Cartilage injury treatment using bone marrow fluid

Abstract

Provided is a cartilage damage treatment composition that is combined with a condensed bone marrow fluid and applied to a damaged cartilage part, that has flowability when applied to the damaged cartilage part, and that contains a monovalent metal salt of alginic acid. Thereby, a novel cartilage damage treatment composition that can be used to restore and / or regenerate cartilage tissue is provided.

Description

[Technical field]

[0001] The present invention relates to a composition and method for treating cartilage damage. [Background technology]

[0002] Joints are surrounded by a joint capsule, the inner surface of which is lined with synovial membrane. The joint cavity between the bones that form the joint is filled with synovial fluid secreted by the synovial membrane. The joint surfaces of the bones are covered with articular cartilage. Articular cartilage has a four-layer structure: superficial, intermediate, deep, and calcified layers. Beneath the deepest calcified layer is bone tissue called subchondral bone, which is firmly connected to the subchondral bone.

[0003] Articular cartilage is hyaline cartilage, consisting of a small number of cells, a collagenous extracellular matrix, a large amount of proteoglycan, and water. In the case of bone, blood vessels and nerve networks are present and it has the ability to repair itself, so even when it is broken, the fractured part is often repaired sufficiently. However, articular cartilage does not have blood vessels or nerve networks. Therefore, it has almost no ability to repair itself, and the cartilage defect is not repaired sufficiently, especially when a large cartilage defect is formed. Even in the repaired part, fibrocartilage with different mechanical properties from hyaline cartilage is formed. For this reason, when a cartilage defect is formed, it leads to joint pain and loss of joint function, and often develops into osteoarthritis. In addition, from the early stage of osteoarthritis, when the surface of the articular cartilage begins to wear down due to aging and overuse of the joint, the disease may progress to a widespread area of ​​cartilage defects.

[0004] Here, attempts are being made to use natural polymers such as collagen, chitosan, agarose, and alginic acid in regenerative medicine for articular cartilage and treatment of joint diseases. For example, it has been proposed to use alginate in regenerating cartilage in joints and treating joint diseases (e.g., Patent Documents 1-4, Non-Patent Document 1). Attempts are also being made to use alginate in combination with cells. For example, it has been reported that embedding bone marrow mesenchymal stem cells in alginate and applying it to a cartilage injury site results in good hyaline cartilage regeneration almost equivalent to that of normal cartilage (e.g., Patent Document 1 and Non-Patent Document 1). In addition, attempts are being made to use growth factors and cytokines together with alginate in cartilage regenerative medicine (Patent Document 3). Furthermore, it has been reported that a combination therapy of alginate and bone marrow stimulation method (BMS) promotes cartilage repair, but does not have a significant promoting effect on the repair of subchondral bone (Non-Patent Document 2).

[0005] In addition, a method of intra-articular injection of autologous concentrated bone marrow aspirate has been reported as a treatment for articular cartilage defects (Non-Patent Documents 3-5). Concentrated bone marrow aspirate is liquid and has fluidity, so if directly transplanted into the cartilage defect, it will not adhere and will flow away with the movement of the joint. Regarding the treatment of transplanting concentrated bone marrow aspirate, there are reports of combined transplantation of concentrated bone marrow aspirate with various biomaterials in order to obtain good adhesion and fixation at the defect site (Non-Patent Documents 6 and 7). However, when transplanting in combination with concentrated bone marrow aspirate, it is necessary to devise measures such as adjusting the shape of the biomaterial to match the shape of the cartilage defect during surgery and fixing it to the defect site. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2008 / 102855 Brochure [Patent Document 2] International Publication No. 2009 / 054181 Brochure [Patent Document 3] International Publication No. 2013 / 027854 Brochure [Patent Document 4] International Publication No. 2017 / 175229 Brochure [Non-patent literature]

[0007] [Non-Patent Document 1] Igarashi et al., J Biomed Mater Res Part A (2012) 100A, 180-187. [Non-Patent Document 2] Baba et al.,Tissue Eng Part C Methods.(2015)21(12),1263-73. [Non-Patent Document 3] Kristin et al.,Recent Adv Arthroplast(2018)2(2),75-81. [Non-Patent Document 4] Kim et al.,Eur J Orthop Surg Traumatol.(2014)24,1505-1511 [Non-Patent Document 5] Shapiro et al.,Am J Sports Med(2017)45,82-90. [Non-Patent Document 6] Gobbi et al., Cartilage (2011)2(3), 286-299. [Non-Patent Document 7] Cavallo et al., J Biomed Mater Res A. (2013) 101(6), 1559-70. Summary of the Invention [Problem to be solved by the invention]

[0008] In the above circumstances, there has been a demand for a new composition for treating cartilage damage that can be used for repairing and / or regenerating damaged cartilage tissue. [Means for solving the problem]

[0009] As a result of extensive research, the inventors discovered that by combining concentrated bone marrow fluid with a monovalent metal salt of alginic acid and applying it to the area of ​​cartilage damage, it is possible to repair and / or regenerate cartilage tissue even in areas of relatively large cartilage damage, and thus completed the present invention. That is, the following is provided herein:

[0010] [1-1] A composition for treating cartilage damage, which is applied to a subject's cartilage damage area in combination with concentrated bone marrow fluid and which has fluidity when applied to the cartilage damage area, and which contains a monovalent metal salt of alginic acid. [1-2] The composition described in [1-1] above, wherein the concentrated bone marrow fluid is derived from a subject. [1-3] The composition according to [1-1] or [1-2] above, wherein the application is carried out by either of the following (1) or (2). (1) mixing a concentrated bone marrow fluid with a composition containing a monovalent metal salt of alginic acid, and applying the resulting mixture to a cartilage injury site of a subject; (2) A composition containing a monovalent metal salt of alginic acid and concentrated bone marrow fluid are each applied to the damaged cartilage of a subject without being mixed with each other prior to application to the damaged cartilage. [1-4] The number of bone marrow mesenchymal stem cells in the concentrated bone marrow fluid is 1x10 2 ~1x10 7 The composition according to any one of the above [1-1] to [1-3], wherein the concentration of each of the composition is 100%. [1-5] The composition according to any one of the above [1-1] to [1-4], wherein the cartilage damage is accompanied by damage to subchondral bone. [1-6] The size of the cartilage damage is 4cm 2 The composition according to any one of the above [1-1] to [1-5]. [1-7] The composition according to any one of the above [1-1] to [1-6], wherein the cartilage damage is in articular cartilage. [1-8] The composition according to any one of the above [1-1] to [1-7], wherein the apparent viscosity of the fluid composition before combination with concentrated bone marrow fluid is 500 mPa s to 10,000 mPa s as measured using a cone-plate viscometer (Sensor 35 / 1), the measurement is performed at a temperature of 20°C, a rotation speed of 0.5 rpm, and a reading time of 2.5 minutes, with the average value being taken from the start of the measurement at 0.5 minutes to 2.5 minutes. [1-9] The composition according to any one of the above [1-1] to [1-8], wherein the monovalent metal salt of alginic acid has a weight average molecular weight (absolute molecular weight) of 80,000 or more as measured by a GPC-MALS method. [1-10] The composition according to any one of the above [1-1] to [1-9], wherein the concentration of the monovalent metal salt of alginic acid before being combined with the concentrated bone marrow fluid is 0.5 w / w% to 5 w / w%. [1-11] The composition described in any one of [1-1] to [1-10] above, wherein the composition is used such that after application to a cartilage injury site, at least a portion of the surface of the composition is brought into contact with a hardening agent. [1-12] The composition described in any one of [1-1] to [1-11] above, wherein the composition does not contain an amount of a hardener that hardens the composition before application to the damaged cartilage area of ​​the subject. [1-13] The composition according to [1-11] or [1-12] above, wherein the curing agent is a divalent or higher metal ion compound. [1-14] The composition according to any one of the above [1-1] to [1-13], wherein the monovalent metal salt of alginic acid is a low-endotoxin monovalent metal salt of alginic acid. [1-15] The composition according to any one of the above [1-1] to [1-14], which is used for treating a cartilage-related disease. [1-16] The composition described in [1-15] above, wherein the cartilage-related disease is at least one cartilage-related disease selected from the group consisting of osteoarthritis, traumatic cartilage defect, peri-defect traumatic cartilage defect, traumatic cartilage damage, osteonecrosis, osteochondritis dissecans, subchondral bone defect, cartilage and / or subchondral bone degeneration, and joint alignment abnormalities. [1-17] The composition described in any one of the above [1-1] to [1-16], which is used for at least one selected from the group consisting of repair of cartilage tissue and / or subchondral bone, regeneration of cartilage tissue and / or subchondral bone, inhibition of the progression of degeneration of cartilage tissue and / or subchondral bone, regeneration of hyaline cartilage, relief of pain, relief of postoperative pain, reduction of functional disorders, alleviation of clinical symptoms, and prevention or inhibition of recurrence of cartilage-related diseases.

[0011] [2-1] A kit for treating cartilage damage, comprising at least the composition described in any one of [1-1] to [1-17] above, and a hardening agent. [2-1A] The kit described in [2-1] above, wherein the kit is used to apply the composition in combination with concentrated bone marrow fluid to a cartilage injury site of a subject. [2-2] The kit described in [2-1] or [2-1A] above, wherein the concentrated bone marrow fluid is derived from a subject. [2-3] The kit according to any one of the above [2-1] to [2-2], wherein the application is carried out by any one of the following (1) and (2). (1) mixing a concentrated bone marrow fluid with a composition containing a monovalent metal salt of alginic acid, and applying the resulting mixture to a cartilage injury site of a subject; (2) A composition containing a monovalent metal salt of alginic acid and concentrated bone marrow fluid are each applied to the damaged cartilage of a subject without being mixed with each other prior to application to the damaged cartilage. [2-4] The number of bone marrow mesenchymal stem cells in the concentrated bone marrow fluid is 1x10 2 ~1x10 7 The kit according to any one of the above [2-1] to [2-3], wherein the concentration of each of the cells is 100% or more. [2-5] The kit according to any one of the above [2-1] to [2-4], wherein the cartilage damage is accompanied by damage to subchondral bone. [2-6] The size of the cartilage damage is 4cm 2 The kit according to any one of the above [2-1] to [2-5]. [2-7] The kit according to any one of the above [2-1] to [2-6], wherein the damaged cartilage site is in articular cartilage. [2-8] The kit according to any one of the above [2-1] to [2-7], wherein the apparent viscosity of the composition having flowability before combination with concentrated bone marrow fluid is 500 mPa s to 10,000 mPa s as measured using a cone-plate viscometer (Sensor 35 / 1), the measurement is performed at a temperature of 20°C, a rotation speed of 0.5 rpm, and a reading time of 2.5 minutes, with the average value being taken from 0.5 minutes to 2.5 minutes after the measurement. [2-9] The kit according to any one of the above [2-1] to [2-8], wherein the monovalent metal salt of alginic acid has a weight average molecular weight (absolute molecular weight) of 80,000 or more as measured by a GPC-MALS method. [2-10] The kit according to any one of the above [2-1] to [2-9], wherein the concentration of the monovalent metal salt of alginic acid in the composition before being combined with the concentrated bone marrow fluid is 0.5 w / w% to 5 w / w%. [2-11] The kit described in any one of the above [2-1] to [2-10], wherein the kit is used so as to contact at least a portion of the surface of the composition with a hardener after applying the composition to a cartilage injury site. [2-12] The kit described in any one of the above [2-1] to [2-11], wherein the composition does not contain an amount of hardener that hardens the composition before application to the damaged cartilage area of ​​the subject. [2-13] The kit according to the above [2-11] or [2-12], wherein the curing agent is a divalent or higher metal ion compound. [2-14] The kit according to any one of the above [2-1] to [2-13], wherein the monovalent metal salt of alginic acid is a low-endotoxin monovalent metal salt of alginic acid. [2-15] The kit according to any one of the above [2-1] to [2-14], which is used for treating a cartilage-related disease. [2-16] The kit described in [2-15] above, wherein the cartilage-related disease is at least one cartilage-related disease selected from the group consisting of osteoarthritis, traumatic cartilage defect, traumatic cartilage defect around the defect, traumatic cartilage damage, osteonecrosis, osteochondritis dissecans, subchondral bone defect, degeneration of cartilage and / or subchondral bone, and alignment abnormalities in the joint. [2-17] The kit described in any one of the above [2-1] to [2-16], which is used for at least one selected from the group consisting of repair of cartilage tissue and / or subchondral bone, regeneration of cartilage tissue and / or subchondral bone, inhibition of the progression of degeneration of cartilage tissue and / or subchondral bone, regeneration of hyaline cartilage, relief of pain, relief of postoperative pain, reduction of functional disorders, relief of clinical symptoms, and prevention or inhibition of recurrence of cartilage-related diseases.

