Modified tussah silk protein and 3D printing ink based on modified tussah silk protein
阅读说明：本技术 改性柞蚕丝蛋白以及基于该柞蚕丝蛋白的3d打印墨水 (Modified tussah silk protein and 3D printing ink based on modified tussah silk protein ) 是由 苟马玲 黄玉兰 杨雄 于 2021-08-18 设计创作，主要内容包括：本发明涉及3D生物打印材料,具体涉及改性柞蚕丝蛋白以及基于该柞蚕丝蛋白的3D打印墨水,属于生物材料和生物医学工程技术领域。本发明提供了一种新型的具有优越力学性能和生物相容性的可光聚合改性柞蚕丝蛋白,该改性柞蚕丝蛋白为甲基丙烯酸酐接枝柞蚕丝蛋白,采用甲基丙烯酸酐与柞蚕丝蛋白反应得到。本发明采用柞蚕丝蛋白为基体,甲基丙烯酸酐接枝柞蚕丝蛋白,得到了一种新型的可用于光固化3D打印的生物材料,该材料具有优异的力学性能、生物相容性以及3D打印特性,其制备和打印方法简单,成本较低,适用于各种细胞,且打印后的支架力学性能和细胞相容性较好,可以让细胞具有较好的存活率和增殖率。(The invention relates to a 3D biological printing material, in particular to modified tussah fibroin and 3D printing ink based on the tussah fibroin, and belongs to the technical field of biological materials and biomedical engineering. The invention provides a novel photopolymerisable modified tussah silk protein with excellent mechanical property and biocompatibility, wherein the modified tussah silk protein is methacrylic anhydride grafted tussah silk protein and is obtained by reacting methacrylic anhydride with tussah silk protein. According to the invention, tussah fibroin is used as a substrate, methacrylic anhydride is grafted on the tussah fibroin to obtain a novel biological material capable of being used for photocuring 3D printing, the material has excellent mechanical property, biocompatibility and 3D printing characteristic, the preparation and printing methods are simple, the cost is lower, the material is suitable for various cells, the mechanical property and the cell compatibility of the printed scaffold are better, and the cells can have better survival rate and proliferation rate.)
1. The modified tussah silk protein is characterized in that: the modified tussah silk protein is formed by grafting methacrylic anhydride onto tussah silk protein and reacting the methacrylic anhydride with the tussah silk protein, wherein the mass ratio of the volume of the methacrylic anhydride to the mass of the tussah silk protein is 0.1-14% mL/g.
2. The modified tussah silk protein of claim 1, wherein: the mass ratio of the volume of the methacrylic anhydride to the tussah silk protein is 2.5-12% mL/g; preferably, the mass ratio of the volume of the methacrylic anhydride to the tussah silk protein is 2.5-10% mL/g; more preferably, the mass ratio of the volume of the methacrylic anhydride to the tussah silk protein is 5-10% mL/g; more preferably, the mass ratio of the volume of the methacrylic anhydride to the tussah silk protein is 10% mL/g.
3. The modified tussah silk protein of claim 1, wherein: the modified tussah silk protein is prepared by the following method:
a. preparing regenerated tussah silk protein: mixing tussah fibroin with molten Ca (NO)3)2·4H2Mixing O according to the weight ratio of 1: 1-50, reacting for 3-5 h, dialyzing for 3-5 days at the temperature of 2-6 ℃, dewatering and drying to obtain regenerated tussah silk protein;
b. preparing modified tussah silk protein: dissolving the regenerated tussah fibroin in a carbonate buffer solution, and controlling the concentration of the tussah fibroin to be 0.1-10 g/mL; adding methacrylic anhydride, stirring for reacting for 4-8 h, dialyzing for 3-5 days at 2-6 ℃, dewatering and drying to obtain modified tussah silk protein;
wherein, the dialysis of step a and step b both adopt MW3500 dialysis bag.
4. The modified tussah silk protein of claim 3, wherein: in the step a, tussah fibroin and molten Ca (NO) are mixed3)2·4H2Mixing O according to the weight ratio of 1:20, and reacting for 4 h; in the step b, the tussah fibroin concentration is 1g/mL, and the stirring reaction is carried out for 6 hours.
