WO2018186611A2 - Bioencre et son procédé de préparation - Google Patents

Bioencre et son procédé de préparation Download PDF

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Publication number
WO2018186611A2
WO2018186611A2 PCT/KR2018/003353 KR2018003353W WO2018186611A2 WO 2018186611 A2 WO2018186611 A2 WO 2018186611A2 KR 2018003353 W KR2018003353 W KR 2018003353W WO 2018186611 A2 WO2018186611 A2 WO 2018186611A2
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WIPO (PCT)
Prior art keywords
polymer
bio
silk fibroin
ink
photoinitiator
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PCT/KR2018/003353
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English (en)
Korean (ko)
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WO2018186611A3 (fr
Inventor
박찬흠
김순희
이정민
연응규
이영진
서예빈
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한림대학교 산학협력단
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Publication of WO2018186611A2 publication Critical patent/WO2018186611A2/fr
Publication of WO2018186611A3 publication Critical patent/WO2018186611A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
    • C09D11/00—Inks
    • C09D11/02—Printing inks
    • C09D11/03—Printing inks characterised by features other than the chemical nature of the binder
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
    • C09D11/00—Inks
    • C09D11/02—Printing inks
    • C09D11/10—Printing inks based on artificial resins
    • C09D11/106—Printing inks based on artificial resins containing macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
    • C09D133/00—Coating compositions based on homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides, or nitriles thereof; Coating compositions based on derivatives of such polymers
    • C09D133/04—Homopolymers or copolymers of esters
    • C09D133/06—Homopolymers or copolymers of esters of esters containing only carbon, hydrogen and oxygen, the oxygen atom being present only as part of the carboxyl radical
    • C09D133/10—Homopolymers or copolymers of methacrylic acid esters

Definitions

  • the present invention uses silk fibroin, one of the natural protein fibers.
  • the present invention relates to a bio ink capable of producing a biological tissue having improved cell compatibility and excellent physical properties using a 3D printer, and a method of manufacturing the same.
  • 3D printing technology is to realize a three-dimensional shape, that is, a three-dimensional shape, based on a three-dimensional drawing, depending on the ink material used for printing, a curing method, and the driving principle of a 3D printer. Among them, they can be classified according to the classification of the American ASTM (Amer i can Society for Testing and Materials) F2792-12a.
  • the photopolymerization method SLACstereo li thography (DCR) method and DLP (digital processing process) method, which uses a photocurable polymer that is cured in response to light as a base material, creates a three-dimensional shape by irradiating and curing light.
  • DCR SLACstereo li thography
  • DLP digital processing process
  • a fused deposi- tion ion modeling (FDM) method of a material extrusion method which is a method of continuously pushing a heated material at high pressure through a nozzle to produce a three-dimensional shape
  • Adhesive spraying method for producing a three-dimensional shape by combining the base material by discharging the adhesive of the liquid form on the base material of the powder form
  • a material injection method for discharging the material in solution form by jet ting and curing with ultraviolet light
  • Powder-laminated melting method for producing a three-dimensional shape by combining the high-energy beam and irradiating it on the powder-based base material
  • Etc A fused deposi- tion ion modeling
  • 3D printing technology gradually develops, it is possible to manufacture more precise and detailed 3D shapes, and it is applied to the medical or bio fields, and fine and large tissue structures that almost mimic medical device parts or actual human tissues are intact. It is used to manufacture human body model, skin tissue and body organ regeneration.
  • the support for tissue engineering is very important for the selection of constituent materials and structural control technology.
  • the supporter plays a role as a bridge connecting the tissue to regenerate the tissue lost through the self-recovery function, it should be excellent in cell affinity so that tissue regeneration is smoothly performed.
  • cells grow in three dimensions
  • the support for tissue engineering must first be physically stable at the implant site, secondly, exhibit physiological activity that can regulate regenerative efficacy, and thirdly, form new tissue and then degrade in vivo. It must not have this toxicity.
  • bioinks which are materials for producing biostructures such as surgical simulations and surgical implants, personalized implants, artificial blood vessels, and artificial organs, have shown many limitations.
  • bio-inks required for 3D bioprinting include, firstly, excellent biocompatibility, and when applied to a 3D printer printed through a nozzle, a fine diameter dispensing nozzle (di spensing nozz le) is smoothly applied. It must have physical properties that can be passed through and printed in the desired pattern. In addition, it should be possible to maintain a mechanical support role while providing cell-specific signals after 3D printing.
