WO2023106746A1 - Composition for manufacturing skeletal muscle formation-promoting support, skeletal muscle formation-promoting support manufactured thereby, and bio-ink for 3d printing - Google Patents

Composition for manufacturing skeletal muscle formation-promoting support, skeletal muscle formation-promoting support manufactured thereby, and bio-ink for 3d printing Download PDF

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WO2023106746A1
WO2023106746A1 PCT/KR2022/019481 KR2022019481W WO2023106746A1 WO 2023106746 A1 WO2023106746 A1 WO 2023106746A1 KR 2022019481 W KR2022019481 W KR 2022019481W WO 2023106746 A1 WO2023106746 A1 WO 2023106746A1
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skeletal muscle
composition
muscle formation
support
mxene
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French (fr)
Korean (ko)
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한동욱
홍석원
이석현
전상헌
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부산대학교 산학협력단
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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
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/446—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with other specific inorganic fillers other than those covered by A61L27/443 or A61L27/46
    • 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
    • A61L27/14—Macromolecular materials
    • A61L27/26—Mixtures of macromolecular compounds
    • 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
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • 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
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3826—Muscle cells, e.g. smooth muscle cells
    • 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
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • 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
    • 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
    • A61L27/58—Materials at least partially resorbable by the body
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y70/00—Materials specially adapted for additive manufacturing
    • B33Y70/10—Composites of different types of material, e.g. mixtures of ceramics and polymers or mixtures of metals and biomaterials
    • 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
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/30—Materials or treatment for tissue regeneration for muscle reconstruction

Definitions

  • the present invention relates to a composition for preparing a support for promoting skeletal muscle formation, and more particularly, to a composition for preparing a support for promoting skeletal muscle formation, a support for promoting skeletal muscle formation prepared therefrom, and a bioink for 3D printing.
  • Tissue engineering is to collect necessary tissues from the patient's body, isolate cells from the tissue pieces, grow the separated cells to the required amount through culture, plant them on a porous nanofiber scaffold, incubate them in vitro for a certain period of time, and then incubate the hybrid cells. is to transplant the implanted nanofiber scaffold back into the human body. After transplantation, cells are supplied with oxygen and nutrients by the diffusion of bodily fluids until new blood vessels are formed in most tissues or organs. It is to apply a technique in which the nanofiber scaffold is formed and removed by decomposition in the meantime.
  • Such a polymer scaffold for tissue engineering provides a topographical environment in which cells grow without dying or losing their shape in the early stages of transplantation into the body, and biodegradable biopolymers are slowly decomposed to provide enough space for cells to grow, so that they can be used as transplanted cells in the future. It can be regenerated into a tissue that has the same form and function as the constructed natural tissue.
  • the polymer scaffold for tissue engineering should be capable of sufficiently adhering and proliferating cells in vitro.
  • porous sponge-type materials or fiber-type materials have been continuously studied.
  • myoblasts refer to a state before a muscle cell of a living organism differentiates into a muscle fiber.
  • Myoblasts unlike cells that have the potential to differentiate into other things, are mononuclear cells that are destined to become muscle fibers.
  • the present inventors prepared a nanofibrous scaffold that promotes the growth and differentiation of myoblasts using MXene nanoparticles for the first time in the world.
  • An object of the present invention is to provide a composition for preparing a scaffold for promoting skeletal muscle formation that promotes the growth and differentiation of myoblasts.
  • Another object of the present invention is to provide a support for promoting skeletal muscle formation prepared from the composition for preparing a support for promoting skeletal muscle formation.
  • Another object of the present invention is to provide a bioink for 3D printing comprising the support for promoting skeletal muscle formation.
  • One embodiment of the present invention for achieving the above object is a bio-polymer compound; and MXene nanoparticles.
  • the biopolymer compound is poly(glycolic acid) (PGA), poly-L-lactide (PLLA), poly ⁇ -caprolactone (poly ⁇ -caprolactone) composed of poly-L-lysine (PLL), polyethylene oxide, polyvinyl alcohol (PVA), polyaniline and polymethylmethacrylate (PMMA). It may be one type alone or a copolymer of two or more types selected from the group. More specifically, the biopolymer compound may be poly(L-lactide-co- ⁇ -caprolactone) (Poly(L-lactide-co- ⁇ -caprolactone; PLCL).
  • the MXene nanoparticles may include titanium-based MXene nanoparticles.
  • Another embodiment of the present invention for achieving the above object may provide a composition for preparing a support for promoting skeletal muscle formation further comprising a substrate.
  • the matrix is any one selected from the group consisting of collagen, gelatin, laminin, fibronectin, Arg-Gly-Asp (RGD) peptide, silk fibroin, albumin, chitosan, heparin, hyaluronic acid, starch, alginic acid, cellulose, and combinations thereof can
  • Another embodiment of the present invention for achieving the above object provides a support for promoting skeletal muscle formation prepared from the composition for preparing a support for promoting skeletal muscle formation.
  • Another embodiment of the present invention for achieving the above object provides a bioink for 3D printing including a support for promoting skeletal muscle formation.
  • a composition for preparing a scaffold for promoting skeletal muscle formation that promotes the growth and differentiation of myoblasts can be provided.
  • a nanofibrous scaffold providing a topography in which myoblasts can grow can be implemented using such a scaffold preparation composition.
  • the nanofiber scaffold as a bioink material for 3D printing, it is possible to expect rapid skeletal muscle regeneration effect by promoting the attachment, proliferation and differentiation of myoblasts.
  • Figure 2a is an immunofluorescence staining photograph of myoblasts cultured in a growth medium on day 3 with different concentrations of MXene nanoparticles.
  • Figure 2b is an immunofluorescence staining photograph of myoblasts cultured in differentiation medium on day 3 with different concentrations of MXene nanoparticles.
  • Figure 2c is an immunofluorescence staining photograph of myoblasts cultured in growth medium on day 7 with different concentrations of MXene nanoparticles.
  • Figure 2d is an immunofluorescence staining photograph of myoblasts cultured in differentiation medium on day 7 with different concentrations of MXene nanoparticles.
  • Figure 3a is a fluorescence image of myoblasts cultured in the random nanofiber scaffold according to Preparation Example 2 in the growth medium on day 5.
  • Figure 3b is a fluorescence image of myoblasts cultured in the random nanofiber scaffold according to Preparation Example 2 in the 10th day growth medium.
  • Figure 4a is a fluorescence image of myoblasts cultured on the aligned nanofiber scaffold according to Preparation Example 3 in the growth medium on day 3.
  • Figure 4b is a fluorescence image of myoblasts cultured on the aligned nanofiber scaffold according to Preparation Example 3 in the growth medium on day 7.
  • One embodiment of the present invention provides a composition for preparing a scaffold for promoting skeletal muscle formation including a biopolymer compound and MXene nanoparticles. According to one aspect of the present invention, it is possible to provide a composition for preparing a scaffold for promoting skeletal muscle formation that promotes the growth and differentiation of myoblasts.
  • a nanofibrous scaffold providing a topography in which myoblasts can grow can be implemented using such a scaffold preparation composition.
  • the composition for preparing a scaffold for promoting skeletal muscle formation according to the present invention includes a bio-polymer compound.
  • the biopolymer compound is a synthetic polymer, and may be a compound that has no immune rejection by satisfying biocompatibility and biodegradability.
  • the biopolymer compound according to the present invention is, for example, poly(glycolic acid) (PGA), poly-L-lactide (PLLA), poly ⁇ -caprolactone (poly ⁇ - caprolactone), poly-L-lysine (PLL), polyethylene oxide and polyvinyl alcohol (PVA), polyaniline and polymethylmethacrylate (PMMA) It may be one alone or two or more copolymers selected from the group consisting of, and specifically poly(L-lactide-co- ⁇ -caprolactone) (Poly(L-lactide-co- ⁇ -caprolactone; PLCL)
  • the technical spirit of the present invention is not limited thereto, and any high molecular compound that satisfies biocompatibility and biodegradability can be applied.
  • the weight average molecular weight of the biopolymer compound may be 400,000 to 800,000 g/mol, preferably 600,000 to 700,000 g/mol.
  • weight average molecular weight of the biopolymer compound exceeds the above range, the viscosity increases excessively, making it difficult to perform electrospinning to form the nanofiber support, and when it is below the above range, weaving may become difficult.
  • the biopolymer compound is poly(L-lactide-co- ⁇ -caprolactone) (Poly(L-lactide-co- ⁇ -caprolactone; PLCL), poly L-
  • the weight ratio of lactide and poly ⁇ -caprolactone may be 50:50 to 90:10, specifically 60:40 to 80:20, and more specifically It may be 70:30 to 80:20
  • the weight ratio of the poly L-lactide and the poly ⁇ -caprolactone is within the above numerical range, the biocompatibility and biodegradability are more excellent and immune rejection is not caused. may not be
  • the composition for preparing a scaffold for promoting skeletal muscle formation according to the present invention includes MXene nanoparticles.
  • the Mxene nanoparticle may be a growth factor that promotes the growth and differentiation of myoblasts on the nanofibrous scaffold.
  • the content of MXene nanoparticles according to the present invention may be 0.2 to 1.5 parts by weight, preferably 0.6 to 1.0 parts by weight, and more preferably 0.75 to 0.85 parts by weight based on 100 parts by weight of the biopolymer compound. .
  • the content of MXene nanoparticles may be 0.40 to 1.05% (w/v), preferably 0.60 to 0.85% (w/v) based on the total volume of the composition (polymer solution) for preparing the support for promoting skeletal muscle formation. /v), more preferably 0.70 to 0.75% (w / v).
  • the content of the MXene nanoparticles is within the above numerical range, the growth and differentiation rates of myoblasts on the nanofibrous scaffold may be fast.
  • MXene nanoparticles according to the present invention may include titanium-based MXene nanoparticles.
  • the titanium-based MXene nanoparticles may be, for example, any one selected from the group consisting of Ti 3 C 2 , Ti 2 C, Ti 3 CN, Ti 3 C 2 Tx, and combinations thereof.
  • T may be any one selected from the group consisting of OH, O, and F, and x may be 1 to 3.
  • Ti 3 C 2 Tx may be Ti 3 C 2 F 2 , Ti 3 C 2 (OH) 2 , Ti 3 C 2 O 2 , or the like.
  • titanium-based MXene nanoparticles have relatively low toxicity to myoblasts than MXene nanoparticles of other series.
  • composition for preparing a support for promoting skeletal muscle formation may further include a substrate.
  • the substrate may be a matrix for adherence of myoblasts.
  • the matrix is, for example, in the group consisting of collagen, gelatin, laminin, fibronectin, Arg-Gly-Asp (RGD) peptide, silk fibroin, albumin, chitosan, heparin, hyaluronic acid, starch, alginic acid, cellulose, and combinations thereof It may be any one selected, preferably collagen.
  • the molecular weight of the collagen may be, for example, 250 to 350 kDa.