5. The modified tussah silk protein of claim 3, wherein: in the step a, the tussah silk protein is prepared by the following method: cleaning tussah cocoons, shearing, removing sericin, and drying to obtain tussah fibroin; preferably, the sericin removal operation is repeated at least once by the following steps: adding Na into Antheraea Pernyi cocoon2CO3Heating the solution to boil for 10-20 min, taking the solid, and washing.
6. 3D prints ink based on modified tussah silk protein, its characterized in that: the 3D printing ink comprises the modified tussah silk protein as claimed in any one of claims 1 to 5, a photoinitiator and a solvent.
7. The modified tussah silk protein-based 3D printing ink of claim 6, wherein: the modified tussah silk protein accounts for 10-30% of the weight of the 3D printing ink, and the photoinitiator accounts for 0.1-2% of the weight of the 3D printing ink; preferably, the modified tussah silk protein accounts for 20% of the weight of the 3D printing ink, and the photoinitiator accounts for 1% of the weight of the 3D printing ink.
8. 3D prints biological ink based on modified tussah silk protein, its characterized in that: the 3D printing biological ink comprises 3D printing ink and cells, wherein the 3D printing ink is the modified tussah silk protein-based 3D printing ink according to claim 6 or 7.
9. The modified tussah silk protein based 3D printing bio-ink according to claim 8, wherein: the density of the cells was 0.5X 106～2×106Per mL; preferably, the density of the cells is 1X 106one/mL.
Technical Field
The invention relates to a 3D biological printing material, in particular to modified tussah fibroin and 3D printing ink based on the tussah fibroin, and belongs to the technical field of biological materials and biomedical engineering.
Background
The 3D bioprinting technology is one of important technical supports for tissue engineering and regenerative medicine for constructing complex tissues and organs in vitro. The 3D bio-printing technology based on Digital Light Processing (DLP) initiates layer-by-layer photocuring of photosensitive liquid through an ultraviolet light projection technology, and compared with other 3D printing technologies such as inkjet, extrusion and laser-assisted printing, the 3D bio-printing technology has the advantages of rapid molding, high printing precision, high cell survival rate and the like, and is widely applied.
The core of the 3D bio-printing technology lies in bio-printing ink, the conventional 3D printing material mainly has a problem of biocompatibility, and silk fibroin is applied to the field of 3D printing due to its good biocompatibility, for example, chinese patent application No. 201610832278.4 discloses a silk protein/gelatin scaffold material based on three-dimensional printing and a preparation method thereof, the scaffold is prepared by adding gelatin into a silk protein solution to form a silk protein/gelatin composite hydrogel, and then extruding the composite hydrogel from a needle of a three-dimensional printer. The Chinese patent application No. 201711362521.1 discloses a paper-mass-shaped graphene-modified photocuring fibroin three-dimensional printing material and a preparation method thereof, and the material comprises paper-mass-shaped graphene, fibroin hydrogel, a photoinitiator and an auxiliary agent. The Chinese patent with application number 201710793524.4 discloses a three-dimensional printing wound coating material based on silk microsphere biological ink and a preparation method thereof, wherein silk microsphere biological ink is prepared from silk protein microspheres containing coated aspirin, a silk protein aqueous solution, an ultraviolet light initiator and polyvinyl alcohol ester. The literature, "precisely printable and biocompatible silk fibroin bio-inks for digital light processing3D printing" (Kim S H, Yeon Y K, Lee J M, et al, Precisely printable and biocompatable silk fibroin bio-oil for digital light processing3D printing [ J ]. Nature Communications,2018,9(1):1620) also reports the reaction of glycidyl methacrylate with the free amino group of fibroin grafted with a double bond for DLP 3D printing (BSF-GMA).
It can be seen that most of the existing bio-inks use Bombyx mori silk fibroin (BSF), which lacks an arginine-glycyl-aspartic acid (RGD) sequence for cell adhesion, thus limiting the application to some extent.
Tussah silk (Antheraea pernyi (A.p.) silk fibrin, ASF) is silk spun by wild tussah silkworm, has the characteristics of acid and alkali resistance, high strength and the like, and the degummed tussah silk protein mainly consists of repeated chain segments of alanine, has stronger hydrophobicity compared with the domestic silk protein and has more regular molecular chain segments; in addition, the tussah silk protein contains an RGD tripeptide sequence combined with an integrin receptor, so that the cell adhesion effect is more obvious, and the tussah silk protein has better application potential when being used for DLP 3D printing.
