WO2015186906A1 - Composition de cellules souches mésenchymateuses autologues et allogéniques du tissu adipeux, pour la cicatrisation d'une lésion d'un tendon ou d'un ligament, et procédé pour sa préparation - Google Patents

Composition de cellules souches mésenchymateuses autologues et allogéniques du tissu adipeux, pour la cicatrisation d'une lésion d'un tendon ou d'un ligament, et procédé pour sa préparation Download PDF

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WO2015186906A1
WO2015186906A1 PCT/KR2015/003939 KR2015003939W WO2015186906A1 WO 2015186906 A1 WO2015186906 A1 WO 2015186906A1 KR 2015003939 W KR2015003939 W KR 2015003939W WO 2015186906 A1 WO2015186906 A1 WO 2015186906A1
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tendon
growth factor
ligament
stem cells
mesenchymal stem
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PCT/KR2015/003939
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English (en)
Korean (ko)
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이성구
김미형
최윤정
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(주)안트로젠
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells

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  • 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.
  • 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.
  • MSCs Mesenchymal stem cells
  • 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.
  • 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 (TGF ⁇ ), 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).
  • TGF ⁇ transforming growth factor beta
  • VEGF vascular endothelial growth factor
  • 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.
  • 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).
  • 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.
  • 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.
  • PDGF platelet-derived growth factor
  • the process is complicated, and the obtained by using recombinant DNA technique is also economical because it has to go through several processes.
  • 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.
  • 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.
  • 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).
  • ASC adipose stem cells
  • the liver cytokine such as HGF is useful for regeneration of liver tissue when transplanted in vivo
  • 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.
  • the literature does not disclose methods for efficiently treating tendon-related diseases using adipose derived mesenchymal stem cells, and optimal culture conditions.
  • 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.
  • 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.
  • 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).
  • FACS Fluorescence Activated Cell Sorter
  • HGF hepatocyte growth factor
  • TGFb transforming growth factor beta
  • IGF-1 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).
  • VEGF vascular endothelial growth factor
  • IGF insulin-like growth factor
  • 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).
  • FIG. 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.
  • the mesenchymal stem cells are of autologous or allogeneic origin.
  • the mesenchymal stem cells may be one passage passage or more.
  • the mesenchymal stem cells are to have at least one of the following features.
  • HGF hepatocyte growth factor
  • IGF insulin-like growth factor
  • TGFb transforming growth factor beta
  • vascular vascular endothelial cell growth factor secrete any one or more growth factors from the group consisting of endothelial growth factor (VEGF).
  • HGF hepatocyte growth factor
  • IGF insulin-like growth factor
  • TGFb transforming growth factor beta
  • vascular vascular endothelial cell growth factor
  • 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.
  • HGF hepatocyte growth factor
  • HGF hepatocyte growth factor
  • 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
  • IGF insulin-like growth factor
  • 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.
  • 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
  • TGFb transforming growth factor beta
  • 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.
  • 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
  • VEGF vascular endothelial growth factor
  • 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).
  • fetal bovine serum FBS fetal bovine serum FBS
  • bFGF basic fibroblast growth factor
  • 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, TGF ⁇ was found to increase more than 1.2 times, more specifically 1.3 times or more.
  • the mesenchymal stem cells may promote cell proliferation and angiogenesis.
  • the mesenchymal stem cells can secrete extracellular matrix protein.
  • the mesenchymal stem cells may secrete collagen.
  • 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- ⁇ 1), 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).
  • FGF fibroblast growth factor
  • EGF epidermal growth factor
  • EGF epidermal growth factor
  • transforming growth factor beta-1 transforming growth factor beta-1
  • TGF- ⁇ 1 platelet-derived growth factor
  • PDGF platelet-derived growth factor
  • VEGF vascular endothelial growth factor
  • HGF hepatocyte growth factor
  • IGF-1 insulin-like growth factor
  • the composition may be injected into the affected area.
  • the affected area may be a bone- tendon or bone-ligament junction.
  • the affected part may be a tendon or ligament.
  • 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.
  • 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.
  • the composition may be administered as a single dose or as a single dose excess.
  • the composition may be administered by a single injection.
  • 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.
  • 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.
  • the composition may further comprise a biodegradable support.
  • 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.
  • the composition may further comprise a pharmaceutically acceptable carrier and diluent.
  • the pharmaceutically acceptable carrier and diluent may be biologically and physiologically friendly to stem cells and recipients to be transplanted thereof.
  • 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, TGF ⁇ - 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.
  • FGF fibroblast growth factor
  • EGF epidermal growth factor
  • 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.
  • 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. .
  • 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 TGF ⁇ than cells cultured in substrate medium.
  • the method for culturing mesenchymal stem cells according to the present invention is as follows:
  • 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 / cm2 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.
  • DMEM or DMEM / F12 Dulbecco's Modified Eagle Medium / Ham's F-12 Nutrient Broth
  • 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.
  • bFGF basic fibroblast growth factor
  • the growth medium is removed and the cells are removed from the culture vessel by trypsin treatment.
  • 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.
  • 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.
  • 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.
  • DMEM Dulbecco's Modified Eagle Medium / Ham's F-12 Nutrient Broth
  • 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.
  • serum such as fetal bovine serum, horse or human
  • 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
  • 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.
  • 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.
  • Tedon 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.
  • ASC Adipose-derived stem cells
  • ADAS adipose-derived adult 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 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.
  • IGF insulin-like growth factor
  • IGF-I insulin-like growth factor
  • IGF-II insulin-like growth factor
  • GH growth hormone
  • 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 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.
  • VEGF Vascular endothelial growth factor
  • 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.
  • 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.
  • 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 °C, 1200 rpm. At this time, the subsided mesenchymal stem cell-vascular fraction, the supernatant was removed.
  • KRB Krebs-Ringer bicarbonate
  • 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.
  • bFGF basic fibroblast growth factor
  • Example 2 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 ⁇ l of FACS staining solution.
  • FACS staining solution phosphate buffer solution containing 1% fetal calf serum
  • 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 ⁇ l of FACS fixative was added to resuspend and analyzed using a flow cytometer.
  • 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.
  • 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).
  • HGF hepatocyte growth factor
  • HGF insulin-like growth factor
  • TGFb transforming growth factor beta
  • 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.
  • HGF hepatocyte growth factor
  • IGF Insulin-like growth factor
  • the mesenchymal stem cells cultured according to the present invention secreted a growth factor beta (transforming growth factor beta, TGF ⁇ ) 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.
  • 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.
  • FIG. 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.
  • Example 2 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 °C, 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.
  • Example 2 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 °C, 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.
  • bFGF basic fibroblast growth factor
  • FIG. 5 is a graph showing quantitative measurement of stem cell proliferation ability in five different lots using tetrazolium salts (WST-1). could confirm.
  • Example 2 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).
  • ELISA enzyme linked immunoassay
  • 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.
  • VEGF vascular endothelial growth factor
  • IGF insulin-like growth factor
  • 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).
  • RT-PCR reverse transcription-polymerase chain reaction
  • 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
  • the fifth lot (lot) of the adipose derived mesenchymal stem cells were confirmed that the most expression of collagen type I.
  • 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.
  • PBS phosphate buffer saline
  • PBS phosphate buffer saline
  • phosphate buffer saline PBS
  • PBS phosphate buffer saline
  • 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
  • ECM extracellular matrix
  • 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.
  • Example 2 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.
  • fetal bovine serum FBS fetal bovine serum FBS
  • bFGF basic fibroblast growth factor
  • 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.
  • VAS Pain Scale
  • 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.