[0012] [3-1] A method for treating cartilage damage in a subject, comprising applying a composition containing a monovalent metal salt of alginic acid in combination with concentrated bone marrow fluid to the subject's cartilage damage area, wherein the composition has fluidity when applied to the cartilage damage area. [3-2] The method according to [3-1] above, wherein the concentrated bone marrow fluid is derived from a subject. [3-3] The method according to [3-1] or [3-2] above, wherein the application is carried out by either of the following (1) or (2). (1) mixing a concentrated bone marrow fluid with a composition containing a monovalent metal salt of alginic acid, and applying the resulting mixture to a cartilage injury site of a subject; (2) A composition containing a monovalent metal salt of alginic acid and concentrated bone marrow fluid are each applied to the damaged cartilage of a subject without being mixed with each other prior to application to the damaged cartilage. [3-4] The number of bone marrow mesenchymal stem cells in the concentrated bone marrow fluid is 1x10 2 ~1x10 7 The method according to any one of the above [3-1] to [3-3], wherein the concentration of each of the cells is 100%. [3-5] The method according to any one of the above [3-1] to [3-4], wherein the cartilage damage is accompanied by damage to subchondral bone. [3-6] The size of the cartilage damage is 4cm 2The method according to any one of the above [3-1] to [3-5]. [3-7] The method according to any one of [3-1] to [3-6] above, wherein the cartilage damage is in articular cartilage. [3-8] The method according to any one of [3-1] to [3-7] above, wherein the apparent viscosity of the composition having flowability before being combined with the concentrated bone marrow fluid is 500 mPa s to 10,000 mPa s as measured using a cone-plate viscometer (sensor 35 / 1), the measurement is performed at a temperature of 20°C, a rotation speed of 0.5 rpm, and a reading time of 2.5 minutes, with the average value being taken from the start of the measurement at 0.5 minutes to 2.5 minutes. [3-9] The method according to any one of [3-1] to [3-8] above, wherein the monovalent metal salt of alginic acid has a weight average molecular weight (absolute molecular weight) of 80,000 or more as measured by a GPC-MALS method. [3-10] The method according to any one of [3-1] to [3-9] above, wherein the concentration of the monovalent metal salt of alginic acid in the composition before being combined with the concentrated bone marrow fluid is 0.5 w / w% to 5 w / w%. [3-11] The method according to any one of [3-1] to [3-10] above, wherein after application of the composition to the damaged cartilage site, a hardener is contacted with at least a portion of the surface of the composition. [3-12] The method according to any one of [3-1] to [3-11] above, wherein the composition does not contain an amount of a hardener that hardens the composition before application to the damaged cartilage area of ​​the subject. [3-13] The method according to [3-11] or [3-12] above, wherein the curing agent is a divalent or higher metal ion compound. [3-14] The method according to any one of the above [3-1] to [3-13], wherein the monovalent metal salt of alginic acid is a low-endotoxin monovalent metal salt of alginic acid. [3-15] The method according to any one of the above [3-1] to [3-14], wherein the cartilage damage is a cartilage-related disease. [3-16] The method according to [3-15] above, wherein the cartilage-related disease is at least one cartilage-related disease selected from the group consisting of osteoarthritis, traumatic cartilage defect, peri-defect traumatic cartilage defect, traumatic cartilage damage, osteonecrosis, osteochondritis dissecans, subchondral bone defect, degeneration of cartilage and / or subchondral bone, and alignment abnormalities in joints. [3-17] The method according to any one of [3-1] to [3-16] above, wherein the treatment of cartilage damage includes at least one selected from the group consisting of repair of cartilage tissue and / or subchondral bone, regeneration of cartilage tissue and / or subchondral bone, inhibition of the progression of degeneration of cartilage tissue and / or subchondral bone, regeneration of hyaline cartilage, relief of pain, relief of postoperative pain, reduction of functional disorders, relief of clinical symptoms, and prevention or inhibition of recurrence of cartilage-related diseases.

[0013] [4-1] A monovalent metal salt of alginic acid for use in treating cartilage damage, comprising: A monovalent metal salt of alginic acid which is applied in combination with concentrated bone marrow fluid to a subject's cartilage injury site and which has flowability when applied to the cartilage injury site. [4-2] The monovalent metal salt of alginic acid described in [4-1] above, wherein the concentrated bone marrow fluid is derived from a subject. [4-3] A monovalent metal salt of alginic acid according to the above [4-1] or [4-2], wherein the application is carried out by either of the following (1) or (2). (1) mixing concentrated bone marrow fluid with a monovalent metal salt of alginic acid and applying the resulting mixture to the cartilage injury site of a subject; (2) A monovalent metal salt of alginic acid and a concentrated bone marrow fluid are each applied to the cartilage injury site of a subject without being mixed with each other prior to application to the cartilage injury site. [4-4] The number of bone marrow mesenchymal stem cells in the concentrated bone marrow fluid is 1x10 2 ~1x10 7 The monovalent metal salt of alginic acid according to any one of the above [4-1] to [4-3], wherein the concentration of the monovalent metal salt is 100% or more. [4-5] The monovalent metal salt of alginic acid according to any one of the above [4-1] to [4-4], wherein the cartilage damage is accompanied by damage to subchondral bone. [4-6] The size of the cartilage damage is 4cm2 The monovalent metal salt of alginic acid according to any one of the above [4-1] to [4-5]. [4-7] The monovalent metal salt of alginic acid according to any one of the above [4-1] to [4-6], wherein the damaged cartilage site is in articular cartilage. [4-8] The monovalent metal salt of alginic acid according to any one of the above [4-1] to [4-7], wherein the apparent viscosity of the monovalent metal salt of alginic acid having fluidity before combination with concentrated bone marrow fluid is 500 mPa s to 10,000 mPa s as measured using a cone-plate viscometer (Sensor 35 / 1), the measurement is performed at a temperature of 20°C, a rotation speed of 0.5 rpm, and a reading time of 2.5 minutes, with the average value being taken from 0.5 minutes to 2.5 minutes after the measurement. [4-9] The monovalent metal salt of alginic acid according to any one of [4-1] to [4-8] above, wherein the monovalent metal salt of alginic acid has a weight average molecular weight (absolute molecular weight) of 80,000 or more as measured by a GPC-MALS method. [4-10] The monovalent metal salt of alginic acid according to any one of the above [4-1] to [4-9], wherein the monovalent metal salt of alginic acid is in the form of a composition having a concentration of 0.5 w / w% to 5 w / w% before being combined with concentrated bone marrow fluid. [4-11] The monovalent metal salt of alginic acid described in any one of [4-1] to [4-10] above, wherein the monovalent metal salt of alginic acid is used such that after application to a cartilage injury site, at least a portion of the surface of the monovalent metal salt of alginic acid is brought into contact with a hardener. [4-12] The monovalent metal salt of alginic acid described in any one of [4-1] to [4-11] above, wherein the monovalent metal salt of alginic acid does not contain an amount of hardener that hardens the monovalent metal salt of alginic acid before application to the damaged cartilage area of ​​the subject. [4-13] A monovalent metal salt of alginic acid according to [4-11] or [4-12] above, wherein the hardener is a divalent or higher metal ion compound. [4-14] The monovalent metal salt of alginic acid according to any one of the above [4-1] to [4-13], wherein the monovalent metal salt of alginic acid is a low-endotoxin monovalent metal salt of alginic acid. [4-15] The monovalent metal salt of alginic acid according to any one of the above [4-1] to [4-14], which is used for treating a cartilage-related disease. [4-16] The monovalent metal salt of alginic acid described in [4-15] above, wherein the cartilage-related disease is at least one cartilage-related disease selected from the group consisting of osteoarthritis, traumatic cartilage defect, peri-defect traumatic cartilage defect, traumatic cartilage damage, osteonecrosis, osteochondritis dissecans, subchondral bone defect, cartilage and / or subchondral bone degeneration, and joint alignment abnormalities. [4-17] The monovalent metal salt of alginic acid according to any one of the above [4-1] to [4-16], wherein the monovalent metal salt of alginic acid is used for at least one selected from the group consisting of repair of cartilage tissue and / or subchondral bone, regeneration of cartilage tissue and / or subchondral bone, inhibition of the progression of degeneration of cartilage tissue and / or subchondral bone, regeneration of hyaline cartilage, relief of pain, relief of postoperative pain, reduction of functional disorders, relief of clinical symptoms, and prevention or inhibition of recurrence of cartilage-related diseases. Effect of the Invention

[0014] A new composition for treating cartilage damage is provided, which can be used for repairing and / or regenerating damaged cartilage tissue. [Brief description of the drawings]

[0015] [Figure 1] 1 shows schematic diagrams and macroscopic photographs of each group of osteochondral defect surgery models. [Diagram 2] Representative macroscopic findings and ICRS score results (E, J) for each group at 4 weeks (A-D) and 16 weeks (F-I) after surgery. The scores are shown as the mean and standard deviation for each group. * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. [Diagram 3]This figure shows histological findings and Niederauer score results 4 and 16 weeks after surgery. Microscopic findings (x20) (A-D) and magnified images (x100) (E-H) are shown 4 weeks after surgery. Microscopic findings (x20) (J-M) and magnified images (x100) (N-Q) are shown 16 weeks after surgery. The histological scores for each group at 4 weeks (I) and 16 weeks (R) after surgery are shown as mean values ​​and standard deviations, with * indicating P<0.05, ** indicating P<0.01, and *** indicating P<0.001. Scale bar indicates 1 mm. [Figure 4] This figure shows the results of evaluating collagen orientation in repair tissue 16 weeks after surgery. A to D are tissue sections immunostained with anti-type II collagen. Scale bar indicates 1 mm. E to P are images of HE-stained tissue sections photographed with a PLM after rotating them at 0 degrees, 45 degrees, and 90 degrees. Q is the average value and standard deviation of each group for degenerative changes in repair tissue. ** indicates P<0.01. [Diagram 5] This figure shows the results of evaluating the amount of subchondral bone repair using micro-CT 16 weeks after surgery. A to D are reconstructed 2D (axial) and 3D-CT composite images of each group 4 weeks after surgery. G to J are reconstructed 2D (axial) and 3D-CT composite images of each group 16 weeks after surgery. The average and standard deviation of the repaired subchondral bone volume (BV) of each group 4 weeks after surgery (F) and 16 weeks after surgery (K) are shown. * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. [Figure 6] This figure shows the results of the evaluation of mechanical properties 16 weeks after surgery. A is a schematic diagram of the mechanical test. The tip is a 2 mm hemispherical shape. B is the appearance of the indentation tester. C is a representative indentation-deformation curve of the repair tissue calculated from each group. D is the average value and standard deviation of the standardized stiffness value of the repair tissue for each group. * indicates P<0.05. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The details will be explained below. 1. The composition Here, a composition is provided that is preferably used for treating cartilage damage. The composition is a composition for treating cartilage damage, which is applied to the cartilage damaged area of ​​a subject in combination with concentrated bone marrow fluid and contains a monovalent metal salt of alginic acid, and has flowability when applied to the cartilage damaged area (sometimes referred to as "the composition" in this specification). The composition is preferably used so that after application to the cartilage damaged area, at least a part of the surface is contacted with a hardening agent.