Disclosure of Invention
Aiming at the defects, the technical problem solved by the invention is to provide a novel modified tussah silk protein which can be used for 3D printing.
The modified tussah silk protein is methacrylic anhydride grafted tussah silk protein and is obtained by reacting methacrylic anhydride with tussah silk protein, wherein the mass ratio of the volume of the methacrylic anhydride to the tussah silk protein is 0.1-14% mL/g.
In one embodiment of the invention, the mass ratio of the volume of the methacrylic anhydride to the tussah silk protein is 2.5-12% mL/g. In one embodiment of the invention, the mass ratio of the volume of the methacrylic anhydride to the tussah silk protein is 2.5-10% mL/g. In a more specific embodiment of the invention, the ratio of the volume of the methacrylic anhydride to the mass of the tussah silk protein is 5% to 10% mL/g. In one embodiment of the invention, the ratio of the volume of the methacrylic anhydride to the mass of the tussah silk protein is 10% mL/g.
Further, in an embodiment of the present invention, the modified tussah silk protein is prepared by the following method:
a. preparing regenerated tussah silk protein: mixing tussah fibroin with molten Ca (NO)3)2·4H2Mixing O according to the weight ratio of 1: 1-50, reacting for 3-5 h, dialyzing for 3-5 days at the temperature of 2-6 ℃, dewatering and drying to obtain regenerated tussah silk protein;
b. preparing modified tussah silk protein: dissolving the regenerated tussah fibroin in a carbonate buffer solution, and controlling the concentration of the tussah fibroin to be 0.1-10 g/mL; adding methacrylic anhydride, stirring for reacting for 4-8 h, dialyzing for 3-5 days at 2-6 ℃, dewatering and drying to obtain modified tussah silk protein;
wherein, the dialysis of step a and step b both adopt MW3500 dialysis bag.
In one embodiment of the present invention, in step a, tussah fibroin is mixed with molten Ca (NO)3)2·4H2Mixing O according to the weight ratio of 1:20, and reacting for 4 h; in the step b, the tussah fibroin concentration is 1g/mL, and the stirring reaction is carried out for 6 hours.
In a specific embodiment of the present invention, in the step a, the tussah silk protein is prepared by the following method: cleaning tussah cocoons, shearing, removing sericin, and drying to obtain tussah fibroin.
In one embodiment of the present invention, the sericin removal operation is repeated at least once by the following steps: adding Na into Antheraea Pernyi cocoon2CO3Heating the solution to boil for 10-20 min, taking the solid, and washing.
The second technical problem solved by the invention is to provide the modified tussah silk protein-based 3D printing ink.
The 3D printing ink based on the modified tussah silk protein comprises the modified tussah silk protein, a photoinitiator and a solvent.
In one embodiment of the invention, the modified tussah silk protein accounts for 10-30% of the weight of the 3D printing ink, and the photoinitiator accounts for 0.1-2% of the weight of the 3D printing ink. In one embodiment of the present invention, the modified tussah silk protein is 20% by weight of the 3D printing ink and the photoinitiator is 1% by weight of the 3D printing ink.
The invention also provides 3D printing biological ink based on the modified tussah silk protein.
The 3D printing biological ink comprises 3D printing ink and cells, wherein the 3D printing ink is the 3D printing ink based on the modified tussah silk protein.
In one embodiment of the invention, the density of cells is 0.5X 106～2×106Per mL; preferably, the density of the cells is 1X 106one/mL.
Compared with the prior art, the invention has the following beneficial effects:
1) according to the invention, tussah fibroin is used as a matrix, methacrylic anhydride is grafted on the tussah fibroin to obtain a novel biological material capable of being used for photocuring 3D printing, and the material has excellent mechanical property, better biocompatibility and 3D printing characteristic, and can be used in the fields of tissue engineering and the like.
2) The 3D printing ink is prepared by using the modified tussah silk protein as a main raw material, can be used for printing various support materials, is simple in preparation and printing method, low in cost, suitable for various cells, good in mechanical property of the printed support, good in cell compatibility and capable of enabling the cells to have good survival rate and proliferation rate.
Drawings
FIG. 1 shows the NMR spectra of methacrylic anhydride grafted tussah silk protein with different degrees of substitution prepared in examples 1 and 2 of the present invention.
