WO2008035832A1 - Composition cellulaire pour régénération osseuse et procédé de fabrication correspondant - Google Patents

Composition cellulaire pour régénération osseuse et procédé de fabrication correspondant Download PDF

Info

Publication number
WO2008035832A1
WO2008035832A1 PCT/KR2006/003791 KR2006003791W WO2008035832A1 WO 2008035832 A1 WO2008035832 A1 WO 2008035832A1 KR 2006003791 W KR2006003791 W KR 2006003791W WO 2008035832 A1 WO2008035832 A1 WO 2008035832A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
osteogenic capacity
hyaluronic acid
bone
cell
Prior art date
Application number
PCT/KR2006/003791
Other languages
English (en)
Inventor
Jae-Deog Jang
Kyoung-Phil Byun
Sae-Bom Lee
Hyun-Shin Park
Jang-Hoon Kim
Cheong-Ho Chang
Original Assignee
Sewon Cellontech Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sewon Cellontech Co., Ltd. filed Critical Sewon Cellontech Co., Ltd.
Publication of WO2008035832A1 publication Critical patent/WO2008035832A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • 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
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis

Definitions

  • the present invention relates to a cell composition for promoting bone formation, and a method for preparing the same. More specifically, the present invention relates to a cell composition for promoting bone formation which is capable of achieving local or systemic bone regeneration by subjecting cells having osteogenic capacity to surface treatment using a hyaluronic acid and injecting the thus-treated cells into a circulation system of target animals.
  • a therapeutic cell composition for bone formation according to the present invention is injected via the blood stream, it is possible to achieve uniform delivery of bone- forming cells to target regions in need of bone formation while not causing destruction of a structure of the remaining bone tissue, and thereby it is possible to treat a variety of bone diseases including osteoporosis in need of extensive bone regeneration. Therefore, the present invention accomplishes remarkably improved quality and reliability of the product and thereby is very useful to enhance customer satisfaction.
  • osteoporosis is the medical term used to describe a condition which undergoes a gradual loss of bone mass and density and as a result, is highly susceptible to bone fracture due to the formation of increased numbers of tiny pores within the bone, as shown in coarse pumice stones or sponges. That is, osteoporosis is a disease of progressive bone loss involving the formation of many tiny holes or pores as compared to normal bone, reduction of bone mass, thinning and weakening of bone microarchitecture, thus causing the bones to become brittle and prone to breaking even with only light impact.
  • osteoporosis progresses silently without subjective pain or symptoms, people might not be aware that they have osteoporosis until they accidentally fall or get hit and in turn easily break a bone. Even with light falls, people having osteoporosis may experience wrist fractures, pelvic fractures and vertebral fractures with accompanying severe pain. In particular, pelvic fractures and vertebral fractures are severely painful and require surgical operations, and the patients have to put up with the hardship of being sick in bed even for several months. Even after complete recovery from the surgery, physical impairment may still remain due to surgical sequelae or complications of osteoporosis.
  • osteoporosis has a relationship with combination of various risk factors such as female gender, a thin and/or small body frame, advanced age, a family medical history of osteoporosis, menopause (including hysterectomy), irregular menstruation (amenorrhoea), neurasthenia, use of adrenocortical hormones or anticonvulsants, hypoandrogenemia in male gender, insufficient exercise, smoking, excessive drinking, Asian and Caucasian nationalities (African and Hispanic- American nationalities are at lower risk), early menopause (before age 45), excessive caffeine and alcohol consumption, and diets low in calcium.
  • risk factors such as female gender, a thin and/or small body frame, advanced age, a family medical history of osteoporosis, menopause (including hysterectomy), irregular menstruation (amenorrhoea), neurasthenia, use of adrenocortical hormones or anticonvulsants, hypoandrogenemia in male gender, insufficient exercise, smoking