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Abstract

La présente invention concerne une composition pour soulager ou traiter une maladie d'un tendon ou d'un ligament, contenant des cellules souches mésenchymateuses autologues et allogéniques du tissu adipeux ; un procédé de préparation de cette composition ; et un procédé pour traiter une maladie des tendons ou des ligaments. En particulier, des effets de soulagement ou de traitement d'une maladie des tendons ou des ligaments sont présentés par sécrétion de collagène, une protéine de la matrice extracellulaire (ECM) et de différents facteurs de croissance lors de l'administration des cellules souches mésenchymateuses du tissu adipeux selon la présente invention à des maladies des tendons telles que la maladie du tendon d'Achille, la maladie du tendon de la rotule, l'épicondylite latérale, l'épicondylite médiale, la fasciite plantaire, la tendinite de la coiffe des rotateurs, la ténosynovite, la tendinopathie, la tendinite, la ténosynovite, la lésion des tendons et la ténolyse ou les maladies ligamentaires telles que la lésion des ligaments croisés, la liaison des ligaments de l'articulation tibiotarsienne, et la lésion des ligaments collatéraux, par administration des cellules souches mésenchymateuses du tissu adipeux, seules ou avec un hydrogel.
PCT/KR2015/003939 2014-06-02 2015-04-20 Composition de cellules souches mésenchymateuses autologues et allogéniques du tissu adipeux, pour la cicatrisation d'une lésion d'un tendon ou d'un ligament, et procédé pour sa préparation WO2015186906A1 (fr)

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Cited By (5)

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Publication number Priority date Publication date Assignee Title
US10513689B2 (en) 2016-04-29 2019-12-24 Hope Biosciences, Llc Culture media for multipotent stem cells
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CN110448732A (zh) * 2019-08-27 2019-11-15 中南大学湘雅医院 区域干细胞诱导活性去细胞骨腱界面书页支架
CN110448732B (zh) * 2019-08-27 2022-03-29 中南大学湘雅医院 区域干细胞诱导活性去细胞骨腱界面书页支架

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