FIG. 2 is a graph of compressive stress-strain of hydrogel prepared in example 3 of the present invention, wherein a) is a graph of compressive stress-strain of hydrogel after PBS soaking; b) the compression stress-strain curve of the hydrogel after the 75% ethanol soaking treatment is shown.
FIG. 3 is a graph showing proliferation rates of different cells in test example 1 of the present invention, wherein a) NIH/3T3 cells; b) s16 cells; c) HUVEC cells.
Fig. 4 shows a 3D printing hollowed-out structure according to test example 2 of the present invention, with a scale of 2 mm.
FIG. 5 is a photograph of the "peace pigeon" tissue constructs printed on a circular slide and observed under a fluorescence microscope on days 1, 2 and 3 of Experimental example 2 of the present invention, with a scale of 500. mu.m.
Detailed Description
The modified tussah silk protein is methacrylic anhydride grafted tussah silk protein and is obtained by reacting methacrylic anhydride with tussah silk protein, wherein the mass ratio of the volume of the methacrylic anhydride to the tussah silk protein is 0.1-14% mL/g.
The modified tussah silk protein is grafted to the tussah silk protein by adopting methacrylic anhydride, and the material has good mechanical property, biocompatibility and 3D printing characteristic, provides a new method and a new choice for photocuring 3D printing biological ink, and has good application prospect in the fields of tissue engineering and the like.
The volume of the methacrylic anhydride and the mass ratio of the tussah silk protein are related to the degree of substitution of methacryloyl groups in the product, and in one embodiment of the invention, the volume of the methacrylic anhydride and the mass ratio of the tussah silk protein are 2.5-12% mL/g. In one embodiment of the invention, the mass ratio of the volume of the methacrylic anhydride to the tussah silk protein is 2.5-10% mL/g. In a more specific embodiment of the invention, the ratio of the volume of the methacrylic anhydride to the mass of the tussah silk protein is 5% to 10% mL/g. In one embodiment of the invention, the ratio of the volume of the methacrylic anhydride to the mass of the tussah silk protein is 10% mL/g.
Further, in an embodiment of the present invention, the modified tussah silk protein is prepared by the following method:
a. preparing regenerated tussah silk protein: mixing tussah fibroin with molten Ca (NO)3)2·4H2Mixing O according to the weight ratio of 1: 1-50, reacting for 3-5 h, dialyzing for 3-5 days at the temperature of 2-6 ℃, dewatering and drying to obtain regenerated tussah silk protein;
b. preparing modified tussah silk protein: dissolving the regenerated tussah fibroin in a carbonate buffer solution, and controlling the concentration of the tussah fibroin to be 0.1-10 g/mL; adding methacrylic anhydride, stirring for reacting for 4-8 h, dialyzing for 3-5 days at 2-6 ℃, dewatering and drying to obtain modified tussah silk protein;
wherein, the dialysis of step a and step b both adopt MW3500 dialysis bag.
In one embodiment of the invention, the water is dialyzed at 4 ℃ for 3 days, and the water is changed every 4 hours during dialysis.
In one embodiment of the present invention, in step a, tussah fibroin is mixed with molten Ca (NO)3)2·4H2Mixing O according to the weight ratio of 1:20, and reacting for 4 h; in the step b, the tussah fibroin concentration is 1g/mL, and the stirring reaction is carried out for 6 hours.
Step a is to prepare the regenerated tussah fibroin, which can be prepared by adopting commercially available tussah fibroin as a raw material, and can also be prepared by self. In one embodiment of the invention, the tussah silk protein is prepared by the following method: cleaning tussah cocoons, shearing, removing sericin, and drying to obtain tussah fibroin.
In one embodiment of the present invention, the sericin removal operation is repeated at least once by the following steps: adding Na into Antheraea Pernyi cocoon2CO3Heating the solution to boil for 10-20 min, taking the solid, and washing.
All drying steps of the present invention may be performed by drying methods conventional in the art, and in one embodiment of the present invention, the drying is performed by freeze-drying. The freeze drying can keep the original chemical composition and physical properties of the dried material.