  • natural or synthetic hydrogel bio inks have been developed and used in the field of 3D bio printing (Republic of Korea Patent Publication No. 10-2017-0001444: 2017.01.04.) There are significant limitations in physical and biological aspects such as suitability, printing suitability, geometrical precision and precision.
  • the present invention provides a bio-ink that can be applied to a 3D printer and a method of manufacturing the same, by producing a bio-ink based on silk fibroin, which is a natural protein polymer as a main skeleton, to maintain excellent mechanical strength and cellular suitability of silk fibroin. I would like to.
  • One embodiment of the present invention for achieving the above object relates to a bio ink that can be used for 3D printing, preferably silk fibroin (Si lk Fibroin) and methacrylate (Methacryl ate) compound Polymerized high polymers; And a photoinitiator.
  • a bio ink that can be used for 3D printing, preferably silk fibroin (Si lk Fibroin) and methacrylate (Methacryl ate) compound Polymerized high polymers; And a photoinitiator.
  • the polymer formed by copolymerizing at least one methacrylate compound with the amino acid residue of silk fibroin is preferable to use.
  • the photoinitiator lithium thienyl phenyl-2,4,6-trimethylbenzoylphosphinate (li thium phenyl- (2,4,6-trimethylbenzoyl) phosphinate, LAP), benzyl dimethyl ketal, acetophenone Acetophenone , Benzoin methyl ether ether), diethoxyacetophenone, benzoyl phosphine oxide and benzoyl phosphine oxide, and may include one or more selected from the group consisting of 1-hydroxycyclohexyl phenyl ketone.
  • a dissolution step of dissolving silk fibroin (Si lk Fibroin) in a solvent Adding a methacrylate compound to a solution in which silk fibroin is dissolved, followed by stirring to prepare a polymer polymer; A drying step of lyophilizing and powdering the solution containing the polymer prepared by the polymerization step; And a mixing step of mixing a powder containing a high molecular polymer with water and a photoinitiator in water.
  • the solution containing the prepared polymer polymer in the dialysis tube After the polymerization step, the solution containing the prepared polymer polymer in the dialysis tube, the dialysis step of removing impurities by immersing in water;
  • the dialysis step, the dialysis step, the solution containing the prepared polymer polymer It is desirable to remove the impurities by immersing in 12 14 kDa cutof f dialysis tube, immersed in water for 3-5 days.
  • the dissolving step after dissolving the silk fibroin at a concentration of 0.05-0.35 g / ml in a solvent, it can be heated to a temperature of 50 ⁇ 70 ° C for 40-80 minutes.
  • methacrylate-based compound is added to the solution in which the silk fibroin is dissolved at a concentration of 141 to 705 mM, and after the methacrylate-based compound is added to the solution in which the silk fibroin is dissolved, the temperature is 50 to 70 ° C. It is preferable to stir at 200 to 400 rpm rotational speed for 2 to 4 hours at.
  • the mixing step 20 to 30 ⁇ % of the powder containing the polymer polymer in water and 0.1 to 0.3% of the photoinitiator are mixed, but it is preferable that the sum of the water, the polymer and the photoinitiator does not exceed 100 wt%. .
  • lithium thiphenyl phenyl-2,4,6-trimethylbenzoylphosphinate (li thium phenyl- (2,4,6-tr imethyl benzoydopinyll) phosphinate, LAP), benzyldimethyl Benzyl dimethyl ketal, acetophenone, benzoin methyl ether, diethoxyacetophenone, benzoyl phosphine oxide and 1-hydroxycyclonucleic phenyl ketone 1-hydroxycyclohexyl phenyl ketone) may be used that includes at least one selected from the group consisting of.
  • the solution containing the polymer polymer is frozen at -90 to -70 ° C for 10 to 14 hours, and then lyophilized for 40 to 60 hours at the same temperature as the freezing temperature.
  • another embodiment of the present invention is the aforementioned bio ink; Or a bio structure manufactured by 3D printing using the bio ink prepared by the aforementioned manufacturing method.
  • the present invention is based on the silk fibroin, one of the natural protein fibers in a liquid state that can cause a gelation or curing reaction upon exposure to light through polymerization reaction.
  • biological tissues having mechanical properties such as moisture absorption, volume expansion ratio, compressive strength, and tensile strength can be prepared.
  • FIG. 1 is a schematic diagram schematically showing a hydrogel state of a bioink which is an embodiment of the present invention.
  • Figure 2 is a schematic diagram showing the manufacturing process of the bio ink of the present invention.
