CN110859994B - Modified tussah silk fibroin 3D printing support and preparation method thereof - Google Patents

Modified tussah silk fibroin 3D printing support and preparation method thereof Download PDF

Info

Publication number
CN110859994B
CN110859994B CN201910773760.9A CN201910773760A CN110859994B CN 110859994 B CN110859994 B CN 110859994B CN 201910773760 A CN201910773760 A CN 201910773760A CN 110859994 B CN110859994 B CN 110859994B
Authority
CN
China
Prior art keywords
silk fibroin
tussah silk
printing
modified
preparation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201910773760.9A
Other languages
Chinese (zh)
Other versions
CN110859994A (en
Inventor
邵惠丽
邹盛之
张耀鹏
王心如
张佳明
范苏娜
胡学超
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Donghua University
Original Assignee
Donghua University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Donghua University filed Critical Donghua University
Priority to CN201910773760.9A priority Critical patent/CN110859994B/en
Publication of CN110859994A publication Critical patent/CN110859994A/en
Application granted granted Critical
Publication of CN110859994B publication Critical patent/CN110859994B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

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
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/222—Gelatin
    • 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
    • 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
    • B33Y10/00—Processes of additive manufacturing
    • 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
    • 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
    • B33Y80/00—Products made by additive manufacturing
    • 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Dermatology (AREA)
  • Medicinal Chemistry (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Manufacturing & Machinery (AREA)
  • Materials Engineering (AREA)
  • Materials For Medical Uses (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention relates to a modified tussah silk fibroin 3D printing bracket and a preparation method thereof, wherein the modified tussah silk fibroin 3D printing bracket is prepared by 3D printing of core part printing ink prepared from chemically modified tussah silk fibroin nano microfibers and shell part printing ink prepared from a chemically modified tussah silk fibroin nano microfiber/gelatin composite system; the modified tussah silk fibroin 3D printing support has a compression modulus of 100-600 MPa after being soaked in genipin with a mass concentration of 0.1-5 wt% for crosslinking reaction for 24 hours, and the survival rate and the proliferation rate of induced pluripotent stem cells are high after being cultured for 10 days; the finally prepared modified tussah silk fibroin 3D printing support is formed by printing lines with a core-shell structure. The preparation method is relatively simple, and the prepared 3D printing support has excellent mechanical property, excellent biocompatibility and good tissue repair capability.

