CN108794771B - Double-network cross-linked cellulose/silk fibroin high-strength hydrogel and preparation and application thereof - Google Patents
Double-network cross-linked cellulose/silk fibroin high-strength hydrogel and preparation and application thereof Download PDFInfo
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Abstract
The invention discloses a double-network cross-linked cellulose/silk fibroin high-strength hydrogel and preparation and application thereof, wherein the preparation method comprises the following steps: (1) uniformly mixing a cellulose solution, a silk fibroin solution and epichlorohydrin, and crosslinking under stirring to obtain a crosslinked solution; (2) centrifuging the crosslinking solution to remove bubbles, pouring the crosslinking solution into a mold, and reacting and curing to obtain the low-density chemical crosslinking cellulose/silk fibroin hydrogel; (3) placing the obtained cellulose/silk fibroin hydrogel in a carbon dioxide incubator for atmosphere physical crosslinking; (4) and washing with running water to obtain the double-network cross-linked cellulose/silk fibroin high-strength hydrogel. The hydrogel with different mechanical strength and hydrophilicity and hydrophobicity can be obtained by adjusting the proportion of the cellulose to the silk fibroin. Experimental results show that the double-network cross-linked high-strength hydrogel has good cell compatibility and blood compatibility, and has great application potential in the field of tissue engineering.
Description
Technical Field
The invention belongs to the cross field of polymer chemistry and physics and tissue engineering, and more particularly relates to a double-network cross-linked cellulose/silk fibroin high-strength hydrogel and a preparation method and application thereof.
Background
With the development of life science, advanced biomaterials such as hydrogel, composite sponge, nano-microspheres and the like are continuously brought out, and a new scheme is provided for solving clinical problems. Hydrogels refer to a class of polymer systems having a three-dimensional network structure formed by physical or chemical crosslinking (adv. mater.,2017,29,1700321). At present, raw materials for preparing hydrogel mainly comprise natural polymers such as cellulose, chitosan and collagen and synthetic polymers such as polyvinyl alcohol, polyethylene glycol and polyethylene lactone (biomacromolecules, 2018,4, 1356-1364; adv.funct.mater, 2017 and 27,1606497). The natural polymer hydrogel has high water content and water retention capacity, and specific microstructure and biological activity of raw materials (biomaterials, 2017,122,34-37), and has wide application prospect in the medical fields of skin tissue engineering, bone tissue engineering, drug release carriers and the like. In practical applications, the hydrogel should have a mechanical strength that matches that of human tissue. However, hydrogel generally has the defects of soft quality, easy breakage and the like (acs. nano.,2017,11,2554-2560), and the improvement of the mechanical properties of hydrogel is closely concerned by research and development personnel.
Cellulose is a natural polymer material which is most widely distributed in the world, and has the advantages of abundant reserves, natural degradation, no cytotoxicity, good processing performance and the like. The invention patent of patent No. 200810197646.8 produced a epichlorohydrin-crosslinked cellulose hydrogel whose use of an excessive amount of a crosslinking agent resulted in an excessively high crosslinking density of the hydrogel and whose compressive strength was only several tens of KPa. Patent No. CN 107417934A discloses a high-strength and high-toughness cellulose hydrogel, which is prepared by reacting a small amount of epichlorohydrin to obtain a cellulose hydrogel with low chemical crosslinking density, and then soaking the cellulose hydrogel in an acidic aqueous solution for rapid physical crosslinking. Experimental results show that the compressive strength of the obtained hydrogel reaches MPa, and the defects are that the cellulose hydrogel has poor bioactivity, the mechanical property is not easy to regulate and control, and the use of an acidic reagent can aggravate environmental pollution.
Silk fibroin is one of the main components of natural silk fibers, and has good water solubility, cell compatibility, blood compatibility and low immunogenicity (acta biomaterials, 2016,31, 1-16). The polypeptide chain of silk fibroin contains multiple chemical active groups and cell active factors, such as cell adhesion factor RGD, etc., so that it has certain bioactivity. Research results show that the degradation products of silk fibroin are beneficial to skin, periodontal and liver tissue regeneration (prog. polym. sci.,2014,39,251-267), and at present, silk fibroin has already achieved good application effects in the fields of bioreactors, tissue engineering organs, drug sustained release carriers and the like.