[0017] Cartilage is found in tubular structures such as joints, thoracic walls, intervertebral discs, menisci, larynx, airways, and ears, and can be classified into three types: hyaline cartilage, elastic cartilage, and fibrocartilage. For example, articular cartilage is hyaline cartilage, which consists of chondrocytes, collagenous extracellular matrix, proteoglycans, and water, and is not distributed with blood vessels. Hyaline cartilage is rich in type II collagen, and is stained with anti-type II collagen antibodies and stains red with safranin-O staining, which stains proteoglycans.

[0018] "Cartilage damage" refers to a state in which cartilage is impaired due to aging, trauma, or various other factors, including a state in which the function of cartilage is impaired, such as when some factor reduces the unique viscoelasticity of cartilage (it slowly shrinks when a load is applied and slowly returns to its original shape when the load is removed), making it difficult to support a load while maintaining mobility. In this specification, the term "damage" also includes "degeneration", unless otherwise specified.

[0019] The term "cartilage defect" refers to a portion where the cartilage layer is missing, including a cavity in the cartilage tissue and the surrounding tissue that forms the cavity. A cartilage defect is one aspect of cartilage damage, and the present composition is preferably used for treating a cartilage defect.

[0020] In addition, in this specification, "damaged area" is used synonymously with "lesion area," and the word "damage" may be replaced with the word "lesion."

[0021] In some aspects, the cartilage injury or defect is in articular cartilage, for example, in the knee or elbow.

[0022] The size of the cartilage damage or defect may be, for example, 1 cm in an adult human. 2 More than 2cm 2 More than 3cm 2 Over 4cm 2 More than 5cm 2 or more, or 6cm 2 The size of the cartilage damage or defect may be, in addition to the lower limit of each of these values, for example, 10 cm 2 Below, 9cm 2 Below, 8m 2 Below, 7cm 2 Below, 6cm 2 Less than or equal to 5cm 2 In some embodiments, the size of the cartilage damage or defect may be less than 4 cm in an adult human. 2 It could be more than that.

[0023] Furthermore, cartilage damage or cartilage defect may involve damage to not only the cartilage but also the subchondral bone. In this case, the present composition may be used for treating the damage to the subchondral bone.

[0024] "Treatment of cartilage damage" refers to promoting the repair and / or regeneration of damaged cartilage tissue and alleviating or curing the disease, damaged state, and clinical symptoms. Furthermore, unless otherwise specified, the term "treatment" includes improvement, alleviation, reduction, inhibition of progression, prevention, and / or inhibition of recurrence of cartilage damage and cartilage-related diseases. In other words, the present composition can be used for improvement, alleviation, reduction, inhibition of progression, prevention, and / or inhibition of recurrence of cartilage-related diseases.

[0025] The composition can also be used for repairing cartilage tissue and / or subchondral bone, regenerating cartilage tissue and / or subchondral bone, inhibiting the progression of degeneration of cartilage tissue and / or subchondral bone, regenerating hyaline cartilage, relieving pain, relieving postoperative pain, reducing functional disorders, and alleviating clinical symptoms.

[0026] "Repair of cartilage tissue (damage)" and "regeneration of cartilage tissue (damage)" refer to bringing damaged or missing cartilage tissue closer to normal cartilage tissue. However, it is not necessary to restore the cartilage to a completely normal state, and it is sufficient that the composition, shape, function, etc. of the cartilage tissue are closer to normal cartilage compared to the damaged state before application of the composition.

[0027] In the present invention, "regeneration of hyaline cartilage" refers to the regeneration of cartilage having a higher ratio of hyaline cartilage compared to fibrocartilage, and is intended to regenerate cartilage tissue rich in type II collagen and proteoglycan. In one preferred embodiment, the composition of the cartilage regenerated by application of the present composition is desirably close to the composition of natural normal cartilage. The present composition may be, for example, a "composition for cartilage repair" or a "composition for hyaline cartilage regeneration".

[0028] "Cartilage-related diseases" refer to diseases or conditions associated with cartilage damage and / or subchondral bone damage, and include, but are not limited to, osteoarthritis, traumatic cartilage defect, traumatic cartilage defect around the defect, traumatic cartilage damage, osteonecrosis, osteochondritis dissecans, subchondral bone defect, degeneration of cartilage and / or subchondral bone, and alignment abnormalities in joints.

[0029] "Combined with concentrated bone marrow fluid" means to use in combination, and when the present composition containing a monovalent metal salt of alginic acid is applied to a cartilage injury site, both the monovalent metal salt of alginic acid (hereinafter, sometimes referred to as "component (a)") and concentrated bone marrow fluid (hereinafter, sometimes referred to as "component (b)") are contained, or both the components (a) and (b) are present in the cartilage injury site after the present composition is applied to the cartilage injury site. There is no particular limitation, for example, (1) the components (a) and (b) may be mixed and then applied to the injury site, or (2) the components (a) and (b) may be applied to the injury site without being mixed. In this case, for example, (2-1) the components (a) and (b) may be applied simultaneously, (2-2) the components (b) may be applied after the application of the components (a), (2-3) the components (b) may be applied after the application of the components (a), or (2-4) at least one of the components (a) and (b) may be applied in multiple separate applications.

[0030] The phrase "in combination with concentrated bone marrow fluid" does not specifically limit the form of distribution, including sales. For example, a composition containing component (a) may be sold, and component (b) may be prepared by collecting bone marrow fluid from a patient and preparing concentrated bone marrow fluid.

[0031] A "subject" is a human or a non-human organism, such as birds and non-human mammals (eg, cows, monkeys, cats, mice, rats, guinea pigs, hamsters, pigs, dogs, rabbits, sheep, and horses).

[0032] "Applying the composition to the cartilage damage" means bringing the composition into contact with the cartilage damage, preferably filling the cartilage damage in an amount sufficient to fill the cartilage damage (or defect). "Applying concentrated bone marrow fluid to the cartilage damaged area" means bringing the concentrated bone marrow fluid into contact with the cartilage damaged area, or bringing the concentrated bone marrow fluid into contact with the present composition which is in contact with the cartilage damaged area.

[0033] The phrase "contacting at least a portion of the surface with a sclerosing agent" refers to contacting at least a portion of the surface of the composition applied to the damaged cartilage site with a sclerosing agent.

[0034] "Containing a monovalent metal salt of alginic acid" means that the composition contains a monovalent metal salt of alginic acid in an amount effective to treat cartilage damage at the site of cartilage damage when applied in combination with concentrated bone marrow fluid.

[0035] The composition may be provided in a solution state using a solvent, or in a dry state such as a lyophilized form (particularly, a lyophilized powder). When provided in a dry state, the composition is used in a state having fluidity such as a solution state using a solvent at the time of application. The solvent is not particularly limited as long as it is a solvent that can be applied to a living body, and examples of the solvent include water for injection, purified water, distilled water, ion-exchanged water (or deionized water), Milli-Q water, physiological saline, phosphate-buffered saline (PBS), etc. Preferred are water for injection, distilled water, physiological saline, etc. that can be used for the treatment of humans and animals.

[0036] Using the present compositions of some embodiments, relatively large cartilage lesions (e.g., lesions up to 4 cm in size in an adult human) can be repaired. 2 The composition can be used for the above conditions, and can provide better therapeutic effects for cartilage damage than the case where a monovalent metal salt of alginic acid is used alone. In addition, by using some embodiments of the composition, both the cartilage and the subchondral bone can be treated even in cases of cartilage damage accompanied by damage to the subchondral bone. The composition has good adhesion to the cartilage damage site, and can be applied with a syringe or the like, so that it is easy to apply to the cartilage damage site. When the cartilage damage site is articular cartilage, the composition can be administered under arthroscopic observation, and extensive incision of the affected area can be avoided.

[0037] 2. Monovalent metal salts of alginic acid "Monovalent metal salts of alginic acid" are salts of alginic acid in which the hydrogen atom of the 6th carboxylate is replaced by Na + Or K +Alginic acid is a water-soluble salt produced by ion exchange with monovalent metal ions such as sodium alginate and potassium alginate. Specific examples of monovalent metal salts of alginic acid include sodium alginate and potassium alginate, with sodium alginate being particularly preferred, which is commercially available. A solution of a monovalent metal salt of alginic acid forms a gel when mixed with a hardener.

[0038] "Alginic acid" is a biodegradable polymeric polysaccharide, which is a polymer formed by linear polymerization of two types of uronic acid, D-mannuronic acid (M) and L-guluronic acid (G). In this specification, the term "alginic acid" may also include alginates such as monovalent metal salts of alginic acid. More specifically, it is a block copolymer in which a homopolymer fraction of D-mannuronic acid (MM fraction), a homopolymer fraction of L-guluronic acid (GG fraction), and a fraction in which D-mannuronic acid and L-guluronic acid are randomly arranged (MG fraction) are arbitrarily bonded. The composition ratio (M / G ratio) of D-mannuronic acid and L-guluronic acid in alginic acid varies mainly depending on the type of organism from which seaweed, etc. are derived, and is also influenced by the growing place and season of the organism, and ranges widely from a high G type with an M / G ratio of about 0.4 to a high M type with an M / G ratio of about 5.

[0039] Alginic acid may be naturally derived or synthetic, but is preferably naturally derived. Examples of naturally derived alginic acid include alginic acid extracted from brown algae. Brown algae containing alginic acid grow abundantly in coastal areas around the world, but the seaweed that can actually be used as a raw material for alginic acid is limited, and representative examples include Lessonia in South America, Macrocystis in North America, Laminaria and Ascophyllum in Europe, and Durvillea in Australia. Examples of brown algae that can be used as a raw material for alginic acid include the genera Lessonia, Macrocystis, Laminaria (kelp), Ascophyllum, Durvillea, Eisenia, and Ecklonia.

[0040] Monovalent metal salts of alginic acid are high molecular weight polysaccharides, and it is difficult to accurately determine the molecular weight. However, if the molecular weight is too low, the viscosity will be low and there is a risk of poor adhesion to the surrounding tissues at the site of application. On the other hand, if the molecular weight is too high, it will be difficult to manufacture and will have problems such as reduced solubility, too high a viscosity when made into a solution making it difficult to handle, and it will be difficult to maintain its physical properties during long-term storage. Therefore, an appropriate molecular weight is desirable.

[0041] On the other hand, it is known that the molecular weight measurement of polymeric substances derived from natural products may result in different values ​​depending on the measurement method. For example, the weight average molecular weight measured by gel permeation chromatography (GPC) or gel filtration chromatography (collectively referred to as size exclusion chromatography) is preferably 100,000 or more, more preferably 500,000 or more, and preferably 5 million or less, more preferably 3 million or less, according to the effects shown in the examples. The preferred range is 100,000 to 5 million, more preferably 500,000 to 3.5 million. In this specification, the numerical range indicated using "to" indicates a range including the numerical values ​​before and after "to" as the minimum and maximum values, respectively.