  • Osteoporosis is estimated to affect more than 28,990,000 American people (80 percent of those affected are women). In the United States, 10 million individuals already have osteoporosis. 18 million more have low bone mass, placing them at increased risk for osteoporosis. One in two women and one in eight men over age 50 will have an osteoporosis-related fracture in their lifetime. One in ten African-American women over age 50 has osteoporosis; an additional one in 3 has low bone density that puts them at risk of developing osteoporosis. Osteoporosis is responsible for more than 1.5 million fractures annually, including: 300,000 hip fractures, 700,000 vertebral fractures, 250,000 wrist fractures and 300,000 fractures at other sites.
  • osteoporosis therapies such as by use of bisphosphonates or selective estrogen receptor modulators (SERMs) primarily focus on the suppression of bone absorption and are known to inhibit a progress of osteoporosis via the prevention of a further loss of bone mass.
  • SERMs selective estrogen receptor modulators
  • bone union or bone regeneration can be achieved in fractures of local lesions caused by various factors including osteoporosis or in target regions requiring bone regeneration.
  • the conventional therapeutic methods block a further progress of osteoporosis by preventing bone absorption via inhibition of the osteoclast activity, and therefore suffer from problems failing to substantially facilitate bone regeneration.
  • use of the above-mentioned bone graft technique or autologous osteoblast-based therapy products is disadvantageous in that it is difficult to achieve biological bone regeneration throughout extensive regions.
  • the bone autograft solves or alleviates such problems suffered by the bone allograft, but has disadvantages such as a difficulty of securing sufficient donor sites to provide bone for bone transplantation, the morbidity of the donor sites and the like.
  • Cell therapy product utilizing autologous osteoblasts is a therapeutic method which was developed to solve the problems of the conventional bone graft techniques, and is known as a technique which is capable of achieving local bone regeneration by mass proliferation of osteoprogenitor cells isolated from bone marrow, differentiation of the osteoprogenitor cells into osteoblasts and transplantation of the osteoblasts into the target sites in need of bone regeneration.
  • the present invention has been made in view of the problems associated with conventional bone graft techniques as discussed above, and it is a first object of the present invention to provide a method for preparing a cell composition for promoting bone formation by isolating cells having osteogenic capacity from bone marrow, preparing a cell suspension having osteogenic capacity and subjecting the cells to surface treatment, thereby preparing a cell therapy product having osteogenic capacity.
  • a second object of the present invention is to provide local or systemic bone regeneration by subjecting cells having osteogenic capacity to surface treatment using a hyaluronic acid and injecting the thus-treated cells into a circulation system of target animals.
  • a third object of the present invention is to achieve uniform delivery of bone-forming cells to target regions in need of bone formation while not causing destruction of the remaining bone tissue structure, by injection of a bone-forming therapeutic agent via the blood stream. Therefore, it is possible to treat various bone diseases including osteoporosis in need of extensive bone regeneration.
  • a fourth object of the present invention is to provide a cell composition for promoting bone formation and a method for preparing the same, which are suited for enhancing customer satisfaction via remarkably improved quality and reliability of the product.
  • a method for preparing a cell composition for promoting bone-formation comprising:
  • a cell composition for promoting bone-formation comprising autologous cells having osteogenic capacity and a low-molecular weight hyaluronic acid, wherein the cells are surface-coated with the hyaluronic acid.
  • the autologous cells having osteogenic capacity are isolated from bone marrow, and are cultured in ⁇ -MEM containing 40 to 60 mg/L of vitamin C (ascorbic acid) to improve functions of osteoprogenitor cells, 7 to 10 days prior to a surgical application.
  • the isolated cells are cultured and proliferated in DMEM (Dulbecco's Modified Eagle's Medium) or ⁇ -MEM (Minimum Essential Medium, Alpha Modification) to prepare a cell suspension having osteogenic capacity, and the resulting cell suspension is mixed with a low-molecular weight hyaluronic acid of 1,000 to 1,000,000 MW as a bio-matrix, the hyaluronic acid being added in a ratio of 0.01 to 0.03% per 10 to 20 mL of the cell suspension containing I xIO 6 to IxIO 7 cells having osteogenic capacity.
  • DMEM Dynamic Eagle's Medium