Description

Modified tussah silk fibroin 3D printing support and preparation method thereof
Technical Field
The invention belongs to the technical field of biological 3D printing, and relates to a modified tussah silk fibroin 3D printing support and a preparation method thereof.
Background
Tissue and organ defects and dysfunction caused by trauma, tumor, aging and the like are important factors harming human health, and repair and functional reconstruction of the defective tissue and organ are the international difficult problems at present. Large-area defects are usually repaired by adopting autologous or allogeneic tissues and organ transplantation, the autologous transplantation has the problem of repairing wounds by using wounds, and the allogeneic transplantation treatment also has the main defects of insufficient donor sources, immunological rejection and the like. The proposal, establishment and development of tissue engineering (tissue engineering) provide a new way for solving the problems of tissue and organ defects and dysfunction. Since the assumption that cells are implanted into degradable biological materials to construct tissues was proposed in the early 80 s of the last century, until the success of applying tissue engineering technology to repair clinical defects in recent years, the tissue engineering technology has proved to be one of effective ways for solving tissue wound repair and functional reconstruction.
In recent years, the rise of biological three-dimensional (3D) printing technology has attracted attention in the field of regenerative reconstruction of human tissues or organs. The biological 3D printing technology simulates different tissues and organs of a human body through a CAD technology and is controlled by a computer to print by using biological materials, seed cells and other biological reagents as ink to reconstruct tissues and organs of the human body. The support structure printed by biological 3D has accuracy and specificity, and can maintain cell activity, thereby meeting the reconstruction requirements of various complex tissues and organs of a human body, and therefore, the biological 3D printing can certainly cause the technical revolution in the field of biomedicine. However, one of the major challenges facing bio-3D printing technology is the choice of bio-ink used. The traditional materials for 3D printing are mainly some synthetic high polymers such as polyethylene glycol (PEG), Polycaprolactone (PCL), polyglycolic acid (PGA), polyvinyl alcohol (PVA), polylactic acid (PLA) and the like, the materials have the advantages of easiness in forming and high resolution, but the defects of high processing temperature and poor cell compatibility exist, toxic substances are generally introduced into the stent prepared by using the materials in the preparation process, and if the toxic substances cannot be well removed, the biocompatibility of the stent is negatively influenced.
Compared with the synthetic materials, silk fibroin is a natural polymer material with better biocompatibility, and a plurality of people have already studied the application of silk fibroin in the biological material, and also apply silk fibroin in the field of 3D printing to prepare the biological scaffold material.
Patent CN108085760A discloses a paper-like graphene-modified photo-cured fibroin three-dimensional printing material and a preparation method thereof, wherein the paper-like graphene-modified photo-cured fibroin three-dimensional printing material comprises ink for 3D printing, and the ink comprises graphene oxide solution and silkworm fibroin solution; patent CN107744601A discloses a three-dimensional printing wound coating material based on silk microsphere biological ink and a preparation method thereof, wherein the three-dimensional printing wound coating material comprises ink for 3D printing, and the ink comprises silkworm fibroin solution and aspirin; patent CN105031728A discloses a low-temperature rapid prototyping three-dimensional printing collagen silk fibroin material, which comprises silk fibroin ink for 3D printing, wherein the ink comprises silkworm silk fibroin, collagen, glycerol and a compatilizer; patent CN105903071A discloses a corneal scaffold material, a preparation method thereof and a 3D printing method of corneal scaffold, wherein silk fibroin ink for 3D printing is contained, the ink comprises silkworm silk fibroin, collagen, graphene oxide and a cross-linking agent; patent CN104958785A discloses a composite bone repair material with a secondary three-dimensional structure and a preparation method thereof, wherein the composite bone repair material comprises silk fibroin ink for 3D printing, the ink comprises silkworm silk fibroin, hydroxyapatite and collagen, and the elastic modulus of a 3D tissue engineering scaffold prepared by the method is in the range of 290-430 kPa.
In the document 3D printing of single-particle-reinforced chitosan hydrogels and the third properties, silkworm silk fibroin (BSF), chitosan and water are used to print, and the maximum compression modulus of the obtained scaffold can reach 5 KPa; the document "structured and functionalized bulk-fiber-collagen using 3D printing prosthesis in surgery and in vivo" uses silkworm silk fibroin (BSF), gelatin and water to make composite biological 3D printing ink for printing, and the elastic modulus of the obtained scaffold can reach 15kPa at most; the document "Precisely printable and Biocompatable Silk Fibroin (BSF) biological 3D printing ink modified by glycidyl methacrylate is used for printing, and the elastic modulus of the obtained scaffold can reach 120KPa at most.
In the above studies, the 3D printing technology is applied to the field of silk fibroin, but the above studies only study the 3D printing of silkworm silk fibroin. The special China tussah silk fibroin molecular chain has RGD tripeptide chain segments which are formed by connecting arginine, glycine and aspartic acid and are not contained in the tussah silk fibroin molecular chain, and the chain segments are proved to have higher cell adhesion, so compared with the tussah silk fibroin, the tussah silk fibroin is expected to have better biocompatibility. In addition, the tussah silk fibroin contains a large number of alanine segments (AAAAAAAAA …) in the molecular chain, and compared with a glycine-alanine-serine alternating segment (GAGAGASGAGAGAS …) contained in a large number in the tussah silk fibroin, the tussah silk fibroin has higher regularity and can endow the printing support with more excellent mechanical properties. Therefore, compared with the silkworm silk fibroin, the tussah silk fibroin has more potential application in the field of 3D printing.
Therefore, patent CN106267370A discloses silk fibroin/cellulose 3D printing ink, wherein the ink comprises water-soluble silk fibroin, water-insoluble cellulose micro/nano materials, non-toxic polyol and water, and the compression modulus of a 3D support printed by using the ink is within the range of 10-50 MPa. The content disclosed by the patent contains the relevant content of the 3D printing ink based on the tussah silk fibroin aqueous solution, but related researchers find that the tussah silk fibroin is easier to form an anti-parallel zigzag structure under the action of hydrogen bonds among molecular chains, is more sensitive to the action of temperature and external force, and the tussah silk fibroin solution is very easy to perform conformational transition under the influence of the action of temperature and external force to form a water-insoluble solid, so that the whole preparation process is influenced.
Moreover, the large number of alanine hydrophobic segments (AAAAAAAAA …) in the tussah silk fibroin molecular chain can also cause the printed scaffold to have low hydrophilicity, thereby causing adverse effects on the biocompatibility of the scaffold.
In order to solve the problem of low hydrophilicity of the printed scaffold, if a tussah silk fibroin molecular chain can be subjected to certain chemical modification and a certain hydrophilic group is introduced on the molecular chain, the biocompatibility of the material is expected to be improved.
In addition, if a scaffold has superior tissue repair ability, it must have superior adhesion and proliferation ability to cells, particularly stem cells, and currently Induced Pluripotent Stem Cells (iPSCs) are the most effective among a variety of stem cells that can be used for tissue engineering, particularly cardiac tissue engineering. At present, many people use the cells in the field of 3D printing to produce 3D scaffolds with tissue repair capacity.
In the document 3D Bioprinting Human Induced branched tissue Stem Cell-derived DNA tissue Using a Novel Lab-on-a-Printer Technology, iPSCs are loaded in a biological composite 3D printing ink prepared by dissolving fibrinogen protein, sodium alginate and genipin in water for printing, and the survival rate of the iPSCs at 7 days after printing can reach about 95% at most; the literature 3D printing human induced pluripotent cells with novel hydroxypropyl chitin bioink that 3D printing is carried out by loading iPSCs in biological 3D printing ink based on hydroxypropyl chitin finds that the survival rate of the iPSCs in one day after printing can reach about 95% at most, the survival rate of the iPSCs in 10 days after printing can reach about 90% and the cell proliferation rate is 3000-4000%; in the literature, "Laser bioprinting of human induced multiplexed cells-the effect of printing and biological cell Survival, multiplexed, and differentiation" iPSCs are loaded in different culture media for 3D printing, and it is found that the survival rate of iPSCs within 3h after printing can reach about 90% at the maximum, and the cell proliferation rate can reach about 429% after printing for 4 days; a plurality of biological 3D printing inks based on chitosan, silkworm fibroin and polyurethane are prepared in a document of 'A simple and effective feeder-free culture system up-scale iPSCs on polymeric material surface for use in 3D bioprinting', and iPSCs are cultured by taking the inks as a substrate, and the result shows that the proliferation rate of the iPSCs after 3 days of culture can reach about 300% at most.
The iPSCs are applied to the field of 3D printing in the above researches, but the mechanical properties of the 3D printing support are not researched, and the mechanical properties of the support are particularly important for certain tissues (such as hearts) needing violent mechanical motion. And aiming at different tissue parts and tissue injuries of different degrees, the tissue engineering scaffold material needs to have a repair speed matched with the tissue engineering scaffold material, and the existing preparation method of the biological material 3D printing scaffold is difficult to simultaneously take the repair speed of the scaffold on the injured tissue and other performances of the scaffold into consideration.
Based on the background, the research on the modified tussah silk fibroin 3D printing bracket which can simultaneously take the repair speed of the bracket on the injured tissue and the mechanical property and biocompatibility of the bracket as well as the adhesion and proliferation capacity of cells into consideration and has the functions of controlling the growth and proliferation speed of the cells and the preparation method thereof has very important significance. If the chemically modified tussah silk fibroin can be prepared into the nanofiber, the conformation can be kept stable in the preparation process of the ink, the preparation process is easier to control, the mechanical property, the biocompatibility and the tissue repair capability of the scaffold can be adjusted by adjusting the content of the tussah silk fibroin nanofiber and the preparation process, and the tissue engineering scaffold with excellent mechanical property and tissue repair capability is expected to be obtained.
Disclosure of Invention
The invention aims to overcome the defects that the mechanical property and biocompatibility of a scaffold and the adhesion and proliferation capacity of cells cannot be simultaneously considered in the prior art, and provides a modified tussah silk fibroin 3D printing scaffold and a preparation method thereof. The scaffold prepared by the method not only has excellent mechanical property and cell adhesion and proliferation capacity, but also has good biocompatibility. In addition, the cell growth and proliferation speed can be controlled.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a preparation method of a modified tussah silk fibroin 3D printing bracket is characterized in that core part printing ink prepared from chemically modified tussah silk fibroin nano microfibers and shell part printing ink prepared from a chemically modified tussah silk fibroin nano microfiber/gelatin composite system are subjected to 3D printing to prepare the modified tussah silk fibroin 3D printing bracket;
