KR20180010948A - Composition including mesenchymal stem cell derived from adipose tissue and hyaluronic acid derivative and method for preparing the same - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating backache comprising mesenchymal stem cells derived from adipose tissue and a hyaluronic derivative solution, and a method for preparing the same. The mesenchymal stem cells having excellent cartilage cell differentiation potency is mixed with a hyaluronic derivative solution of which reaction conditions of hyaluronic derivatives are controlled to be used as a composition for preventing or treating diseases caused by a degenerative change in a disk and backache.

Description

[0001] The present invention relates to a composition comprising adipose tissue-derived mesenchymal stem cells and hyaluronic acid derivatives, and a method for producing the same,

To a pharmaceutical composition for preventing or treating low back pain and a method for producing the same.

Back pain is a common disease that affects 84% of the population at least once in their lifetime. The main causes of back pain are degenerative changes in the intervegetal disc, and degenerative changes in the discs are irreversible diseases without effective treatment. The intervertebral disc consists of nucleus pulosus and anulus fibrosus. The main constituent of the nucleus pulposus cell (nucleus pulposus cell), glycoprotein (proteoglycan) and type 2 collagen (type 2 collagen). Degenerative changes in the disc are caused by a decrease in nucleus pulposus cells and a gradual decrease in glycoprotein and type 2 collagen, and a decrease in water content in the nucleus pulposus. Resulting in a general degenerative change in the posterior spinal joints and vertebrae, resulting in severe pain and limited activity for the patient.

Conservative therapies such as medication and physical therapy are used to treat back pain caused by degenerative discs. If conservative therapy is not effective, surgical treatment such as disc removal, spinal fusion, or artificial intervertebral disc insertion may be considered. Surgical treatment has the effect of reducing the pain for a short period of time, but in the long term, the degenerative changes may worsen and the spinal instability may lead to more severe back pain.

Adipose tissue-derived mesenchymal stem cells can be used as one of the cells capable of regenerating the intervertebral disc. Adipose tissue-derived mesenchymal stem cells have the ability to differentiate into osteoblasts, chondroblasts, or adipocytes in appropriate differentiation conditions. Research is needed on biological and fundamental cell therapy for regenerating disc cartilage using mesenchymal stem cells derived from autologous tissue.

One aspect provides a pharmaceutical composition for preventing or treating low back pain comprising a fat tissue-derived mesenchymal stem cell and a hyaluronic acid derivative solution.

Another aspect provides a method of preparing a pharmaceutical composition for preventing or treating low back pain comprising adipose tissue-derived mesenchymal stem cells and a hyaluronic acid derivative solution.

When the hyaluronic acid derivative solution is administered to the intervertebral disc together with the adipose tissue-derived mesenchymal stem cells, the hyaluronic acid derivative solution which maintains a constant viscosity with the adipose tissue-derived mesenchymal stem cells prevents the stem cells from leaking It is possible to more effectively treat the back pain caused by the degenerative change of the intervertebral disc, and the present invention has been completed.

Hereinafter, the present invention will be described in more detail. All technical terms used in the present invention are meant to be understood by those skilled in the art to which the present invention pertains in general, unless otherwise defined. In addition, preferred methods or samples are described in this specification, but similar or equivalent ones are also included in the scope of the present invention.

One aspect provides a pharmaceutical composition for preventing or treating low back pain comprising a fat tissue-derived mesenchymal stem cell and a hyaluronic acid derivative solution.

The stem cell refers to a cell capable of developing into any tissue. There are two basic features: self-renewal, which creates itself by repeated division, and multipotency, which can differentiate into cells with specific functions depending on the environment. The mesenchymal stem cell (MSC) is a multipotential stem cell capable of differentiating into mesodermal cells such as bone, cartilage, fat, muscle cells, and various mesodermal cells or nerve cells. The adipose tissue-derived mesenchymal stem cells are similar in cell shape and immunophenotype to mesenchymal stem cells derived from bone marrow or umbilical cord blood, and have a higher yield because of their higher frequency of cell clustering than cord blood-derived mesenchymal stem cells. In addition, since the fat tissue is extracted from the autologous fat, there is no danger of immunity rejection, and there is no ethical problem. The fat tissue extraction or inhalation procedure is a method of extracting the mesenchymal stem cells, The bone marrow-derived mesenchymal stem cells are relatively small compared to bone marrow aspirate. The adipose tissue can be obtained from adipose tissues such as humans, mice, rats, dogs, and cows.

The hyaluronic acid is a glycosaminoglycan, which is an essential element of the extracellular matrix (ECM), and is a kind of mucopolysaccharide composed of amino acid and uronic acid. The monomer, N-acetylglucosamine, And D-glucuronic acid are continuously connected to each other in a linear polysaccharide. In addition, hyaluronic acid is a biocompatible substance that is essential for cell morphogenesis, cell differentiation, and cell division, and is a biocompatible substance that helps restore the wound. Hyaluronic acid exhibits excellent viscoelasticity and high water absorbing ability while being insoluble in an aqueous solution through an ether bond, and is maintained in the form of a body for a certain period of time and is decomposed and absorbed in the body. Natural hyaluronic acid is rapidly degraded by hyaluronidase when injected into the body. Therefore, in order to control the rate of such degradation, the hyaluronic acid is crosslinked by various methods, or the hyaluronic acid modified by using a chemical substance such as benzyl alcohol Lonic acid derivative can be used.

The hyaluronic acid may include hyaluronic acid itself, a salt of hyaluronic acid, or a mixture thereof. The salt of hyaluronic acid may be in any salt form suitable for application to a living body and may be in the form of any salt suitable for application to a living body and may be in the form of sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate, tetrabutylammonium hyaluronate, . ≪ / RTI >

The hyaluronic acid derivative may be a hyaluronic acid crosslinked product in which hyaluronic acid, hyaluronic acid salt, or a mixture thereof is crosslinked by a crosslinking agent.

The hyaluronic acid derivative solution may be a solution in which the hyaluronic acid derivative is dissolved in physiological saline solution, phosphate buffered saline solution or biocompatible saline solution.

The pH of the composition may be from 5 to 8.5, or from 5.5 to 7. As the pH of the composition is within the above range, the mesenchymal stem cells can be retained in the intervertebral disc for a long time without affecting the survival of adipose derived mesenchymal stem cells.

When the composition is transplanted into an intervertebral disc, cells of surrounding human tissues migrate into the composition. Although the cells secrete the extracellular matrix and some components of the composition are degraded, the adipocyte-derived mesenchymal stem cells transplanted with the secreted extracellular matrix can sustain cell adhesion, cell survival and regeneration ability.