  • Figure 3 is an embodiment of the present invention, a schematic chemical formula showing the polymerization reaction of silk fibroin and GMA.
  • FIG. 4 is a schematic view showing an embodiment of the present invention in which gel fibroin and GMA polymerized polymer (SGMA) are gelled.
  • SGMA gel fibroin and GMA polymerized polymer
  • Figure 5 is an embodiment of the present invention, FT-IR spectrum of the SGMA according to the concentration of GMA injected into the silk fibroin.
  • 6 is an embodiment of the present invention, which is -NMR spectrum of SGMA according to the concentration of GMA injected into silk fibroin.
  • Figure 7 is a schematic diagram showing a 3D printing process in a DLP method using a bio ink prepared according to an embodiment of the present invention. .
  • Figure 8 is a graph showing the results of measuring the water absorption of the hydrogel printed by 3D printing using a bio ink prepared in another embodiment of the present invention.
  • 9 is a graph showing the results of measuring the volume expansion rate of the hydrogel printed by 3D printing using a bio ink prepared according to another embodiment of the present invention.
  • 10 to 16 are photographs or graphs showing an experimental procedure or test results of measuring the mechanical strength of a hydrogel printed by 3D printing using a bio ink prepared according to another embodiment of the present invention.
  • FIG. 17 is a graph showing storage modulus (G ′) and loss modulus (G ′ 1 ) according to the shear strain of a hydrogel printed by 3D printing using a bio ink prepared according to another embodiment of the present invention.
  • FIG. 18 is a graph showing storage modulus (G ′) and loss modulus (G ′ ′) of a hydrogel printed by 3D printing using a bio ink prepared according to another embodiment of the present invention.
  • FIG. 19 is a graph showing the stiffness of hydrogels printed by 3D printing using bio inks containing different photoinitiator contents according to another embodiment of the present invention.
  • 20 is a graph showing the stiffness of the hydrogel according to the curing time exposed to UV under 3D printing using different SGMA-3 content of different bio inks prepared by another embodiment of the present invention.
  • 21 is a fluorescence micrograph showing the results of cytotoxicity test of the bio ink prepared according to another embodiment of the present invention.
  • FIG. 22 is a cell proliferation experiment of a bio ink prepared by another embodiment of the present invention A graph showing the results.
  • each step the identification code is used for convenience of explanation, and the identification code does not describe the order of each step, and each step may be performed differently from the stated order unless the context clearly indicates a specific order. have. That is, each step may be performed in the same order as specified, may be performed substantially simultaneously, or may be performed in the reverse order.
  • each step may be performed in the same order as specified, may be performed substantially simultaneously, or may be performed in the reverse order.
  • the bio ink of the present invention is a biomaterial capable of printing biostructures that can be utilized in medical and bio fields such as tissue engineering supports, surgical implants, personalized implants, artificial blood vessels, artificial organs, etc., via 3D printers.
  • the biostructure consisting of a hydrogel formed as shown in FIG. 1 can be printed by exposing a liquid bio ink obtained by dissolving a polymer polymerized in a silk fibroin, which is a protein polymer, in water together with a photoinitiator to light (for example, UV). have.
  • bio ink of the present invention may be applied to various types of 3D printers, but may be preferably applied to 3D printers based on digital processing (DLP).
  • DLP digital processing
  • DLP digi- tal process processing
  • DLP type 3D printer
  • It is a shape to be molded by using a beam projector in a tank containing a liquid photocurable resin.
  • It is a 3D printing method of manufacturing a structure by projecting light with light, and curing and laminating resin in the projected shape.
  • This 3D printing technology of the DLP method projects the image from the beam projector in mask units (2D), which enables output without additional materials such as a support, and has excellent surface roughness and no noise during the printing process. There is this.
  • the structure is molded through the surface unit molding method using the resin in the liquid state as the ink, and thus the work speed can be uniformly and rapidly formed, which reduces the damage to the cells, thereby improving the cell compatibility.
