve is compressed. A method of extracting a hernia (the Love method) may be used. A method of reducing the volume of the nucleus pulposus by irradiating the nucleus pulposus with a laser may also be used.
- the composition After application of the composition of the present invention to the nucleus pulposus region, the composition can be partially cured with a cross-linking agent as described above.
- the amount of the composition of the present invention to be applied may be determined according to the volume of the application site of the nucleus pulposus to be applied, and is not particularly limited, but is, for example, 0.01 ml to 10 ml, more preferably 0.1 ml to 5 ml. and more preferably 0.2 ml to 3 ml.
- it is desirable to inject it so as to sufficiently fill the defect volume of the nucleus pulposus region.
- the number and frequency of application of the composition of the present invention can be increased or decreased according to symptoms and effects. For example, it may be applied only once, or may be applied continuously once a month to a year. Since alginic acid is a substance that does not originally exist in the body of animals, animals do not possess an enzyme that specifically decomposes alginic acid. Alginic acid is gradually decomposed in the animal body by normal hydrolysis, but the decomposition in the body is slower than polymers such as hyaluronic acid, and since there are no blood vessels in the nucleus pulposus, When filled, long-term effects can be expected.
- 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 site, the cells, growth factors, cell death inhibitors, and the cell death inhibitor described below may be added to the nucleus pulposus site. Other drugs may be used in combination.
- the composition of the present invention exerts an effect of suppressing degenerative changes in the entire intervertebral disc tissue and nucleus pulposus and promoting regeneration. Therefore, the composition of the present invention is preferably used as a composition for filling the nucleus pulposus of intervertebral discs.
- composition of the present invention is a composition for suppressing intervertebral disc degeneration, more preferably a composition for suppressing degeneration of the intervertebral disc nucleus pulposus.
- “Degeneration of the intervertebral disc or nucleus pulposus” refers to morphological changes caused by deterioration in the number of intervertebral disc cells, water content, extracellular matrix (type II collagen, aggrecan, etc.) due to aging, etc., resulting in decreased function. It is a condition that occurs, and if it progresses, the intervertebral disc will not be able to function as a shock absorber.
- “inhibition of degeneration” does not necessarily mean that degeneration is suppressed, as long as degenerative changes are suppressed as compared to untreated cases.
- composition of the present invention is a composition for nucleus pulposus regeneration.
- the purpose of nucleus pulposus regeneration is to prevent the accumulation of fibroblast-like cells and to regenerate the nucleus pulposus with a high ratio of nucleus pulposus cells. It is intended that The term "nucleus pulposus regeneration" also includes suppression of degeneration of the nucleus pulposus.
- One of the preferred aspects of the present invention is that the composition of the nucleus pulposus regenerated by applying the composition of the present invention is close to the composition of the natural, normal nucleus pulposus.
- composition of a preferred embodiment of the present invention is used for treatment, prevention or suppression of recurrence of intervertebral disc degeneration and/or intervertebral disc injury.
- treatment, prevention or recurrence suppression refers to treatment, prevention, recurrence suppression, reduction, suppression, improvement, elimination, reduction in onset rate, delay in onset time, suppression of progression, reduction in severity, recurrence rate It includes reduction, delay of recurrence time, alleviation of clinical symptoms, etc.
- treatment, prevention or suppression of recurrence includes alleviation of (chronic) pain.
- composition of the present invention is used to suppress (chronic) pain (particularly lumbago) associated with intervertebral disc degeneration and/or intervertebral disc injury. be done.
- Disc degeneration and/or disc injury e.g. disc herniation, disc disease, spinal degenerative spondylolisthesis, suppurative discitis, spondylolisthesis, spinal canal stenosis, lumbar spinal canal stenosis, intervertebral disc injury, lumbar spinal canal stenosis at least one condition or disease selected from the group consisting of intervertebral disc herniation (also referred to as mixed lumbar spinal stenosis) associated with spinal stenosis.
- Disc degeneration and/or disc injury may be associated with back pain.
- the composition of the present invention may be used without hardening the cell-embedding carrier according to the clinical symptoms and the size and shape of the injured area. Used to control associated pain, especially chronic low back pain.
- the present invention provides a method for treating, preventing, or suppressing recurrence of intervertebral disc degeneration and/or intervertebral disc injury using the composition of the present invention.
- the therapeutic method of the present invention is a method for treating, preventing, or suppressing recurrence of intervertebral disc degeneration and/or intervertebral disc damage, and comprises a fluid composition containing a low-endotoxin monovalent metal salt of alginic acid. to the nucleus pulposus region of the intervertebral disc of the subject in need of said treatment, prevention or inhibition of recurrence, and allowing a portion of said applied composition to harden.
- Treatment methods of the present invention may include removing at least a portion of the nucleus pulposus prior to applying the composition of the present invention to the nucleus pulposus site.
- the intervertebral disc degeneration and/or intervertebral disc injury is, for example, at least one selected from the group consisting of intervertebral disc herniation, intervertebral disc disease, degenerative spondylolisthesis, suppurative discitis, spondylolisthesis, spinal canal stenosis, and intervertebral disc injury. is a condition or disease of In the method of treatment of some aspects of the invention, said disc degeneration and/or disc injury is disc herniation, particularly lumbar disc herniation.
- the disc degeneration and/or disc injury is disc herniation associated with lumbar spinal stenosis (also referred to as mixed lumbar spinal stenosis).
- the disc degeneration and/or disc injury may be chronic low back pain.
- Disc degeneration and/or disc injury may be a combination of these conditions or diseases.
- one of several aspects of the present invention provides a method of suppressing degenerative changes in an intervertebral disc using the composition of the present invention.
- a method for regenerating the nucleus pulposus of an intervertebral disc using the composition of the present invention include applying a flowable composition containing mesenchymal stem cells and a monovalent metal salt of low endotoxin alginic acid to the nucleus pulposus region of an intervertebral disc in need of inhibition of intervertebral disc degeneration or regeneration of the nucleus pulposus. and curing a portion of the applied composition.
- the method may comprise removing at least a portion of the nucleus pulposus prior to applying the composition of the present invention to the nucleus pulposus site.
- composition of the present invention the specific application method to the nucleus pulposus region of the intervertebral disc, the method of curing the composition, the meaning of terms, etc. are as described above.
- the therapeutic method of the present invention may be performed by appropriately combining other intervertebral disc therapeutic methods and therapeutic agents.
- Antibiotics such as streptomycin, penicillin, tobramycin, amikacin, gentamicin, neomycin, and amphotericin B, aspirin, non-steroidal antipyretic agents may also be used prior to, concurrently with, or after application of the compositions of the present invention to the nucleus pulposus site.
- Concomitant drugs such as analgesics (NSAIDs), anti-inflammatory drugs such as acetaminophen, proteolytic enzymes, corticosteroids, and HMG-CoA reductase inhibitors such as simvastatin and lovastatin may be filled.
- NSAIDs analgesics
- anti-inflammatory drugs such as acetaminophen, proteolytic enzymes, corticosteroids, and HMG-CoA reductase inhibitors
- simvastatin and lovastatin may be filled.
- the present invention also relates to the use of monovalent metal salts of low endotoxin alginic acid to prepare the compositions of the invention.
- the use of the present invention is the use of a low endotoxin monovalent metal salt of alginic acid for producing a composition for treating, preventing or suppressing recurrence of intervertebral disc degeneration and/or intervertebral disc injury, wherein the composition is a subject It is applied to the nucleus pulposus site of the nucleus and is used to partially harden after application and has flowability when applied to the nucleus pulposus site.
- the present invention further provides a fluid composition containing mesenchymal stem cells and a monovalent metal salt of low endotoxin alginic acid to a subject in need of treatment, prevention or suppression of recurrence of intervertebral disc degeneration and/or intervertebral disc injury.
- the compositions of the invention can be evaluated using the inventors' newly established ovine severe disc degeneration model.
- the severe intervertebral disc degeneration model includes (a) removing an amount of nucleus pulposus tissue corresponding to 0.00004% to 0.00005% of the weight of the sheep from the intervertebral disc of the sheep in the first surgery to prepare a degenerated intervertebral disc; (b) Four weeks after the first surgery, the degenerated intervertebral disc prepared in (a) was further removed from the nucleus pulposus tissue in an amount corresponding to 0.00014% to 0.000175% of the body weight of the sheep, resulting in a severe intervertebral disc.
- a modified sheep model can be generated.
- a severe intervertebral disc degeneration sheep model can be prepared by further removing 70 mg of nucleus pulposus tissue from the degenerated intervertebral disc prepared in (a).
- the composition of the present invention can be evaluated by the following procedures (a) to (d).
- the vertebral body and intervertebral disc collected from the degeneration model are subjected to at least one evaluation method selected from the group consisting of MRI, tissue staining, and immunohistochemical staining (IHC) to regenerate the intervertebral disc. evaluate.
- the intervertebral disc degeneration model was prepared by partially removing the normal intervertebral disc, so there was a possibility that the intervertebral disc would heal spontaneously.
- this animal model in which an already degenerated disc is partially excised, allows a more accurate assessment of the effects of this example on human degenerated discs.
- BMSCs bone marrow-derived mesenchymal stem cells
- UPAL ultra-purified alginate
- NPC rabbit nucleus pulposus cells
- NP nucleus pulposus
- NP samples were obtained from four rabbits after euthanasia via intravenous pentobarbital overdose followed by lumbar IVD (L1/2 to L5/6; A total of 20 IVDs).
- NPCs were isolated from nucleus pulposus (NP) tissue and cultured as previously reported [2,5,7,8]. Specifically, under sterile conditions, gelatinous NP tissue was separated from the annulus fibrosus (AF) using micro forceps.
- Tissue specimens were prepared with 10% fetal bovine serum (Nichirei Bioscience, Tokyo, Japan), 1% penicillin/streptomycin, and 1.25 mg/ml fungizone (Life Technologies, Waltham, Mass., USA) in Dulbecco's modified Eagle medium (Sigma-Aldrich). , St. Louis, MO, USA). No exogenous growth factors were used. Samples were resuspended in medium (Wako Pure Chemical Industries, Osaka, Japan) supplemented with 0.25% collagenase and incubated for 4 hours at 37°C, 20% O2 and 5% CO2 in a shaking incubator to quench the enzyme. Isolated by digestion. Cells detached from NP tissue were grown in culture dishes and cultured in the above medium at 37°C with 20% O2 and 5% CO2 in a humidified atmosphere. Medium was changed twice a week and NPCs were used at passage 2.
- BMSCs Preparation of rabbit allogeneic BMSCs OriCell TM rabbit mesenchymal stem cells purchased from Cyagen (Santa Clara, Calif., USA; catalog number: RBXMX-01,001, lot number: 151114I31) were used as rabbit allogeneic BMSCs. These cells have been tested for characteristics, post-thaw viability, cell cycle, validation of undifferentiated state, and pluripotent differentiation potential along osteogenic, chondrogenic, and adipogenic lineages. BMSCs were cultured according to the manufacturer's instructions, medium was changed twice weekly, and BMSCs were used at passage 2.
- UPAL gel (Mochida Pharmaceutical Co. Ltd., Tokyo, Japan) was used as an alginate scaffold for 3D culture [2].
- the UPAL gel purification process was previously reported [2]. Specifically, alginate in seaweed was extracted by converting it to water-soluble sodium alginate by a clarification procedure [2]. This alginate solution was diluted with a large amount of water due to its high viscosity [2]. The extract was then filtered to separate the sodium alginate solution from the fibrous residue [2]. Acid was added to this solution to isolate high quality alginate [2].
- a 2% (w/v) UPAL solution dissolved in phosphate buffered saline (Wako Pure Chemical Industries) and 102 mM CaCl 2 was prepared for gelation.
- the manufacturer's BMSCs were fluorescently labeled according to the instructions of [9].
- Labeled BMSCs and unlabeled NPCs were then embedded in UPAL fluid at a ratio of 1:1 (1 ⁇ 10 6 cells/ml each) [1,10], resulting in a final cell concentration of 2 ⁇ 10 6 cells/ml. Concentrations were obtained [9,10].
- the UPAL /cell mixture was passed through a 22 gauge needle into 102 mM CaCl2 for gelation.
- RNA extraction and real-time quantitative reverse transcription polymerase chain reaction Collected NPCs and BMSCs were dissolved in 1 ml of TRIzol (TM) (Invitrogen, Carlsbad, Calif., USA), and total RNA was extracted from the samples using the RNeasy Mini kit (Qiagen, Valencia, Calif., USA).
- Real-time qRT-PCR analysis was performed using the TaqMan TM Gene Expression Assay and a custom TaqMan TM Gene Expression Assay (Table 1) (Applied Biosystems, Waltham, Mass., USA). Cycle threshold values (Ct) were obtained for each sample and the relative mRNA expression of each target gene relative to the Ct value of the housekeeping gene GAPDH was calculated using the 2 - ⁇ Ct method [1].
- BMSC Labeling and Embedding in UPAL Solution In the in vivo experiments, OriCell TM rabbit mesenchymal stem cells at passage 2, similar to the in vitro experiments, were used as transplanted cells. BMSCs were labeled with CFDA-SE before transplantation, embedded in 2% UPAL solution, and adjusted to a final cell concentration of 1 ⁇ 10 6 cells/ml [14].
- the IVD defect was filled with 20 ⁇ l of 2% UPAL solution using a microsyringe fitted with a 27-gauge needle (Hamilton Medical, Bonaduz, Switzerland), and in the BMSC+gel group, the IVD defect was filled with UPAL solution containing BMSCs. fulfilled.
- 27-gauge needles were used because 26-gauge needles have been shown to have no effect on cell viability or tissue degeneration [2,7,8,17].
- 1 mL of 102 mM CaCl 2 was sprinkled over the UPAL solution to induce gelation. After 5 minutes, the surgical wound was washed with saline and closed. A sham operation was performed in the puncture group.
- BMSC survival was confirmed based on CFDA-SE fluorescent labeling [18,19] at 4 and 12 weeks post-implantation.
- IVDs intact control group and BMSC+gel group
- DAPI 4′,6-diamidino-2-phenylindole
- MRI Analysis T2-weighted mid-sagittal images of the IVD were obtained using a 7.0-T MR scanner (Varian Unity Inova; Varian Medical Systems, Palo Alto, Calif., USA) at 4 and 12 weeks postoperatively. [2,8,16]. IVD degeneration was graded using the Pfirrmann classification (grade 5 classified as severe degeneration) [20]. Quantitative analysis was also performed using analysis software version 12.0 (AnalyzeDirect, Overland Park, KS, USA) to calculate the MRI index. The MRI index (the product of NP area and mean signal intensity) was applied to quantify degeneration of NPs, and quantitative data were expressed as a percentage of the MRI index obtained with untreated control IVDs (relative MRI index) [2, 8, 16].
- IHC Immunohistochemical staining was performed to detect type I and type II collagen at 4 and 12 weeks postoperative [8], and at 1, 7 and 28 postoperative days. HIF-1 ⁇ , GLUT-1 and Brachyury were detected.
- mouse monoclonal antibodies against type I collagen Sigma-Aldrich; C2456, RRID: AB_476836
- type II collagen Kyowa Pharma Chemical, Toyama, Japan; F-57
- Staining was developed with 3,3'-diaminobenzidine hydrochloride (Dako) and Mayer's hematoxylin (Merck, Darmstadt, Germany) as counterstain.
- DyLight 550-conjugated rabbit polyclonal antibody to HIF-1 ⁇ (Novus Biologicals, Centennial, CO, USA; NB100-479R, RRID: AB_1642267), PE-conjugated rabbit to GLUT-1.
- a polyclonal antibody (LS Bio, Seattle, WA, USA; LS-A109342-100) and an unconjugated rabbit polyclonal antibody against Brachyury (LS Bio; LS-C31179-100, RRID: AB_911118) were applied.
- an Alexa Fluor 594-conjugated goat anti-rabbit polyclonal antibody (Invitrogen; A32740) was used as a secondary antibody for Brachyury. Staining was developed with DAPI as a counterstain.
- Type I and II collagen, HIF-1 ⁇ , GLUT-1 and Brachyury positive cells were counted separately in 5 independent, randomly selected fields [2, 8].
- the field of view spans the width of the NP, including both deep and surface regions. Values are expressed as the percentage of positive cells to the total number of cells for all endpoints and to the number of CFDA-SE positive cells for NPC marker evaluation. All experiments were performed on 8 IVDs for type I and type II collagen assessment and 4 IVDs for NPC marker assessment from each treatment group at each time point.
- NPC and BMSC co-culture promotes differentiation of BMSC to NPC and production of growth factors and ECM.
- Embedded in UPAL gel for 3D culture We harvested both cell types in the co-culture group using a cell sorter. Phosphate-buffered saline/cell suspension analysis was performed using forward and side scatter. A P1 gate was drawn on the 2D dot plot (Fig. 1a) to exclude dead cells and debris. Unlabeled NPCs and CFDA-SE labeled BMSCs were sorted using different gates in fluorescence vs. side scatter dot plots (Fig. 1b).
- the P2 gate was set above unlabeled cells and the P3 gate above CFDA-SE labeled cells, with a gap between the two gates to avoid cross-contamination [1].
- HIF-1 ⁇ , GLUT-1 and Brachyury as NPC markers gene expression of HIF-1 ⁇ , GLUT-1 and Brachyury as NPC markers
- CDMP-1, TGF- ⁇ and IGF-1 extracellular Gene expression of type II collagen and aggrecan as matrix (ECM) was assessed using qRT-PCR.
- Brachyury gene expression showed no statistically significant difference among the three NPCs.
- Brachyury gene expression was observed only in BMSC co-cultures and not in either BMSC control or monocultures (Fig. 1e).
- BMSCs and UPAL gel promotes IVD regeneration after discectomy in degenerative IVDs
- Degenerative changes in treated IVDs were qualitatively analyzed by MRI and T2-weighted midsagittal images were captured (Fig. 3a) .
- the overall structure of the IVD Prior to histological degeneration classification, the overall structure of the IVD, including NP and AF, was evaluated to observe gross differences between groups. Histological evaluation of the IVD revealed that the intact control specimen showed a typical oval shape of the NP tissue without structural disruption of the internal AF (Figs. 4a and b). In the discectomy group, medial AF collapse and fibrotic changes in NP tissue were observed at both 4 and 12 weeks. However, the medial AF in the BMSC_+ gel group appeared to be well preserved with minimal fibrotic changes in the NP tissue at both 4 and 12 weeks, and the medial AF in the gel group also , appeared to be relatively well preserved (Figs. 4a and b).
- the ratio of three types of NPC marker-positive cells to the total number of cells was significantly higher on day 28 compared to days 1 and 7 (p ⁇ 0.0001, p ⁇ 0.0001, Student's t test). In contrast, approximately 20% of the cells were positive at all time points in the discectomy group (Fig. 6d-f). Similarly, the ratio of three types of NPC marker-positive cells to the number of CFDA-SE-positive cells (representing engrafted BMSCs) was significantly higher on day 28 compared to days 1 and 7 (p ⁇ 0 .0001, p ⁇ 0.0001, Student's t-test) (Fig. 6g-i).
- transplanted BMSCs were shown to be positive for HIF-1 ⁇ , GLUT-1 and MMP-2, indicating that BMSCs are cells that express some of the typical phenotypic characteristics of NPCs. [28].
- the gel and BMSC+gel groups significantly decreased collagen I production in NPs, but gel and BMSC+gel filling repaired AF defects due to puncture.
- IVD degeneration is characterized by degradation of the NP extracellular matrix [2], this example mainly focused on NP storage/regeneration.
- Implanted BMSCs can be localized within the cavity of the IVD via embedding in UPAL gel without leakage outside the intervertebral disc.