The volume ratio of the adipose tissue-derived mesenchymal stem cell and the hyaluronic acid derivative solution may be 1: 3 to 3: 1, 1: 2 to 2: 1, 1: 1.5 to 1.5: 1, or about 1: 1. In the case of mixing in the above volume ratio, the swelling ratio of the composition is constant even when the temperature or the pH is changed, so that the composition is structurally stable. Therefore, the cell adhesion, cell survival and regeneration ability of the adipose tissue-derived mesenchymal stem cells can be maintained for a long time even after the composition is transplanted into an intervertebral disc. Alternatively, the composition may be a mixture of pellets of adipose tissue-derived mesenchymal stem cells and the hyaluronic acid derivative solution.

The viscosity of the hyaluronic acid derivative solution may be 150 to 900 Pa.s, 200 to 900 Pa.s, or 250 to 900 Pa.s. Specifically, the viscosity of the hyaluronic acid derivative solution may be from 150 to 900 Pa.s, from 200 to 900 Pa.s, or from 250 to 900 Pa.s, in a shear rate range of from 200 to 1400 1 / s have. The viscosity of the composition may be from 150 to 900 Pa.s, from 160 to 700 Pa.s, from 170 to 500 Pa.s, or from 200 to 300 Pa.s. Specifically, the viscosity of the composition can range from 150 to 900 Pa.s, from 160 to 700 Pa.s, from 170 to 500 Pa.s, or from 200 to 300 Pa.s, at a shear rate range of from 200 to 1400 1 / have. When the composition having the above range of viscosity and elasticity is transplanted into an intervertebral disc, it is possible to prevent cells from leaking out and to prevent abnormal growth of osteophyte growth in the anterolateral intervertebral disc space.

The content of the hyaluronic acid derivative in the hyaluronic acid derivative solution is preferably 0.7 to 3 w / v%, 0.8 to 2.5 w / v%, 0.8 to 2.3 w / v%, 1.0 to 2.1 w / v%, or 1.3 to 2.1 w / v%. Wherein the content of the hyaluronic acid derivative in the composition is 0.7 to 3 w / v%, 0.7 to 2.5 w / v%, 0.7 to 2.3 w / v%, 0.7 to 2.1 w / v%, 0.7 to 2 w / v% To 1.8 w / v%, 0.8 to 1.5 w / v%, 0.9 to 1.5 w / v%, or 0.9 to 1.3 w / v%. When the content of the hyaluronic acid derivative in the composition is less than 0.7, there is a fear that the hyaluronic acid derivative solution can not adhere to the intervertebral disc. When the content of the hyaluronic acid derivative in the composition is more than 3 w / v%, there is a fear that the viscosity and elasticity of the solution are not homogeneous throughout the solution.

The weight average molecular weight of the hyaluronic acid derivative may be from 100 Da to 10,000 kDa, from 1,000 Da to 6,000 kDa, from 6,000 Da to 4,000 kDa, from 100 kDa to 3,000 kDa, or from 300 kDa to 3,000 kDa. In the range of the weight average molecular weight, the viscosity and elasticity of the hyaluronic acid derivative solution can be kept relatively constant even if the hyaluronic acid derivative swells.

The size of the hyaluronic acid derivative may be 100 to 3,000 μm, 100 to 2,000 μm, and 300 to 1,500 μm. The hyaluronic acid derivative may be in the form of particles, microparticles or micro beads.

The degree of crosslinking of the hyaluronic acid derivative may be 10 to 100%, 30 to 90%, or 50 to 90%. This is to match the rate of degradation of hyaluronic acid derivatives to the normal period of cartilage regeneration.

The hyaluronic acid derivative may be one obtained by crosslinking hyaluronic acid, hyaluronic acid salt, or a mixture thereof with a cross-linking agent. In this case, the equivalent ratio of the crosslinking agent may be 0.01 to 500%, 0.1 to 400%, 1 to 300%, 10 to 250%, or 50 (%) relative to the repeating unit of hyaluronic acid, hyaluronic acid salt, To 150%. When the equivalent ratio of the crosslinking agent is less than the above range, hyaluronic acid derivatives in the form of particles may not be formed, and when exceeding the above range, a target viscosity and elasticity may not be obtained . The viscosity and elasticity of the hyaluronic acid derivative solution may be changed by controlling the equivalent ratio of the crosslinking agent.

The crosslinking agent may be an epoxy crosslinking agent having two or more epoxy functional groups, and may be 1,4-butanediol diglycidyl ether (BDDE), ethylene glycol diglycidyl ether (EGDGE) Hexanediol diglycidyl ether, propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene < RTI ID = 0.0 > But are not limited to, polytetramethylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, polyglycidyl ether, glycerol polyglycidyl ether, tri-methylpropane polyglycidyl ether, ether, bis (2,3-epoxypropoxy) ethylene, pentaerythritol polyglycidyl ether, sorbitol polyglycidyl ether ), Or any combination thereof.

The degree of swelling of the hyaluronic acid derivative may be 100 to 3,000%, 100 to 2,000%, or 400 to 1200%. The degree of swelling of the hyaluronic acid derivative may be such that the swelling occurs in the range of the weight average molecular weight and the degree of crosslinking of the hyaluronic acid derivative.

The adipose tissue-derived mesenchymal stem cells may be those having the ^{CD44 +, CD73 +, CD90 +} , CD105 +, CD29 +, CD49 + and HLA (human leukocyte antigen) ⁺ -ABC the immunological properties. In addition, the adipose tissue-derived mesenchymal stem cells may have immunological characteristics such as CD45 ^- and HLA (human leukocyte antigen) -DR ^- .

The adipose tissue-derived mesenchymal stem cells may have immunological properties of TGF-beta receptor ⁺ . The TGF- [beta] receptor may be TGF- [beta] receptor I, TGF- [beta] receptor II or TGF- [beta] receptor III. The adipose-derived mesenchymal stem cells, which express TGF-β receptor, are able to stimulate TGF-β-1 signaling, and may further stimulate the disc cartilage forming ability in response to autologous TGF-β-1.

The adipose tissue-derived mesenchymal stem cells are obtained by separating adipocytes, red blood cells, cell lysates, etc. from adipose tissues obtained by liposuction or the like using a method such as washing and filtration, and then culturing them in a culture medium for stem cell culture have. The adipose tissue-derived mesenchymal stem cells can be obtained by subculturing stem cells isolated from adipose tissue for 3 to 17 passages or 3 to 14 passages. The mesenchymal stem cells derived from adipose tissue cultured in excess of 17 passages may have a tendency to slow the cell proliferation and decrease the cell survival rate.

The adipose tissue-derived mesenchymal stem cells may be in the form of a spindle.

The back pain may occur due to degenerative changes of the disc. Degenerative changes in the disc may be accompanied by physiological and chemical alterations that cause pain in the intervertebral disc. There is no nerve or vascular tissue in the intervertebral disc. However, if the intervertebral disc is degenerated and cracked, the sinuvertebral nerve that is distributed outside of the fibrous ring and the blood vessels grow inward to promote degenerative changes that cause pain, Inflammatory substances such as Substance P are actively released in the intervertebral disc, resulting in intradiscal disruption pain in which a pathogen is generated in the intervertebral disc. Such back pain may also occur in herniated disc, scoliosis or spinal stenosis.

The content of the adipose tissue-derived mesenchymal stem cells may be 1 x 10 ⁶ to 1 x 10 ⁸ cells / ml. The dose of the adipose tissue-derived mesenchymal stem cells may be 5 x 10 ⁵ to 5 x 10 ⁷ cells / individual. Specifically, the dose of the adipose tissue-derived mesenchymal stem cells is 0.1 x 10 ⁵ to 10 x 10 ⁵ cells / kg, 0.5 x 10 ⁵ to 10 x 10 ⁵ cells / kg, 1 x 10 ⁵ to 10 x 10 ⁵ cells / cell / kg, or 2 x 10 ⁵ to 8 x 10 ⁵ may be contained within the cell / kg composition. When the dose is less than 5 x 10 ⁵ cells / individual or 0.1 x 10 ⁵ cells / kg on the basis of one administration, it is difficult to expect an objective disc regeneration effect. Pulmonary embolism may be induced if the dose is 5 x 10 ⁷ cells / individual or 10 x 10 ⁵ cells / kg on a single administration.

The hyaluronic acid derivative solution may be in the form of a hydrogel. The hydrogel refers to a gel containing water as a dispersion medium. The hydrosol may lose its fluidity due to cooling, or may be formed by expanding a hydrophilic polymer having a three-dimensional network structure and a microcrystalline structure by containing water.

Another aspect provides a method for preparing a pharmaceutical composition for preventing or treating low back pain, comprising mixing a fat tissue-derived mesenchymal stem cell and a hyaluronic acid derivative solution.

The volume ratio of the adipose tissue-derived mesenchymal stem cell and the hyaluronic acid derivative solution is as described above. The volume ratio of the adipose tissue-derived mesenchymal stem cell and the hyaluronic acid derivative solution may be 1: 3 to 3: 1, 1: 2 to 2: 1, 1: 1.5 to 1.5: 1, or about 1: 1.

The content of the hyaluronic acid derivative in the hyaluronic acid derivative solution is preferably 0.7 to 3 w / v%, 0.8 to 2.5 w / v%, 0.8 to 2.3 w / v%, 1.0 to 2.1 w / v%, or 1.3 to 2.1 w / v%. The content of the hyaluronic acid derivative in the hyaluronic acid derivative solution and the content of the hyaluronic acid derivative in the composition are as described above.

The weight average molecular weight of the hyaluronic acid derivative may be 100 Da to 10,000 kDa, 1,000 Da to 6,000 kDa, 6,000 Da to 4,000 kDa, 100 kDa to 3,000 kDa, or 300 kDa to 3,000 kD. The weight average molecular weight of the hyaluronic acid derivative is as described above.

The hyaluronic acid, the hyaluronic acid salt, or a mixture thereof is dissolved in an aqueous alkali solution of 0.1 to 0.5 N at a concentration of 50 to 250 mg / ml before the step of mixing the adipose tissue-derived mesenchymal stem cell and the hyaluronic acid derivative solution Thereby preparing a hyaluronic acid solution; Adding a crosslinking agent to the hyaluronic acid solution in an equivalent ratio of 0.01 to 500% to the repeating units of hyaluronic acid, hyaluronic acid, or a mixture thereof to prepare a hyaluronic acid derivative solution.

The hyaluronic acid, hyaluronic acid salt or a mixture thereof may be dissolved in an aqueous alkali solution at a concentration of 50 to 250 mg / ml, 75 to 225 mg / ml, 75 to 210 mg / ml, or 80 to 200 mg / ml . The hyaluronic acid, hyaluronic acid salt, or mixture thereof may be dissolved in an aqueous alkali solution of 0.1 to 0.5 N, 0.15 to 0.4 N, or 0.2 to 0.3 N. The alkali aqueous solution is an aqueous alkali solution having a pH of 9 to 13, which is an aqueous solution of sodium hydroxide, an aqueous solution of potassium hydroxide, or ammonia water. The above-mentioned hyaluronic acid, hyaluronic acid salt, or mixture thereof is as described above.

The crosslinking agent may be added in an equivalent ratio of 0.01 to 500%, 0.1 to 400%, 1 to 300%, 10 to 250%, or 50 to 150% based on the repeating units of hyaluronic acid, the salt of hyaluronic acid, have. The crosslinking agent may be homogeneously mixed with hyaluronic acid, a salt of hyaluronic acid, or a mixture thereof. The equivalent ratio of the crosslinking agent is as described above.

The crosslinking agent may be an epoxy crosslinking agent having two or more epoxy functional groups, as described above.

The adipose tissue-derived mesenchymal stem cells may be those having the ^{CD44 +, CD73 +, CD90 +} , D105 +, CD29 +, CD49 + and HLA (human leukocyte antigen) ⁺ -ABC the immunological properties. In addition, the adipose tissue-derived mesenchymal stem cells may have immunological characteristics such as CD45 ^- and HLA (human leukocyte antigen) -DR ^- . The adipose tissue-derived mesenchymal stem cells may have immunological properties of TGF-beta receptor ⁺ .

The adipose tissue-derived mesenchymal stem cells can be obtained by subculturing stem cells isolated from adipose tissue for 3 to 17 passages or 3 to 14 passages. The passage of the adipose tissue-derived mesenchymal stem cells is as described above.

The adipose tissue-derived mesenchymal stem cells may be in the form of a spindle.

The back pain may be caused by a degenerative change of the disc.

The content of the adipose tissue-derived mesenchymal stem cells may be 1 x 10 ⁶ to 1 x 10 ⁸ cells / ml. The dose of the adipose tissue-derived mesenchymal stem cells may be 5 x 10 ⁵ to 5 x 10 ⁷ cells / individual. Specifically, the dose of the adipose tissue-derived mesenchymal stem cells is 0.1 x 10 ⁵ to 10 x 10 ⁵ cells / kg, 0.5 x 10 ⁵ to 10 x 10 ⁵ cells / kg, 1 x 10 ⁵ to 10 x 10 ⁵ cells / cell / kg, or 2 x 10 ⁵ to 8 x 10 ⁵ may be contained within the cell / kg composition.

A pharmaceutical composition for preventing or treating low back pain comprising a fat tissue-derived mesenchymal stem cell and a hyaluronic acid derivative solution may be provided as a cell therapy agent. The cell treatment agent is a series of cells that are used to multiply, select, or alter biological characteristics of cells in vitro, such as autologous, allogenic, or xenogenic cells in order to restore the functions of cells and tissues It is used for the purpose of treatment, diagnosis, and prevention through the action of somatic cell treatment agent and stem cell treatment agent depending on the type and degree of differentiation of cells. The composition may be provided as a stem cell treatment agent, particularly a mesenchymal stem cell treatment agent.

The composition may further comprise a pharmaceutically acceptable carrier, excipient or diluent depending on the mode of administration. Specifically, it is possible to use a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome and any of the above components, And other additives conventionally used, such as buffers. In addition, depending on the purpose of administration, diluents, dispersants, surfactants, binders, and lubricants can be added to formulate into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets, A target organ or tissue specific antibody or other ligand can be used in combination with the carrier. The carrier, excipient, or additive may be any conventional formulation, and the carrier, excipient, or additive is not limited by the above examples.

Such a composition or mixture may be appropriately administered to a subject according to the purpose or necessity, depending on the conventional methods used in the art, the route of administration, and the dose. Examples of routes of administration include oral, non-oral, subcutaneous, and intraperitoneal. In addition, the appropriate dosage and the frequency of administration can be selected according to methods known in the art, and the amount and the administration frequency of the composition to be actually administered are determined depending on the kind of the prevention or treatment, the administration route, sex, , The age and weight of the individual, and the severity of the disease.

The composition comprising the adipose tissue-derived mesenchymal stem cell and the hyaluronic acid derivative solution is capable of controlling the viscosity of the hyaluronic acid derivative solution according to the reaction conditions of the hyaluronic acid and the hyaluronic acid derivative. The mesenchymal stem cells having remarkable chondrocyte differentiation ability are mixed with the hyaluronic acid derivative solution and transplanted into the intervertebral disc to increase the cell adhesion and cell survival and can be effectively used for the prevention and treatment of diseases and back pain due to the degenerative change of the intervertebral disc have.

Figs. 1A to 1C show the mechanical properties, degree of spread, viscosity, and viscosity using a rheometer of hyaluronic acid derivative solutions of various concentrations.
2 is a flow chart of a method for preparing mesenchymal stem cells derived from adipose tissue for transplantation into a disc.
FIG. 3 is an image showing the morphology of isolated mesenchymal stem cells.
4A to 4C are graphs showing the population doubling level (PDL), the doubling time (dT), and the cell survival rate by cell division of isolated mesenchymal stem cells.
FIG. 5 is an image showing the multipotent ability of adipocytes, osteoblasts and chondrocytes after differentiation inducing substances were added to isolated mesenchymal stem cells.
FIG. 6 is a graph showing the surface expression of the isolated mesenchymal stem cells expressing the mesenchymal stem cell markers.
FIGS. 7A and 7B are graphs showing the expression levels of CD44, CD73, CD90, CD105, CD45, CD29, CD49 and HLA-ABC in isolated mesenchymal stem cells.
FIG. 8 is a graph showing the expression level of TGF-.beta. Receptor III in isolated mesenchymal stem cells. FIG.
FIG. 9 is an image showing the morphology and karyotype of the separated mesenchymal stem cells after subculturing to the 7th passage.
FIG. 10 is a graph showing pfirrmann grade after administration of adipose derived mesenchymal stem cells and hyaluronic acid derivative solution in an animal in which disc degeneration has been induced.
11 is an image showing an experimental procedure according to the tenth embodiment.
12 is a flow chart showing an experimental procedure of a clinical experiment for evaluating disc pain. Under the written consent of the patient to participate in clinical trials, liposuction is performed on the subcutaneous fat layer of the abdomen or thigh of the patient to obtain autologous adipose tissue from the patient. Approximately 3 weeks after the liposuction, the autologous adipose tissue-derived mesenchymal stem cells and hyaluronic acid derivative solution are injected and administered for about 1 week, about 1 month, about 3 months, about 6 months, about 9 months, and about 12 Evaluate the degree of disc herniation over a month.
13A to 13D are MRI images taken before and after transplantation of adipose tissue-derived mesenchymal stem cells and hyaluronic acid derivative solution in Patient 1. FIG. 13 (a) shows the results obtained before transplantation of the adipose tissue-derived mesenchymal stem cell and hyaluronic acid derivative solution into the patient 1, FIG. 13 (b) about 1 month after implantation, FIG. 13c after about 6 months implantation, Image.
14 is a graph showing VAS scores before and after transplantation of adipose tissue-derived mesenchymal stem cells and hyaluronic acid derivative solution in Patients 1 to 10;
15 is a graph showing ODI scores before and after transplantation of adipose tissue-derived mesenchymal stem cells and hyaluronic acid derivative solution in Patients 1 to 10;

Hereinafter, the present invention will be described in further detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.

Example  1: derived from adipose tissue Intermediate lobe  Administration of stem cell and hyaluronic acid derivative solution and confirmation of reduction of intervertebral disc pain

1: Production of hyaluronic acid derivative

A hyaluronic acid derivative solution was prepared as follows. Sodium hyaluronate was dissolved at a concentration of 100 mg / ml in a 0.25 N NaOH solution. To this solution was added BDDE (1,4-butane diol diglycidyl ether) as a crosslinking agent (butanediol diglycidyl ether was added in an equivalent ratio of 100% based on the repeating unit of the hyaluronate) After reaction for 36 hours, the reaction mixture was washed with physiological saline to remove unreacted material. The washed product was pulverized and the particle size was adjusted to prepare a hyaluronic acid derivative (weight average molecular weight: 300 to 3,000 kDa, swelling degree: 400 to 1200%).

2: Preparation of hyaluronic acid derivative solution and its physicochemical properties

2.1: Physical properties of hyaluronic acid derivative solution

The hyaluronic acid derivative of Example 1 was mixed with physiological saline and subjected to differential equilibration at 0.1 w / v% to prepare a hyaluronic acid derivative solution having various concentrations of 0.1 to 4 w / v%. The physical properties of the hyaluronic acid derivative solution were measured, and the suitable physical properties and hyaluronic acid derivative content of the composition for transplanting into the intervertebral disc were determined. The degree of spreading of the hyaluronic acid derivative solution in a general petri dish, the degree of movement of the hyaluronic acid derivative solution after tilting the Petri dish at 45 °, and the rheometer analysis are used to maintain the viscosity against the shear rate Was measured.

As a result, it was confirmed that the hyaluronic acid derivative solution having a concentration of 0.7 w / v% or more takes the form of a gel. At a concentration of 0.7-2 w / v%, the hyaluronic acid derivative solution was maintained at a viscosity of 150-900 Pa.s, confirming that it could be injected into the target disc. Particularly, since the hyaluronic acid derivative solution of 0.9 to 1.3 w / v% has a viscosity range of 200 to 300 Pa.s and an average elasticity of about 250 Pa.s, It was confirmed that the material had optimum physical properties described below. In the above physical properties range, the hyaluronic acid derivative solution is not easily decomposed in vivo and is structurally stable, minimizing the loss of fat stem-derived stem cells lost from the intervertebral disc, It can have an effect of supporting the stem cells stably positioned. On the other hand, when the content of the hyaluronic acid derivative in the hyaluronic acid derivative solution exceeds 3 w / v%, it is confirmed that the viscosity and elasticity of the solution are not very homogeneous.

FIG. 1 represents the physical properties of 0.1, 0.5, 1 and 2 w / v% hyaluronic acid derivative solutions in various concentrations of hyaluronic acid derivative solutions. 1, 2 w / v% of the hyaluronic acid derivative solution and 1 w / v% of the hyaluronic acid derivative solution did not spread when dropped on a Petri dish. On the other hand, it was confirmed that the 0.1% hyaluronic acid derivative solution and the 0.5 w / v% hyaluronic acid derivative solution spread on the petri dish without maintaining the droplet form. In addition, the degree of movement of the hyaluronic acid derivative solution after tilting the Petri dish at 45 ° was checked. As a result, a solution of 2 w / v% of the hyaluronic acid derivative and 1 w / v% of the hyaluronic acid derivative adhered to the petri dish I did.

2.2: pH determination of hyaluronic acid derivative solution

[0156] The hyaluronic acid derivative solution of various concentrations was measured three times in accordance with the pH measurement method of the general test method described in the pharmacopoeia to confirm that the pH of the solution was 5.5 to 8.5.

3: derived from adipose tissue Intermediate lobe  Isolation of stem cells

In 10 patients with degenerative lumbar disc herniation suffering from chronic back pain, adipose tissue was collected from the subcutaneous fat layer of the patient 's abdomen or thigh using liposuction. Cells were proliferated and cultured from the adipose tissues according to the following protocol.

2 is a flow chart of a method for preparing mesenchymal stem cells derived from adipose tissue for transplantation into a disc. After mixing with the same volume of Dulbecco's phosphate buffered saline (DPBS) as the fat tissue, the process of removing the oil from the upper layer by centrifugation was repeated 3 times. Thereafter, the pellet was incubated with an enzyme reaction solution containing HBSS (Hank's balanced salt solution), collagenase I, trypsin, dispase and Dnase I for 1 hour And the cells were separated. Separated cells were placed in a culture medium containing the substrate medium and cultured in an incubator supplied with 5% CO ₂ . The substrate medium was MEM Alpha GlutaMAX containing 10% Fetal Bovine Serum.

When the initially cultured cells reached 70% or more of the area of the culture vessel, TrypLE was evenly distributed on the bottom, reacted for 3 to 5 minutes in an incubator supplied with 5% CO ₂ , and the cells were then removed from the culture vessel . The cells were then cultured in proliferation medium. Growth medium was MEM Alpha GlutaMAX containing 10 ng / ml bFGF and 10% fetal bovine serum. After 3 passages, the cells were reacted with TrypLE, and the cells were separated and suspended in sterilized normal saline.

4: morphology of isolated cells and Proliferative ability  analysis

4.1: Morphology of isolated cells

The morphological changes of the cells were observed under a microscope while the separated cells were continuously subcultured. Fig. 3 is an image showing the morphology of isolated cells. Fig. As a result of continued subculture to the 12th passage, the isolated cells retained the specific shape of spindle fibroblasts with irregular protrusions. The isolated cells showed typical mesenchymal stem cell morphology.

4.2: of isolated cells Proliferative ability  analysis

The isolated cells were continuously subcultured until the level of proliferation of the cells was slowed down. The degree of cell differentiation and cell division time were measured using the trypan blue dye exlusion method at each culture step. FIG. 4 is a graph showing the degree of biodegradation, cell division time, and cell survival rate of isolated cell groups. Cell division time and cell viability did not change until passage 17, but cell proliferation slowed down and cell survival rate decreased to below 95%.

5: Multiparameter  analysis

By adding the differentiation inducing substance to the separated cells, Fat, cartilage and bone.

Cell line
(Cell lineage) Fat cell
(Adipocyte) Cartilage cell
(Chondrocyte) Osteoblast
(Osteoblast) Induction of differentiation
matter Dexamethasone
(Dexamethasone) Ascorbic acid
(Ascorbic acid) Ascorbic acid
(Ascorbic acid) Isobutylmethylate
(Isobutyl methylxanthine) BMP-6 BMP-2 Indomethacin
(Indomethacin) Dexamethasone
(Dexamethasone) Dexamethasone
(Dexamethasone) Insulin Insulin 1,25-dihydroxyvitamin D
(1,25-dihydroxyvitamine D) Thiazolidinedione
(Thiazolidinedione) Transforming growth factor-beta
(Transforming growth factor-β) -

The specimens inducing differentiation into adipocytes were stained with Oil Red O, the samples induced to differentiate into chondrocytes were stained with Alcian blue, and the samples induced to differentiate into osteoblasts were stained with Alizarin Red S. FIG. 5 is an image obtained by inducing differentiation into adipocytes, osteoblasts and cartilage cells, respectively, by adding differentiation inducing substances to the separated cells. Referring to FIG. 5, it can be seen that the isolated cells have differentiation potentials such as mesenchymal stem cells, and that they are induced to differentiate into adipocytes, osteoblasts and chondrocytes, respectively.

6: Intermediate lobe  Stem cell Marker  Confirmation (confirmation of expression of CD44, CD73, CD90, CD105, CD29, CD49 and HLA-ABC)

Flow cytometry was performed to analyze the characteristics of isolated cells and analyzed using FACS accuri C6 instrument (BD Biosciences, Billerica, MA, USA).

FIG. 6 is a graph showing the surface expression of the isolated cells expressing the mesenchymal stem cell marker. Referring to FIG. 6, the isolated cells expressed mesenchymal stem cell markers CD44 and CD73. Moving cells to damaged tissues is a very important factor in cell therapy. CD29 is a homing effect among the surface factors expressed on the cell surface, that is, a factor that helps the cell to move to the infected site or damaged tissue. The degree of expression of CD29 in isolated cells was confirmed. 7a is the result of confirming the expression of CD29 in cells isolated from three individuals. Referring to FIG. 7A, the isolated cells expressed CD29, a mesenchymal stem cell marker. FIG. 7B shows the results of confirming the expression levels of surface factors, CD44, CD73, CD90, CD105, CD45, CD29, CD49 and HLA-ABC in adipose tissue-derived mesenchymal stem cells. Table 2 shows the surface expression rate of mesenchymal stem cells. Expression of CD44, CD73, CD90, CD105, CD29, CD49 and HLA-ABC was high in isolated cell surface. From this, it can be seen that the isolated cells have the characteristics of mesenchymal stem cells.

