WO2015186906A1 - Autologous and allogeneic adipose-derived mesenchymal stem cell composition for healing tendon or ligament injury, and preparation method therefor - Google Patents

Abstract

The present invention relates to: a composition for alleviating or treating a tendon or ligament disease, containing autologous and allogeneic adipose-derived mesenchymal stem cells; a preparation method therefor; and a method for treating a tendon or ligament disease. Particularly, tendon or ligament disease alleviation or treatment effects are exhibited by secreting collagen, an extracellular matrix (ECM) protein and various growth factors when administering the adipose-derived mesenchymal stem cells according to the present invention to tendon diseases such as Achilles tendon disease, patella tendon disease, lateral epicondylitis, medial epicondylitis, plantar fasciitis, rotator cuff tendon disease, tenosynovitis, tendinopathy, tendinitis, tenosynovitis, tendon injury and tenolysis or ligament diseases such as cruciate ligament injury, ankle joint ligament injury and collateral ligament injury by administering the adipose-derived mesenchymal stem cells alone or together with a hydrogel.

Description

Autologous and allograft-derived adipose-derived mesenchymal stem cell compositions for the treatment of tendon or ligament injuries

The present invention relates to a composition containing autologous or allogeneic adipose derived mesenchymal stem cells, a method for preparing the same, and a method of treatment using the same. Specifically, the adipose derived mesenchymal stem cells are administered alone or in combination with a hydrogel. Tendon disease or cruciate ligament injuries, ankle ligament, achilles tendon disease, patellar tendon disease, lateral epicondylitis, medial epicondylitis, plantar fasciitis, rotator cuff tendon disease, tendonitis, tendonitis, tendonitis, hay salt, tendon injury, tendon detachment The present invention relates to compositions for improving or treating ligament diseases such as injury, collateral ligament injury, methods for improving or treating ligament diseases, and methods for their preparation.

Mesenchymal stem cells (MSCs) are cell lines with self-renewal ability, high viability, and potential differentiation into a variety of cells, and have potential as cell therapeutics in regenerative medicine. This regenerative capacity is more influenced by indirect factors such as cytokines and growth factors secreted from mesenchymal stem cells than by the differentiation ability of mesenchymal stem cells directly into tissue cells (Fox, JM et. (2007) Recent advances into the understanding of mesenchymal stem cell trafficking.Br. J. Haematol. 137: 491-502.

On the other hand, mesenchymal stem cells were obtained mainly from bone marrow through invasive methods, but mesenchymal stem cells present in adipose tissues were present at about 1,000 times higher than bone marrow, and the clustering ability of adipose-derived mesenchymal stem cells was bone marrow stem cells. Even higher, there is a difference in growth factor secretion such as transforming growth factor beta (TGFβ), vascular endothelial growth factor (VEGF) (Dmitrieva RI et al. (2012) Bone marrow- and subcutaneous adipose tissue-derived mesenchymal stem cells: differences and similarities.Cell Cycle. 15; 11 (2): 377-83).

Fats also have the advantage of being rich in tissue and very low risk for separation from individuals and can be preserved cold for immediate use or for future autologous or allogeneic applications.

Therefore, in order to obtain secretory substances such as growth factors of mesenchymal stem cells in a pharmaceutically effective amount for therapeutic purposes, a method of culturing them in large quantities using adipose tissue-derived mesenchymal stem cells is required.

Tendon and ligament are fibrous soft tissues, and collagen is the main component, and the bone and bone, bone and muscle, respectively, differ only in attachment point, but also in terms of mechanical properties and structural aspects.

Tendons or ligaments in human tissues are relatively poor in supply of blood flow to other tissues in the body, so once they are damaged, they take considerable time to regenerate and function like normal tendons or ligaments even if they are regenerated and treated. It is not known to recover completely. After regeneration they showed a decrease in biomechanical strength compared to normal tendons or ligaments, and this biomechanical strength was reported to be affected by the collagen constituting these tissues. Meanwhile, various growth factors have been reported for the treatment of tendon and ligament (Molly T. et al. (2003) The roles of growth factors in tendon and ligament healing. Sports Medicine 33, 5: 381-394).

Injuries due to trauma to the tendon or ligament are common in sports, work, and daily life. Inflammation or partial rupture of the tendon due to degenerative changes due to aging can also occur with minor trauma.

However, steroid injections, which are only surgical treatment and symptomatic repair of the broken tendons or ligaments, for the sores, partial ruptures (sprains) or complete ruptures of the tendon resulting from aging (degenerative changes) and trauma, No special treatment has been developed other than physical use.

 Accordingly, there is a need for treatment of a tendon or ligament disease that can regenerate and repair a damaged tendon to substantially ameliorate or treat tendon or ligament disease.

Korean Patent No. 1,219,624 discloses an angiotensin II type 1 receptor blocker and adipose tissue-derived stem cells as active ingredients to inhibit excessive fibrosis or to fusion of damaged muscle fibers or to new muscle fibers during regeneration of damaged muscles. It has been disclosed that the formation of a microenvironment (Niche) of stem cells that regulate differentiation may promote treatment of damaged muscle or regeneration of damaged muscle. However, the inclusion of an angiotensin receptor blocker does not determine the differentiation of stem cells. It is difficult to conclude that the effect is clear when applied to the clinic, and the experimental example on which it is based is only an animal test, and thus the effect cannot be guaranteed.