Specifically, the modified tussah silk protein is prepared by the following method:
a. preparation of regenerated tussah silk protein
Cleaning tussah cocoon, removing dirt, air drying, cutting each cocoon into 4-5 pieces, weighing 40g cocoon, and adding 1L 0.05M Na2CO3Adding the solution, boiling at 100 deg.C for 15min to remove sericin, washing with distilled water for several times, wringing, repeating the previous operation, and adding 1L 0.05M Na2CO3Boiling the solution for 30min, wringing, washing with pure water for several times until no turbid residue is found in the water, and air drying the tussah fibroin at room temperature for later use. 500g Ca (NO) are weighed3)2·4H2Placing O in 500mL round bottom flask, heating at 110 deg.C for melting, slowly adding 25g tussah silk protein, reacting for 4 hr, and placing the reaction solution in MWIn 3500 dialysis bag, dialyzing with pure water at 4 deg.C for 3 days, changing water every 4h, and lyophilizing the dialyzed solution to obtain 20g of regenerated tussah silk protein as soft light brown solid.
b. Preparation of modified tussah silk protein
Weighing 1g of regenerated tussah silk protein, dissolving in 10mL of 0.25M carbonate buffer solution, dropwise adding methacrylic anhydride (MAA) after stirring and dissolving, stirring and reacting for 6 hours, filtering the solution through miraculous filter cloth, placing the solution in a dialysis bag with MW (3500), dialyzing with pure water at 4 ℃ for 3 days, changing water every 4 hours, and freeze-drying the dialyzed solution to obtain the modified tussah silk protein.
The second technical problem solved by the invention is to provide the modified tussah silk protein-based 3D printing ink.
The 3D printing ink based on the modified tussah silk protein comprises the modified tussah silk protein, a photoinitiator and a solvent.
Wherein, the photoinitiator can be the one commonly used in the art, including but not limited to LAP, etc., and the solvent commonly used in the art is also suitable for the present invention, including but not limited to PBS buffer, etc.
In one embodiment of the invention, the modified tussah silk protein accounts for 10-30% of the weight of the 3D printing ink, and the photoinitiator accounts for 0.1-2% of the weight of the 3D printing ink. In one embodiment of the present invention, the modified tussah silk protein is 20% by weight of the 3D printing ink and the photoinitiator is 1% by weight of the 3D printing ink.
The invention also provides 3D printing biological ink based on the modified tussah silk protein.
The 3D printing biological ink comprises 3D printing ink and cells, wherein the 3D printing ink is the 3D printing ink based on the modified tussah silk protein.
The cell may be any existing cell, including but not limited to 3T3, HUVEC, S16, and the like.
In one embodiment of the invention, the density of cells is 0.5X 106～2×106Per mL; preferably, the density of the cells is 1X 106one/mL.
The following examples are provided to further illustrate the embodiments of the present invention and are not intended to limit the scope of the present invention.
Example 1
1. Preparation of regenerated tussah silk protein
Cleaning tussah cocoon, removing dirt, air drying, cutting each cocoon into 4-5 pieces, weighing 40g cocoon, and adding 1L 0.05M Na2CO3Adding the solution, boiling at 100 deg.C for 15min to remove sericin, washing with distilled water for several times, wringing, repeating the previous operation, and adding 1L 0.05M Na2CO3Boiling the solution for 30min, wringing, washing with pure water for several times until no turbid residue is found in the water, and air drying the tussah fibroin at room temperature for later use. 500g Ca (NO) are weighed3)2·4H2Placing O in 500mL round bottom flask, heating at 110 deg.C for melting, slowly adding 25g tussah silk protein, reacting for 4 hr, and placing the solution in MWIn 3500 dialysis bag, dialyzing with pure water at 4 deg.C for 3 days, changing water every 4h, and lyophilizing the dialyzed solution to obtain 20g of regenerated tussah silk protein as soft light brown solid.
2. Preparation of methacrylic anhydride grafted tussah silk protein
Weighing 1g of regenerated tussah silk protein, dissolving in 10mL of 0.25M carbonate buffer solution, slowly dropwise adding 100 mu L MAA after stirring and dissolving, stirring and reacting for 6h, filtering with miraculous filter cloth, and placing in MWDialyzing in 3500 dialysis bag with pure water at 4 deg.C for 3 days, changing water every 4 hr, and lyophilizing the dialyzed solution to obtain 800mg methacrylic anhydride grafted tussah silk protein, and recording as ASF-MA10％By hydrogen nuclear magnetic resonance spectroscopy H1NMR analysis of the results of the synthesis, as shown in FIG. 1.