At present, reports about the preparation of high-strength hydrogel of cellulose and silk fibroin are not found.
Disclosure of Invention
Aiming at the defects or the improvement requirements of the prior art, the invention aims to provide a double-network cross-linked cellulose/silk fibroin high-strength hydrogel and preparation and application thereof2Preparing the double-network crosslinking high-strength hydrogel by processes of atmosphere crosslinking, flushing and the like.
The invention selects cellulose and silk fibroin as main raw materials, not only effectively solves the problem of poor biological activity of pure high-strength cellulose hydrogel, but also realizes simple and convenient regulation and control of mechanical strength. The mechanical strength of the high-strength hydrogel can be adjusted by changing the content of silk fibroin, so that the requirements of different tissues and organs of a human body on the mechanical strength of the hydrogel are met. The invention adopts CO2The atmosphere crosslinking method carries out secondary physical crosslinking on the low-density chemically crosslinked hydrogel, and has the advantages of mild reaction conditions, good formability, no pollution to the environment and the like compared with the prior art such as soaking in ethanol or acidic solution. Obtained byThe double-network cross-linked cellulose/silk fibroin high-strength hydrogel has adjustable compression strength, hydrophilicity and hydrophobicity, excellent cell compatibility and blood compatibility and very potential application in the field of tissue engineering.
In order to achieve the above object, according to one aspect of the present invention, there is provided a method for preparing a double-network cross-linked cellulose/silk fibroin high-strength hydrogel, comprising the steps of:
(1) uniformly mixing a cellulose solution, a silk fibroin solution and epichlorohydrin, and crosslinking under stirring to obtain a crosslinked solution;
(2) centrifuging the crosslinking solution obtained in the step (1) to remove bubbles, pouring the crosslinking solution into a mold, and reacting and curing to obtain the low-density chemical crosslinking cellulose/silk fibroin hydrogel;
(3) placing the cellulose/silk fibroin hydrogel obtained in the step (2) in a carbon dioxide incubator for atmosphere physical crosslinking;
(4) and (4) washing the cellulose/silk fibroin hydrogel obtained in the step (3) with running water to obtain the double-network cross-linked cellulose/silk fibroin high-strength hydrogel.
Preferably, in the step (1), the cellulose solution is prepared by the following method: dissolving 7 parts by weight of sodium hydroxide and 12 parts by weight of urea in 81 parts by weight of deionized water to obtain a sodium hydroxide/urea solution, respectively precooling the cellulose and the sodium hydroxide/urea solution to-12 ℃, then mixing, and violently stirring until the cellulose and the sodium hydroxide/urea solution are dissolved to obtain the cellulose solution.
Preferably, the silk fibroin solution is prepared by the following method: dissolving 7 parts by weight of sodium hydroxide and 12 parts by weight of urea in 81 parts by weight of deionized water to obtain a sodium hydroxide/urea solution, mixing silk fibroin and the sodium hydroxide/urea solution, and mechanically stirring until the silk fibroin and the urea are completely dissolved to obtain the silk fibroin solution.
Preferably, in the crosslinking solution obtained in the step (1), the mass fraction of the cellulose is 1.5-4.0%, the mass fraction of the silk fibroin is 0-2.0%, and the mass fraction of the epichlorohydrin is 0.5-10%.
Preferably, in the step (1), the crosslinking is performed at room temperature, and the crosslinking time is 5-15 min.
Preferably, in the step (2), the centrifugal degassing bubble condition is 1500-3000rpm centrifugation for 5-10 min;
preferably, in the step (4), the washing with running water is specifically washing with deionized water for more than 48 hours.
Preferably, in the step (2), the reaction curing condition is standing for 4-24h at room temperature.