Meanwhile, Korean Patent Publication No. 2013-0000397 discloses that a tendon disease can be treated by administering a composition containing platelet-derived growth factor (PDGF), but the platelet-derived growth factor is separated through biological fluids including blood. The process is complicated, and the obtained by using recombinant DNA technique is also economical because it has to go through several processes. In addition, if the artificial and non-homogenous materials are applied to the clinic, it can not be guaranteed that they will be completely reborn in the individual and perform the expected function without side effects.

The prior art proposes a method for treating dry disease by controlling the differentiation of stem cells or administering growth factors to the dry disease site. However, unless it is a cell or substance derived from autologous and allogeneic, it is difficult to confirm that it can have a clear effect without side effects, and it is economical when a complicated step is required.

On the other hand, International Patent WO2007 / 127698 describes a method for producing liver stem cells derived from stem cells derived from tissues other than the liver, wherein liver stem cells are derived from adipose stem cells, and are derived from adipose stem cells (ASC). It is disclosed that the liver cytokine such as HGF is useful for regeneration of liver tissue when transplanted in vivo, and Korean Patent No. 995,133 discloses a method for producing cell growth factors from adipose tissue-derived stem cells and monocytes, and The use of tissue regeneration of a culture obtained by the production method is disclosed, Korean Patent No. 955,212 discloses a method for producing a large amount of human growth factors using human adipose derived stem cells. However, the literature does not disclose methods for efficiently treating tendon-related diseases using adipose derived mesenchymal stem cells, and optimal culture conditions.

Therefore, there are no side effects that can alleviate all the above disadvantages, and a pharmacologically effective amount can be prepared by a simple method.

An object of the present invention is a variety of tendon diseases or crosses such as Achilles tendon disease, patellar tendon disease, lateral epicondylitis, medial epicondylitis, plantar fasciitis, rotator cuff tendon disease, tendonitis, tendonitis, tendinitis, hay salt, tendon injury, tendon detachment It is to provide a composition, a treatment method, and a method for preparing the same, which can improve or treat ligament diseases such as ligament injury, ankle ligament injury, collateral ligament injury and the like.

In order to achieve the above object, the present invention is a composition for improving or treating tendon or ligament disease containing mesenchymal stem cells derived from autologous or allogeneic adipose tissue and its culture method, mesenchymal derived from autologous or allogeneic adipose tissue Provided are methods for ameliorating or treating tendon or ligament diseases using stem cells.

The composition according to the present invention is cultured in a pharmaceutically effective amount of autologous or allogeneic adipose-derived mesenchymal stem cells and applied to the tendon or ligament disease alone or in combination with a hydrogel to regenerate and reconstruct the tissue at the site of injury without side effects. This can improve or treat tendon or ligament disease.

Figure 1a shows the results of surface phenotypic analysis of fat stem cells.

1B is a graph showing CD29 and CD44 expressed in human adipose-derived mesenchymal stem cells compared with cells cultured in basal medium after confirmation by Fluorescence Activated Cell Sorter (FACS).

2 shows hepatocyte growth factor (HGF), transforming growth factor beta (TGFb), and insulin-like growth factor-1 secreted from human adipose derived mesenchymal stem cells. , IGF-1) is quantified by enzyme linked immunoassay (ELISA) and is a graph showing the comparison with the cells cultured in the basal medium.

Figure 3 is a diagram showing the proliferation of tendon cells when co-cultured in proliferation medium compared to the substrate medium when the fat stem cells co-cultured with the tendon cells.

Figure 4 is a graph showing the growth of tendon cells compared to cells treated with the culture medium cultured in stromal medium when treated with human adipose-derived mesenchymal stem cell culture cultured in growth medium.

5 is a graph showing the cell proliferation rate of the fat stem cells of five different lots (lots) cultured in proliferation medium for 4 days.

Figure 6 shows the amount of vascular endothelial growth factor (VEGF) and insulin-like growth factor (IGF) secreted from human adipose derived mesenchymal stem cells (enzyme linked immunoassay) , Quantified by ELISA).

Figure 7a is a graph measuring the collagen secreted from cells cultured for 4 days in the growth medium.

7B is a graph showing the expression of collagen type I genes of adipose stem cells cultured in proliferation medium.

7C is a graph showing expression of collagen type I protein.

Figure 7d is a fluorescence microscope picture observed by staining adipose derived mesenchymal stem cells with collagen type I (X 40).

8 is a graph showing the proliferation rate of tendon cells when five different lots of fat stem cells cultured in proliferation medium were co-cultured with tendon cells.

9 is a graph showing the efficacy of tendon injury healing after transplantation of adipose stem cells with a biodegradable support.

The present invention relates to a composition for improving or treating tendon or ligament disease, including adipose derived mesenchymal stem cells.

In one embodiment, the mesenchymal stem cells are of autologous or allogeneic origin.

In one embodiment according to the present invention, the mesenchymal stem cells may be one passage passage or more.

In one embodiment according to the invention, the mesenchymal stem cells are to have at least one of the following features.

(a) exhibits positive immunological properties for CD29 and CD44;

(b) hepatocyte growth factor (HGF), insulin-like growth factor (IGF), transforming growth factor beta (TGFb), and vascular endothelial cell growth factor (vascular) secrete any one or more growth factors from the group consisting of endothelial growth factor (VEGF).