  • the bio ink of the present invention can be used as a biomaterial Biocompatible materials (e.g. agarose, fibrinogen, fibroin, methacrylated hyaluronic acid (HAMA), thiolated hyaluronic acid, gelatin, gelatin methacrylate ( GelMA), Silicated Gelatin, Collagen, Alginate, Methyl Cellulose, Chitosan, Chitin, Synthetic Peptide, Polyethylene glycol based hydrogel, Polyglycolic acid (PGA), Poly-lact ic-co-glycolic acid ), Polylactic acid (PLA), poly (L-lactic acid) (PLLA), Polycaprolactone (PCL), PH 1 (Po 1 yhyd roxybutyrate), Polyhydroxyvalerate (PHV), PDO (Po 1 yd i ox anone), PTMC Polytrimethylene carbonate) and the like, and among them, fibroin having excellent biocompatibility and physical properties can be used as
  • Fibroin can maintain rapid cross-link formation and mechanical stiffness, have excellent mechanical properties in terms of stability when formed into a hydrogel, and can create a microenvironment suitable for cell attachment and differentiation after the cells are printed. There is an advantage.
  • the bio ink of the present invention is fibroin.
  • it contains a high molecular polymer prepared from silk fibroin obtained from silkworm cocoon, showing excellent cell compatibility, excellent compressive strength after printing, tensile strength and storage modulus.
  • it can be applied to DLP type 3D printer.
  • the bio ink according to the present invention includes a polymer polymer in which silk fibroin and methacrylate compound are polymerized; And a photoinitiator, wherein the polymer polymer and photoinitiator are dissolved in water to fill a liquid bio ink in a 3D printer, and the bio ink is exposed to light, preferably UV lltraviolet ray ultraviolet rays.
  • the structure may be formed by hydrogel.
  • Silk fibroin which forms the main skeleton of the bio ink of the present invention, is a natural protein polymer obtained by removing sericin from silkworm cocoon, and a polymer polymer can be prepared by polymerizing a single amino acid residue and a methacrylate compound in the silk fibroin.
  • a methacrylate compound is copolymerized to the lysine residue in the amino acid group in the silk fibroin to prepare a polymer polymer in which a methacrylate vinyl group is formed in the amine group of the silk fibroin.
  • the polymer is a methacrylate-based compound as shown in FIG. 3, and polymerized with the silk fibroin by using glycidyl methacrylate (GMA) to meta-amine the amine in the silk fibroin molecule.
  • the acrylate group (metacrylate groLip) may be a polymerized polymer (hereinafter, SGMA).
  • the photoinitiator may cause radical reaction by attacking the polymer when exposed to light, but may be used without particular limitation as long as it is a chemically stable compound that is not toxic to the human body.
  • the photoinitiator included in the bio ink of the present invention is a free radical type photoinitiator, lithium phenyl— 2,4,6-trimethylbenzoylphosphinate (lithium phenyl- (2,4,6-trimethyl benzoyl) phosphinate, LAP), Benzyldimethyl ketal (benzyl dimethyl ketal, acetophenone, benzoin methyl ether, diethethacetophenone, benzoyl phosphine oxide and 1-hydroxycyclonuclear phenyl ketone (l- hydroxycyc lohexyl phenyl ketone) may be used that includes at least one selected from the group consisting of, but is preferably low cytotoxic lithium phenyl-2,4,6-trimethylbenzoylphosphinate (li thium phenyl- (2, 4, 6-tr imethyl benzoyl) phosphinate, LAP) can be used.
  • the photoinitiator attacks the vinyl monomer of the polymer polymer to generate radical reaction, and the free radicals are cured through the polymerization reaction to form a structure.
  • the bio ink according to the present invention may be a cell, growth factor, according to the use or characteristic of the biostructure to be prepared through 3D bioprinting in addition to the polymer and photoinitiator polymerized with the silk fibroin and methacrylate-based compounds mentioned above.
  • Biodegradable polymers and the like may be further included as appropriate.
  • FIG. 2 (a) a method for producing a bio ink as shown in Figure 2 dissolution step of dissolving silk fibroin (Si lk Fibroin) in a solvent (Fig. 2 (a)); After adding methacrylate to a solution in which silk fibroin is dissolved, a polymerization step of preparing a polymer by stirring it (Fig. 2 (b)); Drying step of lyophilizing and drying the solution containing the polymer prepared by the polymerization step (Fig. 2 (d) and (e); and mixing step of mixing the powder and photoinitiator containing the polymer polymer in water) (FIG. 2 (f)) may be used to produce a bio ink.
  • the silk fibroin is a natural protein polymer, rejection reaction and immune reaction does not occur in the body, there is an advantage that the biocompatibility is excellent due to less inflammatory reaction.
  • Silk fibroin is a silkworm cocoon (Bombyx mor i) silkworm cocoon raw silk as it is removed by sericin and impurities through the refining process, the method of refining silkworm cocoon silk is usually boiled over 10 hours with boiling water, diluted alkaline There is a method of treatment with a solution, and the technique for obtaining silk fibroin refined by removing sericin and impurities from silkworm cocoon can be used if it is a widely known method, so a detailed description thereof will be omitted.