[0042] Also, for example, the absolute weight average molecular weight can be measured by the GPC-MALS method, which combines gel permeation chromatography (GPC) and a multi-angle light scattering detector (Multi Angle Light Scattering: MALS). According to the effects shown in the examples, the weight average molecular weight (absolute molecular weight) measured by the GPC-MALS method is preferably 10,000 or more, more preferably 80,000 or more, even more preferably 90,000 or more, and preferably 1,000,000 or less, more preferably 800,000 or less, even more preferably 700,000 or less, and particularly preferably 500,000 or less. The preferred range is 10,000 to 1,000,000, more preferably 800,000 to 800,000, even more preferably 90,000 to 700,000, and particularly preferably 90,000 to 500,000.

[0043] Usually, when the molecular weight of a polymeric polysaccharide is calculated by the above-mentioned method, a measurement error of 10 to 20% or more may occur. For example, if the molecular weight is 400,000, the value may vary within a range of 320,000 to 480,000, if the molecular weight is 500,000, the value may vary within a range of 400,000 to 600,000, and if the molecular weight is 1,000,000, the value may vary within a range of 800,000 to 1,200,000.

[0044] The molecular weight of the monovalent metal salt of alginic acid can be measured by a conventional method.

[0045] Representative conditions for using gel permeation chromatography for molecular weight measurement are as described in the Examples of this specification. For example, a GMPW-XL×2+G2500PW-XL (7.8 mm ID×300 mm) column can be used, for example, a 200 mM sodium nitrate aqueous solution can be used as the eluent, and pullulan can be used as the molecular weight standard.

[0046] Representative conditions for using GPC-MALS for molecular weight measurement are as described in the Examples of this specification. As a detector, for example, an RI detector and a light scattering detector (MALS) can be used.

[0047] The apparent viscosity of the monovalent metal salt of alginic acid is preferably 40 mPa·s to 800 mPa·s, and more preferably 50 mPa·s to 600 mPa·s, when the monovalent metal salt of alginic acid is dissolved in MilliQ water to prepare a solution with a concentration of 1 w / w% and the viscosity is measured at 20°C using a cone-plate viscometer. The apparent viscosity is preferably measured under the conditions described below. In this specification, the "apparent viscosity" may be simply referred to as "viscosity".

[0048] 3. Low endotoxin treatment In some embodiments, the monovalent metal salt of alginic acid is a monovalent metal salt of alginic acid with low endotoxin. Low endotoxin refers to a low endotoxin level that does not substantially cause inflammation or fever. More preferably, the monovalent metal salt of alginic acid is treated to reduce endotoxin.

[0049] The endotoxin reduction treatment can be carried out by a known method or a method similar thereto. For example, the purification of sodium hyaluronate can be carried out by the method of Suga et al. (see, for example, Japanese Patent Application Laid-Open No. 9-324001), the purification of β1,3-glucan by Yoshida et al. (see, for example, Japanese Patent Application Laid-Open No. 8-269102), the purification of biopolymer salts such as alginate and gellan gum by Williams et al. (see, for example, Japanese Translation of PCT International Publication No. 2002-530440), the purification of polysaccharides by James et al. (see, for example, the pamphlet of International Publication No. 93 / 13136), the method of Lewis et al. (see, for example, the specification of U.S. Patent No. 5,589,591), the purification of alginate by Herman Frank et al. (see, for example, Appl Microbiol Biotechnol (1994) 40:638-643), or methods similar thereto. The low endotoxin treatment of the present invention is not limited to these, and may be carried out by known methods such as washing, filtration with a filter (endotoxin removal filter, charged filter, etc.), ultrafiltration, purification using a column (endotoxin adsorption affinity column, gel filtration column, column with ion exchange resin, etc.), adsorption to hydrophobic substances, resins or activated carbon, organic solvent treatment (extraction with organic solvent, precipitation / sedimentation by adding organic solvent, etc.), surfactant treatment (see, for example, JP-A-2005-036036, etc.), or by appropriately combining these. These treatment steps may be appropriately combined with known methods such as centrifugation. It is desirable to appropriately select the method according to the type of alginic acid, etc.

[0050] The endotoxin level can be confirmed by known methods, for example, a method using Limulus Alkaline Lympholysate (LAL) or a method using Entospecy (registered trademark) ES-24S set (Seikagaku Corporation).

[0051] The method for treating endotoxins in monovalent metal salts of alginic acid is not particularly limited, but as a result, when endotoxin is measured using a Limulus reagent (LAL), the endotoxin content of the monovalent metal salts of alginic acid is preferably 500 endotoxin units (EU) / g or less, more preferably 100 EU / g or less, particularly preferably 50 EU / g or less, and particularly preferably 30 EU / g or less. Sodium alginate that has been treated to reduce endotoxins is available, for example, under the trade name Sea Matrix (registered trademark) (Mochida Pharmaceutical Co., Ltd.), PRONOVA TM It is available commercially as a product such as UP LVG (FMC BioPolymer).

[0052] 4. Preparation of Solutions of Monovalent Metal Salts of Alginic Acid The composition may be prepared using a solution of a monovalent metal salt of alginic acid. The solution of a monovalent metal salt of alginic acid can be prepared by a known method or a method similar thereto. That is, the monovalent metal salt of alginic acid used in the present invention can be produced by a known method such as an acid method or a calcium method using the above-mentioned brown algae. Specifically, for example, alginic acid can be obtained by extracting from these brown algae using an alkaline aqueous solution such as an aqueous sodium carbonate solution, and then adding an acid (e.g., hydrochloric acid, sulfuric acid, etc.), and a salt of alginic acid can be obtained by ion exchange of alginic acid. In some embodiments, as described above, a low endotoxin treatment is performed. The solvent for the monovalent metal salt of alginic acid is not particularly limited as long as it is a solvent that can be applied to a living body, and examples thereof include purified water, distilled water, ion-exchanged water, Milli-Q water, physiological saline, and phosphate-buffered saline (PBS). These are preferably sterilized, and are preferably low endotoxin treated. For example, Milli-Q water can be used after sterilization by filtration.

[0053] Even when the present composition is provided in a dry state, such as a lyophilized form, it can be prepared into a flowable solution using the above-mentioned solvent.

[0054] In addition, all operations for obtaining the present composition are desirably performed in an environment with low endotoxin and bacterial levels. For example, the operations are preferably performed on a clean bench using sterilized instruments, and the instruments used may be treated with a commercially available endotoxin remover.

[0055] 5. Apparent Viscosity of the Composition In some embodiments, the composition is in a liquid state with flowability, i.e., in a solution state, when applied to a cartilage injury site. When the composition is mixed with concentrated bone marrow fluid and then applied to a cartilage injury site, it is preferable that the mixture has flowability. When the composition is mixed with a solvent-insoluble substance such as concentrated bone marrow fluid or cells and then applied to the injury site, the apparent viscosity of the composition is preferably the viscosity of a composition containing a monovalent metal salt of alginic acid before being mixed with concentrated bone marrow fluid or the like in order to accurately measure viscosity. When the composition is applied to the injury site without being mixed with concentrated bone marrow fluid before application to the injury site, the apparent viscosity of the composition is the viscosity of a composition containing a monovalent metal salt of alginic acid. The preferred apparent viscosity of the present composition is not particularly limited as long as it can be applied to the cartilage injury site, but considering the adhesion to the surrounding tissues of the applied site, the apparent viscosity is preferably 10 mPa·s or more, more preferably 100 mPa·s or more, even more preferably 200 mPa·s or more, and particularly preferably 500 mPa·s or more, when measured under the measurement conditions using a rotational viscometer (cone-plate type viscometer) described below and at 20°C. In addition, considering the ease of handling, the apparent viscosity of the present composition is preferably 100,000 mPa·s or less, more preferably 50000 mPa·s or less, even more preferably 20000 mPa·s or less, even more preferably 10000 mPa·s or less, and particularly preferably 7000 Pa·s or less. When the apparent viscosity is 20000 mPa·s or less, application with a syringe or the like is easier. However, even if the apparent viscosity is 20000 mPa·s or more, application is possible using a pressurized or electric type filling device or other means. The preferred range of the present composition is 10 mPa·s to 50000 mPa·s, more preferably 100 mPa·s to 30000 mPa·s, even more preferably 200 mPa·s to 20000 mPa·s, even more preferably 500 mPa·s to 20000 mPa·s, and particularly preferably 700 mPa·s to 20000 mPa·s. In another preferred embodiment, the viscosity may be 500 mPa·s to 10000 mPa·s, or 2000 mPa·s to 10000 mPa·s. The present composition in some embodiments has a viscosity that allows it to be applied to a subject using a syringe or the like.

[0056] It is more preferable to measure the apparent viscosity of the composition using a cone-plate viscometer and a sensor 35 / 1 (cone diameter 35 mm, 1°). It is preferable to follow the viscosity measurement method of the Japanese Pharmacopoeia (16th edition). For example, it is preferable to measure under the following measurement conditions. When a solvent is required for viscosity measurement, it is preferable to prepare the sample solution using MilliQ water. The measurement temperature is 20°C. The rotation speed of the cone-plate viscometer is 1 rpm when measuring a 1% solution of monovalent metal alginate, and 0.5 rpm when measuring a 2% solution, and is determined based on this. When measuring a 1% solution of monovalent metal alginate, the measurement is performed for 2 minutes, and the reading time is the average value from 1 minute to 2 minutes. When measuring a 2% solution, the measurement is performed for 2.5 minutes, and the average value from 0.5 minutes to 2.5 minutes is taken. The test value is the average value of three measurements. If the viscosity of the composition is 10,000 mPa s or more, it is also desirable to use a sensor 20 / 1 (cone diameter 20 mm, 1°) and measure at a rotation speed of 0.5 rpm for 2.5 minutes, and to calculate the average value from the start of 0.5 minutes to 2.5 minutes.

[0057] Since the apparent viscosity of a solution of a monovalent metal salt of alginic acid is affected by the M / G ratio, for example, an alginic acid having a preferred M / G ratio can be appropriately selected depending on the viscosity of the solution, etc. The M / G ratio of the alginic acid used in the present invention is about 0.1 to 5.0, preferably about 0.1 to 4.0, and more preferably about 0.2 to 3.5.

[0058] As mentioned above, since the M / G ratio is mainly determined by the type of seaweed, the type of brown algae used as the raw material affects the viscosity of the solution of the monovalent metal salt of alginic acid. The alginic acid used in the present invention is preferably derived from brown algae of the genus Lessonia, Macrocystis, Laminaria, Ascophyllum, or Durvillea, more preferably from brown algae of the genus Lessonia, and particularly preferably from Lessonia nigrescens.

[0059] 6. Concentrated Bone Marrow Aspirate (cBMA) The composition is used in combination with concentrated bone marrow fluid. Concentrated bone marrow aspirate concentrate (cBMA), also known as bone marrow aspirate concentrate (BMAC), is a concentrated cell solution containing bone marrow derived-mesenchymal stem cells (MSCs) and liquid growth factors obtained by centrifugation of bone marrow aspirate. cBMA can be prepared by simply centrifuging bone marrow aspirate. This means that ex vivo cell culture is not required, and it can be used for one-stage treatment. In addition, since a concentration operation is performed during the preparation process, it can contain a sufficient number of cells. In some embodiments, the concentrated cell solution is an autologous concentrated cell solution, i.e., derived from the subject.