  • ⁇ -MEM Minimum Essential Medium, Alpha Modification
  • the low-molecular weight hyaluronic acid is reacted with the cells in a CO 2 incubator at a temperature of 36 to 38 °C and a concentration of 4 to 6% CO 2 for 1 to 2 hours to achieve binding between the hyaluronic acid and CD44 (cluster designation 44) present on the surface of the cells having osteogenic capacity.
  • FIG. 1 is a process flow chart illustrating a method for preparing a cell composition for promoting bone-formation which is applied to the present invention
  • FIG. 2 is a photograph of a cell composition for promoting bone-formation which is applied to the present invention
  • FIG. 3 is an enlarged X-ray photograph of femora and tubercles of rats in Example 1 of the present invention
  • FIG. 4 is a photograph confirming mobility/anchorage of cells having osteogenic capacity injected into blood stream, as in Example 2 of the present invention.
  • FIGS. 1 and 2 A cell composition for promoting bone-formation and a method for preparing the same, which are applied to the present invention, are constituted as shown in FIGS. 1 and 2.
  • the therapeutic method using bone-absorption inhibitors such as bisphosphonates and the like may be applied when extensive bone defects such as osteoporosis have occurred throughout broad regions.
  • a bone allograft technique still suffers from the various problems such as propagation possibility of contagious diseases, insufficient supply of implant materials, occurrence of immune rejection such as graft rejection, and a difficulty in complete regeneration of the implants into autologous tissues.
  • the bone autograft technique solves or alleviates such problems of the bone allograft technique, but also has shortcomings such as a difficulty of securing sufficient donor sites, the morbidity of the donor sites and the like.
  • the autologous osteoblast- based therapy product is a therapeutic method which was developed to solve the problems of such conventional bone graft techniques, and is known as a technique which is capable of achieving local bone regeneration by large-scale growth of osteoprogenitor cells isolated from bone marrow, differentiation of the osteoprogenitor cells into osteoblasts and transplantation of the osteoblasts into the target sites where bone regeneration is sought.
  • the autologous osteoblast-based therapy technique may suffer from the problems associated with probability of reduced viability of the osteoblasts, which are adherent cells, and the problems associated with probability of escape of injected osteoblasts from the desired target site for bone regeneration and then propagation thereof to other sites via the blood stream.
  • the cell therapy product wherein cells are surface-treated by mixing the cells having osteogenic capacity with a bio- matrix
  • the cell therapy product is a more advanced version of an injection method of a liquid osteoblast suspension which is based on cell therapy.
  • the conventional injection method of a liquid osteoblast suspension which involves transplantation of the osteoblasts alone, has suffered from various problems such as decreased viability of the osteoblasts which are adherent cells, and the migration possibility of injected osteoblasts to unwanted regions via the blood stream, rather than toward the desired target sites for bone regeneration.
  • the three- dimensional cell therapy product for promoting bone formation which is composed of a combination of cells having osteogenic capacity and a bio-matrix, in accordance with the present invention, due to combination of the cells having osteogenic capacity with the bio-matrix, can ensure a desired viability of the cells and can migrate only into bone defect regions when it is injected into the blood stream and therefore it is possible to effectively treat extensive bone defects such as osteoporosis.
  • the cell composition can be prepared by the following steps.
  • the present invention provides a step of isolating cells having osteogenic capacity from bone marrow (Step A).
  • the cells having osteogenic capacity used in Step A are preferably employed.
  • the autologous cells having osteogenic capacity are autologous cells cultured in ⁇ -MEM containing 40 to 60 mg/L of vitamin C (ascorbic acid), in order to improve functions of osteoprogenitor cells, 7 to 10 days prior to a surgical application.
  • the present invention provides a step of culturing and proliferating the thus-isolated cells in DMEM (Dulbecco's Modified Eagle's Medium) or ⁇ -MEM (Minimum Essential Medium, Alpha Modification) to thereby prepare a cell suspension of the cells having osteogenic capacity (Step B).