  • Silk fibroin refined through the refining process is insoluble in dilute aqueous solution such as water, dilute acid or dilute base, and in order to dissolve silk fibroin in the solvent in the dissolution step, 50 ⁇ 70 ° C in lithium bromide solution It can be dissolved by heating to a temperature of 40-80 minutes.
  • a lithium bromide (LiBr) solution or calcium chloride (CaCl 2 ) solution of 8.0 to 10.0 M may be used.
  • a lithium bromide solution of 8.7 to 9.8 M may be used.
  • a polymer After adding a methacrylate compound to a solution in which the silk fibroin is prepared through the dissolution step, to prepare a polymer by stirring
  • a polymer may be prepared by polymerizing a methacrylate group to an amino acid residue of silk fibroin, thereby forming a structure made of a hydrogel having improved mechanical properties such as compressive strength, tensile strength, and storage elasticity when exposed to light. have.
  • the amino acid residue refers to a structural unit from which H and 0H are separated from the molecular structure of various amino acids included in silk fibroin, and broadly means each amino acid constituting a peptide or protein.
  • the polymerization step may be prepared by adding a methacrylate compound to a solvent in which the silk fibroin is dissolved, and then stirred at a speed of 200 to 400 rpm for 2 to 4 hours at a temperature of 50 ⁇ 70 ° C. .
  • the polymer included in the bio ink of the present invention is a polymer polymer prepared by copolymerizing silk fibroin (Sl ik Fibtoin, SF) and a methacrylate-based compound, and is a polymer polymer that is a basic skeleton of a biostructure manufactured during 3D printing.
  • One or more methacrylate-based compounds may be copolymerized to the residues of amino acids included in the silk fibroin, thereby forming a polymer by forming a copolymer having a methacrylate group bonded to the residues of the amino acids. .
  • two methacrylate-based compounds may be polymerized to the residue of the amino acid contained in the silk fibroin to prepare a polymer, which may be cured upon exposure to light with a photoinitiator to form a hydrogel. Can be formed.
  • the wavelength of light may be used to irradiate a light wavelength region in which the photoinitiator may initiate a radical reaction, thereby initiating gelation and curing reaction of the bio ink.
  • the polymer copolymer is a combination of silk fibroin (SF) and glycidyl methacrylate (glyc idyl met hacry late, GMA) as shown in Figure 3, the amine in the silk fibroin molecule, preferably As the epoxy ring of glycidyl methacrylate is boiled, a polymer polymer (hereinafter, SGMA) in which a methacrylate group (metacrylate group) is polymerized to an amine of a lysine group in a silk fibroin molecule may be prepared.
  • SGMA polymer polymer polymer
  • the polymer SGMA can be prepared.
  • the mixing ratio of the silk fibroin and the methacrylate-based compound mixed in the polymerization step is most important.
  • a methacrylate-based compound at a concentration of 141 to 705 mM in a solution in which silk fibroin is dissolved in a solvent at a concentration of 0.05 to 0.35 g / ml.
  • a solution containing the polymer prepared in order to remove impurities, which are ions contained in the solution containing the polymer polymer prepared through the polymerization step, that is, ions contained in the solvent used in the dissolution step, is placed in the dialysis stream. It is preferable to further include a dialysis step (FIG. 2 (c)) to be immersed in water. More preferably, before the dialysis step, the solution containing the polymer polymer may be filtered using a filter, and then a dialysis step may be performed to remove ions contained in the solution.
  • the dialysis step may use a dialysis tube that passes the ionic component but does not pass the polymer polymer, preferably in the 12 ⁇ 14 kDa cutof f dialysis tube containing a solution containing the polymer prepared in water It is preferable to immerse for 3-5 days.
  • the dialysis time is less than 3 days, the removal of the ionic components contained in the solution containing the prepared polymer may not be sufficient, resulting in a decrease in biocompatibility or a decrease in mechanical strength of the structure manufactured by printing. If the dialysis time exceeds 5 days, there is no benefit over time, which may lower the economic efficiency.
  • the drying step of lyophilizing and drying the solution containing the polymer polymer passed through the dialysis step, the solution containing the polymer polymer in order to improve the circulation and storage of the liquid bio ink and to impart chemical stability of the bio ink All lyophilization and powdering are preferred.
