CN116726247A - Preparation method and application of 3D printing tenacious silk fibroin scaffold material - Google Patents
Preparation method and application of 3D printing tenacious silk fibroin scaffold material Download PDFInfo
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- CN116726247A CN116726247A CN202310711749.6A CN202310711749A CN116726247A CN 116726247 A CN116726247 A CN 116726247A CN 202310711749 A CN202310711749 A CN 202310711749A CN 116726247 A CN116726247 A CN 116726247A
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- 108010022355 Fibroins Proteins 0.000 title claims abstract description 63
- 238000010146 3D printing Methods 0.000 title claims abstract description 47
- 239000000463 material Substances 0.000 title claims abstract description 47
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 238000000016 photochemical curing Methods 0.000 claims abstract description 45
- 239000000243 solution Substances 0.000 claims abstract description 37
- 239000007864 aqueous solution Substances 0.000 claims abstract description 21
- AMXOYNBUYSYVKV-UHFFFAOYSA-M lithium bromide Chemical compound [Li+].[Br-] AMXOYNBUYSYVKV-UHFFFAOYSA-M 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 16
- -1 methacryloyl phosphate compound Chemical class 0.000 claims abstract description 13
- 239000000835 fiber Substances 0.000 claims abstract description 11
- VOZRXNHHFUQHIL-UHFFFAOYSA-N glycidyl methacrylate Chemical compound CC(=C)C(=O)OCC1CO1 VOZRXNHHFUQHIL-UHFFFAOYSA-N 0.000 claims abstract description 9
- 238000004108 freeze drying Methods 0.000 claims abstract description 8
- 230000004048 modification Effects 0.000 claims abstract description 8
- 238000012986 modification Methods 0.000 claims abstract description 8
- 159000000007 calcium salts Chemical class 0.000 claims abstract description 7
- 238000011534 incubation Methods 0.000 claims abstract description 3
- 238000001125 extrusion Methods 0.000 claims description 23
- 102100028188 Cystatin-F Human genes 0.000 claims description 18
- 101710169749 Cystatin-F Proteins 0.000 claims description 18
- 210000004027 cell Anatomy 0.000 claims description 16
- 102000007000 Tenascin Human genes 0.000 claims description 12
- 108010008125 Tenascin Proteins 0.000 claims description 12
- IGAAQDGISNXKQL-UHFFFAOYSA-L P(=O)(OC(C1=C(C(=C(C=C1C)C)C1=CC=CC=C1)C)=O)([O-])[O-].[Li+].[Li+] Chemical compound P(=O)(OC(C1=C(C(=C(C=C1C)C)C1=CC=CC=C1)C)=O)([O-])[O-].[Li+].[Li+] IGAAQDGISNXKQL-UHFFFAOYSA-L 0.000 claims description 6
- 150000001875 compounds Chemical class 0.000 claims description 6
- CXVZSCNFUYXXIS-UHFFFAOYSA-N phosphono 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OP(O)(O)=O CXVZSCNFUYXXIS-UHFFFAOYSA-N 0.000 claims description 6
- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 claims description 5
- 239000000920 calcium hydroxide Substances 0.000 claims description 5
- 229910001861 calcium hydroxide Inorganic materials 0.000 claims description 5
- 235000011116 calcium hydroxide Nutrition 0.000 claims description 5
- 229910019142 PO4 Inorganic materials 0.000 claims description 4
- 239000010452 phosphate Substances 0.000 claims description 4
- 210000000130 stem cell Anatomy 0.000 claims description 4
- WDFFWUVELIFAOP-UHFFFAOYSA-N 2,6-difluoro-4-nitroaniline Chemical compound NC1=C(F)C=C([N+]([O-])=O)C=C1F WDFFWUVELIFAOP-UHFFFAOYSA-N 0.000 claims description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
- 239000002202 Polyethylene glycol Substances 0.000 claims description 3
- 239000001110 calcium chloride Substances 0.000 claims description 3
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 3
- 229960002713 calcium chloride Drugs 0.000 claims description 3
- 235000011148 calcium chloride Nutrition 0.000 claims description 3
- 229920001223 polyethylene glycol Polymers 0.000 claims description 3
- NXBXJOWBDCQIHF-UHFFFAOYSA-N 2-[hydroxy-[2-(2-methylprop-2-enoyloxy)ethoxy]phosphoryl]oxyethyl 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OCCOP(O)(=O)OCCOC(=O)C(C)=C NXBXJOWBDCQIHF-UHFFFAOYSA-N 0.000 claims description 2