In one embodiment according to the invention, the mesenchymal stem cells may secrete hepatocyte growth factor (HGF) more than 1,000 pg / ml, more specifically, fat-derived medium collected by incubating more than two passages Hepatocyte stem cells were inoculated into 24-well plates in 1.0 × 10 ⁵ volumes, and then 1 mL of growth medium was added to the hepatocyte growth factor as measured in mesenchymal stem cells cultured at 37 ° C. in a 5% CO ₂ incubator for 72 hours. hepatocyte growth factor (HGF) may be to secrete more than 1,000 pg / ml.

In one embodiment according to the invention, the mesenchymal stem cells may be one or more secretion of insulin-like growth factor (insulin-like growth factor, IGF) more than 200 pg / ml, more specifically collected by incubating more than two passages Adipose-derived mesenchymal stem cells were inoculated in 24-well plates in 1.0 × 10 ⁵ volumes, and then insulin was measured in mesenchymal stem cells cultured for 72 hours in a 37 ° C., 5% CO ₂ incubator with 1 mL of growth medium. Insulin-like growth factor (IGF) may secrete more than 200 pg / ml.

In one embodiment according to the invention, the mesenchymal stem cells may be to secrete more than 150 pg / ml of transforming growth factor beta (TGFb), more specifically collected by incubating more than two passages Adipocyte-derived mesenchymal stem cells were inoculated in 24-well plates in 1.0 × 10 ⁵ amounts, and 1 mL of growth medium was added and measured in mesenchymal stem cells cultured at 37 ° C. in a 5% CO ₂ incubator for 72 hours. Transforming growth factor beta (transforming growth factor beta, TGFb) may be to secrete more than 150 pg / ml.

In one embodiment according to the present invention, the mesenchymal stem cells may secrete vascular endothelial growth factor (VEGF) at least 500 pg / ml, more specifically, fat-derived in two or more passages Mesenchymal stem cells were inoculated in 24-well plates in 1.0 × 10 ⁵ amounts, and then contained 10% serum solution (fetal bovine serum FBS) and 1 ng / mL basic fibroblast growth factor (bFGF). When culturing mesenchymal stem cells incubated for 72 hours at 37 ℃, 5% CO ₂ incubator by adding a culture medium may be more than 500 pg / ml secretion of vascular endothelial growth factor (VEGF) .

The results may vary depending on the experimental conditions and the apparatus, but when using the growth medium according to the present invention, the growth factor is three times or more, more specifically 3.5 times or more, for HGF than cells cultured in the substrate medium. In the case of 3 times or more, more specifically 5 times or more, TGFβ was found to increase more than 1.2 times, more specifically 1.3 times or more.

In one embodiment according to the present invention, the mesenchymal stem cells may promote cell proliferation and angiogenesis.

In one embodiment according to the invention, the mesenchymal stem cells can secrete extracellular matrix protein.

In one embodiment according to the invention, the mesenchymal stem cells may secrete collagen.

In one aspect, the adipose derived mesenchymal stem cells are fibroblast growth factor (FGF), epidermal growth factor (Epidermal Growth Factor, EGF), transforming growth factor beta-1 (transforming) growth factor beta-1, TGF-β1), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) And one or more growth factors selected from the group consisting of insulin-like growth factor (IGF-1).

In one aspect according to the invention, the composition may be injected into the affected area.

In one aspect according to the invention, the affected area may be a bone- tendon or bone-ligament junction.

In one aspect according to the invention, the affected part may be a tendon or ligament.

In one aspect according to the present invention, the tendon disease is achilles tendon disease, patellar tendon disease, lateral epicondylitis, medial epicondylitis, plantar fasciitis, rotator cuff tendon disease, tendonitis, tendonitis, tendinitis, hay salt, tendon damage and tendon It may be selected from the group consisting of peeling, but is not limited thereto, wherein the outer epicondylitis may represent tennis elbow, the medial epicondylitis may represent golfers elbow.

In one embodiment according to the present invention, the ligament disease may be selected from the group consisting of cruciate ligament injury, ankle ligament injury, collateral ligament injury, ligament rupture, ligament sprain, but is not limited thereto.

In one aspect according to the invention, the composition may be administered as a single dose or as a single dose excess.

In one aspect according to the invention, the composition may be administered by a single injection.

In one embodiment according to the present invention, the dosage of the composition is appropriately determined in consideration of various factors such as the route of administration, the number of treatments, the age, weight, health condition, sex, severity of the disease, diet and excretion rate of the subject in need of treatment. You can decide.

In one embodiment according to the present invention, the composition may be a reduction in size of the tendon or ligament disease lesion by at least 10% compared to baseline within about 6 weeks of administration.

In one aspect according to the invention, the composition may further comprise a biodegradable support.

In one embodiment according to the invention, the biodegradable support is a hydrogel, which is selected from the group consisting of fibrin glue, hyaluronic acid, gelatin, collagen, alginic acid, cellulose, pectin, chitin, polyglycolic acid, and polylactic acid It may be more than, but is not limited thereto.

In one aspect according to the invention, the composition may further comprise a pharmaceutically acceptable carrier and diluent.

In one embodiment according to the present invention, the pharmaceutically acceptable carrier and diluent may be biologically and physiologically friendly to stem cells and recipients to be transplanted thereof.