- Implanted BMSCs produce growth factors and ECM, leading to activation of pre-existing NPCs.
- Activated NPCs also increase production of growth factors and ECM.
- the implanted BMSCs differentiate into NPCs.
- BMSCs and pre-existing NPCs activate each other resulting in IVD regeneration (Fig. 7).
- BMSCs and NPCs were fused in this in vitro experiment, it would have been impossible to separate the cells, so the possibility that the cell binding action constitutes the main mechanism of action of IVD degeneration is unlikely. think low. In addition, no direct contact was observed between BMSCs and NPCs in vivo. The underlying mechanism of action may therefore be attributed to growth factors and/or some key humoral factors that regulate the interaction between BMSCs and NPCs.
- Injectable hydrogel combined with nucleus pulposus-derived mesenchymal stem cells for the treatment of degenerative interval disc in rats.
- Omlor GW Lorenz S, Nerlich AG, Guehring T, Richter W.; Disc cell therapy with bone-marrow-derived autologous mesenchymal stromal cells in a large porcine disc degeneration model. Eur Spine J 2018;27(10):2639-49.
- Risbud MV Albert TJ, Guttapalli A, et al. Differentiation of mesenchymal stem cells towards a nucleus pulposus-like phenotype in vitro: Implications for cell-based transplantation therapy.
- NPC nucleus pulposus cells
- REC mesenchymal stem cells
- PI-negative live cell populations were developed in FSC/SSC cytograms, gates (P1) were set on the main cell populations, and dust and noise were excluded from analysis targets.
- P1 The cell population in the P1 gate was developed with an FSC histogram, a marker (M1) was set, and the CV value was measured.
- adipogenesis-inducing medium a medium obtained by adding dexamethasone, indomethacin, and IBMX to the above culture medium was used.
- the average growth rate of clones with a CV value of 30% to 35% is 6.4, the average growth rate of clones with a CV value of 25% to 30% is 9.2, and the average growth rate of clones with a CV value of 25% or less is was 15.1.
- the average growth rate of clones with an average cell size of 18 ⁇ m to 20 ⁇ m is 7.0, the average growth rate of clones with an average cell size of 16 ⁇ m to 18 ⁇ m is 12.7, and the average growth rate of clones with an average cell size of 16 ⁇ m or less. The rate was 21.3.
- Intervertebral disc L1/L2 intervertebral disc between the first and second lumbar vertebrae
- Intervertebral disc L2/L3 intervertebral disc between the second and third lumbar vertebrae
- Intervertebral disc L3/L4 between the third and fourth lumbar vertebrae
- intervertebral discs L4/L5 intervertebral discs between the 4th and 5th lumbar vertebrae
- nucleus pulposus tissue was removed from each group's intervertebral disc under anesthesia to induce severe disc degeneration.
- an additional 70 mg of nucleus pulposus tissue was removed under anesthesia and suspended in UPAL at a final cell concentration of 1 ⁇ 10 6 cells/ml (100 ⁇ l) in the intervertebral disc space.
- REC REC prepared in Example 2, Section 1.2
- sheep were euthanized.
- the discs were analyzed using 3 Tesla magnetic resonance imaging (MRI) for quantitative assessment of degeneration of the treated discs, after which discs were evaluated histologically by H&E staining and safranin-0 staining, and Type II by immunohistochemical evaluation. Assessment of collagen expression was performed. All data are shown as mean ⁇ standard error, and differences between groups were tested by one-way analysis of variance (ANOVA) and Tukey-Kramer post hoc test.
- MRI 3 Tesla magnetic resonance imaging
- test design in this example was carried out with the following contents.
- NPC markers including HIF-1 ⁇ , GLUT-1, and brachyury
- growth factors including CDMP-1, TGF- ⁇ , and IGF-1
- ECM components type II (including collagen and aggrecan)
- IVDs were qualitatively analyzed using 3.0-T MRI (2,3,15,37,38). IVDs were then stained with H&E and safranin-0 for histological analysis, and levels of type II and type I collagen were assessed by IHC for analysis of ECM components. Finally, tumorigenesis analysis was performed using tissue specimens (2,3,15).
- BMSCs Preparation of Commercially Available Human BMSCs
- hMSC-BM PromoCell, Heidelberg, Germany; C-12974, lot number: 412Z022.4
- BMSCs were grown in complete culture medium: 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).
- FBS HyClone fetal bovine serum
- penicillin/streptomycin 1.25 mg/mL fungizone
- UPAL gel and 3D culture UPAL gel (Mochida Pharmaceutical Co. Ltd., Tokyo, Japan) was used as an alginate scaffold for 3D culture (2,3).
- RECs or commercially available BMSCs were mixed with UPAL solution at a final cell concentration of 1 ⁇ 10 6 cells/mL (2, 31, 49).
- the cell- UPAL mixed solution was pipetted into the 102 mM CaCl2 solution using a 22 gauge needle for gelling.
- the two types of gel obtained in the form of beads were cultured together with the medium in a humid environment (20% O 2 , 5% CO 2 , 37° C.) for 7 days. Medium was changed every 3 days. To harvest cells after 7 days of 3D culture, they were lysed using 55 mM sodium citrate and centrifuged (110 ⁇ g for 10 min at 4° C.) as previously reported (2, 3, 35). Cells were harvested from the gel beads.
- Td doubling time
- Flow cytometry analysis was performed using the CytoFLEX System (Beckman Coulter) to assess cell viability as well as homogeneity and positivity of each cell surface antigen.
- FlowJo software (Becton Dickinson, Franklin Lakes, NJ, USA) was used for data analysis.
- RECs and commercial human BMSCs were also used for 3D co-culture.
- the two types of cells were cultured according to the manufacturer's instructions in the same manner as the 2D culture described above. Medium was changed twice a week. Both cells from passage 2 (REC, total 8 passages; BMSC, total 4 passages) were used.
- 3D co-culture and mono-culture We prepared a 2% UPAL solution and used CaCl 2 solution (102 mM) for gelation as previously reported (2, 3).
- RECs and BMSCs Prior to 3D culture, RECs and BMSCs were cultured with 20 mM CFDA-SE (CFDA-SE Cell Proliferation Assay Kit; BIO RAD, Hercules, Calif., USA) according to the manufacturer's manual (2, 15, 29). fluorescently labeled. Labeled cells and unlabeled NPCs were then mixed with the UPAL solution in the same ratio (1 ⁇ 10 6 cells/mL for each cell) (2, 31, 49) to give a final cell concentration of 2 ⁇ 10 6 cells/mL.
- CFDA-SE CFDA-SE Cell Proliferation Assay Kit
- the cell- UPAL mixed solution was put into 102 mM CaCl2 solution using a 22 gauge needle and allowed to gel.
- the resulting gels were cultured for 7 days under hypoxic conditions (5% O 2 and 5% CO 2 ) (2, 49).
- hypoxic conditions 5% O 2 and 5% CO 2
- the three types of cells were each separately mixed into the UPAL solution at a concentration of 1 ⁇ 10 6 cells/mL. Cell concentrations were chosen based on previously reported results (2).
- (a) Monoculture NPCs (b) Monoculture BMSCs (c) Monoculture REC (d) Co-cultured NPCs + BMSCs (e) Co-cultured NPCs + RECs.
- RNA extraction and qRT-PCR Ten different cell types were collected (control NPCs, monocultured NPCs, NPCs co-cultured with commercial BMSCs, NPCs co-cultured with RECs, control BMSCs, monocultured BMSCs, co-cultured BMSCs, control RECs, monocultured RECs, and Co-cultured REC) were dissolved in 1 mL of TRIzol® (Invitrogen, Thermo Fisher Scientific) and total RNA was extracted from the samples using the RNeasy Mini kit (Qiagen, Valencia, Calif., USA). Real-time qRT-PCR was performed using TaqMan® Gene Expression Assays (Applied Biosystems, Thermo Fisher Scientific) (Table 3).
- Cycle threshold (Ct) values were obtained for each sample.
- relative mRNA expression levels for each target gene, NPC markers, growth factors, and ECM components were calculated via the 2- ⁇ Ct method (2). Expression levels were normalized by the expression level of the housekeeping gene GAPDH (2,31).
- Anesthesia induction was performed by intramuscular injection of a 4:1 mixture of ketamine (0.2 mg/kg) and xylazine (20 mg/kg) at a rate of 0.5 mL/kg, and anesthesia was maintained by inhalation anesthesia (isoflurane). Achieved.
- Surgery was performed using a right lateral retroperitoneal approach, exposing the vertebral bodies and IVD from L1 to L5.
- a solid cancellous screw (ZIMMER BIOMET, Warsaw, IN, USA) was inserted into the L2 vertebral body as a vertebral landmark.
- 20 mg of NP tissue was excised after AF incision (5 ⁇ 3 mm) to induce IVD degeneration (FIGS. 15, A, B) (3, 8).
- a 102 mM CaCl2 solution was immediately injected onto the surface of the mixture and gelation was confirmed after 5 min.
- the sheep were euthanized with pentobarbital and the lumbar spine was removed en bloc (3).
- MRI T2-weighted midsagittal section images were obtained using a 3.0-T MR scanner (MAGNETOM Prisma; Siemens, Kunststoff, Germany).
- Pfirrmann classification 36
- 5 grades 1: normal to 5: severely degenerated
- MRI index values were measured using Analyze 14.0 software (AnalyzeDirect, Overland Park, KS, USA) to quantify brightness of NP tissue.
- the relative MRI index which is the ratio of the MRI index in the untreated control group, was evaluated in all three treatment groups (2, 3, 15, 37, 38).
- the DHI which is the ratio of disc height to cranial adjacent vertebral body height, was also measured (4, 39).
- Relative DHI which is the percentage value of DHI of the untreated control group, was determined.
- IHC The expression of type II and type I collagen in IVD was determined by IHC. Sections were deparaffinized in xylene and treated with 0.1% trypsin for 30 minutes for antigen activation. Sections were then treated with 3% H 2 O 2 in methanol for 10 minutes, followed by protein blocking for 30 minutes using protein block serum-free solution (DAKO, Agilent, Santa Clara, Calif., USA). rice field. Goat anti-type I collagen (1:40; Southern Biotech, Birmingham, AL, USA) was used with anti-type I collagen antibody, anti-hCL(II) and purified IgG (1:400; Kyowa Pharma Chemical Co., Ltd.). , Toyama, Japan) was used as the primary antibody together with an anti-type II collagen antibody.
- Tumorigenesis Assays To assess tumorigenesis of transplanted and pre-existing cells in the transplanted IVD, (i) invasive growth, (ii) mitosis, (iii) binucleated cells, and (iv) nucleoli were evaluated. rice field. During the 24-week evaluation period, using H&E-stained specimens in the untreated control and REC+gel groups, the total number of positive cells was determined in 15 randomly selected visualization fields (15 visualizations at 400x magnification). Fields; 26.5 visualized fields, totaling 5 mm 2 ) and counts per square millimeter were calculated.
- Results 2.1 Results of 3D co-culture of human NPCs with REC and commercial human BMSCs in vitro Comparison of RECs with commercial human BMSCs Table 2 details the results of stability confirmation for RECs and commercial human BMSCs before and after gel incorporation. and 3.
- REC In a 3D culture environment with alginate, which mimics the cell environment after implantation into the intervertebral disc in vitro, REC maintained the expression of cell surface markers characteristic of mesenchymal stem cells compared to BMSCs. This indicates that REC stably maintains mesenchymal stem cell traits even in an alginate environment.
- NPC markers including HIF-1 ⁇ , GLUT-1, brachyury
- growth factors including CDMP-1, TGF- ⁇ , IGF-1 (including type II collagen and aggrecan)
- ECM components including type II collagen and aggrecan expression were measured by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) (Table 4) (2, 9, 30, 31).
- NPCs co-cultured with REC were higher in NPCs co-cultured with REC than in control NPCs, mono-cultured NPCs, and NPCs co-cultured with commercial BMSCs. significantly higher.
- GLUT-1 expression levels were significantly higher in NPCs co-cultured with commercial BMSCs than in control NPCs (Fig. 13, DF).
- Expression levels of CDMP-1 and IGF-1 were significantly increased in NPCs co-cultured with RECs compared to control NPCs, mono-cultured NPCs, and NPCs co-cultured with commercial BMSCs.
- TGF- ⁇ expression levels were significantly increased in NPCs co-cultured with RECs compared to control and monocultured NPCs.
- NPCs co-cultured with commercially available BMSCs showed significantly increased CDMP-1 and IGF-1 expression compared to control NPCs and monocultured NPCs (FIG. 13, G-I).
- the expression levels of type II collagen and aggrecan were significantly higher in NPCs co-cultured with REC than in control and mono-cultured NPCs, and the expression levels in NPCs co-cultured with commercial BMSCs were also similar to those in control and mono-cultured NPCs. (Fig. 13, J and K).
- NP tissue was removed during the first surgery (Fig. 15, A and B), and based on the results of preliminary experiments, the amount to be removed was set at 20 mg (Fig. 19). ).
- NP tissue was removed again (Fig. 15C).
- the IVD defect was then implanted with UPAL or REC+UPAL solution and exposed to 102 mM CaCl 2 to gel after the second discectomy (Fig. 15D). Implanted lumbar vertebrae were harvested for various analyses, 4 and 24 weeks after implantation.
- Fig. 16A Degenerative changes in the treated IVD were assessed using T2-weighted midsagittal images obtained via MRI.
- Fig. 16A For morphological analysis of IVDs, Pfirrmann score and MRI index were evaluated to assess signal changes in implanted IVDs (2, 3, 15, 36-38), and disc high index (DHI). Based on the Pfirrmann score, the degeneration score of the gel group was significantly lower than the Discectomy group at 24 weeks, and the degeneration score of the REC + gel group was significantly lower than the Discectomy group and the gel group at both 4 and 24 weeks. (Fig. 16B).
- the MRI index was significantly higher in the REC+gel group than in the Discectomy group at 4 weeks.
- the MRI index of the gel group was significantly higher than that of the Discectomy group, and the MRI index of the REC+gel group was significantly higher than that of the Discectomy group and the gel group (Fig. 16C).
- the disc height of the treated IVD was measured using MRI images.
- the ratio of disc height to upper adjacent vertebral body height, or DHI was measured anteriorly and posteriorly to the IVD.
- the relative DHI ie the ratio of the DHI values of the three treatment groups to the DHI values of the untreated control group, was then determined ( Figure 20) (4, 39).
- the DHI values of the three treatment groups were significantly lower than the DHI values of the untreated control group at both 4 and 24 weeks.
- Four weeks after implantation there was no significant difference between the three groups, but 24 weeks after implantation, the DHI of the gel group was significantly higher than the Discectomy group, and the REC + gel group was significantly higher than the Discectomy group and the gel group ( Figure 16D).
- Histological analysis was performed using hematoxylin & eosin (H&E) and safranin-0 staining.
- H&E hematoxylin & eosin
- the IVD showed a spindle shape, no fibrotic changes in the NP tissue, and the annulus fibrosus (AF) was a concentric structure and uniformly stained with safranin-0.
- Fig. 17, A, B marked scar tissue, fibrous changes, tissue defects, and collapse/destruction of the endplates were observed (Fig. 17, A, B), based on the modified Boos' classification of the gel group (3, 40, 41).
- the degree of histological degeneration determined by the method was significantly lower than the Discectomy group at both 4 and 24 weeks, and the REC+gel group was significantly lower than the Discectomy and gel groups at both 4 and 24 weeks (Fig. 17C).
- Type II collagen is an essential ECM component in NP tissue and is replaced by type I collagen as degeneration progresses.
- NP tissue was uniformly stained in the untreated control group, but slightly decreased in the REC+gel group. In the gel group, scattered unstained areas were observed, and in the Discectomy group, extensive unstained areas were observed.
- the proportion of type II collagen-positive cells was significantly higher in the REC+gel group than in the gel group and Discectomy group at both weeks 4 and 24, 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 evaluation period, and significantly lower than in the Discectomy and Gel groups during the 24-week evaluation period. In addition, the ratio of these cells was significantly lower in the gel group than in the Discectomy group at 24 weeks (Fig. 18D).
- the data are the mean standard deviation, and the P value was determined by the Mann-Whitney U test after the Welch test.
- This example demonstrates the superior properties of RECs compared to commercially available human BMSCs in terms of cell proliferative capacity, cell size uniformity, and expression of cell surface antigens. Furthermore, this example demonstrates the expression levels of NPC markers, growth factors, and ECM components, compared to the expression levels observed in 3D co-cultures of NPCs with commercial BMSCs, in 3D co-cultures of human NPCs and RECs. demonstrated a significant increase in Efficacy of combining REC with UPAL gel was observed at the site of IVD degeneration in a sheep lumbar spine model. UPAL gel alone inhibited IVD degeneration compared to the discectomy group, but the combination of REC and gel enhanced IVD regeneration more effectively.
- Example 1 it was suggested that co-culturing RECs with NPCs led to differentiation of RECs into NPCs, thereby improving ECM component production in both cell types. Similar results have been observed with results in rabbits (Example 1). That is, in Example 1, the expression of NPC markers increased after transplantation of BMSCs embedded in UPAL gel, and ECM production increased in the BMSC + UPAL gel group compared with the discectomy group. (2).
- Biomaterials/hydrogels used for IVD repair must be biologically and mechanically compatible.
- the ovine lumbar spine model was selected for implantation of candidate hydrogels into preclinical animal models because the biomechanics and geometry of the lumbar IVD are comparable to those of humans (3,4,34). did.
- UPAL gels implanted in post-discectomy sheep lumbar IVDs exhibit adequate biomechanical properties, do not result in material extrusion, and do not require post-discectomy AF suturing.
- unrestrained compression tests revealed no significant difference in Young's modulus between the UPAL and REC-UPAL groups, indicating that REC embedded in UPAL gel did not change the mechanical properties of the gel. showed that Due to its critical ability to provide rapid healing, the combination of UPAL gel and REC offers clinical advantages in preventing cell leakage without suturing the AF.
- Example 1 demonstrate that BMSCs embedded in UPAL gels suppressed degeneration more effectively (2 ).
- Example 1 also showed a lower histologic grade score for degeneration after BMSC+UPAL gel implantation compared to animals that did not undergo discectomy that created a degenerated IVD via AF needle puncture. This suggests that BMSC transplantation resulted in IVD regeneration (2).
- BMSC transplantation resulted in IVD regeneration (2).
- our findings indicate that transplantation of RECs embedded in UPAL gel enhances IVD regeneration in vivo (2).
- Nonoyama, N.; Iwasaki, H.; Sudo Bone marrow aspirate concentrate combined with in situ forming bioresorbable gel enhances interconnected disc regeneration in rabbits. J. Bone Joint Surg. Am. 103, e31 (2021). 16. K. Ura, K. Yamada, T.; Tsujimoto, D.; Ukeba, N.; Iwasaki, H.; Sudo, Ultra-purified alginate gel implantation decreases inflammation cytokine levels, prevents intervertebral disc degeneration, and reduces acute pain after diagnosis. Sci. Rep. 11, 638 (2021). 17. A. J. Friedenstein, U.S.A.; F. Deriglasova, N.; N.
- rat intervertebral disc puncture degeneration model (Mohd Isa et al. Sci Adv 2018) is used for experiments related to test substance administration (IHC analysis, histological analysis, pain-related behavioral analysis).
- IHC analysis histological analysis, pain-related behavioral analysis.
- a total of 60 12-week-old female SD rats (260-300 g) were divided into a group of skin incision only (sham group), a group of intervertebral disc puncture only (punch group), and a group of implanting UPAL after disc puncture (UPAL group).