the chemically modified tussah silk fibroin nano microfiber is obtained by mixing a tussah silk fibroin nano microfiber/boric acid buffer solution mixed system with a chemical modifier and then carrying out a series of reactions and operations, the adsorption and proliferation speed of cells on the scaffold can be regulated and controlled by adjusting the reaction time of the tussah silk fibroin and the chemical modifier, and the faster the proliferation speed is, the faster the repair speed is, so that the requirement of tissue damage of different degrees on the repair speed is met; the modification reaction time of the tussah silk fibroin molecular chain and the grafted hydrophilic groups are different, so that the scaffold has different cell compatibility, the proliferation rate of cells on the scaffold is different, and the scaffold with controllable repair speed is obtained;
the 3D printing method comprises the steps of drawing a three-dimensional model through a computer, obtaining contour data and filling data of each layer of section in the three-dimensional model, selecting a high polymer material to manufacture a plurality of different 3D printing materials, extruding lines through a printing nozzle with specified size and specification, depositing the extruded lines in specified areas on a working platform along different directions, cooling to enable the extruded lines to be solidified and formed, manufacturing a single-layer section structure, and repeating the manufacturing of stacking the single-layer section structure layer by layer according to different directions to finish the 3D printing of the whole three-dimensional model;
the core part printing ink and the shell part printing ink are extruded from the coaxial nozzle device to form a printing line with a core-shell structure, and for core-shell structure printing, if the core-shell structure printing is not coaxial, the obtained support is not uniform, so that the mechanical property of the support is influenced;
the mass content of the chemically modified tussah silk fibroin nano microfiber in the core part printing ink is more than or equal to 6 wt%, otherwise, the tussah silk fibroin nano microfiber collapses; the mass content of the chemically modified tussah silk fibroin nano microfibers in the shell printing ink is less than or equal to 6 wt%, the tussah silk fibroin nano microfibers also contain gelatin, the concentration of the tussah silk fibroin nano microfibers must be within 6 wt%, and otherwise, the tussah silk fibroin nano microfibers cannot be uniformly mixed with the gelatin due to poor fluidity;
the mass content of the chemically modified tussah silk fibroin nano microfibers in the core part printing ink is more than or equal to 6 wt%, otherwise, the core part printing ink collapses, because the main component of the core part printing ink is the tussah silk fibroin nano microfibers which do not exist in a molecular state and have weak interaction force, so the core part printing ink is easy to fall off; therefore, in order to prevent the ink from falling off, a layer of shell ink which mainly contains molecular components and has strong intermolecular force is coated on the outer surface, so that an tussah silk fibroin nano microfiber/gelatin composite system which mainly contains gelatin components is used in the shell ink;
the chemically modified tussah silk fibroin nano microfiber is grafted with a hydrophilic group, wherein the purpose of chemical modification is to introduce the hydrophilic group to improve the hydrophilicity of the scaffold, and cells grow in an environment with water as a main body, so that after the hydrophilic group is introduced into a molecular chain of the tussah silk fibroin nano microfiber, the cell adhesion and proliferation capacity of the scaffold can be improved, and the biocompatibility of the scaffold can be further improved;
in the prior art, when Induced Pluripotent Stem Cells (iPSCs) are cultured on a 3D printing tissue engineering scaffold, the survival rate of the cells can reach about 90% after ten days of culture, and the proliferation rate of the cells can reach 3000-4000%. After the modified tussah silk fibroin nano microfiber/gelatin composite 3D printing support is inoculated and cultured for 10 days, the survival rate of Induced Pluripotent Stem Cells (iPSCs) is 92.0-99.0%, and the proliferation rate is 9000-25000%.
In addition, in the prior art, the silk fibroin protein based 3D printing ink is used for printing, and the maximum compression modulus of the obtained scaffold can reach 5 kPa; printing a 3D support based on 3D printing ink of tussah silk fibroin aqueous solution, wherein the compression modulus is within the range of 10-50 MPa; the compression modulus of the printed modified tussah silk fibroin 3D bracket after being soaked in genipin with the mass concentration of 0.1-5 wt% for crosslinking reaction for 24h is 100-600 MPa, which is far higher than the compression modulus of the 3D bracket printed by the 3D printing ink based on the tussah silk fibroin and the 3D printing ink based on the tussah silk fibroin aqueous solution in the prior art, and unexpected technical effects are also obtained.
As a preferable scheme:
according to the preparation method of the modified tussah silk fibroin 3D printing support, the extrusion swelling rate of the core part printing ink is 0.10-1.00%, and the dynamic viscosity is 300-500 cP; the self-gelling time of the shell printing ink is 10-60 s, the extrusion swelling rate is 1-30%, and the dynamic viscosity is 1000-5000 cP.
According to the preparation method of the modified tussah silk fibroin 3D printing bracket, the preparation process of the shell printing ink comprises the following steps: firstly, adding chemically modified tussah silk fibroin nano microfibers, cell growth factors and antibiotics into water, mechanically stirring for 1-3 hours, then mixing with gelatin, standing for 1-3 hours, mechanically stirring for 1-5 hours at the temperature of 35-45 ℃, and finally carrying out ultrasonic treatment for 1-60 min;
the shell printing ink comprises the following components in percentage by mass: 1-6 wt% of chemically modified tussah silk fibroin nano microfiber, 14-20 wt% of gelatin, 0.1-1.0 wt% of cell growth factor, 0.1-1.0 wt% of antibiotic and the balance of water;
the preparation process of the core part printing ink comprises the following steps: adding the chemically modified tussah silk fibroin nano microfiber, a cell growth factor and an antibiotic into water, and mechanically stirring for 1-3 hours at the temperature of 1-45 ℃;
the mass content of each component in the core part printing ink is as follows: 6-20 wt% of chemically modified tussah silk fibroin nano microfiber, 0.1-1.0 wt% of cell growth factor, 0.1-1.0 wt% of antibiotic and the balance of water.
Because the stent needs to be used for in vivo tissue engineering and must be nontoxic, the dispersion media in the shell printing ink and the core printing ink adopt water, and other nontoxic solvents can be adopted; the cell growth factor is used for improving the cell growth and proliferation capacity of the material; the antibiotic has the functions of promoting the expression of the iPSCs on the cardiac muscle protein and promoting the transformation of the iPSCs into the cardiac muscle cells.
According to the preparation method of the modified tussah silk fibroin 3D printing bracket, all cell growth factors are fibroblast growth factors 2; all antibiotics are tetracyclines;
all the cell growth factors are not limited to fibroblast growth factor 2, but can be other substances, such as bone morphogenetic protein 4 and the like; all antibiotics are not limited to tetracycline, and other substances such as doxycycline can be used, and the above mentioned cell growth factor and antibiotics can achieve the effect of improving cell proliferation rate, survival rate and transformation to cardiac muscle cells, but the cell growth factor and antibiotics that can be added to the ink used in the scaffold of the present invention are not limited to the above two substances, and the addition of other cell growth factors (such as bone morphogenetic protein 4, etc.) and antibiotics (such as doxycycline, etc.) should be considered to be within the scope of the present invention without departing from the technical principles of the present invention.
According to the preparation method of the modified tussah silk fibroin 3D printing bracket, all the chemically modified tussah silk fibroin nano microfibers are prepared by the following steps:
(1) preparing tussah silk fibroin pulp;
(2) preparing a chemical modifier and a tussah silk fibroin nano microfiber/boric acid buffer solution mixed system;
(2.a) the preparation process of the chemical modifier comprises the following steps: mixing a p-phenylamino organic acetonitrile solution with the concentration of 1 mM-1M (M is mol/L), a p-toluenesulfonic acid aqueous solution with the concentration of 1 mM-2M and a sodium nitrite aqueous solution with the concentration of 1.0 mM-1.0M according to the volume ratio of 1: 0.5-4: 0.5-2 at the temperature of 1-4 ℃ for 5s by vortex, and then mechanically stirring and reacting for 5-30 min at the same temperature to obtain a chemical modifier; the vortex mixing means that liquid to be mixed is put into a test tube or a small flask and put on a common vortex mixer to vibrate;
(2, b) the preparation method of the tussah silk fibroin nano microfiber/boric acid buffer solution mixed system comprises the following steps:
(2, b.1) soaking the tussah silk fibroin pulp in water according to the mass volume ratio of 1g: 50-200 mL, and mechanically stirring to uniformly disperse the tussah silk fibroin pulp;
(2, b.2) adding a NaClO aqueous solution with the mass concentration of 5-35 wt% into an aqueous dispersion system of the tussah silk fibroin pulp according to the mass molar ratio of the tussah silk fibroin pulp to the NaClO of 1g: 0.001-0.050 mol, and mechanically stirring at the temperature of 1-60 ℃, wherein in the process, the pH value of the system is kept between 10.0-10.1 by continuously adding 0.5-5.0M of NaOH aqueous solution until the pH value of the system can be kept between 10.0-10.1 when no NaOH aqueous solution is added;
(2, b.3) transferring the system into a cellulose dialysis bag with the molecular weight cutoff of 14000Da, dialyzing in deionized water for 1-3 days, continuing to dialyze in a boric acid buffer solution for 1 day, and concentrating to obtain a tussah silk fibroin nano microfiber/boric acid buffer solution mixed system with the concentration of 1-20 wt% of the tussah silk fibroin nano microfiber, wherein the boric acid buffer solution mainly comprises boric acid, sodium chloride and water, and the concentrations of the boric acid and the sodium chloride are 100mM and 150mM respectively;
(3) chemical modification;
the method comprises the steps of mixing a 1-20 wt% tussah silk fibroin nano microfiber/boric acid buffer solution mixed system with a chemical modifier according to a volume ratio of 1: 0.1-2, mechanically stirring and reacting in an ice water bath for 1-5 days, dialyzing with deionized water for 1-3 days, purifying, and freeze-drying to obtain the chemically modified tussah silk fibroin nano microfiber with the length-diameter ratio of 100-200 and the diameter of 10-200 nm.
According to the preparation method of the modified tussah silk fibroin 3D printing bracket, the preparation process of the tussah silk fibroin pulp is as follows:
(1.1) immersing the tussah cocoons into Na with the mass concentration of 0.05-5.00 wt% according to the mass-volume ratio of 1g to 50-100 mL2CO3Boiling in the water solution for 1-5 times, each time for 30-60 min, and then washing and drying the boiled tussah cocoons with water to obtain degummed tussah silk fibers;
(1.2) immersing the dried degummed tussah silk fiber into a formic acid aqueous solution with the mass concentration of 60-100 wt% for 1-3 h according to the mass volume ratio of 1g: 15-25 mL;
(1.3) homogenizing at the temperature of 10-60 ℃ for 1-5 min, and crushing, wherein the homogenizing speed is 5000-15000 rpm;
(1.4) repeating the step (1.2) and the step (1.3) for 1-5 times to obtain tussah silk fibroin slurry;
and (1.5) centrifuging, filtering, washing and drying the tussah silk fibroin slurry to obtain the tussah silk fibroin pulp.