[0060] The method for preparing the concentrated bone marrow fluid is not particularly limited, but it can be prepared, for example, as follows.

[0061] Concentrated bone marrow fluid can be prepared according to the protocol described in Chahla J et al., Arthrosc Tech. (2017) 6 (2), e441-e445. Specifically, bone marrow fluid is collected from the subject's iliac bone using a syringe, and the blood clotted in the bone marrow fluid is removed by filtration. The filtered bone marrow fluid is then centrifuged, and the cell fluid is then aspirated, focusing on the monocytic cell layer (Buffy coat) in the middle layer, including a small amount of the plasma component (PRP) just above it, while being careful not to aspirate the RBCs (red blood cells) below as much as possible. This is used as concentrated bone marrow fluid.

[0062] Alternatively, it can be prepared using commercially available equipment for preparing concentrated bone marrow fluid from bone marrow fluid, such as the Bio-Cue System (Biomet Biologics, Mrtin JR et al., 2013;54:219-24) or the CenTrate(R) BMA Device.

[0063] The number of bone marrow mesenchymal stem cells in the concentrated bone marrow fluid is preferably 1×10 1 ~1×10 9 cells / ml, more preferably 1×10 2 ~1×10 7 cells / ml, more preferably 1×10 3 ~1×10 6 In some embodiments, the number of bone marrow mesenchymal stem cells in the concentrated bone marrow fluid is 1×10 3 ~1×10 5 cells / ml.

[0064] 7. Preparation of the Composition This composition contains a monovalent metal salt of alginic acid. The present inventors have found for the first time that when a monovalent metal salt of alginic acid is combined with concentrated bone marrow fluid and applied to a cartilage injury site in a living body, it exerts a regenerative or therapeutic effect on cartilage tissue even when the size of the cartilage injury site is relatively large. Preferably, the monovalent metal salt of alginic acid is contained in this composition in an amount that can exert a regenerative or therapeutic effect on cartilage tissue when the monovalent metal salt of alginic acid is combined with concentrated bone marrow fluid and applied to the affected site, and the amount is preferably at least 0.1 w / v% or more of the entire composition before being combined with concentrated bone marrow fluid, more preferably 0.5 w / v% or more, and even more preferably 1 w / v% or more. The preferred concentration of the monovalent metal salt of alginic acid in the present composition before being combined with the concentrated bone marrow fluid is influenced by the molecular weight, so it cannot be generally stated, but is preferably 0.5 w / v% to 5 w / v%, more preferably 1 w / v% to 5 w / v%, even more preferably 1 w / v% to 4 w / v%, and particularly preferably 1 w / v% to 3 w / v%. In another embodiment, the concentration of the monovalent metal salt of alginic acid in the present composition may be preferably 0.5 w / w% to 5 w / w%, more preferably 1 w / w% to 5 w / w%, even more preferably 1 w / w% to 4 w / w%, and particularly preferably 1 w / w% to 3 w / w%. When the present composition is mixed with the concentrated bone marrow fluid and then applied to the cartilage injury site, it is also desirable to prepare the concentration of the monovalent metal salt of alginic acid in the mixture to be in the above range.

[0065] When a composition is prepared as described above using a monovalent metal salt of alginic acid that has been purified to exhibit a preferred endotoxin level, the endotoxin content of the composition is typically 500 EU / g or less, more preferably 300 EU / g or less, even more preferably 150 EU / g or less, and particularly preferably 100 EU / g or less.

[0066] In one embodiment, the composition does not contain any component that exerts a pharmacological effect on the damaged cartilage site other than the monovalent metal salt of alginic acid, except when the composition is used in combination with concentrated bone marrow fluid. Even in this case, the composition can exert a sufficient regenerative or therapeutic effect on the damaged cartilage site.

[0067] In some embodiments, the composition may contain, as necessary, other medicament active ingredients, or ingredients typically used in pharmaceuticals, such as conventional stabilizers, emulsifiers, osmotic pressure adjusting agents, buffers, isotonicity adjusting agents, preservatives, soothing agents, coloring agents, etc.

[0068] 8. Application of the composition Before applying the composition to the damaged cartilage site, it is desirable to make the damaged cartilage site visible by means of incision, arthroscope, endoscope, etc. An arthroscope is a type of endoscope used to observe the condition of a joint.

[0069] Before applying the composition to the cartilage injury, a step of removing unnecessary tissues from the cartilage injury site and its periphery may be included, if necessary. The "cartilage injury site and unnecessary tissues from its periphery" referred to here includes the diseased tissues of the cartilage injury site, the periphery of the damaged cartilage site that has been repaired by fibrocartilage, and the unstable cartilage site.

[0070] If necessary, the affected area may be washed before applying the composition. "Washing the affected area" refers to removing blood components and other unnecessary tissues from the area to which the composition is to be applied, for example, using physiological saline. After washing the affected area, it is desirable to apply the composition after drying the area by wiping off any remaining unnecessary liquid components.

[0071] Before applying the composition to the cartilage injury, the cartilage injury may be subjected to bone marrow stimulation treatment, if necessary. Bone marrow stimulation is a treatment method in which one or more defects (full-thickness defects) reaching the subchondral bone are created in the injury by a technique such as picking or drilling, and is expected to have the effect of bleeding from the bone marrow and of chondrocytes in the bone marrow migrating to the cartilage injury.

[0072] In the present specification, "applying the composition to the cartilage injury site" means bringing the composition into contact with the cartilage injury site, and preferably filling the cartilage injury site with an amount sufficient to fill the cartilage injury site (or defect site). Alternatively, the composition may be used to fill the hole by forming one or more relatively small holes in the cartilage injury site (or defect site) and injecting the composition into the hole. When applying to the cartilage injury site, it is preferable to inject the composition so as to fully fill the cavity volume of the affected site. It is also preferable to embed the composition until the liquid level of the composition reaches the same height as the cartilage tissue surrounding the cartilage defect site. In the present specification, the term "apply" is used in a sense that includes the term "embed".

[0073] The composition is preferably in the form of a fluid liquid, i.e., a solution, when applied to a cartilage injury site. "Having fluidity" means that the composition has the property of changing its form into an indefinite shape, and does not necessarily have the property of always flowing like a solution. Preferably, the composition has fluidity such that it can be sealed in a syringe or the like and injected into a cartilage injury site. When the composition is mixed with a concentrated bone marrow fluid and then applied to a cartilage injury site, it is preferable that the mixture has fluidity.

[0074] In some embodiments, the composition is preferably fluid enough to be injected into the cartilage injury site with a syringe equipped with a 14G to 26G injection needle after standing at 20°C for 1 hour, more preferably with a 21G injection needle. When the composition is provided in a dry state such as a lyophilized body, a solvent or the like can be used at the time of application to make the composition fluid as described above. Since the composition in some embodiments is in the form of a solution, it can be adapted to any shape of cartilage injury site and can fill the entire cartilage injury site.

[0075] The present composition in solution form can be easily applied to the damaged cartilage area using a syringe, a gel pipette, a special injection syringe, a special injection device, a filling device, or the like.

[0076] When the viscosity of the composition is high, it is difficult to apply the composition with a syringe, so a pressurized or electric syringe may be used. Even if a syringe is not used, the composition may be applied to the damaged cartilage site with a spatula or stick. When injecting the composition with a syringe, it is preferable to use a needle of 14G to 27G or 14G to 26G.

[0077] The amount of the composition to be applied may be determined according to the volume of the application site of the cartilage damage to be applied, and is not particularly limited, but is, for example, 0.1 ml to 10 ml, more preferably 0.2 ml to 8 ml, and even more preferably 0.2 ml to 5 ml. When the composition is applied to the cartilage damage, it is desirable to inject the composition so as to sufficiently fill the volume of the defect in the cartilage damage.

[0078] The amount of concentrated bone marrow fluid to be applied may be determined according to the volume of the application site of the cartilage injury to be applied, and is not particularly limited, but is, for example, preferably 0.1 ml to 10 ml, more preferably 0.2 ml to 8 ml, and even more preferably 0.2 ml to 5 ml. When the concentrated bone marrow fluid is applied separately from the present composition without being mixed, the concentrated bone marrow fluid can be applied to the cartilage injury site using an instrument such as a syringe.

[0079] When combined with concentrated bone marrow fluid, the ratio of the amount of the composition to be applied to the concentrated bone marrow fluid is not particularly limited, but is, for example, the composition (ml): concentrated bone marrow fluid (ml) = 1:5 to 10:1, and more preferably 1:2 to 5:1.

[0080] In another embodiment, it is also desirable that the ratio of the amount of the present composition to the total volume (ml) of the applied amount of the present composition plus the amount of concentrated bone marrow fluid is preferably 20% to 80%, more preferably 30% to 70%.

[0081] In another embodiment, it is also desirable to apply the composition so that the concentration of the monovalent metal salt of alginic acid in the total volume (ml) of the composition to be applied plus the volume of concentrated bone marrow fluid is preferably 0.5 w / w% to 5 w / w%, more preferably 1 w / w% to 5 w / w%, even more preferably 1 w / w% to 4 w / w%, and particularly preferably 1 w / w% to 3 w / w%. Here, the "total amount" may refer to either the total amount of the present composition and concentrated bone marrow fluid in a mixture in which they are mixed, or the total amount of the two in which they are applied separately.

[0082] In another embodiment, when the monovalent metal salt of alginic acid is in a freeze-dried form, it is also desirable to dissolve the freeze-dried form using a solvent such as water for injection and concentrated bone marrow fluid, and prepare the above-mentioned ratios and concentrations.

[0083] The present composition is used such that after application to the cartilage injury site, a hardening agent is brought into contact with at least a portion of the surface of the present composition having fluidity. This process gels the surface of the composition containing a monovalent metal salt of alginic acid, hardening the surface, thereby preventing the present composition from falling off or leaking from the affected site. As long as this effect can be obtained, there is no limitation on the use of the hardening agent, but it is preferable to bring the hardening agent into contact with the entire surface of the composition. It is also preferable that immediately after the hardening agent is brought into contact with the surface of the composition, the entire surface of the composition is hardened, and the area other than the surface is in a sol state.

[0084] The present composition preferably does not contain a hardening agent in an amount that hardens the composition before application to the cartilage injury of the subject. Therefore, the present composition may contain a hardening agent in an amount that does not harden the composition even after a certain period of time has passed. The certain period of time here is not particularly limited, but is preferably about 30 minutes to 12 hours. The fact that the composition "does not contain a hardening agent in an amount that hardens the composition" may be shown, for example, by the fact that the composition can be injected with a syringe equipped with a 21G injection needle after being left to stand at 20°C for 1 hour. Some embodiments of the composition do not contain a hardening agent.

[0085] The hardener is not particularly limited as long as it can crosslink the solution of the monovalent metal salt of alginic acid and fix the surface. 2+ , Mg 2+ , B.A. 2+ , Sr 2+ and crosslinking reagents having 2 to 4 amino groups in the molecule. More specifically, examples of the divalent or higher metal ion compounds include CaCl 2 , MgCl 2 , CaSO 4 , BaCl 2 As a crosslinking reagent having 2 to 4 amino groups in the molecule, 2 )-(CH 2 ) 4 -NH 2 ), that is, diaminoalkanes and their derivatives in which the amino group is replaced with a lysyl group to form a lysylamino group are included. Specific examples include diaminoethane, diaminopropane, and N-(lysyl)-diaminoethane. However, for reasons of availability and gel strength, CaCl 2 It is preferably in the form of a solution.