- SEILKFZTLVMHRR-UHFFFAOYSA-N 2-phosphonooxyethyl 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OCCOP(O)(O)=O SEILKFZTLVMHRR-UHFFFAOYSA-N 0.000 claims description 2
- WOBHKFSMXKNTIM-UHFFFAOYSA-N Hydroxyethyl methacrylate Chemical compound CC(=C)C(=O)OCCO WOBHKFSMXKNTIM-UHFFFAOYSA-N 0.000 claims description 2
- KLGDRWGOXDJNPH-UHFFFAOYSA-N P(=O)(O)(O)O.C1(=CC=CC=C1)C=1C(=C(C(=O)[Li])C(=CC1C)C)C Chemical compound P(=O)(O)(O)O.C1(=CC=CC=C1)C=1C(=C(C(=O)[Li])C(=CC1C)C)C KLGDRWGOXDJNPH-UHFFFAOYSA-N 0.000 claims description 2
- 239000004227 calcium gluconate Substances 0.000 claims description 2
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- 235000013927 calcium gluconate Nutrition 0.000 claims description 2
- 229940095643 calcium hydroxide Drugs 0.000 claims description 2
- MKJXYGKVIBWPFZ-UHFFFAOYSA-L calcium lactate Chemical compound [Ca+2].CC(O)C([O-])=O.CC(O)C([O-])=O MKJXYGKVIBWPFZ-UHFFFAOYSA-L 0.000 claims description 2
- 239000001527 calcium lactate Substances 0.000 claims description 2
- 229960002401 calcium lactate Drugs 0.000 claims description 2
- 235000011086 calcium lactate Nutrition 0.000 claims description 2
- NEEHYRZPVYRGPP-UHFFFAOYSA-L calcium;2,3,4,5,6-pentahydroxyhexanoate Chemical compound [Ca+2].OCC(O)C(O)C(O)C(O)C([O-])=O.OCC(O)C(O)C(O)C(O)C([O-])=O NEEHYRZPVYRGPP-UHFFFAOYSA-L 0.000 claims description 2
- 238000005286 illumination Methods 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 2
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- 238000007639 printing Methods 0.000 abstract description 10
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- 238000002156 mixing Methods 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 230000008569 process Effects 0.000 description 2
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- 238000003892 spreading Methods 0.000 description 2
- VALXVSHDOMUUIC-UHFFFAOYSA-N 2-methylprop-2-enoic acid;phosphoric acid Chemical compound OP(O)(O)=O.CC(=C)C(O)=O VALXVSHDOMUUIC-UHFFFAOYSA-N 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
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- 101800001554 RNA-directed RNA polymerase Proteins 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 229920001872 Spider silk Polymers 0.000 description 1
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- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
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Classifications
-
- 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
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
-
- 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
-
- 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
- A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem 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
- A61L27/50—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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/58—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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
- C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
Abstract
The invention discloses a preparation method and application of a 3D printing tenacious silk fibroin bracket material. The method comprises the following steps: dissolving silk fibroin fibers in a lithium bromide aqueous solution for incubation; freeze-drying after modification by using glycidyl methacrylate; dissolving in a photoinitiator aqueous solution to obtain biological ink; dissolving calcium salt in the methacryloyl phosphate compound to obtain an intermediate solution; dissolving the intermediate solution in the biological ink to obtain photo-curing biological ink; and (3) placing the fiber into a 3D printer for photo-curing and printing to obtain the 3D printing tenacious silk fibroin bracket material. The method has the advantages of good formability, mobility matched with a 3D printer, suitability for various 3D printing modes, rapid photo-curing performance, good biocompatibility and the like, and the prepared printing bracket has better mechanical property and stability and slow degradation rate compared with SilMA, so that the printing bracket can be widely applied to various fields such as bone tissue engineering and the like.