Preferably, in the step (3), the physical crosslinking is performed at room temperature in CO2CO-containing gas having a concentration of 3 to 30 vol%2Treating for 2-12h under atmosphere.
According to another aspect of the invention, the invention provides the double-network cross-linked cellulose/silk fibroin high-strength hydrogel prepared by the preparation method.
According to another aspect of the present invention, the present invention provides an application of the double-network cross-linked cellulose/silk fibroin high-strength hydrogel, which is characterized by being applied to the field of tissue engineering.
In the double-network cross-linked cellulose/silk fibroin high-strength hydrogel obtained by the invention, different compression strengths and hydrophilicity and hydrophobicity can be obtained by adjusting the mass ratio of the cellulose to the silk fibroin.
In vitro cytotoxicity experiments and hemolysis experiments show that the cellulose/silk fibroin high-strength hydrogel has good cell compatibility and blood compatibility. Therefore, the invention has certain application potential in the field of tissue engineering.
The invention has the following advantages and beneficial effects: (1) combined use of low density chemical crosslinking and CO2The mechanical strength of the hydrogel is obviously improved by atmosphere crosslinking; (2) the biological activity of the hydrogel is improved by adding silk fibroin, and the simple regulation and control of the mechanical strength and hydrophilicity and hydrophobicity of the hydrogel are realized; (3) CO 22The reaction conditions of atmosphere crosslinking and soaking in ethanol and an acidic solvent are milder, the gel formability is better, and the environmental pollution is less (4)The obtained cellulose/silk fibroin high-strength hydrogel has good cell compatibility and blood compatibility, and has great potential to be applied to the field of tissue engineering.
Drawings
FIG. 1 is the results of the compression test of the double-network cross-linked high-strength hydrogel obtained in comparative example 1 and example 1.
FIG. 2 is the results of water contact angle measurement experiments of the double-network cross-linked high-strength hydrogel obtained in comparative example 1 and example 1.
FIG. 3 is the results of the in vitro cytotoxicity test of the double-network cross-linked high-strength hydrogel obtained in comparative example 1 and example 1.
FIG. 4 is a result of hemolysis test of the double-network cross-linked high-strength hydrogel obtained in comparative example 1 and example 1, wherein DS represents distilled water (distilled water) and NS represents physiological saline (normal saline).
FIG. 5 is a schematic diagram of the preparation process of the double-network cross-linked cellulose/silk fibroin high-strength hydrogel of the present invention, which respectively corresponds to blending, low-density chemical cross-linking, atmosphere cross-linking, and flushing from left to right and from top to bottom.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
As shown in FIG. 5, the preparation method of the double-network cross-linked cellulose/silk fibroin high-strength hydrogel in the invention is summarized as that cellulose and silk fibroin are separately dissolved in sodium hydroxide/urea solution respectively, and then are subjected to blending, low-density chemical cross-linking and CO2And preparing the double-network cross-linked cellulose/silk fibroin high-strength hydrogel by processes such as atmosphere cross-linking, flushing and the like.
Comparative example 1
7g of sodium hydroxide and 12g of urineThe plain was dissolved in 81g of deionized water to give a sodium hydroxide/urea solution. Respectively precooling the cellulose and the sodium hydroxide/urea solution to-12 ℃, mixing and then violently stirring for 4min to obtain a cellulose solution with the mass fraction of 2.5%. Dropwise adding epichlorohydrin accounting for 100% of the total mass of the cellulose solute into the solution, continuously stirring and crosslinking for 15min, centrifuging at 2000rpm for 10min, pouring the crosslinked solution into a mold, standing at room temperature, reacting and curing for 12h, and transferring to 5 vol% CO2And continuously crosslinking for 6h in the incubator, and washing with deionized water for 48h to obtain the double-network crosslinked cellulose high-strength hydrogel.