[0086] In some embodiments, the timing of contacting the surface of the composition with the hardening agent is preferably after the composition is applied to the cartilage injury site. The method of contacting a part of the composition with the hardening agent (e.g., divalent or higher metal ion) is not particularly limited, but may include, for example, a method of spraying a solution of divalent or higher metal ion on the surface of the composition with a syringe, injector (spray), etc. For example, the hardening agent may be slowly sprayed on the surface of the composition for several seconds to 10-odd seconds. After that, if necessary, a treatment may be added to remove the hardening agent remaining near the cartilage injury site. The hardening agent may be removed by, for example, washing the application site with physiological saline or the like.

[0087] The amount of the hardener used is preferably adjusted appropriately taking into consideration the amount of the composition applied, the size, shape, and location of the cartilage injury. In order to prevent the hardener from strongly affecting the tissue surrounding the cartilage injury, the amount of the hardener used is adjusted so as not to be excessive. The amount of the divalent or higher metal ion used is not particularly limited as long as it is an amount that can harden the surface of the composition containing a monovalent metal salt of alginic acid. However, for example, 100 mM CaCl 2 When using the solution, the size of the cartilage injury is 4cm 2 If the concentration is about 100%, CaCl 2 The amount of solution used is preferably about 0.3 ml to 10 ml, more preferably about 1 ml to 5 ml, and can be increased or decreased as appropriate while observing the condition of the composition at the application site.

[0088] It is known that when calcium is contained in the hardening agent, the higher the calcium concentration, the faster the gelation and the harder the gel that can be formed. However, calcium is cytotoxic, so if the concentration is too high, it may have a negative effect on the cartilage regeneration effect of the composition. Therefore, in order to harden the surface of a composition containing a monovalent metal salt of alginic acid, for example, CaCl 2 When a solution is used, the concentration is preferably 25 mM to 200 mM, and more preferably 50 mM to 150 mM.

[0089] Preferably, after adding the curing agent to the composition, it is desirable to leave it for a certain period of time, and then remove the curing agent remaining at the added site by washing or the like. The certain period of time for leaving it is not particularly limited, but it is desirable to leave it for about 1 minute or more, more preferably for about 4 minutes or more to gel the surface of the composition. Alternatively, it is preferable to leave it for about 1 minute to about 10 minutes, more preferably for about 4 minutes to about 10 minutes, about 4 minutes to about 7 minutes, and even more preferably for about 5 minutes. During this certain period of time, it is desirable to keep the composition and the curing agent in contact with each other, and the curing agent may be appropriately added so that the liquid surface of the composition does not dry out.

[0090] Since the composition is in the form of a solution, it can be easily applied to any shape of application site, and the entire application site can be covered with the composition, and it has good adhesion to the surrounding tissue. The calcium concentration can be kept low in the portion of the composition that contacts the surrounding tissue, and there is little problem of calcium cytotoxicity. Since the portion of the composition that contacts the surrounding tissue is less affected by the hardener, the composition can easily contact the cells and tissues at the application site, and the therapeutic effect of the composition is more potent. Preferably, the composition is fused with the tissue of the living body so that it cannot be distinguished at the application site after about 4 weeks have passed after application to the cartilage injury site, and has high affinity to the living body.

[0091] If necessary, the affected area may be sutured.

[0092] The number and frequency of application of the present composition can be increased or decreased depending on the symptoms and effects. For example, it may be applied only once, or it may be applied continuously once a month to once a year.

[0093] Alginic acid is a substance that does not naturally exist in the bodies of animals, so animals do not possess the enzymes that specifically degrade alginic acid. Although alginic acid is gradually degraded in the animal body by normal hydrolysis, it degrades more slowly in the body than polymers such as hyaluronic acid, and because cartilage does not have blood vessels, it is expected to have a long-lasting effect when filled into damaged cartilage.

[0094] In some embodiments, when the composition is applied to the cartilage injury site, cells, growth factors, etc., in addition to those contained in the concentrated bone marrow fluid, may be used in combination, and cell death inhibitors, other drugs described below, etc. may also be used in combination. In other embodiments, it is also preferable to use the composition without using these cells or factors in combination.

[0095] The cells are not particularly limited as long as they are useful for treating cartilage damage, and examples thereof include chondrocytes, chondrocyte precursor cells, stem cells, stromal cells, mesenchymal stem cells, bone marrow stromal cells, synovial cells, ES cells, iPS cells, etc. The origin is not particularly limited, and examples thereof include bone marrow, adipose tissue, umbilical cord blood, etc. More preferably, the cells are bone marrow mesenchymal stem cells and / or bone marrow stromal cells.

[0096] The composition may contain factors that promote cell growth in addition to those contained in the concentrated bone marrow fluid, such as BMP, FGF, VEGF, HGF, TGF-β, IGF-1, PDGF, CDMP (cartilage-derived-morphogenetic protein), CSF, EPO, IL, PRP (platelet rich plasma), SOX, and IF.

[0097] In addition, before, simultaneously, or after applying the present composition to the damaged cartilage area, concomitant drugs such as antibiotics such as streptomycin, penicillin, tobramycin, amikacin, gentamicin, neomycin, and amphotericin B, aspirin, nonsteroidal antipyretic analgesics (NSAIDs), anti-inflammatory drugs such as acetaminophen, protease, corticosteroids, and HMG-CoA reductase inhibitors such as simvastatin and lovastatin may be filled. These drugs may be mixed into the present composition. Alternatively, they may be administered orally or parenterally in combination. In addition, muscle relaxants, opioid analgesics, neuropathic pain relievers, etc. may be administered orally or parenterally in combination as necessary.

[0098] The preferred embodiments of the composition and the method of using the composition are as described above.

[0099] 9.Treatment method Here, the present invention also provides a method for treating cartilage damage using the present composition.Preferably, the method for treating cartilage damage in a subject is a method for treating cartilage damage in a subject, in which a composition containing a monovalent metal salt of alginic acid is applied to the cartilage damage site of a subject in combination with concentrated bone marrow fluid, and the composition has flowability when applied to the cartilage damage site, and more preferably, the composition is used so that a hardening agent is contacted with at least a part of the surface after application to the cartilage damage site.

[0100] In some embodiments, it may be desirable to include the steps of: (a) making the cartilage defect visible by incision, arthroscope, or endoscope; (b) removing unnecessary tissue from the damaged cartilage area and its surrounding area, if necessary; (c) combining a composition containing a monovalent metal salt of alginic acid with a bone marrow concentrate and applying the combined composition to the damaged cartilage area; (d) contacting the surface of the implanted composition with a hardener; (e) washing the area contacted with the curing agent; and (g) Stuturing, if necessary.

[0101] The preferred embodiment of the composition, the specific method of applying the composition to the damaged cartilage site, the method of hardening the composition, the meanings of the terms, etc. are as described above. The present treatment method may be appropriately combined with other treatment methods or therapeutic agents for cartilage damage.

[0102] In some embodiments, the above-mentioned cells, factors promoting cell growth, other drugs, etc. may be applied to the damaged cartilage site together with the present composition. In some other embodiments, it is also desirable that the present composition is not used in combination with the above-mentioned additional cells, additional factors, or other drugs. In a preferred embodiment, the present composition can promote cartilage regeneration even without the use of these additional cells, additional factors, or other drugs.

[0103] The present invention also relates to the use of a monovalent metal salt of alginic acid for preparing the composition.

[0104] The present use is the use of a monovalent metal salt of alginic acid to manufacture a composition for treating cartilage damage, wherein the composition is applied in combination with concentrated bone marrow fluid to the cartilage damage site of a subject and has flowability when applied to the cartilage damage site.

[0105] The present invention further provides a monovalent metal salt of alginic acid for use in treating cartilage damage, comprising applying a flowable composition containing the monovalent metal salt of alginic acid in combination with concentrated bone marrow fluid to the area of ​​cartilage damage in a subject in need of such treatment.

[0106] The present invention further provides a combination comprising a flowable composition containing a monovalent metal salt of alginic acid and concentrated bone marrow fluid, the combination being intended for application to an area of ​​cartilage damage in a subject in need of treatment for cartilage damage and for use in treating cartilage damage.

[0107] 10. Kit The present invention provides a kit for treating cartilage damage. The kit of the present invention may contain the composition of the present invention. The composition of the present invention to be contained in the kit of the present invention is preferably in a solution state or a dry state. The dry state is preferably a lyophilized form, and particularly preferably a lyophilized powder.

[0108] When the composition of the present invention is in a dry state, it is desirable to include a solvent for dissolution (eg, water for injection), a syringe for dissolution, and an injection needle.

[0109] The kit of the present invention may further include a hardening agent (eg, a calcium chloride solution, etc.), and desirably includes a syringe, injection needle, etc. for the hardening agent.

[0110] The kit of the present invention may include an instruction manual and the like.

[0111] A specific example of a suitable kit for use in the present invention is a kit in which (1) a vial containing a freeze-dried form of low endotoxin sodium alginate, (2) an ampoule containing a solvent such as water for injection as a dissolving solution, (3) an ampoule containing a divalent or higher metal ion compound such as calcium chloride solution as a hardening agent, (4) a syringe, (5) an injection needle, etc. are packaged together. Another example is a kit in which a monovalent metal salt of alginic acid is enclosed in one chamber of a syringe consisting of two chambers separated by a partition wall, and a solvent as a dissolving solution or a solution containing a hardening agent is enclosed in the other chamber, and the partition walls of both chambers are configured to be easily opened when used, so that both can be mixed and dissolved when used. Another example is a kit in which a monovalent metal salt solution of alginic acid is enclosed in a prefilled syringe, and the kit can be filled as it is without any preparation operation when used. Another example is a kit in which an alginic acid solution and a hardening agent are enclosed in separate syringes and packaged together in one pack. Alternatively, a vial filled with a solution of a monovalent metal salt of alginic acid may be used. The "composition of the present invention" and the "hardening agent" are as described above.

[0112] All documents and publications described herein are incorporated herein by reference in their entirety regardless of their purpose. This specification also incorporates by reference the disclosures of the claims, specification, and drawings of Japanese Patent Application No. 2020-005679 (filed January 17, 2020), which is the basis for the priority claim of this application. EXAMPLES

[0113] The present invention will be described in more detail with reference to the following examples, but the present invention should not be understood as being limited to these examples.

[0114] Example 1: Application of sodium alginate solution and concentrated bone marrow fluid to a rabbit osteochondral defect model 1. Method 1.1. Procedure for creating the rabbit osteochondral defect model Fifty-two 24-week-old Japanese white rabbits (104 knees) were placed under general anesthesia and a 3.0 cm skin incision was made to expose the patellofemoral joint and femoral trochlea using a medial parapatellar approach. An osteochondral defect was created in the center of the trochlea using a 5.0 mm diameter electric drill with a special sleeve to a depth of 2.0 mm while cooling with saline. This defect, when extrapolated to a human defect, is 4 cm 2 This corresponds to a defect of 10 mm, which is a relatively large defect.