  • the present invention provides a step of mixing the resulting cell suspension having osteogenic capacity with a bio-matrix to perform cell- surface treatment for improving the viability of the cells having osteogenic capacity and the mobility/anchorage thereof into bone cavities, thereby preparing a cell therapy product having osteogenic capacity (Step C).
  • the present invention is also characterized by using a low-molecular weight hyaluronic acid of 1 ,000 to 1 ,000,000 MW as the bio-matrix in Step C.
  • the present invention provides steps of adding the low-molecular weight hyaluronic acid in a ratio of 0.01 to 0.03% per 10 to 20 mL of the cell suspension containing l*10 6 to IxIO 7 cells having osteogenic capacity; reacting the low-molecular weight hyaluronic acid with the cells in a CO 2 incubator at a concentration of 4 to 6% CO 2 and a temperature of 36 to 38 ° C for 1 to 2 hours, in order to achieve binding between the hyaluronic acid and CD44 (cluster designation 44) present on the surface of the cells having osteogenic capacity; and removing the hyaluronic acid not attached to the cell surface to thereby prepare a cell composition for promoting bone-formation.
  • the present invention provides a cell composition for promoting bone-formation, comprising autologous cells having osteogenic capacity and a low-molecular weight hyaluronic acid wherein the cells are surface-coated with the hyaluronic acid, through the above-mentioned steps.
  • the present invention is characterized by the following: the autologous cells having osteogenic capacity are isolated from bone marrow, and are cultured in ⁇ -MEM containing 40 to 60 mg/L of vitamin C (ascorbic acid) in order to improve functions of osteoprogenitor cells, 7 to 10 days prior to a surgical application.
  • the isolated cells are cultured and proliferated in DMEM (Dulbecco's Modified Eagle's Medium) or ⁇ -MEM (Minimum Essential Medium, Alpha Modification) to thereby prepare a cell suspension having osteogenic capacity, and the resulting cell suspension having osteogenic capacity is mixed with a low-molecular weight hyaluronic acid of 1,000 to 1,000,000 MW as a bio-matrix, the hyaluronic acid being added in a ratio of 0.01 to 0.03% per 10 to 20 mL of the cell suspension containing IxIO 6 to IxIO 7 cells having osteogenic capacity.
  • DMEM Dynamic Eagle's Medium
  • ⁇ -MEM Minimum Essential Medium, Alpha Modification
  • the low-molecular weight hyaluronic acid is reacted with the cells in a CO 2 incubator at a concentration of 4 to 6% CO 2 and a temperature of 36 to 38 ° C for 1 to 2 hours, in order to achieve binding between the hyaluronic acid and CD44 (cluster designation 44) present on the surface of the cells having osteogenic capacity.
  • the hyaluronic acid is attached to the surface of the autologous cells, such that the cell composition for promoting bone- formation can be injected into the blood stream via an artery or vein.
  • attachment of a low-molecular weight hyaluronic acid having 1,000 to 1,000,000 MW to CD44 present on the surface of the cells having osteogenic capacity leads to an increased survival time of cells because the cells having osteogenic capacity which are adherent cells can survive only in the presence of an adequate support material, and also leads to an improved mobility of the cells into the bone cavities when the low-molecular weight hyaluronic acid are attached to the cells having osteogenic capacity.
  • attachment of a high-molecular weight hyaluronic acid to the cells results in an increased volume of the cells and a decreased mobility of the cells in the blood stream due to aggregation thereof.
  • the low-molecular weight hyaluronic acid is added in a ratio of less than 0.01% per 10 to 20 mL of the cell suspension, the amount of the low-molecular weight hyaluronic acid attached to the cells having osteogenic capacity and the number of cells bound to the hyaluronic acid are decreased, thereby resulting in a decrease of an therapeutic efficiency. Therefore, the low-molecular weight hyaluronic acid was added in a ratio of 0.01% per 10 to 20 mL of the cell suspension. In addition, upon binding of the low-molecular weight hyaluronic acid to the cells having osteogenic capacity, the cells should be continuously viable.
  • the cells and hyaluronic acid are reacted in a CO 2 incubator furnished with favorable growth conditions at a concentration of 5% CO 2 and a temperature of 37 ° C .
  • the cells are reacted for an optimal reaction time of 1 to 2 hours, taking into consideration the time necessary for attachment, anchorage and growth of the suspended cells having osteogenic capacity onto a supporting material.