Description
Technical Field
The invention relates to a preparation method of a scaffold material, relates to the field of biological materials, and in particular relates to a preparation method of a 3D printing tenascin protein scaffold material.
Background
Silk protein biomaterials are of great interest because of their excellent mechanical properties, biocompatibility, immune rejection free, degradability and natural protein structural features. Among them, bombyx mori silk fibroin, tussah silk fibroin and spider silk fibroin are representative silk protein biomaterials. The materials can be used for producing biological materials with different shapes and different functions, such as bone tissue repair, wound surface covering materials and medicine slow release materials, so as to meet the diversified demands of human beings on the biological materials.
The 3D printing has the characteristics of strong customization, high precision, strong substitution, high production efficiency, high safety and the like. The 3D printing technology can produce biological materials which are implanted according with the damaged part according to the individual needs of patients so as to realize personalized medical treatment. The shape and size of the material are precisely controlled by 3D printing, thereby producing a precise biomaterial. This is important for complex tissue engineering and the manufacture of medical devices. In addition, 3D printing techniques can produce biological materials similar to human tissue that can be used to repair or replace damaged tissues or organs. Therefore, it can be widely used in the medical field including tissue engineering, surgery, stomatology, etc. The technology can automatically produce biological materials, thereby greatly shortening the production time and the cost. In addition, since the printed material has higher precision than that of manual production, the error of manual operation can be reduced, and thus the safety and reliability of production are improved. This is particularly important for high risk medical applications.
SilMA, which has been developed since 2018 as a bio-ink material for photo-curing 3D printing, has been widely used in biomedical fields. The preparation method has the advantages of simple production process, controllable components, no toxicity or cancerogenic risk, and can be used in human body. Because it can be degraded by enzymes in the body, it can be gradually metabolized and cleared, so that it does not need to be removed by secondary operation, so that it can reduce damage to human body. However, because SilMA is too fluid, it can only be printed by photo-curing 3D printing techniques. In addition, the photo-cured SilMA has general formability and poor stability in aqueous solution, and printing conditions required by different types and even different batches of biological ink materials are different, so that accurate regulation and control and large-scale industrial production are difficult to perform. In addition, for the extrusion type 3D printing technology, the viscosity of the material is required to be moderate, so that the material is not too thick, difficult to extrude, and too thin, and the shape of the material cannot be maintained. However, silMA has too good fluidity to maintain morphology during extrusion, making it unusable in the field of extrusion 3D printing. For the above reasons, silMA has limitations in different modes of 3D printing technology and further limits its application in the biomedical field.
Disclosure of Invention
In order to solve the problems in the background art, the preparation method and the application of the 3D printing tenacious silk fibroin bracket material provided by the invention can meet the actual demands by improving the viscosity before the SilMA is cured and the mechanical strength after the SilMA is cured.
The technical scheme adopted by the invention is as follows:
the preparation method of the 3D printing tenacious silk fibroin scaffold material comprises the following steps:
1) And (3) dissolving silk fibroin fibers obtained after degumming the cocoons in a lithium bromide aqueous solution for incubation to obtain silk fibroin solution.
2) The silk fibroin solution is modified by using glycidyl methacrylate and then freeze-dried to obtain the methacryloylated silk fibroin SilMA.
3) Dissolving the methacryloyl silk fibroin SilMA in an aqueous solution containing a photoinitiator to obtain the biological ink SilMA.