According to the preparation method of the modified tussah silk fibroin 3D printing support, the p-aminobenzoic acid, the 4- (2-ethylamino) aniline, the p-aminoacetophenone or the p-aminophenazone is taken as the p-aminobenzoic acid organic matter.
According to the preparation method of the modified tussah silk fibroin 3D printing support, the diameters of the core part and the shell part of the printing line with the core-shell structure are 50-500 microns and 100-1000 microns respectively, and the diameter of the shell part is larger than that of the core part; during printing, the printing lines with the core-shell structure are deposited on a glass sheet in a layer-by-layer stacking manner; the printing process parameters are as follows: the temperature of the charging barrel and the temperature of the needle head are 25-37 ℃, the extrusion air pressure is 10-500 KPa, the printing speed is 1-10 mm/s, the included angle between two adjacent printing layers formed by the printing lines with the core-shell structure is 30-90 degrees, the line interval is 20-2000 mu m, and the number of deposition layers is 2-10.
The stent manufactured by the printing process conditions has relatively excellent mechanical properties and biocompatibility, but the printing conditions of the stent of the invention are not limited to the above range, and the changes of the basic parameters of the printing process conditions and the shape of the printing stent without departing from the technical principle of the invention are also considered to be within the protection scope of the invention.
The modified tussah silk fibroin 3D printing support prepared by the preparation method of the modified tussah silk fibroin 3D printing support is composed of a printing line with a core-shell structure, wherein the core part of the printing line with the core-shell structure mainly consists of chemically modified tussah silk fibroin nano microfibers, and the shell part mainly consists of chemically modified tussah silk fibroin nano microfibers and gelatin.
As a preferable scheme:
the resolution of the modified tussah silk fibroin 3D printing support is 0.5-10.0 μm. The phenomenon of extrusion swelling and extrusion cracking can occur in the 3D printing process of general high polymer material ink, the thinner the needle is, the more easily the phenomenon occurs, the resolution refers to the minimum diameter of a uniform and stable line which can be obtained through the ink printing, that is, the smaller the resolution value is, the thinner the stable line can be obtained, the more easily the phenomenon of extrusion swelling and extrusion cracking can occur, the higher the printing precision is, the resolution of the 3D printing support prepared by the prior art is about 30 mu m generally, and the resolution of the modified tussah silk fibroin protein 3D printing support prepared finally by the invention is 0.50-10.0 mu m, which proves that the 3D printing support prepared by the invention has higher printing precision.
Has the advantages that:
(1) according to the modified tussah silk fibroin 3D printing bracket, the tussah silk fibroin nano microfiber contained in the bracket can enable the bracket to have excellent mechanical property, and the compression modulus of the bracket can be improved by more than 5 times compared with the existing bracket printed by using silk fibroin-containing ink in a 3D mode;
(2) according to the modified tussah silk fibroin 3D printing support, the tussah silk fibroin in the used ink is from tussah cocoons, the tussah silk fibroin contains an RGD chain segment, and the chain segment has good cell adsorption and proliferation capacity; the tussah silk fibroin has better surface hydrophilicity after being chemically modified. Since cells generally grow in an environment mainly containing water, after iPSCs are inoculated on the scaffold and cultured for 10 days, the survival rate and the proliferation capacity of the iPSCs are improved compared with those of the existing scaffold printed by using silkworm silk fibroin-containing ink 3D, and the scaffold has excellent tissue repair capacity;
(3) according to the preparation method of the modified tussah silk fibroin 3D printing support, in the preparation process of chemically modified tussah silk fibroin nano microfibers, NaClO enables a molecular chain of tussah silk fibroin to generate an oxidation reaction, and the oxidation reaction is mainly located on serine in the molecular chain; the chemical modification reaction on the tussah silk fibroin molecular chain is mainly positioned on tyrosine in the molecular chain, and both the two reactions can not influence the RGD chain segment with good cell compatibility in the tussah silk fibroin molecular chain;
(4) according to the preparation method of the modified tussah silk fibroin 3D printing support, all components of ink used by the preparation method are water as a dispersing agent, so that the modified tussah silk fibroin 3D printing support has excellent biocompatibility and can be well compounded with cells for 3D printing;
(5) according to the modified tussah silk fibroin 3D printing bracket, the tussah silk fibroin contained in the printing ink exists in the form of the nano microfiber, and the conformation of the tussah silk fibroin can be kept stable under wide environmental conditions, so that adverse effects on the stability of the ink due to fluctuation of conditions such as temperature, external force and the like can be avoided in the preparation process;
(6) according to the modified tussah silk fibroin 3D printing bracket, the adsorption and proliferation speed of cells on the bracket can be regulated and controlled by regulating the reaction time of the tussah silk fibroin and a chemical modifier, so that the requirement of tissue damage of different degrees on the repair speed is met;
(7) according to the modified tussah silk fibroin 3D printing support, the printing ink printing process is carried out under the condition from room temperature to physiological temperature, so that the modified tussah silk fibroin 3D printing support is beneficial to loading of various biological factors and embedding of cells to realize active printing.
Detailed Description
The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Example 1
A preparation method of a modified tussah silk fibroin 3D printing bracket mainly comprises the following steps:
(1) preparing tussah silk fibroin pulp:
(1.1) immersing the tussah cocoon in Na with the mass concentration of 0.50 wt% according to the mass-volume ratio of 1g to 50mL2CO3Boiling in water solution for 3 times, each for 45min, washing the boiled tussah cocoon with water, and drying to obtain degummed tussah silk fiber;
(1.2) immersing the degummed tussah silk fiber into 90 wt% formic acid aqueous solution for 2h according to the mass volume ratio of 1g to 20 mL;
(1.3) homogenizing at 25 ℃ for 2min, and crushing, wherein the homogenizing speed is 10000 rpm;
(1.4) repeating the step (1.2) and the step (1.3) for 3 times to obtain tussah silk fibroin slurry;
(1.5) centrifuging, filtering, washing and drying the tussah silk fibroin slurry to obtain tussah silk fibroin pulp;
(2) preparing a p-aminobenzoic acid modifier: mixing a p-aminobenzoic acid acetonitrile solution with the concentration of 0.2M, a p-toluenesulfonic acid aqueous solution with the concentration of 1.6M and a sodium nitrite aqueous solution with the concentration of 0.8M at the volume ratio of 1:0.5:0.5 at the temperature of 2 ℃ for 5s by vortex, and then mechanically stirring and reacting for 15min at the same temperature to obtain a p-aminobenzoic acid modifier;
(3) preparing a tussah silk fibroin nano microfiber/boric acid buffer solution mixed system:
(3.1) soaking the tussah silk fibroin pulp in water according to the mass volume ratio of 1g:100mL, and mechanically stirring to uniformly disperse the tussah silk fibroin pulp;
(3.2) adding a NaClO aqueous solution with the mass concentration of 25 wt% into an aqueous dispersion system of the tussah silk fibroin pulp according to the mass molar ratio of 1g NaClO to 0.015mol, mechanically stirring at the temperature of 25 ℃, and continuously adding a 1.0M NaOH aqueous solution to maintain the pH value of the system between 10.0 and 10.1 in the process until the pH value of the system can be maintained between 10.0 and 10.1 when no NaOH aqueous solution is added;
(3.3) transferring the system into a cellulose dialysis bag with the molecular weight cutoff of 14000Da, dialyzing in deionized water for 2 days, continuing to dialyze in a boric acid buffer solution for 1 day, and concentrating to obtain a tussah silk fibroin nano microfiber/boric acid buffer solution mixed system with the concentration of 10 wt% of the tussah silk fibroin nano microfiber, wherein the boric acid buffer solution mainly comprises boric acid, sodium chloride and water, and the concentrations of the boric acid and the sodium chloride are 100mM and 150mM respectively;
(4) preparing the tussah silk fibroin nano microfiber modified by p-aminobenzoic acid: mixing a 10 wt% tussah silk fibroin nano microfiber/boric acid buffer solution mixed system with a p-aminobenzoic acid modifier according to a volume ratio of 1:0.25, mechanically stirring and reacting for 2 days in an ice water bath, dialyzing for 2 days by using deionized water, purifying, and freeze-drying to obtain the tussah silk fibroin nano microfiber modified by p-aminobenzoic acid, wherein the average length-diameter ratio of the tussah silk fibroin nano microfiber is 150, and the average diameter of the tussah silk fibroin nano microfiber is 80 nm;
(5) preparation of core portion printing ink: adding the p-aminobenzoic acid modified tussah silk fibroin nano microfiber, fibroblast growth factor 2 and tetracycline into water, and mechanically stirring for 3 hours at the temperature of 20 ℃ to prepare the core part printing ink, wherein the core part printing ink comprises the following components in percentage by mass: 14 wt% of p-aminobenzoic acid modified tussah silk fibroin nano microfiber, 0.1 wt% of fibroblast growth factor 2, 0.4 wt% of tetracycline and the balance of water; the extrusion swelling rate of the printing ink of the core part is 0.50 percent, and the dynamic viscosity is 420 cP;
(6) preparation of shell-printing ink: firstly, adding the tussah silk fibroin nano microfiber modified by p-aminobenzoic acid, fibroblast growth factor 2 and tetracycline into water, mechanically stirring for 3 hours, then mixing with gelatin, standing for 2 hours, mechanically stirring for 2 hours at the temperature of 40 ℃, and finally performing ultrasonic treatment for 10min to obtain shell printing ink; wherein the shell part printing ink comprises the following components in percentage by mass: 5 wt% of p-aminobenzoic acid modified tussah silk fibroin nano microfiber, 15 wt% of gelatin, 0.1 wt% of fibroblast growth factor 2, 0.4 wt% of tetracycline and the balance of water; the self-gelling time of the shell printing ink is 38s, the extrusion swelling rate is 10%, and the dynamic viscosity is 3700 cP;
(7) extruding the prepared core part printing ink and shell part printing ink from a coaxial nozzle device to form a printing line with a core-shell structure, wherein the diameters of the core part and the shell part of the printing line with the core-shell structure are respectively 200 micrometers and 400 micrometers, and then carrying out 3D printing to prepare a modified tussah silk fibroin 3D printing bracket; during printing, the printing lines with the core-shell structure are deposited on a glass sheet in a layer-by-layer stacking manner; the printing process parameters are as follows: the temperature of a charging barrel and a needle head is 30 ℃, the extrusion air pressure is 160KPa, the printing speed is 8mm/s, the included angle between two adjacent printing layers formed by the printing lines with the core-shell structure is 90 degrees, the line interval is 400 mu m, and 4 layers are deposited.
The resolution of the finally prepared modified tussah silk fibroin 3D printing bracket is 4.6 mu m; the modified tussah silk fibroin 3D printing bracket has a compressive modulus of 336MPa after being soaked in genipin with a mass concentration of 0.5 wt% for crosslinking reaction for 24 hours;
when 1mL of fetal bovine serum culture containing 10000 second generation iPSCs was inoculated on a 15mm diameter scaffold printed and crosslinked as described above and incubated at 37.0 deg.C and 5.0% CO2After culturing for 1, 3, 4, 7, and 10 days under the conditions, the cell viability was 99.1%, 98.1%, 97.7%, 95.9%, and 93.0%, respectively, by trypan blue staining, and the cell proliferation rate was 183%, 622%, 1269%, 7678%, and 17478%, respectively, by cell counting.
Comparative example 1