In one embodiment according to the present invention, the pharmaceutically acceptable carrier may be used by mixing one or more of saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and these components. If necessary, other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, and the like, but are not limited thereto.

The present invention comprises the steps of (a) separating the mesenchymal stem cell-vascular fraction from adipose tissue and culturing in a matrix medium; (b) The stromal-vascular fraction was converted into fibroblast growth factor (FGF), epidermal growth factor (EGF), and transforming growth factor beta-1 (transforming growth factor beta-1, TGFβ- 1), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and insulin-like growth factor (insulin-) like growth factor, IGF-1), and culturing in a medium containing one or more growth factors selected from the group consisting of (c) subcultured one or more passages of cultured mesenchymal stem cells; It relates to a method for producing a composition comprising adipose tissue-derived mesenchymal stem cells for tendon or ligament disease improvement or treatment.

In one embodiment according to the present invention, the adipose tissue-derived mesenchymal stem cells obtained in step (c) may be characterized in that it is administered by a single injection with a biodegradable support.

In one embodiment according to the present invention, the biodegradable support may be any one or more selected from the group consisting of fibrin glue, hyaluronic acid, gelatin, collagen, alginic acid, cellulose, pectin, chitin, polyglycolic acid and polylactic acid. .

In the case of using the growth medium according to the method according to the present invention, the expression of CD29 is increased by 5% or more and CD44 is increased by 30% or more compared with the culture in the conventional substrate medium, growth Factors were 3.5 times higher for IGF, 5 times higher for HGF, and 1.3 times higher for TGFβ than cells cultured in substrate medium.

More specifically, the method for culturing mesenchymal stem cells according to the present invention is as follows:

(1) Cultivation of mesenchymal stem cells

Mesenchymal stem cells obtained from the tissues are suspended in the substrate medium and inoculated in a culture vessel at a concentration of 10,000-40,000 cells / ㎠ and then cultured. The substrate medium is incubated in DMEM or DMEM / F12 (Dulbecco's Modified Eagle Medium / Ham's F-12 Nutrient Broth) medium containing 10% bovine serum for about 24 hours.

(2) cultivation in expansion media

After removal of the matrix medium, the cells are grown in proliferation medium to proliferate adherent cells. Proliferation medium is DMEM or DMEM / F12 containing 10% bovine serum, basic fibroblast growth factor (bFGF) at a concentration of 0.1-100 ng / ml, and rapidly proliferate adherent adipose derived mesenchymal stem cells to shorten the cell volume. It acts to increase in large quantities.

(3) subculture

When cells fill 80-90% of the bottom of the culture vessel, the growth medium is removed and the cells are removed from the culture vessel by trypsin treatment. For subculture, cells are diluted 1: 3 to 1: 4 and incubated with proliferation medium in a new culture vessel. In this way, additional subcultures are possible.

(4) Collagen and growth factor secretion confirmed

Since allogeneic adipose-derived mesenchymal stem cells have different biological characteristics, depending on their type (individual), the secretion of tendon damage by confirming the secretion of collagen and growth factors in adipose-derived mesenchymal stem cells passaged at least one passage The use of allogeneic adipose-derived mesenchymal stem cells effective for healing is clinically effective compared to autologous cells.

In one embodiment according to the present invention, the cell culture medium may be DMEM or DMEM / F12 (Dulbecco's Modified Eagle Medium / Ham's F-12 Nutrient Broth) medium, but is not limited thereto.

In one embodiment according to the present invention, the cell culture medium may be added one or more accessory ingredients as needed, including serum, such as fetal bovine serum, horse or human, antibiotics and antifungal agents for preventing the contamination of microorganisms Etc. can be used.

In one embodiment according to the present invention, autologous or homogenous adipose derived mesenchymal stem cells are administered to a subject alone or in combination with a hydrogel to treat Achilles tendon disease, patellar tendon disease, lateral epicondylitis, medial epicondylitis, plantar fasciitis There is provided a method for ameliorating or treating a tendon disease such as rotator cuff tendon disease, tendonitis, tendonitis, tendinitis, hay salt, tendon injury, tendon detachment, or ligament disease such as cruciate ligament injury, ankle ligament injury, collateral ligament injury, etc. .

In one embodiment according to the present invention, Achilles tendon disease, patellar tendon disease, lateral epicondylitis, medial epicondylitis, plantar fasciitis, rotator cuff tendon disease, tendonitis, tendonitis, using autologous or allogeneic adipose-derived mesenchymal stem cells Use is provided for the treatment of tendon diseases such as tendonitis, hay salt, tendon injury, tendon detachment, or ligament disease such as cruciate ligament injury, ankle ligament injury, collateral ligament injury and the like.

[Justice]

"Tendon" is a fibrous soft tissue that holds muscles to bones to transfer the contractile force of the muscles so that joint movements can take place, while controlling energy and performing physical stabilization functions to store energy and restore high efficiency. Play a role.

"Ligament" is a fibrous soft tissue that connects bones to bones and is mainly located in the joints, which serves to keep the joints stable.

"Adipose-derived stromal stem cells" means mesenchymal stem cells obtained from the mesenchymal stem cell-vascular fraction. Adipose-derived stem cells (ASC), adipose-derived adult stem cells (ADAS), can be expressed in the same manner as adipose stem cells.

"Allogeneic transplant" is the transplantation of a specific tissue, organ or cell from another person or another individual of the same species, and the transplantation of tissue, organ or cell from another person or other animal if the tissue, organ or cell is not available. Means that.

