CN113730662B - Nanofiber 3D porous aerogel and preparation method and application thereof - Google Patents
Nanofiber 3D porous aerogel and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of tissue engineering scaffolds, and particularly discloses a nanofiber 3D porous aerogel and a preparation method and application thereof, wherein the method comprises the following steps: s1, dissolving a degradable biological piezoelectric material in a solvent to prepare a spinning solution; s2, spraying the spinning solution into a coagulating bath under the action of an electric field, uniformly dispersing, and then drying to obtain nanofiber aerogel; and S3, carrying out cross-linking treatment on the nanofiber aerogel to obtain the nanofiber 3D porous aerogel. Compared with the prior art, the preparation method is simple and feasible, and the surface of the prepared 3D nanofiber is of a bitter gourd-like surface rough structure; the aerogel structure is rich in a large number of pores, is similar to a cell growth microenvironment, and is non-toxic and excellent in biocompatibility; the special piezoelectric effect of the used piezoelectric material can convert external mechanical stimulation into electric energy, and the micro-current stimulation is generated to accelerate cell growth by combining the micro-electric field of the human body.
Description
Technical Field
The invention belongs to the technical field of tissue engineering scaffolds, and particularly relates to a nanofiber 3D porous aerogel and a preparation method and application thereof.
Background
In tissue engineering, artificial tissue is required to be implanted into damaged tissue or organs, and in order to proliferate and differentiate seed cells, a cell scaffold composed of a biomaterial is required to be provided, and the implanted scaffold material has a geometric shape conforming to the damaged tissue or organ, which is equivalent to an artificial extracellular matrix. The tissue engineering scaffold material comprises bone, cartilage, blood vessel, nerve, skin and artificial organs, such as liver, spleen, kidney, bladder, etc.
It has been found that wound-induced current may stimulate tissue growth, a phenomenon known as galvanotaxis. Thus, simulating endogenous current flow at the wound may accelerate wound healing. In addition, a plurality of piezoelectric active substances exist in natural bone tissues of a human body to jointly construct an electrical microenvironment of the bone tissues, and the piezoelectric active bone repair biomembrane is proved to effectively promote the regeneration and healing of the bone tissues by simulating the electrical microenvironment of the bone tissues. The biological piezoelectric material has certain biocompatibility and good signal conduction capability, can make up the defects of the prior biological material in the aspects of regulating and controlling cell differentiation and tissue regeneration, and has good application prospect in the field of tissue engineering. However, the problems that the electrical activity of the piezoelectric material cannot be kept stable for a long time and the surface potential is not improved sufficiently still exist when the piezoelectric material is applied to the field of biomedicine at present.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a nanofiber 3D porous aerogel and a preparation method and application thereof, the 3D porous aerogel is prepared by combining a bio-piezoelectric material with a dry spinning wet-spinning electrostatic spinning process, can output stable current under external stimulation and accelerate cell growth, and aims to solve the problem of unstable electrical activity of the conventional piezoelectric material used in tissue engineering.
In order to achieve the above object, the present invention provides a preparation method of a nanofiber 3D porous aerogel, which comprises the following steps:
s1, dissolving a degradable biological piezoelectric material in a solvent to prepare a spinning solution;
s2, spraying the spinning solution obtained in the step S1 into a coagulating bath under the action of an electric field, uniformly dispersing, and then drying to obtain the nanofiber aerogel;
and S3, carrying out cross-linking treatment on the nanofiber aerogel obtained in the step S2 to obtain the nanofiber 3D porous aerogel.
Preferably, in step S1, the degradable bio-piezoelectric material is one or more of poly- β -hydroxybutyric acid, chitosan, chitin, and cellulose; the solvent is one or more of dimethylformamide, isopropanol, trifluoroethanol, tetrahydrofuran and glacial acetic acid.
Preferably, in step S2, the coagulation bath is an aqueous solution of tert-butanol.
Preferably, the volume ratio of the tert-butyl alcohol to the water in the tert-butyl alcohol aqueous solution is (1-9): 1.
Preferably, in step S2, ultrasonic dispersion in an ice bath is adopted in the dispersion process, and the ultrasonic dispersion time is 0.5h-1h.
Preferably, in step S2, the drying is freeze drying, and the drying time is 24h-48h.
Preferably, in step S3, the crosslinking is chemical crosslinking.
Preferably, the crosslinking agent used for the chemical crosslinking is genipin.