4) Dissolving calcium salt in a methacryloyl phosphate MAP compound to obtain an intermediate solution CMAP; and dissolving the intermediate solution CMAP in the bio-ink SilMA to obtain the photo-curing bio-ink BSC.
5) And (3) placing the photo-curing bio-ink BSC into an extrusion type 3D printer or a digital photo-curing 3D printer, photo-curing under illumination, and performing 3D printing to obtain the 3D printing tenacious silk fibroin bracket material with excellent mechanical properties and biocompatibility.
In the step 1), 20g of silk fibroin fibers obtained after degumming silkworm cocoons are dissolved in 100mL of 9.3M lithium bromide aqueous solution, and incubated for 1h at 60 ℃ to obtain silk fibroin solution.
In the step 2), 3-10mL of glycidyl methacrylate is used for carrying out modification on 100mL of silk fibroin solution at 25-60 ℃ for 1-5h, and then freeze drying is carried out to obtain the methacryloyl silk fibroin SilMA.
In the step 3), the aqueous solution containing the photoinitiator is specifically an aqueous solution of phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate LAP with the concentration of 0.25 wt%.
Dissolving the methacryloyl silk fibroin SilMA in a concentration of 10-20wt% in a lithium phenyl (2, 4, 6-trimethylbenzoyl) phosphate LAP aqueous solution with a concentration of 0.25wt% to obtain the biological ink SilMA.
In the step 4), 0.04-0.05g of calcium salt is dissolved in 1mL of methacryloyl phosphate MAP compound to obtain an intermediate solution CMAP; the intermediate solution CMAP was dissolved in bio-ink SilMA at a concentration of 2-3wt% to obtain a photo-cured bio-ink BSC.
In the step 5), the photo-curing bio-ink BSC is placed into an extrusion type 3D printer or a digital photo-curing 3D printer, and photo-curing is carried out under the irradiation of an ultraviolet light source with the wavelength of 450 nm.
In the step 4), the calcium salt is specifically calcium gluconate, calcium chloride, calcium hydroxide or calcium lactate.
In the step 4), the methacryloyl phosphate MAP compound is specifically 2-hydroxyethyl methacrylate phosphate HEMAP, bis [2- (methacryloyloxy) ethyl ] phosphate BMAP, 2-methacryloyloxy ethyl phosphate or polyethylene glycol methacrylate phosphate.
The invention adopts simple bi-component mixing, and can obtain the bio-ink with good biocompatibility by adding a small amount of MAP into SilMA, the ink has an organic-inorganic composite structure without interphase boundaries in the nano-scale after photo-curing, thereby improving the mechanical strength, the fluidity of the ink is controllable, and the ink can be well printed and molded by using 3D printing technologies in forms of extrusion, photo-curing and the like by adjusting the concentration of MAP.
The 3D printing tenascin protein scaffold material is used for biomedical application in inoculating adult cells and stem cells.
The 3D printing tenacious silk fibroin bracket can be used for experiments with human fibroblasts: human fibroblasts are inoculated into the silk fibroin scaffold, and the cells can grow on the surface and inside the scaffold, so that the human fibroblasts have good cell proliferation capability.
The 3D printing tenacious silk fibroin bracket can be used for experiments with mesenchymal stem cells: human mesenchymal stem cells are inoculated into the regular lamellar structure silk fibroin scaffold, so that the mesenchymal stem cells have a high adherence effect after one day of culture, can promote cells to secrete a large amount of collagen and alkaline phosphatase after two weeks of culture, and have a good capability of promoting directional differentiation of the stem cells.
The silk fibroin material used in the invention is a natural active material, is widely used in the biological medicine industry, and has no toxic reaction to cells or tissues. The addition of MAP makes liquid biological ink become viscous state and keeps good forming property, so that SilMA which cannot be used for extrusion type 3D printing originally can be used for 3D printing forming by an extrusion method, and the printing efficiency of the three-dimensional biological bracket is greatly improved.