Example 1
7g of sodium hydroxide and 12g of urea were dissolved in 81g of deionized water to give a sodium hydroxide/urea solution. Respectively precooling the cellulose and the sodium hydroxide/urea solution to-12 ℃, mixing and then violently stirring for 4min to obtain a cellulose solution with the mass fraction of 3%. Dissolving silk fibroin in a sodium hydroxide/urea solution to obtain a silk fibroin solution with the mass fraction of 10%. Uniformly mixing a cellulose solution and a silk fibroin solution according to a certain proportion, and then adding a small amount of sodium hydroxide/urea solution to adjust the mass fraction of the cellulose to be 2.5 percent, and the mass fraction of the silk fibroin to be 0.5 percent, 1 percent and 1.5 percent in sequence. Adding epichlorohydrin accounting for 100% of total mass of the cellulose and silk fibroin solutes into the mixed solution, continuously stirring and crosslinking for 15min, centrifuging at 2000rpm for 10min, pouring the crosslinked solution into a mold, standing at room temperature, reacting and curing for 12h, and transferring to 5% CO2And continuously crosslinking for 6h in the incubator, and washing with deionized water for 48h to obtain the double-network crosslinked cellulose/silk fibroin high-strength hydrogel. CCS-0, CCS-0.5, CCS-1 and CCS-1.5 respectively represent the final mass fractions of silk fibroin in the hydrogel to be 0%, 0.5%, 1% and 1.5% respectively. CCS-0 corresponds to the product of comparative example 1.
Example 2
The double-network crosslinked high-strength hydrogels obtained in comparative example 1 and example 1 were cut into cylinders having a diameter of 10mm and a height of 8 mm. The compressive strength of the hydrogel is detected by adopting a universal material tester, and the compression rate is 2 mm/min.
FIG. 1 is the results of the compression test of the double-network cross-linked high-strength hydrogel obtained in comparative example 1 and example 1, and as can be seen, the compressive strengths of CCS-0, CCS-0.5, CCS-1, CCS-1.5 are 2.51MPa,1.58MPa, 0.91MPa and 0.55MPa, respectively, indicating that the hydrogel prepared according to the present invention has good mechanical strength. With the increase of the content of silk fibroin, the mechanical strength of the hydrogel is obviously reduced, and the hydrogel has statistical difference (P <0.05) among groups. The mechanical strength of the high-strength hydrogel can be simply and conveniently regulated and controlled by controlling the content of the silk fibroin, so that the requirements of different tissues and organs on the mechanical strength of the hydrogel are met in practical application.
Example 3
The double-network cross-linked high-strength hydrogels obtained in comparative example 1 and example 1 were dried in an oven at 60 ℃ for 24 hours, and then cut into a size of 1cm × 1cm and attached to a glass slide. The hydrophilicity and hydrophobicity of the gel surface were measured by a water contact angle measuring instrument.
FIG. 2 is the results of water contact angle measurement experiments of the double-network cross-linked high-strength hydrogel obtained in comparative example 1 and example 1. As shown in FIG. 2, the water contact angles of CCS-0, CCS-0.5, CCS-1, and CCS-1.5 are 53.8 °, 64.7 °, 85.8 °, and 115.8 °, respectively. With the increase of the content of silk fibroin, the hydrophilic property of the hydrogel is gradually weakened, and the transition from hydrophilicity to hydrophobicity is realized. The adhesion capability of cells on the surface of the material has obvious correlation with the hydrophilicity and hydrophobicity of the material, and the content of the silk fibroin is increased or decreased according to the needs of cells from different tissue sources.
Example 4
drying the double-network cross-linking high-strength hydrogel obtained in comparative example 1 and example 1, cutting into powder, autoclaving, soaking in α -DMEM medium, adding 1mL of medium to every 0.05g of powder, incubating at 37 ℃ for 72h, filtering to obtain hydrogel leaching liquor, detecting the cell compatibility of the hydrogel by using in vitro cytotoxicity test (MTT method), inoculating mouse fibroblast (L929) into a 96-well plate with the inoculation density of 1 × 103cell/well, culturing for 24 hr, removing culture medium, adding 200 μ L material leaching solution, culturing for 24, 48 and 72 hr, culturing with α -DMEM L929 as negative control, and culturing in airWhite plates served as blank control. Add 20. mu.L of thiazole blue (MTT) reagent to each well. After continuing culturing for 4 hours, completely removing the liquid in the culture plate, adding 150 mu L of dimethyl sulfoxide (DMSO) into each hole, shaking in dark for 10min, and detecting the absorbance at 490nm by using a multifunctional microplate reader. The cell proliferation rate of L929 was obtained by statistical analysis.