- the entire tail (Co4/5-Co5/6) is surgically removed and under sterile conditions the soft tissue is removed to obtain only the tail vertebrae and intervertebral discs.
- Harvested discs are fixed with 4% (w/v) paraformaldehyde (48 hours at room temperature) and embedded in paraffin.
- the specimen is traversed mid-disc to obtain a mid-coronal transverse section (5 ⁇ m thick). After the sections are deparaffinized with xylene, they are incubated in proteinase K (Dako, Agilent Technologies, Santa Clara, Calif., USA) (37° C., 15 minutes).
- Anti-TNF- ⁇ mouse monoclonal antibody (ab220210, Abcam), anti-IL-6 mouse monoclonal antibody (ab9324, Abcam), anti-TrkA rabbit monoclonal antibody (ab86474, Abcam) are used.
- Histofine (registered trademark) Fast Red II (Nichirei Bioscience) for TNF- ⁇ analysis
- HistoGreen Substrate kit for Peroxidase Cosmo Bio Co., Ltd., Tokyo, Japan
- TrkA analysis registered trademark DAB (Nichirei Bioscience)
- Counterstaining of cell nuclei is performed to improve visibility, using hematoxylin for TNF- ⁇ or TrkA staining and fast red for IL-6 staining, respectively.
- TNF- ⁇ , IL-6, or TrkA-positive cells were individually counted in 5 randomly selected fields, and positive nucleus pulposus or annulus fibrosus cells in each staining were analyzed. Numbers are calculated as a percentage of the total nucleus pulposus or annulus fibrosus cell number in the field. All assessments are performed by two blinded independent observers. Each observer evaluates one sample three times, calculates the average value for each sample, and compares between groups.
- Hargreaves test was performed on the 2nd preoperative day (Day-2) and 2, 7, 14, 27 postoperative days using the Hargreaves test apparatus (Ugo Basile Biological Instruments, Gemonio, Italy). (Mohd Isa et al. Sci Adv 2018). Rats are placed in individual chambers (with air vents at the top) enclosed on all four sides and above on a glass plate (Ugo Basile Biological Instruments). An infrared beam is applied to the ventral side of the skin incision as a thermal stimulus. The latency to exhibit withdrawal behavior to the thermal stimulus is recorded. The intensity of the beam is set at 50% of maximum power. A cut-off time of 20 seconds is set to prevent tissue damage. Four measurements are performed on the same rat at each time point, with a rest of at least 1 minute between each measurement.
- the von Frey test is performed on the 2nd day before surgery (Day-2) and 2, 7, 14, 27 days after surgery using a dynamic plantar aesthesiometer (Ugo Basile Biological Instruments). Rats are placed in the same cubicle used in the Hargreaves test above wire mesh. A 0.5 mm diameter filament was applied to the ventral side of the skin incision and a linearly increasing force starting at 0 g to 5 g was applied over 10 seconds, followed by a force of 5 g 30 seconds after the start of the test. Apply with a constant force. The latency for the rat to exhibit any escape behavior is recorded. Five measurements are performed on the same rat at each time point, with a rest of at least 10 seconds between each measurement.
- Tail flick test is performed using a heat flux radiometer (manufactured by Ugo Basile Biological Instruments). In order to avoid tissue damage due to excessive heat stimulation due to being performed on the same schedule as the Hargreaves test, it is performed on the 1st day before surgery (Day-1) and 3, 8, 15, and 28 days after surgery (Mohd Isa et al. Sci Adv 2018). After each rat was wrapped in a towel and allowed to settle for 10 minutes, the tail was placed on the device while the body was covered with the towel, and an infrared beam was irradiated on the ventral side of the proximal portion of the tail 5 cm from the distal end of the tail. do. The latency to tail flick response to thermal stimulation is recorded. A cut-off time of 20 seconds is set to prevent tissue damage. Four measurements are performed on the same rat at each time point, with a rest of at least 15 seconds between each measurement.
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Abstract
Description
このため、椎間板組織が修復・再生できるさらなる組成物の開発が必要とされている。
すなわち、本発明は以下の通りである。
[1] 低エンドトキシンアルギン酸の1価金属塩と間葉系幹細胞とを含有する、椎間板再生用組成物。
[1−1] アルギン酸の1価金属塩と間葉系幹細胞とを含有する、椎間板再生用組成物。
[2] 間葉系幹細胞による髄核細胞の活性化及び/又は間葉系幹細胞の髄核細胞への分化を介して、椎間板の髄核再生を促進する、[1]又は[1−1]に記載の組成物。
[3] 前記間葉系幹細胞が、ヒト骨髄由来高純度間葉系幹細胞である[1]~[2]のいずれか1項に記載の組成物。
[4] 前記ヒト骨髄由来高純度間葉系幹細胞は、LNGFR(CD271)が陽性、又はLNGFR(CD271)及びThy−1(CD90)が共陽性の高速増殖性間葉系幹細胞クローンの細胞集団であって、以下の(a)及び(b)の少なくとも1つの特徴を満たす細胞集団である、[3]に記載の組成物。
(a)フローサイトメトリーの前方散乱光の変動係数が35%以下である
(b)平均細胞サイズが20μm以下である
[4−1] 前記ヒト骨髄由来高純度間葉系幹細胞は、LNGFR(CD271)が陽性、又はLNGFR(CD271)及びThy−1(CD90)が共陽性であることを指標に分離された、高速増殖性間葉系幹細胞クローンの細胞集団であって、以下の(a)及び(b)の少なくとも1つの特徴を満たす細胞集団である、[3]に記載の組成物。
(a)フローサイトメトリーの前方散乱光の変動係数が35%以下である
(b)平均細胞サイズが20μm以下である
[4−2] 前記ヒト骨髄由来高純度間葉系幹細胞は、LNGFR(CD271)が陽性、又はLNGFR(CD271)及びThy−1(CD90)が共陽性の細胞に由来する高速増殖性間葉系幹細胞クローンの細胞集団であって、以下の(a)及び(b)の少なくとも1つの特徴を満たす細胞集団である、[3]に記載の組成物。
(a)フローサイトメトリーの前方散乱光の変動係数が35%以下である
(b)平均細胞サイズが20μm以下である
[4−3] 前記ヒト骨髄由来高純度間葉系幹細胞は、LNGFR(CD271)が陽性、又はLNGFR(CD271)及びThy−1(CD90)が共陽性の高速増殖性間葉系幹細胞クローンの細胞集団であって、以下の(a)及び(b)の少なくとも1つの特徴を満たす細胞集団である、[3]に記載の組成物。
(a)フローサイトメトリーの前方散乱光の変動係数が40%以下である
(b)平均細胞サイズが20μm以下である
[4−4] 前記ヒト骨髄由来高純度間葉系幹細胞は、LNGFR(CD271)が陽性、又はLNGFR(CD271)及びThy−1(CD90)が共陽性であることを指標に分離された、高速増殖性間葉系幹細胞クローンの細胞集団であって、以下の(a)及び(b)の少なくとも1つの特徴を満たす細胞集団である、[3]に記載の組成物。
(a)フローサイトメトリーの前方散乱光の変動係数が40%以下である
(b)平均細胞サイズが20μm以下である
[4−5] 前記ヒト骨髄由来高純度間葉系幹細胞は、LNGFR(CD271)が陽性、又はLNGFR(CD271)及びThy−1(CD90)が共陽性の細胞に由来する高速増殖性間葉系幹細胞クローンの細胞集団であって、以下の(a)及び(b)の少なくとも1つの特徴を満たす細胞集団である、[3]に記載の組成物。
(a)フローサイトメトリーの前方散乱光の変動係数が40%以下である
(b)平均細胞サイズが20μm以下である
[5] 対象の椎間板に適用し、当該適用時には流動性を有する、[1]~[4−5]のいずれか1項に記載の組成物。
[5−1] 対象の椎間板髄核部位に適用し、適用後に組成物の表面の少なくとも一部分に架橋剤を接触させるように用いられ、髄核部位への適用時に流動性を有する、[1]~[4]のいずれか1項に記載の組成物。
[5−2] 対象の椎間板髄核部位に適用し、適用後に一部分を硬化するように用いられ、髄核部位への適用時に流動性を有する、[1]~[4]のいずれか1項に記載の組成物。
[6] 前記髄核部位への適用は、髄核欠損部への前記組成物の充填である、[5]~[5−2]のいずれか1項に記載の組成物。
[7] 前記一部分の硬化は、組成物の表面の少なくとも一部分に架橋剤を接触させることにより行われる、[5−2]又は[6]に記載の組成物。
[8] 前記架橋剤が2価以上の金属イオン化合物である、[5−1]又は[7]に記載の組成物。
[8−1] 前記アルギン酸の1価金属塩が、低エンドトキシンアルギン酸1価金属塩である、[1]~[8]のいずれか1項に記載の組成物。
[9] 前記低エンドトキシンアルギン酸の1価金属塩は、GPC−MALS法により測定された重量平均分子量(絶対分子量)が8万以上である、[1]~[8−1]のいずれか1項に記載の組成物。
[10] 低エンドトキシンアルギン酸の1価金属塩の濃度が0.5w/w%~5.0w/w%である、[1]~[9]のいずれか1項に記載の組成物。
[11] 椎間板変性及び/又は椎間板損傷の治療、予防又は再発抑制のために用いられる、[1]~[10]のいずれか1項に記載の組成物。
[12] 前記椎間板変性及び/又は椎間板損傷が、椎間板ヘルニア、椎間板症、脊椎変性辷り症、化膿性椎間板炎、変形性脊椎症、脊柱管狭窄症、腰部脊柱管狭窄症、腰部脊柱管狭窄症を伴う椎間板ヘルニア及び椎間板損傷からなる群から選択される少なくとも1種である、[11]に記載の組成物。
[12−1] 前記椎間板変性及び/又は椎間板損傷が、慢性腰痛を伴う、[11]又は[12]に記載の組成物。
[14] ヒト骨髄由来高純度間葉系幹細胞による髄核細胞の活性化及び/又はヒト骨髄由来高純度間葉系幹細胞の髄核細胞への分化を介して、椎間板の髄核再生を促進する、[13]に記載の組成物。
[15] 前記ヒト骨髄由来高純度間葉系幹細胞が、適用時に未分化状態である及び/又は誘導分化の処置なしに適用される[13]又は[14]に記載の組成物。
[16] 前記ヒト骨髄由来高純度間葉系幹細胞は、LNGFR(CD271)が陽性、又はLNGFR(CD271)及びThy−1(CD90)が共陽性の高速増殖性間葉系幹細胞クローンの細胞集団であって、以下の(a)及び(b)の少なくとも1つの特徴を満たす細胞集団である、[13]~[15]のいずれか1項に記載の組成物。
(a)フローサイトメトリーの前方散乱光の変動係数が35%以下である
(b)平均細胞サイズが20μm以下である
[16−1] 前記ヒト骨髄由来高純度間葉系幹細胞は、LNGFR(CD271)が陽性、又はLNGFR(CD271)及びThy−1(CD90)が共陽性であることを指標に分離された、高速増殖性間葉系幹細胞クローンの細胞集団であって、以下の(a)及び(b)の少なくとも1つの特徴を満たす細胞集団である、[13]~[15]のいずれか1項に記載の組成物。
(a)フローサイトメトリーの前方散乱光の変動係数が35%以下である
(b)平均細胞サイズが20μm以下である
[16−2] 前記ヒト骨髄由来高純度間葉系幹細胞は、LNGFR(CD271)が陽性、又はLNGFR(CD271)及びThy−1(CD90)が共陽性の細胞に由来する高速増殖性間葉系幹細胞クローンの細胞集団であって、以下の(a)及び(b)の少なくとも1つの特徴を満たす細胞集団である、[13]~[15]のいずれか1項に記載の組成物。
(a)フローサイトメトリーの前方散乱光の変動係数が35%以下である
(b)平均細胞サイズが20μm以下である
[16−3] 前記ヒト骨髄由来高純度間葉系幹細胞は、LNGFR(CD271)が陽性、又はLNGFR(CD271)及びThy−1(CD90)が共陽性の高速増殖性間葉系幹細胞クローンの細胞集団であって、以下の(a)及び(b)の少なくとも1つの特徴を満たす細胞集団である、[13]~[15]のいずれか1項に記載の組成物。
(a)フローサイトメトリーの前方散乱光の変動係数が40%以下である
(b)平均細胞サイズが20μm以下である
[16−4] 前記ヒト骨髄由来高純度間葉系幹細胞は、LNGFR(CD271)が陽性、又はLNGFR(CD271)及びThy−1(CD90)が共陽性であることを指標に分離された、高速増殖性間葉系幹細胞クローンの細胞集団であって、以下の(a)及び(b)の少なくとも1つの特徴を満たす細胞集団である、[13]~[15]のいずれか1項に記載の組成物。
(a)フローサイトメトリーの前方散乱光の変動係数が40%以下である
(b)平均細胞サイズが20μm以下である
[16−5] 前記ヒト骨髄由来高純度間葉系幹細胞は、LNGFR(CD271)が陽性、又はLNGFR(CD271)及びThy−1(CD90)が共陽性の細胞に由来する高速増殖性間葉系幹細胞クローンの細胞集団であって、以下の(a)及び(b)の少なくとも1つの特徴を満たす細胞集団である、[13]~[15]のいずれか1項に記載の組成物。
(a)フローサイトメトリーの前方散乱光の変動係数が40%以下である
(b)平均細胞サイズが20μm以下である
[17] 前記組成物が、細胞を包埋するための担体をさらに含む、[13]~[16−5]のいずれか1項に記載の組成物。
[18] 前記担体が、アルギン酸、ヒアルロン酸、コンドロイチン硫酸、デルマタン硫酸、ケラタン硫酸、ヘパリン、ヘパラン硫酸、およびガラクトサミノグリクロングリカン硫酸、並びにこれらの薬学的に許容可能な塩からなる群から選択される、[17]に記載の組成物。
[19] 前記担体が、アルギン酸の1価金属塩である[17]に記載の組成物。
[20] 対象の椎間板髄核部位に適用し、適用後に組成物の表面の少なくとも一部分に架橋剤を接触させるように用いられ、髄核部位への適用時に流動性を有する、[13]~[19]のいずれか1項に記載の組成物。
[21] 対象の椎間板髄核部位に適用し、適用後に一部分を硬化するように用いられ、髄核部位への適用時に流動性を有する、[13]~[19]のいずれか1項に記載の組成物。
[22] 前記髄核部位への適用は、髄核欠損部への前記組成物の充填である、[20]又は[21]に記載の組成物。
[23] 前記架橋剤が2価以上の金属イオン化合物である、[20]~[22]のいずれか1項に記載の組成物。
[24] 前記アルギン酸の1価金属塩は、GPC−MALS法により測定された重量平均分子量(絶対分子量)が8万以上である、[19]~[23]のいずれか1項に記載の組成物。
[25] 前記アルギン酸の1価金属塩の濃度が0.5w/w%~5.0w/w%である、[19]~[24]のいずれか1項に記載の組成物。
[26] 前記アルギン酸の1価金属塩が、低エンドトキシンアルギン酸1価金属塩である、[19]~[25]のいずれか1項に記載の組成物。
[27] 椎間板変性及び/又は椎間板損傷の治療、予防又は再発抑制のために用いられる、[13]~[26]のいずれか1項に記載の組成物。
[28] 前記椎間板変性及び/又は椎間板損傷が、椎間板ヘルニア、椎間板症、脊椎変性辷り症、化膿性椎間板炎、変形性脊椎症、脊柱管狭窄症、腰部脊柱管狭窄症、腰部脊柱管狭窄症に伴う椎間板ヘルニア(混合性腰部脊柱管狭窄症)及び椎間板損傷からなる群から選択される少なくとも1種である、[27]に記載の組成物。