The invention fully develops the improvement of the mechanical properties of the multiple components and can be applied to the field of extrusion type 3D printing. In addition, the material integrates the advantage of good biological compatibility of SilMA, belongs to an ideal three-dimensional support material, and can provide reference information for researching the design and preparation process of the photo-curing 3D printing biological ink.
The beneficial effects of the invention are as follows:
1) Excellent biocompatibility: the components are safe, and the CCK-8 proves that the material has no biological toxicity and no toxic or side effect on organism tissues.
2) No pollution to the environment: the preparation process does not use toxic reagent, the preparation condition is mild, and the product which is toxic to human body and environment is not produced.
3) The process is simple and rapid: and (3) simply mixing the prepared SilMA and CMAP, and placing the mixture into an extrusion type 3D printer or a photo-curing 3D printer to print the bracket.
4) The mechanical property of the bracket is improved: compared with SilMA, the BSC bracket has obviously improved mechanical strength, better swelling resistance, better degradation resistance and better cyclic compression performance.
5) Improving the adhesion of cells to the scaffold: the BSC scaffold has significantly enhanced hardness compared to SilMA, which is advantageous for cell adhesion and spreading.
Therefore, the method provided by the invention has the advantages of good formability, flowability matched with a 3D printer, suitability for various 3D printing modes, rapid photocuring performance, good biocompatibility and the like, and the prepared printing bracket has the advantages of better mechanical property and stability, slow degradation rate and the like compared with SilMA, and the proliferation performance of human cells and the directional differentiation performance of stem cells are obviously improved, so that the bone tissue repairing effect of the biological bracket is improved, the printing bracket can be widely applied to various fields such as bone tissue engineering and the like, and has wide application prospects in the fields of tissue engineering, medicine slow release, hemostatic materials and filtering membranes.
Drawings
Fig. 1 is a scanning electron microscope image of a BSC support after photo-curing and a SilMA support in example 1, wherein a in fig. 1 is a scanning electron microscope image of a BSC support after photo-curing in example 1, and B in fig. 1 is a scanning electron microscope image of a SilMA support after photo-curing in example 1;
fig. 2 is a topography of BSC bio-ink and bio-ink SilMA in example 1 and an extrusion type 3D print of BSC bio-ink and bio-ink SilMA, wherein a of fig. 2 is a topography of BSC bio-ink in example 1, B of fig. 2 is a topography of bio-ink SilMA in example 1, C of fig. 2 is an extrusion type 3D print of BSC bio-ink in example 1, and D of fig. 2 is an extrusion type 3D print of bio-ink SilMA in example 1;
FIG. 3 is a graph showing compression curves and rheological curves of the BSC and the SilMA support after photo-curing in example 3, wherein A in FIG. 3 is a graph showing compression curves of the BSC and the SilMA support after photo-curing in example 3, and B in FIG. 3 is a graph showing rheological curves of the BSC and the SilMA support after photo-curing in example 3;
fig. 4 is a topography of a cell optical microscope on a BSC and SilMA scaffold after photocuring in example 4 of human fibroblasts, wherein a of fig. 4 is a topography of a cell optical microscope on a BSC scaffold after photocuring in example 4 of human fibroblasts, and B of fig. 4 is a topography of a cell optical microscope on a SilMA scaffold of human fibroblasts.
Detailed Description
The invention will be described in further detail with reference to the accompanying drawings and specific examples.