  • the low-molecular weight hyaluronic acid unbound to the cells having osteogenic capacity probably has adverse side effects on the viscosity of the therapeutic agent and the cell viability, due to aggregation of hyaluronic acid which may occur during the production process of the therapeutic agent, the unbound hyaluronic acid is precipitated by centrifugation and is removed from the hyaluronic acid-bound cells by washing the cells twice or more.
  • the thus-washed low- molecular weight hyaluronic acid-bound cells having osteogenic capacity are suspended in an injection solution of DMEM (Dulbecco's Modified Eagle's Medium) containing 12.5 to 25 mM HEPES (N-2-hydroxyethylpiperazine-N'-2- ethanesulfonic acid) serving as a pH buffer.
  • DMEM Dulbecco's Modified Eagle's Medium
  • HEPES N-2-hydroxyethylpiperazine-N'-2- ethanesulfonic acid
  • HBSS Hank's Balanced Salt Solution
  • a transfer bottle is washed with 5 mL of HBSS and transferred to a 50 mL tube.
  • a 50 mL fresh tube is equipped with a sieve.
  • 10 mL of HBSS is added to the tube which is then closed with a stopper and is vigorously tapped 50 times.
  • Type II collagenase is dissolved to a concentration of
  • FBS low-glucose DMEM (with a final concentration of 100 ⁇ g/mL gentamicin): for neutralization and washing.
  • Isolated nucleated cells are cultured and proliferated in 10% FBS Io w- glucose DMEM. Upon performing cell subculture taking a period of 3 to 4 weeks, a predetermined number of cells are stored.
  • Isolated nucleated cells are washed with HBSS three times. 2. The cells are treated with 2 or 4 mL of trypsin and incubated for 3 to 5 min.
  • the cell cultures are treated with 1 or 2 mL of FBS, and are neutralized with shaking. 4.
  • the cells are filtered (through a cell strainer), transferred to a 15 mL tube, washed with 10 mL of a medium, and centrifuged at 1,200 rpm for 5 min.
  • the cells are subjected to suction, tapping and suspension, then collected in a tube, and washed with 5 mL of a medium. 6.
  • the cells are seeded in a flask.
  • the cells are observed under a microscope, QC samples are collected on a clean bench.
  • the culture medium is removed, washed with a given amount of an HBSS solution. This procedure is repeated three times. 4. Each vessel is treated with trypsin and is incubated for 5 min.
  • the cell cultures are neutralized with FBS and shaken. Then, the culture flask is washed with a medium, and the cell cultures are transferred to a 15 mL tube and centrifuged for 5 min.
  • the cells After centrifugation for 5 min, the cells are mixed with a freezing medium and each 1.8 mL aliquot having a cell density of 2X10 6 to 1.5X10 7 cells is stored in vials. 8. Upon performing transplantation, the cells may be stored at a temperature of 4 ° C for up to 2 hours.
  • the cells are freeze-stored in a double-seal container at -70 ° C overnight (up to 72 hours). 10. On the next day, the cells are stored in a liquid nitrogen tank at -196 ° C for a long period of time.
  • the cells are cultured by replacement of a culture medium with 10% FBS ⁇ - MEM containing 50 mg/L of vitamin C (ascorbic acid), 7 to 10 days prior to surgical transplantation.
  • the collected cells are treated with CM-DiI and are aliquoted into Petri dishes having a diameter of 10 cm (25 ⁇ l of DMSO/2 mL of HBSS). 4. The cells are incubated at 37 ° C for 5 min.
  • the cells are incubated at 4 0 C for 15 min. 6. 1 mL of FBS is added to terminate fluorescent labeling. ⁇ Cell-containing Petri dishes are wrapped with aluminum foil to block light, and the following steps are carried out. 7. The cells are centrifuged at 1200 rpm for 5 min, and washed with HBSS.
  • the cells are centrifuged again at 1200 rpm for 5 min, and washed with HBSS.
  • the cells are suspended in 20 mL of a medium (10% FBS low-glucose DMEM) to which 2 mL of HA was added. 10. The cells are incubated at 37 0 C for about 90 min.
  • Centrifugation (1200 rpm, 5 min) is carried out to remove a supernatant, thereby leaving coated cells.
  • DMEM containing 20 mL of 12.5 mM HEPES is added to re-suspend the cells.
  • the cells are washed twice with low-glucose DMEM containing 12.5 mM HEPES.
  • the resulting cell pellets are suspended in low-glucose DMEM containing 400 ⁇ l of 12.5 mM HEPES. 16.
  • the cell suspension is wrapped with foil (to block fluorescent labeling) and transported while maintaining it at a temperature of 4 ° C .