  • the amount of the substrate according to the present invention may be 1 to 30 parts by weight, preferably 5 to 25 parts by weight, more preferably 10 to 20 parts by weight, based on 100 parts by weight of the biopolymer compound.
  • the content of the matrix is within the above numerical range, the adhesion of myoblasts to the matrix may be sufficiently increased.
  • the composition for preparing a support for promoting skeletal muscle formation according to the present invention may further include a solvent.
  • the solvent is a compound that dissolves the biopolymer compound and may be appropriately selected according to the type of the biopolymer compound.
  • the solvent is, for example, tetrahydrofuran, dimethylacetamide, dimethylformamide, chloroform, dimethylsulfoxide, butanol, isopropanol, hexafluoroisopropanol, isobutyl alcohol, tetrabutyl alcohol, acetic acid, 1,4-dioxane , toluene, ortho-xyrene, and dichloromethane, and may be a single solvent or a mixed solvent of two or more selected from the group consisting of.
  • the amount of the solvent may be defined as the amount remaining excluding the biopolymer compound based on the total content of the composition for preparing a support for promoting skeletal muscle formation.
  • the amount of the solvent may be defined as the amount remaining after excluding the sum of the amounts of the biopolymer compound and the substrate.
  • Another embodiment of the present invention provides a scaffold for promoting skeletal muscle formation prepared from a composition for preparing a scaffold for promoting skeletal muscle formation.
  • the scaffold for promoting skeletal muscle formation according to the present invention may be, for example, a scaffold with random or aligned nanofibers, preferably an aligned nanofiber scaffold.
  • the arrangement of the nanofibers may vary according to different electrospinning conditions. Since the aligned nanofiber scaffold has nanofibers arranged in an ordered manner rather than the random nanofiber scaffold, the growth and differentiation of myoblasts on the scaffold can be further promoted.
  • the scaffold for promoting skeletal muscle formation according to the present invention may be prepared by electrospinning a composition for preparing a scaffold for promoting skeletal muscle formation.
  • a conventional electrospinning apparatus in the art may be used.
  • a needle gauge may be 23 to 26 gauge, a voltage may be 14 to 18 kV, and a spinning distance may be 8 to 12 cm.
  • the average diameter of the nanofibers constituting the skeletal muscle formation promoting support may be, for example, 400 to 800 ⁇ m.
  • Another embodiment of the present invention provides a bio-ink for 3D printing including the support for promoting formation of skeletal muscle.
  • the bio-ink for 3D printing according to the present invention may be a material capable of producing a structure required by application to bio-printing technology. Through this, it is possible to expect rapid skeletal muscle regeneration effect by promoting attachment, proliferation and differentiation of myoblasts by using the skeletal muscle formation promoting support as a bioink material for 3D printing.
  • the bioink for 3D printing according to the present invention may include a biocompatible material.
  • the biocompatible material may be, for example, collagen, gelatin, polyethylene glycol diacrylate, or the like. If necessary, the bioink for 3D printing may further include a conventional growth factor.
  • bioink should provide physical properties for 3D processing and a biological environment for cells to perform their intended functions. In order to satisfy the bioink, it must have excellent cell compatibility, and when the printing process is prolonged, nutrients and oxygen necessary for survival of the cells in the cartridge must be properly supplied.
  • bioink must have physical properties required in the printing process, such as repeatability of 3D patterning, productivity, and nozzle clogging.
  • the 3D printing bioink according to the present invention may satisfy all of the above requirements.
  • - Myoblasts mouse-derived cells (C2C12; ATCC ® )
  • - MXene nanoparticles a mixture prepared by slowly adding Ti 3 AlC 2 powder (2 g, 11 Technology Co., Ltd) to a HF solution (20 mL, 50 wt %) in an oil bath at 50° C. for 48 hours Stir. After centrifugation at 3,500 rpm for 3 minutes in the stirred solution until the pH of the supernatant reached 6, the precipitate was washed several times with deionized water to obtain multi-layers of Ti 3 C 2 T x . A previously prepared multilayer of Ti 3 C 2 T x (2 g) was redispersed in deionized water (50 mL), then dimethyl sulfoxide (40 mL) was added and stirred for 24 hours.
  • the Ti 3 C 2 T x means Ti 3 C 2 F 2 .
  • Biopolymer compound PLCL polymerized with a weight average molecular weight of 600,000 g/mol and a weight ratio of poly L-lactide and poly ⁇ -caprolactone (poly L-lactide: poly ⁇ -caprolactone) of 75:25 ( poly(L-lactide-co- ⁇ -caprolactone))
  • Dulbecco's Modified Eagle's Medium (Welgene) supplemented with 2% by volume Horse Serum and 1% by volume Antibiotic-antimycotic solution (Antibiotic-antimycotic solution)
  • CCK-8 cell counting kit-8 assay was used. Specifically, after culturing the myoblasts (1.0 x 10 4 cell/well) according to the Preparation Preparation Example at 37° C. for 24 hours, the absorbance was measured at 450 nm using a micro plate reader.
  • 1 is a result of evaluating the toxicity of MXene nanoparticles to myoblasts. Specifically, compared to the control group (concentration of MXene nanoparticles: 0 ⁇ g/mL) according to the concentration of MXene nanoparticles, cell viability of myoblasts was analyzed.
  • the concentration of MXene nanoparticles increased, the viability of most myoblasts tended to decrease, but it was confirmed that no toxicity to myoblasts was observed when the concentration of MXene nanoparticles was less than 250 ⁇ g/mL. On the other hand, when the concentration of MXene nanoparticles exceeded 250 ⁇ g/mL to 500 ⁇ g/mL, it was confirmed that the viability of myoblasts decreased to 60% or less compared to the control group.
  • MXene nanoparticles according to the preparation preparation example are added to the growth medium and differentiation medium under the above conditions at each concentration Added a lot. Specifically, the MXene nanoparticles were added while varying the concentrations of 0, 5, 10, and 20 ⁇ g/mL, respectively.
  • the growth medium and differentiation medium were replaced at intervals of 2 to 3 days, and at the time of replacement, washing was carried out twice with DPBS (Dulbecco's phosphate-buffered saline). Then, they were cultured for 3 days and 7 days, and images were acquired through immunofluorescence staining.
  • DPBS Denbecco's phosphate-buffered saline
  • Figure 2a is an immunofluorescence staining photograph of myoblasts cultured in a growth medium on day 3 with different concentrations of MXene nanoparticles.
  • Figure 2b is an immunofluorescence staining photograph of myoblasts cultured in differentiation medium on day 3 with different concentrations of MXene nanoparticles.
  • Figure 2c is an immunofluorescence staining photograph of myoblasts cultured in growth medium on day 7 with different concentrations of MXene nanoparticles.
  • Figure 2d is an immunofluorescence staining photograph of myoblasts cultured in differentiation medium on day 7 with different concentrations of MXene nanoparticles.
  • MXene nanoparticles promote the differentiation of myoblasts into skeletal muscle.
  • a tissue engineering scaffold providing a topography environment was prepared by the following method. .
  • a polymer solution for electrospinning was prepared by mixing hexafluoroisopropanol (HFIP) (5 mL) and PLCL (0.25 g) according to the Preparation Preparation Example above. Specifically, the content of PLCL according to the Preparation Preparation Example is 5% (w/v) based on the total volume of the polymer solution.
  • HFIP hexafluoroisopropanol
  • Comparative Example 2 Unlike Comparative Example 1, a composition for preparing a scaffold for promoting skeletal muscle formation further comprising a matrix>
  • a polymer solution (composition for preparing a scaffold) was prepared in the same manner as in Comparative Example 1, but collagen according to the Preparation Preparation Example was additionally added. Specifically, the amount of collagen according to the Preparation Preparation Example is 0.5% (w/v) based on the total volume of the polymer solution.
  • Example 1 Composition for preparing a scaffold for promoting skeletal muscle formation containing MXene nanoparticles>
  • a polymer solution (composition for preparing a support) was prepared in the same manner as in Comparative Example 1, but MXene nanoparticles according to the Preparation Preparation Example were additionally added. Specifically, the content of MXene nanoparticles is 400 ⁇ g/mL.
  • Example 2 Unlike Example 1, a composition for preparing a scaffold for promoting skeletal muscle formation further comprising a substrate>
  • a polymer solution (composition for preparing a support) was prepared in the same manner as in Comparative Example 2, but MXene nanoparticles according to the Preparation Preparation Example were additionally added. Specifically, the content of MXene nanoparticles is 400 ⁇ g/mL.
  • a random nanofiber scaffold was prepared by electrospinning the polymer solution according to Preparation Example 1 under the conditions shown in Table 1 below (RPM condition: 20 RPM).
  • Myoblasts were cultured as follows using the random nanofiber scaffold according to Preparation Example 2.
  • Figure 3a is a fluorescence image of myoblasts cultured in the random nanofiber scaffold according to Preparation Example 2 in the growth medium on day 5.
  • Figure 3b is a fluorescence image of myoblasts cultured in the random nanofiber scaffold according to Preparation Example 2 in the 10th day growth medium.
  • MHC means myosin heavy chain.
  • myoblasts were not generally differentiated on the random nanofiber scaffold for each sample on day 5, but differentiation of myoblasts was confirmed in the sample to which MXene nanoparticles were added on day 10.
  • the present inventors were able to confirm for the first time in the world that the random nanofiber scaffold containing MXene nanoparticles prepared through electrospinning promotes the differentiation of myoblasts into skeletal muscle.
  • Aligned nanofiber scaffolds were prepared by electrospinning the polymer solution according to Preparation Example 1 under the conditions shown in Table 2 below.
  • Myoblasts were cultured as follows using the aligned nanofiber scaffold according to Preparation Example 3.
  • Figure 4a is a fluorescence image of myoblasts cultured on the aligned nanofiber scaffold according to Preparation Example 3 in the growth medium on day 3.
  • Figure 4b is a fluorescence image of myoblasts cultured on the aligned nanofiber scaffold according to Preparation Example 3 in the growth medium on day 7.
  • myoblasts were not generally differentiated on the nanofiber scaffolds aligned for each sample on day 3, but differentiation of myoblasts was confirmed on the scaffolds to which MXene nanoparticles were added on day 7.
  • the present inventors have confirmed that the aligned nanofibrous scaffold containing MXene nanoparticles prepared through electrospinning promotes the differentiation of myoblasts into skeletal muscle more effectively than the random nanofibrous scaffold.

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Abstract

Provided is a composition for manufacturing a skeletal muscle formation-promoting support, which promotes the growth and differentiation of myoblasts. Provided in one embodiment of the present invention is a composition for manufacturing a skeletal muscle formation-promoting support, comprising a biopolymer compound and Mxene nanoparticles.

