CN110859994B - A kind of modified tussah silk fibroin 3D printing scaffold and preparation method thereof - Google Patents

Abstract

The invention relates to a modified tussah silk fibroin 3D printing scaffold and a preparation method thereof. The core part printing ink and the chemically modified tussah silk fibroin nano-microfibers are prepared by using chemically modified tussah silk fibroin nano-microfibers. The modified tussah silk fibroin 3D printing scaffold was prepared by 3D printing with the shell part printing ink prepared by the composite system of gelatin/gelatin; wherein, the modified tussah silk fibroin 3D printing scaffold was soaked in genipin with a mass concentration of 0.1-5 wt%. The compressive modulus after the combined reaction for 24 hours was 100-600 MPa, and the survival rate and proliferation rate of induced pluripotent stem cells were high after 10 days of culture. line composition. The preparation method of the present invention is relatively simple, and the prepared 3D printed scaffold has excellent mechanical properties, excellent biocompatibility and good tissue repair ability.

Description

A kind of modified tussah silk fibroin 3D printing scaffold 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 technique

Tissue and organ defects and dysfunctions caused by trauma, tumors, and aging are important factors that endanger human health, and the repair and functional reconstruction of defective tissues and organs is currently a difficult problem facing the world. Large-scale defects usually need to be repaired by autologous or allogeneic tissue and organ transplantation. Autologous transplantation has the problem of "repairing wounds with wounds", and for allogeneic transplantation, there are also major shortcomings such as insufficient donor source and immune rejection. . The proposal, establishment and development of tissue engineering provides a new way to solve the problems of such tissue and organ defects and dysfunctions. From the idea of implanting cells into degradable biomaterials to construct tissues in the early 1980s, to the success of applying tissue engineering technology to repair clinical defects in recent years, tissue engineering technology has been proved to be the solution to tissue wound repair and functional reconstruction. one of the effective ways.

In recent years, the rise of biological three-dimensional (3D) printing technology has attracted much attention in the regenerative reconstruction of human tissues or organs. Biological 3D printing technology simulates different tissues and organs of the human body through CAD technology and uses biological materials, seed cells and some other biological reagents as inks to print and reconstruct human tissues and organs under computer control. The scaffold structure produced by 3D bioprinting is not only accurate and specific, but also can maintain cell viability, so as to meet the reconstruction needs of various complex tissues and organs in the human body. Therefore, 3D bioprinting will definitely lead to a technological revolution in the field of biomedicine. However, one of the main challenges facing 3D bioprinting technology lies in the selection of the bioink used. The traditional materials used for 3D printing are mainly synthetic polymers such as polyethylene glycol PEG, polycaprolactone PCL, polyglycolic acid PGA, polyvinyl alcohol PVA, polylactic acid PLA, etc. These materials are easy to form. , the advantages of high resolution, but there are also the shortcomings of high processing temperature and poor cell compatibility, and the scaffolds prepared by using these materials will generally introduce some toxic substances during the preparation process. The removal of these substances will negatively affect the biocompatibility of the stent.

Compared with the above synthetic materials, silk fibroin is a natural polymer material with better biocompatibility. At present, many people have carried out research on its application in biological materials, and some people have applied it to 3D. In the field of printing, biological scaffold materials have been prepared.

Patent CN108085760A discloses a paper mass graphene-modified light-cured silk fibroin three-dimensional printing material and a preparation method thereof, including ink for 3D printing, and the ink includes a graphene oxide solution and a silk fibroin solution; Patent CN107744601A discloses a three-dimensional printing wound wrapping material based on silk microsphere bio-ink and its preparation method, including ink for 3D printing, the ink includes silkworm silk protein solution and aspirin; patent CN105031728A discloses a Low temperature rapid prototyping three-dimensional printing collagen silk fibroin material, which contains silk fibroin ink for 3D printing, the ink includes silk fibroin, collagen, glycerol and compatibilizer; Patent CN105903071A discloses a corneal scaffold material and its preparation method and 3D printing method of corneal stent, including silk fibroin ink for 3D printing, said ink comprising silk fibroin, collagen, graphene oxide and cross-linking agent; patent CN104958785A discloses a Composite bone repair material with secondary three-dimensional structure and preparation method thereof, including silk fibroin ink for 3D printing, said ink comprising Bombyx mori silk fibroin, hydroxyapatite and collagen, using the method described in this patent The elastic modulus of the fabricated 3D tissue engineering scaffolds is in the range of 290-430 kPa.

In the document "3D printing of silk particle-reinforced chitosan hydrogelstructures and their properties", a composite bioprinting ink made of Bombyx mori silk fibroin (BSF), chitosan and water was used for printing, and the obtained scaffolds had the highest compressive modulus. Reach 5KPa; the document "Structurally and functionally optimized silk-fibroin–gelatin scaffold using 3D printing torepair cartilage injury in vitro and in vivo" used a composite bio-3D printing ink made of Bombyx mori silk fibroin (BSF), gelatin and water for printing, The elastic modulus of the obtained scaffold can reach a maximum of 15kPa; the document "Precisely printable and biocompatible silk fibroin bioink for digital lightprocessing 3D printing" uses glycidyl methacrylate modified Bombyx mori silk fibroin (BSF) bioprinting ink for printing , the elastic modulus of the obtained stent can reach a maximum of 120KPa.

The above studies have all applied 3D printing technology to the field of silk fibroin, but the above studies only studied the 3D printing of silk fibroin. The unique Chinese tussah silk fibroin molecular chain has an RGD tripeptide chain segment composed of arginine, glycine, and aspartic acid, which is not found in the silk fibroin molecular chain of Bombyx mori. It is proved to have higher cell adhesion, so compared with Bombyx mori silk fibroin, tussah silk fibroin is expected to have better biocompatibility. In addition, the molecular chain of tussah silk fibroin contains a large number of alanine segments (AAAAAAAAA...), which has more glycine-alanine-serine alternating segments (GAGAGASGAGAGAS...) High regularity can endow the printed scaffold with better mechanical properties. Therefore, compared with Bombyx mori silk fibroin, tussah silk fibroin has more potential in the field of 3D printing.

To this end, patent CN106267370A discloses silk fibroin/cellulose 3D printing ink, the ink includes water-soluble silk fibroin, water-insoluble cellulose micro/nano material, non-toxic polyol and water. The compressive modulus of the stent is in the range of 10 to 50 MPa. The content disclosed in the patent includes the related content of 3D printing ink based on tussah silk fibroin aqueous solution, but related researchers found that tussah silk fibroin is more likely to form an anti-parallel zigzag structure under the action of intermolecular hydrogen bonds. It is also more sensitive to temperature and external force. The tussah silk fibroin solution is very easy to undergo conformational transformation under the influence of temperature and external force to form a water-insoluble solid, thereby affecting the entire preparation process.

Not only that, the large amount of alanine hydrophobic segments (AAAAAAAAA...) in the tussah silk fibroin molecular chain will also make the printed scaffolds have low hydrophilicity, which will adversely affect its biocompatibility.