Example 2
Using the method of example 1, methacrylic anhydride (MAA) was added in varying amounts and slowly dropwise adding 10. mu.L, 25. mu.L, 50. mu.L and 150. mu.L MAA, respectively, to obtain methacrylic anhydride grafted tussah silk protein, designated ASF-MA1％、ASF-MA2.5％、ASF-MA5％And ASF-MA15％. Wherein, ASF-MA15％After stirring for 6h, the precipitation was severe, since the degree of substitution was too high to be favorable for the stability of the silk fibroin aqueous solution.
Prepared ASF-MA1％、ASF-MA2.5％、ASF-MA5％Hydrogen spectrum of nuclear magnetic resonance (H)1NMR details are shown in FIG. 1. As shown in fig. 1, the results show that methacryloyl group was successfully grafted to tussah silk protein, and as MAA increased, the intensity of characteristic peak increased and the degree of methacryloyl substitution increased.
Example 3
The obtained ASF-MA2.5％、ASF-MA5％、ASF-MA10％Sequentially and respectively preparing 10 wt%, 20 wt% and 30 wt% of solutions, and adding 1 wt% of photoinitiator LAP to obtain the printing ink.
The printing ink is irradiated for 10s by 405nm ultraviolet light to prepare the hydrogel with different concentrations and substitution degrees. The mechanical property of the alloy is measured by the following method: and adopting a DMA Q800 dynamic mechanical analyzer, and evaluating the compression performance of the hydrogel sample by using a uniaxial compression test method. The test samples were made into 6mm diameter, 4mm height cylindrical hydrogels using DLP 3D printing. The compression performance was measured after PBS soaking and 75% ethanol soaking, respectively. Wherein the PBS soaking treatment is to soak the printed sample for 4h with PBS, and then the soaking treatment is carried out for 1mm & min-1The speed of (2) is compressed to the bottom and the sample is observed. The 75% ethanol soaking treatment comprises soaking printed sample in 75% ethanol solution for 4 hr, washing with PBS and soaking for 3 times, each time for 10min, balancing, and soaking for 2mm min-1Velocity compression measurement of (2). At least three samples per group were prepared for compression testing. The results are shown in FIG. 2. In FIG. 2, 30% ASF-MA10％ASF-MA at a concentration of 30 wt%10％Prepared from solutionHydrogel, analogized, 20% ASF-MA10％ASF-MA at a concentration of 20 wt%10％Hydrogel from solution, 10% ASF-MA10％ASF-MA at a concentration of 10 wt%10％Hydrogel prepared from solution, 30% ASF-MA5％ASF-MA at a concentration of 30 wt%5％Hydrogel prepared from solution, 20% ASF-MA5％ASF-MA at a concentration of 20 wt%5％Hydrogel from solution, 10% ASF-MA5％ASF-MA at a concentration of 10 wt%5％Hydrogel prepared from solution, 30% ASF-MA2.5％ASF-MA at a concentration of 30 wt%2.5％Hydrogel prepared from solution, 20% ASF-MA2.5％ASF-MA at a concentration of 20 wt%2.5％Hydrogel prepared from the solution. Data analysis shows that the compression modulus of the hydrogel after being soaked in 75% ethanol is obviously increased, the hardness is enhanced, and the mechanical property is obviously improved. Due to ASF-MA2.5％Low degree of substitution, 10% ASF-MA2.5％PBS hydrogels were too soft and brittle to perform the compression test.
Test example 1 cellular compatibility of hydrogel
Selecting ASF-MA considering precision and mechanical property of 3D printing10％A cell compatibility study was performed. The control group used was a conventional methacrylic acylated gelatin (GelMA) with the same type of BSF-GMA hydrogel. Among them, BSF-GMA hydrogel was prepared by the method of reference to the reference document [ Kim S H, Yeon Y K, Lee J M, et al].Nature Communications,2018,9(1):1620]The specific experimental operation is as follows: subjecting 20g of silkworm cocoon to 0.05M Na treatment by the same method as that for extracting tussah fibroin2CO3Boiling the solution to remove sericin, extracting the domestic fibroin, and airing and fluffing at room temperature. 2g of bombyx mori fibroin was weighed and dissolved in 10mL of 9.3M lithium bromide solution prepared with precision, and dissolved for 1h at 60 ℃ with stirring. Then, 600. mu.L of Glycidyl Methacrylate (GMA) was slowly added dropwise to the solution, and the reaction was terminated after reacting at 60 ℃ for 3 hours at a stirring speed of 300 rpm. Transferring the reacted solution to MWDialyzing with ultrapure water at low temperature for 7 days in 12400kDa dialysis bag, and transferring the dialyzed solution at 3500rpmAnd (4) quickly centrifuging for 15min, and freeze-drying the supernatant to obtain a white solid BSF-GMA material.