[28−1] 前記椎間板変性及び/又は椎間板損傷が、慢性腰痛を伴う、[27]又は[28]に記載の組成物。
[29] 椎間板性疼痛を抑制するために用いられる、[1]~[28−1]のいずれか1項に記載の組成物。
[30] 前記椎間板性疼痛が、慢性腰痛である[29]に記載の組成物。
[31−1] 対象の椎間板髄核部位に流動性を有する状態で適用された後に、組成物を硬化させる処置を要しない使用のための、上記[31]に記載の椎間板再生用組成物。
[31−2] 対象の椎間板髄核部位に流動性を有する状態で適用された後に、該組成物に架橋剤を接触させることなく用いられる上記[31]又は[31−1]に記載の椎間板再生用組成物。
[32] ヒト骨髄由来高純度間葉系幹細胞による髄核細胞の活性化及び/又はヒト骨髄由来高純度間葉系幹細胞の髄核細胞への分化を介して、椎間板の髄核再生を促進する、[31]~[31−2]のいずれか1項に記載の組成物。
[33] 前記ヒト骨髄由来高純度間葉系幹細胞が、適用時に未分化状態である及び/又は誘導分化の処置なしに適用される[31]~[32]のいずれか1項に記載の組成物。
[34] 前記ヒト骨髄由来高純度間葉系幹細胞は、LNGFR(CD271)が陽性、又はLNGFR(CD271)及びThy−1(CD90)が共陽性の高速増殖性間葉系幹細胞クローンの細胞集団であって、以下の(a)及び(b)の少なくとも1つの特徴を満たす細胞集団である、[31]~[33]のいずれか1項に記載の組成物。
(a)フローサイトメトリーの前方散乱光の変動係数が40%以下である
(b)平均細胞サイズが20μm以下である
[34−1] 前記ヒト骨髄由来高純度間葉系幹細胞は、LNGFR(CD271)が陽性、又はLNGFR(CD271)及びThy−1(CD90)が共陽性であることを指標に分離された、高速増殖性間葉系幹細胞クローンの細胞集団であって、以下の(a)及び(b)の少なくとも1つの特徴を満たす細胞集団である、[31]~[33]のいずれか1項に記載の組成物。
(a)フローサイトメトリーの前方散乱光の変動係数が40%以下である
(b)平均細胞サイズが20μm以下である
[34−2] 前記ヒト骨髄由来高純度間葉系幹細胞は、LNGFR(CD271)が陽性、又はLNGFR(CD271)及びThy−1(CD90)が共陽性の細胞に由来する高速増殖性間葉系幹細胞クローンの細胞集団であって、以下の(a)及び(b)の少なくとも1つの特徴を満たす細胞集団である、[31]~[33]のいずれか1項に記載の組成物。
(a)フローサイトメトリーの前方散乱光の変動係数が40%以下である
(b)平均細胞サイズが20μm以下である
[34−3] 前記変動係数が35%以下である、[34]~[34−2]のいずれか1項に記載の組成物。
[35] 前記髄核部位への適用は、髄核欠損部への前記組成物の充填である、[31]~[34]のいずれか1項に記載の組成物。
[36] 前記アルギン酸の1価金属塩は、GPC−MALS法により測定された重量平均分子量(絶対分子量)が8万以上である、[31]~[35]のいずれか1項に記載の組成物。
[37] 前記アルギン酸の1価金属塩の濃度が0.5w/w%~5.0w/w%である、[31]~[36]のいずれか1項に記載の組成物。
[38] 前記アルギン酸の1価金属塩が、低エンドトキシンアルギン酸1価金属塩である、[31]~[37]のいずれか1項に記載の組成物。
[39] 椎間板変性及び/又は椎間板損傷の治療、予防又は再発抑制のために用いられる、[31]~[38]のいずれか1項に記載の組成物。
[40] 前記椎間板変性及び/又は椎間板損傷が、椎間板ヘルニア、椎間板症、脊椎変性辷り症、化膿性椎間板炎、慢性腰痛、変形性脊椎症、脊柱管狭窄症、腰部脊柱管狭窄症、腰部脊柱管狭窄症に伴う椎間板ヘルニア(混合性腰部脊柱管狭窄症)及び椎間板損傷からなる群から選択される少なくとも1種である、[39]に記載の組成物。
[41] 前記椎間板変性及び/又は椎間板損傷が、慢性腰痛を伴う、[39]又は[40]に記載の組成物。
[42] 椎間板性疼痛を抑制するために用いられる、[31]~[41]のいずれか1項に記載の組成物。
[43] 前記椎間板性疼痛が、慢性腰痛である[42]に記載の組成物。
[44−1] 対象の椎間板髄核部位に流動性を有する状態で適用された後に、組成物を硬化させる処置を要しない使用のための、上記[44]に記載の椎間板性疼痛抑制用組成物。
[44−2] 対象の椎間板髄核部位に流動性を有する状態で適用された後に、該組成物に架橋剤を接触させることなく用いられる上記[44]又は[44−1]に記載の椎間板性疼痛抑制用組成物。
[45] 前記疼痛が、椎間板ヘルニア、椎間板症、脊椎変性辷り症、化膿性椎間板炎、変形性脊椎症、脊柱管狭窄症、腰部脊柱管狭窄症、腰部脊柱管狭窄症に伴う椎間板ヘルニア(混合性腰部脊柱管狭窄症)及び椎間板損傷からなる群から選択される少なくとも1種を伴う疼痛である、[44]~[44−2]のいずれか1項に記載の組成物。
[46] 前記疼痛が、慢性腰痛である、[44]~[45]に記載の組成物。
[47] 前記ヒト骨髄由来高純度間葉系幹細胞が、適用時に未分化状態である及び/又は誘導分化の処置なしに適用される[44]~[46]のいずれか1項に記載の組成物。
[48] 前記ヒト骨髄由来高純度間葉系幹細胞は、LNGFR(CD271)が陽性、又はLNGFR(CD271)及びThy−1(CD90)が共陽性の高速増殖性間葉系幹細胞クローンの細胞集団であって、以下の(a)及び(b)の少なくとも1つの特徴を満たす細胞集団である、[44]~[47]のいずれか1項に記載の組成物。
(a)フローサイトメトリーの前方散乱光の変動係数が40%以下である
(b)平均細胞サイズが20μm以下である
[48−1] 前記ヒト骨髄由来高純度間葉系幹細胞は、LNGFR(CD271)が陽性、又はLNGFR(CD271)及びThy−1(CD90)が共陽性であることを指標に分離された、高速増殖性間葉系幹細胞クローンの細胞集団であって、以下の(a)及び(b)の少なくとも1つの特徴を満たす細胞集団である、[44]~[47]のいずれか1項に記載の組成物。
(a)フローサイトメトリーの前方散乱光の変動係数が40%以下である
(b)平均細胞サイズが20μm以下である
[48−2] 前記ヒト骨髄由来高純度間葉系幹細胞は、LNGFR(CD271)が陽性、又はLNGFR(CD271)及びThy−1(CD90)が共陽性の細胞に由来する高速増殖性間葉系幹細胞クローンの細胞集団であって、以下の(a)及び(b)の少なくとも1つの特徴を満たす細胞集団である、[44]~[47]のいずれか1項に記載の組成物。
(a)フローサイトメトリーの前方散乱光の変動係数が40%以下である
(b)平均細胞サイズが20μm以下である
[48−3] 前記変動係数が35%以下である、[48]~[48−2]のいずれか1項に記載の組成物。
[48−4] 前記髄核部位への適用は、髄核欠損部への前記組成物の充填である、[44]~[48−3]のいずれか1項に記載の組成物。
[49] 前記アルギン酸の1価金属塩は、GPC−MALS法により測定された重量平均分子量(絶対分子量)が8万以上である、[44]~[48−4]のいずれか1項に記載の組成物。
[50] 前記アルギン酸の1価金属塩の濃度が0.5w/w%~5.0w/w%である、[44]~[49]のいずれか1項に記載の組成物。
[51] 前記アルギン酸の1価金属塩が、低エンドトキシンアルギン酸1価金属塩である、[44]~[50]のいずれか1項に記載の組成物。
[52] 対象の椎間板髄核部位に、X線透視下で適用し、髄核部位への適用時に流動性を有し、該組成物に架橋剤を接触させることなく用いられる[31]~[51]のいずれか1項に記載の組成物。
[53] 対象の椎間板髄核部位に、X線透視下で圧力計シリンジを用いて適用し、髄核部位への適用時に流動性を有し、該組成物に架橋剤を接触させることなく用いられる[52]に記載の組成物。
(a1)ヒツジ椎間板からヒツジ体重の0.00004%~0.00005%に相当する量の髄核組織を除去し、変性椎間板を作製する工程
(a2)ヒツジ椎間板から20mgの髄核組織を除去し、変性椎間板を作製する工程
(b1)前記工程(a1)又は(a2)の4週後に、さらにヒツジ体重の0.00014%~0.000175%に相当する量の髄核組織を除去する工程
(b2)前記工程(a1)又は(a2)の4週後に、さらに70mgの髄核組織を除去する工程
[55] 組成物を投与する工程が、工程(b1)又は(b2)の後に行われる、[54]に記載の方法。
[56] 評価は、組成物投与後に、変性モデルから採取した椎体及び椎間板に対して、MRI、組織染色、免疫組織化学染色(IHC)からなる群から選択される少なくとも1つの評価方法により行う、[54]又は[55]に記載の方法。
[57] 椎間板変性および/または椎間板損傷の治療、予防または再発抑制のための方法であって、
アルギン酸の1価金属塩と間葉系幹細胞とを含有する組成物を、前記治療、予防または再発抑制を必要とする対象の椎間板の髄核部位に適用することを含む、前記方法。
[58] 椎間板変性および/または椎間板損傷の治療、予防または再発抑制のための方法であって、
ヒト骨髄由来高純度間葉系幹細胞を、前記治療、予防または再発抑制を必要とする対象の椎間板の髄核部位に適用することを含む、前記方法。
[59] 椎間板変性および/または椎間板損傷の治療、予防または再発抑制のための方法であって、
ヒト骨髄由来高純度間葉系幹細胞及びアルギン酸の1価金属塩を含む組成物を、前記治療、予防または再発抑制を必要とする対象の椎間板の髄核部位に適用することを含み、前記組成物は、当該髄核部位への適用時に流動性を有する、前記方法。
[60] 椎間板性疼痛の治療、予防または再発抑制のための方法であって、
ヒト骨髄由来高純度間葉系幹細胞及びアルギン酸の1価金属塩を含む組成物を、前記治療、予防または再発抑制を必要とする対象の椎間板の髄核部位に適用することを含み、前記組成物は、当該髄核部位への適用時に流動性を有する、前記方法。
[61] アルギン酸の1価金属塩と間葉系幹細胞を含有する組成物を、椎間板変性および/または椎間板損傷の治療、予防または再発抑制を必要とする対象の椎間板の髄核部位に適用し、椎間板変性および/または椎間板損傷の治療、予防または再発抑制において使用されるためのアルギン酸の1価金属塩および間葉系幹細胞。
[62] 椎間板変性および/または椎間板損傷の治療、予防または再発抑制に使用するための、アルギン酸の1価金属塩と間葉系幹細胞とを含有する組成物。
[63] 椎間板変性および/または椎間板損傷の治療、予防または再発抑制に使用するための、ヒト骨髄由来高純度間葉系幹細胞。
[64] 椎間板変性および/または椎間板損傷の治療、予防または再発抑制に使用するための、ヒト骨髄由来高純度間葉系幹細胞及びアルギン酸の1価金属塩を含む組成物であって、当該組成物は、前記治療、予防または再発抑制を必要とする対象の椎間板の髄核部位に適用することを含み、前記組成物は、当該髄核部位への適用時に流動性を有する、前記組成物。
[65] 椎間板性疼痛の治療、予防または再発抑制に使用するための、ヒト骨髄由来高純度間葉系幹細胞及びアルギン酸の1価金属塩を含む組成物であって、当該組成物は、前記治療、予防または再発抑制を必要とする対象の椎間板の髄核部位に適用することを含み、前記組成物は、当該髄核部位への適用時に流動性を有する、前記組成物。
(a):共培養細胞の二次元(2D)ドットプロット。P1ゲートは、単一の生細胞の周りに配置された。
(b):分類されたP2ゲート中の非標識NPC及びP3ゲート中のCFDA−SE標識BMSCの2Dドットプロット。
FSC−A:前方散乱領域;FSC−W:前方散乱幅;SSC−A:側方散乱領域;CFDA−SE−A:CFDA−SE−領域。
各細胞の遺伝子発現をハウスキーピング遺伝子GAPDHを基準とし、対数スケール(y軸)でプロットした。データは、4つの異なるウサギNPC系統の平均値である。
(c):HIF−1α、(d):GLUT−1、(e):Brachyury、(f):CDMP−1、(g):TGF−β、(h):IGF−1、(i):II型コラーゲン、及び(j):アグリカン。
データは平均値±標準誤差で表し、p値はTukey−Kramerの事後検定を用いた一元配置分散分析により求めた。
[図2]骨髄由来間葉系幹細胞(BMSC)が椎間板(IVD)で生存することを示す図。
カルボキシフルオレセインジアセテートスクシンイミジルエステル(CFDA−SE)で標識した移植後の骨由来間葉系幹細胞(BMSC)は、術後4週目及び12週目の椎間板(IVD)で生存する。IVDの凍結切片を4’,6−ジアミノ−2−フェニルインドール(DAPI)で染色した。画像は、4回の反復試験(無傷対照及びBMSC+Gel、n=4;4週間及び12週間)の代表である。スケールバー=50μm。
[図3]骨髄由来間葉系幹細胞(BMSC)と組み合わせたゲルは、椎間板切除後の変性椎間板(IVD)の水分含量を保存することを示す図。
(a):術後4週目と12週目の変性IVDのT2強調、正中矢状断像。画像は、8回の撮像の代表画像である。
(b):IVD変性のPfirrmann分類。データは平均値±標準誤差(無傷の対照、穿刺,椎間板切除,ゲル,及びBMSC+ゲル,n=8;4週及び12週)である。
(c)NPの変性変化に対する磁気共鳴イメージング(MRI)index(髄核(NP)面積×平均信号強度)。数値は、無傷の対照IVDと比較した割合(%)として表される。データは平均値±標準誤差であり、Tukey−Kramer検定を用いて事後解析を伴う一元配置分散分析によりp値を求めた。
[図4]骨髄由来間葉系幹細胞(BMSC)と組み合わせたゲルは、椎間板切除後の椎間板(IVD)変性を予防することを示す図。
(a,b):ヘマトキシリン及びエオシン(H&E)又はサフラニン0で染色されたIVDの正中矢状断切片画像(8回の実験画像の代表例)(無傷対照(Intact control)、穿刺(Puncture)、椎間板切除(Discectomy)、ゲル、BMSC+ゲル、n=8;4及び12週間)。
AF:線維輪、NP:髄核。スケールバー=(A):500μm(上から1番目及び3番目のセクション)、50mm(上から2番目及び4番目のセクション)、及び(B):500μm。
(c):4週目と12週目の組織学的変性度を示す。
データは平均値±標準誤差であり、p値はTukey−Kramerの事後検定を用いた一元配置分散分析により求めた。
[図5]ウサギ髄核(NP)におけるII型コラーゲン陽性細胞を示す図。
(a):ウサギ椎間板(IVD)の正中矢状断切片をII型コラーゲンに対して染色した。画像は、8回の反復実験結果(無傷対照、穿刺、椎間板切除、ゲル、BMSC+ゲル、n=8;4及び12週間)の代表画像である。矢印は、II型コラーゲンに対して陽性の細胞を示す。スケールバー=500μm(上から1番目及び3番目のセクション)及び20μm(上から2番目及び4番目のセクション)。
(b):II型コラーゲン陽性細胞の割合。データは平均値±標準誤差であり、p値はTukey−Kramerの事後検定を用いた一元配置分散分析により求めた。
[図6]ウサギNPにおける髄核(NP)マーカー陽性細胞を示す図。
(a−c):1、7、28日目にHIF−1α、GLUT−1、Brachyury染色したウサギ椎間板(IVD)の水平切片。画像は4回の反復実験(無傷対照、椎間板切除、及びBMSC+ゲル、n=4;day1、7、28)の代表画像である。スケールバー=50μm。
(d−f):全細胞に対するNPマーカー陽性細胞の割合。
(g−i):CFDA−SE陽性細胞(移植されたBMSCを代表する)に対するNPマーカー陽性細胞の割合。データは平均±標準誤差であり、p値は対応のあるt検定を用いて決定した。
[図7]椎間板(IVD)再生のメカニズムを示す図。
移植された骨髄由来間葉系幹細胞(BMSC)は、既存の髄核細胞(NPC)の活性化をもたらす成長因子と細胞外マトリックス(ECM)を産生する。既存のNPC活性化はまた、増殖因子及びECMの産生を増加させる。移植されたBMSCはNPCに分化する。UPAL:超精製アルギン酸塩(低エンドトキシン高純度アルギン酸塩ともいう)。
[図8]RECクローンの網羅的解析結果を示す図である。
[図9]ヒトの健常椎間板髄核細胞(NPC)及び高純度間葉系細胞(REC)における各種遺伝子の発現量を示す図。
[図10]移植後4週目のヒツジモデルにおける椎間板のMRIの結果を示す図。
[図11]移植後4週目のヒツジモデルにおける椎間板の組織学的検査結果を示す図。
[図12]移植後4週目のヒツジモデルにおける椎間板の組織学的検査結果を示す図。
[図13]3D共培養の7日後のヒトNPC及びRECの発現プロフィールを示す図。
(A) REC単離を示す概略図。CD271(LNGFR)及びCD90(THY−1)について染色したヒト骨髄細胞のフローサイトメトリープロフィール。1ウェルから収集した細胞を35mm培養皿に播種し、14日まで増殖させて、高い分化能及び増殖能を有する均一な細胞集団を得る。
(B及びC)細胞選別データを用いて、CFDA−SE標識RECと非標識NPCとを区別する。
(B) P1ゲートは、死細胞及び残屑生細胞を排除する。
(C) P2ゲートでのラベルなしNPCとP3ゲートでのCFDA−SE標識RECを示す2Dドットプロット。
(D~K) 各細胞の遺伝子発現レベルをハウスキーピング遺伝子GAPDHの発現レベルに対して正規化し、対数スケール(y軸)でプロットした。4つの異なるヒトNPC株から得られたデータを平均化した。
(D)HIF−1α,(E)GLUT−1,(F)brachyury,(G)CDMP−1,(H)TGF−β,(I)IGF−1,(J)II型コラーゲン、(K)アグリカン
データは、平均±SD値(n=4)として表す。有意差は、post hoc Tukey−Kramer検定による一元配置分散分析で評価した。
NPC:髄核細胞
REC:急速に増殖するクローン
CFDA−SE:5,6−カボキシフルオレセインジアクタトスクシニミジルエステル
ANOVA:分散分析
FSC−A:前方散乱面積
FSC−W:前方散乱幅
SSC−A:側方散乱面積
CFDA−SE−A:CFDA−SE面積
[図14]2種類のゲルの弾性比を示す図。
(A) CaCl2で誘導させたゲル化(直径、4.5mm;厚さ、2mm)後の円盤状UPAL及びREC+UPALゲルの形成。
(B及びC) 引張圧縮機械試験装置。サンプルを0.5mm/分の一定速度で圧縮した。
(D) 応力歪曲線。ヤング率は、圧縮値10~20%の間の接線の傾きに従って計算した。4つの反復試験を行い、代表的な画像を示す。
(E) 2種類のゲルのヤング率。データは、平均±SD値(n=4)を示す。有意差はWelchの検定後、Student’s t−testにより評価した。
N.S:有意差なし
UPAL:超精製アルギン酸塩(低エンドトキシン高純度アルギン酸塩ともいう)
REC:迅速に増殖するクローン
SD:標準偏差
[図15]各群の実験スケジュール及び治療の詳細を示す図。
(A及びB) 最初の手術で20mgの新鮮なNP組織を処置IVDから除去して、重度の変性IVDモデルを確立した。
(C) 最初の手術の4週間後、70mgの変性NP組織を変性IVDからさらに除去した。
(D) 変性NPの除去後、UPAL又はREC+UPAL溶液を椎間板内の空隙に移植した。
NP:髄核
UPAL:超精製アルギン酸(低エンドトキシン高純度アルギン酸ともいう)
IVD:椎間板
[図16]埋植後4週目及び24週目における治療したIVDのMRI評価を示す図。
(A) ヒツジにおける術後4週目及び24週目のIVDのT2強調、正中矢状断画像。画像は、6又は8回の反復結果の代表である。
(B)PfirrmannグレードのIVD変性。
(C) NPの変性変化に対するMRI指数(NP面積×平均シグナル強度)値。数値は、無処置の対照IVDについての値に対する割合(%)として表される。
(D) IVD治療のための椎間板高さ指数。数値は、無処置の対照IVDの数値に対する割合(%)として示す。データは、平均±SD値として表す(無処置対照,n=6;椎間板切除,n=6;ゲル,n=8;REC+gel,n=8)。有意差は、Tukey‐Kramer検定を用いたpost hoc解析による一元配置ANOVAにより評価した。
NP:髄核
MRI:磁気共鳴画像法
IVD:椎間板
ANOVA:分散分析
SD:標準偏差
[図17]埋植4週後及び24週後の治療したIVDの組織学的評価を行った結果を示す図。