  • the cells When the low-molecular weight hyaluronic acid is bound to the cells having osteogenic capacity, the cells should be continuously viable. In order to ensure that the cells do not stay and adhere to the reaction vessel after attachment of the low-molecular weight hyaluronic acid to the cells, the cells and hyaluronic acid are reacted in a CO 2 incubator furnished with favorable growth conditions at a concentration of 5% CO 2 and a temperature of 37 ° C . In addition, the cells are reacted for an optimal reaction time of 1 to 2 hours, taking into consideration the time for which the suspended cells having osteogenic capacity adhere to a supporting material and stably stay to grow therein.
  • the low-molecular weight hyaluronic acid unbound to the cells having osteogenic capacity may have adverse side effects on the viscosity of the therapeutic agent and the cell viability, due to aggregation of the hyaluronic acid which may occur during the production process of the therapeutic agent, the unbound hyaluronic acid is precipitated by centrifugation and the hyaluronic acid- bound cells are washed twice or more to remove the unbound hyaluronic acid.
  • Example 1 Application of cells having osteogenic capacity to rat
  • Bone marrow was collected from iliac crest of rats as an animal model and was subjected to enzyme treatment, and nucleated cells were isolated. An osteogenic inducer was treated to differentiate the nucleated cells into the cells having osteogenic capacity and the cells were then mass-cultured.
  • a hyaluronic acid for surface treatment of the cells a low-molecular weight hyaluronic acid
  • LMW-HA low-density lipoprotein
  • the hyaluronic acid was added and uniformly distributed in a vessel in which the cells having osteogenic capacity and a culture medium were mixed. The resulting mixture was reacted in a CO 2 incubator at 37 ° C for 90 min to effect surface treatment of the cells.
  • the LMW-HA Low-density lipoprotein
  • HA-surface treated cells having osteogenic capacity were injected into an artery and vein of rats with ovariectomy (OVX)-induced osteoporosis.
  • A represents a normal rat
  • B represents a rat with OVX-induced osteoporosis
  • C represents a rat with OVX-induced osteoporosis in which the cells having osteogenic capacity were injected into the vein
  • D represents a rat with OVX- induced osteoporosis in which LMW-HA-surface treated cells having osteogenic capacity were injected into the vein
  • E represents a rat with OVX-induced osteoporosis in which LMW-HA-surface treated cells having osteogenic capacity were injected into the artery, respectively.
  • Example 2 Application of human-derived cells into NOD/SCID mice (non-obese diabetic severe combined immuno-deficiencv mouse) A low-molecular weight hyaluronic acid (LMW-HA) was added and uniformly distributed in a reaction vessel in which human cells having osteogenic capacity and a culture medium were mixed. The resulting mixture was reacted in a CO 2 incubator at a temperature of 37 ° C and a concentration of 5% CO 2 for 120 min to effect surface treatment of the cells.
  • LMW-HA low-molecular weight hyaluronic acid
  • Fluorescence-labeled, human-derived cells having osteogenic capacity were injected into immunodeficient NOD/SCID mice via the blood stream, and the cell mobility of the human-derived cells and the distribution and anchorage of the cells within the bone were confirmed as shown in results of FIG. 4.
  • the fluorescence-labeled cells (C) were observed by enlarging cross sections (A and B) of the mouse femur under a fluorescence microscope, thus confirming that the fluorescence-labeled, LMW-HA-surface treated cells having osteogenic capacity, which were injected to the blood stream, have migrated and settled into the bone.
  • the present invention enables achievement of bone regeneration throughout extensive regions by delivery of bone-forming cells via the blood stream, using a method for preparing a cell therapy product by isolating cells having osteogenic capacity from bone marrow, preparing a cell suspension having osteogenic capacity and subjecting the cells to surface treatment, thereby preparing a cell therapy product having osteogenic capacity.
  • the present invention can accomplish local or systemic bone regeneration by subjecting cells having osteogenic capacity to surface treatment using a hyaluronic acid and injecting the thus-treated cells into a circulation system of target animals.
  • the present invention can achieve uniform delivery of bone- forming cells to target regions in need of bone formation while not causing destruction of the remaining bone tissue structure, by injection of a bone-forming therapeutic agent via the blood stream. Therefore, it is possible to treat various bone diseases including osteoporosis in need of extensive bone regeneration.
  • the present invention accomplishes remarkably improved quality and reliability of the product and thereby is very useful to enhance customer satisfaction.