  • the dialysis proceeds sufficiently to completely freeze the solution containing the polymer polymer from which the ionic component is removed first at -90 to -70 ° C for 10 to 14 hours, and then for 40 to 60 hours. Freeze drying is preferably performed at the same temperature as the freezing temperature.
  • the solution containing the freeze-dried polymer polymerizer is preferably powdered to have an appropriate size particle size through processing such as crushing and grinding.
  • the powder containing the polymer may be prepared in the bio ink of the present invention through a mixing step of mixing with a photoinitiator in water.
  • a mixing step it is preferable to mix 20-30 wt% of the powder containing the polymer polymer in water and 0.01-1.3 wt% of the photoinitiator, wherein the sum of water, the polymer polymer and the photoinitiator is 100 ⁇ . It is desirable not to exceed ⁇ %.
  • the bio ink of the present invention manufactured as described above may be gelled or cured by exposure to light having a wavelength at which the photoinitiator may initiate photopolymerization reaction as shown in FIG. 4 to form a hydrogel.
  • the bioinks of the present invention prepared by adding LAP, a photoinitiator, to a solution containing SGMA formed by polymerization of silk fibroin and glycidyl methacrylate (GMA) are used as light (ultraviolet, Exposure to UV) causes the photoinitiator LAP to attack the vinyl group of SGMA to generate free radicals, thereby binding within the chain of the double bond portion of the methacryl group of SGMA, between Not only covalent bonds are induced, but also the physical entanglement between the long chain of the polymer can be produced a structure of the hydrogel.
  • LAP light
  • a photoinitiator LAP causes the photoinitiator LAP to attack the vinyl group of SGMA to generate free radicals, thereby binding within the chain of the double bond portion of the methacryl group of SGMA, between Not only covalent bonds are induced, but also the physical entanglement between the long chain of the polymer can be produced a structure of the hydrogel.
  • the bio-structure in the hydrogel state may be manufactured by 3D printing, preferably DLP-type 3D printing using the aforementioned bio ink or the bio ink manufactured by the aforementioned manufacturing method.
  • 3D printing preferably DLP-type 3D printing using the aforementioned bio ink or the bio ink manufactured by the aforementioned manufacturing method.
  • 40 g of the cocoon was immersed in 1 L of 0.05 M sodium carbonate solution, heated to 100 0 C for 30 minutes, and washed several times with distilled water. Then, it was dried at room temperature to obtain 31.1 g (about 80% yield) of silkworm cocoon, ie, silk fibroin, from which sericin was removed.
  • GMG solution (Sigma-Aldrich, St. Louis, Missouri, USA) was added 2, 4, 6, 10 ml, respectively, and stirred for 3 hours at 300 rpm at a temperature of 60 ° C so that the silk fibroin and GMA could be fully polymerized.
  • a 13 kDa cut-off dialysis tube contains a solution containing silk fibroin and SGMA, which is a combination of GMA, and then the dilution water is completely immersed in distilled water. After leaving for 4 days, the ions and impurities contained in the solution were dialyzed and removed (see FIG. 2C).
  • the dialysis solution was frozen for 12 hours at an average temperature of -80 0 C, then lyophilized for 48 hours (see FIG. 2 (d)), and then powdered to give SGMA powder (in order of GMA dosage, SGMA-l , SGMA-2, SGMA-3, SGMA-4) were prepared (see FIG. 2 (e)).
  • All of SGMA-4 showed peaks at amide 1 (1639 cm “1 ), amide ⁇ (1512 cm “ 1 ), and amide ⁇ (1234 cm _1 ), representing 1105 halves of about 5000 amino acids contained in silk fibroin. Male amino acid.
  • the CH-0H peak which is weakly measured at 1238 ⁇ 1 , represents an alcohol group formed by breaking the epoxy ring of GMA, and the peaks of 1165 CITT 1 and 951 cnf 1 show the resonance of CH 2 of the methacrylate vinyl group of GMA. It was expected that the formed by the peak, it was confirmed that the intensity of the peak also increased as the amount of GMA contained.
  • the degree of denaturation of the methylene group of lysine of silk fibroin was estimated to be about 22-42% according to the change of GMA content.
  • Table 1 is a measurement of the width of each peak (vinyl group, lysine, methyl group of methacrylate) observed in FIG. 6 which is an NMR graph, and the degree of methacrylateization is 3 ⁇ 4 NMR of FIG. 6.
  • methacrylate may occur in all secondary amine groups having semi-ungwoong in silk fibroin, but since the representative amino acid of the secondary amine group having semi-ungwoong in silk fibroin is lysine group The degree of methacrylated in silk fibroin was confirmed based on the freshness.