In order to solve the problem of low hydrophilicity of the above-mentioned printed scaffolds, it is envisaged that if the molecular chain of tussah silk fibroin can be chemically modified and a certain hydrophilic group is introduced into the molecular chain, it is expected to improve the biological properties of the material. compatibility.

In addition, if a scaffold has excellent tissue repair ability, it must have relatively good adhesion and proliferation ability to cells, especially stem cells. At present, among many kinds of stem cells that can be used for tissue engineering, especially cardiac tissue engineering, the effect is The best are induced pluripotent stem cells (iPSCs). At present, many people have used the cells in the field of 3D printing to produce 3D scaffolds with tissue repair capabilities.

The paper "3D Bioprinting Human Induced Pluripotent Stem Cell-DerivedNeural Tissues Using a Novel Lab-on-a-Printer Technology" loaded iPSCs into 3D printed biocomposites made of fibronectin, sodium alginate, and genipin dissolved in water The iPSCs were printed in the ink, and the survival rate of iPSCs could reach about 95% on the 7th day after printing. 3D printing was carried out in the bio 3D printing ink of 100% cellulose, and it was found that the survival rate of iPSCs could reach about 95% within one day after printing, and the survival rate of iPSCs could reach about 90% within 10 days after printing, and the cell proliferation rate was 3000%. ～4000%; the document "Laser bioprinting of human induced pluripotent stem cells—the effect of printing and biomaterialson cell survival, pluripotency, and differentiation" loaded iPSCs in different media for 3D printing, and found that iPSCs were 3D within 3 h after printing. The maximum survival rate can reach about 90%, and the cell proliferation rate can reach about 429% 4 days after printing; the document "A simple and efficient feeder-free culture system to up-scale iPSCs on polymeric material surface for use in 3D bioprinting" produced A variety of bio-3D printing inks based on chitosan, silk fibroin and polyurethane were used, and iPSCs were cultured with these inks as substrates. It was found that the proliferation rate of iPSCs reached a maximum of about 300% after 3 days of culture.

The above studies all applied iPSCs to the field of 3D printing, but did not study the mechanical properties of 3D printed scaffolds. For some tissues (such as the heart) that require relatively vigorous mechanical movements, the mechanical properties of scaffolds are particularly important. And for different tissue parts and different degrees of tissue damage, tissue engineering scaffold materials need to have a matching repair speed, and the current preparation method of biomaterial 3D printing scaffolds is difficult to take into account the repair speed and speed of the injured tissue. Other properties of the bracket.

Based on the above background, a modified tussah silkworm that can simultaneously take into account the repair speed of the scaffold to the injured tissue and the mechanical properties, biocompatibility, and cell adhesion and proliferation ability of the scaffold and has the function of controllable cell growth and proliferation speed. Silk fibroin 3D printed scaffolds and their preparation methods are of great significance. If the chemically modified tussah silk fibroin can be made into nanofibers, the conformation can be kept stable during the ink preparation process, making the preparation process easier to control, and the mechanical properties, biocompatibility and tissue repair ability of the scaffold can be improved By adjusting the content of tussah silk fibroin nanofibers and the preparation process, it is expected to obtain tissue engineering scaffolds with excellent mechanical properties and tissue repair ability.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a modified tussah silk fibroin 3D printing scaffold and the its preparation method. The scaffolds produced by this method not only have excellent mechanical properties and cell adhesion and proliferation ability, but also have good biocompatibility. In addition, it also has the function of controllable cell growth and proliferation rate.

For achieving the above object, the technical scheme of the present invention is:

A preparation method of modified tussah silk fibroin 3D printing scaffold, core part printing ink prepared from chemically modified tussah silk fibroin nano-microfibers and chemically modified tussah silk fibroin nano-microfibers/gelatin composite system The obtained shell part printing ink is 3D printed to obtain a modified tussah silk fibroin 3D printing scaffold;

The chemically modified tussah silk fibroin nano-microfibers are obtained by mixing the tussah silk fibroin nano-microfibers/boric acid buffer mixed system with chemical modifiers through a series of reactions and operations. The reaction time of the agent to adjust the adsorption and proliferation rate of cells on the scaffold, the faster the proliferation rate, the faster the repair rate, so as to meet the needs of different degrees of tissue damage for the repair rate; The different hydrophilic groups attached will make the scaffolds have different cytocompatibility, which will make the proliferation rate of cells on the scaffolds different, so as to obtain scaffolds with controllable repair speed;

The 3D printing method of the present invention draws a three-dimensional model through a computer, obtains the contour data and filling data of the cross-section of each layer in the three-dimensional model, selects polymer materials to make multiple copies of different 3D printing materials, and extrudes through a printing nozzle of a specified size specification. The lines are deposited in the designated areas on the working platform in different directions, and the extrusion lines are solidified and formed by cooling down to realize the production of a single-layer cross-sectional structure, and the single-layer cross-sectional structure is repeated in different directions. Print;

Among them, the printing ink of the core part and the printing ink of the shell part are extruded from the coaxial nozzle device to form the printing line with the core-shell structure. For the printing of the core-shell structure, if the axis is different, the obtained scaffold will be uneven, which will affect its mechanical properties;

The mass content of chemically modified tussah silk fibroin nanofibers in the core part printing ink is greater than or equal to 6wt%, otherwise it will collapse; the mass content of chemically modified tussah silk fibroin nanofibers in the shell part printing ink is less than or equal to 6wt% %, and also contains gelatin, the concentration of tussah silk fibroin nano-microfibers must be within 6wt%, otherwise it will not be able to mix with gelatin because of its poor fluidity;

The mass content of chemically modified tussah silk fibroin nano-microfibers in the core part printing ink is greater than or equal to 6wt%, otherwise it will collapse, because its main components are tussah silk fibroin nano-microfibers, tussah silk fibroin nano-microfibers It does not exist in a molecular state, and the interaction force between them is weak, so it is easy to fall apart; so in order to prevent it from falling apart, it is necessary to coat a layer of shell ink with the main components existing in molecular state and strong intermolecular force on the outside, so The tussah silk fibroin nanofiber/gelatin composite system mainly composed of gelatin was used in the printing ink of the shell part;

The chemically modified tussah silk fibroin nanofibers are tussah silk fibroin nanofibers grafted with hydrophilic groups, wherein the purpose of chemical modification is to introduce hydrophilic groups to improve the hydrophilicity of the scaffold. It grows in a water-based environment, so the introduction of hydrophilic groups into the molecular chain of tussah silk fibroin nanofibers will improve its cell adhesion and proliferation ability, thereby improving the biocompatibility of the scaffold;

In the prior art, when induced pluripotent stem cells (iPSCs) are cultured on 3D-printed tissue engineering scaffolds, the highest cell survival rate can reach about 90% after 10 days of culture, and the highest cell proliferation rate can reach 3000%-4000%. However, the survival rate of induced pluripotent stem cells (iPSCs) was 92.0%-99.0% and the proliferation rate was 9000%-25000 after seeding and culturing on the modified tussah silk fibroin nanofiber/gelatin composite 3D printing scaffold of the present invention for 10 days. %, compared with the prior art, the survival rate and proliferation rate of iPSCs after printing with the 3D printing ink of the present invention are significantly improved, and unexpected technical effects are achieved.