Surface factor CD44 CD73 CD90 CD105 CD45 CD29 CD49 HLA-ABC HLA-DR Expression rate (%) 99.9 99.9 99.7 99.5 0.523 99.9 99.9 98.5 0.296

7: TGF -β receptor

The expression of TGF-β receptor was confirmed in isolated cells. 8 is a graph showing the expression level of TGF-β receptor III in cells isolated from three individuals. The expression rate of TGF-β Ⅲ in isolated cells was 98.7 ± 1.4% on average.

8: Confirm chromosome stability

In order to continuously pass the separated cells, the effect of repeated cell division on the chromosome stability of the cells was confirmed. After subculturing to the 7th passage, the proliferated cells were separated and the karyotype was analyzed. Fig. 9 is an image showing the morphology and karyotype of the separated cells after subculturing to the seventh passage. Referring to FIG. 9, it can be seen that even when cell division is repeated, the chromosomes of the separated cells show a normal karyotype, and no specific variation appears on the chromosome.

9: derived from fatty tissue in hyaluronic acid derivative environment Intermediate lobe  Toxicity test of stem cells

CCK-8 (cell counting kit-8) analysis was performed to confirm the proliferation of adipose tissue-derived mesenchymal stem cells in the hyaluronic acid derivative environment. Adipose tissue-derived mesenchymal stem cells were seeded on a 96-well plate, and then 0.7, 1, 1.3, and 2 w / v% of hyaluronic acid derivative solution was added to the cells each day. Cells cultured for 1, 3, and 7 days were washed once with phosphate buffered saline (PBS), and then 1 ml of the culture was added and 100 μl of CCK-8 (Dojindo, Tokyo, Japan) And reacted at 37 캜 for 2 hours. From the culture medium of adipose tissue-derived mesenchymal stem cells to which various concentrations of hyaluronic acid derivative solution had been added, 100 占 퐇 was taken and placed in a 96-well plate. The absorbance of the sample was measured with an Elisa plate reader (Power Wave X340; Bio- Tek Instruments, Inc., Winooski, VT) at a wavelength of 450 nm. The cell proliferation rate was converted to the ratio of absorbance to cell proliferation time (Absorbance / day).

In addition, cytotoxicity of each adipose tissue derived mesenchymal stem cell cultured for 3 days was verified in 2D and 3D environment using Live / Dead analysis. Each of the cultured adipose tissue-derived mesenchymal stem cells was washed three times with phosphate-buffered saline, and 2 μM of calcein AM (Invitrogen) and 4 μM of ethidium homodimer (EthD-1, Invitrogen) For 5 minutes, and then live cells (green) and dead cells (red) were observed through a fluorescence microscope.

The cytotoxicity of the hyaluronic acid derivative solution was confirmed, and no cytotoxicity was observed to affect the viability of the cells.

10: derived from adipose tissue Intermediate lobe  Administration of stem cell and hyaluronic acid derivative solution and confirmation of disc moisture content recovery

An IVD (Intervertebral Disc, degenerative lumbar disc) animal model was prepared according to the method described in SaKai D et al (Biomaterial, 2003). Specifically, 28 rabbits (New Zealand White rabbits) were selected, which were 4 to 5 months old and weigh about 3 kg. IVD animal models were prepared by removing nuclei pulposus (NP) from L3 / 4, L4 / 5 and L5 / 6 from the rabbit. The inhaled intervertebral disc piece was observed under a microscope to confirm that the pulp was inhaled. The average weight of the removed nuclei was about 7 mg.

Among 28 rabbits in total, one control group did not induce disc degeneration and administered physiological saline. Three sham groups induced disc degeneration and were given saline solution. Twenty-four experimental groups were divided into eight groups and samples 1 to 8 of Table 3 were administered to each experimental group. The adipose-derived mesenchymal stem cells were injected with 1 x 10 ⁶ cells or 2 x 10 ⁶ cells per animal, and the content of hyaluronic acid derivatives was adjusted to 0.7 to 2 w / v%.

The method of administration is as follows. The adipose tissue-derived mesenchymal stem cell and / or hyaluronic acid derivative solution of Table 3 was administered once to the rabbit discs under fluoroscopic imaging using a 25 gauge needle (see FIG. 11).

division Derived mesenchymal stem cells
(1 x 10 ⁶ cells / 25 μl) Hyaluronic acid derivative solution Control group - - - Sham - - - Experimental group Sample 1
(Concentration: 2 w / v%) - 50 μl
Sample 2 25 μl - Sample 3 50 μl - Sample 4
(Concentration: 1 w / v%) 25 μl 25 μl
Sample 5
(Concentration: 2 w / v%) 25 μl 25 μl Sample 6
(Concentration: 1.3 w / v%) 25 μl 25 μl
Sample 7
(Concentration: 0.9 w / v%) 25 μl 25 μl
Sample 8
(Concentration: 0.7 w / v%) 25 μl 25 μl

Three weeks after the administration, the presence or absence of the adverse reaction was observed by T2 magnetic resonance imaging (MRI). The degree of recovery of the disc water content was confirmed by MRI at 6 and 12 weeks. In addition, the herniated disc herniation grade was evaluated as pfirrmann grade.

As a result, no adverse reaction was found after administration, and the moisture content in the discs was significantly restored after the administration of Samples 4 to 8 as compared to Sample 1. Before administration of the composition containing the adipose-derived mesenchymal stem cells and the hyaluronic acid derivative solution, the MRI images of the Sham group and the experimental group showed blackness due to lack of moisture. On the other hand, the results of MRI of the experimental group administered with Samples 4 to 8 confirmed that the water content in the disc was recovered and appeared white. This can be expected to reduce pain. In particular, after the administration of Samples 4, 6 and 7, the moisture content in the cuticle was recovered to a very high degree.

FIG. 10 is a graph showing the pfirrmann grade of the experimental group to which the control group, the Sham group, the sample 2, the sample 4, the sample 5, and the sample 6 were administered. In the pfirrmann grade, the control group was 1.00 ± 00, the Sham group was 3.12 ± 0.35, the sample 2 was 2.42 ± 0.3, the sample 5 was 2.11 ± 0.40, the sample 4 was 1.65 ± 0.22, And the group to which Sample 6 was administered showed 1.63 ± 0.41. pfirrmann grade was significantly lower in the group to which the sample 4 or 6 was administered.

11: Selection of patients with degenerative lumbar disc herniation and mesenchymal stem cell transplantation derived from adipose tissue

11.1: Selection of patients with degenerative lumbar disc herniation

The patients with lumbar disc herniation were selected according to the following criteria.

A) Men and women aged 19 and over, under 70 years

B) If there is no response to conservative therapy for 3 months or longer due to back pain or hips that lasted more than 6 months

C) More than 30% of the Oswestry Disability Index (ODI)

D) Visual analogue scale (VAS) of 4 or more

E) Magnetic Resonance Imaging (MRI) grades according to the Pfrrmann classification between lumbar spine # 1 and spine # 1 were graded 3 to 4

F) Discography on a degenerative lumbar disc confirmed by MRI causes pain consistent with usual pain, and one or two discs with pain

G) If you have written an agreement on stem cell transplantation therapy

In order to diagnose intervertebral disc pain of the bar, automatic pressure control discography was used. If a needle (25 gauge, 6 inches) is inserted into the degenerative disc identified by MRI and the needle tip is properly positioned in the center of the nucleus pulposus, contrast medium containing antibiotics may be injected into the discs at a slow rate using a pressure- Respectively. The total injection volume was within 3.5cc and injected at 0.05cc per second. Pressure, contrast agent location and pain response were recorded every 0.5 cc injection of contrast media. Contrast medium injection continued until one of the following conditions was reached:

First, if the VAS score exceeds 10 points out of 6 points,

Second, when the disc internal pressure is increased to 50 psi or more from the opening pressure in the case of grade 3 or more of the fiber damage,

Third, the total amount of injected contrast agent is 3.5 cc.

The intervertebral disc pain was gradually diagnosed by inducing the pain by gradually increasing the pressure of the intervertebral disc and by comparing the point at which the induced pain was consistent with the usual pain.

Based on the selection criteria, ten patients with degenerative lumbar disc herniation with sex and age in Table 4 were selected.

division gender age Patient 1 Woman 37 Patient 2 Woman 42 Patient 3 Woman 49 Patient 4 man 42 Patient 5 man 44 Patient 6 man 41 Patient 7 Woman 30 Patient 8 man 32 Patient 9 man 64 Patient 10 man 54

11.2: Derived from adipose tissue Intermediate lobe  Stem cell and hyaluronic acid derivative solution administration

Ten patients were subjected to liposuction and 20 to 30 ml of autologous adipose tissue was collected from each of 10 patients. After 3 weeks of liposuction, autologous adipose tissue-derived mesenchymal stem cells and hyaluronic acid derivative solution (performed in a volume ratio of 1: 3 to 3: 1 according to clinical judgment, see Table 5 below) A single injection was made with a spinal needle.

The area where the spinal needle enters is inserted 10 ~ 14 cm from the midline according to the target patient, and the tip of the spinal needle is inserted in the center of the disc using a C-shaped projector. In order to minimize the loss of stem cells, the needles were pulled out for about 5 minutes without moving the needles.

division Derived mesenchymal stem cells
Hyaluronic acid derivative solution Sample 1
(Concentration: 1 w / v%) 500 μl
(2 x 10 < ⁷ > cells) 1.5 ml
(Concentration: 1.3 w / v%) Sample 2
(Concentration: 1 w / v%) 1 ml
(4 x 10 < ⁷ > cells) 1 ml
(Concentration: 2 w / v%)

At all time points from about 1 week, about 1 month, about 3 months, about 6 months, about 9 months, and about 12 months after administration of autologous adipose tissue-derived mesenchymal stem cells and hyaluronic acid derivative solution, No adverse reactions were observed with the derived mesenchymal stem cells and the hyaluronic acid derivative solution.

12: derived from fat tissue Intermediate lobe  Stem cell and hyaluronic acid derivative solution and MRI image confirmed disc reduction of disc

The discs were observed through MRI before and 1 month and 6 months after transplantation, and the results of observation before and after transplantation of patient 1 are shown in FIG. 13 as a representative example. The MRI technique is capable of imaging with a T2 map, a diffusion weighted image, and an apparent diffusion coefficient (ADC) map. MRI photographs of the patient 1 receiving the sample 1 confirmed that the water content in the discs increased. This means that the degenerative disc has been reproduced. Similar results were observed in the other patients 2 to 10.

After autologous adipose tissue-derived mesenchymal stem cell transplantation, the extracellular matrix can be restored to recover moisture content before autologous adipose tissue-derived mesenchymal stem cell transplantation, degenerative disc herniation is regenerated, degenerative disc herniation is reduced ≪ / RTI >

13: Fat tissue origin Intermediate lobe  Stem cell and hyaluronic acid derivative solution and Visual Analogue Scale (VAS)

The VAS was assessed for the intervertebral discs before implantation, and about 1 week, about 1 month, about 3 months, about 6 months, about 9 months, and about 12 months after transplantation and the VAS score before and after implantation for patients 1 to 10 Shown in Table 6 and Fig. The VAS assessment noted that no pain was present at one end of the line, the most severe pain imaginable at the end of the other, and the degree of pain felt by the patient was written on the line.

As the autologous adipose tissue derived mesenchymal stem cells and hyaluronic acid derivative solution were administered, the pain experienced by each of the 10 patients gradually decreased over time. If grade 4 or higher, it can be judged that there is moderate pain. About 12 months after transplantation, 7 out of 10 patients showed VAS grade 0-3.

Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8 Patient 9 Patient 10 Before implantation 9 7 6 7 4 7 6 6 6 7 transplantation 9 9 8 9 10 9 8 7 3 8 1 week 5 7 7 9 7 5 3 7 3 7 1 month 4 6 5 5 5 5 3 7 3 8 3 months 2 6 5 7 5 5 3 2 4 4.5 6 months 3 3 5 0 4 3 3 3 3 5 9 months 2 2 6 One 3 6 3 3 3 2 12 months 3 0 4 One 2 6 3 3 3 4

14: derived from fat tissue Intermediate lobe  Administration of stem cell and hyaluronic acid derivative solution and Oswestry  Disability Index ( Oswestry  Disability Index: ODI ) To confirm disc pain reduction

ODI was assessed for the discs prior to transplantation and at about 1 week, about 1 month, about 3 months, about 6 months, about 9 months, and about 12 months after transplantation and ODI scores before and after transplantation for patients 1 to 10 Are shown in Table 7 and Fig. In the ODI evaluation, items such as the degree of pain, personal hygiene (washing, dressing), lifting, walking, sitting, standing, sleeping, social life (friendship, hobbies) 0-20% are minimal disability, 21-40% are moderate disability, 41-60% are severe disability, 61-80% are cripping back pain, Between 81 and 100% were divided into bed-bound or exaggerated patients who were lying alone.

Over time, the degree of impairment felt by each of the 10 patients gradually decreased as the autologous adipose tissue-derived mesenchymal stem cells and hyaluronic acid derivative solution were administered. About 12 months after transplantation, 7 out of 10 patients showed a mild level of disability ranging from 0 to 20%.

Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8 Patient 9 Patient 10 Before implantation 32 34 30 50 32 72 54 32 32 60 transplantation 92 34 90 62 88 98 78 70 34 64 1 week 58 26 58 62 58 66 52 58 32 64 1 month 23 20 20 32 20 60 52 24 20 30 3 months 24 18 22 58 22 50 46 22 24 31 6 months 30 12 24 9 22 30 22 13 26 24 9 months 20 11 24 11 14 46 26 13 18 16 12 months 30 2.2 26 8.9 8 31.1 15.6 12 14 20

Claims (28)

A pharmaceutical composition for preventing or treating low back pain comprising an adipose tissue-derived mesenchymal stem cell and a hyaluronic acid derivative solution. The pharmaceutical composition for preventing or treating low back pain according to claim 1, wherein the content of the hyaluronic acid derivative in the hyaluronic acid derivative solution is 0.7 to 3 w / v%. The pharmaceutical composition for preventing or treating low back pain according to claim 1, wherein the content of the hyaluronic acid derivative in the composition is 0.7 to 2.5 w / v%. The pharmaceutical composition according to claim 1, wherein the hyaluronic acid derivative solution has a viscosity of 150 to 900 Pa.s. The pharmaceutical composition according to claim 1, wherein the volume ratio of the adipose tissue-derived mesenchymal stem cell and the hyaluronic acid derivative solution is 1: 3 to 3: 1. The pharmaceutical composition for preventing or treating low back pain according to claim 2, wherein the hyaluronic acid derivative has a weight average molecular weight of 100 Da to 10,000 kDa. [3] The hyaluronic acid derivative according to claim 2, wherein the hyaluronic acid derivative is crosslinked with a crosslinking agent, wherein the equivalent ratio of the hyaluronic acid, hyaluronic acid salt, unit is 0.01 to 500% relative to the total weight of the composition. [Claim 7] The pharmaceutical composition for preventing or treating low back pain according to claim 7, wherein the hyaluronic acid derivative has a degree of crosslinking of 10 to 100%. [7] The method of claim 7, wherein the crosslinking agent is selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), ethylene glycol diglycidyl ether (EGDGE), hexanediol diglycidyl ether 1,6-hexanediol diglycidyl ether, propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, diglycidyl ether, diglycidyl ether, neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether, Glycerol polyglycidyl ether, tri-methylpropane polyglycidyl ether, 1,2- bis (2,3- hydroxypropyl methylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxypropyl cellulose, hydroxypropyl cellulose, hydroxypropyl cellulose, hydroxypropyl cellulose, hydroxypropyl cellulose, hydroxypropyl cellulose, hydroxypropyl cellulose, epoxypropoxy ethylene, Composition. The method according to claim 1, wherein the adipose tissue-derived mesenchymal stem cells are ^{CD44 +, CD73 +, CD90 +} , D105 +, CD29 +, CD49 + and HLA (human leukocyte antigen) -ABC ^+,
CD45 ^- and HLA (human leukocyte antigen) -DR ^- and,
TGF-beta receptor < / RTI ^& gt ^{; +} . The method according to claim 1, wherein the adipose tissue-derived mesenchymal stem cells are subcultured for 3 to 17 passages of stem cells isolated from adipose tissue. The pharmaceutical composition according to claim 1, wherein the adipose tissue-derived mesenchymal stem cell is in the form of a spindle. The pharmaceutical composition according to claim 1, wherein the back pain is caused by a degeneracy change of the disc. The pharmaceutical composition according to claim 1, wherein the content of the adipose tissue-derived mesenchymal stem cells is 1 x 10 ⁶ to 1 x 10 ⁸ cells / ml. The pharmaceutical composition according to claim 1, wherein the dose of the adipose tissue-derived mesenchymal stem cells is 0.1 x 10 ⁵ to 10 x 10 ⁵ cells / kg. A method for producing a pharmaceutical composition for preventing or treating low back pain, which comprises mixing a fat tissue-derived mesenchymal stem cell and a hyaluronic acid derivative solution. [Claim 16] The method according to claim 16, wherein the content of the hyaluronic acid derivative in the hyaluronic acid derivative solution is 0.7 to 3 w / v%. [Claim 16] The method according to claim 16, wherein the content of the hyaluronic acid derivative in the composition is 0.7 to 2.5 w / v%. [Claim 16] The pharmaceutical composition according to claim 16, wherein the volume ratio of the adipose tissue-derived mesenchymal stem cell and the hyaluronic acid derivative solution is 1: 3 to 3: 1. [Claim 17] The method according to claim 17, wherein the hyaluronic acid derivative has a weight average molecular weight of 100 Da to 10,000 kDa. [Claim 16] The method according to claim 16, wherein before mixing the adipose tissue-derived mesenchymal stem cell and the hyaluronic acid derivative solution,
Dissolving hyaluronic acid, hyaluronate, or a mixture thereof in an aqueous alkali solution of 0.1 to 0.5 N at a concentration of 50 to 250 mg / ml to prepare a hyaluronic acid solution;
Preparing a hyaluronic acid derivative solution by adding to the hyaluronic acid solution a cross-linking agent at an equivalent ratio of 0.01 to 500% to the repeating units of hyaluronic acid, hyaluronic acid salt or a mixture thereof to prepare a hyaluronic acid derivative solution; ≪ / RTI > wherein the pharmaceutical composition is administered to a patient. The method of claim 21, wherein the cross-linking agent is selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), ethylene glycol diglycidyl ether (EGDGE), hexanediol diglycidyl ether 1,6-hexanediol diglycidyl ether, propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, diglycidyl ether, diglycidyl ether, neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether, Glycerol polyglycidyl ether, tri-methylpropane polyglycidyl ether, bis-epoxypropylene (1,2- (bis (2,3 polyoxyethylene-ethoxypropoxy) ethylene, pentaerythritol polyglycidyl ether, sorbitol polyglycidyl ether, or any combination thereof. A method of making a pharmaceutical composition. The method according to claim 16, wherein the adipose tissue-derived mesenchymal stem cells are ^{CD44 +, CD73 +, CD90 +} , D105 +, CD29 +, CD49 + and HLA (human leukocyte antigen) -ABC ^+,
CD45 ^- and HLA (human leukocyte antigen) -DR ^- and,
Lt; RTI ID = 0.0 & gt ^; TGF-s < / RTI ^& gt ^; receptor ⁺ . [Claim 16] The method according to claim 16, wherein the adipose tissue-derived mesenchymal stem cells are subcultured for 3 to 17 passages of stem cells isolated from adipose tissue. [Claim 16] The method according to claim 16, wherein the adipose tissue-derived mesenchymal stem cell is in the form of a spindle. 17. The method of claim 16, wherein the back pain is caused by a degenerative change in the disc. [Claim 16] The method according to claim 16, wherein the content of the adipose tissue-derived mesenchymal stem cells is 1 x 10 ⁶ to 1 x 10 ⁸ cells / ml. The method according to claim 16, wherein the adipose tissue-derived mesenchymal stem cells The dose of 0.1 x 10 ⁵ to 10 x 10 ⁵ in one of the cells / kg, method for preparing a pharmaceutical composition for preventing or treating low back pain.

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