  • SGMA-3 powder and lithium phenyl-2,4,6-trimethylbenzoylphosphinate powder (hereinafter referred to as LAP powder; Tokyo chemical industry, Tokyo, Japan) prepared in Preparation Example 1 were added to 1 L of water, respectively. After adding together, the SGMA '3 powder and the LAP powder were completely dissolved to prepare a bio ink.
  • the specimens printed with 10 10 X 2mm 3 hexahedron were 0.3 0.5, 1, respectively in 37 0 C PBS (phosphate reduced saline, ⁇ 7 ⁇ 4). After soaking for 2, 3, 4, 5 hours, the weight was measured (W swollen ), which was lyophilized to measure the weight (W dry ). The weight of the lyophilized SGMA powder was measured and the weight of the water absorbed state was measured based on this (100%), and the water absorption capacity (Q) was derived through the following Equation (2). Table 3 shows.
  • the prepared specimen was 10% SGMA-3.
  • cylindrical specimens (diameter; 6 mm, height; 12 mm) by 3D printing using bio inks of Examples 1 to 3 ((10% SGMA-3 to 30% SGMA-3) to measure mechanical strength. It was manufactured and pressured at a displacement rate of 5 ⁇ / min using a universal test ing device (QM100S, QMESYS, South Korea) equipped with a lO kgf load shell (FIG. 10) compressive stress, modulus of elasticity (el ast ic modul us) was measured, and the tensile stress (tensi le stress) was measured by setting the elongation rate 3 ⁇ 4! im / min at room temperature, and the Young's modulus was calculated as the slope of 50% strain deformation. The results of the experiment are shown in Table 3 and FIGS. 10 to 16.
  • the measured values also increase as the content ratio of SGMA-3 increases in all measurement results such as compressive stress, compressive strain, elastic modulus, tensile stress, elongation, and Young's modulus.
  • Example 3 containing 30% SGMA-3 it can be seen that the compression force is increased by about 2 times compared to Example 2.
  • Example 1 which is 10% SGMA-3, was too soft, The result was not obtained, but as the SGMA-3 content increased, it was confirmed that the measured values of tensile stress, elongation and Young's modulus increased.
  • Figure 16 is to confirm the strength or elasticity of the hydrogel, when a 7kg kettle bell on top of Example 3 which is 30% SGMA-3, Example 3 is a hydrogel weight of the In addition to supporting, after removing Kerbell after a certain period of time, it returned to the same shape as before the measurement.
  • the rheological properties were measured by using 0.1% strain and Anton Paar MCR 302 (Anton Paar, Zofingen, Swi tzer land) at 1 Hz frequency. 4 and FIGS. 17 and 18.
  • the storage modulus (G ') is about 6 times greater than the loss modulus (G' '), so the hydrogels of Examples 1 to 3 can be expected to behave in an elastomeric form. .
  • the phase change angle represents the state of the material to 6.4 ⁇ 9.1 ° (behavior like a solid '0 0 ', behave like a liquid '90 ⁇ ')
  • Example 1 (10% SGMA-3) to 3 It can be seen that the hydrogel prepared through 3D printing using (30% SGMA-3) bio ink has a solid state. Therefore, when combining the results of Experimental Example 2 (results of FIGS.
  • the measured value gradually increases with increasing SGMA content. It could be predicted that the silk fibroin polymer and GMA were polymerized by light irradiation and had excellent physical properties due to the entanglement between molecules and molecules. As a result, the reduction of the expansion rate according to the increase of the SGMA content in the bio ink could be expected to increase the degree of crystallization by increasing the chemical bond 1 ⁇ 2 degree in the molecule and increasing the rigidity and elasticity of the hydrogel.
  • Example 3 in particular SGMA-3 of 30> has the highest form stability.
  • the storage modulus was measured according to the change of SGMA-3 and LAP content of photoinitiator. 19 and FIG. 20.
  • FIG. 19 shows a tendency of stiffness of the hydrogel according to the change in the content of LAP, a photoinitiator, and the curing time of SGMA-3 (UV exposure for 4 seconds) of Examples 1 to 3 and 30, respectively, through FIG. 20.
  • the storage modulus (G ′) of the hydrogel was increased. This could be expected to stabilize the hydrophobi c domain in which silk fibroin (SF) methacrylated by GMA with increasing exposure to UV light.
  • SF silk fibroin
  • the storage modulus (G ′) rapidly decreased.