[0115] 1.2. Preparation of sodium alginate solution The following sodium alginate was used. The endotoxin content was less than 50 EU / g, which was low endotoxin sodium alginate. The apparent viscosity and weight average molecular weight of each sodium alginate are shown in the table below. The apparent viscosity of sodium alginate was measured using a rotational viscometer method (cone-plate type rotational viscometer) according to the viscosity measurement method of the Japanese Pharmacopoeia (16th edition). The specific measurement conditions are as follows. The sample solution was prepared using MilliQ water. The measurement equipment used was a cone-plate type rotational viscometer (viscosity viscoelasticity measuring device Rheostress RS600 (Thermo Haake GmbH) sensor: 35 / 1). The rotation speed was 1 rpm when measuring 1 w / w% sodium alginate solution and 0.5 rpm when measuring 2 w / w% sodium alginate solution. The reading time was 2 minutes for 1 w / w% solution measurement, and the average value from 1 to 2 minutes was used. For 2 w / w% solution measurement, the reading time was 2.5 minutes, and the average value from 0.5 to 2.5 minutes was used. The average value of three measurements was used. The measurement temperature was 20°C. The weight-average molecular weight of each sodium alginate was measured by two methods, gel permeation chromatography (GPC) and GPC-MALS, under the following measurement conditions:

[0116] [Pretreatment method] The sample was dissolved in an eluent and then filtered through a 0.45 μm membrane filter to prepare a measurement solution.

[0117] (1) Gel permeation chromatography (GPC) measurement [Measurement conditions (relative molecular weight distribution measurement)] Column: TSKgel GMPW-XL x 2 + G2500PW-XL (7.8 mm ID x 300 mm x 3) Eluent: 200mM sodium nitrate aqueous solution Flow rate: 1.0mL / min Concentration: 0.05% Detector: RI detector Column temperature: 40℃ Injection volume: 200μL Molecular weight standards: standard pullulan, glucose

[0118] (2)GPC-MALS measurement [Refractive index increment (dn / dc) measurement (measurement conditions)] Suggestive refractometer: Optilab T-rEX Measurement wavelength: 658nm Measurement temperature: 40℃ Solvent: 200mM sodium nitrate aqueous solution Sample concentration: 0.5-2.5mg / mL (5 concentrations)

[0119] [Measurement conditions (absolute molecular weight distribution measurement)] Column: TSKgel GMPW-XL x 2 + G2500PW-XL (7.8 mm ID x 300 mm x 3) Eluent: 200mM sodium nitrate aqueous solution Flow rate: 1.0mL / min Concentration: 0.05% Detector: RI detector, light scattering detector (MALS) Column temperature: 40℃ Injection volume: 200μL

[0120] [Table 1]

[0121] The low endotoxin sodium alginate was dissolved in physiological saline to prepare 2 w / v % and 4 w / v % solutions.

[0122] 1.3. Preparation of a mixed solution of transplant cells (bone marrow mesenchymal stem cells: MSCs) or bone marrow aspirate concentrate (BMAC) and sodium alginate

[0123] BMACs were prepared according to the BMAC preparation protocol (Chahla J et al., Arthrosc Tech. (2017) 6 (2), e441-e445.). First, after anesthesia induction in rabbits (intravenous administration of 0.05 mg / kg pentobarbital), bone marrow fluid was collected from both iliac bones of the rabbits using an 18G needle-equipped syringe containing 0.5 ml of heparin (Novo-heparin 5000 units / 5 mL, Mochida Pharma.Co.Ltd., Tokyo, Japan), and passed through a strainer with a pore size of 200 μm to remove blood clots in the bone marrow fluid. Five ml of the filtered bone marrow fluid was transferred and centrifuged at 800 g for 5 min at 4°C. Using a pipette tip, the cell fluid was aspirated from the monocytic cell layer (Buffy coat) in the middle layer, including a small amount of plasma component (PRP) immediately above it, while being careful not to aspirate the RBCs below as much as possible. This was used as the BMAC.

[0124] Allogeneic MSCs for transplantation were prepared by expanding BMAC purified by the same method as above in MSC medium on a plate. The cells after two passages were used as MSCs for transplantation (Passage 3). Mix MSCs with endotoxin-free sodium alginate solution (sometimes called "UPAL") to a final MSC concentration of 2 × 10 4 The UPAL+MSC group was used as a transplant solution for transplantation into the defect area (allogeneic transplantation).

[0125] For the BMAC for transplantation, the purified BMAC cell solution was mixed with an equal amount of 4% UPAL (final MSCs concentration was 2 × 10 4cells / ml, final UPAL concentration was 2%) and transplanted (autologous transplant) into the defect as a transplant solution for the UPAL+BMAC group.

[0126] Here, MSC medium was prepared by adding 10% FBS (10828028SP, Gibco) and antibiotics (penicillin G: 100 U / ml and streptomycin: 0.1 mg / ml) to DMEM-High Glucose (044-29765, WAKO).

[0127] Each knee was randomly assigned to one of four groups: no treatment (Defect group), UPAL transplantation alone (UPAL group), UPAL and MSCs combined transplantation (UPAL+MSC group), or UPAL and BMAC combined transplantation (UPAL+BMAC group) (Figure 1).

[0128] In the UPAL, UPAL+MSC, and UPAL+BMAC groups, an 18G needle was used to carefully fill the defect with the graft solution so that it was at the same height as the cartilage surrounding the graft, and a 100 mM calcium chloride solution was dripped onto the surface for 30 to 40 seconds to gel the surface of the sodium alginate solution. The surgical field was thoroughly washed with saline, and the joint capsule and skin were sutured with 4-0 nylon thread to complete the surgery. After waking up from anesthesia, the animals were allowed to move freely in the cage without any particular restrictions. The animals were euthanized 4 and 16 weeks after surgery, and the knees were removed.

[0129] Four weeks after surgery, ten knees from each group were evaluated macroscopically and histologically. Micro-CT measurements were also performed to evaluate the amount of subchondral bone repair. Sixteen weeks after surgery, in addition to the same evaluations as four weeks after surgery, ten knees from each group were also evaluated immunohistochemically and for collagen orientation. Furthermore, six weeks after surgery, mechanical properties were evaluated for six knees from each group.

[0130] 1.4. Methods of macroscopic, histological and immunohistochemical evaluation After euthanasia at 4 and 16 weeks after surgery, the knee joints, including the femoral trochlea, were excised and the defects were photographed with a digital camera. The repair tissue in each group was evaluated macroscopically using the ICRS scoring system (Brittberg M et al., ICRS Newsletter (1998) 1, 5-8.) (Table 2) (n=10).

[0131] [Table 2]

[0132] To use the same specimens for histological evaluation, samples were fixed in 10% paraformaldehyde, decalcified with 10% EDTA, and then 5-μm-thick paraffin sections were prepared at the sagittal plane passing through the center of the defect. The sections were stained with Hematoxylin Eosin (HE) and Safranin-O (Saf-O), and the repair tissues of each group were histologically evaluated using the Niederauer scoring system (Niederauer et al., Biomaterials (2000) 21, 2561-2574.) (Table 3) (n=10).

[0133] [Table 3]

[0134] Sixteen weeks after surgery, the tissue was stained with type II collagen antibody (FujiPharma Co., Ltd., Toyama, Japan) for immunohistochemical evaluation.

[0135] 1.3. Evaluation method of collagen orientation using a polarizing microscope At 16 weeks after surgery, HE-stained sections were observed using a polarized light microscope (PLM) to evaluate collagen orientation in the repaired tissue (Roberts et al., 2009; Ross et al., 2013). Collagen orientation has also been reported to be related to the mechanical strength of articular cartilage (Rieppo et al., 2003). To confirm collagen orientation in the repaired cartilage tissue, the tissue sections themselves were rotated 0°, 45°, and 90° to observe subtle changes. Images taken with a digital camera (DS-5M-L1; Nikon) were evaluated using the PLM qualitative scoring system (Changoor et al., Osteoarthritis Cartilage. (2011) 19(1), 126-135) (Table 4) (n=10).

[0136] [Table 4]

[0137] 1.5.Method of evaluating the amount of subchondral bone repair using Micro-CT As described above, the above-mentioned excised knee samples were macroscopically evaluated, and then, in order to evaluate the subchondral bone repair status of the repair tissue, the subchondral bone volume was quantitatively measured by micro-CT (R_mCT2; Rigaku, Tokyo, Japan) at 4 and 16 weeks after surgery (n = 10). CT scans of the specimens were taken for each knee sample. The imaging method and conditions were as described in Baba et al., Tissue Eng Part C Methods. (2015) 21 (12), 1263-73.; and Hishimura et al., The American Journal of Sports Medicine (2019) e47 (2), 468-478. Among the images obtained, the osteochondral defect of the actual defect (cylindrical shape with a diameter of 5 mm and a depth of 2 mm) was set as the target area, and the amount of calcified bone within that range was calculated as the subchondral bone repair volume (Bone Volume, hereafter referred to as BV) (mm 3) and calculated using Image J software (National Institutes of Health, Bethesda, MD).

[0138] 1.6.Measuring methods for mechanical properties To measure the mechanical properties of the repair tissue in the defect of each knee sample 16 weeks after surgery, we used a mechanical testing machine (Autograph AG-X, Shimadzu Co., Tokyo, Japan) owned by our research facility (Sadia et al., Macromolecules (2016) 49, 5630-5636.). Each knee sample was fixed on a base of dental self-polymerizing resin (PMMA) so that it faced perpendicularly to the indenter (hemispherical shape with a diameter of 2 mm) to be measured (Wada et al., Acta Biomater. (2016) 15, 44, 125-34.), and the indenter was pressed into the measurement site at a speed of 0.5 mm / min (Figure 6A). In addition to the center of the repair tissue, the normal cartilage proximal to the defect was also measured as a reference value for the measurement (Figure 6B). The load-deformation curve was plotted, and the slope of the first part of the curve that approximated the straight line, which reflected the minute deformation of the surface of the measured tissue, was calculated as stiffness (N / mm). The calculated stiffness of each sample was converted into a percentage based on the normal cartilage and compared as standardized stiffness.

[0139] 1.7.Statistical analysis Data are expressed as mean ± standard deviation (SD). Steel-Dwass test was used to compare data between four groups, and Mann-Whitney U test was used to compare data between two groups. Analysis was performed using statistical analysis software JMP Pro 14.1 (SAS Institute, Cary, NC), and the significance level was set to less than 0.05.

[0140] 2.Results 2.1. Results of visual evaluation No findings suggestive of postoperative infection were observed in samples from either group at 4 or 16 weeks.

[0141] Four weeks after surgery, the defect in the Defect group was depressed and incompletely covered with opaque fibrous tissue with an irregular surface (Fig. 2A). The defect in the UPAL and UPAL+MSC groups was somewhat flattened, but was partially covered with translucent tissue with visible cracks or small depressions on the surface or in the surrounding fusion area (Fig. 2B, C). In the UPAL+BMAC group, the defect was almost flat and completely covered with translucent hyaline cartilage-like tissue with only slight cracks on the surface (Fig. 2D). The overall macroscopic scoring (ICRS score) was significantly higher in the UPAL+BMAC group (8.00±1.83) than in the Defect group (mean±SD: 1.70±1.06, P<0.01), UPAL group (5.1±1.45, P<0.05), and UPAL+MSC group (5.5±1.35, P<0.05) (Fig. 2E).

[0142] At 16 weeks after surgery, the defect in the Defect group had become somewhat flat, but had been repaired with opaque tissue with large cracks or depressions visible on the surface or at the periphery (Fig. 2F). The defect in the UPAL and UPAL+MSC groups had become almost flat, and was completely covered with translucent tissue with small cracks or depressions visible on the surface (Fig. 2G,H). In the UPAL+BMAC group, the defect had become almost similar in shape to the surrounding normal cartilage, and was completely covered with smooth, transparent hyaline cartilage-like tissue on the surface (Fig. 2I). Overall macroscopic scoring (ICRS score) was significantly higher in the UPAL+BMAC group (11.20±0.63) than in the Defect group (4.30±1.42, p<0.01), UPAL group (7.90±1.29, p<0.01), and UPAL+MSC group (9.20±1.03, p<0.01) at 4 weeks (Fig. 2J).