A preparation method of a modified tussah silk fibroin 3D printing bracket, which has the basically same steps as the embodiment 1, and is different from the preparation method in that an aminobenzoic acid modifier is not modified,
the length-diameter ratio of the finally prepared modified tussah silk fibroin nano microfiber is 50, and the average diameter is 400 nm.
The core portion printing ink had an extrusion swell of 4.00% and a dynamic viscosity of 100 cP.
The self-gelling time of the shell printing ink is 60s, the extrusion swelling rate is 40%, and the dynamic viscosity is 600 cP.
The resolution of the finally prepared modified tussah silk fibroin 3D printing bracket is 30 μm; the modified tussah silk fibroin 3D printing bracket has a compression modulus of 80MPa after being soaked in genipin with the mass concentration of 0.5 wt% for crosslinking reaction for 24 hours;
when 1mL of fetal bovine serum culture containing 10000 second generation iPSCs was inoculated on a 15mm diameter scaffold printed and crosslinked as described above and incubated at 37.0 deg.C and 5.0% CO2After culturing for 1, 3, 4, 7, and 10 days under the conditions, the cell viability was 98.6%, 97.6%, 97.2%, 95.4%, and 91.5%, respectively, by trypan blue staining, and the cell proliferation rate was 125%, 256%, 468%, 1789%, and 4958%, respectively, by cell counting.
Comparing example 1 with comparative example 1, it can be seen that the survival rate and proliferation capacity of the cells on the modified tussah silk fibroin 3D printing scaffold of example 1 are higher than those of comparative example 1 after 1, 3, 4, 7, 10 days, and the mechanical properties are also improved compared with comparative example 1, because the hydrophilicity and the intermolecular action of the tussah silk fibroin are greatly improved after the chemical modification, the cellular compatibility is correspondingly improved along with the improvement of the hydrophilicity of the scaffold, and the ability of the scaffold to resist external acting force is correspondingly improved along with the improvement of the intermolecular action of the scaffold, so that the cellular compatibility and the mechanical properties of the modified tussah silk fibroin 3D printing scaffold obtained by the invention can be further improved compared with the unmodified tussah silk fibroin-based 3D printing scaffold.
Example 2
The preparation method of the modified tussah silk fibroin 3D printing bracket is basically the same as that in the embodiment 1, and the difference is that a p-aminoacetophenone modifier is used for replacing a p-aminobenzoic acid modifier.
The aspect ratio of the finally prepared modified tussah silk fibroin nano microfiber is 146, and the average diameter is 86 nm.
The core portion printing ink had an extrusion swell of 0.52% and a dynamic viscosity of 390 cP.
The self-gelling time of shell printing ink is 39s, the extrusion swelling rate is 12%, and the dynamic viscosity is 3500 cP.
The resolution of the finally prepared modified tussah silk fibroin 3D printing bracket is 5.6 mu m, and the compression modulus is 319MPa after the bracket is soaked in 0.5 wt% genipin aqueous solution for crosslinking reaction for 24 h;
when 1mL of fetal bovine serum culture containing 10000 second generation iPSCs was inoculated on a 15mm diameter scaffold printed and crosslinked as described above and incubated at 37.0 deg.C and 5.0% CO2After culturing under these conditions for 1, 3, 4, 7, and 10 days, the cell viability was 98.9%, 97.9%, 97.5%, 95.7%, and 92.8%, respectively, by trypan blue staining, and the cell proliferation rate was 175%, 536%, 1045%, 5704%, and 14484%, respectively, by cytometric method.
Example 3
The preparation method of the modified tussah silk fibroin 3D printing bracket is basically the same as the embodiment 2 in step, and is different in that a 4- (2-ethylamino) aniline modifier is used for replacing a p-aminoacetophenone modifier.
The aspect ratio of the finally prepared modified tussah silk fibroin nano microfiber is 111, and the average diameter is 126 nm.
The core portion printing ink had an extrusion swell of 0.62% and a dynamic viscosity of 350 cP.
The shell portion printing ink had a self-gelling time of 42s, an extrusion swell of 16% and a dynamic viscosity of 2900 cP.
The resolution of the finally prepared modified tussah silk fibroin 3D printing bracket is 8.2 mu m, and the compression modulus is 289MPa after the bracket is soaked in 0.5 wt% genipin aqueous solution for crosslinking reaction for 24 hours;
if 1mL of fetal calf serum containing 10000 second-generation iPSCs is culturedThe nutrient solution was inoculated onto a 15mm diameter scaffold printed and crosslinked as described above and at 37.0 ℃ with 5.0% CO2After culturing for 1, 3, 4, 7, and 10 days under the conditions, the cell viability was 99.4%, 98.4%, 98.0%, 96.2%, and 92.3%, respectively, by trypan blue staining, and the cell proliferation rate was 163%, 407%, 709%, 2743%, and 9993%, respectively, by cell counting.
Example 4
A preparation method of a modified tussah silk fibroin 3D printing bracket, which has the steps basically the same as the embodiment 3, and is characterized in that a p-aminophenyl heptanoid modifier is used for replacing a 4- (2-ethylamino) aniline modifier,
the aspect ratio of the finally prepared modified tussah silk fibroin nano microfiber is 132, and the average diameter is 93 nm.
The core portion printing ink extrusion swelling rate was 0.56%, and the dynamic viscosity was 380 cP.
The self-gelling time of the shell printing ink is 41s, the extrusion swelling rate is 15%, and the dynamic viscosity is 3200 cP.
The resolution of the finally prepared modified tussah silk fibroin 3D printing bracket is 6.5 mu m, and the compression modulus is 301MPa after the bracket is soaked in 0.5 wt% genipin aqueous solution for crosslinking reaction for 24 hours;
when 1mL of fetal bovine serum culture containing 10000 second generation iPSCs was inoculated on a 15mm diameter scaffold printed and crosslinked as described above and incubated at 37.0 deg.C and 5.0% CO2After culturing under these conditions for 1, 3, 4, 7, and 10 days, the cell viability was 98.8%, 97.8%, 97.4%, 95.6%, and 92.7%, respectively, by trypan blue staining, and the cell proliferation rate was 171%, 493%, 933%, 4717%, and 12987%, respectively, by cytometric method.
Example 5
A preparation method of a modified tussah silk fibroin 3D printing bracket, which has the basically same steps as the embodiment 1, and is characterized in that in the step (4), the mechanical stirring reaction is carried out in an ice water bath for 3 days,
the length-diameter ratio of the finally prepared modified tussah silk fibroin nano microfiber is 158, and the average diameter is 67 nm.
The core portion printing ink had a die swell of 0.46% and a dynamic viscosity of 460 cP.
The self-gelling time of the shell printing ink is 34s, the extrusion swelling rate is 8%, and the dynamic viscosity is 4000 cP.
The resolution of the finally prepared modified tussah silk fibroin 3D printing bracket is 2.9 mu m, and the compression modulus is 354MPa after the bracket is soaked in 0.5 wt% genipin aqueous solution for crosslinking reaction for 24 hours;
when 1mL of fetal bovine serum culture containing 10000 second generation iPSCs was inoculated on a 15mm diameter scaffold printed and crosslinked as described above and incubated at 37.0 deg.C and 5.0% CO2After culturing under these conditions for 1, 3, 4, 7, and 10 days, the cell viability was 99.2%, 98.2%, 97.8%, 96.0%, and 93.1%, respectively, by trypan blue staining, and the cell proliferation rate was 187%, 665%, 1381%, 8665%, and 18975%, respectively, by cytometric method.
Example 6
The preparation method of the modified tussah silk fibroin 3D printing bracket is basically the same as the embodiment 5 in the difference that the mechanical stirring reaction is carried out in an ice-water bath for 5 days in the step (4).
The length-diameter ratio of the finally prepared modified tussah silk fibroin nano microfiber is 185, and the average diameter is 45 nm.
The core portion printing ink had a die swell of 0.42% and a dynamic viscosity of 490 cP.
The shell-printed ink had a self-gelling time of 30s, an extrusion swell of 5% and a dynamic viscosity of 4200 cP.
The resolution of the finally prepared modified tussah silk fibroin 3D printing bracket is 1.2 mu m, and the compression modulus is 443MPa after the bracket is soaked in 0.5 wt% genipin aqueous solution for crosslinking reaction for 24 hours;
when 1mL of fetal bovine serum culture containing 10000 second generation iPSCs was inoculated on a 15mm diameter scaffold printed and crosslinked as described above and incubated at 37.0 deg.C and 5.0% CO2After culturing for 1, 3, 4, 7 and 10 days under the condition, the cell survival rate is respectively 99.3%, 98.3%, 97.9%, 96.1% and 93.3% by trypan blue staining method. Using cell counting, cell proliferation rates were measured at 191%, 708%, 1493%, 9652%, 20473%, respectively.
Example 7
The preparation method of the modified tussah silk fibroin 3D printing bracket is basically the same as that in the embodiment 1, except that in the step (6), the shell part printing ink is prepared, the concentration of the tussah silk fibroin nano microfiber modified by para aminobenzoic acid is 3 wt%, the concentration of gelatin is 17 wt%, and the diameters of the core part and the shell part of the printing line of the core-shell structure used in printing are 400 micrometers and 800 micrometers respectively; the printing process parameters are as follows: the temperature of the cylinder and the needle head is 32 ℃, the extrusion air pressure is 130KPa, the printing speed is 8mm/s, and the line spacing is 800 mu m.
The self-gelling time of the shell printing ink was 46s, the extrusion swelling rate was 22%, and the dynamic viscosity was 2000 cP.
The resolution of the finally prepared modified tussah silk fibroin 3D printing bracket is 7.0 mu m, and the compression modulus is 243MPa after the bracket is soaked in 0.5 wt% genipin aqueous solution for crosslinking reaction for 24 hours;
when 1mL of fetal bovine serum culture containing 10000 second generation iPSCs was inoculated on a 15mm diameter scaffold printed and crosslinked as described above and incubated at 37.0 deg.C and 5.0% CO2After culturing under the conditions for 1, 3, 4, 7, 10 days, the cell viability was measured to be 99.0%, 98.0%, 97.6%, 95.8%, 92.9% by trypan blue staining method, respectively. The cell proliferation rates were found to be 179%, 579%, 1157%, 6691%, 15981% respectively using cell counting method.
Example 8
The preparation method of the modified tussah silk fibroin 3D printing bracket is basically the same as that in the embodiment 1, except that in the step (6), the shell part printing ink is prepared, the concentration of the tussah silk fibroin nano microfiber modified by para aminobenzoic acid is 1 wt%, the concentration of gelatin is 19 wt%, and the diameters of the core part and the shell part of the printing line of the core-shell structure used in printing are respectively 500 micrometers and 1000 micrometers; the printing process parameters are as follows: the temperature of the cylinder and the needle head is 37 ℃, the extrusion air pressure is 100KPa, the printing speed is 8mm/s, and the line spacing is 2000 mu m.
The self-gelling time of the shell-printed ink was 50s, the extrusion swell was 26%, and the dynamic viscosity was 1400 cP.
The resolution of the finally prepared modified tussah silk fibroin 3D printing bracket is 9.0 mu m, and the compression modulus is 196MPa after the bracket is soaked in 0.5 wt% genipin aqueous solution for crosslinking reaction for 24 hours;
when 1mL of fetal bovine serum culture containing 10000 second generation iPSCs was inoculated on a 15mm diameter scaffold printed and crosslinked as described above and incubated at 37.0 deg.C and 5.0% CO2After culturing under the conditions for 1, 3, 4, 7 and 10 days, the cell viability was determined to be 98.7%, 97.7%, 97.3%, 95.5% and 92.6% by trypan blue staining, respectively. The cell proliferation rates were 167%, 450%, 821%, 3730% and 11490%, respectively, using a cell counting method.
Example 9
A preparation method of a modified tussah silk fibroin 3D printing bracket mainly comprises the following steps:
(1) preparing tussah silk fibroin pulp:
(1.1) immersing the tussah cocoon in Na with the mass concentration of 5.00 wt% according to the mass-volume ratio of 1g to 100mL2CO3Boiling in water solution for 5 times, each time for 60min, washing the boiled tussah cocoon with water, and drying to obtain degummed tussah silk fiber;
(1.2) immersing the degummed tussah silk fiber into 100 wt% formic acid aqueous solution for 3h according to the mass volume ratio of 1g:25 mL;