(A及びB) H&E又はサフラニン−0染色によって染色された処理IVDの代表的な正中矢状切片(無処置対照、n=6;椎間板切除術、n=6;ゲル、n=8;REC+ゲル、n=8)。スケールバー(A);50μm(上から2番目及び4番目の部分)又は1mm(上から1番目及び3番目の部分)(B);1mm。
(C) 組織学的グレードは、改変Boos分類を介して決定した。データは平均値±標準偏差で表す。有意差はpost hoc Tukey−Kramer検定による一元配置分散分析で評価した。
IVD:椎間板
H&E:ヘマトキシリン&エオシン
SD:標準偏差
AF:線維輪
NP:髄核
[図18]埋植4週後及び24週後の処置IVDにおけるII型又はI型コラーゲン陽性細胞を示す図。
(A及びB) II型コラーゲン又はI型コラーゲンについて染色された処置IVDの代表的な正中矢状切片(無処置対照、n=6;椎間板切除術、n=6;ゲル、n=8;REC+ゲル、n=8)。スケールバー、1mm(第1及び第3のライン)又は50μm(第2及び第4のライン)。
(C及びD) 処置されたIVD中の総細胞に対するII型又はI型コラーゲン陽性細胞の割合。データは平均値±標準偏差で表す。有意差はpost hoc Tukey−Kramer検定による一元配置分散分析で評価した。
IVD:椎間板
REC:急速増殖クローン
SD:標準偏差
ANOVA:分散分析
[図19]NP組織除去後4週目の組織学的評価を示す図。
(A) IVDから除去したNP組織(無処置対照、又は20、50、100、200mg除去)の中矢状部(H&E又はサフラニン−0で染色)。ヒツジモデルにおいて、4週間後にNP組織の20mg以上の除去に伴ってIVD変性が生じた。スケールバー;1mm(全て)。
(B)組織学的グレードは、改変Boos分類を介して決定した。データは平均値±標準偏差。
NP:髄核
IVD:椎間板
H&E:ヘマトキシリン及びエオシン
SD:標準偏差
[図20]T2強調中間矢状画像を用いた、隣接する椎骨の椎間板高さに対する椎間板高さの測定結果を示す図。
BCとEFはそれぞれ前方及び後方椎間板高であり、ABとDEは頭側の隣接椎体高である。椎間板高さ(BC+EF)と椎体高さ(AB+DE)との比としてDHI値を計算し、無処置対照IVDのDHIに対する処置IVDのDHIの比として相対DHI値を計算した。
DHI:椎間板高さ指数
IVD:椎間板
本発明は、アルギン酸の1価金属塩と間葉系幹細胞(例えばヒト骨髄由来高純度間葉系幹細胞)とを含有する、椎間板再生用組成物に関する。本発明の一態様において、本発明は、低エンドトキシンアルギン酸の1価金属塩と間葉系幹細胞(例えばヒト骨髄由来高純度間葉系幹細胞)とを含有する、椎間板再生用組成物に関する。本発明の組成物は、間葉系幹細胞による髄核細胞の活性化及び/又は間葉系幹細胞の髄核細胞への分化を介して、椎間板の髄核再生を促進する。本発明の組成物は、対象の髄核部位に適用し、適用後に一部分を硬化するように用いられる。本発明のいくつかの態様では、本発明の組成物は、対象の髄核部位に適用し、表面の少なくとも一部分に架橋剤を接触させるように用いられる。
「低エンドトキシン」、「アルギン酸の1価金属塩」は後述する。
「椎間板」は、脊椎に連なる椎骨と椎骨との間にある円柱状の組織である。椎間板は、円板状の無血管組織であり、髄核を中心にして周りを線維輪が取り巻き、さらに上下に終板が配置された構造をしている。
「髄核」は、椎間板の中心に存在するゲル状の組織であり、髄核細胞と、主にプロテオグリカンとII型コラーゲンで構成される細胞外基質と、水を主として含有する。髄核には、自己修復能・再生能が著しく低いと考えられている。
「髄核部位」とは、髄核が存在する部位、髄核の変性若しくは縮小が生じている部位、又は、髄核の少なくとも一部を除去することで形成された髄核の欠損部をいい、髄核が存在する部位の周辺部も含む。
「適用」とは、本発明の組成物を椎間板の髄核部位の変性分、縮小分、除去分、欠損部などを埋めるのに十分な量で髄核部位に充填することを意味する。
「包埋する」とは、生体適合性材料、好ましくは、アルギン酸の1価金属塩の溶液に細胞を懸濁又は混合することをいう。
「一部分を硬化する」とは、後述の通りである。
「流動性を有する」とは、後述の通りである。
「椎間板変性および/または椎間板損傷」、「治療、予防または再発抑制」とは、後述の通りである。
「椎間板性疼痛」とは、椎間板に起因して生じる疼痛のことを意味する。
「腰痛」とは、椎間板の周囲に生じる疼痛であり、背部痛・臀部痛を含む。
「慢性腰痛」とは、腰痛の発症から12週以上続く腰痛を意味する。
本発明において、担体として使用される「アルギン酸の1価金属塩」は、アルギン酸の6位のカルボン酸の水素原子を、Na+やK+などの1価金属イオンとイオン交換することでつくられる水溶性の塩である。アルギン酸の1価金属塩としては、具体的には、アルギン酸ナトリウム、アルギン酸カリウムなどを挙げることができるが、特に、市販品により入手可能なアルギン酸ナトリウムが好ましい。アルギン酸の1価金属塩の溶液は、架橋剤と混合したときにゲルを形成する。
分子量測定にゲル浸透クロマトグラフィーを用いる場合の代表的な条件は、本明細書の実施例に記載のとおりである。カラムは、例えば、GMPW−XL×2+G2500PW−XL(7.8mm I.D.×300mm)を用いることができ、溶離液は、例えば、200mM硝酸ナトリウム水溶液とすることができ、分子量標準としてプルランを用いることができる。
本発明で用いるアルギン酸の1価金属塩は特に限定されるものではなく、例えば低エンドトキシンのアルギン酸の1価金属塩である。本発明の一態様では、細胞を包埋するための担体は、低エンドトキシンであることが好ましい。すなわち、本発明で用いるアルギン酸の1価金属塩は、低エンドトキシンのアルギン酸の1価金属塩であることが好ましい。低エンドトキシンとは、実質的に炎症、または発熱を惹起しない程度にまでエンドトキシンレベルが低いことをいう。より好ましくは、低エンドトキシン処理されたアルギン酸の1価金属塩であることが望ましい。
このように低エンドトキシン処理されたアルギン酸ナトリウムを、本明細書では「高純度アルギン酸ナトリウム」(UPAL)ともいう。
本発明の組成物は、アルギン酸の1価金属塩の溶液を用いて調製してもよい。アルギン酸の1価金属塩の溶液は、公知の方法またはそれに準じる方法により調製することができる。すなわち、本発明で用いられるアルギン酸の1価金属塩は、前述の褐藻類を用いて、酸法、カルシウム法など公知の方法により製造することができる。具体的には、例えば、これらの褐藻類から、炭酸ナトリウム水溶液などのアルカリ水溶液を用いて抽出した後、酸(例えば、塩酸、硫酸など)を添加することによってアルギン酸を得ることができ、アルギン酸のイオン交換によりアルギン酸の塩を得ることができる。前述のとおり、低エンドトキシン処理を行う。アルギン酸の1価金属塩の溶媒は、生体へ適用可能な溶媒であれば特に限定されないが、例えば、精製水、蒸留水、イオン交換水、ミリQ水、生理食塩水、リン酸緩衝生理食塩水(PBS)などが挙げられる。これらは、滅菌されていることが好ましく、低エンドトキシン処理されたものが好ましい。例えば、ミリQ水をろ過滅菌して用いることができる。
また、本発明の組成物を得るための操作は全てエンドトキシンレベル、および、細菌レベルの低い環境下で行うことが望ましい。例えば、操作はクリーンベンチで、滅菌器具を使用して行うことが好ましく、使用する器具を市販のエンドトキシン除去剤で処理してもよい。
本発明のいくつかの態様の組成物は、流動性のある液体状、すなわち、溶液状である。本発明の組成物は、髄核部位への適用時に流動性を有する。本発明の態様の1つでは、好ましくは、本発明の組成物は、組成物を20℃で1時間静置した後に、21Gの注射針で注入できる流動性を有する。この態様の本発明の組成物の見掛け粘度は、本発明の効果が得られれば、特に限定されないが、粘度が低すぎると適用した部位の周辺組織への密着性が弱くなる恐れがあるため、好ましくは10mPa・s以上、より好ましくは100mPa・s以上、さらに好ましくは200mPa・s以上、とりわけ好ましくは500mPa・s以上である。見掛け粘度が高すぎると取扱性が悪くなる恐れがあるため、好ましくは50000mPa・s以下、より好ましくは20000mPa・s以下であり、さらに好ましくは10000mPa・s以下である。見掛け粘度が20000mPa・s以下のときシリンジ等での適用がより容易に行える。しかし、見掛け粘度が20000mPa・s以上であっても加圧型や電動型の充填器具やその他の手段を用いて適用可能である。本発明の組成物の好ましい範囲は、10mPa・s~50000mPa・s、より好ましくは、100mPa・s~30000mPa・s、さらに好ましくは200mPa・s~20000mPa・s、またさらに好ましくは500mPa・s~20000mPa・s、とりわけ好ましくは700mPa・s~20000mPa・sである。別の好ましい態様では、500mPa・s~10000mPa・s、あるいは2000mPa・s~10000mPa・sであってもよい。本発明のいくつかの態様の組成物は、シリンジ等で対象に適用することもできる粘度である。
アルギン酸の1価金属塩の溶液の見掛け粘度は、溶液中のアルギン酸1価金属塩濃度が高い場合に粘度が高く、濃度が低い場合に粘度が低くなる。またアルギン酸1価金属塩の分子量が大きい場合に粘度が高く、分子量が小さい場合に粘度が低くなる。
前述のように、M/G比が主に海藻の種類によって決まることなどから、原料として用いられる褐藻類の種類はアルギン酸の1価金属塩の溶液の粘度に影響を及ぼす。本発明で用いられるアルギン酸としては、好ましくは、レッソニア属、マクロシスティス属、ラミナリア属、アスコフィラム属、ダービリア属の褐藻由来であり、より好ましくはレッソニア属の褐藻由来であり、特に好ましくはレッソニア・ニグレッセンズ(Lessonia nigrescens)由来である。
本発明において使用される間葉系幹細胞は、中胚葉性組織(間葉)に由来する体性幹細胞であり、骨や血管、心筋の再構築などの再生医療への応用が期待されている。
間葉系幹細胞は、骨髄、脂肪組織、胎盤組織、歯髄又は臍帯組織等の種々の組織から取得できる。その精製プロセスは、例えば以下のとおりである。
ヒト又は非ヒト哺乳動物(例えば、ウシ、サル、ネコ、マウス、ラット、モルモット、ハムスター、ブタ、イヌ、ウサギ、ヒツジ、およびウマヤギ、ウサギ等)から採取した少量の脂肪片を酵素処理して得られる細胞型の混合集団から、遠心分離によって浮遊性の脂肪細胞集団を分離し、培養液を満たした培養器の天井面に接触させた状態で静置したときに下床面に沈降して増殖する線維芽様細胞を継代培養によって増殖させる。
また、本発明においては、iPS細胞由来の間葉系幹細胞や、市販の間葉系幹細胞を使用することも可能である。本発明において、間葉系幹細胞は、好ましくは未分化状態である及び/又は誘導分化の処置なしに髄核に適用される。未分化状態とは、分化能を有する幹細胞が分化していない状態が維持されていることをいう。「分化誘導の処置なしに」とは、例えば、分化能を有する幹細胞を分化誘導培地を用いて特定の細胞に分化させる等の処置をしないことを指す。
(1)RECクローン
本発明者は、以前の研究により、LNGFR(CD271)が陽性の間葉系幹細胞(CD271+細胞)、又はLNGFR(CD271)及びThy−1(CD90)が共陽性の間葉系幹細胞(CD271+CD90+細胞)の中から、増殖が早い高速増殖性の細胞クローン(Rapidly Expanding Clone:REC)を単離することに成功した。
本発明においては、上記RECクローンのうち、分化能や増殖能においてばらつきの少ない細胞クローンを使用することができる。
(a)フローサイトメトリーの前方散乱光の変動係数が40%以下である。
(b)平均細胞サイズが20μm以下である。
本発明において、LNGFR(CD271)が陽性、又はLNGFR(CD271)及びThy−1(CD90)が共陽性の間葉系幹細胞を得るには、例えばWO2009/31678号に記載の方法に従うことができる。
その方法の概要は以下の通りである。
この細胞集団を得るための材料は特に限定されないが、例えば、骨髄、脂肪組織、臍帯血、末梢血(G−CSF投与後の末梢血を含む)等が挙げられる。なお、骨髄は、脊椎、胸骨、腸骨等の骨髄を用いればよい。また、細胞として、ES細胞およびiPS細胞を挙げることもできる。
上記の通り調製された細胞集団を用い、CD271+細胞又はCD271+CD90+細胞を選別する。
抗体は、CD271+細胞又はCD271+CD90+細胞を選別することが可能な、抗CD271抗体及び/又は抗CD90抗体である。選別にフローサイトメトリーを用いる場合には、FITC、PE、APC等の異なる蛍光色素で標識された抗CD271抗体、又は抗CD271抗体と抗CD90抗体を、適宜組み合わせて用いることにより、生細胞を短時間で選別することが可能になる。また、フローサイトメトリー以外にも、磁気ビーズを用いる方法やアフィニティー・クロマトグラフィーを用いる方法によって、CD271+CD90+細胞を選別することが可能である。
なお、これらの方法を用いる前に、予め、死細胞を染色する蛍光色素(例えば、PI)を細胞集団と反応させ、蛍光で染色された細胞を除去することにより、死細胞を除去してもよい。
次に、選別したLNGFR陽性細胞、又はLNGFR及びThy1共陽性細胞を単一細胞(クローン)培養し、増殖が速いロットを選択することで、増殖能・分化能・遊走能にすぐれた高純度ヒト間葉系幹細胞(REC:Rapidly Expanding Clone)を得る。
本発明においては、個々のロットのRECクローンについて、細胞の増殖能、脂肪分化能、REC特異的マーカー発現量、細胞サイズの均一性を調べ、それぞれの相関性を解析することにより、高純度でより細胞性能の高いRECを選別することが可能となった。
本発明においては、前方散乱光の変動係数(Coefficient of Variation:CV値)及び細胞の平均サイズを選別の指標とする。
前方散乱光(Forward Scatter)とは、レーザー光の軸に対して前方向の小さい角度で散乱する光である。前方散乱光は、細胞表面で生じるレーザー光の散乱光、回折光及び屈折光からなり、サンプルの大きさに関する情報が得られる。
本発明においては、上記CV値が40%以下、好ましくは35%以下のものを選別する。CV値が40%以下の細胞集団、より好ましくは35%以下の細胞集団は、大きさが均一な細胞により構成されている細胞集団である。好ましくは、CV値は30%以下、25%以下、又は20%以下である。 また、本発明により選別された細胞集団における細胞の平均サイズは、20μm以下である。好ましくは18μm以下であり、14μm~18μmの範囲の大きさである。
(a)フローサイトメトリーの前方散乱光の変動係数が40%以下である。
(b)平均細胞サイズが20μm以下である。
このようにして評価及び選別された細胞集団は、当該集団を構成する細胞クローンの数が限定されるものではなく、例えば1mlの溶液に0.8X107~1.2X107個程度の細胞を有する。
(a)フローサイトメトリーの前方散乱光の変動係数が35%以下である。
(b)平均細胞サイズが20μm以下である。
このようにして評価及び選別された細胞集団は、当該集団を構成する細胞クローンの数が限定されるものではなく、例えば1mlの溶液に0.8X107~1.2X107個程度の細胞を有する。
本発明の組成物は、例えば低エンドトキシンアルギン酸の1価金属塩、及び上記間葉系幹細胞を有効成分として含有することを特徴とする。本発明者らは、本発明の組成物を生体の髄核部位に充填した場合に、アルギン酸の1価金属塩自体が髄核組織の再生または治療効果を発揮することを初めて見出した。有効成分として含有するとは、低エンドトキシンアルギン酸の1価金属塩が患部に適用された際に、髄核組織の再生または治療効果を発揮できる量で含有されていればよく、少なくとも、組成物全体の0.1w/v%以上であることが好ましく、より好ましくは0.5w/v%以上、さらに好ましくは、1w/v%である。本発明の組成物中の好ましいアルギン酸の1価金属塩濃度は、好ましくは0.5w/v%~5w/v%、より好ましくは1w/v%~5w/v%であり、さらに好ましくは、1w/v%~3w/v%で、とりわけ好ましくは1.5w/v%~2.5w/v%である。また、別の態様では、本発明の組成物中のアルギン酸の1価金属塩濃度は、好ましくは、0.5w/w%~5w/w%、より好ましくは1w/w%~5w/w%であり、さらに好ましくは、1w/w%~3w/w%で、とりわけ好ましくは1.5w/w%~2.5w/w%であってもよい。
本発明のいくつかの態様では、必要に応じて、他の医薬活性成分や、慣用の安定化剤、乳化剤、浸透圧調整剤、緩衝剤、等張化剤、保存剤、無痛化剤、着色剤等、通常医薬に用いられる成分を本発明の組成物に含有させることもできる。
本発明の組成物は、髄核部位への適用後に一部分を硬化するように用いられる。
「一部分を硬化する」とは、流動性を有する本発明の組成物の一部分に架橋剤を接触させて、架橋剤と接触した組成物の全体ではなく一部分をゲル化し、固めることをいう。好ましくは、流動性を有する本発明の組成物の表面の少なくとも一部分に架橋剤を接触させることで本発明の組成物の一部分を硬化する。本発明では、使用する担体に適した架橋剤や硬化手段を選択できる。
本発明の組成物は、ヒト、またはヒト以外の生物、例えば、トリおよび非ヒト哺乳動物(例えば、ウシ、サル、ネコ、マウス、ラット、モルモット、ハムスター、ブタ、イヌ、ウサギ、ヒツジ、およびウマ)の椎間板の髄核部位に適用し、髄核の再生を促進するために用いられる。
本発明の組成物の粘度が高い場合には、シリンジで適用するのが困難になるため、加圧型や電動型などのシリンジを用いてもよい。シリンジなどを使用しなくても、例えば、へら、棒などで髄核部位の欠損部へ適用してもよい。シリンジで注入する場合、例えば、14G~27Gまたは14G~26Gの針を使用するのが好ましい。
本発明の組成物の適用量は、適用する対象の髄核の適用部位の容積に応じて決めれば良く、特に限定されないが、例えば、0.01ml~10ml、より好ましくは、0.1ml~5mlであり、さらに好ましくは0.2ml~3mlである。本発明の組成物を髄核欠損部に適用する場合は、髄核部位の欠損部容積を十分に満たすように注入されるのが望ましい。
アルギン酸は動物の体内に元来存在しない物質であるため、動物はアルギン酸を特異的に分解する酵素を保有していない。アルギン酸は動物体内においては、通常の加水分解により徐々に分解されるが、ヒアルロン酸等のポリマーに比べ体内の分解が緩やかであり、また髄核内には血管が存在しないため、髄核内に充填した場合、長期間の効果持続が期待できる。
本発明の組成物は、髄核部位へ適用することにより、椎間板組織全体及び髄核の変性変化を抑制し、再生を促進する効果を発揮する。そのため、本発明の組成物は、椎間板の髄核補填用組成物として好ましく用いられる。
椎間板変性および/または椎間板損傷は、例えば、椎間板ヘルニア、椎間板症、脊椎変性辷り症、化膿性椎間板炎、変形性脊椎症、脊柱管狭窄症、腰部脊柱管狭窄症、椎間板損傷、腰部脊柱管狭窄症に伴う椎間板ヘルニア(混合性腰部脊柱管狭窄症ともいう)からなる群から選択される少なくとも1種の状態または疾患である。椎間板変性及び/又は椎間板損傷が、腰痛を伴うものであってもよい。
本発明の組成物は、臨床症状や損傷部の大きさ・形状に合わせて、細胞を包埋する担体を硬化せず用いてもよく、そのような態様において、椎間板変性および/または椎間板損傷に伴う疼痛、特に慢性的な腰痛を抑制するために用いられる。
本発明は、前記本発明の組成物を用いる、椎間板変性および/または椎間板損傷の治療、予防または再発抑制のための方法を提供する。好ましくは、本発明の治療方法は、椎間板変性および/または椎間板損傷の治療、予防または再発抑制のための方法であって、低エンドトキシンアルギン酸の1価金属塩を含有し、流動性を有する組成物を、前記治療、予防または再発抑制を必要とする対象の椎間板の髄核部位に適用し、適用した前記組成物の一部分を硬化することを含む。