FIG. 3 is the results of the in vitro cytotoxicity test of the double-network cross-linked high-strength hydrogel obtained in comparative example 1 and example 1. As seen from the figure, the cell proliferation rate of the control group was set to 100%, and compared to the control group, the cell proliferation rates of CCS-0, CCS-0.5, CCS-1, CCS-1.5 were significantly greater than 80% on days 1 to 3, exhibiting good cell compatibility.
Example 5
The double-network crosslinked high-strength hydrogel obtained in comparative example 1 and example 1 was cut into 1cm × 1cm samples, and placed in a 15mL centrifuge tube, and rinsed 3 times with physiological saline for 30min each. Adding 10mL of physiological saline into each centrifuge tube, carrying out constant-temperature water bath at 37 ℃ for 30min, adding 0.2mL of diluted whole blood with the mass ratio of the whole blood to the physiological saline of 1:1.25, uniformly mixing, carrying out constant-temperature water bath at 37 ℃ for 60min, and centrifuging at 1500rpm for 10 min. A positive control was obtained by diluting 0.2mL of whole blood with 10mL of distilled water, and a negative control was obtained by diluting 0.2mL of whole blood with 10mL of physiological saline. And finally, detecting the absorbance of the supernatant at the wavelength of 545nm by using a multifunctional microplate reader. The hemolysis rate is obtained by statistical analysis.
FIG. 4 is a result of the hemolysis ratio of the double-network cross-linked high-strength hydrogel obtained in comparative example 1 and example 1. As can be seen, the hemolysis rate of blood in deionized water is defined as 100% and the hemolysis rate in physiological saline is defined as 0%. The hemolysis rates of CCS-0, CCS-0.5, CCS-1, CCS-1.5 are 0.12%, 2.66%, 0.11% and 1.20%, respectively, all significantly lower than the 5% hemolysis rate (P <0.05) prescribed by the United states pharmacopoeia. The above experimental results show that: the double-network cross-linking high-strength hydrogel meets the general requirements of the hemolytic performance of medical devices.
In the cross-linking solution formed by blending, the mass ratio of the cellulose to the silk fibroin is preferably controlled to be 5:0 to 5: 3.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (8)
1. A process for preparing the dual-network cross-linked high-strength hydrogel of cellulose/silk fibroin includes such steps as dissolving cellulose and silk fibroin in solution of sodium hydroxide/urea, mixing, low-density chemical cross-linking, and CO2Carrying out atmosphere crosslinking and flushing to prepare the double-network crosslinking high-strength hydrogel; the method specifically comprises the following steps:
(1) uniformly mixing a cellulose solution, a silk fibroin solution and epichlorohydrin, and crosslinking under stirring to obtain a crosslinked solution;
(2) centrifuging the crosslinking solution obtained in the step (1) to remove bubbles, pouring the crosslinking solution into a mold, and reacting and curing to obtain the low-density chemical crosslinking cellulose/silk fibroin hydrogel;
(3) placing the cellulose/silk fibroin hydrogel obtained in the step (2) in a carbon dioxide incubator for atmosphere physical crosslinking;
(4) washing the cellulose/silk fibroin hydrogel obtained in the step (3) with running water to obtain double-network cross-linked cellulose/silk fibroin high-strength hydrogel;
wherein in the crosslinking solution obtained in the step (1), the mass fraction of cellulose is 1.5-4.0%, the mass fraction of silk fibroin is not more than 2.0%, and the mass fraction of epichlorohydrin is 0.5-10%;
in the step (1), the crosslinking is carried out at room temperature, and the crosslinking time is 5-15 min;
in the step (3), the physical crosslinking is carried out at room temperature in CO2Treating for 2-12h under the atmosphere with the gas concentration of 3-30 vol%.