In addition, in the prior art, the 3D printing ink based on Bombyx mori silk fibroin is printed, and the compressive modulus of the obtained scaffold can reach up to 5kPa; the 3D scaffold printed with the 3D printing ink based on the tussah silk fibroin aqueous solution has a compressive modulus in In the range of 10-50MPa; while the modified tussah silk fibroin 3D scaffold printed by the present invention has a compressive modulus of 100-600MPa after being soaked and cross-linked in genipin with a mass concentration of 0.1-5wt% for 24h. In the prior art, the 3D printing ink based on Bombyx mori silk fibroin and the 3D printing ink based on tussah silk fibroin aqueous solution printed the compressive modulus of the 3D scaffold, which also achieved unexpected technical results.

As a preferred solution:

A method for preparing a modified tussah silk fibroin 3D printing scaffold as described above, the extrusion expansion rate of the printing ink of the core part is 0.10-1.00%, and the dynamic viscosity is 300-500 cP; the self-gelling of the printing ink of the shell part The melting time is 10-60s, the extrusion expansion rate is 1-30%, and the dynamic viscosity is 1000-5000cP.

The above-mentioned preparation method of a modified tussah silk fibroin 3D printing scaffold, the preparation process of the shell part printing ink is as follows: first, chemically modified tussah silk fibroin nanofibers, cell growth factors and antibiotics are added into water, Mechanical stirring for 1-3 hours, then mixing with gelatin, after standing for 1-3 hours, mechanically stirring for 1-5 hours at a temperature of 35-45°C, and finally ultrasonically treating for 1-60 minutes;

The mass content of each component in the shell part printing ink is: chemically modified tussah silk fibroin nano-microfibers 1-6 wt %, gelatin 14-20 wt %, cell growth factor 0.1-1.0 wt %, antibiotic 0.1-1.0 wt % , the balance is water;

The preparation process of the core part printing ink is as follows: adding chemically modified tussah silk fibroin nanofibers, cell growth factors and antibiotics into water, and mechanically stirring for 1-3 hours at a temperature of 1-45°C;

The mass content of each component in the core part printing ink is: chemically modified tussah silk fibroin nano-microfiber 6-20 wt%, cell growth factor 0.1-1.0 wt%, antibiotic 0.1-1.0 wt%, and the balance is water.

Since the scaffold of the present invention needs to be used for in vivo tissue engineering, it must be non-toxic, so the dispersion medium in the printing ink of the shell part and the printing ink of the core part is water, and of course other non-toxic solvents can also be used; In the nuclear part printing ink, the function of cell growth factor is to improve the cell growth and proliferation ability of the material; the function of antibiotics is to promote the expression of cardiac proteins in iPSCs cells, and promote the transformation of iPSCs cells into cardiomyocytes.

The above-mentioned preparation method of a modified tussah silk fibroin 3D printing scaffold, all cell growth factors are fibroblast growth factor 2; all antibiotics are tetracycline;

All cell growth factors are not limited to fibroblast growth factor 2, but can also be other substances, such as bone morphogenetic protein 4; all antibiotics are not limited to tetracycline, but can also be other substances, such as doxycycline etc., the above-mentioned cell growth factors and antibiotics can achieve the effect of improving cell proliferation rate, survival rate and transformation to cardiomyocytes, but the cell growth factors and antibiotics that can be added to the ink used in the stent of the present invention are not limited to the above two. The addition of other cell growth factors (such as bone morphogenetic protein 4, etc.) and antibiotics (such as doxycycline, etc.) should also be considered within the protection scope of the present invention without departing from the technical principle of the present invention.

The above-mentioned preparation method of a modified tussah silk fibroin 3D printing scaffold, the preparation steps of all chemically modified tussah silk fibroin nano-fibers are as follows:

(1) prepare tussah silk fibroin pulp;

(2) Preparation of chemical modifier and tussah silk fibroin nanofiber/boric acid buffer mixed system;

(2.a) The preparation process of the chemical modifier is as follows: acetonitrile solution of p-aniline-based organic compounds with a concentration of 1mM to 1M (M=mol/L), an aqueous solution of p-toluenesulfonic acid with a concentration of 1mM to 2M, and a concentration of It is 1.0mM～1.0M sodium nitrite aqueous solution according to the volume ratio of 1:0.5～4:0.5～2, after mixing by vortex for 5s under the temperature condition of 1～4℃, under the same temperature condition, mechanical stirring reaction 5 The chemical modifier is obtained in ~30min; the vortex mixing means that the liquid to be mixed is put into a test tube or a small flask and shaken on a commonly used vortex mixer;

(2.b) The preparation steps of the tussah silk fibroin nanofiber/boric acid buffer mixed system are as follows:

(2.b.1) Immerse the tussah silk fibroin pulp in water at a mass-to-volume ratio of 1 g:50 to 200 mL, and mechanically stir to disperse it evenly;

(2.b.2) According to the mass molar ratio of tussah silk fibroin pulp:NaClO=1g:0.001～0.050mol, add the NaClO aqueous solution with a mass concentration of 5～35wt% to the water dispersion system of tussah silk fibroin pulp , mechanically stirring at a temperature of 1-60 ° C. During this process, the pH value of the system is maintained between 10.0 and 10.1 by continuously adding 0.5-5.0M NaOH aqueous solution until the system pH value is not added when the NaOH aqueous solution is not added. The value can also be maintained between 10.0 and 10.1;

(2.b.3) Transfer the system to a cellulose dialysis bag with a molecular weight cut-off of 14000 Da, dialyze it in deionized water for 1 to 3 days, continue to dialyze it in boric acid buffer for 1 day, and then concentrate to obtain tussah silk fibroin The tussah silk fibroin nanofiber/boric acid buffer mixed system with the nanofiber concentration of 1-20wt%, wherein the boric acid buffer is mainly composed of boric acid, sodium chloride and water, and the concentrations of boric acid and sodium chloride are 100mM and 100mM respectively. 150mM;

(3) chemical modification;

First, the tussah silk fibroin nanofiber/boric acid buffer mixed system with a concentration of 1-20wt% was mixed with the chemical modifier in a volume ratio of 1:0.1-2, and the reaction was mechanically stirred in an ice-water bath for 1-5 days, and then Dialysis with deionized water for 1-3 days, purification and freeze-drying to obtain chemically modified tussah silk fibroin nanofibers with an aspect ratio of 100-200 and a diameter of 10-200 nm.

The above-mentioned preparation method of a modified tussah silk fibroin 3D printing scaffold, the preparation process of the tussah silk fibroin pulp is as follows:

(1.1) Immerse the tussah cocoons in a Na ₂ CO ₃ aqueous solution with a mass concentration of 0.05-5.00 wt % at a mass-to-volume ratio of 1 g:50-100 mL, boil 1-5 times for 30-60 min each time, and then boil the boiled cocoons The tussah cocoons are washed with water and dried to obtain degummed tussah silk fibers;

(1.2) immersing the dried degummed tussah silk fibers in an aqueous formic acid solution with a mass concentration of 60 to 100 wt % for 1 to 3 hours at a mass volume ratio of 1 g:15 to 25 mL;

(1.3) Under the temperature condition of 10～60℃, homogenize for 1～5min and pulverize, and the homogenization rate is 5000～15000rpm;

(1.4) repeating step (1.2) and step (1.3) 1 to 5 times to obtain tussah silk fibroin protein slurry;

(1.5) Centrifuging, filtering, washing and drying the tussah silk fibroin pulp to obtain the tussah silk fibroin pulp.

The preparation method of a modified tussah silk fibroin 3D printing scaffold as described above, the p-aniline-based organic compound is p-aminobenzoic acid, 4-(2-ethylamino)aniline, p-aminoacetophenone or p-aminophenheptyl ether.

The above-mentioned preparation method of a modified tussah silk fibroin 3D printing scaffold, the diameters of the core part and the shell part of the printing lines with the core-shell structure are respectively 50-500 μm and 100-1000 μm, and the diameter of the shell part is larger than that of the core Part of the diameter; when printing, the printing lines with core-shell structure are deposited on the glass sheet in a layer-by-layer manner; the printing process parameters are: barrel and needle temperature 25 ~ 37 ℃, extrusion pressure 10 ~ 500KPa, printing speed 1 to 10 mm/s, the angle between two adjacent printing layers formed by printing lines with a core-shell structure is 30 to 90°, the line spacing is 20 to 2000 μm, and the number of deposition layers is 2 to 10.

The stent obtained by the above printing process conditions has relatively excellent mechanical properties and biocompatibility, but the printing conditions of the stent of the present invention are not limited to the above range. Changes to the basic parameters of the printing process conditions and the shape of the printing support should also be considered within the protection scope of the present invention.

The modified tussah silk fibroin 3D printing scaffold prepared by the method for preparing a modified tussah silk fibroin 3D printing scaffold as described in any one of the above is composed of printing lines with a core-shell structure, and a The core part of the printed lines is mainly composed of chemically modified tussah silk fibroin nanofibers, and the shell part is mainly composed of chemically modified tussah silk fibroin nanofibers and gelatin.

As a preferred solution:

The modified tussah silk fibroin 3D printed scaffold as described above has a resolution of 0.5-10.0 μm. Generally, the phenomenon of extrusion swelling and extrusion rupture will occur in the 3D printing process of polymer inks. The thinner the needle, the more likely this phenomenon occurs. The resolution refers to the minimum diameter of uniform and stable lines that can be printed by this ink. That is to say, the smaller the resolution value is, the thinner the stable lines can be obtained, the less likely to cause extrusion swelling and extrusion cracking, and the higher the printing accuracy. The resolution of the 3D printing bracket prepared by the prior art is generally average. It is about 30 μm, and the resolution of the modified tussah silk fibroin 3D printing scaffold finally prepared by the present invention is 0.50-10.0 μm, which proves that the 3D printing scaffold prepared by the present invention has higher printing accuracy.

Beneficial effects:

(1) A modified tussah silk fibroin 3D printing scaffold of the present invention, the tussah silk fibroin nano-microfibers contained in the scaffold can make the scaffold have excellent mechanical properties, and the compressive modulus of the scaffold is compared with the existing use 3D printed scaffolds containing silk fibroin ink can be increased by more than 5 times;

(2) A modified tussah silk fibroin 3D printing scaffold of the present invention, the tussah silk fibroin in the ink used is from the cocoon of the tussah silk fibroin, and the tussah silk fibroin contains RGD segments, which have better cell adsorption and proliferation ability; after chemical modification of tussah silk fibroin, it has better surface hydrophilicity. Since cells are generally grown in a water-based environment, after inoculating iPSCs on the scaffold of the present invention for 10 days, their survival rate and proliferation ability are compared with those of existing 3D-printed cells containing Bombyx mori silk fibroin ink. The scaffolds are all improved, and it can be seen that the scaffold of the present invention has excellent tissue repair ability;

(3) In the preparation method of a modified tussah silk fibroin 3D printing scaffold of the present invention, in the preparation process of the chemically modified tussah silk fibroin nanofibers, NaClO will cause the tussah silk fibroin molecular chain to undergo an oxidation reaction , the oxidation reaction is mainly located on the serine in the molecular chain; while the chemical modification reaction on the tussah silk fibroin molecular chain is mainly located on the tyrosine in the molecular chain, neither of these two reactions will affect the tussah silk fibroin molecular chain. Influenced by the RGD segment with good cytocompatibility;

(4) In the preparation method of a modified tussah silk fibroin 3D printing scaffold of the present invention, each component of the ink used in the ink uses water as a dispersant, so it has excellent biocompatibility and can be well compounded with cells. 3D printing;

(5) In a modified tussah silk fibroin 3D printing scaffold of the present invention, the tussah silk fibroin contained in the printing ink exists in the form of nano-fibers, and its conformation can be kept stable under wider environmental conditions, Therefore, the adverse effects on the stability of the ink due to fluctuations in temperature and external force can be avoided during the preparation process;

(6) A modified tussah silk fibroin 3D printing scaffold of the present invention can adjust the adsorption and proliferation speed of cells on the scaffold by adjusting the reaction time of tussah silk fibroin and chemical modifiers, so as to adapt to different degrees of tissue Damage demand for speed of repair;

(7) In the modified tussah silk fibroin 3D printing scaffold of the present invention, the printing process of the printing ink is carried out under the conditions of room temperature to physiological temperature, which is conducive to loading various biological factors and embedding cells to realize active printing.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Example 1

A preparation method of a modified tussah silk fibroin 3D printing scaffold, the main steps are as follows:

(1) Preparation of tussah silk fibroin pulp:

(1.1) Immerse the tussah cocoons in a Na ₂ CO ₃ aqueous solution with a mass concentration of 0.50 wt % at a mass volume ratio of 1 g:50 mL, boil 3 times for 45 min each time, and then wash the boiled tussah cocoons with water and dry to obtain Degummed tussah silk fibers;

(1.2) immersing the degummed tussah silk fiber in an aqueous formic acid solution with a mass concentration of 90wt% for 2h at a mass-volume ratio of 1g:20mL;

(1.3) Under the temperature condition of 25 ℃, homogenize for 2min and pulverize, and the homogenization rate is 10000rpm;

(1.4) repeat step (1.2) and step (1.3) 3 times to obtain tussah silk fibroin slurry;

(1.5) tussah silk fibroin pulp is centrifuged, filtered, washed and dried to obtain tussah silk fibroin pulp;

(2) Preparation of p-aminobenzoic acid modifier: acetonitrile solution of p-aminobenzoic acid with a concentration of 0.2M, an aqueous solution of p-toluenesulfonic acid with a concentration of 1.6M and an aqueous solution of sodium nitrite with a concentration of 0.8M in a ratio of 1:0.5 A volume ratio of 0.5 was mixed by vortexing for 5s under the temperature condition of 2°C, and the p-aminobenzoic acid modifier was obtained by mechanical stirring reaction for 15min under the same temperature condition;

(3) Preparation of tussah silk fibroin nanofiber/boric acid buffer mixed system:

(3.1) The tussah silk fibroin pulp is immersed in water at a mass volume ratio of 1g:100mL, and mechanically stirred to make it evenly dispersed;

(3.2) According to the mass molar ratio of tussah silk fibroin pulp: NaClO=1g:0.015mol, in the water dispersion system of tussah silk fibroin pulp, add a NaClO aqueous solution with a mass concentration of 25wt%, and under the temperature condition of 25°C Mechanical stirring. During this process, the pH value of the system is maintained between 10.0 and 10.1 by continuously adding 1.0M aqueous NaOH solution, until the pH value of the system can also be maintained between 10.0 and 10.1 when no aqueous NaOH solution is added;

(3.3) Transfer the system to a cellulose dialysis bag with a molecular weight cut-off of 14,000 Da, dialyze it in deionized water for 2 days, continue to dialyze it in boric acid buffer for 1 day, and then concentrate to obtain tussah silk fibroin nanofibers with a concentration of 10wt % Tussah silk fibroin nanofiber/boric acid buffer mixed system, wherein the boric acid buffer is mainly composed of boric acid, sodium chloride and water, and the concentrations of boric acid and sodium chloride are 100mM and 150mM respectively;

(4) Preparation of p-aminobenzoic acid-modified tussah silk fibroin nanofibers: first, the concentration of 10wt% tussah silk fibroin nano-fibers/boric acid buffer mixed system and p-aminobenzoic acid modifier were 1:0.25 The volume ratio was mixed, the reaction was mechanically stirred in an ice-water bath for 2 days, and then purified by deionized water dialysis for 2 days and freeze-dried to obtain p-aminobenzoic acid-modified tussah silk fibroin with an average aspect ratio of 150 and an average diameter of 80 nm. protein nanofibers;

(5) Preparation of nuclear part printing ink: adding p-aminobenzoic acid-modified tussah silk fibroin nanofibers, fibroblast growth factor 2 and tetracycline into water, and mechanically stirring at 20°C for 3 hours to prepare the core part The printing ink, wherein the mass content of each component in the printing ink of the core part is: 14wt% of tussah silk fibroin nanofibers modified by p-aminobenzoic acid, 0.1wt% of fibroblast growth factor 2, and 0.4 wt% of tetracycline wt%, the balance is water; the extrusion swelling rate of the printing ink in the core part is 0.50%, and the dynamic viscosity is 420cP;

(6) Preparation of printing ink for shell part: First, add p-aminobenzoic acid-modified tussah silk fibroin nanofibers, fibroblast growth factor 2 and tetracycline into water, stir mechanically for 3 hours, and then mix with gelatin and let stand for 2 hours. , mechanically stirred for 2 hours at a temperature of 40 °C, and finally ultrasonically treated for 10 minutes to obtain a shell part printing ink; wherein, the mass content of each component in the shell part printing ink is: p-aminobenzoic acid modified tussah silk fibroin nanometer Microfiber 5wt%, gelatin 15wt%, fibroblast growth factor 2 is 0.1wt%, tetracycline is 0.4wt%, and the balance is water; the self-gelling time of the shell part printing ink is 38s, and the extrusion swelling rate is 10%, the dynamic viscosity is 3700cP;

(7) First, the core part printing ink and the shell part printing ink prepared above are extruded from the 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 The modified tussah silk fibroin 3D printing scaffolds were obtained by 3D printing; when printing, the printing lines with core-shell structure were deposited on the glass sheet in a layer-by-layer manner; the printing process parameters were: material The temperature of the cylinder and needle head is 30°C, the extrusion pressure is 160KPa, the printing speed is 8mm/s, the angle between two adjacent printing layers formed by printing lines with a core-shell structure is 90°, the line spacing is 400 μm, and 4 layers are deposited.

The final resolution of the modified tussah silk fibroin 3D printed scaffold was 4.6 μm; the compressive modulus of the modified tussah silk fibroin 3D printed scaffold after immersion and cross-linking reaction in genipin with a mass concentration of 0.5 wt% for 24 h is 336MPa;

If 1 mL of fetal bovine serum culture medium containing 10,000 second-generation iPSCs was inoculated on a 15 mm diameter scaffold printed and cross-linked as described above, and cultured at 37.0°C, 5.0% CO ₂ for 1, 3, 4 , 7 and 10 days later, using trypan blue staining method, the cell viability was measured to be 99.1%, 98.1%, 97.7%, 95.9%, and 93.0%, respectively. 622%, 1269%, 7678%, 17478%.

Comparative Example 1

A preparation method of a modified tussah silk fibroin 3D printing scaffold, the steps are basically the same as those in Example 1, the difference is that it is not modified by a p-aminobenzoic acid modifier,

The finally prepared modified tussah silk fibroin nanofibers have an aspect ratio of 50 and an average diameter of 400 nm.

The core part printing ink has an extrusion swell rate of 4.00% and a dynamic viscosity of 100 cP.

The self-gelling time of the shell part printing ink is 60s, the extrusion swelling rate is 40%, and the dynamic viscosity is 600cP.

The resolution of the finally prepared modified tussah silk fibroin 3D printed scaffold was 30 μm; the compressive modulus of the modified tussah silk fibroin 3D printed scaffold after immersion and cross-linking reaction in genipin with a mass concentration of 0.5 wt% for 24 h was 80MPa;

If 1 mL of fetal bovine serum culture medium containing 10,000 second-generation iPSCs was inoculated on a 15 mm diameter scaffold printed and cross-linked as described above, and cultured at 37.0°C, 5.0% CO ₂ for 1, 3, 4 , 7 and 10 days later, using trypan blue staining, the cell viability was measured to be 98.6%, 97.6%, 97.2%, 95.4%, and 91.5%, respectively. 256%, 468%, 1789%, 4958%.

Comparing Example 1 with Comparative Example 1, it can be seen that the survival rate and proliferation ability of the cells on the modified tussah silk fibroin 3D printing scaffold of Example 1 were higher than those of the control group after 1, 3, 4, 7, and 10 days. Example 1, and the mechanical properties are also improved compared to Comparative Example 1, this is because after chemical modification of tussah silk fibroin, its hydrophilicity and inter-molecular chain interactions are greatly improved. , its cytocompatibility will also be correspondingly improved, with the improvement of the inter-chain interaction of scaffold molecules, its ability to resist external forces will also be correspondingly improved. Therefore, the modified tussah silk fibroin 3D obtained by the present invention Compared with 3D printed scaffolds based on unmodified tussah silk fibroin, the cytocompatibility and mechanical properties of the printed scaffolds can be further improved.

Example 2

A method for preparing a modified tussah silk fibroin 3D printing scaffold, the steps of which are basically the same as those in Example 1, except that the p-aminobenzoic acid modifier is replaced by a p-aminoacetophenone modifier.

The finally obtained modified tussah silk fibroin nanofibers had an aspect ratio of 146 and an average diameter of 86 nm.

The core part printing ink has an extrusion swell rate of 0.52% and a dynamic viscosity of 390cP.

The self-gelling time of the shell part printing ink is 39s, the extrusion swelling rate is 12%, and the dynamic viscosity is 3500cP.

The final obtained modified tussah silk fibroin 3D printing scaffold had a resolution of 5.6 μm, and a compressive modulus of 319 MPa after immersion and cross-linking reaction in 0.5 wt% genipin aqueous solution for 24 h;

If 1 mL of fetal bovine serum culture medium containing 10,000 second-generation iPSCs was inoculated on a 15 mm diameter scaffold printed and cross-linked as described above, and cultured at 37.0°C, 5.0% CO ₂ for 1, 3, 4 , 7 and 10 days later, using trypan blue staining, the cell viability was measured to be 98.9%, 97.9%, 97.5%, 95.7%, and 92.8%, respectively. 536%, 1045%, 5704%, 14484%.

Example 3

A method for preparing a modified tussah silk fibroin 3D printing scaffold, the steps of which are basically the same as those in Example 2, except that the 4-(2-ethylamino)aniline modifier is used to replace the p-aminoacetophenone modifier.

The aspect ratio of the final modified tussah silk fibroin nanofibers was 111, and the average diameter was 126 nm.

The core part printing ink has an extrusion swell of 0.62% and a dynamic viscosity of 350cP.

The self-gelling time of the shell part printing ink is 42s, the extrusion swelling rate is 16%, and the dynamic viscosity is 2900cP.

The final obtained modified tussah silk fibroin 3D printed scaffold had a resolution of 8.2 μm, and a compressive modulus of 289 MPa after immersion and cross-linking reaction in 0.5 wt% genipin aqueous solution for 24 h;

If 1 mL of fetal bovine serum culture medium containing 10,000 second-generation iPSCs was inoculated on a 15 mm diameter scaffold printed and cross-linked as described above, and cultured at 37.0°C, 5.0% CO ₂ for 1, 3, 4 , 7 and 10 days later, using trypan blue staining method, the cell viability was measured to be 99.4%, 98.4%, 98.0%, 96.2%, and 92.3%, respectively. 407%, 709%, 2743%, 9993%.

Example 4

A method for preparing a modified tussah silk fibroin 3D printing scaffold, the steps of which are basically the same as those in Example 3, except that the 4-(2-ethylamino)aniline modifier is replaced by a p-aminophenheptyl ether modifier,

The finally prepared modified tussah silk fibroin nanofibers had an aspect ratio of 132 and an average diameter of 93 nm.

The extrusion swelling rate of the core part printing ink is 0.56%, and the dynamic viscosity is 380cP.

The self-gelling time of the shell part printing ink is 41s, the extrusion swelling rate is 15%, and the dynamic viscosity is 3200cP.

The resolution of the finally prepared modified tussah silk fibroin 3D printing scaffold was 6.5 μm, and the compressive modulus was 301 MPa after immersion and cross-linking reaction in 0.5 wt% genipin aqueous solution for 24 h;

If 1 mL of fetal bovine serum medium containing 10,000 second-generation iPSCs was inoculated on a 15 mm diameter scaffold printed and cross-linked as described above, and cultured at 37.0°C, 5.0% CO ₂ for 1, 3, 4 , 7 and 10 days later, using trypan blue staining, the cell viability was measured to be 98.8%, 97.8%, 97.4%, 95.6%, and 92.7%, respectively. 493%, 933%, 4717%, 12987%.

Example 5

A method for preparing a modified tussah silk fibroin 3D printing scaffold, the steps of which are basically the same as those in Example 1, except that in step (4), the reaction is mechanically stirred in an ice-water bath for 3 days,

The aspect ratio of the final modified tussah silk fibroin nanofibers was 158, and the average diameter was 67 nm.

The core part printing ink has an extrusion swell rate of 0.46% and a dynamic viscosity of 460cP.

The self-gelling time of the shell part printing ink is 34s, the extrusion swelling rate is 8%, and the dynamic viscosity is 4000cP.

The final obtained modified tussah silk fibroin 3D printed scaffold had a resolution of 2.9 μm, and a compressive modulus of 354 MPa after immersion and cross-linking reaction in 0.5 wt% genipin aqueous solution for 24 h;

If 1 mL of fetal bovine serum medium containing 10,000 second-generation iPSCs was inoculated on a 15 mm diameter scaffold printed and cross-linked as described above, and cultured at 37.0°C, 5.0% CO ₂ for 1, 3, 4 , 7 and 10 days later, using trypan blue staining method, the cell viability was measured to be 99.2%, 98.2%, 97.8%, 96.0%, and 93.1%, respectively. 665%, 1381%, 8665%, 18975%.

Example 6

A method for preparing a modified tussah silk fibroin 3D printing scaffold, the steps of which are basically the same as those in Example 5, except that in step (4), the reaction is mechanically stirred in an ice-water bath for 5 days.

The finally prepared modified tussah silk fibroin nanofibers had an aspect ratio of 185 and an average diameter of 45 nm.

The extrusion swell of the core part printing ink was 0.42%, and the dynamic viscosity was 490cP.

The self-gelling time of the shell part printing ink is 30s, the extrusion expansion rate is 5%, and the dynamic viscosity is 4200cP.

The resolution of the final modified tussah silk fibroin 3D printed scaffold was 1.2 μm, and the compressive modulus was 443 MPa after immersion and cross-linking reaction in 0.5 wt% genipin aqueous solution for 24 h;

If 1 mL of fetal bovine serum medium containing 10,000 second-generation iPSCs was inoculated on a 15 mm diameter scaffold printed and cross-linked as described above, and cultured at 37.0°C, 5.0% CO ₂ for 1, 3, 4 , 7 and 10 days later, using trypan blue staining, the cell viability was measured to be 99.3%, 98.3%, 97.9%, 96.1% and 93.3%, respectively. Using the cell counting method, the cell proliferation rates were measured to be 191%, 708%, 1493%, 9652%, and 20473%, respectively.

Example 7

A method for preparing a modified tussah silk fibroin 3D printing scaffold, the steps of which are basically the same as those in Example 1, the difference is that in the preparation of the shell part printing ink in step (6), the tussah silk fibroin modified by p-aminobenzoic acid is The concentration of protein nanofibers is 3wt%, the concentration of gelatin is 17wt%, and the diameters of the core part and the shell part of the printed lines of the core-shell structure used in printing are 400 μm and 800 μm respectively; the printing process parameters are: barrel And the needle temperature is 32℃, the extrusion pressure is 130KPa, the printing speed is 8mm/s, and the line spacing is 800μm.

The self-gelling time of the shell part printing ink is 46s, the extrusion swelling rate is 22%, and the dynamic viscosity is 2000cP.

The final obtained modified tussah silk fibroin 3D printed scaffold had a resolution of 7.0 μm, and a compressive modulus of 243 MPa after immersion and cross-linking reaction in 0.5 wt% genipin aqueous solution for 24 h;

If 1 mL of fetal bovine serum culture medium containing 10,000 second-generation iPSCs was inoculated on a 15 mm diameter scaffold printed and cross-linked as described above, and cultured at 37.0°C, 5.0% CO ₂ for 1, 3, 4 , 7 and 10 days later, using trypan blue staining, the cell viability was measured to be 99.0%, 98.0%, 97.6%, 95.8% and 92.9%, respectively. Using the cell counting method, the cell proliferation rates were measured to be 179%, 579%, 1157%, 6691%, and 15981%, respectively.

Example 8

A method for preparing a modified tussah silk fibroin 3D printing scaffold, the steps of which are basically the same as those in Example 1, the difference is that in the preparation of the shell part printing ink in step (6), the tussah silk fibroin modified by p-aminobenzoic acid is The concentration of protein nanofibers is 1 wt%, the concentration of gelatin is 19 wt%, and the diameters of the core part and the shell part of the printed lines of the core-shell structure used in printing are 500 μm and 1000 μm respectively; the printing process parameters are: barrel And the needle temperature is 37℃, the extrusion pressure is 100KPa, the printing speed is 8mm/s, and the line spacing is 2000μm.

The self-gelling time of the shell part printing ink is 50s, the extrusion expansion rate is 26%, and the dynamic viscosity is 1400cP.

The resolution of the final modified tussah silk fibroin 3D printed scaffold was 9.0 μm, and the compressive modulus was 196 MPa after immersion and cross-linking reaction in 0.5 wt% genipin aqueous solution for 24 h;

If 1 mL of fetal bovine serum medium containing 10,000 second-generation iPSCs was inoculated on a 15 mm diameter scaffold printed and cross-linked as described above, and cultured at 37.0°C, 5.0% CO ₂ for 1, 3, 4 , 7 and 10 days later, using trypan blue staining, the cell viability was measured to be 98.7%, 97.7%, 97.3%, 95.5%, and 92.6%, respectively. Using the cell counting method, the cell proliferation rates were measured to be 167%, 450%, 821%, 3730%, and 11490%, respectively.

Example 9

A preparation method of a modified tussah silk fibroin 3D printing scaffold, the main steps are as follows:

(1) Preparation of tussah silk fibroin pulp:

(1.1) Immerse the tussah cocoons in a Na ₂ CO ₃ aqueous solution with a mass concentration of 5.00 wt % at a mass-to-volume ratio of 1 g:100 mL, boil 5 times for 60 min each time, and then wash the boiled tussah cocoons with water and dry to obtain Degummed tussah silk fiber;

(1.2) immersing the degummed tussah silk fiber in an aqueous formic acid solution with a mass concentration of 100 wt % for 3 h at a mass volume ratio of 1 g:25 mL;

(1.3) Under the temperature condition of changing to 60 ℃, homogenize treatment for 5min and pulverize, and the homogenization rate is 15000rpm;

(1.4) repeat step (1.2) and step (1.3) 5 times to obtain tussah silk fibroin slurry;

(1.5) tussah silk fibroin pulp is centrifuged, filtered, washed and dried to obtain tussah silk fibroin pulp;

(2) Preparation of chemical modifier: 4-(2-ethylamino)aniline acetonitrile solution with a concentration of 1 mM, p-toluenesulfonic acid aqueous solution with a concentration of 1 mM and sodium nitrite aqueous solution with a concentration of 1 mM in a ratio of 1:4: After the volume ratio of 2 was mixed by vortex for 5s under the temperature condition of 1℃, the chemical modifier was obtained by mechanical stirring reaction for 5min under the same temperature condition;

(3) Preparation of tussah silk fibroin nanofiber/boric acid buffer mixed system:

(3.1) Immerse the tussah silk fibroin pulp in water at a mass-to-volume ratio of 1g:200mL, and mechanically stir to disperse it evenly;

(3.2) According to the mass molar ratio of tussah silk fibroin pulp: NaClO=1 g: 0.001 mol, a NaClO aqueous solution with a mass concentration of 5 wt% was added to the water dispersion system of tussah silk fibroin pulp, and at a temperature of 1 °C Mechanical stirring. During this process, the pH value of the system is maintained between 10.0 and 10.1 by continuously adding 0.5M aqueous NaOH solution, until the pH value of the system can also be maintained between 10.0 and 10.1 when no aqueous NaOH solution is added;

(3.3) Transfer the system to a cellulose dialysis bag with a molecular weight cut-off of 14,000 Da, dialyze it in deionized water for 1 day, continue dialysis in boric acid buffer for 1 day, and then concentrate to obtain tussah silk fibroin nanofibers with a concentration of 1wt % Tussah silk fibroin nanofiber/boric acid buffer mixed system, wherein the boric acid buffer is mainly composed of boric acid, sodium chloride and water, and the concentrations of boric acid and sodium chloride are 100mM and 150mM respectively;

(4) Preparation of tussah silk fibroin nanofibers modified with 4-(2-ethylamino)aniline: First, the tussah silk fibroin nanofibers/boric acid buffer mixed system with a concentration of 1 wt% was mixed with 4-(2 -Ethylamino)aniline modifier was mixed in a volume ratio of 12, reacted with mechanical stirring in an ice-water bath for 1 day, and then dialyzed with deionized water for 1 day to purify and freeze-dry to obtain an average length-diameter ratio of 100 and an average diameter of 200nm. Tussah silk fibroin nanofibers modified by 4-(2-ethylamino)aniline;

(5) Preparation of nuclear part printing ink: tussah silk fibroin nanofibers modified with 4-(2-ethylamino)aniline, fibroblast growth factor 2 and tetracycline were added to water, and the mechanical The core part printing ink was prepared by stirring for 1 hour, wherein the mass content of each component in the core part printing ink was: the tussah silk fibroin nano-microfibers modified by 4-(2-ethylamino)aniline were 6 wt %, and the fibrous The cell growth factor 2 is 0.1wt%, the tetracycline is 0.1wt%, and the balance is water; the extrusion swelling rate of the core part printing ink is 1.00%, and the dynamic viscosity is 300cP;

(6) Preparation of shell part printing ink: firstly add tussah silk fibroin nanofibers modified with 4-(2-ethylamino)aniline, fibroblast growth factor 2 and tetracycline into water, stir mechanically for 1 h, then mix with gelatin Mixing, after standing for 1 h, mechanically stirring for 1 h at a temperature of 35 ° C, and finally ultrasonically treating for 1 min to obtain the shell part printing ink; wherein, the mass content of each component in the shell part printing ink is: after 4-(2 -Ethylamino)aniline-modified tussah silk fibroin nanofibers were 1wt%, gelatin was 14wt%, fibroblast growth factor 2 was 0.1wt%, tetracycline was 0.1wt%, and the balance was water; The self-gelling time is 60s, the extrusion expansion rate is 30%, and the dynamic viscosity is 1000cP;

(7) First, the core part printing ink and the shell part printing ink prepared above are extruded from the 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 The modified tussah silk fibroin 3D printing scaffolds were obtained by 3D printing; when printing, the printing lines with core-shell structure were deposited on the glass sheet in a layer-by-layer manner; the printing process parameters were: material The temperature of the barrel and needle head was 25°C, the extrusion pressure was 10KPa, the printing speed was 10mm/s, the angle between two adjacent printing layers formed by printing lines with a core-shell structure was 30°, the line spacing was 2000 μm, and 2 layers were deposited.