"CD29 and CD44" are markers of mesenchymal stem cells and allow mesenchymal stem cells to be identified through positive reactions by immunofluorescence and immunocytochemistry.

“Hepatocyte growth factor (HGF) is known as a scattering factor and is a multifunctional cytokine that promotes mitosis, migration, invasion and morphogenesis. HGF-dependent signaling regulates integrin function by promoting aggregation and cell adhesion. HGF / SF inducing effects occur through signaling of the MET tyrosine kinase receptor after ligand binding.

“Insulin-like growth factor” (IGF) is IGF-I and IGF-II that share 50% structural homology with insulin. IGF acts as an mitotic stimulator of cell proliferation as well as an inhibitor of cellular apoptosis pathway. Under the control of growth hormone (GH), the liver is a major site of IGF production. IGF-I levels are also affected by impact and developmental stages. Autoclean and paracrine production of IGF contributes to the level of IFG available for growth regulation.

“Transforming growth factor beta (TGFβ)” is a 55 kDa 391 amino acid (aa consisting of 23 aa signal sequences, 256 aa pro-regions and 112 aa mature segments ) Preproprotein. Prior to secretion, the pro-regions are cleaved at the RxxR site by purine-like proteases. This results in an unglycosylated 25 kDa disulfide bonded mature dimer that covalently attaches with a previously attached disulfide bonded pro-region to form a “latent complex.” The complex is secreted. Almost certainly occurs extracellularly under various conditions via transmembrane serine / threonine kinase, initiating an intracellular signal cascade mediated by the Smad family of transcription factors.

"Vascular endothelial growth factor" (VEGF) was first identified as a growth and survival factor for endothelial cells. It induces endothelial cell proliferation and regulates angiogenesis and angiogenesis. VEGF is a heparin binding glycoprotein secreted as a 45 kDa homodimer. Typically, many cell types that are not endothelial cells themselves secrete VEGF.

"Extracellular matrix protein" is a protein such as collagen, hyaluronic acid, elastin, elastin, and binds to and buffers cells.

"Treatment" refers to therapeutic treatment and preventive or preventive means. Thus, those in need of treatment include those who already have tendon or ligament related diseases as well as those who wish to prevent tendon or ligament related diseases. The method of the present invention is not limited to, but can be used to treat any mammal in need of treatment, including humans, primates, livestock and breeding, pet or sport animals such as dogs, horses, cats, sheep, pigs, cattle, and the like. Can be.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, this is to help the understanding of the present invention, not intended to limit the scope of the present invention to this.

EXAMPLE

Example 1: Cultivation method of human adipose derived mesenchymal stem cells

Adipose-derived mesenchymal stem cells were isolated from adipose tissue obtained by liposuction (fat tissue can usually be obtained by liposuction, but not limited to this). It was washed 3-4 times with Krebs-Ringer bicarbonate (KRB) solution. The same volume of collagenase solution as adipose tissue was added and reacted in a 37 ° C. water bath. This was transferred to a centrifuge tube and centrifuged at 20 ° C. and 1200 rpm for 10 minutes. The supernatant fat layer was removed, and the lower collagenase solution was carefully separated so as not to shake. After the substrate medium was suspended and centrifuged for 5 minutes at 20 ℃, 1200 rpm. At this time, the subsided mesenchymal stem cell-vascular fraction, the supernatant was removed.

Mesenchymal stem cell-vascular fractions were suspended in substrate medium and inoculated in culture vessels and incubated in 37 ° C., 5% CO ₂ incubator for 24 hours. After removal of the culture solution, the cells were washed with phosphate buffer, and then grown on the substrate medium or on the growth medium containing basic fibroblast growth factor (bFGF) at a concentration of 1 ng / ml. When adipose derived mesenchymal stem cells were grown to about 80-90% of the culture vessel, they were obtained by separating them into single cells by trypsin treatment. The obtained cells were diluted 1: 3 to 1: 4 with proliferation medium to carry out passage culture.

Experimental Example

Experimental Example 1 Surface Type Analysis of Human Adipose-derived Mesenchymal Stem Cells

Collect the fat-derived mesenchymal stem cells cultured in two passages or more in Example 1, transfer to 1.5 ml centrifuge tube and add 1 ml of FACS staining solution (phosphate buffer solution containing 1% fetal calf serum) and mix well. , Centrifuged at 10,000 rpm for 5 seconds. The supernatant was removed, resuspended in 1 ml of FACS staining solution, centrifuged at 10,000 rpm for 5 seconds, the supernatant was removed and resuspended in 300 µl of FACS staining solution. Depending on the number of samples, a new tube was dispensed to contain about 2 × 10 ⁵ cells per centrifuge tube, the antibody was added, and reacted for 30 minutes on ice. Resuspended in 1 ml FACS staining solution, centrifuged at 10,000 rpm for 5 seconds to remove supernatant. 400-500 μl of FACS fixative was added to resuspend and analyzed using a flow cytometer.

As a result, as shown in Figures 1a and 1b, it was confirmed that the adipocyte stem cells according to the present invention has an immunological characteristic that increases the expression of CD29, CD44 compared to the cells cultured in the matrix medium.