[0143] 2.2. Results of histological evaluation Four weeks after surgery, fibrous scar tissue was observed in the defect area in the defect group (Fig. 3A). In the UPAL and UPAL+MSC groups, hyaline-like cartilage tissue that was partially stained with Safranin-O was observed in the defect area (Fig. 3B, C). On the other hand, in the UPAL+BMAC group, hyaline-like cartilage tissue that was slightly thicker than the surrounding area and stained well with Safranin-O was observed in the defect area (Fig. 3D). In the magnified image (x100), moderate inflammatory cell infiltration was observed in the repair tissue in the defect group, whereas almost no signs of inflammation were observed in the UPAL, UPAL+MSC, and UPAL+BMAC groups (Fig. 3E-H). The Niederauer score was significantly higher in the UPAL+BMAC group (16.70±1.83) than in the Defect group (4.70±1.89, P<0.001), UPAL group (10.0±3.33, P<0.01), and UPAL+MSC group (12.2±2.90, P<0.05) (Figure 3I).

[0144] Next, at 16 weeks after surgery, hyaline-like cartilage tissue was observed in the repair tissue in the defect group from 4 weeks after surgery, but more than half of it was fibrous tissue that did not stain with Safranin-O, and Safranin-O staining of the normal cartilage around the defect was also reduced, suggesting degeneration (Fig. 3J). In the UPAL and UPAL+MSC groups, half to more than half of the repair tissue was made up of hyaline-like cartilage stained with Safranin-O (Fig. 3K, L). In contrast, in the UPAL+BMAC group, the defect was covered with hyaline-like cartilage tissue that was strongly stained with Safranin-O, which had almost the same properties and thickness as the normal cartilage around the defect (Fig. 3M). In addition, no obvious inflammation was observed in all groups in the magnified image (×100). In the defect, UPAL, and UPAL+MSC groups, moderate to mild formation of high-density spindle cells and cell clusters was observed in the repair tissue, while in the UPAL+BMAC group, it was filled with normal-density round cells, and cell clusters were hardly observed. In addition, in the repair of subchondral bone, while almost no repair was observed in the defect group, insufficient repair of subchondral bone and very irregular cartilage / subchondral bone interface were observed in the UPAL group and the UPAL+MSC group, and extensive repair of subchondral bone with regular cartilage / subchondral bone interface was observed in the UPAL+BMAC group (Fig. 3N-Q). The Niederauer score was significantly higher in the UPAL+BMAC group (24.40±1.65) than in the defect group (9.00±3.65, P<0.001), UPAL group (14.20±3.88, P<0.01), and UPAL+MSC group (16.30±3.62, P<0.01) (Fig. 3R). The results of the histological scores by item are shown (Table 5).

[0145] [Table 5]

[0146] 2.3. Evaluation results of collagen orientation At 16 weeks after surgery, the repair tissues in each group were evaluated using the PLM qualitative score as described above. In the defect group, the deep layer had almost no vertical alignment and the other two layers had little alignment (Fig. 4E, I, M). In the UPAL and UPAL+MSC groups, the deep layer had only partial vertical alignment, and the other two layers were slightly seen, but the thickness of each layer was irregular (Fig. 4F, J, N and Fig. 4G, K, O). On the other hand, in the UPAL+BMAC group, the deep layer had a vertical alignment and the transition zone, and the superficial layer had a horizontal alignment, with each layer having a somewhat regular thickness (Fig. 4H, L, P). The UPAL+BMAC group had a significantly higher score (2.00 ± 0.82) than the defect group (0.20 ± 0.63, P < 0.01) and the UPAL group (0.70 ± 0.67, P < 0.01). There was no significant difference from the UPAL+MSC group (1.40±1.07, P=0.73) (Figure 4Q).

[0147] 2.4. Results of subchondral bone repair Four weeks after surgery, almost no subchondral bone repair was observed in the defect group, and only slight or partial repair was observed in the UPAL and UPAL+MSC groups. In the UPAL+BMAC group, most of the bone had been repaired, with only a few cracks remaining (Figures 5A-D). The amount of repaired subchondral bone quantitatively measured using micro-CT was significantly greater in the UPAL+BMAC group (6.28 ± 1.98 mm 3 ) is the defect group (2.68±0.89mm 3 , P < 0.01) and the UPAL group (3.59 ± 0.88 mm 3 , P<0.05). 3 , P = 0.387). The amount of subchondral bone repair in all groups was significantly different from that in the normal group (Fig. 5E) (normal group: 18.17 ± 0.17 mm 3 , P < 0.01 for all groups) (Figure 5F).

[0148] At 16 weeks after surgery, partial repair of the subchondral bone was observed in the Defect and UPAL groups, with large or moderate defects remaining. In the UPAL+MSC group, repair was observed to the extent that small cracks remained in the subchondral bone. In contrast, the subchondral bone in the UPAL+BMAC group was almost completely repaired, and a regenerated bone structure with good continuity with the surrounding subchondral bone was observed, suggesting bone union. No excessive bone formation was observed in any of the specimens (Fig. 5G-J). Quantitative measurement of the amount of subchondral bone repair showed that the UPAL+BMAC group (17.28 ± 0.77 mm) was the same as at 4 weeks. 3 ) is the defect group (11.37±1.20mm 3 , P < 0.01) and the UPAL group (12.92 ± 1.50 mm 3 , P<0.01). 3 , P=0.579), there was a tendency for the average bone mass to increase, but no clear significant difference was observed between the two groups. 3 ), the defect group (P<0.01), UPAL group (P<0.01), and UPAL+MSC group (P<0.05) had significantly lower values ​​than the UPAL+BMAC group (17.28±0.77mm 3 , P = 0.12) and the significant difference between the normal group disappeared (Figure 5K).

[0149] 2.5.Evaluation results of mechanical properties At 16 weeks after surgery, mechanical tests were performed using an indentation tester to evaluate the mechanical properties of the repair tissue. Representative indentation-deformation curves for the repair tissue in each group are shown (Figure 6C). The stiffness values ​​(N / mm) were calculated from the region where the slope of the curve first became constant (0-0.08 mm) and standardized as described above (Defect group R 2 =0.986±0.018, UPAL group R 2 =0.995±0.004, UPAL+MSC group R 2 =0.996±0.001, UPAL+BMAC group R 2= 0.987 ± 0.018). The standardized stiffness value (stiffness%) was significantly higher in the UPAL+BMAC group (70.69% ± 5.75%) than in the defect group (20.70% ± 3.71%, P < 0.05), UPAL group (34.19% ± 4.63%, P < 0.05), and UPAL+MSC group (42.89% ± 7.43%, P < 0.05) (Figure 6D).

[0150] 3. Discussion Macroscopic, histological, and mechanical aspects showed that the repair tissue was significantly improved in the UPAL gel and BMAC combination group compared to the UPAL alone group and the UPAL gel and MSCs combination group. These results suggest that the synergistic effect of UPAL gel and BMAC promotes better repair with hyaline cartilage and subchondral bone when transplanted together. In particular, the subchondral bone volume in the UPAL gel and BMAC combination group 16 weeks after surgery was restored to a level that was not different from that of the normal group. It is generally believed that subchondral bone is difficult to treat, so the fact that such an effect was obtained was unexpected.

[0151] These results suggest that the combined implantation therapy of UPAL gel and BMAC may have a good therapeutic effect on large osteochondral defects.

[0152] The combination of UPAL gel, a locally injectable biomaterial, and concentrated bone marrow aspirate may be an effective one-stage treatment for large articular cartilage or osteochondral defects. This treatment may also be a new one-stage treatment that can be performed arthroscopically, instead of autologous osteochondrocyte implantation (ACI) or autologous osteochondral trabecular transplantation (AOT), which are the main treatments for severe cartilage or osteochondral defects in Japan.

Claims

1. A composition for treating cartilage damage, which is applied to a subject's cartilage damaged area in combination with concentrated bone marrow fluid and which has flowability when applied to the cartilage damaged area, contains a monovalent metal salt of alginic acid.

2. The composition of claim 1 , wherein the concentrated bone marrow fluid is derived from a subject.

3. The composition according to claim 1 or 2, wherein the application is carried out by either of the following (1) or (2). (1) mixing a concentrated bone marrow fluid with a composition containing a monovalent metal salt of alginic acid, and applying the resulting mixture to a cartilage injury site of a subject; (2) A composition containing a monovalent metal salt of alginic acid and concentrated bone marrow fluid are each applied to the damaged cartilage site of a subject without being mixed with each other prior to application to the damaged cartilage site.

4. The number of bone marrow mesenchymal stem cells in the concentrated bone marrow fluid is 1 x 10 ２ ~1x10 ７ The composition according to any one of claims 1 to 3, wherein the concentration is 100% by weight per mL.

5. The composition according to any one of claims 1 to 4, wherein the cartilage damage is accompanied by damage to the subchondral bone.

6. The size of the cartilage damage is 4 cm ２ The composition according to any one of claims 1 to 5.

7. The composition according to any one of claims 1 to 6, wherein the cartilage damage is in articular cartilage.

8. The composition according to any one of claims 1 to 7, wherein the apparent viscosity of the flowable composition before being combined with concentrated bone marrow fluid is 500 mPa·s to 10,000 mPa·s as measured using a cone-plate viscometer (Sensor 35 / 1), the measurement is performed at a temperature of 20°C, a rotation speed of 0.5 rpm, and a reading time of 2.5 minutes, with the average value being taken from the start of the measurement at 0.5 minutes to 2.5 minutes.

9. The composition according to any one of claims 1 to 8, wherein the monovalent metal salt of alginic acid has a weight average molecular weight (absolute molecular weight) of 80,000 or more as measured by a GPC-MALS method.

10. The composition according to any one of claims 1 to 9, wherein the concentration of the monovalent metal salt of alginic acid in the composition before being combined with the concentrated bone marrow fluid is 0.5 w / w % to 5 w / w %.

11. The composition according to any one of claims 1 to 10, wherein the composition is used so as to contact a hardening agent with at least a portion of a surface of the composition after application to a cartilage injury site.

12. The composition according to any one of claims 1 to 11, wherein the composition does not contain an amount of a hardening agent that hardens the composition prior to application to the cartilage injury of the subject.

13. The composition according to claim 11 or 12, wherein the curing agent is a divalent or higher metal ion compound.

14. The composition according to any one of claims 1 to 13, wherein the monovalent metal salt of alginic acid is a low-endotoxin monovalent metal salt of alginic acid.

15. The composition according to any one of claims 1 to 14, wherein the composition is used for the treatment of a cartilage-related disease.

16. The composition according to claim 15, wherein the cartilage-related disease is at least one cartilage-related disease selected from the group consisting of osteoarthritis, traumatic cartilage defect, peri-defect traumatic cartilage defect, traumatic cartilage damage, osteonecrosis, osteochondritis dissecans, subchondral bone defect, degeneration of cartilage and / or subchondral bone, and alignment abnormalities in joints.

17. The composition according to any one of claims 1 to 16, wherein the composition is used for at least one selected from the group consisting of repair of cartilage tissue and / or subchondral bone, regeneration of cartilage tissue and / or subchondral bone, inhibition of the progression of degeneration of cartilage tissue and / or subchondral bone, regeneration of hyaline cartilage, relief of pain, relief of postoperative pain, reduction of functional disorders, relief of clinical symptoms, and prevention or inhibition of recurrence of cartilage-related diseases.

18. A kit for treating cartilage damage, comprising at least the composition according to any one of claims 1 to 17 and a hardening agent.

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