Specific embodiments of the invention are as follows:
example 1:
20g silk fibroin fiber obtained after degumming silkworm cocoons is dissolved in 100mL of 9.3M lithium bromide aqueous solution, and incubated for 1h at 60 ℃ to obtain silk fibroin solution; 3mL of glycidyl methacrylate is used for carrying out modification on 100mL of silk fibroin solution at 60 ℃ for 3 hours, and then freeze drying is carried out to obtain methacryloylated silk fibroin SilMA; dissolving methacryloyl silk fibroin SilMA in a concentration of 15wt% in a lithium phenyl (2, 4, 6-trimethylbenzoyl) phosphate LAP aqueous solution with a concentration of 0.25wt% to obtain biological ink SilMA; 0.05g of calcium chloride CaCl 2 Dissolved in 1mL of bis [2- (methacryloyloxy) ethyl]Obtaining an intermediate solution CMAP from phosphate BMAP; dissolving an intermediate solution CMAP in biological ink SilMA at a concentration of 3wt% to obtain a photo-cured biological ink BSC; placing viscous light-cured biological ink BSC into an extrusion type 3D printer or digital light-cured 3D printer, and performing light curing under irradiation of ultraviolet light source with wavelength of 450nm to obtain 3And D printing to obtain the 3D printing tenacious silk fibroin bracket material with excellent mechanical property and biocompatibility. The viscosity of the photo-curable bio-ink BSC increases with increasing CMAP content of the intermediate solution.
The scanning electron microscope of the bracket after the light-cured bio-ink BSC in the embodiment 1 is shown as A in fig. 1, and the scanning electron microscope of the bracket after the bio-ink SilMA is cured is shown as B in fig. 1; the optical topography of the photo-curing bio-ink BSC and the photo-curing bio-ink SilMA are shown as A in FIG. 2 and B in FIG. 2, and the printing process of the photo-curing bio-ink BSC and the photo-curing bio-ink SilMA in the extrusion type 3D printer is shown as C in FIG. 2 and D in FIG. 2 respectively; the roughness of BSC and SilMA is obviously improved. BSC can be used for extrusion type 3D printing, while SilMA has too low viscosity and high fluidity to be molded by an extrusion type 3D printer, i.e. the printing accuracy of SilMA under extrusion type 3D printing is poor.
Example 2:
20g silk fibroin fiber obtained after degumming silkworm cocoons is dissolved in 100mL of 9.3M lithium bromide aqueous solution, and incubated for 1h at 60 ℃ to obtain silk fibroin solution; 6mL of glycidyl methacrylate is used for carrying out modification on 100mL of silk fibroin solution for 4 hours at 60 ℃ and then freeze-drying is carried out to obtain methacryloylated silk fibroin SilMA; dissolving methacryloyl silk fibroin SilMA in a concentration of 10wt% in a lithium phenyl (2, 4, 6-trimethylbenzoyl) phosphate LAP aqueous solution with a concentration of 0.25wt% to obtain biological ink SilMA; 0.04g of calcium hydroxide Ca (OH) 2 An intermediate solution CMAP was obtained by dissolving 1mL of 2-hydroxyethyl methacrylate phosphate HEMAP; dissolving an intermediate solution CMAP in biological ink SilMA at a concentration of 3wt% to obtain a photo-cured biological ink BSC; and (3) placing the photo-curing bio-ink BSC into an extrusion type 3D printer or a digital photo-curing 3D printer, performing photo-curing under the irradiation of an ultraviolet light source with the wavelength of 450nm, and performing 3D printing to obtain the 3D printing tenascin protein scaffold material with excellent mechanical properties and biocompatibility.
Example 3:
20g silk fibroin fiber obtained after degumming silkworm cocoon is dissolved in 100mL of 9.3M lithium bromide water solution, and incubated at 60 DEG C1h, obtaining a silk fibroin solution; performing modification on 100mL of silk fibroin solution for 1h at 30 ℃ by using 10mL of glycidyl methacrylate, and performing freeze drying to obtain methacryloylated silk fibroin SilMA; dissolving methacryloyl silk fibroin SilMA in a concentration of 20wt% in a lithium phenyl (2, 4, 6-trimethylbenzoyl) phosphate LAP aqueous solution with a concentration of 0.25wt% to obtain biological ink SilMA; 0.05g of calcium hydroxide Ca (OH) 2 Dissolved in 1mL of bis [2- (methacryloyloxy) ethyl]Obtaining an intermediate solution CMAP from phosphate BMAP; dissolving an intermediate solution CMAP in biological ink SilMA at a concentration of 2wt% to obtain a photo-cured biological ink BSC; pouring the photo-curing bio-ink BSC into a cylindrical mold, and photo-curing under the irradiation of an external ultraviolet light source with the wavelength of 450nm to obtain the tenacious silk fibroin bracket material.