Description

골격근 형성 촉진 지지체 제조용 조성물, 이로부터 제조된 골격근 형성 촉진 지지체 및 3D 프린팅용 바이오잉크Composition for preparing scaffold for promoting skeletal muscle formation, scaffold for promoting skeletal muscle formation prepared therefrom, and bioink for 3D printing
본 발명은 골격근 형성 촉진 지지체 제조용 조성물에 관한 것으로, 보다 구체적으로 골격근 형성 촉진 지지체 제조용 조성물, 이로부터 제조된 골격근 형성 촉진 지지체 및 3D 프린팅용 바이오잉크에 관한 것이다.The present invention relates to a composition for preparing a support for promoting skeletal muscle formation, and more particularly, to a composition for preparing a support for promoting skeletal muscle formation, a support for promoting skeletal muscle formation prepared therefrom, and a bioink for 3D printing.
최근 생명공학 분야 중에서도 조직의 치료 및 재생을 위한 조직공학(tissue engineering) 분야가 발달하고 있다. 조직공학이란 환자의 몸에서 필요한 조직을 채취하고 그 조직편으로부터 세포를 분리한 다음 분리된 세포의 배양을 통하여 필요한 양만큼 증식시키고 다공성을 가지는 나노섬유 지지체에 심어 일정기간 체외 배양한 뒤 이 하이브리드형 세포가 이식된 나노섬유 지지체를 다시 인체 내에 이식하는 것이다. 이식 후 세포들은 대부분의 조직이나 장기의 경우 신생 혈관이 형성될 때까지는 체액의 확산에 의해 산소와 영양분을 공급받다가 인체 내의 혈관이 들어와 혈액의 공급이 이루어지면 세포들이 증식 분화하여 새로운 조직 및 장기를 형성하고 나노섬유 지지체는 그동안 분해되어 제거되는 기법을 응용하는 것이다.BACKGROUND OF THE INVENTION Recently, among the fields of biotechnology, the field of tissue engineering for the treatment and regeneration of tissues is developing. Tissue engineering is to collect necessary tissues from the patient's body, isolate cells from the tissue pieces, grow the separated cells to the required amount through culture, plant them on a porous nanofiber scaffold, incubate them in vitro for a certain period of time, and then incubate the hybrid cells. is to transplant the implanted nanofiber scaffold back into the human body. After transplantation, cells are supplied with oxygen and nutrients by the diffusion of bodily fluids until new blood vessels are formed in most tissues or organs. It is to apply a technique in which the nanofiber scaffold is formed and removed by decomposition in the meantime.
따라서 이러한 조직공학 연구를 위해서는 우선 생체 조직과 유사한 생분해성 고분자 지지체를 제조하는 일이 중요하다. 이러한 조직공학용 지지체의 재료의 주된 요건은 생분해성과 생체적합성의 조건을 만족하여야 하며 면역거부반응이 없어야 한다. Therefore, for such tissue engineering research, it is important to first prepare a biodegradable polymer scaffold similar to living tissue. The main requirements for the material of such a scaffold for tissue engineering are to satisfy the conditions of biodegradability and biocompatibility and to have no immune rejection.
이러한 조직공학용 고분자 지지체는 세포들이 체내에 이식된 초기에 사멸되거나 형태를 잃지 않고 자라날 지형학적 환경을 마련해 주게 되고 생분해성 바이오 고분자가 천천히 분해되어 세포가 자라날 공간을 충분히 내주게 됨으로써, 향후 이식세포로 구성된 자연조직과 동일한 형태와 기능을 지닌 조직으로 재생될 수 있다. 또한, 상기 조직공학용 고분자 지지체는 체외에서 세포가 충분히 접착되고 증식될 수 있어야 한다. 이러한 형태학적인 유사함을 가지는 지지체로 다공성을 지닌 스펀지 형태의 재료나 섬유 형태의 재료가 지속적으로 연구되고 있다.Such a polymer scaffold for tissue engineering provides a topographical environment in which cells grow without dying or losing their shape in the early stages of transplantation into the body, and biodegradable biopolymers are slowly decomposed to provide enough space for cells to grow, so that they can be used as transplanted cells in the future. It can be regenerated into a tissue that has the same form and function as the constructed natural tissue. In addition, the polymer scaffold for tissue engineering should be capable of sufficiently adhering and proliferating cells in vitro. As a support having such morphological similarities, porous sponge-type materials or fiber-type materials have been continuously studied.
한편, 근아세포는 생물의 한 근세포가 근육 섬유로 분화하기 전의 상태를 의미한다. 근아세포는 다른 것으로 분화될 가능성이 있는 세포들과 달리, 근육 섬유가 될 것이 정해져 있는 단핵세포이다. On the other hand, myoblasts refer to a state before a muscle cell of a living organism differentiates into a muscle fiber. Myoblasts, unlike cells that have the potential to differentiate into other things, are mononuclear cells that are destined to become muscle fibers.
하기 비특허문헌(MXene Composite Nanofibers for Cell Culture and Tissue Engineering, ACS Applied Bio Materials, 2020 3 (4), 2125-2131)은 인간의 골수유래 중간 엽 줄기세포(bone marrow-mesenchymal stem cells; hBM-MSCs)의 활성을 높이고 생체호환성을 개선하기 위해, 맥신 나노입자와 PLLA(Poly-L-lactic acid)-PHA(poly(hydroxyalkanoate))를 혼합한 조성물을 전기방사하여 나노섬유를 제조하는 기술사상을 구체적으로 제시하고 있다. 하지만, 상기 비특허문헌은 골격근을 이루는 근아세포의 성장 및 분화를 촉진하는 나노섬유 지지체를 전혀 개시하지 못하고 있고, 폴리(L-락타이드-코-ε-카프로락톤)(Poly(L-lactide-co-ε-caprolactone; PLCL)를 전혀 개시하지 못하고 있다.The following non-patent document (MXene Composite Nanofibers for Cell Culture and Tissue Engineering, ACS Applied Bio Materials, 2020 3 (4), 2125-2131 ) describes human bone marrow-mesenchymal stem cells (hBM-MSCs). ) In order to increase the activity and improve biocompatibility, the technical idea of manufacturing nanofibers by electrospinning a composition in which MXene nanoparticles and PLLA (Poly-L-lactic acid) -PHA (poly (hydroxyalkanoate)) are mixed is specified. is presented as However, the above non-patent literature does not disclose a nanofiber scaffold that promotes the growth and differentiation of myoblasts constituting skeletal muscle at all, and poly(L-lactide-co-ε-caprolactone) (Poly(L-lactide- co-ε-caprolactone; PLCL) was not initiated at all.
본 발명자들은 끊임없는 연구 끝에, 세계 최초로 맥신 나노입자를 이용하여 근아세포의 성장과 분화를 촉진하는 나노섬유 지지체를 제조하였다.After endless research, the present inventors prepared a nanofibrous scaffold that promotes the growth and differentiation of myoblasts using MXene nanoparticles for the first time in the world.
본 발명의 목적은, 근아세포의 성장 및 분화를 촉진하는 골격근 형성 촉진 지지체 제조용 조성물을 제공하는 것이다.An object of the present invention is to provide a composition for preparing a scaffold for promoting skeletal muscle formation that promotes the growth and differentiation of myoblasts.
본 발명의 다른 목적은, 상기 골격근 형성 촉진 지지체 제조용 조성물로 제조된 골격근 형성 촉진 지지체를 제공하는 것이다.Another object of the present invention is to provide a support for promoting skeletal muscle formation prepared from the composition for preparing a support for promoting skeletal muscle formation.
본 발명의 또 다른 목적은, 상기 골격근 형성 촉진 지지체를 포함하는 3D 프린팅용 바이오잉크를 제공하는 것이다.Another object of the present invention is to provide a bioink for 3D printing comprising the support for promoting skeletal muscle formation.
본 발명의 목적들은 이상에서 언급한 목적으로 제한되지 않으며, 언급되지 않은 본 발명의 다른 목적 및 장점들은 하기의 설명에 의해서 이해될 수 있고, 본 발명의 실시예에 의해 보다 분명하게 이해될 것이다. 또한, 본 발명의 목적 및 장점들은 청구범위에 나타낸 수단 및 그 조합에 의해 실현될 수 있음을 쉽게 알 수 있을 것이다.The objects of the present invention are not limited to the above-mentioned objects, and other objects and advantages of the present invention not mentioned above can be understood by the following description and will be more clearly understood by the examples of the present invention. It will also be readily apparent that the objects and advantages of the present invention may be realized by means of the instrumentalities and combinations indicated in the claims.
상기 목적을 달성하기 위한 본 발명의 일 실시예는 바이오 고분자 화합물; 및 맥신 나노입자(Mxene nanoparticle)를 포함하는 골격근 형성 촉진 지지체 제조용 조성물을 제공한다.One embodiment of the present invention for achieving the above object is a bio-polymer compound; and MXene nanoparticles.
상세하게는, 상기 바이오 고분자 화합물은, 폴리글리콜릭산(poly(glycolic acid); PGA), 폴리-L-락타이드(poly-L-lactide; PLLA), 폴리 ε-카프로락톤(poly ε-caprolactone), 폴리-L-라이신(poly-L-lysine; PLL), 폴리에틸렌 옥사이드(polyethylene oxide), 폴리비닐알코올(polyvinyl alcohol; PVA), 폴리아닐린(polyaniline) 및 폴리메타크릴산메틸(polymethylmethacrylate, PMMA)로 이루어진 군에서 선택된 1종 단독 또는 2종 이상의 공중합체일 수 있다. 더욱 상세하게는 상기 바이오 고분자 화합물은, 폴리(L-락타이드-코-ε-카프로락톤)(Poly(L-lactide-co-ε-caprolactone; PLCL)일 수 있다.Specifically, the biopolymer compound is poly(glycolic acid) (PGA), poly-L-lactide (PLLA), poly ε-caprolactone (poly ε-caprolactone) composed of poly-L-lysine (PLL), polyethylene oxide, polyvinyl alcohol (PVA), polyaniline and polymethylmethacrylate (PMMA). It may be one type alone or a copolymer of two or more types selected from the group. More specifically, the biopolymer compound may be poly(L-lactide-co-ε-caprolactone) (Poly(L-lactide-co-ε-caprolactone; PLCL).
상기 맥신 나노입자는, 타이타늄 계열 맥신 나노입자를 포함할 수 있다. The MXene nanoparticles may include titanium-based MXene nanoparticles.
상기 목적을 달성하기 위한 본 발명의 또 다른 실시예는 기질을 더 포함하는 골격근 형성 촉진 지지체 제조용 조성물을 제공할 수 있다.Another embodiment of the present invention for achieving the above object may provide a composition for preparing a support for promoting skeletal muscle formation further comprising a substrate.