(1.3) homogenizing at 60 deg.C for 5min, and pulverizing at 15000 rpm;
(1.4) repeating the step (1.2) and the step (1.3) for 5 times to obtain tussah silk fibroin slurry;
(1.5) centrifuging, filtering, washing and drying the tussah silk fibroin slurry to obtain tussah silk fibroin pulp;
(2) preparing a chemical modifier: mixing 4- (2-ethylamino) aniline acetonitrile solution with the concentration of 1mM, p-toluenesulfonic acid aqueous solution with the concentration of 1mM and sodium nitrite aqueous solution with the concentration of 1mM according to the volume ratio of 1:4:2 at the temperature of 1 ℃ for 5s by vortex, and then mechanically stirring and reacting for 5min at the same temperature to obtain a chemical modifier;
(3) preparing a tussah silk fibroin nano microfiber/boric acid buffer solution mixed system:
(3.1) soaking the tussah silk fibroin pulp in water according to the mass volume ratio of 1g:200mL, and mechanically stirring to uniformly disperse the tussah silk fibroin pulp;
(3.2) adding a NaClO aqueous solution with the mass concentration of 5 wt% into an aqueous dispersion system of the tussah silk fibroin pulp according to the mass molar ratio of 1g NaClO to 0.001mol, mechanically stirring at the temperature of 1 ℃, and continuously adding a 0.5M NaOH aqueous solution to maintain the pH value of the system between 10.0 and 10.1 in the process until the pH value of the system can be maintained between 10.0 and 10.1 when no NaOH aqueous solution is added;
(3.3) transferring the system into a cellulose dialysis bag with the molecular weight cutoff of 14000Da, dialyzing in deionized water for 1 day, continuing to dialyze in a boric acid buffer solution for 1 day, and concentrating to obtain a tussah silk fibroin nano microfiber/boric acid buffer solution mixed system with the concentration of 1 wt% of the tussah silk fibroin nano microfiber, wherein the boric acid buffer solution mainly comprises boric acid, sodium chloride and water, and the concentrations of the boric acid and the sodium chloride are 100mM and 150mM respectively;
(4) preparing the tussah silk fibroin nano microfiber modified by 4- (2-ethylamino) aniline: firstly, mixing a 1 wt% tussah silk fibroin nano microfiber/boric acid buffer solution mixed system with a 4- (2-ethylamino) aniline modifier according to a volume ratio of 12, mechanically stirring and reacting for 1 day in an ice water bath, dialyzing for 1 day by using deionized water, purifying, and freeze-drying to obtain the 4- (2-ethylamino) aniline modified tussah silk fibroin nano microfiber with an average length-diameter ratio of 100 and an average diameter of 200 nm;
(5) preparation of core portion printing ink: adding the tussah silk fibroin nano microfiber modified by 4- (2-ethylamino) aniline, fibroblast growth factor 2 and tetracycline into water, and mechanically stirring for 1h at the temperature of 1 ℃ to prepare the core part printing ink, wherein the mass content of each component in the core part printing ink is as follows: 6 wt% of tussah silk fibroin nano microfiber modified by 4- (2-ethylamino) aniline, 0.1 wt% of fibroblast growth factor 2, 0.1 wt% of tetracycline, and the balance of water; the extrusion swelling rate of the printing ink of the core part is 1.00 percent, and the dynamic viscosity is 300 cP;
(6) preparation of shell-printing ink: firstly, adding tussah silk fibroin nano-microfiber modified by 4- (2-ethylamino) aniline, fibroblast growth factor 2 and tetracycline into water, mechanically stirring for 1h, then mixing with gelatin, standing for 1h, mechanically stirring for 1h at the temperature of 35 ℃, and finally performing ultrasonic treatment for 1min to obtain shell printing ink; wherein the shell part printing ink comprises the following components in percentage by mass: 1 wt% of tussah silk fibroin nano microfiber modified by 4- (2-ethylamino) aniline, 14 wt% of gelatin, 0.1 wt% of fibroblast growth factor 2, 0.1 wt% of tetracycline, and the balance of water; the self-gelling time of the shell printing ink is 60s, the extrusion swelling rate is 30%, and the dynamic viscosity is 1000 cP;
(7) extruding the prepared core part printing ink and shell part printing ink from a coaxial nozzle device to form a printing line with a core-shell structure, wherein the diameters of the core part and the shell part of the printing line with the core-shell structure are respectively 500 micrometers and 1000 micrometers, and then carrying out 3D printing to prepare a modified tussah silk fibroin 3D printing support; during printing, the printing lines with the core-shell structure are deposited on a glass sheet in a layer-by-layer stacking manner; the printing process parameters are as follows: the temperature of a charging barrel and a needle head is 25 ℃, the extrusion air pressure is 10KPa, the printing speed is 10mm/s, the included angle between two adjacent printing layers formed by the printing lines with the core-shell structure is 30 degrees, the line interval is 2000 mu m, and 2 layers are deposited.
The resolution of the finally prepared modified tussah silk fibroin 3D printing bracket is 10.0 μm; the modified tussah silk fibroin 3D printing bracket has the compression modulus of 100MPa after being soaked in genipin with the mass concentration of 5.0 wt% for crosslinking reaction for 24 hours;
when 1mL of fetal bovine serum culture containing 10000 second generation iPSCs was inoculated on a 15mm diameter scaffold printed and crosslinked as described above and incubated at 37.0 deg.C and 5.0% CO2Culturing under the condition of 1, 3, 4,7. After 10 days, cell viability was determined to be 98.7%, 97.6%, 97.3%, 95.5% and 92.0% using trypan blue staining, and cell proliferation rates were determined to be 150%, 344%, 564%, 3158% and 9000% using cell counting.
Example 10
A preparation method of a modified tussah silk fibroin 3D printing bracket mainly comprises the following steps:
(1) preparing tussah silk fibroin pulp:
(1.1) immersing the tussah cocoon in Na with the mass concentration of 0.05 wt% according to the mass-volume ratio of 1g:50mL2CO3Boiling in water solution for 1 time, each time for 30min, washing the boiled tussah cocoon with water, and drying to obtain degummed tussah silk fiber;
(1.2) immersing the degummed tussah silk fiber into 60 wt% formic acid aqueous solution for 1h according to the mass volume ratio of 1g:15 mL;
(1.3) homogenizing at 10 ℃ for 1min, and crushing, wherein the homogenizing speed is 5000 rpm;
(1.4) repeating the step (1.2) and the step (1.3) for 1 time to obtain tussah silk fibroin slurry;
(1.5) centrifuging, filtering, washing and drying the tussah silk fibroin slurry to obtain tussah silk fibroin pulp;
(2) preparing a chemical modifier: vortex mixing a 1M p-aminobenzoic acid acetonitrile solution, a 2M p-methylbenzenesulfonic acid aqueous solution and a 1M sodium nitrite aqueous solution at a volume ratio of 1:2:2 at a temperature of 4 ℃ for 5s, and then mechanically stirring and reacting at the same temperature for 30min to obtain a chemical modifier;
(3) preparing a tussah silk fibroin nano microfiber/boric acid buffer solution mixed system:
(3.1) soaking the tussah silk fibroin pulp in water according to the mass volume ratio of 1g:50mL, and mechanically stirring to uniformly disperse the tussah silk fibroin pulp;
(3.2) adding a NaClO aqueous solution with the mass concentration of 35 wt% into an aqueous dispersion system of the tussah silk fibroin pulp according to the mass molar ratio of 1g NaClO to 0.050mol, mechanically stirring at the temperature of 60 ℃, and continuously adding a 5.0M NaOH aqueous solution to maintain the pH value of the system between 10.0 and 10.1 in the process until the pH value of the system can be maintained between 10.0 and 10.1 when no NaOH aqueous solution is added;
(3.3) transferring the system into a cellulose dialysis bag with the molecular weight cutoff of 14000Da, dialyzing in deionized water for 3 days, continuing to dialyze in a boric acid buffer solution for 1 day, and concentrating to obtain a tussah silk fibroin nano microfiber/boric acid buffer solution mixed system with the concentration of the tussah silk fibroin nano microfiber being 20 wt%, wherein the boric acid buffer solution mainly comprises boric acid, sodium chloride and water, and the concentrations of the boric acid and the sodium chloride are 100mM and 150mM respectively;
(4) preparing the tussah silk fibroin nano microfiber modified by p-aminobenzoic acid: mixing a 20 wt% tussah silk fibroin nano microfiber/boric acid buffer solution mixed system with a p-aminobenzoic acid modifier according to a volume ratio of 1:0.1, mechanically stirring and reacting for 5 days in an ice water bath, dialyzing for 3 days by using deionized water, purifying, and freeze-drying to obtain the tussah silk fibroin nano microfiber modified by p-aminobenzoic acid, wherein the average length-diameter ratio of the tussah silk fibroin nano microfiber is 200, and the average diameter of the tussah silk fibroin nano microfiber is 10 nm;
(5) preparation of core portion printing ink: adding the p-aminobenzoic acid modified tussah silk fibroin nano microfiber, fibroblast growth factor 2 and tetracycline into water, and mechanically stirring for 3 hours at the temperature of 45 ℃ to prepare the core part printing ink, wherein the mass content of each component in the core part printing ink is as follows: 20 wt% of p-aminobenzoic acid modified tussah silk fibroin nano microfiber, 1 wt% of fibroblast growth factor 2, 1 wt% of tetracycline and the balance of water; the extrusion swelling rate of the printing ink of the core part is 0.10 percent, and the dynamic viscosity is 500 cP;
(6) preparation of shell-printing ink: firstly, adding the tussah silk fibroin nano microfiber modified by p-aminobenzoic acid, fibroblast growth factor 2 and tetracycline into water, mechanically stirring for 3 hours, then mixing with gelatin, standing for 3 hours, mechanically stirring for 5 hours at the temperature of 45 ℃, and finally performing ultrasonic treatment for 60 minutes to obtain shell printing ink; wherein the shell part printing ink comprises the following components in percentage by mass: 6 wt% of p-aminobenzoic acid modified tussah silk fibroin nano microfiber, 20 wt% of gelatin, 1 wt% of fibroblast growth factor 2, 1 wt% of tetracycline, and the balance of water; the self-gelling time of the shell printing ink is 10s, the extrusion swelling rate is 1%, and the dynamic viscosity is 5000 cP;
(7) extruding the prepared core part printing ink and shell part printing ink from a coaxial nozzle device to form a printing line with a core-shell structure, wherein the diameters of the core part and the shell part of the printing line with the core-shell structure are respectively 50 micrometers and 100 micrometers, and then carrying out 3D printing to prepare a modified tussah silk fibroin 3D printing support; during printing, the printing lines with the core-shell structure are deposited on a glass sheet in a layer-by-layer stacking manner; the printing process parameters are as follows: the temperature of a charging barrel and a needle head is 37 ℃, the extrusion air pressure is 500KPa, the printing speed is 1mm/s, the included angle between two adjacent printing layers formed by the printing lines with the core-shell structure is 90 degrees, the line interval is 20 mu m, and 10 layers are deposited.
The resolution of the finally prepared modified tussah silk fibroin 3D printing bracket is 0.5 mu m; the modified tussah silk fibroin 3D printing bracket has a compression modulus of 600MPa after being soaked in genipin with a mass concentration of 0.1 wt% for crosslinking reaction for 24 hours;
when 1mL of fetal bovine serum culture containing 10000 second generation iPSCs was inoculated on a 15mm diameter scaffold printed and crosslinked as described above and incubated at 37.0 deg.C and 5.0% CO2After culturing under the conditions for 1, 3, 4, 7, and 10 days, the cell viability was measured to be 99.9%, 99.7%, 99.5%, 99.2%, and 99.0%, respectively, using trypan blue staining, and the cell proliferation rate was measured to be 240%, 864%, 1821%, 11775%, and 25000%, respectively, using cytometric method.
The foregoing is merely an example of embodiments of the present invention. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. For example: changes in the printing ink formulation and preparation process conditions, changes in the printing process (needle diameter, printing temperature, extrusion pressure, printing speed, inter-layer angle, line spacing, number of deposited layers, etc.), changes in the printing support treatment, etc., should also be considered as within the scope of the present invention.