Abstract

La présente invention concerne une composition cellulaire pour favoriser la formation osseuse, capable de donner une régénération osseuse locale ou systémique. En l'occurrence, on prend des cellules ostéogènes et on en traite la surface à l'acide hyaluronique et on les injecte dans la circulation sanguine d'animaux. L'invention concerne également un procédé d'élaboration correspondant. Ce procédé consiste à isoler des cellules ostéogènes extraite de moelle osseuse, à les mettre en culture et à les faire proliférer dans un milieu de culture de type DMEM (Dulbecco's Modified Eagle's Medium) ou α-MEM (Minimum Essential Medium, Alpha Modification) de façon à obtenir une suspension ostéogène de cellules. On prend cette suspension ostéogène de cellule, et on la mélange à une matrice biologique pour exécuter un traitement de la surface des cellules visant à améliorer la viabilité des cellules ostéogènes ainsi que leur mobilité et ancrage dans les cavités osseuses, ce qui permet d'élaborer un produit de thérapie cellulaire ostéogène.
PCT/KR2006/003791 2006-09-20 2006-09-25 Composition cellulaire pour régénération osseuse et procédé de fabrication correspondant WO2008035832A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020060091325A KR100834718B1 (ko) 2006-09-20 2006-09-20 뼈 재생을 도모하는 뼈 형성 촉진 세포조성물 및 그 제조방법
KR10-2006-0091325 2006-09-20

Publications (1)

Publication Number Publication Date
WO2008035832A1 true WO2008035832A1 (fr) 2008-03-27

Family

ID=39200645

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2006/003791 WO2008035832A1 (fr) 2006-09-20 2006-09-25 Composition cellulaire pour régénération osseuse et procédé de fabrication correspondant

Country Status (2)

Country Link
KR (1) KR100834718B1 (fr)
WO (1) WO2008035832A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016042572A1 (fr) * 2014-09-19 2016-03-24 Jaswantrai Doshi Vishal Méthode d'induction de la formation osseuse par mise en culture d'ostéoblastes ex vivo à implanter

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20100007180A (ko) * 2008-07-11 2010-01-22 세원셀론텍(주) 뼈 재생용 콜라겐 젤 조성물의 제조방법
KR101037002B1 (ko) * 2008-09-30 2011-05-25 세원셀론텍(주) 연골세포 조기배양을 위한 연골세포 특이적 배양방법

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5902741A (en) * 1986-04-18 1999-05-11 Advanced Tissue Sciences, Inc. Three-dimensional cartilage cultures
WO2003015802A1 (fr) * 2001-08-14 2003-02-27 Medipost, Co., Ltd. Composition destinee au traitement d'un cartilage articulaire abime
EP1338291A2 (fr) * 2002-02-21 2003-08-27 Ed. Geistlich Söhne Ag Für Chemische Industrie matrice extracellulaire résorbable pour le reconstruction d'os
KR20030081821A (ko) * 2002-04-13 2003-10-22 주식회사 셀론텍 뼈세포의 인공 배양물

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100443413B1 (ko) * 2001-07-30 2004-08-09 김민지 유색불꽃 알코올 램프의 제조방법