前記椎間板変性および/または椎間板損傷は、例えば、椎間板ヘルニア、椎間板症、脊椎変性辷り症、化膿性椎間板炎、変形性脊椎症、脊柱管狭窄症、椎間板損傷からなる群から選択される少なくとも1種の状態または疾患である。本発明のいくつかの態様の治療方法では、前記椎間板変性および/または椎間板損傷は、椎間板ヘルニアであり、特には、腰椎椎間板ヘルニアである。本発明のいくつかの態様の治療方法では、前記椎間板変性および/または椎間板損傷は、腰部脊柱管狭窄症に伴う椎間板ヘルニア(混合性腰部脊柱管狭窄症ともいう)である。椎間板変性及び/又は椎間板損傷が、慢性腰痛であってもよい。椎間板変性及び/又は椎間板損傷が、これらの状態または疾患の組み合わせであってもよい。
これらの方法は、間葉系幹細胞及び低エンドトキシンアルギン酸の1価金属塩を含有し、流動性を有する組成物を、椎間板変性抑制または髄核再生を必要とする対象の椎間板の髄核部位に適用し、適用した組成物の一部を硬化することを含む。前記の方法は、本発明の組成物を髄核部位へ適用する前に、髄核の少なくとも一部を除去する工程を含んでもよい。
本発明の使用は、椎間板変性および/または椎間板損傷の治療、予防または再発抑制のための組成物を製造するための低エンドトキシンアルギン酸の1価金属塩の使用であって、前記組成物が、対象の髄核部位に適用し、適用後に一部分を硬化するように用いられ、髄核部位への適用時に流動性を有する。
本発明の組成物は、発明者らが新たに確立したヒツジ重度椎間板変性モデルを用いて評価することができる。重度椎間板変性モデルは、(a)1回目の手術において、ヒツジ椎間板からヒツジ体重の0.00004%~0.00005%に相当する量の髄核組織を除去し、変性椎間板を作製すること、および(b)1回目の手術の4週間後に、(a)で作製した変性椎間板からさらにヒツジ体重の0.00014%~0.000175%に相当する量の髄核組織を除去することで、重度椎間板変性ヒツジモデルを作製することができる。
本発明の組成物は、下記の(a)~(d)の手順で評価することができる。(a)1回目の手術において、ヒツジ椎間板から20mgの髄核組織を除去し、変性椎間板を作製すること、(b)2回目の手術として、1回目の手術の4週間後に、(a)で作製した変性椎間板からさらに70mgの髄核組織を除去することで重度椎間板変性ヒツジモデルを作製すること(c)2回目の手術の後、生じた空隙に対象の組成物を投与すること、および(d)組成物の投与後に、変性モデルから採取した椎体及び椎間板に対してMRI、組織染色、免疫組織染色(IHC)からなる群から選択される少なくとも1つの評価方法にて、椎間板の再生を評価する。
以下、実施例により本発明をさらに具体的に説明する。但し、本発明の範囲はこれらの実施例により限定されるものではない。
[実施例1]
本実施例では、骨髄由来間葉系幹細胞(BMSC)と生体吸収性超精製アルギン酸塩(低エンドトキシン高純度アルギン酸塩、UPALともいう)ゲルとを含む組成物を用いて、椎間板(IVD)を切除した後の変性IVDに対するBMSCの移植の再生効果を試験した。
1.材料及び方法
1.1.動物実験
すべての動物に対する処置は、北海道大学の動物管理使用委員会(承認番号:13−0051)によって承認され、承認されたガイドラインに従って実施した。オスの日本白色ウサギ(20週齢、3.2~3.5kg)は、三共ラボサービス株式会社(東京、日本)から入手した。
髄核(NP)試料は、4匹のウサギを、静脈内ペントバルビタール過剰投与を介して安楽死させた後、腰部IVD(L1/2からL5/6まで;全部で20のIVD)から得た。NPCを髄核(NP)組織から単離し、既報の方法で培養した[2,5,7,8]。具体的には、無菌条件下で、ゼラチン状NP組織を、マイクロ鉗子を用いて線維輪(AF)から分離した。組織標本は、10%ウシ胎仔血清(Nichirei Bioscience,Tokyo,Japan)、1%ペニシリン/ストレプトマイシン、及び1.25mg/mlファンギゾン(Life Technologies,Waltham,MA,USA)をダルベッコ改変イーグル培地(Sigma−Aldrich,St.Louis,MO,USA)を含有する培養培地に入れた。外因性成長因子は使用しなかった。試料を、0.25%コラゲナーゼを補充した培地(和光純薬工業、大阪、日本)に再懸濁し、振盪インキュベーター中、37℃、20% O2及び5% CO2で4時間インキュベートし、酵素消化により単離した。NP組織から分離した細胞を培養皿中で増殖させ、加湿大気中、20% O2及び5% CO2を含む37℃で上記培地で培養した。培地は週2回交換し、NPCは継代2で使用した。
ウサギ同種BMSCとして、Cyagen(Santa Clara,CA,USA;カタログ番号:RBXMX−01,001、ロット番号:151114I31)から購入したOriCellTMウサギ間葉系幹細胞を使用した。これらの細胞は、特徴、解凍後の生存性、細胞周期、未分化状態の検証、並びに骨形成、軟骨形成、及び脂肪形成系統に沿った多能性分化能について試験されている。BMSCは製造業者の説明書に従って培養し、培地は週2回交換し、BMSCを継代2で使用した。
本実施例では、UPALゲル(Mochida Pharmaceutical Co.Ltd.,Tokyo,Japan)を3D培養のためのアルギン酸塩足場として使用した[2]。UPALゲルの精製プロセスは、既報の通りである[2]。具体的には、海藻中のアルギン酸塩を、清澄化手順[2]によって水溶性アルギン酸ナトリウムに変換することによって抽出した。このアルギン酸塩溶液は、高粘性であったため大量の水で希釈した[2]。次に、抽出物を濾過して、繊維状残渣からアルギン酸ナトリウム溶液を分離した[2]。高品質のアルギン酸を単離するため、この溶液に酸を添加した[2]。
回収したNPC及びBMSCを1mlのTRIzol(TM)(Invitrogen,Carlsbad,CA,USA)に溶解し、RNeasy Miniキット(Qiagen,Valencia,CA,USA)を用いてサンプルから全RNAを抽出した。TaqManTM遺伝子表現アッセイとカスタムTaqManTM遺伝子表現アッセイ(表1)(Applied Biosystems,Waltham,MA,USA)を用いて、リアルタイムqRT−PCR解析を行った。各試料についてサイクル閾値(Ct)を得、2−ΔCt方法を用いて、ハウスキーピング遺伝子GAPDHのCt値を基準とした各標的遺伝子の相対的mRNA発現を計算した[1]。
In vivo試験には合計48匹のウサギを用いた。サンプルサイズは、採用した2つの時点の各々について、以前の報告[2,7,8]に基づいて決定した。48匹のウサギのうち32匹を無作為に選択して、IVD変性の定性分析(磁気共鳴画像(MRI)、組織学、免疫組織化学(IHC))を行い、計80のIVDを、無傷対照(Intact control)群、穿刺(Puncture)群(変性作成のための穿刺のみ)、椎間板切除(Discectomy)群(変性IVDに対して空洞を作るための部分的椎間板切除)、ゲル群(変性IVDに対する部分的椎間板切除及びUPALゲル埋植)、BMSC+ゲル群(変性IVDに対する部分椎間板切除及びBMSCとUPALゲルの埋植)に、無作為に割り付けた(各群8のIVD)。
In vivo実験において、移植細胞としてin vitro実験と同様のOriCellTMウサギ間葉系幹細胞を継代2で用いた。BMSCは、移植前にCFDA−SEで標識し、2% UPAL液中に包埋し、1×106細胞/mlの最終細胞濃度に調製した[14]。
20週齢ウサギは、高齢のヒト集団の状態を模倣するのに十分に老化しておらず、均一な老化を有する高齢ウサギを得ることは困難である。そこで本実施例では、変性IVDを得るためにウサギ椎間板の線維輪(AF)穿刺モデルを使用した[8,15,16]。ケタミン(10mg/kg)及びキシラジン(3mg/kg)の静脈内注射により全身麻酔を行い、自発換気でセボフルラン(2~3%)と混合したO2及び空気(3.01/分)で麻酔維持した。IVD変性は、L2/3及びL4/5 IVDで18ゲージ針を使用するAF穿刺によって作成した。L3/4 IVDは、対照として無処置とした。
埋植後4週目と12週目に、CFDA−SE蛍光標識[18,19]に基づいて埋植BMSCの生存確認した。IVD(無傷対照群及びBMSC+ゲル群)を半分に水平切断し、液体窒素中で凍結し、5mmスライスに切片化し、次いで対比染色として4’,6−ジアミジノ−2−フェニルインドール(DAPI;Invitrogen;P36935)で染色した。特に、DAPI陽性細胞は生存対死滅のNPC及びBMSCを特定できないため、移植BMSCの生存率を定量することはできなかった。
術後4週目と12週目に、7.0−T MRスキャナー(Varian Unity Inova;Varian Medical Systems,Palo Alto,CA,USA)を用いてIVDのT2強調正中矢状断像を得た[2,8,16]。Pfirrmann分類(グレード5は重度変性として分類した)を用いて、IVD変性をグレード化した[20]。MRIindexを算出するために、分析ソフトウェアバージョン12.0(AnalyzeDirect,Overland Park,KS,USA)を用いて定量分析も行った。MRIindex(NP面積と平均信号強度との積)をNPの変性の定量化に適用し、定量データは、未処置対照IVDで得られたMRIindex(相対MRIindex)に対するパーセンテージとして表した[2,8,16]。
MRI分析後、各IVDを組織学的染色のために処理した。プロテオグリカン発現を評価するため、正中矢状断切片(5mm厚)をヘマトキシリンとエオシン(H&E)並びにサフラニンO−ファストグリーンによって染色した[8]。IVDの半定量分析を行い、0(正常)から5(高度変性)に等級分けした[21,22]。具体的には、この組織学的尺度はAF構造の形態学的変化に焦点を当てている。
術後4週目及び12週目にI型及びII型コラーゲンを検出するために免疫組織化学(IHC)染色を行い[8]、術後1日目、7日目、28日目にHIF−1α、GLUT−1及びBrachyuryを検出した。I型及びII型コラーゲン染色には、I型コラーゲン(Sigma−Aldrich;C2456,RRID:AB_476836)及びII型コラーゲン(Kyowa Pharma Chemical,Toyama,Japan;F−57)に対するマウスモノクローナル抗体を適用した。染色は、対比染色として3,3’−ジアミノベンジジン塩酸塩(Dako)及びMayer’sヘマトキシリン(Merck,Darmstadt,Germany)を用いて展開した。HIF−1α、GLUT−1、及びBrachyury染色については、HIF−1αに対するDyLight 550結合ウサギポリクローナル抗体(Novus Biologicals,Centennial,CO,USA;NB100−479R,RRID:AB_1642267)、GLUT−1に対するPE結合ウサギポリクローナル抗体(LS Bio,Seattle,WA,USA;LS−A109342−100)、及びBrachyuryに対する非結合ウサギポリクローナル抗体(LS Bio;LS−C31179−100,RRID:AB_911118)を適用した。さらに、Alexa Fluor 594結合ヤギ抗ウサギポリクローナル抗体(Invitrogen;A32740)をBrachyuryの二次抗体として使用した。染色は、対比染色としてDAPIを用いて展開した。
全てのデータは、平均±標準誤差(SE)として示される。多群比較についてはOne−way分散分析(ANOVA)及びTukey−Kramer事後検定を行った。2群比較についてはPaired t検定を行った。すべての統計計算は、有意閾値p<0.05で、JMP Pro−version 14.0統計ソフトウェア(SAS Institute,Cary,NC,USA)を用いて行った。
2.1.NPC及びBMSC共培養は、BMSCのNPCへの分化並びに成長因子及びECMの産生を促進する
各細胞型に対するNPC及びBMSC共培養の効果を調べるために、非標識NPC及びCFDA−SE標識BMSCを、3D培養のためにUPALゲル中に包埋した。本発明者は、共培養群において、細胞ソーターを用いて両方の細胞型を収集した。リン酸緩衝食塩水/細胞懸濁液分析は、前方散乱及び側方散乱を用いて行った。P1ゲートは2Dドットプロット(図1a)で描き、死細胞及び残渣を除外した。非標識NPC及びCFDA−SE標識BMSCを、蛍光対側方散乱ドットプロットにおいて異なるゲートを用いて選別した(図1b)。従って、P2ゲートを非標識細胞の上に設定し、P3ゲートをCFDA−SE標識細胞の上に設定し、交差汚染を回避するために2つのゲートの間に間隙を設けた[1]。ゲル溶解及び細胞選別に続いて、(a)NPC対照(day0)、(b)NPC単培養、(c)NPC共培養、(d)BMSC対照(day0)、(e)BMSC単培養、及び(f)BMSC共培養の6つのタイプの細胞を得た。BMSC分化の解析については、6種類の細胞において、NPCマーカーとしてHIF−1α、GLUT−1及びBrachyuryの遺伝子発現、成長因子としてCDMP−1、TGF−β及びIGF−1の遺伝子発現、そして細胞外マトリックス(ECM)としてタイプIIコラーゲン及びアグリカンの遺伝子発現を、qRT−PCRを用いて評価した。
NPC共培養におけるGLUT−1の発現は、NPC対照(p<0.0001、Tukey−Kramer試験)及び単培養(p<0.0001、Tukey−Kramer試験)と比較して有意な増加を示し、NPC単培養では、NPC対照(p<0.0001、Tukey−Kramer試験)と比較して有意な増加を示した。BMSC共培養では、BMSC対照(p=0.0189、Tukey−Kramer検定)と比較してGLUT−1発現の有意な増加を示した(図1d)。これに対し、Brachyuryの遺伝子発現は、3種類のNPC間で統計的有意差を示さなかった。さらに、Brachyuryの遺伝子発現は、BMSC共培養においてのみ観察され、BMSC対照及び単培養のいずれにおいても観察されなかった(図1e)。
In vivo試験において、BMSC+ゲル群ではCFDA−SE標識BMSCが観察されたが、術後4週目と12週目では無傷対照群では観察されなかった(図2)。ヒトBMSC群もBMSC+ゲル群(ウサギBMSC)と同様の所見を示し、移植されたBMSCが移植後12週目にIVDで生存したことが確認された。切開すると、ゲルの押し出しは認められなかった。
治療されたIVDにおける変性変化をMRIによって定性的に分析し、T2強調正中矢状断画像を捕捉した(図3a)。BMSC+ゲル群におけるPfirrmannグレードは、4週目に椎間板切除群より有意に低く(p=0.003、Tukey−Kramer検定)、12週目に穿刺群と椎間板切除群で有意に低かった(p=0.0009,p<0.0001,Tukey−Kramer検定)。さらに、ゲル群の変性度も12週目に椎間板切除群より有意に低かった(p=0.0028、Tukey−Kramer検定)(図3b)。ゲル群とBMSC+ゲル群との間に有意差は観察されなかった。BMSC+ゲル群のMRIindexは、4週で椎間板切除群(p=0.0085、Tukey−Kramer試験)及び12週での穿刺、椎間板切除及びゲル群(p=0.0002,p<0.0001,p=0~0248,Tukey−Kramer試験)よりも有意に高かった。また、12週後ではゲル群のindexが椎間板切除群よりも有意に高かった(p=0.0092、Tukey−Kramer検定)(図3c)。特に、ウサギBMSC群とヒトBMSC群との間では、Pfirrmannグレード又はMRIindexのいずれにおいても有意差は観察されなかった。
次に、本発明者はIVDにおけるECM産生を評価した。特に、II型コラーゲンはIVD機能に必要な必須成分であるが、IVD変性過程ではI型コラーゲン合成の増加が観察される[23]。II型コラーゲン陽性細胞の割合は、BMSC+ゲル群では4週目において穿刺群及び椎間板切除群に比べ有意に高く(p=0.0001,p<0.0001,Tukey−Kramer検定)、12週目において穿刺群、椎間板切除群及びゲル群よりも有意に高かった(p<0.0001,p<0.0001,p<0.0001,Tukey−Kramer検定)。また、ゲル群は、4週目において椎間板切除群と比較して有意に高い割合を示し(p<0.0001,Tukey−Kramer検定)、12週目において穿刺群及び椎間板切除群と比較して有意に高かった(p=0.0231,p<0.0001,Tukey−Kramer検定)(図5)。
最後に、HIF−1α、GLUT−1及びBrachyury陽性細胞を経時的に観察し、in vivoにおいて移植BMSCがNPCに分化するメカニズムを検討した(図6a−c)。無傷対照群では、ほとんどすべての細胞がすべての時点で3つのNPCマーカーについて陽性であった。BMSC+ゲル群ではHIF−1α、GLUT−1及びBrachyury陽性細胞が1日目に低レベルで観察されたが、陽性細胞数は経時的に増加した。全細胞数に対する3種類のNPCマーカー陽性細胞の割合は、1日目及び7日目と比較して28日目で有意に高かった(p<0.0001,p<0.0001,Studentのt検定)。これに対し、椎間板切除群では、約20%の細胞が全ての時点で陽性であった(図6d−f)。同様に、CFDA−SE陽性細胞数(移植BMSCを表す)に対する3種類のNPCマーカー陽性細胞の割合は、1日目及び7日目と比較して28日目で有意に高かった(p<0.0001、p<0.0001、Student’s t−test)(図6g−i)。
IVDの再生能は著しく低いため、椎間板切除により生じた欠損は組織修復が不十分であり、さらなるIVD変性につながる可能性がある[2]。本実施例において、UPALゲル単独でも椎間板切除後と比較してIVD変性を抑制したが、BMSCとUPALゲルとを併用すると、IVD再生を誘導するより強力な効果を発揮した。さらに、ヒトBMSCとウサギBMSCでは、IVD再生は両群間で同等に観察された。いくつかの以前の研究においても、変性IVDにおける種々のBMSCの再生能力が実証されている[18、24、25]。さらに、UPALゲルは、高い生体適合性及び十分な生体力学的特性を示し内因性修復治療戦略を支持するため[2]、変性IVD部位にBMSCを移植した後のIVD再生及びさらなる変性予防のための最適な環境を提供する。
1)埋植されたBMSCは、UPALゲル中への包埋を介して、椎間板外へ漏れることなくIVDの空洞内に局在化することができる。
2)埋植されたBMSCは、成長因子及びECMを産生し、既存のNPCの活性化をもたらす。
3)活性化したNPCはまた、成長因子及びECMの産生を増加させる。
4)その結果、埋植されたBMSCはNPCに分化する。
5)さらに、BMSC及び既存のNPCは互いに活性化し、IVD再生を生じる(図7)。
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[実施例2]
1.材料と方法
1.1.ヒトの健常椎間板髄核細胞(NPC)
ヒトの健常椎間板髄核細胞(NPC)は、北海道大学病院における思春期特発性側弯症の若年患者(15.3±3.3歳)の脊椎前方固定術の際に得られた細胞を用いた。
(1)フローサイトメトリーによる前方散乱光(FSC)のCV値測定
公知方法(WO2016/17795)により予め調製しておいた複数のRECクローンを、フローサイトメトリーによる前方散乱光のCV値測定に用いた。
フローサイトメトリーにおけるFSCは細胞の表面積又は大きさに比例する。本実施例では、FSCのCV値を指標として細胞サイズのばらつきを評価した。
(a)個々のRECクローンに対してPI染色を行い、PI陰性の生細胞集団にゲートを設定して、死細胞を解析の対象から除外した。
(b)PI陰性の生細胞集団をFSC/SSCのサイトグラムで展開し、メインの細胞集団にゲート(P1)を設定して、ゴミやノイズを解析の対象から除外した。
(c)P1ゲート内の細胞集団をFSCのヒストグラムで展開し、マーカー(M1)を設定して、CV値を計測した。
細胞増殖能の評価のため、1x105個のREC細胞を100mm培養皿に播種して、37℃、5%CO2環境下で5日間培養し、その後、セルカウンターを用いて細胞数及び平均細胞サイズを計測した。
培養液は、FBS、basic FGF、Hepes、Penicillin−Streptomycinを添加したDMEM培地(富士フイルム和光純薬社)を用いた。