상기 기질은 콜라겐, 젤라틴, 라미닌, 피브로넥틴, Arg-Gly-Asp(RGD) 펩타이드, 실크피브로인, 알부민, 키토산, 헤파린, 히알루론산, 녹말, 알긴산, 셀룰로오스 및 이들의 조합으로 이루어진 군에 선택된 어느 하나일 수 있다.The matrix is any one selected from the group consisting of collagen, gelatin, laminin, fibronectin, Arg-Gly-Asp (RGD) peptide, silk fibroin, albumin, chitosan, heparin, hyaluronic acid, starch, alginic acid, cellulose, and combinations thereof can
상기 목적을 달성하기 위한 본 발명의 또 다른 실시예는 상기 골격근 형성 촉진 지지체 제조용 조성물로 제조된 골격근 형성 촉진 지지체를 제공한다.Another embodiment of the present invention for achieving the above object provides a support for promoting skeletal muscle formation prepared from the composition for preparing a support for promoting skeletal muscle formation.
상기 목적을 달성하기 위한 본 발명의 또 다른 실시예는 골격근 형성 촉진 지지체를 포함하는 3D 프린팅용 바이오 잉크를 제공한다.Another embodiment of the present invention for achieving the above object provides a bioink for 3D printing including a support for promoting skeletal muscle formation.
상기 과제의 해결 수단은, 본 발명의 특징을 모두 열거한 것은 아니다. 본 발명의 다양한 특징과 그에 따른 장점과 효과는 아래의 구체적인 실시예를 참조하여 보다 상세하게 이해될 수 있을 것이다.The solution to the above problems does not enumerate all the features of the present invention. Various features of the present invention and the advantages and effects thereof will be understood in more detail with reference to the following specific examples.
본 발명의 일 실시예에 따르면, 근아세포의 성장 및 분화를 촉진하는 골격근 형성 촉진 지지체 제조용 조성물을 제공할 수 있다. 이러한 지지체 제조용 조성물을 이용하여 근아세포가 자라날 수 있는 지형학적 환경(topography)을 제공하는 나노섬유 지지체를 구현할 수 있다.According to one embodiment of the present invention, a composition for preparing a scaffold for promoting skeletal muscle formation that promotes the growth and differentiation of myoblasts can be provided. A nanofibrous scaffold providing a topography in which myoblasts can grow can be implemented using such a scaffold preparation composition.
본 발명의 다른 실시예에 따르면, 상기 나노섬유 지지체를 3D 프린팅용 바이오잉크 소재로 이용하여 근아세포의 부착, 증식 및 분화를 촉진하여 빠른 골격근 재생 효과를 기대할 수 있다.According to another embodiment of the present invention, by using the nanofiber scaffold as a bioink material for 3D printing, it is possible to expect rapid skeletal muscle regeneration effect by promoting the attachment, proliferation and differentiation of myoblasts.
상술한 효과와 더불어 본 발명의 구체적인 효과는 이하 발명을 실시하기 위한 구체적인 내용을 설명하면서 함께 기술한다.In addition to the above effects, specific effects of the present invention will be described together while explaining specific details for carrying out the present invention.
도 1은 근아세포에 대한 맥신 나노입자의 독성 평가를 나타낸 결과이다.1 is a result of evaluating the toxicity of MXene nanoparticles to myoblasts.
도 2a는 맥신 나노입자의 농도를 달리하여 3일차 성장배지에서 배양한 근아세포의 면역형광염색 사진이다. Figure 2a is an immunofluorescence staining photograph of myoblasts cultured in a growth medium on day 3 with different concentrations of MXene nanoparticles.
도 2b는 맥신 나노입자의 농도를 달리하여 3일차 분화배지에서 배양한 근아세포의 면역형광염색 사진이다. Figure 2b is an immunofluorescence staining photograph of myoblasts cultured in differentiation medium on day 3 with different concentrations of MXene nanoparticles.
도 2c는 맥신 나노입자의 농도를 달리하여 7일차 성장배지에서 배양한 근아세포의 면역형광염색 사진이다. Figure 2c is an immunofluorescence staining photograph of myoblasts cultured in growth medium on day 7 with different concentrations of MXene nanoparticles.
도 2d는 맥신 나노입자의 농도를 달리하여 7일차 분화배지에서 배양한 근아세포의 면역형광염색 사진이다. Figure 2d is an immunofluorescence staining photograph of myoblasts cultured in differentiation medium on day 7 with different concentrations of MXene nanoparticles.
도 3a는 5일차 성장배지 내 제조예 2에 따른 랜덤 나노섬유 지지체에서 배양된 근아세포의 형광 이미지이다. Figure 3a is a fluorescence image of myoblasts cultured in the random nanofiber scaffold according to Preparation Example 2 in the growth medium on day 5.
도 3b는 10일차 성장배지 내 제조예 2에 따른 랜덤 나노섬유 지지체에서 배양된 근아세포의 형광 이미지이다.Figure 3b is a fluorescence image of myoblasts cultured in the random nanofiber scaffold according to Preparation Example 2 in the 10th day growth medium.
도 4a는 3일차 성장배지 내 제조예 3에 따른 정렬 나노섬유 지지체에서 배양된 근아세포의 형광 이미지이다.Figure 4a is a fluorescence image of myoblasts cultured on the aligned nanofiber scaffold according to Preparation Example 3 in the growth medium on day 3.
도 4b는 7일차 성장배지 내 제조예 3에 따른 정렬 나노섬유 지지체에서 배양된 근아세포의 형광 이미지이다.Figure 4b is a fluorescence image of myoblasts cultured on the aligned nanofiber scaffold according to Preparation Example 3 in the growth medium on day 7.
이하, 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있도록 본 발명의 각 구성을 보다 상세히 설명하나, 이는 하나의 예시에 불과할 뿐, 본 발명의 권리범위가 다음 내용에 의해 제한되지 아니한다.Hereinafter, each configuration of the present invention will be described in more detail so that those skilled in the art can easily practice it, but this is only one example, and the scope of the present invention is Not limited.
본 발명의 일 실시예는 바이오 고분자 화합물 및 맥신 나노입자(Mxene nanoparticle)를 포함하는 골격근 형성 촉진 지지체 제조용 조성물을 제공한다. 본 발명의 일 측면에 따르면, 근아세포의 성장 및 분화를 촉진하는 골격근 형성 촉진 지지체 제조용 조성물을 제공할 수 있다. 이러한 지지체 제조용 조성물을 이용하여 근아세포가 자라날 수 있는 지형학적 환경(topography)을 제공하는 나노섬유 지지체를 구현할 수 있다.One embodiment of the present invention provides a composition for preparing a scaffold for promoting skeletal muscle formation including a biopolymer compound and MXene nanoparticles. According to one aspect of the present invention, it is possible to provide a composition for preparing a scaffold for promoting skeletal muscle formation that promotes the growth and differentiation of myoblasts. A nanofibrous scaffold providing a topography in which myoblasts can grow can be implemented using such a scaffold preparation composition.
이하에서는, 본 발명의 구성을 보다 상세히 설명한다.Hereinafter, the configuration of the present invention will be described in more detail.
1. 골격근 형성 촉진 지지체 제조용 조성물1. Composition for preparing a support for promoting skeletal muscle formation
본 발명에 따른 골격근 형성 촉진 지지체 제조용 조성물은 바이오 고분자 화합물을 포함한다. 상기 바이오 고분자 화합물은 합성 고분자로, 생체적합성 및 생분해성을 만족하여 면역 거부 반응이 없는 화합물일 수 있다.The composition for preparing a scaffold for promoting skeletal muscle formation according to the present invention includes a bio-polymer compound. The biopolymer compound is a synthetic polymer, and may be a compound that has no immune rejection by satisfying biocompatibility and biodegradability.
본 발명에 따른 바이오 고분자 화합물은 예를 들어, 폴리글리콜릭산(poly(glycolic acid); PGA), 폴리-L-락타이드(poly-L-lactide; PLLA), 폴리 ε-카프로락톤(poly ε-caprolactone), 폴리-L-라이신(poly-L-lysine; PLL), 폴리에틸렌 옥사이드(polyethylene oxide) 및 폴리비닐알코올(polyvinyl alcohol; PVA), 폴리아닐린(polyaniline) 및 폴리메타크릴산메틸(polymethylmethacrylate, PMMA)로 이루어진 군에서 선택된 1종 단독 또는 2종 이상의 공중합체일 수 있고, 구체적으로 폴리(L-락타이드-코-ε-카프로락톤)(Poly(L-lactide-co-ε-caprolactone; PLCL)일 수 있다. 다만 본 발명의 기술사상이 이에 제한되는 것은 아니고, 생체적합성 및 생분해성을 만족하는 고분자 화합물이면 모두 적용될 수 있다.The biopolymer compound according to the present invention is, for example, poly(glycolic acid) (PGA), poly-L-lactide (PLLA), poly ε-caprolactone (poly ε- caprolactone), poly-L-lysine (PLL), polyethylene oxide and polyvinyl alcohol (PVA), polyaniline and polymethylmethacrylate (PMMA) It may be one alone or two or more copolymers selected from the group consisting of, and specifically poly(L-lactide-co-ε-caprolactone) (Poly(L-lactide-co-ε-caprolactone; PLCL) However, the technical spirit of the present invention is not limited thereto, and any high molecular compound that satisfies biocompatibility and biodegradability can be applied.
상기 바이오 고분자 화합물의 중량평균분자량은 400,000 내지 800,000 g/mol일 수 있고, 바람직하게는 600,000 내지 700,000 g/mol 일 수 있다. 상기 바이오 고분자 화합물의 중량평균분자량이 상기 수치 범위를 초과할 경우 점도가 지나치게 상승하여 나노섬유 지지체를 형성하기 위한 전기방사가 어려워질 수 있고, 상기 수치 범위 미만일 경우 직조가 어려워질 수 있다.The weight average molecular weight of the biopolymer compound may be 400,000 to 800,000 g/mol, preferably 600,000 to 700,000 g/mol. When the weight average molecular weight of the biopolymer compound exceeds the above range, the viscosity increases excessively, making it difficult to perform electrospinning to form the nanofiber support, and when it is below the above range, weaving may become difficult.
본 발명의 또 다른 실시예에 따르면, 상기 바이오 고분자 화합물이 폴리(L-락타이드-코-ε-카프로락톤)(Poly(L-lactide-co-ε-caprolactone; PLCL)일 경우, 폴리 L-락타이드 및 폴리ε-카프로락톤의 중량비(폴리 L-락타이드: 폴리ε-카프로락톤)는 50:50 내지 90:10일 수 있고, 구체적으로 60:40 내지 80:20일 수 있고, 더욱 구체적으로 70:30 내지 80:20일 수 있다. 상기 폴리 L-락타이드 및 폴리ε-카프로락톤의 중량비가 상기 수치 범위 내를 만족할 때, 생체적합성 및 생분해성이 더욱 우수하여 면역 거부 반응이 초래되지 않을 수 있다.According to another embodiment of the present invention, when the biopolymer compound is poly(L-lactide-co-ε-caprolactone) (Poly(L-lactide-co-ε-caprolactone; PLCL), poly L- The weight ratio of lactide and polyε-caprolactone (poly L-lactide: polyε-caprolactone) may be 50:50 to 90:10, specifically 60:40 to 80:20, and more specifically It may be 70:30 to 80:20 When the weight ratio of the poly L-lactide and the polyε-caprolactone is within the above numerical range, the biocompatibility and biodegradability are more excellent and immune rejection is not caused. may not be
본 발명에 따른 골격근 형성 촉진 지지체 제조용 조성물은 맥신 나노입자(Mxene nanoparticle)를 포함한다. 상기 맥신 나노입자(Mxene nanoparticle)는 나노섬유 지지체에서 근아세포의 성장 및 분화를 촉진하는 성장인자일 수 있다.The composition for preparing a scaffold for promoting skeletal muscle formation according to the present invention includes MXene nanoparticles. The Mxene nanoparticle may be a growth factor that promotes the growth and differentiation of myoblasts on the nanofibrous scaffold.