  • the gelation point of SGMA according to the change of the content of SGMA, a polymer polymer in bio ink, and LAP, a photoinitiator, is defined as a gelation point by defining the intersection point of storage modulus (G ') and loss elasticity (G ") Measured.
  • Cells used for cytotoxicity and cell proliferation experiments were HeLa cells and NIH / 3T3 cells, which were purchased from ATCC (Manassas, Virginia).
  • Cytotoxicity experiments were conducted using a LIVE / DEAD analysis kit (Life Technologies, USA), and observed with a fluorescence microscope to identify the cells with green fluorescence (calcein), that is, the living cells as shown in FIG.
  • the cell proliferation experiment was carried out through CC-8 analysis (Do jindo molecular technologt, Rockville, USA) and the results are shown in FIG.
  • GelMA Comparative Example 1
  • 10% SGMA-3 Example 1 measured with HeLa cells, as shown in FIG. 21, most cells showed green fluorescence on Day 1, indicating that living cells could be identified. have.
  • Figure 22 is a graph showing the rate of cell proliferation, based on the number of cells of GelMA (Comparative Example 1) and 10% SGMA-3C Example 1) per day GelMA (Comparative Example 1) cells gradually over time While the number of cells decreased, 10% SGMA-3 Example 1) showed a slight increase in the number of cells until about 7 days, but after 14 days, the number of cells increased significantly, indicating that the cells proliferated. It was confirmed in all 3T3 cells. . Therefore, summarizing the results of Experimental Example 2 and Experimental Example 3, the polymer polymer formed by polymerizing silk fibroin into a basic skeleton can cross-link upon exposure to light together with a photoinitiator. 3D printing can be used to produce 3D biostructures with excellent cell suitability as well as excellent physical properties.
  • the present invention is a polymer polymer in which the silk fibroin (Si lk Fibroin) and the methacrylate-based compound, which is a natural protein polymer, are polymerized; And photoinitiator]; and suggests a bio ink composition and a method of manufacturing the same, which can be applied to a 3D printer while maintaining excellent mechanical strength and cellular suitability of silk fibroin at an equivalent or higher.
  • Absorption, volume expansion rate, compressive strength Since almost no immune reaction occurs in a living tissue having mechanical properties such as tensile strength or in the body, an excellent biocompatibility can be manufactured through 3D printing, and thus there is industrial applicability.

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Abstract

La présente invention concerne une bioencre qui est préparée à partir de fibroïne de soie, une fibre de protéine naturelle, et à partir de laquelle des tissus biologiques ayant une cytocompatibilité améliorée et des propriétés mécaniques exceptionnelles peuvent être préparés à l'aide d'une imprimante 3D, et un procédé de préparation associé. Par impression 3D, la présente invention peut préparer un tissu biologique ayant des propriétés mécaniques telles que le pouvoir absorbant d'humidité, le taux d'expansion de volume, la résistance à la compression, la résistance à la traction, etc. De plus, la préparation d'une bioencre comprenant de la fibroïne de soie en tant que squelette de base permet la construction d'une biostructure qui ne provoque pratiquement aucune réponse immunitaire dans le corps et a donc une excellente biocompatibilité.
PCT/KR2018/003353 2017-04-04 2018-03-22 Bioencre et son procédé de préparation WO2018186611A2 (fr)

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CN112516330A (zh) * 2020-12-15 2021-03-19 上海交通大学医学院附属第九人民医院 丝素蛋白与甲状旁腺激素偶联接枝的方法及其应用
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US11891529B2 (en) 2019-03-12 2024-02-06 Unist(Ulsan National Institute Of Science And Technology) Ink composition for bioprinting and hydrogel formed from the same
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KR102180865B1 (ko) 2019-09-30 2020-11-19 한림대학교 산학협력단 전기전도성을 갖는 광가교 바이오 잉크 조성물 및 이의 제조방법
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KR102415399B1 (ko) 2021-03-24 2022-07-01 한림대학교 산학협력단 다중 세포 및 재료의 프린팅이 가능한, 마그네틱 체결 수조가 포함된 바이오 DLP(digital lighting processing) 3D 프린터
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CN113956506A (zh) * 2020-07-03 2022-01-21 中国科学院苏州纳米技术与纳米仿生研究所 一种双网络水凝胶及其制备方法与应用
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CN113713179B (zh) * 2021-09-06 2022-08-02 山东大学 高综合性能光固化生物3d打印复合水凝胶及其制备方法和应用
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