Comparative example of example 3:
pouring the biological ink SilMA of the embodiment 3 into a cylindrical mold, and carrying out photo-curing under the irradiation of an external ultraviolet light source with the wavelength of 450nm to obtain a contrast curing material, namely a control material for the tenacious silk fibroin scaffold material.
Performing compression test experiments on the photo-cured and molded contrast curing material and the tough silk fibroin bracket material by using a universal tester, wherein the obtained compression stress-strain curve is shown as A in figure 3; the results of the change (rheological properties) of the storage modulus and the loss modulus of the bio-ink SilMA and the photo-curing bio-ink BSC under ultraviolet irradiation are shown in the diagram of FIG. 3B; the graph shows that the BSC and the SilMA have obviously improved mechanical strength and photo-curing rate.
Example 4:
20g silk fibroin fiber obtained after degumming silkworm cocoons is dissolved in 100mL of 9.3M lithium bromide aqueous solution, and incubated for 1h at 60 ℃ to obtain silk fibroin solution; 6mL of glycidyl methacrylate is used for carrying out modification on 100mL of silk fibroin solution at 25 ℃ for 5 hours, and then freeze drying is carried out to obtain methacryloylated silk fibroin SilMA; dissolving methacryloyl silk fibroin SilMA in a concentration of 15wt% in a lithium phenyl (2, 4, 6-trimethylbenzoyl) phosphate LAP aqueous solution with a concentration of 0.25wt% to obtain biological ink SilMA; will be0.05g of calcium hydroxide Ca (OH) 2 Dissolving in 1mL of polyethylene glycol methacrylic acid phosphate to obtain an intermediate solution CMAP; dissolving an intermediate solution CMAP in biological ink SilMA at a concentration of 3wt% to obtain a photo-cured biological ink BSC; the photo-curing bio-ink BSC is coated in a 24-hole plate, photo-cured under the irradiation of an ultraviolet light source with the wavelength of 450nm, and then is put into human fiber cells for culturing.
Comparative example of example 4:
the bio-ink SilMA of example 4 was applied to a 24-well plate, photo-cured under irradiation of an ultraviolet light source having a wavelength of 450nm, and then cultured in human fibroblasts.
After 7 days of culture, the morphology of the cells of human fibroblasts on the cured BSC scaffold obtained in example 4 is shown in FIG. 4A. The morphology of the cells on the SilMA scaffold after solidification of human fibroblasts is shown in fig. 4B. Cell culture results show that the human fibroblasts can adhere and proliferate on the BSC bracket at an early stage, and have good biocompatibility; the SilMA control group bracket has smaller cell quantity and smaller cell spreading area.
In summary, the novel silk fibroin biological ink is prepared by the embodiment of the invention, so that the viscosity, the formability and the mechanical properties of the SilMA are greatly improved, and the curing time of the SiLMA is shortened, so that the SiLMA which cannot be used for extrusion type 3D printing can be molded by an extrusion method. The mass production efficiency of the silk protein 3D printing material is greatly improved. Cells can adhere, proliferate and differentiate well on the BSC scaffolds. The improvement of the mechanical strength can also accelerate the osteogenic differentiation efficiency of the cells. Therefore, the method can prepare the biological scaffold which has good biocompatibility and obvious cell culture effect and can induce the differentiation of the mesenchymal stem cells.