2. The method for preparing the double-network cross-linked cellulose/silk fibroin high-strength hydrogel according to claim 1, wherein in the step (1), the cellulose solution is prepared by the following method: dissolving 7 parts by weight of sodium hydroxide and 12 parts by weight of urea in 81 parts by weight of deionized water to obtain a sodium hydroxide/urea solution, respectively precooling the cellulose and the sodium hydroxide/urea solution to-12 ℃, then mixing, and violently stirring until the cellulose and the sodium hydroxide/urea solution are dissolved to obtain the cellulose solution.
3. The method for preparing the double-network cross-linked cellulose/silk fibroin high-strength hydrogel according to claim 1, wherein the silk fibroin solution is prepared by the following method: dissolving 7 parts by weight of sodium hydroxide and 12 parts by weight of urea in 81 parts by weight of deionized water to obtain a sodium hydroxide/urea solution, mixing silk fibroin and the sodium hydroxide/urea solution, and mechanically stirring until the silk fibroin and the urea are completely dissolved to obtain the silk fibroin solution.
4. The method for preparing the double-network cross-linked cellulose/silk fibroin high-strength hydrogel according to claim 1, wherein in the step (2), the centrifugation and deaeration conditions are 1500-3000rpm centrifugation for 5-10 min.
5. The method for preparing the double-network cross-linked cellulose/silk fibroin high-strength hydrogel according to claim 1, wherein in the step (4), the washing with running water is specifically carried out for more than 48 hours by using deionized water.
6. The method for preparing the double-network cross-linked cellulose/silk fibroin high-strength hydrogel according to claim 1, wherein in the step (2), the reaction curing condition is standing at room temperature for 4-24 h.
7. The double-network cross-linked cellulose/silk fibroin high-strength hydrogel prepared by the preparation method of any one of claims 1-6.
8. The use of the double-network cross-linked cellulose/silk fibroin high-strength hydrogel according to claim 7, characterized by being applied in the field of tissue engineering for non-disease diagnosis and treatment methods.
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| CN110078974A (en) * | 2019-03-22 | 2019-08-02 | 昆明理工大学 | Sesbania gum/cellulose composite water conservation hydrogel preparation method |
| CN112062904B (en) * | 2020-08-28 | 2022-03-29 | 华南理工大学 | Genipin cross-linked antibacterial peptide/quaternized cellulose composite hydrogel bacteriostatic agent and preparation method and application thereof |
| CN112957525B (en) * | 2021-02-07 | 2022-10-21 | 东北林业大学 | Nano-hydroxyapatite/silk fibroin/cellulose composite aerogel and preparation method thereof |
| CN114539564B (en) * | 2022-03-30 | 2023-09-12 | 武汉纺织大学 | Preparation method of rapid-gel silk fibroin-based strong hydrogel |
| CN114958009B (en) * | 2022-05-24 | 2024-01-16 | 浙江理工大学 | Preparation method of silk-based high-strength ionic gel flexible sensing material |
| CN115429934B (en) * | 2022-09-20 | 2023-09-01 | 东华大学 | 3D printing strain sensing tissue engineering scaffold and preparation method thereof |
| CN115895666A (en) * | 2022-09-21 | 2023-04-04 | 华中科技大学 | Method for curing sandy soil through cooperation of MICP (micro-emulsion-modified fibroin) protein |
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| ITVR20010098A1 (en) * | 2001-09-11 | 2003-03-11 | Consorzio Per Gli Studi Uni | PROCEDURE FOR OBTAINING SILK FIBROIN HYDROGELS. |
| CN100560641C (en) * | 2007-08-31 | 2009-11-18 | 武汉大学 | Mierocrystalline cellulose/soy protein composite sponge and preparation method thereof |
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