また、脂肪分化能の評価のため、5x104個のREC細胞を24穴プレートに播種して、37℃、5%CO2環境下で2日間培養し、その後、脂肪分化誘導培地に置換して、さらに14日間培養した。培養14日後にオイルレッド0染色を行い、画像解析により脂肪滴面積を算出した。なお、脂肪分化誘導培地は、上記培養培地にDexamethasone、Indomethacin、IBMXを添加した培地を用いた。
複数のRECクローンについて、FSCのCV値、細胞増殖率(増殖能)、平均細胞サイズ、脂肪分化能を調べたところ、FSCのCV値が低いRECクローン(CV=35%以下)は、増殖能、分化能が共に高く、平均細胞サイズが小さいことがわかった(平均細胞サイズ:20μm以下)(図8)。
CV値30%~35%のクローンの平均増殖率は6.4であり、CV値25%~30%のクローンの平均増殖率は9.2、CV値25%以下のクローンの平均増殖率は15.1であった。また平均細胞サイズが18μm~20μmのクローンの平均増殖率は7.0であり、平均細胞サイズが16μm~18μmのクローンの平均増殖率は12.7、平均細胞サイズが16μm以下のクローンの平均増殖率は21.3であった。
以下、本実施例ではFSCのCV値が低いRECクローン(CV=25%以下)を使用した。
NPCと蛍光標識された高純度間葉系細胞(REC)を各単独群と混合群に群分けし、超高純度アルギン酸(UPAL:持田製薬提供)の3次元ゲル中で共培養又は単独培養し、培養前(day0)と培養7日後に、3次元ゲルを溶解後、セルソーターで下記の細胞に分離した。
(a)NPC対照群(day0)
(b)NPC単独培養群
(c)NPC共培養群
(d)REC対照群(day0)
(e)REC単独培養群
(f)REC共培養群
髄核細胞のマーカー
HIF−1α
GLUT−1
Brachyury
成長因子
CDMP−1
TGF−β
IGF−1
細胞外基質
TypeII collagen
aggrecan
遺伝子発現量の測定法は、実施例1に準じて行った。
RECと髄核細胞を共培養すると、単独培養した場合と比較して分化指標(HIF−1α、GLUT−1,Brachrury)が髄核細胞、間葉系幹細胞の双方で上昇した(図9)。
成長因子(CDMP−1,TGFβ、IGF−1)、及び細胞外基質(TypeII collagen,Aggrecan)についても同様であった。
髄核細胞と高純度間葉系幹細胞は相互作用して活性化し、間葉系幹細胞は髄核細胞へと分化していくことが示唆される。
[実施例3]
1.材料と方法
7頭のヒツジ(40−50kg)はGLP準拠で施術した。
椎間板L1/L2(第1腰椎と第2腰椎の間の椎間板)、椎間板L2/L3(第2腰椎と第3腰椎の間の椎間板)、椎間板L3/L4(第3腰椎と第4腰椎の間の椎間板)、および椎間板L4/L5(第4腰椎と第5腰椎の間の椎間板)を以下の4群に分けた。
対照群(n=6):無処置群
Discectomy群(n=6):髄核組織の摘出のみ
ゲル群(n=8):髄核組織の摘出後、アルギン酸ゲルを埋植
REC+ゲル群(n=8):髄核組織の摘出後、RECとアルギン酸ゲルを埋植
最初の髄核組織摘出から4週間後、さらに70mgの髄核組織を麻酔下に摘出し、椎間板内の空隙に、最終細胞濃度1×106cell/ml(100μl)でUPALに懸濁されたREC(実施例2、1.2項で調製したREC)を注入した。
埋植手術の4週間後、ヒツジを安楽死させた。
処置椎間板の変性定量評価目的に3テスラの磁気共鳴画像法(MRI)を用いて分析し、その後、椎間板はH&E染色およびサフラニン−0染色による組織学的評価、また免疫組織化学的評価によるType II collagen発現の評価を行った。
すべてのデータは平均±標準誤差で示し、各群間の差は一元配置分散分析(ANOVA)とTukey−Kramer post hoc testで検定した。
ヒツジ椎間板の患部にゲルおよびREC+ゲルを埋植後、4週間後にMRIで椎間板変性を評価した結果、Pfirrmann分類では無治療群(Discectomy群)に比較してゲルおよびREC+ゲル群で有意に低く、またMRI indexでは無治療群(Discectomy群)に比較してREC+ゲル群で有意に高かった(図10)。組織染色による組織学的変性スコア(Boos分類)は無治療群(Discectomy群)に比較してゲルおよびREC+ゲル群で有意に低く、またゲル群とREC+ゲル群の比較ではREC+ゲル群が有意に低かった(図11)。免疫組織化学分析では、II型コラーゲン陽性細胞の割合が無治療群(Discectomy群)およびゲル群よりもREC+ゲル群で有意に高かった(図12)。
[実施例4]
1.1.試験デザイン
本実施例における試験デザインは、以下の内容で行った。
(i)in vitroでのヒトNPCとRECの3D共培養を介したIVD再生のメカニズムの検討
(ii)REC及び市販ヒトBMSCとNPCとの3D共培養を介した、NPCに対するRECと市販ヒトBMSCの効果の比較
(iii)UPALとREC+UPALゲルの力学特性の測定
(iv)in vivoでの椎間板切除後の重度変性IVDの再生能力の評価
(v)移植IVDにおける腫瘍形成の評価
北海道大学大学院医学研究科の倫理委員会により、健康なヒトIVDの使用が承認された。動物実験手順は、北海道大学及びHamri株式会社(茨城県)の動物実験委員会(Institutional Animal Care and Use Committee)によって承認され、これらの委員会によって推奨されるガイドラインに従って実施した。
無処置対照群(4週間、n=6;24週間、n=6):無処置群
Discectomy群(4週間、n=6;24週間、n=6):髄核組織の摘出のみ、椎間板切除群ともいう
ゲル群(4週間、n=8;24週間、n=8):髄核組織の摘出後、アルギン酸ゲルを埋植
REC+ゲル群(4週間、n=8;24週間、n=8):髄核組織の摘出後、RECとアルギン酸ゲルを埋植
定量データのためのサンプルサイズは、Tukey−Kramer検定を用いて、αレベル0.05及び0.8のパワーでパワー分析によって決定した。統計解析はJMP Pro version 14.0ソフトウェア(SAS Institute,Cary,NC,USA)を用いて行い、値は、p<0.05である場合に有意であるとみなした。全てのデータは、平均±SD値として示される。多群比較のために一元配置分散分析(ANOVA)とTukey−Kramer事後検定を行った。2群間比較にはStudentのt検定又はMann−WhitneyのU検定及びWelchの検定を用いた。本発明者らは試料の無作為化を行い、試験される試料に対して盲検化した。
RECの調製
本試験では、株式会社ジャパン・ティッシュ・エンジニアリングから提供された凍結GMP準拠RECを使用した。REC及び市販のヒトBMSCの比較分析において、RECの3つのクローン、すなわちREC‐02プロトタイプ#003‐P6‐191220、REC‐02 HLWF‐1‐P6‐210114、及びREC‐02 QRKF‐1‐P6‐210210を継代6で調製した。細胞は、3D培養の前に37℃の温浴中で解凍した。
市販のヒトBMSC(hMSC−BM;PromoCell,Heidelberg,Germany;C−12974、ロット番号:412Z022.4)は、既報に従い入手した(2)。骨形成、軟骨形成及び脂肪生成系統に属するこれらの細胞の特性、解凍後の生存性、細胞周期、未分化状態、多分化能を試験した。BMSCは、完全培養培地、すなわち、20% HyCloneウシ胎仔血清(FBS;Cytiva,Tokyo,Japan)、1%ペニシリン/ストレプトマイシン、1.25mg/mLファンギゾン(Life Technologies,Thermo Fisher Scientific,Waltham,MA,USA)、1% HEPES(Life Technologies,Thermo Fisher Scientific)、及び0.1% bFGF(Kaken Pharmaceutical Co.,Ltd.,Tokyo,Japan)を補充した、L−グルタミン及びフェノールレッド(DMEM;FUJIFILM Wako Pure Chemical Corporation,Osaka,Japan)を含有するダルベッコ改変イーグル培地(低グルコースレベル;2mg/mL)を用い、製造業者の指示書に従って培養した。培地は週に2回交換し、第4継代プロセスからのBMSCを使用した(合計6継代はRECと同様であった)。
UPALゲル(Mochida Pharmaceutical Co.Ltd.,Tokyo,Japan)は、3D培養のためのアルギン酸塩足場として使用した(2,3)。
リン酸緩衝食塩水(PBS;FUJIFILM Wako Pure Chemical Industries)に溶解した2%(w/v)UPAL溶液を調製し、CaCl2溶液(102mM)を用いてゲル化した。REC又は市販のBMSCを、1×106細胞/mL(2、31、49)の最終細胞濃度でUPAL溶液と混合した。細胞−UPAL混合溶液を、ゲル化のために22ゲージ針を使用して、102mM CaCl2溶液中にピペットで入れた。ビーズ状に得られた2種類のゲルを、培地とともに加湿環境(20% O2、5% CO2、37℃)中で7日間培養した。培地は3日毎に交換した。3D培養の7日後に細胞を回収するために、既報の通り(2、3、35)、55mMクエン酸ナトリウムを使用して溶解し、遠心分離(4℃で10分間110×g)した後、ゲルビーズから細胞を回収した。さらに、前述のように、正常条件下で7日間平面培養(2D培養)として調製したREC及び市販のBMSCを対照群として使用した(すなわち、以下の4群を実験群として設定した:1)2D培養REC、2)3D培養REC、3)2D培養BMSC、及び4)3D培養BMSC)。
2D又は3D培養の7日後、得られた4種類の細胞を二次培養のために再度播種した。細胞を集密状態となった段階で回収し、細胞数が倍加するのに必要な時間として定義される倍加時間(Td)に基づいて細胞増殖能を評価した。Tdは、以下の式を用いて計算した。
Td=(t2−t1)×ln(2)/ln(N2/N1)
(ここで、N2及びN1は時間t2及びt1におけるセル数である。)
PBS(2%のFBSを含む)を4つのタイプの細胞の各々に添加して、1×106細胞/mLの最終密度で細胞懸濁液を調製した。次いで、細胞を抗CD90抗体(PE抗ヒトCD90(Thy1)、BioLegend、San Diego、CA、USA)及び抗CD45抗体(FITC抗ヒトCD45、Beckman Coulter、Brea、CA、USA)で染色した。また、陰性対照としてマウスIgG1抗体(マウスIgG1−PE,BioLegend;mouse IgG1−FITC,Beckman Coulter)を用いて染色を行った。ヨウ化プロピジウム(PI)を用いて死細胞を検出した。フローサイトメトリー分析は、CytoFLEX System(Beckman Coulter)を使用して行い、細胞生存率ならびに各細胞表面抗原の均一性及び陽性を評価した。データの分析にはFlowJoソフトウェア(Becton Dickinson,Franklin Lakes,NJ,USA)を使用した。
北海道大学病院で思春期特発性側弯症のため脊椎前方固定術を受けた9人の患者(平均年齢±SD、15.3±3.3歳)からヒトNPサンプルを得た。本研究に参加した参加者からインフォームドコンセントを取得し、同意書を作成し、保存した。サンプルは、外科手術の際に取得した。すべてのIVDはMRIを介して手術前に分析し、Pfirrmann分類(36)を用いて変性変化について評価した。全てのIVDはグレード1に分類され、全てのサンプルが非変性IVDであることが示唆された。
上記REC(REC−02_プロトタイプ#003−P6−191220)及び市販のヒトBMSCもまた、3D共培養に使用した。2つのタイプの細胞を、上記2D培養と同じ方法により製造業者の指示に従って培養した。培地は週に2回交換した。第2継代由来の細胞(REC、合計8継代;BMSC、合計4継代)の両方を使用した。
本発明者らは2% UPAL溶液を調製し、既報の通り(2、3)、ゲル化のためにCaCl2溶液(102mM)を使用した。3D培養の前に、REC及びBMSCを、製造業者のマニュアル(2、15、29)を参照して、20mM CFDA−SE(CFDA−SE細胞増殖アッセイキット;BIO RAD,Hercules,CA,USA)で蛍光標識した。その後、標識細胞及び非標識NPCをUPAL溶液と同じ比率(各細胞1×106細胞/mL)(2、31、49)で混合し、最終細胞濃度を2×106細胞/mLとした。細胞−UPAL混合溶液は、22ゲージ針を用いて102mMのCaCl2溶液に入れてゲル化させた。得られたゲルを、低酸素条件(5% O2及び5% CO2)(2、49)下で7日間培養した。さらに、3つのタイプの細胞を、1×106細胞/mLの濃度でそれぞれ別々にUPAL溶液中に混合した。細胞濃度は、既報の結果(2)に基づいて選択した。ゲル化後、ゲルビーズを低酸素条件下で上記と同じ方法で培養し、以下の実験群を使用した(1群あたりn=4)。
(a)単培養NPC
(b)単培養BMSC
(c)単培養REC
(d)共培養NPC+BMSC
(e)共培養NPC+REC。
回収した10種類の細胞タイプ(対照NPC、単培養NPC、市販のBMSCと共培養したNPC、RECと共培養したNPC、対照BMSC、単培養BMSC、共培養BMSC、対照REC、単培養REC、及び共培養REC)を、1mLのTRIzol(登録商標)(Invitrogen,Thermo Fisher Scientific)に溶解し、RNeasy Miniキット(Qiagen,Valencia,CA,USA)を用いて試料から全RNAを抽出した。リアルタイムqRT−PCRは、TaqMan(登録商標)遺伝子発現アッセイ(Applied Biosystems,Thermo Fisher Scientific)(表3)を用いて行った。各サンプルについてサイクル閾値(Ct)値を得た。さらに、各標的遺伝子、NPCマーカー、増殖因子、及びECM成分の相対的mRNA発現レベルを2−ΔCt法(2)を介して計算した。発現レベルは、ハウスキーピング遺伝子GAPDH(2,31)の発現量により標準化した。
UPAL及びREC−UPALゲルの力学特性を、非拘束圧縮試験を用いて評価した。直径4.5mm、厚さ2mmの2種類の円盤状ゲルを作製した(図14(a))。サンプルを引張−圧縮機械試験機(Autograph AG−X;Shimadzu Corporation,Kyoto,Japan)に入れ、ゲルが潰れるまで、100 Nロードセルを用いて0.5mm/分の一定速度で圧縮した(図14、B及びC)(3、15)。ヤング率は、得られた応力−歪曲線(図14D)に基づき、圧縮値10~20%(n=4ゲル/群)の間の近似直線を用いて計算した(図14E)(3、15、32、33)。
本実施例では、ヒツジモデルを含む全ての手順を、医薬品GLP適合実験室(Hamri Co.,Ltd.)で行った(3)。14頭の雄、サフォークヒツジ(2歳、体重40~60kg)を用い、IVD変性の定性分析を行った(3)。全部で56のIVDを無作為に無処置対照(4週、n=6;24週、n=6)、椎間板切除(4週、n=6;24週、n=6)、ゲル(4週、n=8;24週、n=8)、及びREC+ゲル(4週、n=8;24週、n=8)群に割り当てた。まず、NP組織除去手術を行い、重度に変性したIVDモデルを作成した。ケタミン(0.2mg/kg)及びキシラジン(20mg/kg)の4:1混合物を0.5mL/kgの速度で筋肉内注射することによって麻酔誘導を行い、吸入麻酔(イソフルラン)によって麻酔の維持を達成した。手術は右側方後腹膜アプローチで行い、L1からL5までの椎体とIVDを露出した。固形海綿質スクリュー(ZIMMER BIOMET,Warsaw,IN,USA)を、椎骨目印としてL2椎体に挿入した。椎間板切除群、ゲル群、REC+ゲル群ではAF切開(5×3mm)後にNP組織20mgを摘出し、IVD変性を惹起した(図15,A,B)(3,8)。
初回手術から4週間後、同じアプローチを用いて椎体とIVDを露出した。本発明者らはさらに、上記のように、AF切開後にIVD空洞を作製するために、3つの処置群から得られた変性IVDから70mgのNP組織を抽出した(図15C)。椎間板切除後、椎間板内の空隙にゲル群には2% UPAL溶液110~120μLを移植し、REC+ゲル群には、RECとUPAL溶液との混合物(最終濃度、1×106細胞/mL)110~120μLを移植した(図15D)(2)。直ちに102mMのCaCl2溶液を混合物の表面に注入し、5分後にゲル化を確認した。埋植4週及び24週後に、ペントバルビタールを用いてヒツジを安楽死させ、腰部脊柱を一塊として摘出した(3)。
3.0−T MRスキャナ(MAGNETOM Prisma;Siemens,Munich,Germany)を用いて、T2強調正中矢状切片画像を得た。処置されたIVDのシグナル変化を評価するために、本発明者らは、5つのグレード(1:正常~5:高度に変性)を含むPfirrmann分類(36)を使用して、IVD変性の程度をスコア化した。さらに、Analyze 14.0ソフトウェア(AnalyzeDirect,Overland Park,KS,USA)を使用して、MRI指数値(NPの平均シグナル強度とNP面積との積)を測定し、NP組織の輝度を定量化した。さらに、3つの処置群(2、3、15、37、38)のすべてにおいて、無処置対照群におけるMRIインデックスの比率である相対的MRIインデックスを評価した。頭蓋側の隣接する椎体の高さに対する椎間板の高さの比であるDHIも測定した(4、39)。無処置対照群のDHIのパーセンテージ値である相対DHIを決定した。
MRI撮影後、サンプルを10%ホルムアルデヒド中で固定し、10% EDTA(pH7.5)で脱塩し、パラフィン中に包埋した。矢状5マイクロメートル厚のパラフィン切片をキシレンで脱パラフィンし、アルコールで処理し、水ですすぎ、H&E及びサフラニン−0で染色した。IVD変性の程度を、改変Boos’分類(3、40、41)を使用して、0(正常)から36(高度に変性)までスコア化した。
IVD中のII型及びI型コラーゲンの発現は、IHCにより決定した。切片をキシレン中で脱パラフィン化し、抗原活性化のために0.1%トリプシンで30分間処理した。次いで、切片をメタノール中の3% H2O2で10分間処理し、続いて、タンパク質ブロック無血清溶液(DAKO,Agilent,Santa Clara,CA,USA)を使用して30分間タンパク質のブロッキングを行った。ヤギ抗I型コラーゲン(1:40;Southern Biotech,Birmingham,AL,USA)を抗I型コラーゲン抗体と共に使用し、抗hCL(II)及び精製IgG(1:400;Kyowa Pharma Chemical Co.,Ltd.,Toyama,Japan)を抗II型コラーゲン抗体と共に一次抗体として使用した。細胞をPBSで洗浄した後、II型コラーゲンのためのEnVision+System−HRP標識ポリマー抗マウス(DAKO)、及びI型コラーゲンのためのHistofine(登録商標)Simple Stain Max PO(G)(Nichirei Biosciences,Tokyo,Japan)を二次抗体として使用した。最後に、切片をDAB(DAKO)及びヘマトキシリンで染色した。II型及びI型コラーゲン陽性細胞の数を、5つの無作為に選択した視野で決定し、全細胞における陽性細胞率を計算した(2、3、15)。
移植IVDにおける移植細胞及び既存細胞の腫瘍形成を評価するために、(i)浸潤性増殖、(ii)核分裂、(iii)二核細胞、及び(iv)核小体の評価を行った。
24週間の評価期間中、無処置対照及びREC+ゲル群におけるH&E染色標本を使用して、陽性細胞の総数を、15の無作為に選択された視覚化視野(400倍の倍率で15の視覚化視野;26.5の視覚化視野、総計5mm2)において決定し、平方ミリメートル当たりの数を計算した。
2.1.in vitroでのREC及び市販ヒトBMSCとヒトNPCの3D共培養の結果
RECと市販のヒトBMSCとの比較
ゲルに組み込む前後のREC及び市販のヒトBMSCについての安定性確認の結果の詳細を表2及び3に示す。