Mixing conventional methacrylic acid acylated gelatin (GelMA), BSF-GMA material, and ASF-MA10％Respectively preparing printing ink with the concentration of 20%, adding the solution into a 96-well plate, spreading the solution at the bottom of each well by 30 mu l per well, irradiating by 405nm ultraviolet light to form hydrogel, changing the buffer solution by PBS for three times, soaking and balancing by a culture medium, and respectively carrying out 8 × 10 culture of S16 cells, NIH/3T3 cells and HUVEC cells in logarithmic growth phase21 x 10 per hole31 x 10 per hole3And (3) inoculating the density of each hole into a 96-well plate, replacing a culture medium once every 2 days to ensure normal proliferation of the cells, adding a certain amount of the culture medium and the CCK-8 reagent according to the requirements of the specification after incubation for 2 hours at 37 ℃ in the dark by adopting the CCK-8 reagent on the 1 st, 3 rd, 5 th and 7 th days, sucking 100 mu L of each hole, placing the hole into the 96-well plate, detecting the absorbance at 450nm by adopting an enzyme labeling instrument, and analyzing the proliferation condition of the cells. The three cells can normally proliferate on the GelMA hydrogel, and the BSF-GMA hydrogel is lack of a cell adhesion sequence RGD, so that the three cells can not well adhere to a substrate material and are easy to fall off, so that the detection numerical value is low, and obvious proliferation can be detected along with the increase of proliferation quantity on the 7 th day; cells can grow in an adhesive manner in two ASF-MA 10% hydrogels, and the proliferation rate of the cells is similar to that of GelMA hydrogel. Meanwhile, according to the proliferation conditions of three cells on the surfaces of ASF-MA 10% PBS and ASF-MA 10% EtOH hydrogel, the proliferation rate of the cells on the hydrogel soaked in ethanol is relatively slow, but the cells still have a good proliferation rate.
Test example 23D printing
Adopting DLP 3D printing technology, setting corresponding program, splitting and cutting the hollowed-out structure layer by layer, importing the cut hollowed-out structure into the program, processing the data, and projecting the processed data to a printing platform, ASF-MA10％The printing ink is subjected to ultraviolet light photocuring to form a hollow structure as shown in figure 4, the height of the hollow structure is about 5mm, and the prepared ASF-MA is proved10％The material has better 3D printing characteristics.
On the other hand, red fluorescence in logarithmic growth phaseDigesting mouse colon cancer cells (CT26-RFP) marked by photoprotein with pancreatin, centrifuging, resuspending, mixing, counting with an automatic counter, centrifuging a certain amount of cells, discarding supernatant, adding a certain amount of 20% ASF-MA filtered by a sterile filter membrane of 0.22 μm10％PBS solution to make the density of cells 1X 106At one mL, mix it with cells immediately. Printing on a circular glass slide with the diameter of 16mm under aseptic condition, transferring the printed sample into a 12-pore plate, washing and soaking with sterile PBS, removing non-crosslinked monomers, cells not in hydrogel and photoinitiator LAP, timely replacing with DMEM complete culture medium, balancing for 15min, replacing with fresh DMEM complete culture medium, and adding 5% CO2Saturated humidity, 37 ℃ incubator, 1, 2 and 3 days under fluorescence microscope observation of cells and fluorescence intensity, see figure 5. As can be seen from the figure, the fluorescence intensity of the cells gradually increased, indicating that the cells can have better cell viability inside the 3D printed hydrogel.
In conclusion, the tussah silk protein-based 3D printing ink has good biocompatibility and compressibility, and has a certain application prospect in the fields of tissue engineering and the like.