According to another aspect of the present invention, there is also provided a nanofiber 3D porous aerogel prepared according to the method of the present invention, wherein the nanofibers 3D porous aerogel have a network structure formed by interlacing between the fibers, and the surface of the fibers is rough and has a plurality of through holes.
According to another aspect of the invention, the application of the nanofiber 3D porous aerogel in a tissue engineering scaffold material is also provided.
Generally, compared with the prior art, the above technical solution conceived by the present invention has the following beneficial effects:
(1) The aerogel prepared by using the degradable biological piezoelectric material through the dry spinning wet-spinning electrostatic spinning process is of a 3D (three-dimensional) nanofiber porous structure, the fiber surface is rough, and the aerogel is rich in pores; the used piezoelectric material can generate micro-current under the action of external stimulation to act on cells, and through the construction of the hierarchical porous morphology, the porous structure is utilized to promote the transmission of external mechanical stimulation, and the deformation of the material is increased, so that the piezoelectric effect is enhanced, a larger and more stable piezoelectric electric field is output, and the accelerated growth of the cells is stimulated.
(2) The method for preparing the 3D porous aerogel is simple and feasible in treatment process, has no special requirements on equipment, is high in production efficiency, uses non-toxic and easily-obtained materials, is excellent in biocompatibility, and has a good application prospect in the field of tissue engineering scaffolds.
(3) The nanofiber 3D porous aerogel provided by the invention has a three-dimensional structure, fibers are staggered to form a net structure similar to a bird nest, the surfaces of the fibers are bitter gourd-like epidermis rough structures with micro-nano-scale pores, the inner connectivity of the aerogel is high, the pore sizes are uniform, the appearance and the size of the aerogel well simulate natural extracellular matrix, and the free migration and growth of cells in the aerogel are facilitated.
(4) The nanofiber 3D porous aerogel provided by the invention has a large surface area/volume ratio as a tissue engineering scaffold material, is beneficial to adhesion of a large number of cells, transportation of nutrient substances and discharge of metabolic waste, and can output stable current under external stimulation to accelerate cell growth, so that tissue regeneration and healing are promoted.
Drawings
FIG. 1 is a flow chart of the preparation of nanofiber 3D porous aerogel provided by the present invention;
FIG. 2 is an electron microscope image of a nanofiber 3D porous aerogel prepared in example 1 of the present invention;
FIG. 3 is a staining pattern of the nanofiber 3D porous aerogel cultured cells prepared in example 1 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in 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 do not limit the invention.
As shown in fig. 1, the preparation method of the nanofiber 3D porous aerogel provided by the present invention comprises the following steps:
s1, dissolving a degradable biological piezoelectric material serving as a raw material in a benign solvent to prepare a homogeneous spinning solution;
s2, spraying the spinning solution obtained in the step S1 into a coagulating bath under the action of an electric field, wherein the degradable biological piezoelectric material in the spinning solution is insoluble in the coagulating bath, the coagulating bath has stronger intersolubility with a benign solvent in the spinning solution, the benign solvent in the spinning solution is extracted out to form a two-phase structure with the degradable biological piezoelectric material as a continuous phase and the solvent as a dispersed phase, the two-phase structure is uniformly dispersed, and then drying is carried out to obtain the nanofiber aerogel;
and S3, carrying out cross-linking treatment on the nanofiber aerogel obtained in the step S2 to generate a cross-linked network, so as to obtain the nanofiber 3D porous aerogel.
According to the method, the specific piezoelectric effect of a piezoelectric material is utilized, external mechanical stimulation can be converted into electric energy, the micro-electric field of a human body is combined, the growth of cells is accelerated by generating micro-current stimulation, the spinning solution is prepared by taking a degradable biological piezoelectric material as a raw material, and the electrostatic spinning fiber is received by a non-solvent coagulating bath; then drying the spinning product to obtain a three-dimensional aerogel structure, well simulating the structure of an extracellular matrix, and being more beneficial to cell proliferation and growth; meanwhile, the mechanical strength of the aerogel is improved by performing crosslinking treatment on the aerogel, so that the piezoelectric material can output stable current under external stimulation, the cell growth is accelerated, and the tissue regeneration is effectively promoted.
In some embodiments, the degradable bio-piezoelectric material may be one or more of poly- β -hydroxybutyrate (PHB), chitosan, chitin, cellulose. The solvent with good solubility for the degradable biological piezoelectric material can be one or more of Dimethylformamide (DMF), isopropanol, trifluoroethanol (TFE), tetrahydrofuran and glacial acetic acid.