Claims (10)

1. A preparation method of a modified tussah silk fibroin 3D printing support is characterized by comprising the following steps: performing 3D printing on core part printing ink prepared from the chemically modified tussah silk fibroin nano microfiber and shell part printing ink prepared from a chemically modified tussah silk fibroin nano microfiber/gelatin composite system to obtain a modified tussah silk fibroin 3D printing support;
the core part printing ink and the shell part printing ink are extruded from the coaxial nozzle device to form a printing line with a core-shell structure;
the mass content of the chemically modified tussah silk fibroin nano microfibers in the core part printing ink is more than or equal to 6 wt%, and the mass content of the chemically modified tussah silk fibroin nano microfibers in the shell part printing ink is less than or equal to 6 wt%;
the chemically modified tussah silk fibroin nano microfiber is tussah silk fibroin nano microfiber grafted with a hydrophilic group;
the modified tussah silk fibroin 3D printing support has the compression modulus of 100-600 MPa after being soaked in genipin with the mass concentration of 0.1-5 wt% for crosslinking reaction for 24 hours, the survival rate of induced pluripotent stem cells after being cultured for 10 days is 92.0-99.0%, and the proliferation rate is 9000-25000%.
2. The preparation method of the modified tussah silk fibroin 3D printing bracket as claimed in claim 1, wherein the extrusion swelling rate of the core printing ink is 0.10-1.00%, and the dynamic viscosity is 300-500 cP; the self-gelling time of the shell printing ink is 10-60 s, the extrusion swelling rate is 1-30%, and the dynamic viscosity is 1000-5000 cP.
3. The preparation method of the modified tussah silk fibroin 3D printing bracket as claimed in claim 2, wherein the shell printing ink is prepared by the following steps: firstly, adding chemically modified tussah silk fibroin nano microfibers, cell growth factors and antibiotics into water, mechanically stirring for 1-3 hours, then mixing with gelatin, standing for 1-3 hours, mechanically stirring for 1-5 hours at the temperature of 35-45 ℃, and finally carrying out ultrasonic treatment for 1-60 min;
the shell printing ink comprises the following components in percentage by mass: 1-6 wt% of chemically modified tussah silk fibroin nano microfiber, 14-20 wt% of gelatin, 0.1-1.0 wt% of cell growth factor, 0.1-1.0 wt% of antibiotic and the balance of water;
the preparation process of the core part printing ink comprises the following steps: adding the chemically modified tussah silk fibroin nano microfiber, a cell growth factor and an antibiotic into water, and mechanically stirring for 1-3 hours at the temperature of 1-45 ℃;
the mass content of each component in the core part printing ink is as follows: 6-20 wt% of chemically modified tussah silk fibroin nano microfiber, 0.1-1.0 wt% of cell growth factor, 0.1-1.0 wt% of antibiotic and the balance of water.
4. The method for preparing the modified tussah silk fibroin 3D printing scaffold as claimed in claim 3, wherein all cell growth factors are fibroblast growth factor 2; all antibiotics were tetracyclines.
5. The preparation method of the modified tussah silk fibroin 3D printing scaffold as claimed in claim 1, wherein all chemically modified tussah silk fibroin nano-microfibers are prepared by the following steps:
(1) preparing tussah silk fibroin pulp;
(2) preparing a chemical modifier and a tussah silk fibroin nano microfiber/boric acid buffer solution mixed system;
(2.a) the preparation process of the chemical modifier comprises the following steps: carrying out vortex mixing on a p-phenylenediamine group organic acetonitrile solution with the concentration of 1 mM-1M, a p-toluenesulfonic acid aqueous solution with the concentration of 1 mM-2M and a sodium nitrite aqueous solution with the concentration of 1.0 mM-1.0M for 5s at the temperature of 1-4 ℃ according to the volume ratio of 1: 0.5-4: 0.5-2, and then carrying out mechanical stirring reaction for 5-30 min at the same temperature to obtain a chemical modifier;
(2, b) the preparation method of the tussah silk fibroin nano microfiber/boric acid buffer solution mixed system comprises the following steps:
(2, b.1) soaking the tussah silk fibroin pulp in water according to the mass volume ratio of 1g: 50-200 mL, and mechanically stirring to uniformly disperse the tussah silk fibroin pulp;
(2, b.2) adding a NaClO aqueous solution with the mass concentration of 5-35 wt% into an aqueous dispersion system of the tussah silk fibroin pulp according to the mass molar ratio of the tussah silk fibroin pulp to the NaClO of 1g: 0.001-0.050 mol, and mechanically stirring at the temperature of 1-60 ℃, wherein in the process, the pH value of the system is kept between 10.0-10.1 by continuously adding 0.5-5.0M of NaOH aqueous solution until the pH value of the system can be kept between 10.0-10.1 when no NaOH aqueous solution is added;
(2, b.3) transferring the system into a cellulose dialysis bag with the molecular weight cutoff of 14000Da, dialyzing in deionized water for 1-3 days, continuing to dialyze in a boric acid buffer solution for 1 day, and concentrating to obtain a tussah silk fibroin nano microfiber/boric acid buffer solution mixed system with the concentration of 1-20 wt% of the tussah silk fibroin nano microfiber, wherein the boric acid buffer solution mainly comprises boric acid, sodium chloride and water, and the concentrations of the boric acid and the sodium chloride are 100mM and 150mM respectively;
(3) chemical modification;
the method comprises the steps of mixing a 1-20 wt% tussah silk fibroin nano microfiber/boric acid buffer solution mixed system with a chemical modifier according to a volume ratio of 1: 0.1-2, mechanically stirring and reacting in an ice water bath for 1-5 days, dialyzing with deionized water for 1-3 days, purifying, and freeze-drying to obtain the chemically modified tussah silk fibroin nano microfiber with the length-diameter ratio of 100-200 and the diameter of 10-200 nm.
6. The preparation method of the modified tussah silk fibroin 3D printing bracket as claimed in claim 5, wherein the tussah silk fibroin pulp is prepared by the following steps:
(1.1) immersing the tussah cocoons into Na with the mass concentration of 0.05-5.00 wt% according to the mass-volume ratio of 1g to 50-100 mL2CO3Aqueous solutionBoiling for 1-5 times, each time for 30-60 min, and then washing and drying the boiled tussah cocoons with water to obtain degummed tussah silk fibers;
(1.2) immersing the dried degummed tussah silk fiber into a formic acid aqueous solution with the mass concentration of 60-100 wt% for 1-3 h according to the mass volume ratio of 1g: 15-25 mL;
(1.3) homogenizing at the temperature of 10-60 ℃ for 1-5 min, and crushing, wherein the homogenizing speed is 5000-15000 rpm;
(1.4) repeating the step (1.2) and the step (1.3) for 1-5 times to obtain tussah silk fibroin slurry;
and (1.5) centrifuging, filtering, washing and drying the tussah silk fibroin slurry to obtain the tussah silk fibroin pulp.
7. The preparation method of the modified tussah silk fibroin 3D printing support according to claim 5, wherein the para-anilino organic compound is para-aminobenzoic acid, 4- (2-ethylamino) aniline, para-aminoacetophenone or para-aminophenazone.
8. The preparation method of the modified tussah silk fibroin 3D printing bracket according to claim 1, wherein the diameters of the core part and the shell part of the printing line with the core-shell structure are 50-500 μm and 100-1000 μm respectively, and the diameter of the shell part is larger than that of the core part; during printing, the printing lines with the core-shell structure are deposited on a glass sheet in a layer-by-layer stacking manner; the printing process parameters are as follows: the temperature of the charging barrel and the temperature of the needle head are 25-37 ℃, the extrusion air pressure is 10-500 KPa, the printing speed is 1-10 mm/s, the included angle between two adjacent printing layers formed by the printing lines with the core-shell structure is 30-90 degrees, the line interval is 20-2000 mu m, and the number of deposition layers is 2-10.
9. The modified tussah silk fibroin 3D printing bracket prepared by the preparation method of the modified tussah silk fibroin 3D printing bracket as claimed in any one of claims 1 to 8, is characterized in that: the printing line is composed of a printing line with a core-shell structure, wherein the core part of the printing line with the core-shell structure mainly consists of chemically modified tussah silk fibroin nano microfibers, and the shell part mainly consists of chemically modified tussah silk fibroin nano microfibers and gelatin.
10. The modified tussah silk fibroin 3D printing scaffold of claim 9, wherein the resolution of the modified tussah silk fibroin 3D printing scaffold is 0.5-10.0 μm.
CN201910773760.9A 2019-08-21 2019-08-21 Modified tussah silk fibroin 3D printing support and preparation method thereof Active CN110859994B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910773760.9A CN110859994B (en) 2019-08-21 2019-08-21 Modified tussah silk fibroin 3D printing support and preparation method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910773760.9A CN110859994B (en) 2019-08-21 2019-08-21 Modified tussah silk fibroin 3D printing support and preparation method thereof