本発明者らは、RECとヒトNPCとの共培養の効果、及び異なる細胞型である市販のヒトBMSCとヒトNPCとの共培養の効果を検討し、両者を比較した。5,6−カボキシフルオレセインジアセテートスクシンイミジルエステル(CFDA−SE)で標識したREC又はBMSC、及び非標識NPCを、UPALゲルに包埋し、3次元(3D)培養を行った(2,15,27)。7日間の培養後、ゲルを溶解し、細胞選別機を用いて残渣及び死細胞を除去した。次いで、細胞をCFDA−SE陽性細胞、REC、BMSC、非標識細胞、及びNPCに分類した。細胞選別後、以下の10種類の細胞を得た。
(a)対照NPC(非培養)
(b)単培養NPC
(c)市販のBMSCと共培養したNPC
(d)RECと共培養したNPC
(e)対照BMSC(非培養)
(f)単培養BMSC
(g)共培養BMSC
(h)対照REC(非培養)
(i)単培養REC
(j)共培養REC
UPALゲルに埋め込まれたRECの効果
REC−UPAL及びUPALゲルの力学特性を評価及び比較するために、非拘束圧縮試験を行った(3、15、32、33)。2種類の円筒状ゲルサンプル(直径4.5mm、厚さ2mm)を調製し、軸圧縮応力対歪みの比(図14、A~C)を計算した。圧縮レベル10~20%で、ヤング率は、UPAL及びREC−UPALゲルでそれぞれ18.0±3.5kPa及び18.8±2.1kPaであり、2群間で力学特性に有意差はなかった(p=0.6942)(図14、D及びE)。さらに、これらの2つの値は正常なヒトNP組織の値(3、32)と同等であった。
RECとUPALゲルとの併用はIVD再生を高める
IVDの再生能力は、ゲル、又はREC+ゲルの移植後のヒツジモデルで評価した(図15)。ヒツジは、生体材料を含む椎間板切除後のIVD研究に広く使用されている(3、34、35)。14頭のヒツジ由来の56のIVDを以下の群に分けた。
無処置対照群(n=6 IVD)
Discectomy群(椎間板切除群)(n=6 IVD)
ゲル群(n=8 IVD)
REC+ゲル群(n=8 IVD)
さらに、治療されたIVDの椎間板高さを、MRI画像を用いて測定した。椎間板の高さの、上部隣接椎体の高さに対する比、すなわちDHIを、IVDの前方及び後方で測定した。次いで、相対DHI、すなわち無処置対照群のDHI値に対する3つの処置群のDHI値の比を決定した(図20)(4、39)。3つの処置群のDHI値は、4週目及び24週目の両方で、無処置対照群のDHI値よりも有意に低かった。埋植4週後では3群間に有意差はなかったが、埋植24週後ではゲル群のDHIはDiscectomy群より有意に高く、REC+ゲル群はDiscectomy群、ゲル群より有意に高かった(図16D)。
24週間の評価期間中、無処置対照とREC+ゲル群の組織学的標本で腫瘍形成を分析した。両群の全標本において、核分裂とともに浸潤性増殖を評価し、二核細胞数と平方ミリメートルあたりの核小体数を測定した。両群とも浸潤性増殖、核分裂像、核小体は認められなかった。無処置対照群及びREC+ゲル群の二核細胞数はそれぞれ0.03±0.07及び0.23±0.25であった。すなわち、値は<1/mm2であり、群間に有意差は観察されなかった(p=0.3311)(表5)。
本実施例では、細胞増殖能、細胞サイズ均一性、及び細胞表面抗原の発現に関して、市販品として入手可能なヒトBMSCと比較して、RECの優れた特性が示された。さらに、本実施例は、NPCマーカー、成長因子、及びECM成分の発現レベルに関し、市販のBMSCとのNPCの3D共培養において観察される発現レベルと比較して、ヒトNPC及びRECの3D共培養において有意に増加したことを実証した。RECとUPALゲルとの併用の有効性は、ヒツジ腰椎モデルのIVD変性部位で観察された。UPALゲル単独では、椎間板切除群と比較してIVD変性を抑制したが、RECとゲルとの併用はIVD再生をより効果的に増強した。
1)RECがゲルに包埋されてIVD欠損に移植され得る。
2)RECは成長因子及びECM成分を産生し、それによって、既存のNPCを活性化する(パラクリンメカニズム)。
3)NPCはより多くのECM及び成長因子を産生する。
4)結果として、RECはNPCに直接分化する。
5)最後に、REC及びNPCはIVD再生をもたらす正の細胞間フィードバックループを有する。
本実施例では、腰部IVDの生体力学と幾何学的配置がヒトのものと同等であることから(3,4,34)、候補ヒドロゲルを前臨床動物モデルに移植するためのヒツジ腰椎モデルを選択した。本発明者らは、これまでに、椎間板切除後のヒツジ腰部IVDに埋め込んだUPALゲルが適切な生体力学的特性を示し、材料の突出をもたらさず、椎間板切除後のAFの縫合を必要としないことを示した(3)。本実施例において、非拘束圧縮試験はUPAL群とREC−UPAL群との間のヤング率に有意差がないことを明らかにし、UPALゲルに埋め込まれたRECがゲルの力学特性を変化させなかったことを示した。迅速な治癒をもたらすその決定的な能力のために、UPALゲルとRECとの組み合わせは、AFを縫合することなく細胞漏出を防ぐという点で臨床的利点を提供する。
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[実施例5]
1.ラット椎間板穿刺変性モデルの作成
ラット椎間板穿刺変性モデル(Mohd Isa et al.Sci Adv 2018)を被験物質投与に係る実験(IHC分析、組織学的分析、疼痛関連行動分析)に使用する。計60匹の12週齢雌SDラット(260−300g)を、皮膚切開のみの群(sham群)、椎間板穿刺のみの群(punch群)、椎間板穿刺後にUPALを埋植する群(UPAL群)、椎間板穿刺後にUPALとRECを埋植する群(REC群)に無作為に割り付ける(n=3、各時点・各群)。5% isoflurane吸入による麻酔導入の後に、ketamineとmedetomidineの混合物(ketamine:medetomidine=75mg/kg:0.5mg/kg)の腹腔内注射によって麻酔を維持する。鉗子を用いた尾部つまみ試験で麻酔深度を確認した後、Co4/5 −5/6の背側皮膚を切開する。結合組織を剥離してCo4/5 −5/6を露出し、19 G針を用いてCo4/5 −5/6の椎間板を損傷する(径1mm、深さ2mm)。
ラット椎間板の免疫組織学的評価を実施し、術後1、4、7および28日後のTNF−α、IL−6およびTrkA陽性細胞を検出する。ラット(各時点・各群n=3)をisoflurane吸入によって深く麻酔し、頸椎脱臼で安楽死させる。
術後28日の椎間板組織変性を評価する。ラット(n=3)を免疫組織学的評価と同じ方法で安楽死させ、椎間板を採取する。採取した椎間板を4%(w/v)paraformaldehydeで48時間固定し、Kristensen脱灰液で2週間脱灰した後、水道水で24時間洗浄し、パラフィン包埋する(Mohd Isa et al.Sci Adv 2018)。ラット椎間板の矢状断正中標本(厚さ5μm)を用いて組織学的スコア(Rutges et al.Osteoarth Carti 2013)を評価するためにヘマトキシリン&エオジン、サフラニン0、または、アルシアンブルー(AB)で染色する。AB染色は点数に直接関与しないが、細胞外基質の評価に適した染色であるため補助目的に実施する。全ての評価は、盲検化された2人の独立した観察者によって行う。各観察者は、1検体について3回の評価を実施して個体の平均値を算出した上で、各群間を比較する。
免疫組織学的評価で術後28日目まで生存させたラット12匹(各群n=3)、および、組織学的分析に使用したラット12匹(各群n=3)の、計24匹のラット(各群n=6)を疼痛関連行動分析に使用する。全ラットにHargreaves、von Frey、tail flick試験を実施する。各試験の24時間前および試験直前に、20分間各ラットを個別に試験環境で過ごさせて環境に慣れさせる。全ての試験は、盲検化された同一の検者によって行う。各ラットで複数回の測定を行って平均値を算出し、得られた結果を群間で比較する。
Hargreaves試験は、Hargreaves試験装置(Ugo Basile Biological Instruments、Gemonio、Italy)を使用して、術前2日目(Day−2)と術後2、7、14、27日目に実施する(Mohd Isa et al.Sci Adv 2018)。ラットはガラス板(Ugo Basile Biological Instruments)上に四方および上方を囲われた個別の小部屋(上方に空気孔あり)に入れる。熱刺激として赤外線ビームを皮膚切開部の腹側に照射する。熱刺激に対する逃避行動を示すまでの潜時を記録する。ビームの強度は最大出力の50%に設定する。組織損傷を防ぐ目的で、カットオフ時間を20秒間に設定する。同一ラットにおいて各時点で4回の測定を実施するが、各測定間に少なくとも1分の休憩を挟む。
von Frey試験は、dynamic plantar aesthesiometer(Ugo Basile Biological Instruments)を用いて、術前2日目(Day−2)と術後2、7、14、27日目に実施する。金網の上にHargreaves試験で用いたものと同じ小部屋を設置してラットを入れる。直径0.5mmのフィラメントを皮膚切開部の腹側に当て、0gから開始して5gまで直線的に増加する力を10秒間かけて加えていき、その後は5gの力を試験開始から30秒後まで一定の力で加える。ラットが何らかの逃避行動を示すまでの潜時を記録する。同一ラットにおいて各時点で5回の測定を実施するが、各測定間に少なくとも10秒の休憩を挟む。
tail flick試験はheat flux radiometer(Ugo Basile Biological Instruments社製)を用いて行う。Hargreaves試験と同一日程で施行されたことによる過度の熱刺激による組織損傷を避けるために、術前1日目(Day−1)と術後3、8、15、28日目に実施する(Mohd Isa et al.Sci Adv 2018)。各ラットをタオルにくるんだ状態で10分間落ち着かせた後、体部はタオルで覆ったまま尾だけを装置の上に置き、尾の遠位端から5cm近位部腹側に赤外線ビームを照射する。熱刺激に対する尾振り払い反応までの潜時を記録する。組織損傷を防ぐ目的で、カットオフ時間を20秒間に設定する。同一ラットにおいて各時点で4回の測定を実施するが、各測定間に少なくとも15秒の休憩を挟む。
全てのデータは、平均±標準誤差(SE)として表記する。多群間比較にはone−way ANOVAを使用する。2群比較には対応のないStudent−t検定を用いる。すべてのANOVAの結果は、Tukey−Kramer post−hoc検定またはKruskal−Wallis検定を用いてさらに評価する。差については、5%の有意水準で統計的に有意とみなす(P<0.05)。
UPALとRECを埋植する群(REC群)において、疼痛抑制効果が確認されうる。
Claims (27)
- アルギン酸の1価金属塩と間葉系幹細胞とを含有する、椎間板再生用組成物。
- 間葉系幹細胞による髄核細胞の活性化及び/又は間葉系幹細胞の髄核細胞への分化を介して、椎間板の髄核再生を促進する、請求項1に記載の組成物。
- 前記間葉系幹細胞が、ヒト骨髄由来高純度間葉系幹細胞である請求項1又は2に記載の組成物。
- 前記ヒト骨髄由来高純度間葉系幹細胞は、LNGFR(CD271)が陽性、又はLNGFR(CD271)及びThy−1(CD90)が共陽性の高速増殖性間葉系幹細胞クローンの細胞集団であって、以下の(a)及び(b)の少なくとも1つの特徴を満たす細胞集団である、請求項3に記載の組成物。
(a)フローサイトメトリーの前方散乱光の変動係数が40%以下である
(b)平均細胞サイズが20μm以下である - 前記ヒト骨髄由来高純度間葉系幹細胞は、LNGFR(CD271)が陽性、又はLNGFR(CD271)及びThy−1(CD90)が共陽性であることを指標に分離された高速増殖性間葉系幹細胞クローンの細胞集団であって、以下の(a)及び(b)の少なくとも1つの特徴を満たす細胞集団である、請求項3又は4に記載の組成物。
(a)フローサイトメトリーの前方散乱光の変動係数が40%以下である
(b)平均細胞サイズが20μm以下である - 対象の椎間板に適用し、適用後に組成物の表面の少なくとも一部分に架橋剤を接触させるように用いられ、当該適用時には流動性を有する、請求項1~5のいずれか1項に記載の組成物。
- 椎間板の髄核部位に適用するための請求項6に記載の組成物。
- 前記髄核部位への適用は、髄核欠損部への前記組成物の充填である、請求項7に記載の組成物。
- 前記架橋剤が2価以上の金属イオン化合物である、請求項6~8のいずれか1項に記載の組成物。
- 前記アルギン酸の1価金属塩は、低エンドトキシンアルギン酸1価金属塩である、請求項1~9のいずれか1項に記載の組成物。
- 前記アルギン酸の1価金属塩は、GPC−MALS法により測定された重量平均分子量(絶対分子量)が8万以上である、請求項1~10のいずれか1項に記載の組成物。
- アルギン酸の1価金属塩の濃度が0.5w/w%~5.0w/w%である、請求項1~11のいずれか1項に記載の組成物。
- 椎間板変性及び/又は椎間板損傷の治療、予防又は再発抑制のために用いられる、請求項1~12のいずれか1項に記載の組成物。
- 前記椎間板変性及び/又は椎間板損傷が、椎間板ヘルニア、椎間板症、脊椎変性辷り症、化膿性椎間板炎、変形性脊椎症、脊柱管狭窄症、及び椎間板損傷からなる群から選択される少なくとも1種である、請求項13に記載の組成物。
- アルギン酸の1価金属塩とヒト骨髄由来高純度間葉系幹細胞とを含む椎間板再生用組成物であって、対象の椎間板髄核部位に流動性を有する状態で適用されるための椎間板再生用組成物。
- 対象の椎間板髄核部位に流動性を有する状態で適用された後に、該組成物に架橋剤を接触させることなく用いられる請求項15に記載の椎間板再生用組成物。
- 前記ヒト骨髄由来高純度間葉系幹細胞による髄核細胞の活性化及び/又は前記ヒト骨髄由来高純度間葉系幹細胞の髄核細胞への分化を介して、椎間板の髄核再生を促進する、請求項15又は16に記載の組成物。
- 前記ヒト骨髄由来高純度間葉系幹細胞が、適用時に未分化状態である及び/又は誘導分化の処置なしに適用される請求項15~17のいずれか1項に記載の組成物。
- 前記ヒト骨髄由来高純度間葉系幹細胞は、LNGFR(CD271)が陽性、又はLNGFR(CD271)及びThy−1(CD90)が共陽性の高速増殖性間葉系幹細胞クローンの細胞集団であって、以下の(a)及び(b)の少なくとも1つの特徴を満たす細胞集団である、請求項15~18のいずれか1項に記載の組成物。
(a)フローサイトメトリーの前方散乱光の変動係数が40%以下である
(b)平均細胞サイズが20μm以下である - 前記ヒト骨髄由来高純度間葉系幹細胞は、LNGFR(CD271)が陽性、又はLNGFR(CD271)及びThy−1(CD90)が共陽性であることを指標に分離された、高速増殖性間葉系幹細胞クローンの細胞集団であって、以下の(a)及び(b)の少なくとも1つの特徴を満たす細胞集団である、請求項15~19のいずれか1項に記載の組成物。
(a)フローサイトメトリーの前方散乱光の変動係数が40%以下である
(b)平均細胞サイズが20μm以下である - 前記アルギン酸の1価金属塩が、低エンドトキシンアルギン酸1価金属塩である、請求項15~20のいずれか1項に記載の組成物。
- 前記アルギン酸の1価金属塩は、GPC−MALS法により測定された重量平均分子量(絶対分子量)が8万以上である、請求項15~21のいずれか1項に記載の組成物。
- 前記アルギン酸の1価金属塩の濃度が0.5w/w%~5.0w/w%である、請求項15~22のいずれか1項に記載の組成物。
- 椎間板変性及び/又は椎間板損傷の治療、予防又は再発抑制のために用いられる、請求項15~23のいずれか1項に記載の組成物。
- 前記椎間板変性及び/又は椎間板損傷が、椎間板ヘルニア、椎間板症、脊椎変性辷り症、化膿性椎間板炎、慢性腰痛、変形性脊椎症、脊柱管狭窄症、腰部脊柱管狭窄症、腰部脊柱管狭窄症に伴う椎間板ヘルニア(混合性腰部脊柱管狭窄症)及び椎間板損傷からなる群から選択される少なくとも1種である、請求項24に記載の組成物。
- 前記椎間板変性及び/又は椎間板損傷が、慢性腰痛を伴う、請求項24又は25に記載の組成物。
- 椎間板性疼痛を抑制するために用いる、請求項15~23のいずれか1項に記載の組成物。
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Citations (4)
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WO2004076652A1 (ja) * | 2003-02-25 | 2004-09-10 | Tokai University | 椎間板再生用の幹細胞用培地及び幹細胞を用いた椎間板の再生 |
CN103013910A (zh) * | 2012-10-26 | 2013-04-03 | 中国人民解放军第三军医大学第二附属医院 | 人退变椎间盘软骨终板干细胞、制备方法及其应用 |
WO2016017795A1 (ja) * | 2014-08-01 | 2016-02-04 | 有未 伊谷 | ヒト間葉系幹細胞の品質評価方法、及び、そのためのモノクローナル抗体 |
WO2017163603A1 (ja) * | 2016-03-23 | 2017-09-28 | 国立大学法人北海道大学 | 椎間板治療用組成物 |
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KR20160017795A (ko) | 2014-08-05 | 2016-02-17 | 삼성디스플레이 주식회사 | 박막 트랜지스터 기판, 이의 제조 방법, 및 박막 트랜지스터 기판을 포함하는 표시 장치 |
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WO2004076652A1 (ja) * | 2003-02-25 | 2004-09-10 | Tokai University | 椎間板再生用の幹細胞用培地及び幹細胞を用いた椎間板の再生 |
CN103013910A (zh) * | 2012-10-26 | 2013-04-03 | 中国人民解放军第三军医大学第二附属医院 | 人退变椎间盘软骨终板干细胞、制备方法及其应用 |
WO2016017795A1 (ja) * | 2014-08-01 | 2016-02-04 | 有未 伊谷 | ヒト間葉系幹細胞の品質評価方法、及び、そのためのモノクローナル抗体 |
WO2017163603A1 (ja) * | 2016-03-23 | 2017-09-28 | 国立大学法人北海道大学 | 椎間板治療用組成物 |
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UKEBA DAISUKE, SUDO HIDEKI, TSUJIMOTO TAKERU, URA KATSURO, YAMADA KATSUHISA, IWASAKI NORIMASA: "Bone marrow mesenchymal stem cells combined with ultra-purified alginate gel as a regenerative therapeutic strategy after discectomy for degenerated intervertebral discs", EBIOMEDICINE, ELSEVIER BV, NL, vol. 53, 1 March 2020 (2020-03-01), NL , pages 102698, XP055955060, ISSN: 2352-3964, DOI: 10.1016/j.ebiom.2020.102698 * |
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CA3209669A1 (en) | 2022-08-04 |
JPWO2022163872A1 (ja) | 2022-08-04 |
BR112023014896A2 (pt) | 2023-10-31 |
US20240100099A1 (en) | 2024-03-28 |
AU2022214480A1 (en) | 2023-08-17 |
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