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5902741A (en) * 1986-04-18 1999-05-11 Advanced Tissue Sciences, Inc. Three-dimensional cartilage cultures
WO2003015802A1 (fr) * 2001-08-14 2003-02-27 Medipost, Co., Ltd. Composition destinee au traitement d'un cartilage articulaire abime
EP1338291A2 (fr) * 2002-02-21 2003-08-27 Ed. Geistlich Söhne Ag Für Chemische Industrie matrice extracellulaire résorbable pour le reconstruction d'os
KR20030081821A (ko) * 2002-04-13 2003-10-22 주식회사 셀론텍 뼈세포의 인공 배양물

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016042572A1 (fr) * 2014-09-19 2016-03-24 Jaswantrai Doshi Vishal Méthode d'induction de la formation osseuse par mise en culture d'ostéoblastes ex vivo à implanter
US10577587B2 (en) 2014-09-19 2020-03-03 Regrow Biosciences Private Limited Method of inducing bone formation by ex-vivo osteoblast culturing for implantation

Also Published As

Publication number Publication date
KR100834718B1 (ko) 2008-06-02
KR20080026378A (ko) 2008-03-25

Similar Documents

Publication Publication Date Title
Yin et al. Induction of mesenchymal stem cell chondrogenic differentiation and functional cartilage microtissue formation for in vivo cartilage regeneration by cartilage extracellular matrix-derived particles
Arinzeh et al. Allogeneic mesenchymal stem cells regenerate bone in a critical-sized canine segmental defect
JP6130588B2 (ja) 皮膚再生または傷治癒のための間葉系幹細胞−ヒドロゲル−生分解性または間葉系幹細胞−ヒドロゲル−非分解性支持体造成物
JP3808900B2 (ja) 結合組織細胞に部分的又は完全に分化した骨髄幹細胞の有効培養物及びヒアルロン酸誘導体より成る三次元の生体親和性で且つ生分解性のマトリックスから構成される生物学的物質
US11801331B2 (en) Composition for cartilage regeneration and preparing thereof
JP2001502905A (ja) ホウォートンゼリーから分離した細胞を用いた軟骨組織の形成
US7416889B2 (en) Methods and compositions for repairing cartilage
JP2003517259A (ja) 骨および前駆体軟骨細胞の単離方法
CZ20032082A3 (cs) Zlepšený způsob kultivace savčích buněk in vitro pro autologní způsoby implantace a transplantace
Strong et al. Obesity inhibits the osteogenic differentiation of human adipose-derived stem cells
JP5554002B2 (ja) 軟骨組織再生シートの作製方法
US20160046903A1 (en) Method for preparing chondrocytes
US20160067377A1 (en) Stem Cell Seeded Natural Substrates and Methods Relating Thereto
WO2013146992A1 (fr) Procédé de fabrication de cellules souches pluripotentes dérivées de la pulpe dentaire
JP5890222B2 (ja) 培養軟骨組織材料
KR20170108325A (ko) 성체줄기세포의 연골세포 분화용 조성물 및 분화 방법
JP7273419B2 (ja) 脂肪由来幹細胞を含む生体材料およびその作製方法
JP7273418B2 (ja) 脂肪由来幹細胞を含む無足場構造物用生体材料およびその作製方法
WO2008035832A1 (fr) Composition cellulaire pour régénération osseuse et procédé de fabrication correspondant
JP7350344B2 (ja) ヒト誘導万能幹細胞から軟骨細胞のペレットを製造する方法およびその用途
Yoshikawa et al. Bone and soft tissue regeneration by bone marrow mesenchymal cells
Bohlouli et al. Tissue buccal fat pad–stromal vascular fraction as a safe source in maxillofacial bone regeneration: A clinical pilot study
KR20110032433A (ko) 세포이식술을 위한 혼합세포복합체인 세포스페로이드의 제조방법 및 이의 이용방법
EP3795677A1 (fr) Composition pour favoriser la différenciation de cellules souches, comprenant une solution de culture de cellules progénitrices et un film de graphène multicouche, et son utilisation
WO2016088373A1 (fr) Feuille de cellules cultivées pour l'utilisation en transplantation, procédé de production d'une feuille de cellules cultivées pour l'utilisation en transplantation, et procédé de production de tissu osseux pour l'utilisation en transplantation

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 06798874

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 06798874

Country of ref document: EP

Kind code of ref document: A1