Publications (2)

Publication Number Publication Date
CN110859994A CN110859994A (en) 2020-03-06
CN110859994B true CN110859994B (en) 2020-10-30

Family

ID=69652390

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910773760.9A Active CN110859994B (en) 2019-08-21 2019-08-21 Modified tussah silk fibroin 3D printing support and preparation method thereof

Country Status (1)

Country Link
CN (1) CN110859994B (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111454614B (en) * 2020-05-28 2022-05-24 苏州大学 3D biological printing ink and preparation method and application thereof
CN113274555B (en) * 2021-05-31 2022-05-03 清华大学 Artificial ventricle with bionic spiral orientation microstructure and preparation method thereof

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102827376A (en) * 2012-09-10 2012-12-19 江苏科技大学 Preparation method of sulfated silk fibroin material
WO2016019078A1 (en) * 2014-07-30 2016-02-04 Tufts University Three dimensional printing of bio-ink compositions
CN107041971A (en) * 2016-09-19 2017-08-15 盐城工业职业技术学院 A kind of fibroin based on 3 D-printing/gelatin timbering material and preparation method thereof
CN107551321A (en) * 2017-09-22 2018-01-09 广州迈普再生医学科技有限公司 Organizational project Bone Defect Repari fiber and bone repairing support and preparation method thereof
WO2018089515A1 (en) * 2016-11-09 2018-05-17 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services 3d vascularized human ocular tissue for cell therapy and drug discovery
CN108215165A (en) * 2018-01-25 2018-06-29 清华大学 Intelligent pattern of skin-core structure fiber and printing method and application thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102827376A (en) * 2012-09-10 2012-12-19 江苏科技大学 Preparation method of sulfated silk fibroin material
WO2016019078A1 (en) * 2014-07-30 2016-02-04 Tufts University Three dimensional printing of bio-ink compositions
CN107041971A (en) * 2016-09-19 2017-08-15 盐城工业职业技术学院 A kind of fibroin based on 3 D-printing/gelatin timbering material and preparation method thereof
WO2018089515A1 (en) * 2016-11-09 2018-05-17 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services 3d vascularized human ocular tissue for cell therapy and drug discovery
CN107551321A (en) * 2017-09-22 2018-01-09 广州迈普再生医学科技有限公司 Organizational project Bone Defect Repari fiber and bone repairing support and preparation method thereof
CN108215165A (en) * 2018-01-25 2018-06-29 清华大学 Intelligent pattern of skin-core structure fiber and printing method and application thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
3D-printed scaffolds with synergistic effect of hollow-pipe structure and bioactive ions for vascularized bone regeneration;Wenjie Zhang 等;《biomaterials》;20170503;第135卷;第85-95页 *
A Gelatin-sulfonated Silk Composite Scaffold based on 3D Printing Technology Enhances Skin Regeneration by Stimulating Epidermal Growth and Dermal Neovascularization;Si Xiong 等;《scientific reports》;20170627;第7卷;第4288页 *
Bioprintable, cell-laden silk fibroin–gelatin hydrogel supporting multilineage differentiation of stem cells for fabrication of three-dimensional tissue constructs;Sanskrita Das 等;《Acta Biomaterialia》;20140919;第11卷;第233-246页 *
丝蛋白材料的三维打印;孙放;《现代丝绸科学与技术》;20181231;第33卷(第2期);第35-38页 *

Also Published As

Publication number Publication date
CN110859994A (en) 2020-03-06

Similar Documents

Publication Publication Date Title
Ferreira et al. Porous nanocellulose gels and foams: Breakthrough status in the development of scaffolds for tissue engineering
Hu et al. Biocompatible fibroin blended films with recombinant human-like collagen for hepatic tissue engineering
CN100453124C (en) Porous, laminated, tri-dimensional multiple-grade structure tissue stent material and its preparation method
CN107744601B (en) Three-dimensional printing wound coating material based on silk microsphere biological ink and preparation method thereof
Jiang et al. Preparation of cellulose nanofiber-reinforced gelatin hydrogel and optimization for 3D printing applications
CN111574816A (en) High-strength biomedical hydrogel material and bath-supported hydrogel 3D printing method
CN106310380A (en) Nano-fibrosis silk fibroin gel and preparation method thereof
CN109758611B (en) Method for preparing active biological tissue engineering scaffold by solvent spraying
CN115054728B (en) Bionic bone tissue engineering scaffold material and preparation method thereof
CN111825859A (en) Bionic electronic skin medical stent material with self-repairing function and preparation method thereof
CN110859994B (en) Modified tussah silk fibroin 3D printing support and preparation method thereof
Li et al. Hierarchical porous bacterial cellulose scaffolds with natural biomimetic nanofibrous structure and a cartilage tissue-specific microenvironment for cartilage regeneration and repair
CN113150561B (en) Collagen-based biological ink for 3D biological printing and preparation method and application thereof
CN112999428B (en) Cell-loaded polymer porous microsphere three-dimensional biological printing ink for auricle reconstruction and manufacturing method and application thereof
Li et al. Fabrication, characterization, and in vitro evaluation of biomimetic silk fibroin porous scaffolds via supercritical CO2 technology
CN102493021A (en) Method for preparing cellulose nanocrystal reinforced polyhydroxybutyrate-hydroxyvalerate (PHBV) nanofiber
Chen et al. Bio-inspired hydrogels with fibrous structure: A review on design and biomedical applications
Taneja et al. Hydrogel based 3D printing: Bio ink for tissue engineering
CN111617319B (en) Composite hydrogel, preparation method and application thereof
CN106390208A (en) Three-dimensional support material containing hierarchical porous structures and preparation and application
Wang et al. Biomimetic bioinks of nanofibrillar polymeric hydrogels for 3D bioprinting
Wang et al. Elastic fiber-Reinforced silk fibroin scaffold with a Double-Crosslinking network for human ear-shaped cartilage regeneration
CN110755685A (en) 3D printing guar gum gel bracket and preparation method thereof
CN109943974B (en) Preparation method of nerve conduit material based on polyhydroxyalkanoate/gelatin electrospun nanofiber
CN110680954A (en) 3D printing xanthan gum hydrogel support and preparation method thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant