WO2011081328A2 - Construct for tissue reconstruction and kit for producing a construct for tissue reconstruction - Google Patents

Construct for tissue reconstruction and kit for producing a construct for tissue reconstruction Download PDF

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Publication number
WO2011081328A2
WO2011081328A2 PCT/KR2010/008969 KR2010008969W WO2011081328A2 WO 2011081328 A2 WO2011081328 A2 WO 2011081328A2 KR 2010008969 W KR2010008969 W KR 2010008969W WO 2011081328 A2 WO2011081328 A2 WO 2011081328A2
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Prior art keywords
compartment
growth factor
coating layer
cells
tissue regeneration
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PCT/KR2010/008969
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French (fr)
Korean (ko)
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WO2011081328A3 (en
Inventor
이수홍
강선웅
김진수
한인보
신동아
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차의과학대학교 산학협력단
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Priority claimed from KR1020090133762A external-priority patent/KR101162269B1/en
Priority claimed from KR1020090133756A external-priority patent/KR101141547B1/en
Application filed by 차의과학대학교 산학협력단 filed Critical 차의과학대학교 산학협력단
Publication of WO2011081328A2 publication Critical patent/WO2011081328A2/en
Publication of WO2011081328A3 publication Critical patent/WO2011081328A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/28—Materials for coating prostheses
    • A61L27/34—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/64—Animal cells

Definitions

  • the present invention relates to a kit for forming a tissue regeneration structure and a structure for tissue regeneration. More specifically, the present invention relates to a structure for tissue regeneration comprising a coating layer formed by coating hyaluronic acid or a salt thereof and fibrinogen on the surface of a frame of a polymer support having pores. The present invention further relates to a kit for forming a structure for tissue regeneration crosslinked by thrombin and having a pore of 10 ⁇ m to 1,000 ⁇ m.
  • Tissue engineering is a multidisciplinary study that applies existing scientific fields such as cytology, life sciences, engineering, and medicine to understand the correlation between structure and function of living tissues, and to repair damaged tissues or organs as normal tissues. It is a new convergence discipline to replace and regenerate. That is, it aims to maintain, improve or restore the functions of our bodies by making artificial tissues implantable in the body.
  • the tissue is collected from the patient's body to separate the cells, and then the separated cells are expanded by the necessary amount through the culture.
  • the proliferated cells are planted in a porous biodegradable polymer support and the cell / support complex obtained by in vitro culture for a period of time is transplanted back into the body.
  • the transplanted cell / support complex is supplied with oxygen and nutrients by the diffusion of body fluids until new blood vessels are formed, and blood vessels grow in the body and proliferate when blood is supplied to form new tissues and organs.
  • the support for tissue regeneration may be divided into a case of using itself and a case of containing cells according to the purpose of tissue regeneration.
  • tissue growth factors or differentiation-inducing factors are introduced to induce cell migration from surrounding tissues during transplantation to regenerate tissues.
  • the cells are included, autologous or homologous cells are used, and recently, efforts are being made to induce tissue regeneration more smoothly using stem cells.
  • the scaffold for tissue engineering is one of the essential elements for effectively inducing tissue regeneration.
  • polyglycolic acid (PGA), polylactic acid (PLA), polycaprolactone (PCL) -based polymers and copolymers thereof are prepared in the form of structures having pores.
  • the prepared supports all have hydrophobic properties due to the properties of these materials. Therefore, in tissue engineering applications, the polymer support has a problem of low cell and tissue suitability when transplanted into human tissue or when culturing tissue cells.
  • the present inventors have studied a hydrogel surface treatment method for enhancing cell affinity to solve the disadvantage that the cell adhesion is not efficiently achieved in the tissue engineering support showing hydrophobicity and the disadvantage that the cell growth factors are not easily introduced.
  • the present inventors form a coating layer by coating a hyaluronic acid or a salt thereof and fibrinogen on a surface of a frame of a polymer support having pores by lyophilization or the like to form a coating layer, thereby greatly improving cell affinity and
  • the researchers found that cell growth factors can be introduced very efficiently. That is, when seeding cells and / or cell growth factors, etc.
  • the cells and cell growth factors can be induced to adhere to the surface of the support at a high speed. Furthermore, by inducing crosslinking through thrombin and keeping the resulting structure intact, the body fluids can be smoothly accessed through the pores and the supply of oxygen and nutrients is smooth, as well as through the pores. It was found that continuous and stable release of cell growth factors from the constructs allows tissue cells to adhere and proliferate uniformly and efficiently.
  • the present invention provides a structure for tissue regeneration in which cells and / or cell growth factors are introduced onto a polymer support at a high adhesion rate or loading rate by having high hydrophilicity and cell affinity.
  • the present invention also provides a kit for forming the structure for tissue regeneration.
  • a structure for tissue regeneration comprising a coating layer formed by coating hyaluronic acid or a salt thereof and fibrinogen on a surface of a frame of a polymer support having pores.
  • the coating layer is a cell; Cell growth factor; Or cells and cell growth factors,
  • the coating layer is crosslinked with thrombin, and
  • the structure has a pore of 10 ⁇ m to 1,000 ⁇ m.
  • a structure-compartment comprising a coating layer formed by coating hyaluronic acid or a salt thereof and fibrinogen on a surface of a structure of a polymer support having voids;
  • a thrombin-compartment comprising a solution comprising thrombin and calcium salt, forming a structure for tissue regeneration crosslinked by thrombin and having pores of 10 ⁇ m to 1,000 ⁇ m
  • a kit for is provided.
  • a structure-compartment comprising a coating layer formed by coating hyaluronic acid or a salt thereof and fibrinogen on a surface of a structure of a polymer support having voids;
  • Thrombin-compartment comprising a solution comprising thrombin and calcium salt;
  • one or more aqueous solution-compartments for the provision of cells or cell growth factors, for tissue regeneration crosslinked by thrombin and having pores of 10 ⁇ m to 1,000 ⁇ m Kits for forming structures are provided.
  • a structure-compartment comprising a coating layer formed by coating hyaluronic acid or a salt thereof and fibrinogen on a surface of a structure of a polymer support having voids; Cell growth factor; And a cell growth factor-thrombin-buffer compartment (compartment (b ')) comprising an aqueous solution containing thrombin and calcium salt; And optionally, a cell-aqueous solution-compartment containing a cell [compartment (c ')], which is crosslinked by thrombin and forms a structure for tissue regeneration having pores of 10 ⁇ m to 1,000 ⁇ m
  • a kit for is provided.
  • a structure-compartment comprising a coating layer formed by coating hyaluronic acid or a salt thereof and fibrinogen on a surface of a structure of a polymer support having voids;
  • Thrombin-compartment comprising a solution comprising thrombin and calcium salt;
  • tissue regeneration crosslinked by thrombin and having pores of 10 ⁇ m to 1,000 ⁇ m, including at least one aqueous solution-compartment containing the cell or cell growth factor [compartment (c ′′)]
  • a kit is provided for forming a structure for use.
  • a structure for tissue regeneration formed from the kit of the present invention includes a coating layer formed by coating hyaluronic acid or a salt thereof and fibrinogen on a surface of a frame of a polymer support having pores, and thus obtained By maintaining the pores intact, the structure provides smooth access to body fluids through the pores and facilitates the supply of oxygen and nutrients, as well as continuous and stable release of cell growth factors from the constructs through the pores. Therefore, the tissue cells can adhere and proliferate uniformly and efficiently.
  • the tissue regeneration structure of the present invention can form a uniform coating layer without the use of a chemical catalyst, thereby having excellent safety as well as having high hydrophilicity and cell affinity to cells, cells Growth factors can be introduced at high induction or loading rates.
  • the cell growth factor introduced from the structure for tissue regeneration formed from the kit of the present invention shows a sustained release pattern, it is possible to stably induce differentiation of stem cells. Therefore, the tissue regeneration structure according to the present invention may be implanted into a living body and used as various tissue engineering materials including musculoskeletal regeneration, blood vessel regeneration, and skin regeneration.
  • the kit according to the present invention can be usefully used for forming various tissue regeneration structures including musculoskeletal regeneration, blood vessel regeneration, skin regeneration and the like.
  • FIG. 1 is a result of observing the form of the polymer support (left side of FIG. 1, A) and the tissue regeneration support body (right side of FIG. 1, B) obtained according to the present invention before forming the coating layer.
  • the upper part is a photograph measured by an optical microscope, and the lower part is a photograph measured by a scanning electron microscope (SEM).
  • Figure 2 is a support for tissue regeneration according to the present invention (right side) into which human adipose derived stem cells were introduced; And the support (left) of the comparative example prepared by incubating the human body-derived stem cells in the same manner after sufficiently hydrated the polymer support without the coating layer in a cell culture medium containing 10% of the right serum for 3 days. (Confocal Laser Scanning Microscope, CLSM) and the result of measuring the number of cells attached to the support.
  • CLSM Confocal Laser Scanning Microscope
  • Figure 3 is the result of measuring the release behavior of BMP-2 from the support for tissue regeneration according to the present invention in which bone growth factor was introduced by ELISA.
  • Figure 4 quantifies the degree of bone differentiation of adherent adipose stem cells when the cultured human adipose derived stem cells in the presence of the support for tissue regeneration according to the present invention introduced bone growth factor by alkaline phosphatase activity (alkaline phosphatase activity) The result of the analysis.
  • a coated support without BMP-2 NO BMP-2
  • a coated support BMP-2 addition day 0
  • FIG. 5 is a photograph of qualitative results observed through optical microscope after Goldner-Trichrome staining of regenerated bone tissue on the support surface into which extracellular matrix is introduced.
  • Figure 6 shows the results of the Goldner-Trichrome staining in the third and fourth groups.
  • 7 is a result of quantifying the regenerated bone tissue area in each group.
  • the upper part is the soft X-ray result through animal experiment, and the lower part is the calcium ion quantification result of bone tissue regenerated through animal experiment.
  • tissue regeneration construct refers to a structure that is implanted in or around a damaged tissue in the body to rebuild the damaged tissue and then separate or completely disintegrate.
  • the tissue regeneration construct may be induced in vitro to differentiate and may be directly implanted in vivo without inducing differentiation so that differentiation may be induced in vivo.
  • the tissue regeneration structure is implanted into the body and receives oxygen and nutrients through the body fluid until new blood vessels are formed, and also proliferates as blood is supplied through the formation of blood vessels (angiogenesis) to form new tissues or organs. do.
  • Cell growth factors are also referred to as differentiation-inducing factors and refer to factors involved in cell growth and / or stem cell differentiation.
  • the cell growth factor includes all factors that induce cell migration from surrounding tissues to regenerate tissues or promote stem cell differentiation to regenerate tissues.
  • bone morphogenetic protein 2 BMP-2
  • VEGF vascular endothelial growth factor
  • EGF epidermal growth factor
  • IGF insulin-like growth factor
  • NGF nerve growth factor
  • PDGF platelet-derived PDGF
  • growth factor transforming growth factor- ⁇ (TGF- ⁇ ), basic fibroblast growth factor (bFGF), and the like, but are not limited thereto.
  • the present invention provides a construct for tissue-reconstruction comprising a coating layer formed by coating hyaluronic acid or a salt thereof and fibrinogen on a surface of a frame of a polymer support having pores, wherein (i) the Coating layer of cells; Cell growth factor; Or cells and cell growth factors, (ii) the coating layer is crosslinked with thrombin, and (iii) the structure has a pore of 10 ⁇ m to 1,000 ⁇ m.
  • the polymer support is a matrix made of a polymer and refers to a matrix having pores.
  • the polymer support may be preferably formed from a biodegradable polymer, and the biodegradable polymer may be a biodegradable polymer commonly used in medicine or pharmaceutical fields.
  • the polymer support is polylactide, polyglycolide, polycaprolactone, a copolymer of lactide and glycolide, a copolymer of lactide and caprolactone, and a copolymer of glycolide and caprolactone It may be formed from at least one biodegradable polymer selected from the group consisting of, more preferably a copolymer of D, L-lactide and glycolide [eg, poly (lactic acid-co-glycolic acid) (poly (D, L-lactic acid-co-glycolic acid (PLGA)) and polycaprolactone (poly ( ⁇ -caprolactone), PCL) can be formed from the above.
  • a biodegradable polymer selected from the group consisting of, more preferably a copolymer of D, L-lactide and glycolide
  • poly (lactic acid-co-glycolic acid) poly (D, L-lactic acid-co-glycolic acid (PLGA)
  • biodegradable polymers From biodegradable polymers, it can be produced by conventional methods such as salt extraction, foaming, emulsion freezing, thermal bonding, three-dimensional printing, CAD-CAM, etc.
  • the method of Kim et al. Biofabrication 2009; 1: 1 It is possible to form a polymer support having voids according to a solid free-form fabrication method according to -7)
  • the shape of the voids may be various shapes such as circular, square, etc.
  • the size may range from about 10 ⁇ m to 1,000 ⁇ m, wherein the size of the pore means, for example, a diameter in the case of a circle or a length of a width or a length of a rectangle.
  • the hyaluronic acid is a hydrophilic polysaccharide present in the human body, and a long chain of basic units (Disaccharide, molecular weight 379) of D-glucoronic acid (D-glucoronic acid) and N-glucosamine (N-glucosamine) It exists in the form of a high molecular weight hetero-polysaccharide, and is responsible for various functions in the extratracelluar matrix such as cell growth, differentiation, and migration.
  • D-glucoronic acid D-glucoronic acid
  • N-glucosamine N-glucosamine
  • the salts of hyaluronic acid may be in various salt forms, for example, may be in the form of organic salts such as cobalt hyaluronic acid, magnesium hyaluronate, zinc hyaluronate, calcium hyaluronate, potassium hyaluronate, sodium hyaluronate and the like, and hyaluronic acid tetrabutylammonium.
  • hyaluronate can be used.
  • the tissue regeneration structure of the present invention may include the salts of hyaluronic acid or hyaluronic acid, respectively, or may contain a mixture of hyaluronic acid and a salt of hyaluronic acid.
  • the weight average molecular weight of the hyaluronic acid or a salt thereof is not particularly limited, and for example, the average molecular weight of the hyaluronic acid or a salt thereof may be 1,000 to 4,000,000 Daltons, and preferably 1,000 to 1,500,000 Daltons.
  • the content of hyaluronic acid or a salt thereof in the coating layer may be in the range of 10 to 90% by weight, and the content of fibrinogen in the coating layer may be in the range of 10 to 90% by weight.
  • the weight ratio of the hyaluronic acid or a salt thereof and fibrinogen in the coating layer is not particularly limited, and may be controlled in view of the release rate of cell growth factors.
  • Coating of hyaluronic acid or a salt thereof and fibrinogen on the surface of the frame of the polymer support is immersed in the aqueous solution containing hyaluronic acid and fibrinogen, and then the support is taken out of the aqueous solution and lyophilized. It may be carried out by performing the procedure at least once.
  • the lyophilization may be performed repeatedly, for example, may be performed repeatedly 2 to 20 times, but is not limited thereto.
  • the immersion may be carried out at 4 to 50 °C, preferably 4 to 20 °C.
  • the immersion time is not particularly limited, and may be performed for a short time of about 30 seconds or, if necessary, for a longer time, for example, 12 to 48 hours.
  • by further comprising drying the support taken out of the aqueous solution at room temperature (about 25 °C) before lyophilization after the immersion it is possible to prevent the clogging in the manufacturing process.
  • the polymer support having hyaluronic acid or a salt thereof and fibrinogen coated on the surface of the frame has a high hydrophilicity and cell affinity, thereby resulting in high adhesion rate of cells and / or cell growth factors. or loading rate) may be introduced onto the polymer support.
  • the introduction of the cell growth factor may be performed by dissolving the cell growth factor in an aqueous solution, for example, phosphate-buffered saline, adding it to the coating layer and drying (preferably lyophilization).
  • an aqueous solution for example, phosphate-buffered saline
  • cell growth factors may be uniformly dispersed in the entire coating layer or evenly embedded in the surface of the coating layer.
  • introduction of the cell growth factor may be introduced before the crosslinking of the coating layer, simultaneously with the crosslinking or after the crosslinking.
  • the cell growth factor may be introduced into the coating layer by additionally containing a cell growth factor in an aqueous solution containing hyaluronic acid or a salt thereof and fibrinogen, and forming a coating layer in the same manner as described above. have.
  • an aqueous solution containing cell growth factor is added to the coating layer formed by coating the hyaluronic acid or its salt and fibrinogen (if further dried, preferably after lyophilization, if necessary), and thrombin.
  • the cell growth factor may be introduced into the coating layer by drying by adding a solution containing calcium salt.
  • cell growth factor may be introduced into the coating layer.
  • a solution containing thrombin and calcium salt is added to the coating layer formed by coating the hyaluronic acid or a salt thereof and fibrinogen, followed by drying, and then an aqueous solution containing cell growth factor (additionally, if necessary, Preferably, by lyophilization), the cell growth factor can be introduced into the coating layer.
  • the drying may be preferably performed by lyophilization
  • the concentration of the cell growth factor in the aqueous solution may be in the range of 10 pg / mL to 100 mg / mL.
  • the introduction of the cells is a dispersion layer obtained by dispersing cells such as stem cells in an aqueous medium (for example, a conventional cell culture medium such as phosphate buffered saline, DMEM, etc.) coating layer or a coating layer into which the cell growth factor is introduced. It can be performed by adding to.
  • aqueous medium for example, a conventional cell culture medium such as phosphate buffered saline, DMEM, etc.
  • the method further comprises seeding an aqueous dispersion containing cells on the surface of the coating layer of the tissue regeneration structure into which the cell growth factor obtained as described above is introduced. It may be introduced into the coating layer.
  • the concentration of cells in the aqueous dispersion may range from 10 3 to 10 10 cells / mL.
  • a solution containing thrombin and calcium salt is added to the coating layer formed by coating the hyaluronic acid or a salt thereof and fibrinogen, followed by drying, followed by addition of an aqueous dispersion containing cells, Cells may be introduced into the coating layer alone without introduction of cell growth factors.
  • the drying may be performed by lyophilization, and the concentration of cells in the aqueous dispersion may range from 10 3 to 10 10 cells / mL.
  • the thrombin and calcium salts perform a crosslinking function of fibrinogen, and the amount of the thrombin and the calcium salt is used to achieve a desired degree of crosslinking (for example, to achieve a desired release rate of cell growth factor). Can be selected and used in an appropriate range according to the degree required). Preferably the concentration of thrombin and calcium salt may be in the range of 10 to 90% by weight, but is not limited thereto.
  • the cells may be used without limitation, the cells used for the purpose of tissue regeneration, and may typically be tissue cells, adult stem cells, embryonic stem cells, or induced pluripotent stem cells.
  • the loading amount of the cells may be appropriately selected according to necessity, and typically about 1 ⁇ 10 5 to 5 ⁇ 10 7 cells per tissue regeneration construct or May contain stem cells.
  • the tissue cells can be preferably used cells isolated from the patient-derived cells, cultured and propagated in vitro, that is, cells derived from the patient in vitro.
  • the adult stem cells may be derived from various tissues or cells, for example, bone-derived stem cells, umbilical cord blood-derived stem cells, placental stem cells, muscle-derived stem cells, adipose derived stem cells and the like.
  • Adipose-derived stem cells Adipose-derived stem cells (ASCs) that can be obtained by using adipose tissue discarded in a commonly performed liposuction process may be preferably used.
  • the fat-derived stem cells may be those obtained according to a known method from the fat of the patient or another person of the same phenotype.
  • the fat includes, without limitation, subcutaneous fat tissue, bone marrow adipose tissue, mesenteric adipose tissue, gastrointestinal adipose tissue and posterior peritoneal adipose tissue.
  • the cell growth factors may also be used without limitation cell growth factors used for the purpose of tissue regeneration, for example, bone morphogenetic protein 2 (BMP-2), vascular endothelial growth factor (VEGF), Epidermal growth factor (EGF), insulin-like growth factor (IGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor- ⁇ (TGF- ⁇ ), and basic fibroblast growth factor (bFGF)
  • BMP-2 bone morphogenetic protein 2
  • VEGF vascular endothelial growth factor
  • EGF Epidermal growth factor
  • IGF insulin-like growth factor
  • NGF nerve growth factor
  • PDGF platelet-derived growth factor
  • TGF- ⁇ transforming growth factor- ⁇
  • bFGF basic fibroblast growth factor
  • the loading amount of the cell growth factor may be appropriately selected according to the purpose.
  • a cell growth factor is loaded by adding a solution of cell growth factor at a concentration of about 10 pg / mL to 100 mg / mL in an aqueous medium such as phosphate buffered saline to a unit-form tissue regeneration construct and drying it. can do.
  • an aqueous medium such as phosphate buffered saline
  • the tissue regeneration construct of the present invention may contain cells (eg, tissue cells, stem cells, etc.) and cell growth factors in a separate tissue regeneration construct as described above, and a combination of these may be used as needed. It may be contained in a structure for tissue regeneration.
  • BMP-2 as a cell growth factor and adipose derived stem cells as cells may be contained in one tissue regeneration structure, and the tissue regeneration structure obtained therefrom may be usefully used for bone tissue regeneration.
  • tissue regeneration constructs containing BMP-2 and adipose derived stem cells can be induced in vitro to induce bone differentiation, or in vivo to induce bone differentiation by implanting in vivo without inducing differentiation. It may be.
  • the present invention also provides a kit for forming the structure for tissue regeneration.
  • the present invention provides a structure-compartment (compartment (a)) comprising a coating layer formed by coating hyaluronic acid or a salt thereof and fibrinogen on a surface of a structure of a polymer support having voids; And a thrombin-compartment (compartment (b)) comprising a solution comprising thrombin and calcium salt, forming a structure for tissue regeneration crosslinked by thrombin and having pores of 10 ⁇ m to 1,000 ⁇ m It provides a kit for.
  • a structure-compartment comprising a coating layer formed by coating hyaluronic acid or a salt thereof and fibrinogen on a surface of a structure of a polymer support having voids
  • a thrombin-compartment comprising a solution comprising thrombin and calcium salt, forming a structure for tissue regeneration crosslinked by thrombin and having pores of 10 ⁇ m to 1,000 ⁇ m It provides a kit for.
  • Kits according to the invention may further comprise an aqueous solution compartment for the provision of the tissue regeneration factor.
  • the kit according to the present invention comprises a structure-compartment (compartment (a)) comprising a coating layer formed by coating hyaluronic acid or a salt thereof and fibrinogen on the surface of the structure of the polymer support having pores; Thrombin-compartment (compartment (b)) comprising a solution comprising thrombin and calcium salt; And one or more aqueous solution-compartments (compartments (c)) for the provision of cells or cell growth factors.
  • the aqueous solution of compartment (c) comprises an aqueous medium such as a conventional cell culture medium such as phosphate buffered saline, DMEM or the like.
  • the kit according to the present invention forms a structure for tissue regeneration having pores of 10 ⁇ m to 1,000 ⁇ m by introducing a cell or cell growth factor into the compartment (a) and crosslinking by adding the compartment (b).
  • the polymer support having hyaluronic acid or a salt thereof and fibrinogen coated on the surface of the frame has a high hydrophilicity and cell affinity, thereby resulting in high adhesion rate of cells and / or cell growth factors. or loading rate) may be introduced onto the polymer support.
  • introduction of cell growth factors may be achieved by dissolving the cell growth factors in an aqueous solution, such as phosphate-buffered saline (eg, compartment (c)), adding them to the coating layer and drying. (Preferably lyophilization).
  • cell growth factors may be uniformly dispersed in the entire coating layer or evenly embedded in the surface of the coating layer.
  • introduction of the cell growth factor may be introduced before the crosslinking of the coating layer, simultaneously with the crosslinking or after the crosslinking.
  • the cell growth factor may be introduced into the coating layer by further containing a cell growth factor in an aqueous solution containing hyaluronic acid or a salt thereof and fibrinogen to form a coating layer.
  • an aqueous solution containing cell growth factors e.g., compartment (c)
  • the cell growth factor can be introduced into the coating layer by drying, preferably after lyophilization), followed by drying by addition of a solution containing thrombin and calcium salt.
  • cell growth factor may be introduced into the coating layer.
  • a solution containing thrombin and calcium salt is added to the coating layer formed by coating the hyaluronic acid or a salt thereof and fibrinogen, followed by drying, and then an aqueous solution containing cell growth factor (additionally, if necessary, Preferably, by lyophilization), the cell growth factor can be introduced into the coating layer.
  • the drying may be preferably carried out by lyophilization, the concentration of the cell growth factor in the aqueous solution (eg, compartment (c)) is 10 pg / mL to 100 mg / may be in the range of mL.
  • the introduction of the cells is obtained by dispersing cells such as stem cells in an aqueous medium (for example, conventional cell culture medium such as phosphate buffered saline, DMEM, etc.) (for example, compartment (c)).
  • the dispersion may be carried out by adding the coating layer or the coating layer into which the cell growth factor is introduced.
  • the cells are introduced into the coating layer as described above, the cells are adhered to the coating layer with high adhesion efficiency.
  • the method further comprises seeding an aqueous dispersion containing cells on the surface of the coating layer of the tissue regeneration structure into which the cell growth factor obtained as described above is introduced. It may be introduced into the coating layer.
  • the concentration of cells in the aqueous dispersion [eg, compartment (c)] can range from 10 3 to 10 10 cells / mL.
  • a solution containing thrombin and calcium salt is added to the coating layer formed by coating the hyaluronic acid or a salt thereof and fibrinogen, followed by drying, followed by an aqueous dispersion containing cells [eg , Compartment (c)], the cells can be introduced into the coating layer alone without introducing the cell growth factor.
  • the drying may be performed by lyophilization, and the concentration of cells in the aqueous dispersion may range from 10 3 to 10 10 cells / mL.
  • the kit for forming a tissue regeneration structure according to the present invention can be produced in the form of a pre-made kit (ready-made kit) containing the cells and / or cell growth factors in advance. That is, the tissue regeneration factor necessary for tissue regeneration in advance in the form of a kit, can be used as a kit that can be implanted by forming a structure for tissue regeneration when applied to the patient.
  • the use of the pre-made kit is as described above.
  • the pre-fabricated kit comprises a structure-compartment comprising a coating layer formed by coating hyaluronic acid or a salt thereof and fibrinogen on the surface of the structure of the polymer support having voids [compartment (a) ]; Cell growth factor; And a cell growth factor-thrombin-buffer compartment (compartment (b ')) comprising an aqueous solution containing thrombin and calcium salt; And optionally, a cell-aqueous solution-compartment containing the cells (compartment (c ')).
  • the cell and / or cell regeneration factor may be produced in a form containing a separate compartment, specifically, hyaluronic acid or a salt thereof and fibrinogen on the surface of the structure of the polymer support having pores
  • a structure-compartment [compartment (a)] comprising a coating layer formed by coating;
  • Thrombin-compartment (compartment (b)) comprising a solution comprising thrombin and calcium salt;
  • one or more aqueous solution-compartments (compartments (c '')) containing cells or cell growth factors are examples of cells or cell growth factors.
  • Compartment (c '') may be composed of two or more plurality of compartments, if necessary, for example, physiologically such as an aqueous solution containing cell growth factor, preferably phosphate buffered saline. Suitable buffers; And a plurality of compartments of a dispersion containing cells.
  • an aqueous solution containing cell growth factors is added to the compartment (a) and dried (preferably Lyophilization), followed by addition of compartment (b) to induce crosslinking, followed by culturing the crosslinked-induced construct in a dispersion containing cells (e.g., stem cell dispersion).
  • a dispersion containing cells e.g., stem cell dispersion
  • the polymer support, hyaluronic acid, cells, cell growth factors, etc. are the same as described with reference to the tissue regeneration structure.
  • the content of hyaluronic acid or a salt thereof in the coating layer may be in the range of 10 to 90% by weight, and the content of fibrinogen in the coating layer is 10 to 90 It may range from weight percent.
  • the weight ratio of the hyaluronic acid or a salt thereof and fibrinogen in the coating layer is not particularly limited, and may be controlled in view of the release rate of cell growth factors.
  • Coating of hyaluronic acid or a salt thereof and fibrinogen on the surface of the frame of the polymer support is immersed in the aqueous solution containing hyaluronic acid and fibrinogen, and then the support is taken out of the aqueous solution and lyophilized. It may be carried out by performing the procedure at least once.
  • the lyophilization may be performed repeatedly, for example, may be performed repeatedly 2 to 20 times, but is not limited thereto.
  • the immersion may be carried out at 4 to 50 °C, preferably 4 to 20 °C.
  • the immersion time is not particularly limited, and may be performed for a short time of about 30 seconds or, if necessary, for a longer time, for example, 12 to 48 hours.
  • by further comprising drying the support taken out of the aqueous solution at room temperature (about 25 °C) before lyophilization after the immersion it is possible to prevent the clogging in the manufacturing process.
  • the thrombin and calcium salts perform the crosslinking function of fibrinogen, the amount of which is necessary to achieve the desired degree of crosslinking (eg, to achieve a desired release rate of cell growth factor). Can be selected and used in an appropriate range.
  • concentration of the thrombin and calcium salt in the compartment (b) or the compartment (b ') may be in the range of 10 to 90% by weight, but is not limited thereto.
  • the concentration of the cell growth factor in the compartment (b ') or the compartment (c'') may be in the range of 10 pg / mL to 100 mg / mL, the compartment (c') or The concentration of the cells in the part (c '') may range from 10 3 to 10 10 cells / mL.
  • Kit according to the present invention can be used to prepare a separate tissue regeneration structure using cells (eg, tissue cells, stem cells, etc.), cell growth factors, or a combination of these as needed for one tissue regeneration It can also be used to contain in a structure.
  • BMP-2 as a cell growth factor and adipose derived stem cells as cells can be contained in a structure for tissue regeneration.
  • the tissue regeneration structure formed from the kit according to the present invention for example, tissue regeneration structure containing BMP-2 and adipose derived stem cells can be transplanted in vivo by inducing bone differentiation in vitro, or a separate Bone differentiation may be induced in vivo by implantation in vivo without inducing differentiation.
  • Polymer supports formed from poly (lactic acid-co-glycolic acid) (PLGA) and polycaprolactone (PCL) were prepared according to Kim et al. (Biofabrication 2009; 1: 1-7). form fabrication method).
  • the obtained polymer support was 4 mm x 4 mm x 2 mm, the pore size was about 250 ⁇ m, and the porosity was about 65 to 70%.
  • Fibrinogen solution dissolved in 1 mL apronitine using Green Cross, GreenPlast 1 mL kit
  • an aqueous solution of 200 mg of hyaluronic acid molecular weight 1,500,000, manufactured by LG Life Sciences
  • the polymer support prepared above was immersed in the obtained fibrinogen / hyaluronic acid solution and stirred at 4 ° C. for 24 hours.
  • the polymer support was taken out and the pores were dried at room temperature for 1 hour and then lyophilized for 24 hours.
  • the dipping and lyophilization processes were repeated three times to obtain a polymer support having a coating layer of hyaluronic acid and fibrinogen formed on the surface of the framework.
  • BMP-2 (Chinese hamster ovary cell-derived recombinant human BMP-2, R & D Systems, Minneapolis, MN) was dissolved in phosphate buffered saline at a concentration of 50 ⁇ g / mL, and then 20 ⁇ l of the resulting BMP-2 solution.
  • 32 ⁇ l of an aqueous calcium chloride solution (ie, compartment (b)) in which thrombin (Green Cross, GreenPlast 1 mL kit) was dissolved was added to the freeze-dried support to induce crosslinking, and then lyophilized to regenerate tissue.
  • a support was prepared.
  • the results of observing the shape of the polymer support (left side of FIG. 1) and the obtained tissue regeneration support (right side of FIG. 1) before forming the coating layer are as shown in FIG. 1.
  • the upper part is a photograph measured by an optical microscope, and the lower part is a photograph measured by a scanning electron microscope (SEM). 1, it can be seen that the hyaluronic acid / fibrin coating layer is formed evenly on the surface of the frame of the porous support without clogging the pores.
  • adipose tissue Human adipose tissue was obtained by liposuction with the consent of the Ethics Committee of the College of Medicine and Medical Sciences. To remove contaminated blood, the resulting adipose tissue was washed three times with phosphate buffered saline (PBS) (Sigma, St. Louis, MO). Adipose tissue was then digested with PBS containing 0.2 w / v% bovine albumin and 2 mg / mL type II collagenase (Sigma) at 37 ° C. for 45 minutes. Filtration was performed with a 70 ⁇ m filter, and the filtrate was centrifuged to remove floating adipocytes.
  • PBS phosphate buffered saline
  • ASCs Isolated adipose tissue-derived stem cells
  • a polymer support having a coating layer obtained in Example 2 (2) in suspension (1 ⁇ 10 6 cells / mL) (ie, compartment (c ′)) of human adipose derived stem cells [ie, Compartment (a)] was immersed, incubated in a 37 ° C. incubator for 4 hours, washed three times with PBS to remove cells not attached to the scaffold, and the cell adhesion capacity of the scaffold for tissue regeneration was evaluated.
  • the polymer support without coating layer was sufficiently hydrated in cell culture medium containing 10% of bovine serum for 3 days, and then the suspension of human adipose derived stem cells (1 ⁇ 10 6 cells / mL) in (1). Immersed in, incubated in a 37 ° C. incubator for 4 hours, then washed three times with PBS to remove cells that did not adhere to the support (Comparative Example).
  • BMP-2 (1 ⁇ g) -introduced tissue regeneration support prepared in the same manner as in Example 1 was subjected to immunoassay (ELISA) five times for the release of BMP-2 in PBS at 1.0 mL 37 ° C and PBS. Measured.
  • ELISA plates (NUNC, Polylabo, France) were applied with a capture monoclonal antibody and blocked for 1 hour with 1 w / v% bovine serum albumin and 5 w / v% sucrose. I was. Diluted samples were added to ELISA plates and bound BMP-2 was measured using anti-human BMP-2 polyclonal antibody.
  • streptavidin-horseradish peroxidase complex (streptavidin-conjugated horseradish peroxidase) was added to the plate, peroxidase and tetramethylbenzidine were added and reacted for 20 minutes. After stopping the enzyme reaction by adding an acid solution, the absorbance of the sample was measured at 450 nm using an ELISA plate reader (PowerWave X340, Bio-Tek Instruments, Inc., Winooski, VT). The result is shown in FIG. 3. 3, it can be seen that BMP-2 is continuously released from the scaffold for tissue regeneration according to the present invention for more than 3 days.
  • a composite support having a coating layer not loaded with BMP-2 ie, a support having a coating layer prepared in Example (1)
  • NO BMP-22 a support having a coating layer prepared in Example 1
  • BMP-2 addition day 0 BMP-2 addition day 0
  • DMEM medium added with 10% v / v FBS, 100 units / mL penicillin and 0.1 mg / mL streptomycin was used as the culture medium and exchanged every 3 days.
  • Alkali phosphatase activity was measured by ELISA on the 3rd, 7th and 10th day of the experiment.
  • Each scaffold (all scaffolds collected on days 3, 7 and 10) was added to 50 ⁇ l lysis buffer (Cell Culture Lysis Reagent 5X, Promega) and cell lysate was added 13000 for 10 minutes. It was removed by ultracentrifugation (Micro 17R, Hanil Science Industrial, Seoul, Korea) at rpm.
  • Each sample was added with 150 ⁇ l p-nitrophenylphosphate (pNPP, Sigma) and then incubated at 37 ° C. for 15 minutes. The reaction was stopped by addition of 0.2 N NaOH and the absorbance of each sample was measured at 405 nm using an ELISA plate reader.
  • Example 2 About 1 ⁇ 10 6 cells of ASCs were loaded per BMP-2 (1 ⁇ g) -introduced tissue regeneration support prepared in the same manner as in Example 1. The loading was performed by dispersing about 5 ⁇ 10 4 cells of ASCs obtained in Example 2 (1) in 32 ⁇ L of DMEM medium, and then spraying the dispersion solution onto the tissue regeneration support into which BMP-2 (1 ⁇ g) was introduced. It was later implanted into the back of immunodeficient mice.
  • mice BALB / c-nu, 7 weeks old, female, SLC, Tokyo, Japan
  • Each experimental group was as follows: The first group was a group implanted with a polymer support that did not form a coating layer (G1), and the second group formed a hyaluronic acid / fibrin coating layer (ie, a crosslinked coating layer with thrombin).
  • Group 3 was implanted with a polymer support (G2), and group 3 was a group with a polymer support formed with a hyaluronic acid / fibrin coating layer (ie, a crosslinked crosslinked layer with thrombin) and a BMP-2 implanted from the outside (G3).
  • the fourth group is a group transplanted with the support for tissue regeneration prepared in the above (1) (G4).
  • Each scaffold was implanted in the dorsal subcutaneous of athymic rats (BALB / c-nu).
  • Figure 5 is a photograph of the cross-section of the extracted tissue observed with an optical microscope after Goldner-Trichrome staining. Where the green part and B represent the regenerated bone area, the arrow represents the support, and the scale bar is 100 ⁇ m. 5, it can be seen that the bones were hardly reproduced in the first group, and the bones were reproduced to some extent in the second and third groups. In contrast, bone regeneration was very good in the fourth group.
  • Figure 6 is a photograph of the cross-section taken after the implantation in the third group and the fourth group was observed with an optical microscope after Goldner-Trichrome staining. The scale bar here is 30 ⁇ m. From the results of FIG.
  • osteoblasts osteoblasts (osteoblast, black arrow) and osteoblasts (osteocyte, red arrow) differentiated in the fourth group can be observed.
  • 7 is a graph showing the bone formation area ratio calculated by (bone area / total area) ⁇ 100%, with an asterisk (*) indicating statistical significance (* p ⁇ 0.05). From the results of FIG. 7, the bone formation area ratios of 1.9 ⁇ 0.4% in the first group and 5.5 ⁇ 2.2 and 7.8 ⁇ 1.7% in the second and third groups, respectively. On the contrary, in the fourth group, 19.6 ⁇ 5.0%, when the composite support for bone regeneration according to the present invention is implanted, bone regeneration is very excellent.
  • Soft x-rays of the implanted scaffolds and the amount of calcium were measured to confirm mineralized bone tissue formation.
  • the amount of calcium deposited was measured by the method described in Jeon et al., Biomaterials 2007; 28 (17): 2763-2771. That is, after 8 weeks of transplantation, the tissues were gently washed with phosphate buffer solution, and then 0.6 N hydrochloric acid was added and then finely chopped and stirred in a stirrer for 12 hours. It was then centrifuged at 1,000 g for 5 minutes. The amount of calcium in the supernatant was measured according to the manufacturer's instructions using a commercially available calcium quantification kit (Bioassays).
  • Figure 8 is a graph showing the measurement of the amount of calcium in the implant, asterisk (*) indicates statistical significance (* p ⁇ 0.05). From the results of FIG. 8, the soft x-ray shows the brightest light in Group 4, which indicates that mineralized bone tissue was formed, and the calcium quantification result was also the highest in the fourth group with 23.7 ⁇ g / mg dry tissue. High.

Abstract

The present invention provides a construct for tissue reconstruction, comprising a coating layer formed by coating a surface of a frame of a polymer support having voids, with hyaluronic acids or salts thereof and fibrinogens, wherein (i) the coating layer comprises: cells; cell growth factors; or cells and cell growth factors, (ii) the coating layer is cross-linked by thrombin, and (iii) the construct has voids of 10 μm to 1,000 μm. In addition, the present invention provides a kit for producing a construct for tissue reconstruction, which is cross-linked by thrombin and has voids of 10 μm to 1,000 μm.

Description

조직재생용 구조체 및 조직재생용 구조체 형성을 위한 키트Kit for forming tissue regeneration structure and structure for tissue regeneration
본 발명은 조직재생용 구조체 및 조직재생용 구조체 형성을 위한 키트에 관한 것이다. 더욱 상세하게는, 본 발명은 공극을 갖는 고분자 지지체(polymer support)의 구조틀(frame) 표면 상에 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층을 포함하는 조직재생용 구조체에 관한 것이다. 또한, 본 발명은 트롬빈에 의해 가교화되고 또한 10 ㎛ ∼ 1,000 ㎛의 공극을 갖는 조직재생용 구조체 형성을 위한 키트에 관한 것이다.The present invention relates to a kit for forming a tissue regeneration structure and a structure for tissue regeneration. More specifically, the present invention relates to a structure for tissue regeneration comprising a coating layer formed by coating hyaluronic acid or a salt thereof and fibrinogen on the surface of a frame of a polymer support having pores. The present invention further relates to a kit for forming a structure for tissue regeneration crosslinked by thrombin and having a pore of 10 µm to 1,000 µm.
조직공학(tissue engineering)은 세포학, 생명과학, 공학, 의학 등의 기존의 과학 영역을 응용하는 다학제간 학문으로 생체조직의 구조와 기능 사이의 상관관계를 이해하고, 손상된 조직이나 장기를 정상적인 조직으로 대체, 재생시키기 위한 새로운 융합 학문분야이다. 즉, 체내에 이식가능한 인공생체조직을 만들어 우리 몸의 기능을 유지, 향상 또는 복원하는 것을 목적으로 한다. 이를 위해 우선적으로, 환자의 몸에서 필요한 조직을 채취하여 세포를 분리한 후, 분리된 세포를 배양을 통해 필요한 양만큼 증식시킨다. 증식된 세포를 다공성 생분해 고분자 지지체(support)에 심어 일정기간 동안 체외 배양하여 얻어지는 세포/지지체 복합물을 다시 체내에 이식한다. 이식된 세포/지지체 복합물은 신생 혈관이 형성될 때까지는 체액의 확산에 의해 산소와 영양분을 공급받으며, 체내에 혈관이 자라서 들어와 혈액의 공급이 이루어지면 증식하여 새로운 조직 및 장기를 형성하게 된다. Tissue engineering is a multidisciplinary study that applies existing scientific fields such as cytology, life sciences, engineering, and medicine to understand the correlation between structure and function of living tissues, and to repair damaged tissues or organs as normal tissues. It is a new convergence discipline to replace and regenerate. That is, it aims to maintain, improve or restore the functions of our bodies by making artificial tissues implantable in the body. To this end, first, the tissue is collected from the patient's body to separate the cells, and then the separated cells are expanded by the necessary amount through the culture. The proliferated cells are planted in a porous biodegradable polymer support and the cell / support complex obtained by in vitro culture for a period of time is transplanted back into the body. The transplanted cell / support complex is supplied with oxygen and nutrients by the diffusion of body fluids until new blood vessels are formed, and blood vessels grow in the body and proliferate when blood is supplied to form new tissues and organs.
조직재생용 지지체는 조직재생의 목적에 따라 세포를 포함하지 않고 그 자체를 사용하는 경우와 세포를 포함하는 경우로 나눌 수 있다. 세포를 포함하지 않는 경우에는 세포성장인자(cell growth factors) 또는 분화인자(differentiation-inducing factors)를 도입하여 이식시 주변조직으로부터 세포이동을 유도하여 조직을 재생시킨다. 한편, 세포를 포함하는 경우에는 자가 혹은 동종의 세포를 이용하며, 최근에는 줄기세포를 이용해 조직재생을 보다 원할하게 유도하려는 노력이 진행 중이다. 이와 같이, 세포의 사용 유무와 관계없이 조직공학용 지지체는 조직재생을 효과적으로 유도하는데 필수적인 요소의 하나이다. The support for tissue regeneration may be divided into a case of using itself and a case of containing cells according to the purpose of tissue regeneration. When cells are not included, cell growth factors or differentiation-inducing factors are introduced to induce cell migration from surrounding tissues during transplantation to regenerate tissues. On the other hand, when the cells are included, autologous or homologous cells are used, and recently, efforts are being made to induce tissue regeneration more smoothly using stem cells. As such, with or without cells, the scaffold for tissue engineering is one of the essential elements for effectively inducing tissue regeneration.
조직공학용 지지체로는 폴리글리콜산(PGA), 폴리락틱산(PLA), 폴리카프로락톤(PCL)을 기반으로 하는 고분자와 이들의 공중합체로부터 공극을 갖는 구조의 형태로 제조된다. 제조된 지지체는 이들 재료의 특성으로 인해 모두 소수성의 성질을 가지고 있다. 따라서, 조직 공학적 응용에 있어서 고분자 지지체는 인체조직으로 이식시 혹은 조직세포를 배양하는 경우, 세포 및 조직 적합성이 낮다는 점이 문제이다. 또한, 지지체의 제조시 유기용매 사용 또는 열처리 때문에 성장 혹은 분화인자를 직접적으로 담체하기가 어렵거나 담체 효율이 낮은 경우가 많다. 이러한 문제점을 개선하기 위하여 최근 여러 가지 방법으로 지지체의 표면을 개질하려는 노력이 진행되고 있다.As a support for tissue engineering, polyglycolic acid (PGA), polylactic acid (PLA), polycaprolactone (PCL) -based polymers and copolymers thereof are prepared in the form of structures having pores. The prepared supports all have hydrophobic properties due to the properties of these materials. Therefore, in tissue engineering applications, the polymer support has a problem of low cell and tissue suitability when transplanted into human tissue or when culturing tissue cells. In addition, in the preparation of the support, it is often difficult to directly support the growth or differentiation factor due to the use of an organic solvent or heat treatment, or the carrier efficiency is low. In order to improve this problem, efforts have recently been made to modify the surface of the support in various ways.
친수성 및 생체적합성을 향상시키기 위한 고분자 지지체 표면 개질 방법으로서 산성 및 염기성 용액을 사용해 표면 부식을 통한 표면적 증가와 하이록실기와 카르복실기를 유도하는 방법이 보고된 바 있다(Gao J, Niklason L, Langer R., Surface hydrolysis of poly(glycolic acid) meshes increases the seeding density of vascular smooth muscle cells, J Biomed Mater Res. (1998) 5;42(3), 417-24 ; I. K. Kwon, K. D. Park, S. W. Choi, S-H. Lee, E. B. Lee, J. S. Na, S. H. Kim, and Y. H. Kim, Fibroblast Culture on Surface-modified Poly(glycolide-co-caprolactone) Scaffold for Soft Tissue Regeneration, J. Biomaterials Sci., Polymer Ed, (2001) 12(10), 1147-1160 ; Perego G, Preda P, Pasquinelli G, Curti T, Freyrie A, Cenni E., Functionalization of poly-(L-lactic-co-epsilon-caprolactone): effects of surface modification on endothelial cell proliferation and hemocompatibility. J Biomater Sci Polym Ed. (2003) 14(10), 1057-75). 또한, 세포외기질의 성분 중 하나인 교원질을 PGA, PLA 또는 PLGA와 혼합하여 하이브리드 형태로 지지체를 제조하는 방법 등이 보고된 바 있다 (Chen G, Ushida T, Tateishi T., Poly(DL-lactic-co-glycolic acid) sponge hybridized with collagen microsponges and deposited apatite particulates, J Biomed Mater Res. (2001) 57(1), 8-14 ; Chen G, Sato T, Ushida T, Hirochika R, Tateishi T., Redifferentiation of dedifferentiated bovine chondrocytes when cultured in vitro in a PLGA-collagen hybrid mesh, FEBS Lett. (2003) 8;542(1-3), 95-9). 그러나, 상기와 같은 종래의 방법은 산성 또는 알칼리 용액에 의하여 고분자의 주쇄사슬이 분해되어 분자량이 감소하고, 결국 지지체의 기계적 물성이 크게 저하된다는 단점이 있다. 더욱이, 교원질을 사용하는 경우는 이종의 것을 사용하게 되므로 면역반응의 문제가 발생할 수 있다는 단점이 있다. 이밖에, 플라즈마 처리 및 친수성 중합 단량체로 다공성 고분자 지지체의 표면을 개질을 하려는 연구가 진행되었으나(Yang J, Bei J, Wang S. Enhanced cell affinity of poly (D,L-lactide) by combining plasma treatment with collagen anchorage, Biomaterials (2002) 23(12), 2607-1 ; Shen H, Hu X, Yang F, Bei J, Wang S., Combining oxygen plasma treatment with anchorage of cationized gelatin for enhancing cell affinity of poly(lactide-co-glycolide). Biomaterials (2007) 28(29), 4219-30; Kuo YC, Ku IN., Application of polyethyleneimine-modified scaffolds to the regeneration of cartilaginous tissue. Biotechnol Prog. (2009) 25(5), 1459-67), 화학적 작용기 및 친수성 물질에 의한 세포친화력 증대는 한계가 있는 것으로 평가되고있다. As a method for surface modification of a polymer support to improve hydrophilicity and biocompatibility, an increase in surface area through surface corrosion and derivation of hydroxyl and carboxyl groups using acidic and basic solutions has been reported (Gao J, Niklason L, Langer R.). , Surface hydrolysis of poly (glycolic acid) meshes increases the seeding density of vascular smooth muscle cells, J Biomed Mater Res. (1998) 5; 42 (3), 417-24; IK Kwon, KD Park, SW Choi, SH. Lee, EB Lee, JS Na, SH Kim, and YH Kim, Fibroblast Culture on Surface-modified Poly (glycolide-co-caprolactone) Scaffold for Soft Tissue Regeneration, J. Biomaterials Sci., Polymer Ed , (2001) 12 (10 ), 1147-1160; Perego G, Preda P, Pasquinelli G, Curti T, Freyrie A, Cenni E., Functionalization of poly- (L-lactic-co-epsilon-caprolactone): effects of surface modification on endothelial cell proliferation and hemocompatibility.J Biomater Sci Polym Ed. (2003) 14 (10), 1057-75). In addition, a method of preparing a support in a hybrid form by mixing collagen, which is one of the components of the extracellular matrix, with PGA, PLA, or PLGA has been reported (Chen G, Ushida T, Tateishi T., Poly (DL-lactic-). co-glycolic acid) sponge hybridized with collagen microsponges and deposited apatite particulates, J Biomed Mater Res. (2001) 57 (1), 8-14; Chen G, Sato T, Ushida T, Hirochika R, Tateishi T., Redifferentiation of dedifferentiated bovine chondrocytes when cultured in vitro in a PLGA-collagen hybrid mesh, FEBS Lett. (2003) 8; 542 (1-3), 95-9). However, the conventional method as described above has the disadvantage that the main chain of the polymer is decomposed by an acidic or alkaline solution, thereby reducing the molecular weight, and ultimately, the mechanical properties of the support are greatly reduced. Moreover, when using collagen, there is a disadvantage that a problem of an immune response may occur because it uses a heterogeneous one. In addition, research has been conducted to modify the surface of the porous polymer support with plasma treatment and hydrophilic polymerization monomer (Yang J, Bei J, Wang S. Enhanced cell affinity of poly (D, L-lactide) by combining plasma treatment with collagen anchorage, Biomaterials (2002) 23 (12), 2607-1; Shen H, Hu X, Yang F, Bei J, Wang S., Combining oxygen plasma treatment with anchorage of cationized gelatin for enhancing cell affinity of poly (lactide- . co-glycolide) Biomaterials (2007 ) 28 (29), 4219-30;... Kuo YC, Ku IN, Application of polyethyleneimine-modified scaffolds to the regeneration of cartilaginous tissue Biotechnol Prog (2009) 25 (5), 1459 -67), the increase in cell affinity by chemical functional groups and hydrophilic substances is evaluated to be limited.
최근 펩타이드 혹은 세포외기질(extracellular matrix, ECM) 물질을 하이드로겔 형태로 지지체 표면에 도입함으로써 세포 점착성을 증가시키는 다양한 방법들이 시도되고 있다 (Mann BK, West JL. Cell adhesion peptides alter smooth muscle cell adhesion, proliferation, migration, and matrix protein synthesis on modified surfaces and in polymer scaffolds. J Biomed Mater Res. (2002) 60(1) 86-93 ; Zhu Y, Gao C, He T, Shen J. Endothelium regeneration on luminal surface of polyurethane vascular scaffold modified with diamine and covalently grafted with gelatin. Biomaterials (2004) 25(3), 423-30). 이중 대표적으로는 파이브로넥틴(fibronectin)으로 고분자 표면을 도포하는 방법(Harding SI, Afoke A, Brown RA, MacLeod A, Shamlou PA, Dunnill P., Engineering and cell attachment properties of human fibronectin-fibrinogen scaffolds for use in tissue engineered blood vessels. Bioprocess Biosyst Eng. (2002) 25(1), 53-9), 하이드록시아파타이트 결정과 콜라겐을 혼합하여 복합체를 제조하는 방법(Itoh S, Kikuchi M, Takakuda K, Koyama Y, Matsumoto HN, Ichinose S, Tanaka J, Kawauchi T, Shinomiya K., The biocompatibility and osteoconductive activity of a novel hydroxyapatite/collagen composite biomaterial, and its function as a carrier of rhBMP-2. J Biomed Mater Res. (2001) 5;54(3), 445-53; Zhang R, Ma PX, Biomimetic polymer/apatite composite scaffolds for mineralized tissue engineering, Macromol Biosci. (2004) 20;4(2), 100-11) 등을 예로 들 수 있다. 실제로, 세포외기질 물질 (extracellular matrix)이나 특정 조직성장인자들은 생체내에서 손상된 조직재생에 탁월한 효능이 있는 것으로 보고되고 있으며(Schleicher I, Parker A, Leavesley D, Crawford R, Upton Z, Xiao Y., Surface modification by complexes of vitronectin and growth factors for serum-free culture of human osteoblasts. Tissue Eng. (2005) 11(11-12), 1688-98; Ma Z, Gao C, Gong Y, Shen J., Cartilage tissue engineering PLLA scaffold with surface immobilized collagen and basic fibroblast growth factor, Biomaterials (2005) 26(11), 1253-9; Hosseinkhani H, Hosseinkhani M, Khademhosseini A, Kobayashi H., Bone regeneration through controlled release of bone morphogenetic protein-2 from 3-D tissue engineered nano-scaffold. J Control Release. (2007) 26;117(3), 380-6)), 실제 임상에서도 이들의 우수한 조직재생력이 다수의 결과에서 보고된 바 있다. 그러나, 상기 방법들로 이루어진 지지체는 수화된 하이드로겔로 인해 지지체 표면 공극이 막히는 현상과 하이드로젤내에 세포성장물질 도입 효율이 매우 낮은 단점이 있다. Recently, various methods of increasing cell adhesion by introducing a peptide or extracellular matrix (ECM) material into the surface of the support in the form of a hydrogel have been attempted (Mann BK, West JL. Cell adhesion peptides alter smooth muscle cell adhesion, proliferation, migration, and matrix protein synthesis on modified surfaces and in polymer scaffolds.J Biomed Mater Res. (2002) 60 (1) 86-93; Zhu Y, Gao C, He T, Shen J. Endothelium regeneration on luminal surface of polyurethane vascular scaffold modified with diamine and covalently grafted with gelatin. Biomaterials (2004) 25 (3), 423-30). Among them, a method of coating a polymer surface with fibronectin (Harding SI, Afoke A, Brown RA, MacLeod A, Shamlou PA, Dunnill P., Engineering and cell attachment properties of human fibronectin-fibrinogen scaffolds for use) in tissue engineered blood vessels.Bioprocess Biosyst Eng. (2002) 25 (1), 53-9), Method for preparing complexes by mixing hydroxyapatite crystals and collagen (Itoh S, Kikuchi M, Takakuda K, Koyama Y, Matsumoto HN, Ichinose S, Tanaka J, Kawauchi T, Shinomiya K., The biocompatibility and osteoconductive activity of a novel hydroxyapatite / collagen composite biomaterial, and its function as a carrier of rhBMP-2.J Biomed Mater Res. (2001) 5 54 (3), 445-53; Zhang R, Ma PX, Biomimetic polymer / apatite composite scaffolds for mineralized tissue engineering, Macromol Biosci. (2004) 20; 4 (2), 100-11), and the like. . Indeed, extracellular matrix or specific tissue growth factors have been reported to have excellent efficacy in tissue regeneration in vivo (Schleicher I, Parker A, Leavesley D, Crawford R, Upton Z, Xiao Y.). , Surface modification by complexes of vitronectin and growth factors for serum-free culture of human osteoblasts.Tissue Eng. (2005) 11 (11-12), 1688-98; Ma Z, Gao C, Gong Y, Shen J., Cartilage tissue engineering PLLA scaffold with surface immobilized collagen and basic fibroblast growth factor, Biomaterials (2005) 26 (11), 1253-9; Hosseinkhani H, Hosseinkhani M, Khademhosseini A, Kobayashi H., Bone regeneration through controlled release of bone morphogenetic protein- 2 from 3-D tissue engineered nano-scaffold.J Control Release. (2007) 26; 117 (3), 380-6)) and their excellent tissue regeneration in actual clinical practice has been reported in a number of results. However, the support made of the above methods has the disadvantage of blocking the surface pores of the support due to the hydrated hydrogel and very low efficiency of cell growth material introduction into the hydrogel.
일반적으로 대부분의 세포외기질 및 세포성장인자들은 상대적으로 고가이고, 분자량이 수십 kDa에 이르는 고분자량의 단백질로서 생체 내에서는 불안정하여 활성이 떨어진다. 체내에서 이식될 경우에도 수 분 이내로 방출, 소실되므로 원하는 위치에서 치료효과를 얻기 위해서는 고가의 세포성장인자를 대용량으로 투여해야 하는 점과 다량의 투여로 인한 부작용 유발의 가능성도 제기되고 있다. 최근 고분자 지지체에 골성장인자들을 함유시켜 서방출함으로써 단순 적용에 따른 단점을 경감하고자 하는 시도가 이루어져 왔으며 어느 정도 그 효과도 입증 되었다. 그러나 이들 골이식재나 지지체 자체에 의해서는 조직성장인자가 물리적으로 혼합되어 있는 상태로서 초기 적용시 초기방출 (burst release)이 일어나 치료기간동안 유효농도의 유지가 어렵다는 단점이 있다. 따라서, 지지체 표면에 생체활성이 뛰어난 세포외기질물질을 안정적으로 도포하고, 세포성장인자를 효율적으로 도입하여 안정적으로 방출되도록 유도하는 방법의 개발은 조직재생 분야에서 매우 중요하다.In general, most extracellular matrix and cell growth factors are relatively expensive, high molecular weight proteins of several tens of kDa, unstable in vivo and inferior in activity. Even when implanted in the body, it is released and lost within a few minutes, so that a large amount of expensive cell growth factor should be administered to obtain a therapeutic effect at a desired position, and the possibility of causing side effects due to a large amount of administration has been raised. Recently, attempts have been made to alleviate the shortcomings of simple application by slow release by incorporating bone growth factors into the polymer support, and the effects have been proved to some extent. However, these bone grafts or scaffolds themselves are in a state in which tissue growth factors are physically mixed, so that initial release occurs during initial application, which makes it difficult to maintain effective concentrations during treatment. Therefore, the development of a method of stably applying the extracellular matrix material having excellent bioactivity to the surface of the support and inducing the cell growth factor to be stably released is very important in the field of tissue regeneration.
본 발명자들은 소수성을 나타내는 조직공학용 지지체에서 세포점착이 효율적으로 이루어지지 못하는 단점과 세포 성장인자들의 도입이 용이하지 않은 단점을 해결하기 위해, 세포친화성을 증진시키는 하이드로겔 표면처리 방법에 대한 연구를 수행하였다. 본 발명자들은 공극을 갖는 고분자 지지체(polymer support)의 구조틀(frame) 표면 상에 히아루론산 또는 그의 염 및 피브리노오겐을 동결건조 등의 방법으로 코팅하여 코팅층을 형성시킴으로써, 세포친화력을 크게 증진시켜 세포점착력을 크게 향상시킬 수 있을 뿐 아니라, 세포성장인자의 도입도 매우 효율적으로 이루어질 수 있다는 것을 발견하였다. 즉, 표면이 건조된 하이드로겔 코팅층에 세포 및/또는 세포성장인자 등을 파종할 경우, 빠른 속도로 세포와 세포성장인자를 지지체 표면에 점착하도록 유도할 수 있다는 것을 발견하였다. 더욱이, 트롬빈을 통하여 가교화를 유도하고, 얻어지는 구조체가 공극을 그대로 유지하도록 함으로써, 공극을 통한 체액(body fluid)의 원활한 접근이 가능하고 산소 및 영양분의 공급이 원활할 뿐만 아니라, 상기 공극을 통하여 구조체로부터의 세포성장인자의 지속적이고 안정적인 방출이 가능하므로, 조직세포들이 균일하게 효율적으로 점착, 증식될 수 있다는 것을 발견하였다. The present inventors have studied a hydrogel surface treatment method for enhancing cell affinity to solve the disadvantage that the cell adhesion is not efficiently achieved in the tissue engineering support showing hydrophobicity and the disadvantage that the cell growth factors are not easily introduced. Was performed. The present inventors form a coating layer by coating a hyaluronic acid or a salt thereof and fibrinogen on a surface of a frame of a polymer support having pores by lyophilization or the like to form a coating layer, thereby greatly improving cell affinity and In addition to significantly improving adhesion, the researchers found that cell growth factors can be introduced very efficiently. That is, when seeding cells and / or cell growth factors, etc. in the hydrogel coating layer is dried surface, it was found that the cells and cell growth factors can be induced to adhere to the surface of the support at a high speed. Furthermore, by inducing crosslinking through thrombin and keeping the resulting structure intact, the body fluids can be smoothly accessed through the pores and the supply of oxygen and nutrients is smooth, as well as through the pores. It was found that continuous and stable release of cell growth factors from the constructs allows tissue cells to adhere and proliferate uniformly and efficiently.
따라서, 본 발명은 높은 친수성 및 세포친화성을 가짐으로써, 세포 및/또는 세포성장인자가 높은 점착효율(introduction rate or loading rate)로 고분자 지지체 상에 도입된 조직재생용 구조체를 제공한다.Accordingly, the present invention provides a structure for tissue regeneration in which cells and / or cell growth factors are introduced onto a polymer support at a high adhesion rate or loading rate by having high hydrophilicity and cell affinity.
또한, 본 발명은 상기 조직재생용 구조체 형성을 위한 키트를 제공한다.The present invention also provides a kit for forming the structure for tissue regeneration.
본 발명의 일 태양에 따라, 공극을 갖는 고분자 지지체(polymer support)의 구조틀(frame) 표면 상에 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층을 포함하는 조직재생용 구조체(construct for tissue-reconstruction)로서, (i) 상기 코팅층이 세포; 세포성장인자; 또는 세포 및 세포성장인자를 포함하고, (ii) 상기 코팅층이 트롬빈에 의해 가교화되어 있으며, 또한 (iii) 상기 구조체가 10 ㎛ ∼ 1,000 ㎛의 공극을 갖는, 조직재생용 구조체가 제공된다.According to one aspect of the present invention, a structure for tissue regeneration comprising a coating layer formed by coating hyaluronic acid or a salt thereof and fibrinogen on a surface of a frame of a polymer support having pores. -reconstruction, wherein (i) the coating layer is a cell; Cell growth factor; Or cells and cell growth factors, (ii) the coating layer is crosslinked with thrombin, and (iii) the structure has a pore of 10 µm to 1,000 µm.
본 발명의 다른 태양에 따라, 공극을 갖는 고분자 지지체의 구조틀 표면 상에 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층을 포함하는 구조체-컴파트먼트[컴파트먼트 (a)]; 및 트롬빈 및 칼슘염을 포함하는 용액을 포함하는 트롬빈-컴파트먼트[컴파트먼트 (b)]를 포함하는, 트롬빈에 의해 가교화되고 또한 10 ㎛ ∼ 1,000 ㎛의 공극을 갖는 조직재생용 구조체 형성을 위한 키트가 제공된다.According to another aspect of the present invention, there is provided a structure-compartment (compartment (a)) comprising a coating layer formed by coating hyaluronic acid or a salt thereof and fibrinogen on a surface of a structure of a polymer support having voids; And a thrombin-compartment (compartment (b)) comprising a solution comprising thrombin and calcium salt, forming a structure for tissue regeneration crosslinked by thrombin and having pores of 10 μm to 1,000 μm A kit for is provided.
본 발명의 또다른 태양에 따라, 공극을 갖는 고분자 지지체의 구조틀 표면 상에 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층을 포함하는 구조체-컴파트먼트[컴파트먼트 (a)]; 트롬빈 및 칼슘염을 포함하는 용액을 포함하는 트롬빈-컴파트먼트[컴파트먼트 (b)]; 및 세포 또는 세포성장인자의 제공을 위한 1종 이상의 수성 용액-컴파트먼트[컴파트먼트 (c)]를 포함하는, 트롬빈에 의해 가교화되고 또한 10 ㎛ ∼ 1,000 ㎛의 공극을 갖는 조직재생용 구조체 형성을 위한 키트가 제공된다.According to another aspect of the present invention, there is provided a structure-compartment (compartment (a)) comprising a coating layer formed by coating hyaluronic acid or a salt thereof and fibrinogen on a surface of a structure of a polymer support having voids; Thrombin-compartment (compartment (b)) comprising a solution comprising thrombin and calcium salt; And one or more aqueous solution-compartments [compartment (c)] for the provision of cells or cell growth factors, for tissue regeneration crosslinked by thrombin and having pores of 10 μm to 1,000 μm Kits for forming structures are provided.
본 발명의 또다른 태양에 따라, 공극을 갖는 고분자 지지체의 구조틀 표면 상에 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층을 포함하는 구조체-컴파트먼트[컴파트먼트 (a)]; 세포성장인자; 및 트롬빈과 칼슘염을 함유하는 수성 용액을 포함하는 세포성장인자-트롬빈-완충액 컴파트먼트[컴파트먼트 (b')]; 및 선택적으로, 세포를 함유하는 세포-수성 용액-컴파트먼트[컴파트먼트 (c')]를 포함하는, 트롬빈에 의해 가교화되고 또한 10 ㎛ ∼ 1,000 ㎛의 공극을 갖는 조직재생용 구조체 형성을 위한 키트가 제공된다.According to another aspect of the present invention, there is provided a structure-compartment (compartment (a)) comprising a coating layer formed by coating hyaluronic acid or a salt thereof and fibrinogen on a surface of a structure of a polymer support having voids; Cell growth factor; And a cell growth factor-thrombin-buffer compartment (compartment (b ')) comprising an aqueous solution containing thrombin and calcium salt; And optionally, a cell-aqueous solution-compartment containing a cell [compartment (c ')], which is crosslinked by thrombin and forms a structure for tissue regeneration having pores of 10 μm to 1,000 μm A kit for is provided.
본 발명의 또다른 태양에 따라, 공극을 갖는 고분자 지지체의 구조틀 표면 상에 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층을 포함하는 구조체-컴파트먼트[컴파트먼트 (a)]; 트롬빈 및 칼슘염을 포함하는 용액을 포함하는 트롬빈-컴파트먼트[컴파트먼트 (b)]; 및 세포 또는 세포성장인자를 함유하는 1종 이상의 수성 용액-컴파트먼트[컴파트먼트 (c'')]를 포함하는, 트롬빈에 의해 가교화되고 또한 10 ㎛ ∼ 1,000 ㎛의 공극을 갖는 조직재생용 구조체 형성을 위한 키트가 제공된다.According to another aspect of the present invention, there is provided a structure-compartment (compartment (a)) comprising a coating layer formed by coating hyaluronic acid or a salt thereof and fibrinogen on a surface of a structure of a polymer support having voids; Thrombin-compartment (compartment (b)) comprising a solution comprising thrombin and calcium salt; And tissue regeneration crosslinked by thrombin and having pores of 10 μm to 1,000 μm, including at least one aqueous solution-compartment containing the cell or cell growth factor [compartment (c ″)] A kit is provided for forming a structure for use.
본 발명에 따른 조직재생용 구조체; 및 본 발명의 키트로부터 형성되는 조직재생용 구조체는 공극을 갖는 고분자 지지체(polymer support)의 구조틀(frame) 표면 상에 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층을 포함하며, 따라서 얻어지는 구조체가 공극을 그대로 유지함으로써, 공극을 통한 체액(body fluid)의 원활한 접근이 가능하고 산소 및 영양분의 공급이 원활할 뿐만 아니라, 상기 공극을 통하여 구조체로부터의 세포성장인자의 지속적이고 안정적인 방출이 가능하므로, 조직세포들이 균일하게 효율적으로 점착, 증식될 수 있다. 또한, 본 발명의 조직재생용 구조체; 및 본 발명의 키트에 함유되는 코팅층을 갖는 구조체 즉, 구조체 컴파트먼트는 화학적인 촉매의 사용 없이 균일한 코팅층 형성이 가능하므로, 우수한 안전성을 가질 뿐만 아니라 높은 친수성 및 세포친화성를 가짐으로써 세포, 세포성장인자가 높은 점착효율(introduction rate or loading rate)로 도입될 수 있다. 또한, 본 발명의 키트로부터 형성되는 조직재생용 구조체로부터, 도입된 세포성장인자가 지속적인 방출 패턴을 나타내므로, 줄기세포의 분화를 안정적으로 유도할 수 있다. 따라서, 본 발명에 따른 조직재생용 구조체는 생체에 이식되어 근골격재생, 혈관재생, 피부재생 등을 포함한 다양한 조직공학적 소재로 사용될 수 있다. 또한, 본 발명에 따른 키트는 근골격재생, 혈관재생, 피부재생 등을 포함한 다양한 조직재생용 구조체 형성에 유용하게 사용될 수 있다.Structure for tissue regeneration according to the present invention; And a structure for tissue regeneration formed from the kit of the present invention includes a coating layer formed by coating hyaluronic acid or a salt thereof and fibrinogen on a surface of a frame of a polymer support having pores, and thus obtained By maintaining the pores intact, the structure provides smooth access to body fluids through the pores and facilitates the supply of oxygen and nutrients, as well as continuous and stable release of cell growth factors from the constructs through the pores. Therefore, the tissue cells can adhere and proliferate uniformly and efficiently. In addition, the tissue regeneration structure of the present invention; And the structure having the coating layer included in the kit of the present invention, that is, the structure compartment can form a uniform coating layer without the use of a chemical catalyst, thereby having excellent safety as well as having high hydrophilicity and cell affinity to cells, cells Growth factors can be introduced at high induction or loading rates. In addition, since the cell growth factor introduced from the structure for tissue regeneration formed from the kit of the present invention shows a sustained release pattern, it is possible to stably induce differentiation of stem cells. Therefore, the tissue regeneration structure according to the present invention may be implanted into a living body and used as various tissue engineering materials including musculoskeletal regeneration, blood vessel regeneration, and skin regeneration. In addition, the kit according to the present invention can be usefully used for forming various tissue regeneration structures including musculoskeletal regeneration, blood vessel regeneration, skin regeneration and the like.
도 1은 코팅층을 형성하기 전의 고분자 지지체(도 1의 좌측, A) 및 본 발명에 따라 얻어진 조직재생용 지지체(도 1의 우측, B)의 형태를 관찰한 결과이다. 상단은 광학현미경으로 측정한 사진이고, 하단은 주사전자현미경(Scanning electron microscope, SEM)을 통해 측정한 사진이다.1 is a result of observing the form of the polymer support (left side of FIG. 1, A) and the tissue regeneration support body (right side of FIG. 1, B) obtained according to the present invention before forming the coating layer. The upper part is a photograph measured by an optical microscope, and the lower part is a photograph measured by a scanning electron microscope (SEM).
도 2는 인간 지방유래 줄기세포가 도입된 본 발명에 따른 조직재생용 지지체(우측); 및 코팅층 형성되지 않은 고분자 지지체를 3일 동안 우혈청이 10% 포함된 세포 배양 배지에서 충분히 수화 시킨 후 인간 지방유래 줄기세포를 동일한 방법으로 인큐베이션하여 제작한 비교예의 지지체(좌측)를 공초점 형광현미경(Confocal Laser Scanning Microscope, CLSM)을 이용하여 측정한 결과 및 지지체에 부착된 세포의 갯수를 측정한 결과이다.Figure 2 is a support for tissue regeneration according to the present invention (right side) into which human adipose derived stem cells were introduced; And the support (left) of the comparative example prepared by incubating the human body-derived stem cells in the same manner after sufficiently hydrated the polymer support without the coating layer in a cell culture medium containing 10% of the right serum for 3 days. (Confocal Laser Scanning Microscope, CLSM) and the result of measuring the number of cells attached to the support.
도 3은 골성장인자가 도입된 본 발명에 따른 조직재생용 지지체로부터의 BMP-2의 방출 거동을 ELISA를 통하여 측정한 결과이다.Figure 3 is the result of measuring the release behavior of BMP-2 from the support for tissue regeneration according to the present invention in which bone growth factor was introduced by ELISA.
도 4는 골성장인자가 도입된 본 발명에 따른 조직재생용 지지체 존재하에서 인간 지방유래 줄기세포를 배양하였을 때 점착된 지방줄기세포의 골분화 정도를 알카리 포스파타아제 활성(alkaline phosphatase activity)으로 정량분석한 결과이다. 대조군으로서 BMP-2가 로딩 안된 코팅된 지지체(NO BMP-2) 및 시험시작일에 BMP-2 1 ㎍를 배양 배지 안에 첨가한 코팅된 지지체(BMP-2 addition day 0)를 사용하였다.Figure 4 quantifies the degree of bone differentiation of adherent adipose stem cells when the cultured human adipose derived stem cells in the presence of the support for tissue regeneration according to the present invention introduced bone growth factor by alkaline phosphatase activity (alkaline phosphatase activity) The result of the analysis. As a control, a coated support without BMP-2 (NO BMP-2) and a coated support (BMP-2 addition day 0) in which 1 μg of BMP-2 was added to the culture medium at the start of the test were used.
도 5 내지 도 7은 각각 조직염색을 통한 골조직재생을 평가한 결과를 나타낸다. 도 5 내지 도 7에서, 제1군은 코팅층을 형성시키지 않은 고분자 지지체(G1), 제2군은 히아루론산/피브린 코팅층을 형성시킨 고분자 지지체(G2), 제3군은 히아루론산/피브린 코팅층을 형성시킨 고분자 지지체 및 외부에서 BMP-2를 첨가한 군(G3), 제4군은 상기 (1)에서 제조한 조직재생용 지지체(G4)를 각각 이식하였을 때를 나타낸다. 도 5는 세포외기질이 도입된 지지체 표면에서 재생된 골조직의 골드너-트리크롬(Goldner-Trichrome) 염색 후 정성적 결과를 광학현미경을 통해 관찰한 사진이다. 도 6은 제3군과 제4군에서 골드너-트리크롬(Goldner-Trichrome) 염색 한 결과를 나타낸다. 도 7은 각각의 군에서 재생된 골조직 면적을 정량한 결과이다.5 to 7 show the results of evaluating bone tissue regeneration through tissue staining, respectively. 5 to 7, the first group is a polymer support (G1) without forming a coating layer, the second group is a polymer support (G2) formed a hyaluronic acid / fibrin coating layer, the third group is a hyaluronic acid / fibrin coating layer The group (G3) and the fourth group to which BMP-2 was added from the polymer scaffold and the outside are implanted with the tissue regeneration scaffold (G4) prepared in (1), respectively. FIG. 5 is a photograph of qualitative results observed through optical microscope after Goldner-Trichrome staining of regenerated bone tissue on the support surface into which extracellular matrix is introduced. Figure 6 shows the results of the Goldner-Trichrome staining in the third and fourth groups. 7 is a result of quantifying the regenerated bone tissue area in each group.
도 8은 세포외기질이 도입된 지지체 표면에서의 골재생 정도의 평가를 위한 동물실험 결과이다. 상단은 동물실험을 통한 soft X-ray 결과이며, 하단은 동물실험을 통해 재생된 골조직의 칼슘이온정량 결과이다.8 is an animal test result for evaluation of the degree of bone regeneration on the surface of the support in which the extracellular matrix is introduced. The upper part is the soft X-ray result through animal experiment, and the lower part is the calcium ion quantification result of bone tissue regenerated through animal experiment.
본 명세서에서, "조직재생용 구조체(construct for tissue-reconstruction)"라 함은 체내의 손상된 조직 또는 그 주변에 이식되어 손상된 조직을 재생시킨 후 다시 분리되거나, 혹은 완전히 분해되어 없어지게 되는 구조체를 말한다. 상기 조직재생용 구조체는 체외에서 분화를 유도하여 생체내에 이식되거나, 혹은 별도의 분화 유도 없이 생체내에 직접 이식되어 생체내에서 분화가 유도되도록 할 수 있다. 상기 조직재생용 구조체는 체내에 이식되어 신생 혈관이 형성될 때까지는 체액을 통하여 산소와 영양분을 공급받으며, 또한 혈관의 생성(angiogenesis)을 통하여 혈액의 공급이 이루어지면서 증식하여 새로운 조직이나 장기로 형성된다.As used herein, the term "construct for tissue-reconstruction" refers to a structure that is implanted in or around a damaged tissue in the body to rebuild the damaged tissue and then separate or completely disintegrate. . The tissue regeneration construct may be induced in vitro to differentiate and may be directly implanted in vivo without inducing differentiation so that differentiation may be induced in vivo. The tissue regeneration structure is implanted into the body and receives oxygen and nutrients through the body fluid until new blood vessels are formed, and also proliferates as blood is supplied through the formation of blood vessels (angiogenesis) to form new tissues or organs. do.
또한, "세포성장인자(cell growth factors)"라 함은 분화인자(differentiation-inducing factors)라고도 칭해지며, 세포의 성장 및/또는 줄기세포의 분화에 관여되는 인자를 말한다. 상기 세포성장인자는 생체내에 이식되었을 때, 주변조직으로부터 세포이동을 유도하여 조직을 재생시키거나, 줄기세포의 분화를 촉진시켜 조직을 재생시키는 역할을 하는 모든 인자들을 포함한다. 예를 들어,BMP-2(bone morphogenetic protein 2), VEGF(vascular endothelial growth factor), EGF(epidermal growth factor), IGF(insulin-like growth factor), NGF(nerve growth factor), PDGF(platelet-derived growth factor), TGF-β(transforming growth factor-β), bFGF(basic fibroblast growth factor) 등을 포함하지만, 이에 제한되는 것은 아니다."Cell growth factors" are also referred to as differentiation-inducing factors and refer to factors involved in cell growth and / or stem cell differentiation. When transplanted in vivo, the cell growth factor includes all factors that induce cell migration from surrounding tissues to regenerate tissues or promote stem cell differentiation to regenerate tissues. For example, bone morphogenetic protein 2 (BMP-2), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), nerve growth factor (NGF), and platelet-derived PDGF (PDGF). growth factor), transforming growth factor-β (TGF-β), basic fibroblast growth factor (bFGF), and the like, but are not limited thereto.
본 발명은 공극을 갖는 고분자 지지체의 구조틀(frame) 표면 상에 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층을 포함하는 조직재생용 구조체(construct for tissue-reconstruction)로서, (i) 상기 코팅층이 세포; 세포성장인자; 또는 세포 및 세포성장인자를 포함하고, (ii) 상기 코팅층이 트롬빈에 의해 가교화되어 있으며, 또한 (iii) 상기 구조체가 10 ㎛ ∼ 1,000 ㎛의 공극을 갖는, 조직재생용 구조체를 제공한다.The present invention provides a construct for tissue-reconstruction comprising a coating layer formed by coating hyaluronic acid or a salt thereof and fibrinogen on a surface of a frame of a polymer support having pores, wherein (i) the Coating layer of cells; Cell growth factor; Or cells and cell growth factors, (ii) the coating layer is crosslinked with thrombin, and (iii) the structure has a pore of 10 µm to 1,000 µm.
상기 고분자 지지체(polymer support)는 고분자로 이루어진 매트릭스로서 공극을 갖는 매트릭스를 말한다. 상기 고분자 지지체는 생분해성 고분자로부터 바람직하게 형성될 수 있으며, 상기 생분해성 고분자는 의약 또는 약학 분야에서 통상적으로 사용되는 생분해성 고분자일 수 있다. 바람직하게는, 상기 고분자 지지체는 폴리락타이드, 폴리글리콜라이드, 폴리카프로락톤, 락타이드와 글리콜라이드와의 공중합체, 락타이드와 카프로락톤과의 공중합체, 및 글리콜라이드와 카프로락톤과의 공중합체으로 이루어진 군으로부터 1종 이상 선택된 생분해성 고분자로부터 형성될 수 있으며, 더욱 바람직하게는 D,L-락타이드와 글리콜라이드와의 공중합체[예를 들어, 폴리(락트산-co-글리콜산)(poly(D,L-lactic acid-co-glycolic acid, PLGA)] 및 폴리카프로락톤(poly(ε-caprolactone), PCL)의 혼합물로부터 형성될 수 있다. 상기 공극을 갖는 고분자 지지체는 예를 들어 상기한 생분해성 고분자로부터 통상의 방법, 예를 들어 염추출법, 발포법, 유화동결법, 열접착법, 3차원 프린팅, CAD-CAM 등의 방법에 의해 제조될 수 있다. 예를 들어, Kim 등의 방법(Biofabrication 2009; 1: 1-7)에 따라 자유 임의 형상 제작 방법(solid free-form fabrication method)에 따라 공극을 갖는 고분자 지지체를 형성시킬 수 있다. 상기 공극의 형상은 원형, 사각형 등의 다양한 형태일 수 있다. 상기 공극의 크기는 약 10 ㎛ ∼ 1,000 ㎛ 범위일 수 있으며, 이때 상기 공극의 크기는 예를 들어 원형일 경우 직경, 사각형일 경우 가로 또는 세로의 길이를 의미한다. The polymer support is a matrix made of a polymer and refers to a matrix having pores. The polymer support may be preferably formed from a biodegradable polymer, and the biodegradable polymer may be a biodegradable polymer commonly used in medicine or pharmaceutical fields. Preferably, the polymer support is polylactide, polyglycolide, polycaprolactone, a copolymer of lactide and glycolide, a copolymer of lactide and caprolactone, and a copolymer of glycolide and caprolactone It may be formed from at least one biodegradable polymer selected from the group consisting of, more preferably a copolymer of D, L-lactide and glycolide [eg, poly (lactic acid-co-glycolic acid) (poly (D, L-lactic acid-co-glycolic acid (PLGA)) and polycaprolactone (poly (ε-caprolactone), PCL) can be formed from the above. From biodegradable polymers, it can be produced by conventional methods such as salt extraction, foaming, emulsion freezing, thermal bonding, three-dimensional printing, CAD-CAM, etc. For example, the method of Kim et al. Biofabrication 2009; 1: 1 It is possible to form a polymer support having voids according to a solid free-form fabrication method according to -7) The shape of the voids may be various shapes such as circular, square, etc. The size may range from about 10 μm to 1,000 μm, wherein the size of the pore means, for example, a diameter in the case of a circle or a length of a width or a length of a rectangle.
상기 히아루론산은 인체 내에 존재하는 친수성 다당체로서, D-글루코론산(D-glucoronic acid)과 N-글루고스아민(N-glucosamine)의 이당체(Disaccharide, 분자량 379)의 기본 단위(Unit)가 긴 사슬처럼 연결된 고분자(High molecular weight hetero-polysaccharide)의 형태로 존재하며, 세포의 성장, 분화, 및 이동(migration) 등 세포외 간질(extracelluar matrix) 내에서 다양한 기능을 담당한다. 히아루론산의 염은 다양한 염 형태일 수 있으며, 예를 들어, 히아루론산 코발트, 히아루론산 마그네슘, 히아루론산 아연, 히아루론산 칼슘, 히아루론산 칼륨, 히아루론산 나트륨 등의 무기염 및 히아루론산 테트라부틸암모늄 등의 유기염 형태일 수 있다. 바람직하게는 히아루론산 나트륨을 사용할 수 있다. 본 발명의 조직재생용 구조체는 상기 히아루론산 또는 히아루론산의 염을 각각 포함하거나, 히아루론산과 히아루론산의 염을 혼합하여 포함할 수 있다. 상기 히아루론산 또는 그의 염의 중량평균분자량은 특별히 제한된 것은 아니며, 예를 들어, 상기 히아루론산 또는 그의 염의 평균분자량은 1,000 ∼ 4,000,000 달톤일 수 있고, 바람직하게는 1,000 ∼ 1,500,000 달톤일 수 있다.The hyaluronic acid is a hydrophilic polysaccharide present in the human body, and a long chain of basic units (Disaccharide, molecular weight 379) of D-glucoronic acid (D-glucoronic acid) and N-glucosamine (N-glucosamine) It exists in the form of a high molecular weight hetero-polysaccharide, and is responsible for various functions in the extratracelluar matrix such as cell growth, differentiation, and migration. The salts of hyaluronic acid may be in various salt forms, for example, may be in the form of organic salts such as cobalt hyaluronic acid, magnesium hyaluronate, zinc hyaluronate, calcium hyaluronate, potassium hyaluronate, sodium hyaluronate and the like, and hyaluronic acid tetrabutylammonium. Preferably sodium hyaluronate can be used. The tissue regeneration structure of the present invention may include the salts of hyaluronic acid or hyaluronic acid, respectively, or may contain a mixture of hyaluronic acid and a salt of hyaluronic acid. The weight average molecular weight of the hyaluronic acid or a salt thereof is not particularly limited, and for example, the average molecular weight of the hyaluronic acid or a salt thereof may be 1,000 to 4,000,000 Daltons, and preferably 1,000 to 1,500,000 Daltons.
본 발명의 조직재생용 구조체에 있어서, 상기 코팅층 중의 히아루론산 또는 그의 염의 함량은 10 ∼ 90 중량%의 범위일 수 있으며, 또한 상기 코팅층 중의 피브리노오겐의 함량은 10 ∼ 90 중량%의 범위일 수 있다. 상기 코팅층 중의 상기 히아루론산 또는 그의 염 및 피브리노오겐의 중량비는 크게 제한되는 것은 아니며, 세포성장인자의 방출속도 등을 감안하여 조절될 수 있다.In the structure for tissue regeneration of the present invention, the content of hyaluronic acid or a salt thereof in the coating layer may be in the range of 10 to 90% by weight, and the content of fibrinogen in the coating layer may be in the range of 10 to 90% by weight. have. The weight ratio of the hyaluronic acid or a salt thereof and fibrinogen in the coating layer is not particularly limited, and may be controlled in view of the release rate of cell growth factors.
고분자 지지체의 구조틀(frame) 표면 상에 히아루론산 또는 그의 염 및 피브리노오겐의 코팅은 히아루론산 및 피브리노오겐을 함유하는 수용액 중에 상기 지지체를 침지한 다음, 상기 지지체를 상기 수용액으로부터 꺼내어 동결건조하는 과정을 적어도 1회 이상 수행함으로써 수행될 수 있다. 상기 동결건조는 반복하여 수행될 수 있으며, 예를 들어 2회 내지 20회 반복하여 수행될 수 있으나, 이에 제한되는 것은 아니다. 상기 침지는 4 ∼ 50 ℃, 바람직하게는 4 ∼ 20 ℃에서 수행될 수 있다. 상기 침지 시간은 특별히 제한되는 것은 아니며, 약 30초의 단시간 동안 수행되거나 필요에 따라 그 보다 장시간 예를 들어 12 ∼ 48 시간 동안 수행될 수 있다. 또한, 필요할 경우, 상기 침지 후 동결건조 전에, 상기 수용액으로부터 꺼낸 지지체를 상온(약 25 ℃)에서 건조하는 것을 추가로 포함함으로써 제조과정에서 공극이 막히는 것을 방지할 수 있다. Coating of hyaluronic acid or a salt thereof and fibrinogen on the surface of the frame of the polymer support is immersed in the aqueous solution containing hyaluronic acid and fibrinogen, and then the support is taken out of the aqueous solution and lyophilized. It may be carried out by performing the procedure at least once. The lyophilization may be performed repeatedly, for example, may be performed repeatedly 2 to 20 times, but is not limited thereto. The immersion may be carried out at 4 to 50 ℃, preferably 4 to 20 ℃. The immersion time is not particularly limited, and may be performed for a short time of about 30 seconds or, if necessary, for a longer time, for example, 12 to 48 hours. In addition, if necessary, by further comprising drying the support taken out of the aqueous solution at room temperature (about 25 ℃) before lyophilization after the immersion, it is possible to prevent the clogging in the manufacturing process.
상기와 같이 히아루론산 또는 그의 염 및 피브리노오겐이 구조틀(frame) 표면 상에 코팅되어 있는 고분자 지지체는 높은 친수성 및 세포친화성을 가짐으로써, 세포 및/또는 세포성장인자를 높은 점착효율(introduction rate or loading rate)로 고분자 지지체 상에 도입시킬 수 있다.As described above, the polymer support having hyaluronic acid or a salt thereof and fibrinogen coated on the surface of the frame has a high hydrophilicity and cell affinity, thereby resulting in high adhesion rate of cells and / or cell growth factors. or loading rate) may be introduced onto the polymer support.
예를 들어 세포성장인자의 도입은 수성 용액, 예를 들어 인산완충생리식염수(phosphate-buffered saline) 중에 세포성장인자를 용해시켜 상기 코팅층에 가하고 건조(바람직하게는 동결건조)시킴으로써 수행될 수 있다. 상기와 같이 건조된 코팅층에는 세포성장인자가 코팅층 전체에 균일하게 분산되어 있거나 코팅층 표면에 골고루 박혀있는 형태로 존재하게 된다. 또한, 세포성장인자의 도입은 상기 코팅층을 가교화시키 전, 가교화와 동시에, 혹은 가교화시킨 후에 도입할 수 있다. For example, the introduction of the cell growth factor may be performed by dissolving the cell growth factor in an aqueous solution, for example, phosphate-buffered saline, adding it to the coating layer and drying (preferably lyophilization). In the dried coating layer as described above, cell growth factors may be uniformly dispersed in the entire coating layer or evenly embedded in the surface of the coating layer. In addition, introduction of the cell growth factor may be introduced before the crosslinking of the coating layer, simultaneously with the crosslinking or after the crosslinking.
구체적으로는, 상기 히아루론산 또는 그의 염 및 피브리노오겐을 함유하는 수용액에 세포성장인자를 추가로 함유시켜, 상기한 바와 동일한 방법으로 코팅층을 형성시킴으로써, 상기 세포성장인자가 상기 코팅층에 도입될 수 있다. 또한, 세포성장인자를 함유하는 수성 용액(aqueous solution)을 상기 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층에 가한 후(필요할 경우 추가로 건조, 바람직하게는 동결건조 한 후), 트롬빈 및 칼슘염을 함유하는 용액을 가하여 건조시킴으로써 상기 세포성장인자가 상기 코팅층에 도입될 수 있다. 또한, 세포성장인자; 및 트롬빈과 칼슘염을 함유하는 수성 용액을 상기 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층에 가하여 건조시킴으로써, 상기 세포성장인자가 상기 코팅층에 도입될 수 있다. 또한, 트롬빈 및 칼슘염을 함유하는 용액을 상기 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층에 가하여 건조시킨 다음, 세포성장인자를 함유하는 수성 용액을 가함으로써(필요할 경우 추가로 건조, 바람직하게는 동결건조함으로써), 상기 세포성장인자가 상기 코팅층에 도입될 수 있다. 상기 구현예에 있어서, 상기 건조는 동결건조에 의해 바람직하게 수행될 수 있으며, 상기 수성 용액 중의 세포성장인자의 농도는 10 pg/mL ∼ 100 mg/mL의 범위일 수 있다.Specifically, the cell growth factor may be introduced into the coating layer by additionally containing a cell growth factor in an aqueous solution containing hyaluronic acid or a salt thereof and fibrinogen, and forming a coating layer in the same manner as described above. have. In addition, an aqueous solution containing cell growth factor is added to the coating layer formed by coating the hyaluronic acid or its salt and fibrinogen (if further dried, preferably after lyophilization, if necessary), and thrombin. And the cell growth factor may be introduced into the coating layer by drying by adding a solution containing calcium salt. In addition, cell growth factor; And by adding an aqueous solution containing thrombin and calcium salt to the coating layer formed by coating the hyaluronic acid or a salt thereof and fibrinogen, the cell growth factor may be introduced into the coating layer. In addition, a solution containing thrombin and calcium salt is added to the coating layer formed by coating the hyaluronic acid or a salt thereof and fibrinogen, followed by drying, and then an aqueous solution containing cell growth factor (additionally, if necessary, Preferably, by lyophilization), the cell growth factor can be introduced into the coating layer. In the above embodiment, the drying may be preferably performed by lyophilization, the concentration of the cell growth factor in the aqueous solution may be in the range of 10 pg / mL to 100 mg / mL.
또한, 세포의 도입은 줄기세포 등의 세포를 수성 매질(예를 들어, 인산완충식염수, DMEM 등과 같은 통상의 세포배양용 배지 등)에 분산시켜 얻어진 분산액을 상기 코팅층 또는 세포성장인자가 도입된 코팅층에 가함으로써 수행될 수 있다. 상기와 같이 세포를 코팅층에 도입하면, 세포가 높은 점착효율로 상기 코팅층에 점착되게 된다. In addition, the introduction of the cells is a dispersion layer obtained by dispersing cells such as stem cells in an aqueous medium (for example, a conventional cell culture medium such as phosphate buffered saline, DMEM, etc.) coating layer or a coating layer into which the cell growth factor is introduced. It can be performed by adding to. When the cells are introduced into the coating layer as described above, the cells are adhered to the coating layer with high adhesion efficiency.
일 구현예에서, 상기한 바와 같이 얻어진 세포성장인자가 도입된 조직재생용 구조체의 코팅층 표면 상에, 세포를 함유하는 수성 분산액(aqueous dispersion)을 파종하는 단계를 추가로 포함으로써, 추가적으로 상기 세포가 상기 코팅층에 도입될 수 있다. 상기 수성 분산액 중의 세포의 농도는 103 ∼ 1010 cells/mL의 범위일 수 있다. In one embodiment, the method further comprises seeding an aqueous dispersion containing cells on the surface of the coating layer of the tissue regeneration structure into which the cell growth factor obtained as described above is introduced. It may be introduced into the coating layer. The concentration of cells in the aqueous dispersion may range from 10 3 to 10 10 cells / mL.
또다른 구현에에서, 트롬빈 및 칼슘염을 함유하는 용액을 상기 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층에 가하여 건조시킨 다음, 세포를 함유하는 수성 분산액(aqueous dispersion)을 가함으로써, 세포성장인자의 도입없이 세포가 단독으로 상기 코팅층에 도입될 수 있다. 상기 건조는 동결건조에 의해 수행될 수 있으며, 상기 수성 분산액 중의 세포의 농도는 103 ∼ 1010 cells/mL의 범위일 수 잇다.In another embodiment, a solution containing thrombin and calcium salt is added to the coating layer formed by coating the hyaluronic acid or a salt thereof and fibrinogen, followed by drying, followed by addition of an aqueous dispersion containing cells, Cells may be introduced into the coating layer alone without introduction of cell growth factors. The drying may be performed by lyophilization, and the concentration of cells in the aqueous dispersion may range from 10 3 to 10 10 cells / mL.
본 발명의 조직재생용 구조체에 있어서, 상기 트롬빈 및 칼슘염은 피브리노오겐의 가교화 기능을 수행하며, 그 사용량은 원하는 가교화의 정도(예를 들어 세포성장인자의 원하는 방출속도를 달성하기 위하여 필요한 정도)에 따라 적절한 범위로 선택하여 사용될 수 있다. 바람직하게는 상기 트롬빈 및 칼슘염의 농도는 10 ∼ 90 중량%의 범위일 수 있으나, 이에 제한되는 것은 아니다. In the tissue regeneration structure of the present invention, the thrombin and calcium salts perform a crosslinking function of fibrinogen, and the amount of the thrombin and the calcium salt is used to achieve a desired degree of crosslinking (for example, to achieve a desired release rate of cell growth factor). Can be selected and used in an appropriate range according to the degree required). Preferably the concentration of thrombin and calcium salt may be in the range of 10 to 90% by weight, but is not limited thereto.
상기 세포는 조직재생의 목적에 따라 사용되는 세포를 제한 없이 사용될 수 있으며, 전형적으로는 조직세포, 성체줄기세포, 배아줄기세포, 또는 유도만능줄기세포(induced pluripotent stem cells)일 수 있다. 본 발명의 조직재생용 구조체에 있어서, 상기 세포의 로딩(loading) 양은 필요에 따라 적절히 선택될 수 있으며, 전형적으로는 조직재생용 구조체 당 약 1×105 내지 5×107 cells의 조직세포 또는 줄기세포를 함유할 수 있다. 상기 조직세포는 환자유래의 세포를 분리한 후, 이를 체외에서 배양/증식하여 얻어진 세포 즉, 환자유래의 체외 증식된 세포를 바람직하게 사용할 수 있다. 또한, 상기 성체줄기세포는 다양한 조직 또는 세포로부터 유래될 수 있으며, 예를 들어 골유유래 줄기세포, 제대혈유래 줄기세포, 태반유래 줄기세포, 근육유래 줄기세포, 지방유래 줄기세포 등을 모두 포함한다. 예를 들어, 통상적으로 흔히 시행되는 지방흡입 과정에서 폐기되는 지방조직을 사용하여 얻을 수 있는 지방유래 줄기세포(Adipose-derived stem cells, ASCs)가 바람직하게 사용될 수 있다. 상기 지방유래 줄기세포는 환자 자신 또는 표현형이 일치하는 타인의 지방으로부터 공지의 방법에 따라 얻어진 것을 사용할 수 있다. 상기 지방은 피하지방조직, 골수지방조직, 장간막 지방조직, 위장 지방조직 및 후복막 지방조직 등을 제한없이 포함한다. The cells may be used without limitation, the cells used for the purpose of tissue regeneration, and may typically be tissue cells, adult stem cells, embryonic stem cells, or induced pluripotent stem cells. In the tissue regeneration construct of the present invention, the loading amount of the cells may be appropriately selected according to necessity, and typically about 1 × 10 5 to 5 × 10 7 cells per tissue regeneration construct or May contain stem cells. The tissue cells can be preferably used cells isolated from the patient-derived cells, cultured and propagated in vitro, that is, cells derived from the patient in vitro. In addition, the adult stem cells may be derived from various tissues or cells, for example, bone-derived stem cells, umbilical cord blood-derived stem cells, placental stem cells, muscle-derived stem cells, adipose derived stem cells and the like. For example, Adipose-derived stem cells (ASCs) that can be obtained by using adipose tissue discarded in a commonly performed liposuction process may be preferably used. The fat-derived stem cells may be those obtained according to a known method from the fat of the patient or another person of the same phenotype. The fat includes, without limitation, subcutaneous fat tissue, bone marrow adipose tissue, mesenteric adipose tissue, gastrointestinal adipose tissue and posterior peritoneal adipose tissue.
상기 세포성장인자(cell growth factors) 또한, 조직재생의 목적에 따라 사용되는 세포성장인자를 제한 없이 사용될 수 있으며, 예를 들어 BMP-2(bone morphogenetic protein 2), VEGF(vascular endothelial growth factor), EGF(epidermal growth factor), IGF(insulin-like growth factor), NGF(nerve growth factor), PDGF(platelet-derived growth factor), TGF-β(transforming growth factor-β), bFGF(basic fibroblast growth factor) 등을 1 종 이상 사용할 수 있으나, 이에 제한되는 것은 아니다. 또한, 본 발명의 조직재생용 구조체에 있어서, 상기 세포성장인자의 로딩(loading) 양은 목적에 따라 적절히 선택될 수 있다. 예를 들어 인산완충식염수 등의 수성 매질 중 약 10 pg/mL ∼ 100 mg/mL 농도의 세포성장인자의 용액을 단위 형태의(unit-form) 조직재생용 구조체에 가하여 건조시킴으로써 세포성장인자를 로딩할 수 있다.The cell growth factors may also be used without limitation cell growth factors used for the purpose of tissue regeneration, for example, bone morphogenetic protein 2 (BMP-2), vascular endothelial growth factor (VEGF), Epidermal growth factor (EGF), insulin-like growth factor (IGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), and basic fibroblast growth factor (bFGF) One or more of these may be used, but is not limited thereto. In addition, in the tissue regeneration construct of the present invention, the loading amount of the cell growth factor may be appropriately selected according to the purpose. For example, a cell growth factor is loaded by adding a solution of cell growth factor at a concentration of about 10 pg / mL to 100 mg / mL in an aqueous medium such as phosphate buffered saline to a unit-form tissue regeneration construct and drying it. can do.
본 발명의 조직재생용 구조체는 상기한 바와 같이 세포(예를 들어, 조직세포, 줄기세포 등), 세포성장인자를 별도의 조직재생용 구조체에 함유할 수 있으며, 필요에 따라 이들을 조합하여 하나의 조직재생용 구조체에 함유할 수도 있다. 예를 들어, 세포성장인자로서 BMP-2 및 세포로서 지방유래 줄기세포를 하나의 조직재생용 구조체에 함유할 수 있으며, 이로부터 얻어진 조직재생용 구조체는 골조직 재생에 유용하게 사용될 수 있다. 또한, BMP-2 및 지방유래 줄기세포를 함유하는 조직재생용 구조체는 체외에서 골분화를 유도하여 생체내에 이식될 수 있거나, 혹은 별도의 분화 유도 없이 생체내에 이식함으로써 생체내에서 골분화를 유도할 수도 있다.The tissue regeneration construct of the present invention may contain cells (eg, tissue cells, stem cells, etc.) and cell growth factors in a separate tissue regeneration construct as described above, and a combination of these may be used as needed. It may be contained in a structure for tissue regeneration. For example, BMP-2 as a cell growth factor and adipose derived stem cells as cells may be contained in one tissue regeneration structure, and the tissue regeneration structure obtained therefrom may be usefully used for bone tissue regeneration. In addition, tissue regeneration constructs containing BMP-2 and adipose derived stem cells can be induced in vitro to induce bone differentiation, or in vivo to induce bone differentiation by implanting in vivo without inducing differentiation. It may be.
본 발명은 또한 상기 조직재생용 구조체 형성을 위한 키트를 제공한다.The present invention also provides a kit for forming the structure for tissue regeneration.
일 태양에서, 본 발명은 공극을 갖는 고분자 지지체의 구조틀 표면 상에 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층을 포함하는 구조체-컴파트먼트[컴파트먼트 (a)]; 및 트롬빈 및 칼슘염을 포함하는 용액을 포함하는 트롬빈-컴파트먼트[컴파트먼트 (b)]를 포함하는, 트롬빈에 의해 가교화되고 또한 10 ㎛ ∼ 1,000 ㎛의 공극을 갖는 조직재생용 구조체 형성을 위한 키트를 제공한다.In one aspect, the present invention provides a structure-compartment (compartment (a)) comprising a coating layer formed by coating hyaluronic acid or a salt thereof and fibrinogen on a surface of a structure of a polymer support having voids; And a thrombin-compartment (compartment (b)) comprising a solution comprising thrombin and calcium salt, forming a structure for tissue regeneration crosslinked by thrombin and having pores of 10 μm to 1,000 μm It provides a kit for.
본 발명에 따른 키트는 상기 조직재생인자의 제공을 위한 수성 용액 컴파트먼트를 추가로 포함할 수 있다. 즉, 본 발명에 따른 키트는 공극을 갖는 고분자 지지체의 구조틀 표면 상에 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층을 포함하는 구조체-컴파트먼트[컴파트먼트 (a)]; 트롬빈 및 칼슘염을 포함하는 용액을 포함하는 트롬빈-컴파트먼트[컴파트먼트 (b)]; 및 세포 또는 세포성장인자의 제공을 위한 1종 이상의 수성 용액-컴파트먼트[컴파트먼트 (c)]를 포함할 수 있다. 상기 컴파트먼트 (c)의 수성 용액은 수성 매질 예를 들어, 인산완충식염수, DMEM 등과 같은 통상의 세포배양용 배지 등을 포함한다.Kits according to the invention may further comprise an aqueous solution compartment for the provision of the tissue regeneration factor. That is, the kit according to the present invention comprises a structure-compartment (compartment (a)) comprising a coating layer formed by coating hyaluronic acid or a salt thereof and fibrinogen on the surface of the structure of the polymer support having pores; Thrombin-compartment (compartment (b)) comprising a solution comprising thrombin and calcium salt; And one or more aqueous solution-compartments (compartments (c)) for the provision of cells or cell growth factors. The aqueous solution of compartment (c) comprises an aqueous medium such as a conventional cell culture medium such as phosphate buffered saline, DMEM or the like.
본 발명에 따른 상기 키트는 세포 또는 세포성장인자를 상기 컴파트먼트 (a)에 도입하고, 컴파트먼트 (b)를 가하여 가교화함으로써 10 ㎛ ∼ 1,000 ㎛의 공극을 갖는 조직재생용 구조체를 형성시킬 수 있다. 상기와 같이 히아루론산 또는 그의 염 및 피브리노오겐이 구조틀(frame) 표면 상에 코팅되어 있는 고분자 지지체는 높은 친수성 및 세포친화성을 가짐으로써, 세포 및/또는 세포성장인자를 높은 점착효율(introduction rate or loading rate)로 고분자 지지체 상에 도입시킬 수 있다.The kit according to the present invention forms a structure for tissue regeneration having pores of 10 μm to 1,000 μm by introducing a cell or cell growth factor into the compartment (a) and crosslinking by adding the compartment (b). You can. As described above, the polymer support having hyaluronic acid or a salt thereof and fibrinogen coated on the surface of the frame has a high hydrophilicity and cell affinity, thereby resulting in high adhesion rate of cells and / or cell growth factors. or loading rate) may be introduced onto the polymer support.
예를 들어 세포성장인자의 도입은 수성 용액, 예를 들어 인산완충생리식염수(phosphate-buffered saline)[예를 들어, 컴파트먼트 (c)] 중에 세포성장인자를 용해시켜, 상기 코팅층에 가하고 건조(바람직하게는 동결건조)시킴으로써 수행될 수 있다. 상기와 같이 건조된 코팅층에는 세포성장인자가 코팅층 전체에 균일하게 분산되어 있거나 코팅층 표면에 골고루 박혀있는 형태로 존재하게 된다. 또한, 세포성장인자의 도입은 상기 코팅층을 가교화시키 전, 가교화와 동시에, 혹은 가교화시킨 후에 도입할 수 있다. For example, introduction of cell growth factors may be achieved by dissolving the cell growth factors in an aqueous solution, such as phosphate-buffered saline (eg, compartment (c)), adding them to the coating layer and drying. (Preferably lyophilization). In the dried coating layer as described above, cell growth factors may be uniformly dispersed in the entire coating layer or evenly embedded in the surface of the coating layer. In addition, introduction of the cell growth factor may be introduced before the crosslinking of the coating layer, simultaneously with the crosslinking or after the crosslinking.
구체적으로는, 상기 히아루론산 또는 그의 염 및 피브리노오겐을 함유하는 수용액에 세포성장인자를 추가로 함유시켜 코팅층을 형성시킴으로써, 상기 세포성장인자가 상기 코팅층에 도입될 수 있다. 또한, 세포성장인자를 함유하는 수성 용액(aqueous solution)[예를 들어, 컴파트먼트 (c)]을 상기 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층에 가한 후(필요할 경우 추가로 건조, 바람직하게는 동결건조 한 후), 트롬빈 및 칼슘염을 함유하는 용액을 가하여 건조시킴으로써 상기 세포성장인자가 상기 코팅층에 도입될 수 있다. 또한, 세포성장인자; 및 트롬빈과 칼슘염을 함유하는 수성 용액을 상기 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층에 가하여 건조시킴으로써, 상기 세포성장인자가 상기 코팅층에 도입될 수 있다. 또한, 트롬빈 및 칼슘염을 함유하는 용액을 상기 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층에 가하여 건조시킨 다음, 세포성장인자를 함유하는 수성 용액을 가함으로써(필요할 경우 추가로 건조, 바람직하게는 동결건조함으로써), 상기 세포성장인자가 상기 코팅층에 도입될 수 있다. 상기 구현예에 있어서, 상기 건조는 동결건조에 의해 바람직하게 수행될 수 있으며, 상기 수성 용액[예를 들어, 컴파트먼트 (c)] 중의 세포성장인자의 농도는 10 pg/mL ∼ 100 mg/mL의 범위일 수 있다.Specifically, the cell growth factor may be introduced into the coating layer by further containing a cell growth factor in an aqueous solution containing hyaluronic acid or a salt thereof and fibrinogen to form a coating layer. In addition, an aqueous solution containing cell growth factors (e.g., compartment (c)) is added to the coating layer formed by coating the hyaluronic acid or its salt and fibrinogen (if further The cell growth factor can be introduced into the coating layer by drying, preferably after lyophilization), followed by drying by addition of a solution containing thrombin and calcium salt. In addition, cell growth factor; And by adding an aqueous solution containing thrombin and calcium salt to the coating layer formed by coating the hyaluronic acid or a salt thereof and fibrinogen, the cell growth factor may be introduced into the coating layer. In addition, a solution containing thrombin and calcium salt is added to the coating layer formed by coating the hyaluronic acid or a salt thereof and fibrinogen, followed by drying, and then an aqueous solution containing cell growth factor (additionally, if necessary, Preferably, by lyophilization), the cell growth factor can be introduced into the coating layer. In this embodiment, the drying may be preferably carried out by lyophilization, the concentration of the cell growth factor in the aqueous solution (eg, compartment (c)) is 10 pg / mL to 100 mg / may be in the range of mL.
또한, 세포의 도입은 줄기세포 등의 세포를 수성 매질(예를 들어, 인산완충식염수, DMEM 등과 같은 통상의 세포배양용 배지 등)[예를 들어, 컴파트먼트 (c)]에 분산시켜 얻어진 분산액을 상기 코팅층 또는 세포성장인자가 도입된 코팅층에 가함으로써 수행될 수 있다. 상기와 같이 세포를 코팅층에 도입하면, 세포가 높은 점착효율로 상기 코팅층에 점착되게 된다.In addition, the introduction of the cells is obtained by dispersing cells such as stem cells in an aqueous medium (for example, conventional cell culture medium such as phosphate buffered saline, DMEM, etc.) (for example, compartment (c)). The dispersion may be carried out by adding the coating layer or the coating layer into which the cell growth factor is introduced. When the cells are introduced into the coating layer as described above, the cells are adhered to the coating layer with high adhesion efficiency.
일 구현예에서, 상기한 바와 같이 얻어진 세포성장인자가 도입된 조직재생용 구조체의 코팅층 표면 상에, 세포를 함유하는 수성 분산액(aqueous dispersion)을 파종하는 단계를 추가로 포함으로써, 추가적으로 상기 세포가 상기 코팅층에 도입될 수 있다. 상기 수성 분산액[예를 들어, 컴파트먼트 (c)] 중의 세포의 농도는 103 ∼ 1010 cells/mL의 범위일 수 있다. In one embodiment, the method further comprises seeding an aqueous dispersion containing cells on the surface of the coating layer of the tissue regeneration structure into which the cell growth factor obtained as described above is introduced. It may be introduced into the coating layer. The concentration of cells in the aqueous dispersion [eg, compartment (c)] can range from 10 3 to 10 10 cells / mL.
또다른 구현에에서, 트롬빈 및 칼슘염을 함유하는 용액을 상기 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층에 가하여 건조시킨 다음, 세포를 함유하는 수성 분산액(aqueous dispersion)[예를 들어, 컴파트먼트 (c)]을 가함으로써, 세포성장인자의 도입없이 세포가 단독으로 상기 코팅층에 도입될 수 있다. 상기 건조는 동결건조에 의해 수행될 수 있으며, 상기 수성 분산액 중의 세포의 농도는 103 ∼ 1010 cells/mL의 범위일 수 잇다.In another embodiment, a solution containing thrombin and calcium salt is added to the coating layer formed by coating the hyaluronic acid or a salt thereof and fibrinogen, followed by drying, followed by an aqueous dispersion containing cells [eg , Compartment (c)], the cells can be introduced into the coating layer alone without introducing the cell growth factor. The drying may be performed by lyophilization, and the concentration of cells in the aqueous dispersion may range from 10 3 to 10 10 cells / mL.
또한, 본 발명에 따른 조직재생용 구조체 형성을 위한 키트는 세포 및/또는 세포성장인자를 미리 함유하는 미리-제작된 키트(ready-made kit) 형태로 제작될 수 있다. 즉, 조직재생에 필요한 조직재생인자를 사전에 키트 형태로 제작하여, 환자에게 적용시 조직재생용 구조체를 형성시켜 이식될 수 있는 키트로서 사용될 수 있다. 상기 미리-제작된 키트의 사용방법은 상기에서 설명한 바와 같다.In addition, the kit for forming a tissue regeneration structure according to the present invention can be produced in the form of a pre-made kit (ready-made kit) containing the cells and / or cell growth factors in advance. That is, the tissue regeneration factor necessary for tissue regeneration in advance in the form of a kit, can be used as a kit that can be implanted by forming a structure for tissue regeneration when applied to the patient. The use of the pre-made kit is as described above.
구체적으로는, 상기 미리-제작된 키트는 공극을 갖는 고분자 지지체의 구조틀 표면 상에 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층을 포함하는 구조체-컴파트먼트[컴파트먼트 (a)]; 세포성장인자; 및 트롬빈과 칼슘염을 함유하는 수성 용액을 포함하는 세포성장인자-트롬빈-완충액 컴파트먼트[컴파트먼트 (b')]; 및 선택적으로, 세포를 함유하는 세포-수성 용액-컴파트먼트[컴파트먼트 (c')]를 포함할 수 있다.Specifically, the pre-fabricated kit comprises a structure-compartment comprising a coating layer formed by coating hyaluronic acid or a salt thereof and fibrinogen on the surface of the structure of the polymer support having voids [compartment (a) ]; Cell growth factor; And a cell growth factor-thrombin-buffer compartment (compartment (b ')) comprising an aqueous solution containing thrombin and calcium salt; And optionally, a cell-aqueous solution-compartment containing the cells (compartment (c ')).
또한, 상기 세포 및/또는 세포재생인자를 별도의 컴파트먼트로서 포함하는 형태로 제작될 수 있으며, 구체적으로는, 공극을 갖는 고분자 지지체의 구조틀 표면 상에 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층을 포함하는 구조체-컴파트먼트[컴파트먼트 (a)]; 트롬빈 및 칼슘염을 포함하는 용액을 포함하는 트롬빈-컴파트먼트[컴파트먼트 (b)]; 및 세포 또는 세포성장인자를 함유하는 1종 이상의 수성 용액-컴파트먼트[컴파트먼트 (c'')]를 포함할 수 있다.In addition, the cell and / or cell regeneration factor may be produced in a form containing a separate compartment, specifically, hyaluronic acid or a salt thereof and fibrinogen on the surface of the structure of the polymer support having pores A structure-compartment [compartment (a)] comprising a coating layer formed by coating; Thrombin-compartment (compartment (b)) comprising a solution comprising thrombin and calcium salt; And one or more aqueous solution-compartments (compartments (c '')) containing cells or cell growth factors.
컴파트먼트 (c'')은 필요에 따라, 2개 이상의 복수 컴파트먼트로 구성될 수 있으며, 예를 들어, 세포성장인자를 포함하는 수성 용액, 바람직하게는 인산완충식염수와 같은 생리학적으로 적합한 완충액; 및 세포를 포함하는 분산액의 복수 컴파트먼트로 구성될 수 있다. 일 구현예에서, 컴파트먼트 (c'')를 상기한 바와 같이 2종의 컴파트먼트로 구성할 경우, 세포성장인자를 함유하는 수성 용액을 상기 컴파트먼트 (a)에 가하여 건조(바람직하게는 동결건조)시킨 다음, 컴파트먼트 (b)를 가하여 가교화를 유도한 후, 다시 세포를 포함하는 분산액(예를 들어, 줄기세포 분산액) 중에서 가교화가 유도된 구조체를 배양함으로써, 조직재생용 구조체를 제작할 수 있다.Compartment (c '') may be composed of two or more plurality of compartments, if necessary, for example, physiologically such as an aqueous solution containing cell growth factor, preferably phosphate buffered saline. Suitable buffers; And a plurality of compartments of a dispersion containing cells. In one embodiment, when the compartment (c '') consists of two compartments as described above, an aqueous solution containing cell growth factors is added to the compartment (a) and dried (preferably Lyophilization), followed by addition of compartment (b) to induce crosslinking, followed by culturing the crosslinked-induced construct in a dispersion containing cells (e.g., stem cell dispersion). The structure for this can be produced.
이하, 본 발명에 따른 키트의 제작방법을 설명한다. Hereinafter, a method for manufacturing a kit according to the present invention will be described.
상기 키트의 제작방법에 있어서, 상기 고분자 지지체(polymer support), 히아루론산, 세포, 세포성장인자(cell growth factors) 등은 상기 조직재생용 구조체와 관련하여 설명한 바와 같다.In the method of manufacturing the kit, the polymer support, hyaluronic acid, cells, cell growth factors, etc. are the same as described with reference to the tissue regeneration structure.
본 발명에 따른 키트 중 상기 컴파트먼트 (a)에 있어서, 상기 코팅층 중의 히아루론산 또는 그의 염의 함량은 10 ∼ 90 중량%의 범위일 수 있으며, 또한 상기 코팅층 중의 피브리노오겐의 함량은 10 ∼ 90 중량%의 범위일 수 있다. 상기 코팅층 중의 상기 히아루론산 또는 그의 염 및 피브리노오겐의 중량비는 크게 제한되는 것은 아니며, 세포성장인자의 방출속도 등을 감안하여 조절될 수 있다.In the compartment (a) of the kit according to the present invention, the content of hyaluronic acid or a salt thereof in the coating layer may be in the range of 10 to 90% by weight, and the content of fibrinogen in the coating layer is 10 to 90 It may range from weight percent. The weight ratio of the hyaluronic acid or a salt thereof and fibrinogen in the coating layer is not particularly limited, and may be controlled in view of the release rate of cell growth factors.
고분자 지지체의 구조틀(frame) 표면 상에 히아루론산 또는 그의 염 및 피브리노오겐의 코팅은 히아루론산 및 피브리노오겐을 함유하는 수용액 중에 상기 지지체를 침지한 다음, 상기 지지체를 상기 수용액으로부터 꺼내어 동결건조하는 과정을 적어도 1회 이상 수행함으로써 수행될 수 있다. 상기 동결건조는 반복하여 수행될 수 있으며, 예를 들어 2회 내지 20회 반복하여 수행될 수 있으나, 이에 제한되는 것은 아니다. 상기 침지는 4 ∼ 50 ℃, 바람직하게는 4 ∼ 20 ℃에서 수행될 수 있다. 상기 침지 시간은 특별히 제한되는 것은 아니며, 약 30초의 단시간 동안 수행되거나 필요에 따라 그 보다 장시간 예를 들어 12 ∼ 48 시간 동안 수행될 수 있다. 또한, 필요할 경우, 상기 침지 후 동결건조 전에, 상기 수용액으로부터 꺼낸 지지체를 상온(약 25 ℃)에서 건조하는 것을 추가로 포함함으로써 제조과정에서 공극이 막히는 것을 방지할 수 있다. Coating of hyaluronic acid or a salt thereof and fibrinogen on the surface of the frame of the polymer support is immersed in the aqueous solution containing hyaluronic acid and fibrinogen, and then the support is taken out of the aqueous solution and lyophilized. It may be carried out by performing the procedure at least once. The lyophilization may be performed repeatedly, for example, may be performed repeatedly 2 to 20 times, but is not limited thereto. The immersion may be carried out at 4 to 50 ℃, preferably 4 to 20 ℃. The immersion time is not particularly limited, and may be performed for a short time of about 30 seconds or, if necessary, for a longer time, for example, 12 to 48 hours. In addition, if necessary, by further comprising drying the support taken out of the aqueous solution at room temperature (about 25 ℃) before lyophilization after the immersion, it is possible to prevent the clogging in the manufacturing process.
본 발명에 따른 키트에 있어서, 상기 트롬빈 및 칼슘염은 피브리노오겐의 가교화 기능을 수행하며, 그 사용량은 원하는 가교화의 정도(예를 들어 세포성장인자의 원하는 방출속도를 달성하기 위하여 필요한 정도)에 따라 적절한 범위로 선택하여 사용될 수 있다. 바람직하게는 컴파트먼트 (b) 또는 컴파트먼트 (b') 중의 상기 트롬빈 및 칼슘염의 농도는 10 ∼ 90 중량%의 범위일 수 있으나, 이에 제한되는 것은 아니다.In the kit according to the invention, the thrombin and calcium salts perform the crosslinking function of fibrinogen, the amount of which is necessary to achieve the desired degree of crosslinking (eg, to achieve a desired release rate of cell growth factor). Can be selected and used in an appropriate range. Preferably the concentration of the thrombin and calcium salt in the compartment (b) or the compartment (b ') may be in the range of 10 to 90% by weight, but is not limited thereto.
또한, 컴파트먼트 (b') 또는 컴파트먼트 (c'') 중의 상기 세포성장인자의 농도는 10 pg/mL ∼ 100 mg/mL의 범위일 수 있으며, 컴파트먼트 (c') 또는 컴파트먼트 (c'') 중의 상기 세포의 농도는 103 ∼ 1010 cells/mL의 범위일 수 있다.In addition, the concentration of the cell growth factor in the compartment (b ') or the compartment (c'') may be in the range of 10 pg / mL to 100 mg / mL, the compartment (c') or The concentration of the cells in the part (c '') may range from 10 3 to 10 10 cells / mL.
본 발명에 따른 키트는 세포(예를 들어, 조직세포, 줄기세포 등), 세포성장인자를 별도로 사용하여 별도의 조직재생용 구조체로 제조되도록 사용하거나, 필요에 따라 이들을 조합하여 하나의 조직재생용 구조체에 함유되도록 사용할 수도 있다. 예를 들어, 본 발명에 따른 키트를 사용하여 세포성장인자로서 BMP-2 및 세포로서 지방유래 줄기세포를 하나의 조직재생용 구조체에 함유시킬 수 있다. 또한, 본 발명에 따른 키트로부터 형성된 조직재생용 구조체, 예를 들어 BMP-2 및 지방유래 줄기세포를 함유하는 조직재생용 구조체는 체외에서 골분화를 유도하여 생체내에 이식될 수 있거나, 혹은 별도의 분화 유도 없이 생체내에 이식함으로써 생체내에서 골분화를 유도할 수도 있다.Kit according to the present invention can be used to prepare a separate tissue regeneration structure using cells (eg, tissue cells, stem cells, etc.), cell growth factors, or a combination of these as needed for one tissue regeneration It can also be used to contain in a structure. For example, using the kit according to the present invention, BMP-2 as a cell growth factor and adipose derived stem cells as cells can be contained in a structure for tissue regeneration. In addition, the tissue regeneration structure formed from the kit according to the present invention, for example, tissue regeneration structure containing BMP-2 and adipose derived stem cells can be transplanted in vivo by inducing bone differentiation in vitro, or a separate Bone differentiation may be induced in vivo by implantation in vivo without inducing differentiation.
이하, 실시예를 통하여 본 발명을 더욱 상세하게 설명한다. 그러나, 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.
실시예 1. 골성장인자가 도입된 조직재생용 지지체의 제작Example 1 Preparation of Support for Tissue Regeneration Incorporating Bone Growth Factor
(1) 고분자 지지체의 구조틀(frame) 표면 상에 히아루론산 및 피브리노오겐의 코팅층 형성[컴파트먼트 (a)의 제작](1) Formation of a coating layer of hyaluronic acid and fibrinogen on the surface of a frame of a polymer support [production of compartment (a)]
폴리(락트산-co-글리콜산)(PLGA) 및 폴리카프로락톤(PCL)로부터 형성된 고분자 지지체를 Kim 등의 방법(Biofabrication 2009; 1: 1-7)에 따라, 자유 임의 형상 제작 방식(solid free-form fabrication method)을 사용하여 제작하였다. 얻어진 고분자 지지체의 4mm x 4mm x 2mm이고, 공극의 크기는 약 250 μm 이었으며, 공극률은 약 65∼70% 이었다. 1 mL 아프로니틴에 용해된 피브리노겐 용액(녹십자사, 그린플라스트 1 mL 키트 사용)과 삼차 증류수 10 mL에 히아루론산(분자량 1,500,000, LG생명과학제조) 200 mg을 용해시킨 수용액을 볼륨기준으로 동일 비율로 혼합하였다. 얻어진 피브리노겐/히아루론산 용액에 상기에서 제조한 고분자 지지체를 침지하여 4℃에서 24시간 동안 교반하였다. 상기 고분자 지지체를 꺼내어 공극이 1시간 동안 실온에서 건조한 후, 24 시간 동안 동결건조하였다. 상기 침지 및 동결건조 과정을 3회 반복하여 구조틀 표면 상에 히아루론산 및 피브리노오겐의 코팅층이 형성된 고분자 지지체를 얻었다.Polymer supports formed from poly (lactic acid-co-glycolic acid) (PLGA) and polycaprolactone (PCL) were prepared according to Kim et al. (Biofabrication 2009; 1: 1-7). form fabrication method). The obtained polymer support was 4 mm x 4 mm x 2 mm, the pore size was about 250 μm, and the porosity was about 65 to 70%. Fibrinogen solution dissolved in 1 mL apronitine (using Green Cross, GreenPlast 1 mL kit), and an aqueous solution of 200 mg of hyaluronic acid (molecular weight 1,500,000, manufactured by LG Life Sciences) in 10 mL of tertiary distilled water in equal proportions Mixed. The polymer support prepared above was immersed in the obtained fibrinogen / hyaluronic acid solution and stirred at 4 ° C. for 24 hours. The polymer support was taken out and the pores were dried at room temperature for 1 hour and then lyophilized for 24 hours. The dipping and lyophilization processes were repeated three times to obtain a polymer support having a coating layer of hyaluronic acid and fibrinogen formed on the surface of the framework.
(2) 골성장인자가 도입된 조직재생용 지지체의 제작(2) Preparation of the support for tissue regeneration in which bone growth factor was introduced
골성장인자(BMP-2)(Chinese hamster ovary cell-derived recombinant human BMP-2, R&D Systems, Minneapolis, MN)를 인산완충식염수에 50 μg/mL의 농도로 녹인 후, 얻어진 BMP-2 용액 20 ㎕를 실시예 1에서 얻어진 코팅층이 형성된 고분자 지지체[즉, 컴파트먼트 (a)]에 가하여 흡수시킨 다음, 즉시 동결건조하였다. 동결건조된 지지체에 트롬빈(녹십자사, 그린플라스트 1 mL 키트 사용)을 용해시킨 염화칼슘 수용액[즉, 컴파트먼트 (b)] 32 ㎕를 가하여 가교화를 유도한 후, 다시 동결건조하여 조직재생용 지지체를 제작하였다. BMP-2 (Chinese hamster ovary cell-derived recombinant human BMP-2, R & D Systems, Minneapolis, MN) was dissolved in phosphate buffered saline at a concentration of 50 μg / mL, and then 20 μl of the resulting BMP-2 solution. Was added to the polymer support (ie, compartment (a)) having the coating layer obtained in Example 1 and absorbed, and immediately lyophilized. 32 μl of an aqueous calcium chloride solution (ie, compartment (b)) in which thrombin (Green Cross, GreenPlast 1 mL kit) was dissolved was added to the freeze-dried support to induce crosslinking, and then lyophilized to regenerate tissue. A support was prepared.
(3) 조직재생용 지지체의 표면 형태 관찰(3) Observation of surface shape of scaffold for tissue regeneration
상기에서 코팅층을 형성하기 전의 고분자 지지체(도 1의 좌측) 및 얻어진 조직재생용 지지체(도 1의 우측)의 형태를 관찰한 결과는 도 1과 같다. 상단은 광학현미경으로 측정한 사진이고, 하단은 주사전자현미경(Scanning electron microscope, SEM)을 통해 측정한 사진이다. 도 1로부터, 다공성 지지체의 구조틀(frame) 표면 상에 히아루론산/피브린 코팅층이 공극의 막힘 없이 고르게 형성되어 있음을 알 수 있다.The results of observing the shape of the polymer support (left side of FIG. 1) and the obtained tissue regeneration support (right side of FIG. 1) before forming the coating layer are as shown in FIG. 1. The upper part is a photograph measured by an optical microscope, and the lower part is a photograph measured by a scanning electron microscope (SEM). 1, it can be seen that the hyaluronic acid / fibrin coating layer is formed evenly on the surface of the frame of the porous support without clogging the pores.
실시예 2. 인간 지방유래 줄기세포가 도입된 조직재생용 지지체의 평가Example 2 Evaluation of Support for Tissue Regeneration Incorporating Human Adipose-Derived Stem Cells
(1) 인간 지방유래 줄기세포의 분리 및 배양(1) Isolation and Culture of Human Adipose-derived Stem Cells
인간 지방조직은 차의과학대학 부속병원의 윤리위원회의 허가 아래 동의를 받고 지방흡입술에 의해 얻었다. 오염된 혈액을 제거하기 위해, 얻어진 지방 조직을 인산완충식염수(PBS)(Sigma, St. Louis, MO)를 이용하여 3회 세척하였다. 이어서 지방조직을 0.2 w/v% 소혈청알부민 함유 PBS와 2 mg/mL 타입 II 콜라게네이즈(Sigma)로 45분 동안 37℃로 소화시켰다. 70 μm 필터로 여과하고, 여액을 원심분리한 후 부유하는 지방세포를 제거하였다. 분리된 지방조직 유래 줄기세포 (ASCs)을 100 U/mL의 페니실린, 100 μg/mL의 스트렙토마이신, 그리고 10%의 우태아 혈청(Invitrogen, USA)이 첨가된 Dulbecco's modified Eagle medium(DMEM, Gibco BRL,Gaithersburg, MD)에서 배양하였다. 3회 또는 4회째 계대 배양된 세포를 하기 단계에서 사용하였다.Human adipose tissue was obtained by liposuction with the consent of the Ethics Committee of the College of Medicine and Medical Sciences. To remove contaminated blood, the resulting adipose tissue was washed three times with phosphate buffered saline (PBS) (Sigma, St. Louis, MO). Adipose tissue was then digested with PBS containing 0.2 w / v% bovine albumin and 2 mg / mL type II collagenase (Sigma) at 37 ° C. for 45 minutes. Filtration was performed with a 70 μm filter, and the filtrate was centrifuged to remove floating adipocytes. Isolated adipose tissue-derived stem cells (ASCs) were prepared using Dulbecco's modified Eagle medium (DMEM, Gibco BRL) with 100 U / mL penicillin, 100 μg / mL streptomycin, and 10% fetal calf serum (Invitrogen, USA). (Gaithersburg, MD). Cells passaged 3 or 4 were used in the following steps.
(2) 지방유래 줄기세포가 도입된 조직재생용 지지체의 세포점착능 평가(2) Evaluation of Cell Adhesion of Tissue Regenerated Scaffold Incorporated with Adipose-derived Stem Cell
상기 (1)에서 인간 지방유래 줄기세포의 현탁액(1×106 cells/mL)[즉, 컴파트먼트 (c')]에 실시예 1의 (2)에서 얻어진 코팅층이 형성된 고분자 지지체[즉, 컴파트먼트 (a)]를 침지하고, 4시간 동안 37℃ 인큐베이터에 배양한 다음, PBS로 3회 세척하여 지지체에 부착되지 않은 세포를 제거하여 조직재생용 지지체의 세포점착능을 평가하였다. 비교를 위하여, 코팅층 형성되지 않은 고분자 지지체를 3일 동안 우혈청이 10% 포함된 세포 배양 배지에서 충분히 수화 시킨 후 상기 (1)에서 인간 지방유래 줄기세포의 현탁액(1×106 cells/mL)에 침지하고, 4시간 동안 37℃ 인큐베이터에 배양한 다음, PBS로 3회 세척하여 지지체에 부착되지 않은 세포를 제거하였다(비교예). In (1), a polymer support having a coating layer obtained in Example 2 (2) in suspension (1 × 10 6 cells / mL) (ie, compartment (c ′)) of human adipose derived stem cells [ie, Compartment (a)] was immersed, incubated in a 37 ° C. incubator for 4 hours, washed three times with PBS to remove cells not attached to the scaffold, and the cell adhesion capacity of the scaffold for tissue regeneration was evaluated. For comparison, the polymer support without coating layer was sufficiently hydrated in cell culture medium containing 10% of bovine serum for 3 days, and then the suspension of human adipose derived stem cells (1 × 10 6 cells / mL) in (1). Immersed in, incubated in a 37 ° C. incubator for 4 hours, then washed three times with PBS to remove cells that did not adhere to the support (Comparative Example).
상기에서 제작된 조직재생용 지지체 및 비교예의 지지체에 대하여 세포점착성을 다음과 같이 평가하였다. 즉, 각각의 지지체에 트립신을 가하여 지지체에 부착된 세포만을 수거한 다음, 수거된 세포의 개수를 세포측정기(Marienfeld GMbH & Co.KG, Germany)를 이용하여 계수하였다. 또한, 각각의 지지체를 FDA(fluorescein diacetate)로 염색한 후 공초점 형광현미경(Confocal Laser Scanning Microscope, CLSM)을 이용하여 세포의 부착 모습을 관찰하였다. 그 결과는 도 2와 같다. 도 2로부터 확인할 수 있는 바와 같이, 코팅층을 갖지 않은 고분자 지지체는 낮은 세포점착을 보인 반면, 본 발명에 따른 조직재생용 지지체는 높은 세포점착율을 나타냈다. Cell adhesion was evaluated for the support for tissue regeneration and the support of Comparative Example prepared above as follows. That is, trypsin was added to each support to collect only the cells attached to the support, and then the number of collected cells was counted using a cytometer (Marienfeld GMbH & Co.KG, Germany). In addition, each support was stained with a fluorescein diacetate (FDA) and observed for cell attachment using a confocal laser scanning microscope (CLSM). The result is shown in FIG. As can be seen from Figure 2, the polymer support having no coating layer showed a low cell adhesion, whereas the support for tissue regeneration according to the present invention showed a high cell adhesion rate.
실시예 3. 조직재생용 지지체로부터의 BMP-2 방출Example 3. BMP-2 Release from Support for Tissue Regeneration
실시예 1과 동일한 방법으로 제조한 BMP-2 (1 ㎍)가 도입된 조직재생용 지지체를 1.0 mL 37 ℃, PBS에서 BMP-2의 방출량을 5회에 걸쳐 면역효소측정법(ELISA)를 이용하여 측정하였다. ELISA 플레이트(NUNC, Polylabo, Strasbourg, France)는 포획 단원 항체(capture monoclonal antibody)로 도포시켰으며, 1 w/v % 소혈청알부민 (bovine serum albumin)과 5 w/v % 수크로오스로 1시간 동안 차단시켰다. 희석된 샘플을 ELISA 플레이트에 첨가한 후 결합된 BMP-2를 항-인간 BMP-2 폴리클론 항체를 이용하여 측정하였다. 이어서 스트렙타비딘-호스래디쉬 퍼옥시다제 복합체(streptavidin-conjugated horseradish peroxidase)를 플레이트에 가한 다음, 퍼옥시다제와 테트라메틸벤지딘을 가하고 20분간 반응시켰다. 효소반응을 산 용액을 추가하여 중지시킨 후 샘플의 흡광도를 ELISA plate reader(PowerWave X340, Bio-Tek Instruments, Inc., Winooski, VT)를 이용하여 450 nm에서 측정하였다. 그 결과는 도 3과 같다. 도 3의 결과로부터, 본 발명에 따른 조직재생용 지지체로부터 BMP-2가 3일 이상 지속적으로 방출됨을 알 수 있다.BMP-2 (1 μg) -introduced tissue regeneration support prepared in the same manner as in Example 1 was subjected to immunoassay (ELISA) five times for the release of BMP-2 in PBS at 1.0 mL 37 ° C and PBS. Measured. ELISA plates (NUNC, Polylabo, Strasbourg, France) were applied with a capture monoclonal antibody and blocked for 1 hour with 1 w / v% bovine serum albumin and 5 w / v% sucrose. I was. Diluted samples were added to ELISA plates and bound BMP-2 was measured using anti-human BMP-2 polyclonal antibody. Subsequently, streptavidin-horseradish peroxidase complex (streptavidin-conjugated horseradish peroxidase) was added to the plate, peroxidase and tetramethylbenzidine were added and reacted for 20 minutes. After stopping the enzyme reaction by adding an acid solution, the absorbance of the sample was measured at 450 nm using an ELISA plate reader (PowerWave X340, Bio-Tek Instruments, Inc., Winooski, VT). The result is shown in FIG. 3. 3, it can be seen that BMP-2 is continuously released from the scaffold for tissue regeneration according to the present invention for more than 3 days.
실시예 4. 생체외 (in vitro) 지방줄기세포의 골분화도 평가Example 4 Evaluation of Bone Differentiation of In Vitro Adipose Stem Cells
(1) BMP-2 및 지방유래 줄기세포가 도입된 조직재생용 지지체의 제작(1) Preparation of tissue regeneration scaffold into which BMP-2 and adipose derived stem cells were introduced
실시예 1과 동일한 방법으로 제조한 BMP-2 (1 ㎍)가 도입된 조직재생용 지지체 당 ASCs 약 5×104 cells를 로딩하였다. 상기 로딩은 실시예 2의 (1)에서 얻은 ASCs 약 5×104 cells을 DMEM 배지 32 μL에 분산시켜 얻어진 분산액을 상기 BMP-2 (1 ㎍)가 도입된 조직재생용 지지체에 뿌려주었다.About 5 × 10 4 cells of ASCs were loaded per BMP-2 (1 μg) -introduced tissue regeneration support prepared in the same manner as in Example 1. In the loading, the dispersion obtained by dispersing about 5 × 10 4 cells of ASCs obtained in Example 2 (1) in 32 μL of DMEM medium was sprayed onto the tissue regeneration support into which BMP-2 (1 μg) was introduced.
대조군으로 BMP-2가 로딩 안된 코팅층을 갖는 복합지지체(즉, 실시예 1의 (1)에서 제조된 코팅층을 갖는 지지체)(NO BMP-2)와 시험시작일에 BMP-2 1 ㎍를 배양 배지 안에 첨가한 코팅층을 갖는 지지체(즉, 실시예 1의 (1)에서 제조된 코팅층을 갖는 지지체)(BMP-2 addition day 0)를 설정하여 ASCs를 배양하였다. 배양 배지로 10% v/v FBS, 100 units/mL 페니실린과 0.1 mg/mL 스트렙토마이신을 첨가한 DMEM 배지를 사용하였고, 3일 마다 교환하였다.As a control, a composite support having a coating layer not loaded with BMP-2 (ie, a support having a coating layer prepared in Example (1)) (NO BMP-2) and 1 μg of BMP-2 at the start of the test were added to the culture medium. ASCs were cultured by setting a support (ie, a support having a coating layer prepared in (1) of Example 1) (BMP-2 addition day 0) having an added coating layer. DMEM medium added with 10% v / v FBS, 100 units / mL penicillin and 0.1 mg / mL streptomycin was used as the culture medium and exchanged every 3 days.
(2) 골분화도 평가(2) bone differentiation evaluation
실험시작일, 3, 7과 10일에 알칼리 포스파타제 활성을 ELISA법으로 측정하였다. 각각의 지지체(3, 7과 10일에 수거한 모든 그룹의 지지체)를 50 ㎕ 라이시스 버퍼(lysis buffer, Cell Culture Lysis Reagent 5X, Promega)에 첨가하고, 세포 용해물(lysate)을 10분간 13000 rpm에서 초원심분리(Micro 17R, Hanil Science Industrial, Seoul, Korea)하여 제거하였다. 각각의 샘플은 150 ㎕ p-니트로페닐포스페이트(pNPP, Sigma)를 첨가한 다음, 37℃에서 15분간 배양하였다. 0.2 N NaOH를 첨가하여 반응을 중지시키고, 각 샘플의 흡광도를 ELISA plate reader를 사용하여 405 nm에서 측정하였다. 그 결과는 도 4와 같다. 도 4의 결과로부터 알 수 있는 바와 같이, BMP-2가 존재하지 않는 경우(NO BMP-2), 전체 배양기간 동안 알칼리 포스파타제의 활성이 증가하지 않았다. BMP-2 addition on day 0의 경우, 배양시작 7일 동안 알칼리 포스파타제의 활성이 증가하였으나 그 후는 감소 하였다. 반면, BMP-2가 도입된 조직재생용 지지체의 경우에는 전체 배양기간 동안 알칼리 포스파타제의 활성이 지속적으로 증가하였다.Alkali phosphatase activity was measured by ELISA on the 3rd, 7th and 10th day of the experiment. Each scaffold (all scaffolds collected on days 3, 7 and 10) was added to 50 μl lysis buffer (Cell Culture Lysis Reagent 5X, Promega) and cell lysate was added 13000 for 10 minutes. It was removed by ultracentrifugation (Micro 17R, Hanil Science Industrial, Seoul, Korea) at rpm. Each sample was added with 150 μl p-nitrophenylphosphate (pNPP, Sigma) and then incubated at 37 ° C. for 15 minutes. The reaction was stopped by addition of 0.2 N NaOH and the absorbance of each sample was measured at 405 nm using an ELISA plate reader. The result is shown in FIG. 4. As can be seen from the results of FIG. 4, in the absence of BMP-2 (NO BMP-2), the activity of alkaline phosphatase did not increase during the entire culture period. In the case of BMP-2 addition on day 0, the activity of alkaline phosphatase was increased during 7 days of incubation, but then decreased. On the other hand, in the case of the tissue regeneration scaffold introduced with BMP-2, the activity of alkaline phosphatase continuously increased during the entire culture period.
실시예 5. 생체내(in vivo) 골조직재생 평가 Example 5 Evaluation of In Vivo Bone Tissue Regeneration
(1) BMP-2 및 지방유래 줄기세포가 도입된 조직재생용 지지체의 제작(1) Preparation of tissue regeneration scaffold into which BMP-2 and adipose derived stem cells were introduced
실시예 1과 동일한 방법으로 제조한 BMP-2 (1 ㎍)가 도입된 조직재생용 지지체 당 ASCs 약 1×106 cells를 로딩하였다. 상기 로딩은 실시예 2의 (1)에서 얻은 ASCs 약 5×104 cells을 DMEM 배지 32 μL에 분산시켜 얻어진 분산액을 상기 BMP-2 (1 ㎍)가 도입된 조직재생용 지지체에 뿌려준 다음 한 시간 후에 면역결핍쥐의 등에 이식하였다.About 1 × 10 6 cells of ASCs were loaded per BMP-2 (1 μg) -introduced tissue regeneration support prepared in the same manner as in Example 1. The loading was performed by dispersing about 5 × 10 4 cells of ASCs obtained in Example 2 (1) in 32 μL of DMEM medium, and then spraying the dispersion solution onto the tissue regeneration support into which BMP-2 (1 μg) was introduced. It was later implanted into the back of immunodeficient mice.
(2) 지지체의 이식(2) implantation of support
무흉선 암컷 마우스(BALB/c-nu, 7 weeks old, female, SLC, Tokyo, Japan)를 4개의 실험군으로 나누고(n=6), 실라진(xylazine) 1.15 mg/kg과 케타민 8 mg/kg으로 마취시켰다. 각각의 실험군은 다음과 같다: 제1군은 코팅층을 형성시키지 않은 고분자 지지체를 이식한 군이고(G1), 제2군은 히아루론산/피브린 코팅층(즉, 트롬빈을 가하여 가교화시킨 코팅층)을 형성시킨 고분자 지지체를 이식한 군이고(G2), 제3군은 히아루론산/피브린 코팅층(즉, 트롬빈을 가하여 가교화시킨 코팅층)을 형성시킨 고분자 지지체 및 외부에서 BMP-2를 별도로 이식한 군이고(G3), 제4군은 상기 (1)에서 제조한 조직재생용 지지체를 이식한 군이다(G4). 각각의 지지체는 무흉선쥐(BALB/c-nu)의 등쪽 피하에 이식하였다.Athymic female mice (BALB / c-nu, 7 weeks old, female, SLC, Tokyo, Japan) were divided into four experimental groups (n = 6), 1.15 mg / kg of silazine (xylazine) and 8 mg / kg of ketamine Anesthetized with. Each experimental group was as follows: The first group was a group implanted with a polymer support that did not form a coating layer (G1), and the second group formed a hyaluronic acid / fibrin coating layer (ie, a crosslinked coating layer with thrombin). Group 3 was implanted with a polymer support (G2), and group 3 was a group with a polymer support formed with a hyaluronic acid / fibrin coating layer (ie, a crosslinked crosslinked layer with thrombin) and a BMP-2 implanted from the outside (G3). , The fourth group is a group transplanted with the support for tissue regeneration prepared in the above (1) (G4). Each scaffold was implanted in the dorsal subcutaneous of athymic rats (BALB / c-nu).
(3) 조직염색을 통한 골조직재생평가(3) Evaluation of bone tissue regeneration through tissue staining
이식 8주 후에 실험동물들을 치사시켜 이식된 조직을 적출하여 파라핀에 포매하고(embedding), 4 ㎛의 두께로 절단한 뒤 Weber, F. E., 등의 방법[Biochem. Biophys Res. Commun. 286 (2001) 554-558]에 따라, 골드너-트리크롬(Goldner-Trichrome) 염색을 실시하였다. 골 재생 면적은 광학 현미경과 연결된 image analysis system(KS400, Zeiss, Munich, Germany)를 이용하여 분석하였고, 전체 횡단면에 대한 골 형성 면적비[(골 면적/전체 면적)×100%]로 정량화하였다.After 8 weeks of transplantation, experimental animals were killed and the transplanted tissues were extracted, embedded in paraffin, cut into 4 μm thicknesses, and then treated by Weber, F. E., et al. [Biochem. Biophys Res. Commun. 286 (2001) 554-558, Goldner-Trichrome staining was performed. The bone regeneration area was analyzed using an image analysis system (KS400, Zeiss, Munich, Germany) connected with an optical microscope and quantified by the ratio of bone formation area ((bone area / total area) × 100%) to the total cross section.
도 5는 적출된 조직의 단면을 골드너-트리크롬(Goldner-Trichrome) 염색 후 광학현미경으로 관찰한 사진이다. 여기서 녹색 부분과 B는 재생된 골 부위, 화살표는 지지체를 나타내며, 스케일 바는 100 ㎛이다. 도 5의 결과로부터, 제1군의 경우 거의 골이 재생되지 않았고, 제2군 및 제3군의 경우에는 어느 정도 골이 재생되었음을 알 수 있다. 이에 반해, 제4군의 경우 골 재생이 매우 우수하였다. 도 6은 제3군 및 제4군에서 지지체 이식 후 적출된 단면을 골드너-트리크롬(Goldner-Trichrome) 염색 후 광학현미경으로 관찰한 사진이다. 여기서 스케일 바는 30 ㎛이다. 도 6의 결과로부터, 제4군에서 분화된 조골세포(osteoblast, 검은 화살표) 와 골세포(osteocyte, 빨간 화살표)를 관찰할 수 있다. 도 7은 (골 면적/전체 면적)×100%에 의해 계산된 골 형성 면적비를 나타낸 그래프로, 별표(*)는 통계학적 유의성을 나타낸다(*p<0.05). 도 7의 결과로부터, 제1군의 경우 1.9±0.4%, 제2군 및 제3군의 경우 5.5±2.2와 7.8±1.7%의 골 형성 면적비를 각각 나타내었다. 이에 반해, 제4군의 경우 19.6±5.0%로, 본 발명에 따른 골 재생용 복합 지지체를 이식한 경우 골 재생이 매우 우수함을 알 수 있다.Figure 5 is a photograph of the cross-section of the extracted tissue observed with an optical microscope after Goldner-Trichrome staining. Where the green part and B represent the regenerated bone area, the arrow represents the support, and the scale bar is 100 μm. 5, it can be seen that the bones were hardly reproduced in the first group, and the bones were reproduced to some extent in the second and third groups. In contrast, bone regeneration was very good in the fourth group. Figure 6 is a photograph of the cross-section taken after the implantation in the third group and the fourth group was observed with an optical microscope after Goldner-Trichrome staining. The scale bar here is 30 μm. From the results of FIG. 6, osteoblasts (osteoblast, black arrow) and osteoblasts (osteocyte, red arrow) differentiated in the fourth group can be observed. 7 is a graph showing the bone formation area ratio calculated by (bone area / total area) × 100%, with an asterisk (*) indicating statistical significance (* p <0.05). From the results of FIG. 7, the bone formation area ratios of 1.9 ± 0.4% in the first group and 5.5 ± 2.2 and 7.8 ± 1.7% in the second and third groups, respectively. On the contrary, in the fourth group, 19.6 ± 5.0%, when the composite support for bone regeneration according to the present invention is implanted, bone regeneration is very excellent.
(4) 미네랄화된 골 조직 형성 평가(4) evaluation of mineralized bone tissue formation
미네랄화된 골 조직 형성을 확인하기 위해 이식된 지지체의 soft x-ray촬영과 칼슘의 양을 측정하였다. 침착된 칼슘의 양은 Jeon et al., Biomaterials 2007;28(17):2763-2771에 기술된 방법으로 측정하였다. 즉, 이식 8주 후 적출된 조직을 인산완충용액을 이용하여 부드럽게 세척한 후 0.6 N 염산을 첨가한 후 잘게 자르고 교반기에서 12시간 동안 교반하였다. 이후 이를 1,000 g에서 5분간 원심분리하였다. 상층액에 있는 칼슘의 양은 상용화 된 칼슘 정량 키트(Bioassays)를 사용하여 제조사의 지시에 따라 측정하였다. 도 8는 이식체에서의 칼슘의 양을 측정하여 나타낸 그래프로, 별표(*)는 통계학적 유의성을 나타낸다(*p<0.05). 도 8의 결과로부터, soft x-ray의 경우 Group 4에서 가장 밝은 빛을 나타내며, 이는 미네랄화된 골조직이 형성됐음을 알 수 있고, 칼슘 정량 결과도 제4군에서 23.7 ㎍/㎎ dry tissue로 가장 높게 나타났다.Soft x-rays of the implanted scaffolds and the amount of calcium were measured to confirm mineralized bone tissue formation. The amount of calcium deposited was measured by the method described in Jeon et al., Biomaterials 2007; 28 (17): 2763-2771. That is, after 8 weeks of transplantation, the tissues were gently washed with phosphate buffer solution, and then 0.6 N hydrochloric acid was added and then finely chopped and stirred in a stirrer for 12 hours. It was then centrifuged at 1,000 g for 5 minutes. The amount of calcium in the supernatant was measured according to the manufacturer's instructions using a commercially available calcium quantification kit (Bioassays). Figure 8 is a graph showing the measurement of the amount of calcium in the implant, asterisk (*) indicates statistical significance (* p <0.05). From the results of FIG. 8, the soft x-ray shows the brightest light in Group 4, which indicates that mineralized bone tissue was formed, and the calcium quantification result was also the highest in the fourth group with 23.7 μg / mg dry tissue. High.

Claims (43)

  1. 공극을 갖는 고분자 지지체의 구조틀 표면 상에 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층을 포함하는 조직재생용 구조체로서, (i) 상기 코팅층이 세포; 세포성장인자; 또는 세포 및 세포성장인자를 포함하고, (ii) 상기 코팅층이 트롬빈에 의해 가교화되어 있으며, 또한 (iii) 상기 구조체가 10 ㎛ ∼ 1,000 ㎛의 공극을 갖는, 조직재생용 구조체.A structure for tissue regeneration comprising a coating layer formed by coating hyaluronic acid or a salt thereof and fibrinogen on a surface of a structure of a polymer support having pores, the coating layer comprising: a cell; Cell growth factor; Or cells and cell growth factors, (ii) the coating layer is crosslinked by thrombin, and (iii) the structure has pores of 10 µm to 1,000 µm.
  2. 제1항에 있어서, 상기 고분자 지지체가 폴리락타이드, 폴리글리콜라이드, 폴리카프로락톤, 락타이드와 글리콜라이드와의 공중합체, 락타이드와 카프로락톤과의 공중합체, 및 글리콜라이드와 카프로락톤과의 공중합체으로 이루어진 군으로부터 1종 이상 선택된 생분해성 고분자로부터 형성된 것임을 특징으로 하는 조직재생용 구조체.The method of claim 1, wherein the polymer support is polylactide, polyglycolide, polycaprolactone, a copolymer of lactide and glycolide, a copolymer of lactide and caprolactone, and a glycolide and caprolactone Structure for regenerating tissue, characterized in that formed from at least one biodegradable polymer selected from the group consisting of copolymers.
  3. 제2항에 있어서, 상기 고분자 지지체가 D,L-락타이드와 글리콜라이드와의 공중합체 및 폴리카프로락톤의 혼합물로부터 형성된 것임을 특징으로 하는 조직재생용 구조체.The structure of claim 2, wherein the polymer support is formed from a copolymer of D, L-lactide with glycolide and a mixture of polycaprolactone.
  4. 제1항에 있어서, 상기 고분자 지지체가 10 ㎛ ∼ 1,000 ㎛ 범위의 공극을 갖는 것을 특징으로 하는 조직재생용 구조체.The structure of claim 1, wherein the polymer support has pores in the range of 10 μm to 1,000 μm.
  5. 제1항에 있어서, 상기 히아루론산 또는 그의 염의 중량평균분자량이 1,000 ∼ 4,000,000 달톤의 범위인 것을 특징으로 하는 조직재생용 구조체.The structure for tissue regeneration according to claim 1, wherein the weight average molecular weight of hyaluronic acid or a salt thereof is in the range of 1,000 to 4,000,000 Daltons.
  6. 제1항에 있어서, 상기 코팅층 중의 히아루론산 또는 그의 염의 함량이 10 ∼ 90 중량%의 범위인 것을 특징으로 하는 조직재생용 구조체.The structure for tissue regeneration according to claim 1, wherein the content of hyaluronic acid or a salt thereof in the coating layer is in the range of 10 to 90% by weight.
  7. 제1항에 있어서, 상기 코팅층 중의 피브리노오겐의 함량이 10 ∼ 90 중량%의 범위인 것을 특징으로 하는 조직재생용 구조체.The structure for tissue regeneration according to claim 1, wherein the content of fibrinogen in the coating layer is in the range of 10 to 90% by weight.
  8. 제1항에 있어서, 상기 코팅이 히아루론산 또는 그의 염 및 피브리노오겐을 함유하는 수용액 중에 상기 지지체를 침지한 다음, 상기 지지체를 상기 수용액으로부터 꺼내어 동결건조하는 과정을 적어도 1회 이상 수행되는 것을 특징으로 하는 조직재생용 구조체.The method of claim 1, wherein the coating is performed by immersing the support in an aqueous solution containing hyaluronic acid or a salt thereof and fibrinogen, and then removing the support from the aqueous solution and performing lyophilization. Structure for tissue regeneration.
  9. 제8항에 있어서, 상기 과정을 2회 내지 20회 반복 수행하는 것을 특징으로 하는 조직재생용 구조체.The structure for tissue regeneration according to claim 8, wherein the process is repeated 2 to 20 times.
  10. 제8항에 있어서, 상기 침지가 4∼50 ℃에서 수행되는 것을 특징으로 하는 조직재생용 구조체.The structure for tissue regeneration according to claim 8, wherein the dipping is performed at 4 to 50 ° C.
  11. 제8항에 있어서, 상기 침지 후 동결건조 전에, 상기 수용액으로부터 꺼낸 지지체를 약 25 ℃에서 건조하는 것을 추가로 포함하는 것을 특징으로 하는 조직재생용 구조체.The structure for tissue regeneration according to claim 8, further comprising drying the support taken out of the aqueous solution at about 25 ° C. before lyophilization after the immersion.
  12. 제8항에 있어서, 상기 히아루론산 또는 그의 염 및 피브리노오겐을 함유하는 수용액에 세포성장인자를 추가로 함유시킴으로써, 상기 세포성장인자가 상기 코팅층에 도입되는 것을 특징으로 하는 조직재생용 구조체.The structure for tissue regeneration according to claim 8, wherein the cell growth factor is introduced into the coating layer by further containing a cell growth factor in the aqueous solution containing hyaluronic acid or a salt thereof and fibrinogen.
  13. 제1항에 있어서, 세포성장인자를 함유하는 수성 용액(aqueous solution)을 상기 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층에 가한 후, 트롬빈 및 칼슘염을 함유하는 용액을 가하여 건조시킴으로써, 상기 세포성장인자가 상기 코팅층에 도입되는 것을 특징으로 하는 조직재생용 구조체.The method of claim 1, wherein the aqueous solution containing the cell growth factor is added to the coating layer formed by coating the hyaluronic acid or a salt thereof and fibrinogen, followed by drying by adding a solution containing thrombin and calcium salt. , The cell growth factor is introduced into the coating layer structure for tissue regeneration.
  14. 제1항에 있어서, 세포성장인자; 및 트롬빈과 칼슘염을 함유하는 수성 용액을 상기 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층에 가하여 건조시킴으로써, 상기 세포성장인자가 상기 코팅층에 도입되는 것을 특징으로 하는 조직재생용 구조체.According to claim 1, Cell growth factor; And drying the aqueous solution containing thrombin and calcium salt to the coating layer formed by coating the hyaluronic acid or its salt and fibrinogen, thereby introducing the cell growth factor into the coating layer.
  15. 제1항에 있어서, 트롬빈 및 칼슘염을 함유하는 용액을 상기 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층에 가하여 건조시킨 다음, 세포성장인자를 함유하는 수성 용액을 가함으로써, 상기 세포성장인자가 상기 코팅층에 도입되는 것을 특징으로 하는 조직재생용 구조체.The method of claim 1, wherein the solution containing thrombin and calcium salt is added to the coating layer formed by coating the hyaluronic acid or its salt and fibrinogen and dried, and then the aqueous solution containing cell growth factor is added to the cells. Structure for tissue regeneration, characterized in that the growth factor is introduced into the coating layer.
  16. 제12항 내지 제15항 중 어느 한 항에 있어서, 상기 건조가 동결건조에 의해 수행되는 것을 특징으로 하는 조직재생용 구조체.The structure for tissue regeneration according to any one of claims 12 to 15, wherein the drying is performed by lyophilization.
  17. 제12항 내지 제15항 중 어느 한 항에 있어서, 상기 수성 용액 중의 세포성장인자의 농도가 10 pg/mL ∼ 100 mg/mL의 범위인 것을 특징으로 하는 조직재생용 구조체.The structure for tissue regeneration according to any one of claims 12 to 15, wherein the concentration of cell growth factor in the aqueous solution is in the range of 10 pg / mL to 100 mg / mL.
  18. 제12항 내지 제15항 중 어느 한 항에 있어서, 얻어진 조직재생용 구조체의 코팅층 표면 상에, 세포를 함유하는 수성 분산액(aqueous dispersion)을 파종하는 단계를 추가로 포함으로써, 상기 세포가 상기 코팅층에 도입되는 것을 특징으로 하는 조직재생용 구조체.The method according to any one of claims 12 to 15, further comprising the step of seeding an aqueous dispersion containing cells on the surface of the coating layer of the structure for tissue regeneration obtained, wherein the cells are coated with the coating layer. Structure for tissue regeneration, characterized in that it is introduced into.
  19. 제18항에 있어서, 상기 수성 분산액 중의 세포의 농도가 103 ∼ 1010 cells/mL의 범위인 것을 특징으로 하는 조직재생용 구조체.19. The tissue regeneration construct according to claim 18, wherein the concentration of cells in the aqueous dispersion is in the range of 10 3 to 10 10 cells / mL.
  20. 제1항에 있어서, 트롬빈 및 칼슘염을 함유하는 용액을 상기 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층에 가하여 건조시킨 다음, 세포를 함유하는 수성 분산액(aqueous dispersion)을 가함으로써, 상기 세포가 상기 코팅층에 도입되는 것을 특징으로 하는 조직재생용 구조체.The solution of claim 1, wherein the solution containing thrombin and calcium salt is added to the coating layer formed by coating the hyaluronic acid or its salt and fibrinogen, followed by addition of an aqueous dispersion containing cells, Structure for regenerating tissue, characterized in that the cells are introduced into the coating layer.
  21. 제20항에 있어서, 상기 건조가 동결건조에 의해 수행되는 것을 특징으로 하는 조직재생용 구조체.The structure for tissue regeneration according to claim 20, wherein said drying is performed by lyophilization.
  22. 제20항에 있어서, 상기 수성 분산액 중의 세포의 농도가 103 ∼ 1010 cells/mL의 범위인 것을 특징으로 하는 조직재생용 구조체.The structure for tissue regeneration according to claim 20, wherein the concentration of cells in the aqueous dispersion is in the range of 10 3 to 10 10 cells / mL.
  23. 제1항에 있어서, 상기 세포가 조직세포, 성체줄기세포, 배아줄기세포, 또는 유도만능줄기세포(induced pluripotent stem cells)인 것을 특징으로 하는 조직재생용 구조체.The structure for tissue regeneration according to claim 1, wherein the cells are tissue cells, adult stem cells, embryonic stem cells, or induced pluripotent stem cells.
  24. 제1항에 있어서, 상기 세포성장인자가 BMP-2(bone morphogenetic protein 2), VEGF(vascular endothelial growth factor), EGF(epidermal growth factor), IGF(insulin-like growth factor), NGF(nerve growth factor), PDGF(platelet-derived growth factor), TGF-β(transforming growth factor-β), 및 bFGF(basic fibroblast growth factor)로 이루어진 군으로부터 1 종 이상 선택되는 것을 특징으로 하는 조직재생용 구조체.According to claim 1, wherein the cell growth factor BMP-2 (bone morphogenetic protein 2), VEGF (vascular endothelial growth factor), EGF (epidermal growth factor), IGF (insulin-like growth factor), NGF (nerve growth factor) ), Platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), and basic fibroblast growth factor (bFGF) is selected from the group consisting of one or more structures for tissue regeneration.
  25. 공극을 갖는 고분자 지지체의 구조틀 표면 상에 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층을 포함하는 구조체-컴파트먼트[컴파트먼트 (a)]; 및 A structure-compartment (compartment (a)) comprising a coating layer formed by coating hyaluronic acid or a salt thereof and fibrinogen on the surface of the structure of the polymer support having voids; And
    트롬빈 및 칼슘염을 포함하는 용액을 포함하는 트롬빈-컴파트먼트[컴파트먼트 (b)]를 포함하는, Comprising a thrombin-compartment [compartment (b)] comprising a solution comprising thrombin and calcium salt,
    트롬빈에 의해 가교화되고 또한 10 ㎛ ∼ 1,000 ㎛의 공극을 갖는 조직재생용 구조체 형성을 위한 키트.A kit for forming a structure for tissue regeneration crosslinked by thrombin and having a pore of 10 μm to 1,000 μm.
  26. 공극을 갖는 고분자 지지체의 구조틀 표면 상에 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층을 포함하는 구조체-컴파트먼트[컴파트먼트 (a)]; A structure-compartment (compartment (a)) comprising a coating layer formed by coating hyaluronic acid or a salt thereof and fibrinogen on the surface of the structure of the polymer support having voids;
    트롬빈 및 칼슘염을 포함하는 용액을 포함하는 트롬빈-컴파트먼트[컴파트먼트 (b)]; 및 Thrombin-compartment (compartment (b)) comprising a solution comprising thrombin and calcium salt; And
    세포 또는 세포성장인자의 제공을 위한 1종 이상의 수성 용액-컴파트먼트[컴파트먼트 (c)]를 포함하는, Comprising at least one aqueous solution-compartment [compartment (c)] for the provision of cells or cell growth factors,
    트롬빈에 의해 가교화되고 또한 10 ㎛ ∼ 1,000 ㎛의 공극을 갖는 조직재생용 구조체 형성을 위한 키트.A kit for forming a structure for tissue regeneration crosslinked by thrombin and having a pore of 10 μm to 1,000 μm.
  27. 공극을 갖는 고분자 지지체의 구조틀 표면 상에 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층을 포함하는 구조체-컴파트먼트[컴파트먼트 (a)]; A structure-compartment (compartment (a)) comprising a coating layer formed by coating hyaluronic acid or a salt thereof and fibrinogen on the surface of the structure of the polymer support having voids;
    세포성장인자; 및 트롬빈과 칼슘염을 함유하는 수성 용액을 포함하는 세포성장인자-트롬빈-완충액 컴파트먼트[컴파트먼트 (b')]; 및Cell growth factor; And a cell growth factor-thrombin-buffer compartment (compartment (b ')) comprising an aqueous solution containing thrombin and calcium salt; And
    선택적으로, 세포를 함유하는 세포-수성 용액-컴파트먼트[컴파트먼트 (c')]를 포함하는, Optionally, comprising a cell-aqueous solution-compartment containing the cells [compartment (c ')],
    트롬빈에 의해 가교화되고 또한 10 ㎛ ∼ 1,000 ㎛의 공극을 갖는 조직재생용 구조체 형성을 위한 키트.A kit for forming a structure for tissue regeneration crosslinked by thrombin and having a pore of 10 μm to 1,000 μm.
  28. 공극을 갖는 고분자 지지체의 구조틀 표면 상에 히아루론산 또는 그의 염 및 피브리노오겐이 코팅되어 형성된 코팅층을 포함하는 구조체-컴파트먼트[컴파트먼트 (a)]; A structure-compartment (compartment (a)) comprising a coating layer formed by coating hyaluronic acid or a salt thereof and fibrinogen on the surface of the structure of the polymer support having voids;
    트롬빈 및 칼슘염을 포함하는 용액을 포함하는 트롬빈-컴파트먼트[컴파트먼트 (b)]; 및 Thrombin-compartment (compartment (b)) comprising a solution comprising thrombin and calcium salt; And
    세포 또는 세포성장인자를 함유하는 1종 이상의 수성 용액-컴파트먼트[컴파트먼트 (c'')]를 포함하는, Comprising at least one aqueous solution-compartment containing the cell or cell growth factor [compartment (c '')],
    트롬빈에 의해 가교화되고 또한 10 ㎛ ∼ 1,000 ㎛의 공극을 갖는 조직재생용 구조체 형성을 위한 키트.A kit for forming a structure for tissue regeneration crosslinked by thrombin and having a pore of 10 μm to 1,000 μm.
  29. 제25항 내지 제28항 중 어느 항에 있어서, 상기 고분자 지지체가 폴리락타이드, 폴리글리콜라이드, 폴리카프로락톤, 락타이드와 글리콜라이드와의 공중합체, 락타이드와 카프로락톤과의 공중합체, 및 글리콜라이드와 카프로락톤과의 공중합체으로 이루어진 군으로부터 1종 이상 선택된 생분해성 고분자로부터 형성된 것임을 특징으로 하는 키트.29. The method according to any one of claims 25 to 28, wherein the polymeric support is polylactide, polyglycolide, polycaprolactone, a copolymer of lactide with glycolide, a copolymer of lactide with caprolactone, and And a biodegradable polymer selected from the group consisting of copolymers of glycolide and caprolactone.
  30. 제29항에 있어서, 상기 고분자 지지체가 D,L-락타이드와 글리콜라이드와의 공중합체 및 폴리카프로락톤의 혼합물로부터 형성된 것임을 특징으로 하는 키트.30. The kit of claim 29, wherein the polymeric support is formed from a mixture of polycaprolactone and a copolymer of D, L-lactide with glycolide.
  31. 제25항 내지 제28항 중 어느 항에 있어서, 상기 고분자 지지체가 10 ㎛ ∼ 1,000 ㎛ 범위의 공극을 갖는 것을 특징으로 하는 키트.29. The kit according to any one of claims 25 to 28, wherein said polymeric support has pores in the range of 10 microns to 1,000 microns.
  32. 제25항 내지 제28항 중 어느 항에 있어서, 상기 히아루론산 또는 그의 염의 중량평균분자량이 1,000 ∼ 4,000,000 달톤의 범위인 것을 특징으로 하는 키트.The kit according to any one of claims 25 to 28, wherein the weight average molecular weight of the hyaluronic acid or a salt thereof is in the range of 1,000 to 4,000,000 Daltons.
  33. 제25항 내지 제28항 중 어느 항에 있어서, 상기 코팅층 중의 히아루론산 또는 그의 염의 함량이 10 ∼ 90 중량%의 범위인 것을 특징으로 하는 키트.29. The kit according to any one of claims 25 to 28, wherein the content of hyaluronic acid or a salt thereof in the coating layer is in the range of 10 to 90% by weight.
  34. 제25항 내지 제28항 중 어느 항에 있어서, 상기 코팅층 중의 피브리노오겐의 함량이 10 ∼ 90 중량%의 범위인 것을 특징으로 하는 키트.29. The kit according to any one of claims 25 to 28, wherein the content of fibrinogen in the coating layer is in the range of 10 to 90% by weight.
  35. 제25항 내지 제28항 중 어느 항에 있어서, 상기 코팅이 히아루론산 또는 그의 염 및 피브리노오겐을 함유하는 수용액 중에 상기 지지체를 침지한 다음, 상기 지지체를 상기 수용액으로부터 꺼내어 동결건조하는 과정을 적어도 1회 이상 수행되는 것을 특징으로 하는 키트.29. The process according to any one of claims 25 to 28, wherein the coating is immersed in the aqueous solution containing hyaluronic acid or a salt thereof and fibrinogen, and then the support is removed from the aqueous solution and lyophilized. Kit, characterized in that carried out one or more times.
  36. 제35항에 있어서, 상기 과정을 2회 내지 20회 반복 수행하는 것을 특징으로 하는 키트.36. The kit of claim 35, wherein the process is repeated 2 to 20 times.
  37. 제35항에 있어서, 상기 침지가 4∼50 ℃에서 수행되는 것을 특징으로 하는 키트.36. The kit of claim 35, wherein said dipping is performed at 4-50 &lt; 0 &gt; C.
  38. 제35항에 있어서, 상기 침지 후 동결건조 전에, 상기 수용액으로부터 꺼낸 지지체를 약 25 ℃에서 건조하는 것을 추가로 포함하는 것을 특징으로 하는 키트.36. The kit of claim 35, further comprising drying the support taken out of the aqueous solution at about 25 [deg.] C. after the immersion and before lyophilization.
  39. 제25항 내지 제28항 중 어느 항에 있어서, 컴파트먼트 (b) 또는 컴파트먼트 (b') 중의 상기 트롬빈 및 칼슘염의 농도가 10 ∼ 90 중량%의 범위인 것을 특징으로 하는 키트.The kit according to any one of claims 25 to 28, wherein the concentration of the thrombin and calcium salt in the compartment (b) or the compartment (b ') is in the range of 10 to 90% by weight.
  40. 제27항 또는 제28항에 있어서, 컴파트먼트 (b') 또는 컴파트먼트 (c'') 중의 상기 세포성장인자의 농도가 10 pg/mL ∼ 100 mg/mL의 범위인 것을 특징으로 하는 키트.The concentration of the cell growth factor in the compartment (b ') or the compartment (c' ') is in the range of 10 pg / mL to 100 mg / mL. Kit.
  41. 제27항 또는 제28항에 있어서, 컴파트먼트 (c') 또는 컴파트먼트 (c'') 중의 상기 세포의 농도가 103 ∼ 1010 cells/mL의 범위인 것을 특징으로 하는 키트.29. The kit according to claim 27 or 28, wherein the concentration of said cells in compartment (c ') or compartment (c ") is in the range of 10 3 to 10 10 cells / mL.
  42. 제27항 또는 제28항에 있어서, 상기 세포가 조직세포, 성체줄기세포, 배아줄기세포, 또는 유도만능줄기세포(induced pluripotent stem cells)인 것을 특징으로 하는 키트. 29. The kit of claim 27 or 28, wherein said cells are tissue cells, adult stem cells, embryonic stem cells, or induced pluripotent stem cells.
  43. 제27항 또는 제28항에 있어서, 상기 세포성장인자가 BMP-2(bone morphogenetic protein 2), VEGF(vascular endothelial growth factor), EGF(epidermal growth factor), IGF(insulin-like growth factor), NGF(nerve growth factor), PDGF(platelet-derived growth factor), TGF-β(transforming growth factor-β), 및 bFGF(basic fibroblast growth factor)로 이루어진 군으로부터 1 종 이상 선택되는 것을 특징으로 하는 키트.The method according to claim 27 or 28, wherein the cell growth factor is BMP-2 (bone morphogenetic protein 2), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), NGF The kit is selected from the group consisting of (nerve growth factor), platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), and basic fibroblast growth factor (bFGF).
PCT/KR2010/008969 2009-12-30 2010-12-15 Construct for tissue reconstruction and kit for producing a construct for tissue reconstruction WO2011081328A2 (en)

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KR1020090133756A KR101141547B1 (en) 2009-12-30 2009-12-30 Construct for tissue-reconstruction comprising coating layer formed by coating hyaluronic acid or its salt and fibrinogen on surface of support frame

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Publication number Priority date Publication date Assignee Title
KR20010027979A (en) * 1999-09-17 2001-04-06 오석송 A guided tissue regeneration membrane
KR20010044624A (en) * 2001-03-12 2001-06-05 정재호 A process for preparing the scaffold and tissue engineered cartilage made from the scaffold
KR20030032420A (en) * 2001-10-18 2003-04-26 한국과학기술연구원 Porous Scaffold Made of Biodegradable Polymer for Reconstructing Damaged Ocular Tissue
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KR20010027979A (en) * 1999-09-17 2001-04-06 오석송 A guided tissue regeneration membrane
KR20010044624A (en) * 2001-03-12 2001-06-05 정재호 A process for preparing the scaffold and tissue engineered cartilage made from the scaffold
KR20030032420A (en) * 2001-10-18 2003-04-26 한국과학기술연구원 Porous Scaffold Made of Biodegradable Polymer for Reconstructing Damaged Ocular Tissue
KR20060091301A (en) * 2003-09-19 2006-08-18 각고호우징 게이오기주크 Composition for coating support for preparation of cell sheet, support for preparation of cell sheet and process for producing cell sheet
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