Claims (10)
1. A preparation method of a 3D printing tenacious silk fibroin scaffold material is characterized by comprising the following steps: the method comprises the following steps:
1) Dissolving silk fibroin fibers in a lithium bromide aqueous solution for incubation to obtain a silk fibroin solution;
2) Modification of the silk fibroin solution by using glycidyl methacrylate, and freeze drying to obtain the methacryloylated silk fibroin SilMA;
3) Dissolving the methacryloyl silk fibroin SilMA in a water solution containing a photoinitiator to obtain biological ink SilMA;
4) Dissolving calcium salt in a methacryloyl phosphate MAP compound to obtain an intermediate solution CMAP; dissolving an intermediate solution CMAP in biological ink SilMA to obtain photo-curing biological ink BSC;
5) And (3) placing the photo-curing bio-ink BSC into an extrusion type 3D printer or a digital photo-curing 3D printer, and performing photo-curing and 3D printing under illumination to obtain the 3D printing tenacious silk fibroin bracket material.
2. The method for preparing the 3D printing tenascin protein scaffold material, which is characterized by comprising the following steps of: in the step 1), 20g of silk fibroin fibers are dissolved in 100mL of 9.3M lithium bromide aqueous solution, and incubated for 1h at 60 ℃ to obtain silk fibroin solution.
3. The method for preparing the 3D printing tenascin protein scaffold material, which is characterized by comprising the following steps of: in the step 2), 3-10mL of glycidyl methacrylate is used for carrying out modification on 100mL of silk fibroin solution at 25-60 ℃ for 1-5h, and then freeze drying is carried out to obtain the methacryloyl silk fibroin SilMA.
4. The method for preparing the 3D printing tenascin protein scaffold material, which is characterized by comprising the following steps of: in the step 3), the aqueous solution containing the photoinitiator is specifically an aqueous solution of phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate LAP with the concentration of 0.25 wt%;
dissolving the methacryloyl silk fibroin SilMA in a concentration of 10-20wt% in a lithium phenyl (2, 4, 6-trimethylbenzoyl) phosphate LAP aqueous solution with a concentration of 0.25wt% to obtain the biological ink SilMA.
5. The method for preparing the 3D printing tenascin protein scaffold material, which is characterized by comprising the following steps of: in the step 4), 0.04-0.05g of calcium salt is dissolved in 1mL of methacryloyl phosphate MAP compound to obtain an intermediate solution CMAP; the intermediate solution CMAP was dissolved in bio-ink SilMA at a concentration of 2-3wt% to obtain a photo-cured bio-ink BSC.
6. The method for preparing the 3D printing tenascin protein scaffold material, which is characterized by comprising the following steps of: in the step 5), the photo-curing bio-ink BSC is placed into an extrusion type 3D printer or a digital photo-curing 3D printer, and photo-curing is carried out under the irradiation of an ultraviolet light source with the wavelength of 450 nm.
7. The method for preparing the 3D printing tenascin protein scaffold material, which is characterized by comprising the following steps of: in the step 4), the calcium salt is specifically calcium gluconate, calcium chloride, calcium hydroxide or calcium lactate.
8. The method for preparing the 3D printing tenascin protein scaffold material, which is characterized by comprising the following steps of: in the step 4), the methacryloyl phosphate MAP compound is specifically 2-hydroxyethyl methacrylate phosphate HEMAP, bis [2- (methacryloyloxy) ethyl ] phosphate BMAP, 2-methacryloyloxy ethyl phosphate or polyethylene glycol methacrylate phosphate.
9. The 3D printing tenascin protein scaffold material prepared by the preparation method of the 3D printing tenascin protein scaffold material according to any one of claims 1 to 8.
10. The use of the 3D printed tenascin-scaffold material prepared by the preparation method of the 3D printed tenascin-scaffold material according to any one of claims 1-8 for seeding of adult cells and stem cells.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117180506A (en) * | 2023-09-19 | 2023-12-08 | 广州贝奥吉因生物科技股份有限公司 | Bracket for treating infectious bone defect and preparation method and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117180506A (en) * | 2023-09-19 | 2023-12-08 | 广州贝奥吉因生物科技股份有限公司 | Bracket for treating infectious bone defect and preparation method and application thereof |
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