Experimental Example 2: Growth Factor Secretion of Human Adipose-derived Mesenchymal Stem Cells

Adipose-derived mesenchymal stem cells cultured in two passages or more were collected in Example 1, 1.0 × 10 ⁵ cells were inoculated into 24-well plates, and then 1 mL of substrate medium or growth medium was added. After 72 hours of incubation at 37 ° C. in a 5% CO ₂ incubator, the supernatant was collected, followed by hepatocyte growth factor (HGF) and insulin-like growth factor (HGF). like growth factor-1, IGF-1) and transforming growth factor beta (TGFb) were measured using enzyme linked immunoassay (ELISA).

As a result, as shown in Figure 2, the cells cultured in the growth medium is hepatocyte growth factor (HGF) of 3,000 ~ 5,000 pg / mL (10 times higher concentration than the substrate medium) and 357 ~ 378 pg / mL Insulin-like growth factor (IGF) (more than 3.5 times higher concentration than substrate medium) was secreted. In addition, the mesenchymal stem cells cultured according to the present invention secreted a growth factor beta (transforming growth factor beta, TGFβ) 1.3-fold growth factor compared to the substrate medium at 200 ~ 214 pg / mL. Therefore, it was confirmed that applying mesenchymal stem cells cultured according to the present invention to the tendon injury site can facilitate the healing of wounds by continuously releasing various growth factors that promote cell proliferation and healing.

Experimental Example 3: Tendon cell proliferation of allogeneic adipose derived mesenchymal stem cells 1

In Example 1, the fat-derived mesenchymal stem cells cultured in two passages or more were collected and 5,000 cells per cm ² were co-cultured with tendon cells in a transwell plate for 72 hours at 37 ° C. in a 5% CO ₂ incubator. After incubation, the growth of tendon cells was confirmed.

3 is a graph showing increased proliferation of tendon cells compared to stem cells co-cultured with adipose derived stem cells cultured in stromal medium when co-cultured with adipose derived stem cells cultured in tendon cells for 7 days. Tensile cells co-cultured with stem cells cultured in stromal media increased 1.53-fold, whereas tendon cells co-cultured with stem cells cultured in proliferation medium increased 2.35-fold.

Experimental Example 4: Tendon cell proliferation of allogeneic adipose derived mesenchymal stem cells

After collecting the fat-derived mesenchymal stem cells cultured in two passages or more in Example 1 and inoculated 5,000 cells per cm ² in a 96-well plate, and cultured after 72 hours in 37 ℃, 5% CO ₂ incubator By diluting to 25, 50, 75% using a substrate medium or proliferation medium, the cells were treated with tendon cells, and cultured for 7 days, the growth of tendon cells was confirmed.

4 shows that when tendon cells were cultured with adipose derived stem cell culture for 7 days, they were increased by concentrations of 1.09, 1.30, and 1.34 times at 25, 50, and 75%, respectively, compared with the culture medium cultured in substrate medium. In the case of treating the culture medium cultured in the substrate medium, the change according to the concentration could not be confirmed.

Experimental Example 5: Proliferation of Human Adipose-derived Mesenchymal Stem Cells

Five lots of fat-derived mesenchymal stem cells cultured in two passages or more were collected in Example 1, and 5,000 cells per cm ² were inoculated into 96-well plates, followed by 10% serum solution (fetal bovine serum FBS). , 1 ng / mL basic fibroblast growth factor (bFGF) containing a culture medium was added and then incubated for 4 days in 37 ℃, 5% CO ₂ incubator. On day 4 of culture, tetrazolium salts (WST-1) were added to measure the degree of cell proliferation between 5 lots.

FIG. 5 is a graph showing quantitative measurement of stem cell proliferation ability in five different lots using tetrazolium salts (WST-1). Could confirm.

Experimental Example 6: Secretion of Growth Factors in Human Adipose-derived Mesenchymal Stem Cells

After collecting five lots of fat-derived mesenchymal stem cells cultured in two passages or more in Example 1 and inoculating 1.0 × 10 ⁵ cells in a 24-well plate, 10% serum solution (fetal bovine serum FBS) A culture medium containing 1 ng / mL basic fibroblast growth factor (bFGF) was added. After culturing for 72 hours in a 37 ° C., 5% CO ₂ incubator, the supernatant was collected and vascular endothelial growth factor (VEGF) and insulin-like growth factor, which are representative growth factors secreted from mesenchymal stem cells. The amount of (insulin-like growth factor-1, IGF-1) was measured using enzyme linked immunoassay (ELISA).

As a result, as shown in Figure 6, the cells are 619.9 ~ 1641.2 pg / mL vascular endothelial growth factor (VEGF) and 37 ~ 402 pg / mL insulin-like growth factor (insulin-like growth factor) , IGF) was secreted. Vascular endothelial growth factor (VEGF) and insulin-like growth factor secreted from lot 5 of 5 different lots of fat-derived mesenchymal stem cells It was confirmed that a large amount of IGF) secretion.

That is, when the mesenchymal stem cells cultured according to the present invention applied to the tendon injury site, it was confirmed that various growth factors that promote cell proliferation and angiogenesis can be continuously secreted to facilitate wound healing.

Experimental Example 7: Expression of Collagen Type I in Human Adipose-derived Mesenchymal Stem Cells

In Example 1, the fat-derived mesenchymal stem cells of five lots (lots) cultured in two passages or more were collected to confirm the degree of collagen secretion and expression.