The resolution of the final modified tussah silk fibroin 3D printed scaffold was 10.0 μm; the compressive modulus of the modified tussah silk fibroin 3D printed scaffold after immersion and cross-linking reaction in genipin with a mass concentration of 5.0 wt% for 24 h is 100MPa;

If 1 mL of fetal bovine serum medium containing 10,000 second-generation iPSCs was inoculated on a 15 mm diameter scaffold printed and cross-linked as described above, and cultured at 37.0°C, 5.0% CO ₂ for 1, 3, 4 , 7 and 10 days later, using trypan blue staining, the cell viability was measured to be 98.7%, 97.6%, 97.3%, 95.5% and 92.0%, respectively. 344%, 564%, 3158% and 9000%.

Example 10

A preparation method of a modified tussah silk fibroin 3D printing scaffold, the main steps are as follows:

(1) Preparation of tussah silk fibroin pulp:

(1.1) Immerse the tussah cocoons in a Na ₂ CO ₃ aqueous solution with a mass concentration of 0.05 wt % at a mass volume ratio of 1 g:50 mL, boil once for 30 min each time, and then wash the boiled tussah cocoons with water and dry to obtain Degummed tussah silk fibers;

(1.2) immersing the degummed tussah silk fiber in an aqueous formic acid solution with a mass concentration of 60wt% for 1h at a mass volume ratio of 1g:15mL;

(1.3) Under the temperature condition of 10 ℃, homogenize for 1min and pulverize, and the homogenization rate is 5000rpm;

(1.4) repeating step (1.2) and step (1.3) once to obtain tussah silk fibroin protein slurry;

(1.5) tussah silk fibroin pulp is centrifuged, filtered, washed and dried to obtain tussah silk fibroin pulp;

(2) Preparation of chemical modifier: 1M p-aminobenzoic acid acetonitrile solution, 2M p-toluenesulfonic acid aqueous solution and 1M sodium nitrite aqueous solution in a volume ratio of 1:2:2 After vortex mixing for 5s under the temperature condition of 4℃, the chemical modifier was obtained by mechanical stirring reaction for 30min under the same temperature condition;

(3) Preparation of tussah silk fibroin nanofiber/boric acid buffer mixed system:

(3.1) Immerse the tussah silk fibroin pulp in water at a mass-volume ratio of 1g:50mL, and mechanically stir to disperse it evenly;

(3.2) According to the mass molar ratio of tussah silk fibroin pulp: NaClO=1g:0.050mol, in the water dispersion system of tussah silk fibroin pulp, add a NaClO aqueous solution with a mass concentration of 35wt%, and under the temperature condition of 60 ° C Mechanical stirring. During this process, the pH value of the system is maintained between 10.0 and 10.1 by continuously adding 5.0M aqueous NaOH solution, until the pH value of the system can also be maintained between 10.0 and 10.1 when no aqueous NaOH solution is added;

(3.3) Transfer the system to a cellulose dialysis bag with a molecular weight cut-off of 14,000 Da, dialyze it in deionized water for 3 days, continue dialysis in boric acid buffer for 1 day, and then concentrate to obtain tussah silk fibroin nanofibers with a concentration of 20wt % Tussah silk fibroin nanofiber/boric acid buffer mixed system, wherein the boric acid buffer is mainly composed of boric acid, sodium chloride and water, and the concentrations of boric acid and sodium chloride are 100mM and 150mM respectively;

(4) Preparation of p-aminobenzoic acid-modified tussah silk fibroin nanofibers: first, mix the tussah silk fibroin nanofibers/boric acid buffer mixture system with a concentration of 20wt% and p-aminobenzoic acid modifier at a ratio of 1:0.1 The volume ratio was mixed, and the reaction was mechanically stirred in an ice-water bath for 5 days, and then dialyzed with deionized water for 3 days to purify and freeze-dry to obtain p-aminobenzoic acid-modified tussah silk with an average aspect ratio of 200 and an average diameter of 10 nm. protein nanofibers;

(5) Preparation of nuclear part printing ink: adding p-aminobenzoic acid-modified tussah silk fibroin nanofibers, fibroblast growth factor 2 and tetracycline into water, and mechanically stirring at 45°C for 3 hours to prepare the nucleus Part of the printing ink, wherein the mass content of each component in the core part of the printing ink is: 20wt% of tussah silk fibroin nanofibers modified with p-aminobenzoic acid, 1wt% of fibroblast growth factor 2, and 1wt% of tetracycline %, the balance is water; the extrusion expansion rate of the printing ink in the core part is 0.10%, and the dynamic viscosity is 500cP;

(6) Preparation of printing ink for shell part: first, add p-aminobenzoic acid-modified tussah silk fibroin nanofibers, fibroblast growth factor 2 and tetracycline into water, stir mechanically for 3 hours, and then mix with gelatin and let stand for 3 hours. , mechanically stirred for 5 hours at a temperature of 45°C, and finally ultrasonically treated for 60 minutes to obtain a shell part printing ink; wherein, the mass content of each component in the shell part printing ink is: tussah silk fibroin nanometer modified with p-aminobenzoic acid Microfibers are 6wt%, gelatin is 20wt%, fibroblast growth factor 2 is 1wt%, tetracycline is 1wt%, and the balance is water; the self-gelling time of the shell part printing ink is 10s, and the extrusion swelling ratio is 1 %, the dynamic viscosity is 5000cP;

(7) First, the core part printing ink and the shell part printing ink prepared above are extruded from the 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 The modified tussah silk fibroin 3D printing scaffolds were obtained by 3D printing; when printing, the printing lines with core-shell structure were deposited on the glass sheet in a layer-by-layer manner; the printing process parameters were: material The temperature of the barrel and needle head was 37°C, the extrusion pressure was 500KPa, the printing speed was 1mm/s, the angle between two adjacent printing layers formed by printing lines with a core-shell structure was 90°, the line spacing was 20 μm, and 10 layers were deposited.

The resolution of the final modified tussah silk fibroin 3D printed scaffold was 0.5 μm; the compressive modulus of the modified tussah silk fibroin 3D printed scaffold after immersion and cross-linking reaction in genipin with a mass concentration of 0.1 wt% for 24 h is 600MPa;

If 1 mL of fetal bovine serum culture medium containing 10,000 second-generation iPSCs was inoculated on a 15 mm diameter scaffold printed and cross-linked as described above, and cultured at 37.0°C, 5.0% CO ₂ for 1, 3, 4 , 7 and 10 days later, using trypan blue staining method, the cell viability was measured to be 99.9%, 99.7%, 99.5%, 99.2% and 99.0%, respectively. 864%, 1821%, 11775% and 25000%.

The above are merely examples of embodiments of the present invention. For those skilled in the art, several improvements and modifications can still be made without departing from the technical principles of the present invention. For example: change of printing ink formulation and preparation process conditions, change of printing process (needle diameter, printing temperature, extrusion pressure, printing speed, interlayer angle, line spacing, number of deposited layers, etc.), change of printing support processing method etc., these should also be regarded as the protection 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.