본 발명에 따른 맥신 나노입자의 함량은, 상기 바이오 고분자 화합물 100 중량부를 기준으로 0.2 내지 1.5 중량부일 수 있고, 바람직하게는 0.6 내지 1.0 중량부일 수 있고, 더욱 바람직하게는 0.75 내지 0.85 중량부일 수 있다. 달리 설명하면, 상기 맥신 나노입자의 함량은 상기 골격근 형성 촉진 지지체 제조용 조성물(고분자 용액)의 전체 부피를 기준으로 0.40 내지 1.05 %(w/v)일 수 있고, 바람직하게는 0.60 내지 0.85 %(w/v)일 수 있고, 더욱 바람직하게는 0.70 내지 0.75 %(w/v)일 수 있다. 상기 맥신 나노입자의 함량이 상기 수치 범위 내를 만족할 때, 나노섬유 지지체 상의 근아세포의 성장 및 분화 속도가 빠를 수 있다. The content of MXene nanoparticles according to the present invention may be 0.2 to 1.5 parts by weight, preferably 0.6 to 1.0 parts by weight, and more preferably 0.75 to 0.85 parts by weight based on 100 parts by weight of the biopolymer compound. . In other words, the content of MXene nanoparticles may be 0.40 to 1.05% (w/v), preferably 0.60 to 0.85% (w/v) based on the total volume of the composition (polymer solution) for preparing the support for promoting skeletal muscle formation. /v), more preferably 0.70 to 0.75% (w / v). When the content of the MXene nanoparticles is within the above numerical range, the growth and differentiation rates of myoblasts on the nanofibrous scaffold may be fast.
본 발명에 따른 맥신 나노입자는 타이타늄 계열 맥신 나노입자를 포함할 수 있다. 구체적으로, 상기 타이타늄 계열 맥신 나노입자는 예를 들어, Ti3C2, Ti2C, Ti3CN, Ti3C2Tx 및 이들의 조합으로 이루어진 군에서 선택된 어느 하나일 수 있다. 상기 Ti3C2Tx에서 상기 T는 OH, O 및 F로 이루어진 군에서 선택된 어느 하나일 수 있고, x는 1 내지 3일 수 있다. 예를 들어 Ti3C2Tx는 Ti3C2F2, Ti3C2(OH)2, Ti3C2O2 등 일 수 있다. 특히, 타이타늄 계열 맥신 나노입자는 상대적으로 다른 계열의 맥신 나노입자보다 근아세포에 대한 독성이 낮은 특징을 가지고 있다.MXene nanoparticles according to the present invention may include titanium-based MXene nanoparticles. Specifically, the titanium-based MXene nanoparticles may be, for example, any one selected from the group consisting of Ti 3 C 2 , Ti 2 C, Ti 3 CN, Ti 3 C 2 Tx, and combinations thereof. In the Ti 3 C 2 Tx, T may be any one selected from the group consisting of OH, O, and F, and x may be 1 to 3. For example, Ti 3 C 2 Tx may be Ti 3 C 2 F 2 , Ti 3 C 2 (OH) 2 , Ti 3 C 2 O 2 , or the like. In particular, titanium-based MXene nanoparticles have relatively low toxicity to myoblasts than MXene nanoparticles of other series.
본 발명의 또 다른 실시예에 따른 골격근 형성 촉진 지지체 제조용 조성물은 기질을 더 포함할 수 있다. 상기 기질은 근아세포의 점착을 위한 매트릭스(matrix)일 수 있다.The composition for preparing a support for promoting skeletal muscle formation according to another embodiment of the present invention may further include a substrate. The substrate may be a matrix for adherence of myoblasts.
상기 기질은 예를 들어, 콜라겐, 젤라틴, 라미닌, 피브로넥틴, Arg-Gly-Asp (RGD) 펩타이드, 실크피브로인, 알부민, 키토산, 헤파린, 히알루론산, 녹말, 알긴산, 셀룰로오스 및 이들의 조합으로 이루어진 군에 선택된 어느 하나일 수 있고, 바람직하게는 콜라겐일 수 있다. 상기 콜라겐의 분자량은 예를 들어, 250 내지 350 kDa일 수 있다.The matrix is, for example, in the group consisting of collagen, gelatin, laminin, fibronectin, Arg-Gly-Asp (RGD) peptide, silk fibroin, albumin, chitosan, heparin, hyaluronic acid, starch, alginic acid, cellulose, and combinations thereof It may be any one selected, preferably collagen. The molecular weight of the collagen may be, for example, 250 to 350 kDa.
본 발명에 따른 기질의 함량은 상기 바이오 고분자 화합물 100 중량부를 기준으로 1 내지 30 중량부일 수 있고, 바람직하게는 5 내지 25 중량부, 더욱 바람직하게는 10 내지 20 중량부일 수 있다. 상기 기질의 함량이 상기 수치 범위 내일 때, 근아세포의 기질에 대한 점착성이 충분히 높아질 수 있다.The amount of the substrate according to the present invention may be 1 to 30 parts by weight, preferably 5 to 25 parts by weight, more preferably 10 to 20 parts by weight, based on 100 parts by weight of the biopolymer compound. When the content of the matrix is within the above numerical range, the adhesion of myoblasts to the matrix may be sufficiently increased.
본 발명에 따른 골격근 형성 촉진 지지체 제조용 조성물은 용매를 더 포함할 수 있다. 구체적으로 상기 용매는 상기 바이오 고분자 화합물을 용해시키는 화합물로 바이오 고분자 화합물의 종류에 따라 적절히 선택될 수 있다.The composition for preparing a support for promoting skeletal muscle formation according to the present invention may further include a solvent. Specifically, the solvent is a compound that dissolves the biopolymer compound and may be appropriately selected according to the type of the biopolymer compound.
상기 용매는 예를 들어, 테트라하이드로퓨란, 디메틸아세트아미드, 디메틸포름아미드, 클로로포름, 디메틸술폭사이드, 부탄올, 이소프로판올, 헥사플루오로이소프로판올, 이소부틸알콜, 테트라부틸알콜, 아세틱산, 1,4-다이옥산, 톨루엔, 오르쏘-자이렌 및 디클로로메탄으로 이루어진 군으로부터 선택되는 1종 단독 또는 2종 이상의 혼합 용매일 수 있다. The solvent is, for example, tetrahydrofuran, dimethylacetamide, dimethylformamide, chloroform, dimethylsulfoxide, butanol, isopropanol, hexafluoroisopropanol, isobutyl alcohol, tetrabutyl alcohol, acetic acid, 1,4-dioxane , toluene, ortho-xyrene, and dichloromethane, and may be a single solvent or a mixed solvent of two or more selected from the group consisting of.
본 발명의 일 실시예에 따르면, 상기 용매의 함량은 골격근 형성 촉진 지지체 제조용 조성물의 전체 함량을 기준으로, 상기 바이오 고분자 화합물을 제외한 나머지 함량으로 정의될 수 있다. According to one embodiment of the present invention, the amount of the solvent may be defined as the amount remaining excluding the biopolymer compound based on the total content of the composition for preparing a support for promoting skeletal muscle formation.
본 발명의 다른 실시예에 따르면, 상기 용매의 함량은 상기 바이오 고분자 화합물 및 상기 기질의 함량의 합을 제외한 나머지 함량으로 정의될 수 있다.According to another embodiment of the present invention, the amount of the solvent may be defined as the amount remaining after excluding the sum of the amounts of the biopolymer compound and the substrate.
2. 골격근 형성 촉진 지지체2. Support for promoting skeletal muscle formation
본 발명의 또 다른 실시예는 골격근 형성 촉진 지지체 제조용 조성물로 제조된 상기 골격근 형성 촉진 지지체를 제공한다.Another embodiment of the present invention provides a scaffold for promoting skeletal muscle formation prepared from a composition for preparing a scaffold for promoting skeletal muscle formation.
본 발명에 따른 골격근 형성 촉진 지지체는 예를 들어, 나노섬유가 랜덤 또는 정렬 나노섬유 지지체일 수 있고, 바람직하게는 정렬 나노섬유 지지체일 수 있다. 상기 나노섬유의 배열 형태는 전기방사 조건을 달리함에 따라 달라질 수 있다. 상기 정렬 나노섬유 지지체는 상기 랜덤 나노섬유 지지체보다 나노섬유가 정렬적으로 배열되어 있기 때문에, 지지체 상의 근아세포의 성장 및 분화가 더욱 촉진될 수 있다. The scaffold for promoting skeletal muscle formation according to the present invention may be, for example, a scaffold with random or aligned nanofibers, preferably an aligned nanofiber scaffold. The arrangement of the nanofibers may vary according to different electrospinning conditions. Since the aligned nanofiber scaffold has nanofibers arranged in an ordered manner rather than the random nanofiber scaffold, the growth and differentiation of myoblasts on the scaffold can be further promoted.
본 발명에 따른 골격근 형성 촉진 지지체는 골격근 형성 촉진 지지체 제조용 조성물을 전기방사하여 제조된 것일 수 있다. 구체적으로, 상기 골격근 형성 촉진 지지체 제조용 조성물을 전기방사하기 위해, 해당 기술분야의 통상적인 전기방사장치가 이용될 수 있다.The scaffold for promoting skeletal muscle formation according to the present invention may be prepared by electrospinning a composition for preparing a scaffold for promoting skeletal muscle formation. Specifically, in order to electrospin the composition for preparing the support for promoting skeletal muscle formation, a conventional electrospinning apparatus in the art may be used.
상기 골격근 형성 촉진 지지체 제조용 조성물을 전기방사하기 위해, 니들게이지(needle gauge)는 23 내지 26 gauge일 수 있고, 전압은 14 내지 18 kV, 방사거리는 8 내지 12 cm일 수 있다.To electrospin the composition for preparing the support for promoting skeletal muscle formation, a needle gauge may be 23 to 26 gauge, a voltage may be 14 to 18 kV, and a spinning distance may be 8 to 12 cm.
상기 골격근 형성 촉진 지지체를 이루는 나노섬유의 평균직경은 예를 들어, 400 내지 800 ㎛일 수 있다.The average diameter of the nanofibers constituting the skeletal muscle formation promoting support may be, for example, 400 to 800 μm.
3. 3D 프린팅용 바이오잉크3. Bioink for 3D printing
본 발명의 또 다른 실시예는 상기 골격근 형성 촉진 지지체를 포함하는 3D 프린팅용 바이오잉크(bio-ink)를 제공한다.Another embodiment of the present invention provides a bio-ink for 3D printing including the support for promoting formation of skeletal muscle.
본 발명에 따른 3D 프린팅용 바이오잉크(bio-ink)는 바이오 프린팅 기술에 응용하여 필요로 하는 구조물을 제작할 수 있는 소재일 수 있다. 이를 통해, 상기 골격근 형성 촉진 지지체를 3D 프린팅용 바이오잉크 소재로 이용하여 근아세포의 부착, 증식 및 분화를 촉진하여 빠른 골격근 재생 효과를 기대할 수 있다.The bio-ink for 3D printing according to the present invention may be a material capable of producing a structure required by application to bio-printing technology. Through this, it is possible to expect rapid skeletal muscle regeneration effect by promoting attachment, proliferation and differentiation of myoblasts by using the skeletal muscle formation promoting support as a bioink material for 3D printing.
본 발명에 따른 3D 프린팅용 바이오잉크는, 생체적합성 재료를 포함할 수 있다. 상기 생체적합성 재료는 예를 들어 콜라겐, 젤라틴, 폴리에틸렌글리콜 디아크릴레이트 등일 수 있다. 필요에 따라, 상기 3D 프린팅용 바이오잉크는 통상적인 성장인자를 더 포함할 수 있다.The bioink for 3D printing according to the present invention may include a biocompatible material. The biocompatible material may be, for example, collagen, gelatin, polyethylene glycol diacrylate, or the like. If necessary, the bioink for 3D printing may further include a conventional growth factor.
한편, 바이오잉크는 3차원 가공을 위한 물리적 성질과 세포가 목적된 기능을 수행하게 하기 위한 생물학적 환경을 제공해야 한다. 바이오잉크를 만족하기 위해서는 우수한 세포친화성을 가져야 하고, 프린팅 공정이 길어질 때에는 카트리지 내 세포의 생존에 필요한 영양분과 산소의 공급이 적절히 이루어져야 한다.On the other hand, bioink should provide physical properties for 3D processing and a biological environment for cells to perform their intended functions. In order to satisfy the bioink, it must have excellent cell compatibility, and when the printing process is prolonged, nutrients and oxygen necessary for survival of the cells in the cartridge must be properly supplied.
또한, 3D 프린팅 과정에서 발생하는 물리적 스트레스로부터 세포를 보호할 수 있어야 한다. 그 외에도 바이오잉크는 3차원 패터닝의 반복성, 생산성, 노즐의 막힘이 없어야 하는 등 프린팅 공정상에서 필요로 하는 물리적 성질을 가져야 한다. In addition, cells must be protected from physical stress generated during the 3D printing process. In addition, the bioink must have physical properties required in the printing process, such as repeatability of 3D patterning, productivity, and nozzle clogging.
본 발명에 따른 3D 프린팅 바이오잉크는 상기와 같은 요건을 모두 만족할 수 있다.The 3D printing bioink according to the present invention may satisfy all of the above requirements.
이하, 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있도록 본 발명의 실시예에 대하여 상세히 설명하나, 이는 하나의 예시에 불과할 뿐, 본 발명의 권리범위가 다음 내용에 의해 제한되지 아니한다. Hereinafter, an embodiment of the present invention will be described in detail so that those skilled in the art can easily practice it, but this is only one example, and the scope of the present invention is Not limited.
[제조준비예: 재료 및 배지 조건][Manufacturing preparation example: materials and medium conditions]
- 근아세포: 생쥐 유래 세포(C2C12; ATCC®)- Myoblasts: mouse-derived cells (C2C12; ATCC ® )
- 맥신 나노입자: Ti3AlC2 분말(2g, 11 Technology Co., Ltd)을 HF 용액(20mL, 50wt%)에 천천히 첨가하여 제조된 혼합물을 오일 배스(oil bath)에서 50℃에서 48시간 동안 교반하였다. 교반된 용액에서 상층액의 pH가 6에 도달할 때까지 3분 동안 3,500rpm으로 원심분리한 후, 침전물을 탈이온수로 여러 번 세척하여 다층의 Ti3C2Tx를 수득하였다. 미리 제조된 다층의 Ti3C2Tx(2g)을 탈이온수(50mL)에서 재분산시킨 다음, 디메틸설폭사이드(40mL)를 첨가한 후, 이들을 24시간 동안 교반하였다. 이후, 교반된 결과물을 1시간 동안 초음파 처리하고 3,500rpm에서 30분 동안 원심분리한 후, 상층액을 수집하고 박리된 Ti3C2Tx 용액을 수득하였다. 상기 Ti3C2Tx은 Ti3C2F2을 의미한다.- MXene nanoparticles: a mixture prepared by slowly adding Ti 3 AlC 2 powder (2 g, 11 Technology Co., Ltd) to a HF solution (20 mL, 50 wt %) in an oil bath at 50° C. for 48 hours Stir. After centrifugation at 3,500 rpm for 3 minutes in the stirred solution until the pH of the supernatant reached 6, the precipitate was washed several times with deionized water to obtain multi-layers of Ti 3 C 2 T x . A previously prepared multilayer of Ti 3 C 2 T x (2 g) was redispersed in deionized water (50 mL), then dimethyl sulfoxide (40 mL) was added and stirred for 24 hours. Thereafter, the agitated product was sonicated for 1 hour and centrifuged at 3,500 rpm for 30 minutes, and the supernatant was collected to obtain a Ti 3 C 2 T x solution exfoliated. The Ti 3 C 2 T x means Ti 3 C 2 F 2 .
- 바이오 고분자 화합물: 중량평균분자량이 600,000 g/mol이고, 폴리 L-락타이드 및 폴리ε-카프로락톤의 중량비(폴리 L-락타이드: 폴리ε-카프로락톤)가 75:25로 중합된 PLCL(poly(L-lactide-co-ε-caprolactone))- Biopolymer compound: PLCL polymerized with a weight average molecular weight of 600,000 g/mol and a weight ratio of poly L-lactide and polyε-caprolactone (poly L-lactide: polyε-caprolactone) of 75:25 ( poly(L-lactide-co-ε-caprolactone))
- 기질: 300 kDa의 콜라겐(collagen)- Substrate: Collagen of 300 kDa
- 성장배지 조건: 10 부피% 소태아혈청(Fetal Bovine Serum) 및 1 부피% 안티바이오틱-안티마이코틱 용액(Antibiotic-antimycotic solution)이 첨가된 돌베코 개질 이글 배지(Dulbecco's Modified Eagle's Medium; Welgene) - Growth medium condition: Dolbecco's Modified Eagle's Medium (Welgene) supplemented with 10 vol% Fetal Bovine Serum and 1 vol% Antibiotic-antimycotic solution
- 분화배지 조건: 2 부피% 말 혈청(Horse Serum) 및 1 부피% 안티바이오틱-안티마이코틱 용액(Antibiotic-antimycotic solution)이 첨가된 돌베코 개질 이글 배지(Dulbecco's Modified Eagle's Medium; Welgene) - Differentiation medium condition: Dulbecco's Modified Eagle's Medium (Welgene) supplemented with 2% by volume Horse Serum and 1% by volume Antibiotic-antimycotic solution (Antibiotic-antimycotic solution)
[실험예 1: 근아세포에 대한 맥신 나노입자의 독성 평가][Experimental Example 1: Toxicity evaluation of MXene nanoparticles on myoblasts]
상기 제조준비예에 따른 근아세포에 대한 맥신 나노입자의 독성 평가를 진행하기 위하여, CCK-8(cell counting kit-8) 분석법을 사용하였다. 구체적으로 상기 제조준비예에 따른 근아세포(1.0 x 104 cell/well)를 37℃에서 24시간 동안 배양시킨 후, 450 nm에서 마이크로 플레이트 리더(micro plate reader)를 이용하여 흡광도를 측정하였다. In order to evaluate the toxicity of MXene nanoparticles to myoblasts according to the preparation preparation example, CCK-8 (cell counting kit-8) assay was used. Specifically, after culturing the myoblasts (1.0 x 10 4 cell/well) according to the Preparation Preparation Example at 37° C. for 24 hours, the absorbance was measured at 450 nm using a micro plate reader.
도 1은 근아세포에 대한 맥신 나노입자의 독성 평가를 나타낸 결과이다. 구체적으로, 맥신 나노입자의 농도에 따른 대조군(맥신 나노입자의 농도: 0μg/mL) 대비, 근아세포의 생존도(cell viability)를 분석하였다. 1 is a result of evaluating the toxicity of MXene nanoparticles to myoblasts. Specifically, compared to the control group (concentration of MXene nanoparticles: 0 μg/mL) according to the concentration of MXene nanoparticles, cell viability of myoblasts was analyzed.
맥신 나노입자의 농도가 증가함에 따라, 대부분 근아세포의 생존도가 감소하는 경향성을 보였지만, 맥신 나노입자의 농도가 250 μg/mL이하일 때 근아세포에 대한 독성을 보이지 않음을 확인하였다. 반면에, 맥신 나노입자의 농도가 250 μg/mL를 초과한 500μg/mL일 때, 대조군 대비 근아세포의 생존도가 60% 이하로 감소함을 확인하였다.As the concentration of MXene nanoparticles increased, the viability of most myoblasts tended to decrease, but it was confirmed that no toxicity to myoblasts was observed when the concentration of MXene nanoparticles was less than 250 μg/mL. On the other hand, when the concentration of MXene nanoparticles exceeded 250 μg/mL to 500 μg/mL, it was confirmed that the viability of myoblasts decreased to 60% or less compared to the control group.
이를 통해, 맥신 나노입자의 농도가 적정 범위 내일 때, 근아세포에 대한 독성을 보이지 않음을 유추할 수 있었다.Through this, it was inferred that no toxicity to myoblasts was shown when the concentration of MXene nanoparticles was within the appropriate range.
[실험예 2: 맥신 나노입자를 처리한 근아세포의 면역형광염색 사진][Experimental Example 2: Immunofluorescent staining of myoblasts treated with MXene nanoparticles]
24 웰 플레이트(24 well plate)에 3x104cell/mL의 용량으로 이식한 후 밀집(confluency)이 50% 되었을 때 상기 조건의 성장배지 및 분화배지에 상기 제조준비예에 따른 맥신 나노입자를 각 농도별로 첨가하였다. 구체적으로 상기 맥신 나노입자의 농도를 각각 0, 5, 10, 20μg/mL로 달리하면서 첨가하였다. 상기 성장배지 및 분화배지는 2~3일 간격으로 교체하였으며, 교체 시 DPBS(Dulbecco's phosphate-buffered saline)로 2회 세척을 진행하였다. 이후, 3일, 7일 동안 배양하였으며 면역형광염색을 통해 이미지를 획득하였다.After transplantation in a 24 well plate at a capacity of 3x10 4 cells/mL, when the confluency is 50%, MXene nanoparticles according to the preparation preparation example are added to the growth medium and differentiation medium under the above conditions at each concentration Added a lot. Specifically, the MXene nanoparticles were added while varying the concentrations of 0, 5, 10, and 20 μg/mL, respectively. The growth medium and differentiation medium were replaced at intervals of 2 to 3 days, and at the time of replacement, washing was carried out twice with DPBS (Dulbecco's phosphate-buffered saline). Then, they were cultured for 3 days and 7 days, and images were acquired through immunofluorescence staining.
도 2a는 맥신 나노입자의 농도를 달리하여 3일차 성장배지에서 배양한 근아세포의 면역형광염색 사진이다. 도 2b는 맥신 나노입자의 농도를 달리하여 3일차 분화배지에서 배양한 근아세포의 면역형광염색 사진이다. 도 2c는 맥신 나노입자의 농도를 달리하여 7일차 성장배지에서 배양한 근아세포의 면역형광염색 사진이다. 도 2d는 맥신 나노입자의 농도를 달리하여 7일차 분화배지에서 배양한 근아세포의 면역형광염색 사진이다.Figure 2a is an immunofluorescence staining photograph of myoblasts cultured in a growth medium on day 3 with different concentrations of MXene nanoparticles. Figure 2b is an immunofluorescence staining photograph of myoblasts cultured in differentiation medium on day 3 with different concentrations of MXene nanoparticles. Figure 2c is an immunofluorescence staining photograph of myoblasts cultured in growth medium on day 7 with different concentrations of MXene nanoparticles. Figure 2d is an immunofluorescence staining photograph of myoblasts cultured in differentiation medium on day 7 with different concentrations of MXene nanoparticles.
도 2a 내지 2d를 참고하면, 맥신 나노입자가 근아세포의 골격근 분화를 촉진하는 것을 확인할 수 있다. 골격근을 이루는 근아세포는 적절한 성장인자 및 지형학적 환경(topography)에서 분화되어 근관(myotube)을 형성하는 사실에 기반하여, 하기의 방법으로 지형학적 환경을 제공하는 조직공학적 지지체(scaffold)를 제조하였다. Referring to FIGS. 2A to 2D , it can be confirmed that MXene nanoparticles promote the differentiation of myoblasts into skeletal muscle. Based on the fact that myoblasts constituting skeletal muscle are differentiated in appropriate growth factors and topography to form a myotube, a tissue engineering scaffold providing a topography environment was prepared by the following method. .
[제조예 1: 랜덤한 골격근 형성 촉진 지지체 제조용 조성물의 제조][Preparation Example 1: Preparation of a composition for preparing a scaffold for promoting random skeletal muscle formation]
<비교예 1: 골격근 형성 촉진 지지체 제조용 조성물><Comparative Example 1: Composition for preparing a support for promoting skeletal muscle formation>
헥사플루오로이소프로판올(hexafluoroisopropanol(HFIP); 5 mL)와 상기 제조준비예에 따른 PLCL(0.25g)를 혼합하여 전기방사용 고분자 용액을 제조하였다. 구체적으로, 상기 제조준비예에 따른 PLCL의 함량은 상기 고분자 용액의 전체 부피를 기준으로 5%(w/v)이다.A polymer solution for electrospinning was prepared by mixing hexafluoroisopropanol (HFIP) (5 mL) and PLCL (0.25 g) according to the Preparation Preparation Example above. Specifically, the content of PLCL according to the Preparation Preparation Example is 5% (w/v) based on the total volume of the polymer solution.
<비교예 2: 비교예 1과 달리 기질을 더 포함하는 골격근 형성 촉진 지지체 제조용 조성물><Comparative Example 2: Unlike Comparative Example 1, a composition for preparing a scaffold for promoting skeletal muscle formation further comprising a matrix>
비교예 1과 동일한 방법으로 고분자 용액(지지체 제조용 조성물)을 제조하되, 추가적으로 상기 제조준비예에 따른 콜라겐을 첨가하였다. 구체적으로, 상기 제조준비예에 따른 콜라겐의 함량은 상기 고분자 용액의 전체 부피를 기준으로 0.5%(w/v)이다.A polymer solution (composition for preparing a scaffold) was prepared in the same manner as in Comparative Example 1, but collagen according to the Preparation Preparation Example was additionally added. Specifically, the amount of collagen according to the Preparation Preparation Example is 0.5% (w/v) based on the total volume of the polymer solution.
<실시예 1: 맥신 나노입자를 포함하는 골격근 형성 촉진 지지체 제조용 조성물><Example 1: Composition for preparing a scaffold for promoting skeletal muscle formation containing MXene nanoparticles>
비교예 1과 동일한 방법으로 고분자 용액(지지체 제조용 조성물)을 제조하되, 추가적으로 상기 제조준비예에 따른 맥신 나노입자를 첨가하였다. 구체적으로, 상기 맥신 나노입자의 함량은 400 μg/mL이다.A polymer solution (composition for preparing a support) was prepared in the same manner as in Comparative Example 1, but MXene nanoparticles according to the Preparation Preparation Example were additionally added. Specifically, the content of MXene nanoparticles is 400 μg/mL.
<실시예 2: 실시예 1과 달리 기질을 더 포함하는 골격근 형성 촉진 지지체 제조용 조성물><Example 2: Unlike Example 1, a composition for preparing a scaffold for promoting skeletal muscle formation further comprising a substrate>
비교예 2와 동일한 방법으로 고분자 용액(지지체 제조용 조성물)을 제조하되, 추가적으로 상기 제조준비예에 따른 맥신 나노입자를 첨가하였다. 구체적으로, 상기 맥신 나노입자의 함량은 400 μg/mL이다.A polymer solution (composition for preparing a support) was prepared in the same manner as in Comparative Example 2, but MXene nanoparticles according to the Preparation Preparation Example were additionally added. Specifically, the content of MXene nanoparticles is 400 μg/mL.
[제조예 2: 랜덤 나노섬유 지지체의 제조][Preparation Example 2: Preparation of Random Nanofiber Support]
상기 제조예 1에 따른 고분자 용액을 하기 표 1과 같은 조건 하에 전기방사하여 랜덤 나노섬유 지지체를 제조하였다(RPM 조건: 20 RPM).A random nanofiber scaffold was prepared by electrospinning the polymer solution according to Preparation Example 1 under the conditions shown in Table 1 below (RPM condition: 20 RPM).
지지체 시료support sample Experimental
GroupExperimental
Group 		Compositioncomposition NeedleNeedle DistanceDistance Flow rateFlow rate VoltageVoltage SolventSolvent
비교예
3
(P)comparative example
3
(P) 		PLCLPLCL 5 w/v%5w/v% 25 G25G 9 cm9cm 0.2 mL/h0.2 mL/h 16kV16kV HFIPHFIP
비교예
4
(PC)comparative example
4
(PC) 		PLCL/ColPLCL/Col 5, 0.5 w/v%5, 0.5 w/v%
실시예 3
(PM)Example 3
(PM) 		PLCL/MXenePLCL/MXene 5 w/v%,
400 μg/mL5 w/v%,
400 µg/mL
실시예 4
(PCM)Example 4
(PCM) 		PLCL/Col/MXenePLCL/Col/MXene 5, 0.5 w/v%
400 μg/mL5, 0.5 w/v%
400 µg/mL
[실험예 3: 제조예 2에 따른 랜덤 나노섬유 지지체에서 배양된 근아세포의 형광 이미지][Experimental Example 3: Fluorescence image of myoblasts cultured on the random nanofiber scaffold according to Preparation Example 2]
상기 제조예 2에 따른 랜덤 나노섬유 지지체를 이용하여 다음과 같이 근아세포를 배양하였다.Myoblasts were cultured as follows using the random nanofiber scaffold according to Preparation Example 2.
1) 상기 제조예 2에 따른 랜덤 나노섬유 지지체를 지름이 1cm의 크기로 펀칭하여 샘플링 작업을 수행하였다. 그 후, 각각의 샘플링을 24 웰 플레이트(24 well plate)에 부착하였다.1) Sampling was performed by punching the random nanofiber support according to Preparation Example 2 to a size of 1 cm in diameter. After that, each sample was attached to a 24 well plate.
2) 자외선을 이용하여 멸균 과정을 거친 뒤 1.5 x 104 cell/mL의 용량으로 각 샘플 별 랜덤 나노섬유 지지체 위에 근아세포를 이식한 후 성장배지만을 이용하여 배양하였다.2) After undergoing a sterilization process using ultraviolet rays, myoblasts were transplanted onto the random nanofiber scaffold for each sample at a capacity of 1.5 x 10 4 cell/mL, and then cultured using only the growth medium.
3) 성장배지는 2~3일 간격으로 교체하였으며, 배지 교체 시 DPBS(dulbecco's phosphate-buffered saline)로 2회 세척을 수행하였다.3) The growth medium was replaced every 2-3 days, and washing was performed twice with DPBS (dulbecco's phosphate-buffered saline) when replacing the medium.
4) 이후 5일, 10일 동안 배양하였으며 면역형광염색을 통해 이미지를 획득하였다.4) After culturing for 5 days and 10 days, images were acquired through immunofluorescence staining.
도 3a는 5일차 성장배지 내 제조예 2에 따른 랜덤 나노섬유 지지체에서 배양된 근아세포의 형광 이미지이다. 도 3b는 10일차 성장배지 내 제조예 2에 따른 랜덤 나노섬유 지지체에서 배양된 근아세포의 형광 이미지이다.Figure 3a is a fluorescence image of myoblasts cultured in the random nanofiber scaffold according to Preparation Example 2 in the growth medium on day 5. Figure 3b is a fluorescence image of myoblasts cultured in the random nanofiber scaffold according to Preparation Example 2 in the 10th day growth medium.
MHC는 미오신 중쇄(myosin heavy chain)을 의미한다. MHC means myosin heavy chain.
도 3a 및 3b를 참고하면, 5일차 각 시료별 랜덤 나노섬유 지지체에서 전반적으로 근아세포가 분화되지 않았으나, 10일차 맥신 나노입자가 첨가된 시료에서 근아세포의 분화를 확인할 수 있었다.Referring to FIGS. 3A and 3B , myoblasts were not generally differentiated on the random nanofiber scaffold for each sample on day 5, but differentiation of myoblasts was confirmed in the sample to which MXene nanoparticles were added on day 10.
이를 통해, 본 발명자는 전기방사를 통해 제조된 맥신 나노입자를 함유하는 랜덤한 나노섬유 지지체가 근아세포의 골격근 분화를 촉진하는 것을 세계 최초로 확인할 수 있었다.Through this, the present inventors were able to confirm for the first time in the world that the random nanofiber scaffold containing MXene nanoparticles prepared through electrospinning promotes the differentiation of myoblasts into skeletal muscle.
[제조예 3: 정렬 나노섬유 지지체의 제조][Preparation Example 3: Preparation of Aligned Nanofiber Support]
상기 제조예 1에 따른 고분자 용액을 하기 표 2와 같은 조건 하에 전기방사하여 정렬 나노섬유 지지체를 제조하였다.Aligned nanofiber scaffolds were prepared by electrospinning the polymer solution according to Preparation Example 1 under the conditions shown in Table 2 below.
지지체 시료support sample Experimental
GroupExperimental
Group 		Compositioncomposition RPMRPM NeedleNeedle DistanceDistance Flow rateFlow rate VoltageVoltage SolventSolvent
비교예
5
(P)comparative example
5
(P) 		PLCLPLCL 5 w/v%5w/v%




3,000




3,000 		25 G25G 8 cm8cm 0.2 mL/h0.2 mL/h 16kV16kV HFIPHFIP
비교예
6
(PC)comparative example
6
(PC) 		PLCL/ColPLCL/Col 5, 0.5 w/v%5, 0.5 w/v%
실시예 5
(PM)Example 5
(PM) 		PLCL/MXenePLCL/MXene 5 w/v%,
400 μg/mL5 w/v%,
400 µg/mL
실시예 6
(PCM)Example 6
(PCM) 		PLCL/Col/MXenePLCL/Col/MXene 5, 0.5 w/v%
400 μg/mL5, 0.5 w/v%
400 µg/mL
[실험예 4: 제조예 3에 따른 정렬 나노섬유 지지체에서 배양된 근아세포의 형광 이미지][Experimental Example 4: Fluorescence image of myoblasts cultured on the aligned nanofibrous scaffold according to Preparation Example 3]
상기 제조예 3에 따른 정렬 나노섬유 지지체를 이용하여 다음과 같이 근아세포를 배양하였다.Myoblasts were cultured as follows using the aligned nanofiber scaffold according to Preparation Example 3.
1) 상기 제조예 3에 따른 정렬 나노섬유 지지체를 지름이 1cm의 크기로 펀칭하여 샘플링 작업을 수행하였다. 그 후, 각각의 샘플링을 24 웰 플레이트(24 well plate)에 부착하였다.1) Sampling was performed by punching the aligned nanofiber support according to Preparation Example 3 to a size of 1 cm in diameter. After that, each sample was attached to a 24 well plate.
2) 자외선을 이용하여 멸균 과정을 거친 뒤 1.5 x 104 cell/mL의 용량으로 각 샘플 별 정렬 나노섬유 지지체 위에 근아세포를 이식한 후 성장배지만을 이용하여 배양하였다.2) After undergoing a sterilization process using ultraviolet light, myoblasts were transplanted onto the aligned nanofiber scaffold for each sample at a capacity of 1.5 x 10 4 cell/mL, and then cultured using only a growth medium.
3) 성장배지는 2~3일 간격으로 교체하였으며, 배지 교체 시 DPBS(dulbecco's phosphate-buffered saline)로 2회 세척을 수행하였다.3) The growth medium was replaced every 2-3 days, and washing was performed twice with DPBS (dulbecco's phosphate-buffered saline) when replacing the medium.
4) 이후 3일, 7일 동안 배양하였으며 면역형광염색을 통해 이미지를 획득하였다.4) After culturing for 3 days and 7 days, images were obtained through immunofluorescence staining.
도 4a는 3일차 성장배지 내 제조예 3에 따른 정렬 나노섬유 지지체에서 배양된 근아세포의 형광 이미지이다. 도 4b는 7일차 성장배지 내 제조예 3에 따른 정렬 나노섬유 지지체에서 배양된 근아세포의 형광 이미지이다. Figure 4a is a fluorescence image of myoblasts cultured on the aligned nanofiber scaffold according to Preparation Example 3 in the growth medium on day 3. Figure 4b is a fluorescence image of myoblasts cultured on the aligned nanofiber scaffold according to Preparation Example 3 in the growth medium on day 7.
도 4a 및 4b를 참고하면, 3일차 각 시료 별 정렬된 나노섬유 지지체에서 전반적으로 근아세포가 분화되지 않았지만, 7일차 맥신 나노입자가 첨가된 지지체에서 근아세포의 분화를 확인할 수 있었다. Referring to FIGS. 4A and 4B , myoblasts were not generally differentiated on the nanofiber scaffolds aligned for each sample on day 3, but differentiation of myoblasts was confirmed on the scaffolds to which MXene nanoparticles were added on day 7.
본 발명자는 전기방사를 통해 제조된 맥신 나노입자를 함유하는 정렬 나노섬유 지지체가 랜덤 나노섬유 지지체 대비, 더 효과적으로 근아세포의 골격근 분화를 촉진하는 것을 확인할 수 있었다.The present inventors have confirmed that the aligned nanofibrous scaffold containing MXene nanoparticles prepared through electrospinning promotes the differentiation of myoblasts into skeletal muscle more effectively than the random nanofibrous scaffold.
이상에서 본 발명의 바람직한 실시예들에 대하여 상세하게 설명하였지만 본 발명의 권리범위는 이에 한정되는 것은 아니고 다음의 청구범위에서 정의하고 있는 본 발명의 기본 개념을 이용한 당업자의 여러 변형 및 개량 형태 또한 본 발명의 권리범위에 속하는 것이다. Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements of those skilled in the art using the basic concept of the present invention defined in the following claims are also present. belong to the scope of the invention.

Claims (10)

  1. 바이오 고분자 화합물; 및biomolecular compounds; and
    맥신 나노입자(Mxene nanoparticle); 를 포함하는Mxene nanoparticle; containing
    골격근 형성 촉진 지지체 제조용 조성물. A composition for preparing a support for promoting skeletal muscle formation.
  2. 제1항에 있어서,According to claim 1,
    상기 바이오 고분자 화합물은,The biopolymer compound,
    폴리글리콜릭산(poly(glycolic acid); PGA), 폴리-L-락타이드(poly-L-lactide; PLLA), 폴리 ε-카프로락톤(poly ε-caprolactone), 폴리-L-라이신(poly-L-lysine; PLL), 폴리에틸렌 옥사이드(polyethylene oxide) 및 폴리비닐알코올(polyvinyl alcohol; PVA), 폴리아닐린(polyaniline) 및 폴리메타크릴산메틸(polymethylmethacrylate, PMMA)로 이루어진 군에서 선택된 1종 단독 또는 2종 이상의 공중합체인,poly(glycolic acid) (PGA), poly-L-lactide (PLLA), poly ε-caprolactone, poly-L-lysine (poly-L -lysine; PLL), polyethylene oxide and polyvinyl alcohol (PVA), polyaniline, and polymethylmethacrylate (PMMA). copolymer,
    골격근 형성 촉진 지지체 제조용 조성물. A composition for preparing a support for promoting skeletal muscle formation.
  3. 제2항에 있어서,According to claim 2,
    상기 바이오 고분자 화합물은,The biopolymer compound,
    폴리(L-락타이드-코-ε-카프로락톤)(Poly(L-lactide-co-ε-caprolactone; PLCL)인,Poly (L-lactide-co-ε-caprolactone) (Poly (L-lactide-co-ε-caprolactone; PLCL),
    골격근 형성 촉진 지지체 제조용 조성물. A composition for preparing a support for promoting skeletal muscle formation.
  4. 제1항에 있어서,According to claim 1,
    상기 맥신 나노입자의 함량은,The content of the MXene nanoparticles,
    상기 바이오 고분자 화합물 100 중량부를 기준으로 0.2 내지 1.5 중량부인,0.2 to 1.5 parts by weight based on 100 parts by weight of the biopolymer compound,
    골격근 형성 촉진 지지체 제조용 조성물. A composition for preparing a support for promoting skeletal muscle formation.
  5. 제1항에 있어서,According to claim 1,
    상기 맥신 나노입자는,The MXene nanoparticles,
    타이타늄 계열 맥신 나노입자를 포함하는containing titanium-based MXene nanoparticles.
    골격근 형성 촉진 지지체 제조용 조성물.A composition for preparing a support for promoting skeletal muscle formation.
  6. 제5항에 있어서,According to claim 5,
    상기 타이타늄 계열 맥신 나노입자는,The titanium-based MXene nanoparticles,
    Ti3C2, Ti2C, Ti3CN, Ti3C2Tx 및 이들의 조합으로 이루어진 군에서 선택된 어느 하나이고,any one selected from the group consisting of Ti 3 C 2 , Ti 2 C, Ti 3 CN, Ti 3 C 2 T x and combinations thereof;
    T는 OH, O 및 F로 이루어진 군에서 선택된 어느 하나이고, x는 1 내지 3인,T is any one selected from the group consisting of OH, O and F, x is 1 to 3,
    골격근 형성 촉진 지지체 제조용 조성물.A composition for preparing a support for promoting skeletal muscle formation.
  7. 제1항에 있어서,According to claim 1,
    기질을 더 포함하는more temperamental
    골격근 형성 촉진 지지체 제조용 조성물.A composition for preparing a support for promoting skeletal muscle formation.
  8. 제7항에 있어서,According to claim 7,
    상기 기질은,The substrate is
    콜라겐, 젤라틴, 라미닌, 피브로넥틴, Arg-Gly-Asp(RGD) 펩타이드, 실크피브로인, 알부민, 키토산, 헤파린, 히알루론산, 녹말, 알긴산, 셀룰로오스 및 이들의 조합으로 이루어진 군에 선택된 어느 하나인,Any one selected from the group consisting of collagen, gelatin, laminin, fibronectin, Arg-Gly-Asp (RGD) peptide, silk fibroin, albumin, chitosan, heparin, hyaluronic acid, starch, alginic acid, cellulose, and combinations thereof,
    골격근 형성 촉진 지지체 제조용 조성물.A composition for preparing a support for promoting skeletal muscle formation.
  9. 제1항에 따른 골격근 형성 촉진 지지체 제조용 조성물로 제조된 골격근 형성 촉진 지지체.A scaffold for promoting skeletal muscle formation prepared from the composition for preparing a scaffold for promoting skeletal muscle formation according to claim 1.
  10. 제9항에 따른 골격근 형성 촉진 지지체를 포함하는 3D 프린팅용 바이오 잉크.A bioink for 3D printing comprising the support for promoting skeletal muscle formation according to claim 9.
PCT/KR2022/019481 2021-12-09 2022-12-02 Composition for manufacturing skeletal muscle formation-promoting support, skeletal muscle formation-promoting support manufactured thereby, and bio-ink for 3d printing WO2023106746A1 (en)

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