FIG. 7 a shows that the culture solution of the adipose derived mesenchymal stem cells on the 4th day of culture was measured by the Sircol assay, and the lot of 5.7 ug / ml of collagen was secreted at lot 5.

Figure 7b was extracted from the adipose-derived mesenchymal stem cells of culture day 4 and confirmed the collagen mRNA expression level using the polymerase chain reaction (Polymerase Chain Reaction, PCR) technique. Collected cells cultured for 4 days, primers (hcol IF 5 ') that specifically react with human-derived gene expression of collagen type I (COLI) in substrates secreted from adipose-derived mesenchymal stem cells -CAGCCGCTTCACCTACAGC, hcol IR 5'-TTTTGTATTCAATCACTGTC) was confirmed by reverse transcription-polymerase chain reaction (RT-PCR).

Figure 7c is a collection of cells cultured for 4 days to confirm the expression of collagen type I (Collagen Type I; COLI) in the substrate secreted from adipose derived mesenchymal stem cells using an antibody that specifically reacts with human-derived protein In the fifth lot (lot) of the adipose derived mesenchymal stem cells, the fifth lot (lot) of the adipose derived mesenchymal stem cells were confirmed that the most expression of collagen type I.

In addition, the expression level of collagen type I was observed by fluorescence immunostaining method. Five lots of adipose derived mesenchymal stem cells cultured as in Example 1 were fixed in phosphate buffer saline (PBS) containing 3.7% formaldehyde for 30 minutes. Wash three times with phosphate buffer saline (PBS) and infiltrate with phosphate buffer saline (PBS) containing 5% normal goat serum and 0.1% triton-X100 for 30 minutes ( permeablization) and blocking were performed. After adding phosphate buffer saline (PBS) containing a primary antibody, and reacted at 37 ℃ for 1 hour, washed three times with phosphate buffer saline (PBS), secondary antibody is added After reaction at room temperature for 30 minutes. After washing three times with phosphate buffer saline (phosphate buffer saline, PBS) was mounted and observed by fluorescence microscope.

Figure 7d is a photograph showing the secretion capacity of collagen type I, a protein constituting the tendon 95% of the extracellular matrix (ECM) of adipose derived mesenchymal stem cells (x 40), as shown here Adipose-derived mesenchymal stem cells prepared according to the invention showed a positive response to collagen type I as a whole. That is, the adipose-derived mesenchymal stem cells prepared according to the present invention not only secrete a large amount of extracellular matrix proteins essential for healing tendon damage, but also facilitate tendon damage healing by providing various substrates when secreted collagen is transplanted into the body. We confirmed that we could.

Experimental Example 8: Tendon cell proliferation of allogeneic adipose derived mesenchymal stem cells

Five lots of fat-derived mesenchymal stem cells cultured in two passages or more were collected in Example 1 and co-cultured with tendon cells in a transwell plate to prepare a 10% serum solution (fetal bovine serum FBS), After adding a culture medium containing 1 ng / mL basic fibroblast growth factor (bFGF), the growth of tendon cells was confirmed after 72 hours of incubation in a 37 ° C., 5% CO ₂ incubator.

8 is 1.67- to 2.2-fold increase in tendon cells co-cultured with adipose-derived stem cells for 7 days compared to the control group in which only tendon cells were cultured and the fifth adipose-derived mesenchymal stem cells out of five lots. Was found to increase the proliferation of tendon cells most.

Experimental Example 9 Treatment of Tendon Damage of Allogeneic Adipose-derived Mesenchymal Stem Cells

In order to clinically apply the adipose derived mesenchymal stem cells cultured by the method of Example 1, a clinical trial was conducted in a patient diagnosed with lateral epicondylitis. The prepared cells were passaged for one or more passages, and the cells of two concentrations, Group 1 (1 × 10 ⁶ cells / mL) and Group 2 (10 × 10 ⁶ cells / mL), were dosed with 1 mL at the site of tendon injury with fibrin glue. Implanted while observing with ultrasound.

FIG. 9A is a graph showing tendon injury healing and pain degree at 6 and 12 weeks after transplantation, with VAS score and modified Mayo activity index. Pain Scale (VAS) at rest and activity (pain indicators were significantly reduced at 6 and 12 weeks compared to pre-injection in both Group 1 and Group 2, and modified Mayo, a measure of a comprehensive assessment of pain and functional status) The activity index was also statistically significantly improved from 6 weeks.

Figure 9b is a photograph of the lesion size observed by ultrasound image to investigate the anatomical structure of the tendon defect in the clinical subjects, the size of the lesion site at 6 weeks post-injection compared to before the injection in both Group 1 and Group 2 At 12 weeks, the size decreased significantly. In particular, dense tissue was observed at the lesion site on the ultrasound at 12 weeks after the injection, and the anatomical damage was improved by regeneration and reconstruction.

By culturing autologous or allogeneic adipose-derived mesenchymal stem cells according to the present invention in a pharmaceutically effective amount or applying to a tendon or ligament disease alone or in combination with a hydrogel, the tissue at the site of injury is regenerated and reconstructed without side effects. It is very useful in industry to improve or treat ligament diseases.

Claims (25)

1. Composition for improving or treating tendon or ligament disease, including fat-derived mesenchymal stem cells.
2. According to claim 1, The fat-derived mesenchymal stem cells, characterized in that the autologous or allogeneic, composition for improving or treating tendon or ligament disease.
3. According to claim 1, The fat-derived mesenchymal stem cells, characterized in that passaged more than one passage, composition for improving or treating tendon or ligament disease.
4. The composition for ameliorating or treating tendon or ligament disease according to claim 1, wherein the adipose derived mesenchymal stem cells have at least one of the following characteristics: (a) positive for CD29 and CD44 Exhibit immunological properties; (b) hepatocyte growth factor (HGF), insulin-like growth factor (IGF), transforming growth factor beta (TGFb), and vascular endothelial cell growth factor (vascular) secrete any one or more growth factors from the group consisting of endothelial growth factor (VEGF).
5. The composition for ameliorating or treating tendon or ligament disease according to claim 4, wherein the adipose derived mesenchymal stem cells secrete at least 1,000 pg / ml of hepatocyte growth factor (HGF).
6. The composition for ameliorating or treating tendon or ligament disease according to claim 4, wherein the adipose derived mesenchymal stem cells secrete at least 200 pg / ml of insulin-like growth factor (IGF).
7. The method for ameliorating or treating tendon or ligament disease according to claim 4, wherein the adipose derived mesenchymal stem cells secrete at least 150 pg / ml of transforming growth factor beta (TGF-β). Composition.
8. The composition for ameliorating or treating tendon or ligament disease according to claim 4, wherein the adipose derived mesenchymal stem cells secrete vascular endothelial growth factor (VEGF) at least 500 pg / ml.
9. According to claim 1, The fat-derived mesenchymal stem cells, characterized in that to promote cell proliferation and angiogenesis, composition for improving or treating tendon or ligament disease.
10. According to claim 1, The fat-derived mesenchymal stem cells, characterized in that the secretion of extracellular matrix protein, composition for improving or treating tendon or ligament disease.
11. According to claim 1, The fat-derived mesenchymal stem cells, characterized in that to secrete collagen, composition for improving or treating tendon or ligament disease.
12. The composition for ameliorating or treating tendon or ligament disease according to claim 1, wherein the composition is injected into the affected area.
13. 13. The composition for ameliorating or treating tendon or ligament disease according to claim 12, wherein the affected part is a bone-tendon junction or a bone-ligament junction.
14. The composition for ameliorating or treating a tendon or ligament disease according to claim 12, wherein the affected part is a tendon or ligament.
15. 15. The method according to any one of claims 1 to 14, wherein the tendon disease is achilles tendon disease, patellar tendon disease, lateral epicondylitis, medial epicondylitis, plantar fasciitis, rotator cuff tendon disease, tendonitis, tendonitis, tendinitis, Composition for improvement or treatment of tendon or ligament disease, characterized in that selected from the group consisting of hay salt, gun damage and gun peeling.
16. The method of claim 1, wherein the ligament disease is selected from the group consisting of cruciate ligament injury, ankle ligament injury, collateral ligament injury, ligament rupture, and ligament sprain. Improvement or Therapeutic Composition.
17. 15. The method according to any one of claims 1 to 14, wherein the adipose derived mesenchymal stem cells are fibroblast growth factor (FGF), epidermal growth factor (EGF), transforming growth factor. Transforming growth factor beta-1 (TGF-β1), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor ( improvement of a tendon or ligament disease characterized by culturing in a medium containing one or more growth factors selected from the group consisting of hepatocyte growth factor (HGF) and insulin-like growth factor (IGF-1). Therapeutic composition.
18. The composition for ameliorating or treating tendon or ligament disease according to claim 1, wherein the composition is administered as a single dose or as a single dose excess.
19. The composition for ameliorating or treating tendon or ligament disease according to claim 1, wherein the composition is administered by a single injection.
20. The composition for improving or treating tendon or ligament disease of claim 1, wherein the composition has a 10% or more reduction in the size of the tendon or ligament disease lesion compared to baseline within about 6 weeks of administration.
21. The method of claim 1, further comprising a biodegradable support, composition for improving or treating tendon or ligament disease.
22. 22. The gun of claim 21, wherein the biodegradable support is at least one selected from the group consisting of fibrin glue, hyaluronic acid, gelatin, collagen, alginic acid, cellulose, pectin, chitin, polyglycolic acid, and polylactic acid. Or a composition for improving or treating ligament disease.
23. (a) separating the mesenchymal stem cell-vascular fraction from adipose tissue and culturing in matrix medium; (b) The stromal-vascular fraction was converted into fibroblast growth factor (FGF), epidermal growth factor (EGF), and transforming growth factor beta-1 (transforming growth factor beta-1, TGF- β1), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and insulin-like growth factor (insulin-) like growth factor, IGF-1), and culturing in a medium containing one or more growth factors selected from the group consisting of (c) subcultured one or more passages of cultured mesenchymal stem cells; Method for producing a composition for improving or treating tendon or ligament disease, including adipose tissue-derived mesenchymal stem cells.
24. The method of claim 23, wherein the adipose tissue-derived mesenchymal stem cells obtained in step (c) are administered by a single injection together with a biodegradable support. .
25. 25. The gun of claim 24, wherein the biodegradable support is at least one selected from the group consisting of fibrin glue, hyaluronic acid, gelatin, collagen, alginic acid, cellulose, pectin, chitin, polyglycolic acid, and polylactic acid. Or a method for producing a composition for improving or treating ligament disease.

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