In some embodiments, the coagulation bath may be selected from an aqueous solution of t-butanol which is capable of displacing the benign solvent of the spinning solution well, and which is similar to water in nature and suitable for use as a lyophilizate in a freeze-drying process, and further preferably, the volume ratio of t-butanol to water in the aqueous solution of t-butanol is (1-9): 1. If the water content in the coagulation bath is too low, the coagulation bath solution is volatile in the spinning process; if the moisture content is too much, the spinning product is not easy to immerse in the coagulating bath, and the spinning product is easy to float on the surface of the coagulating bath, so that the solvent replacement effect is influenced, and the formation of aerogel pores is influenced.
In some embodiments, the dispersion process in step S2 adopts ultrasonic dispersion in an ice bath, and the fibers in the liquid are uniformly dispersed by using the liquid as a medium through the "cavitation" action of ultrasonic waves in the liquid, so that the dispersion quality is high, the speed is high, and the ultrasonic dispersion time can be specifically 0.5h to 1h. The drying is preferably freeze drying for 24h-48h to form a good 3D porous aerogel structure. When the freeze drying process is adopted, the mixed solution of the coagulating bath is preferably filtered after electrostatic spinning is finished, the upper-layer spinning gel is taken, the coagulating bath is used for cleaning for multiple times, and then ultrasonic dispersion is carried out in the coagulating bath, so that the aim of thoroughly removing benign solvents among gel fibers is fulfilled, a good freeze drying effect is favorably achieved, and meanwhile, organic solvents in the gel are thoroughly removed, so that cell culture is more favorably.
In some embodiments, the crosslinking in step S3 is preferably chemical crosslinking, which is more friendly to the bio-piezoelectric material. Meanwhile, genipin is preferably used as a cross-linking agent, is an excellent natural biological cross-linking agent, can be cross-linked with protein, collagen, gelatin, chitosan and the like to prepare biological materials, such as artificial bones, wound dressing materials and the like, and has far lower toxicity than glutaraldehyde and other common chemical cross-linking agents. The temperature adopted in the crosslinking process is 20-50 ℃, and the crosslinking time is 1-5 h.
According to the nanofiber 3D porous aerogel prepared by the method, the fibers are staggered to form a net-shaped structure, the surfaces of the fibers are rough and are provided with a plurality of through holes, the mutually communicated net-shaped porous structure is close to a microenvironment for cell growth, and meanwhile, the porous structure enhances the piezoelectric effect of the piezoelectric material, so that the piezoelectric material can generate more stable micro-current under external stimulation and can stimulate cells to accelerate growth.
The invention also provides application of the nanofiber 3D porous aerogel in a tissue engineering scaffold material, the nanofiber 3D porous aerogel has a large surface area/volume ratio, can well simulate a natural extracellular matrix in appearance and size, is used for culturing cells, has good biocompatibility and has no toxicity to the cells, and the piezoelectric effect specific to the adopted biological piezoelectric material can convert external mechanical stimulation into electric energy, and can generate stable micro-current by combining with a micro-electric field of a human body, so that the scaffold material can promote cell adhesion, proliferation, migration and differentiation when in mutual contact with the cells, and is suitable for repairing wounds in tissue engineering.
The technical solution described above is explained in detail below with reference to specific examples.
Example 1
1. Ultrasonically dispersing PHB and chitosan in an isopropanol solution to prepare 8% (m/v) of spinning solution, transferring the spinning solution into a 10mL syringe, spinning under the conditions that the ambient temperature is 25 +/-3 ℃, the humidity is 50% +/-5%, the spinning voltage is 15kV, the spinning speed is 8mL/min and the receiving distance is 15cm, and collecting the nanofibers by adopting a coagulation bath comprising tert-butyl alcohol and water (v/v) = 1.
2. The collected nanofibers were transferred to a beaker and sonicated for 1h on an ice bath.
3. And freezing the nanofiber mixed solution obtained by ultrasonic dispersion by using liquid nitrogen, putting the frozen nanofiber mixed solution into a freeze dryer, and freeze-drying for 48 hours to prepare the 3D porous aerogel.
4. And (3) soaking the aerogel obtained by using 1% genipin, and crosslinking for 2h under the heating condition of 50 ℃ to obtain the nanofiber 3D porous aerogel.
In the embodiment, PHB and chitosan are adopted as the degradable piezoelectric bio-material, so that the biodegradable piezoelectric bio-material has excellent biocompatibility and higher strength and toughness. Observing the aerogel prepared in the embodiment under an electron microscope, as shown in fig. 2, fibers inside the aerogel are staggered to form a porous network structure similar to a bird nest, and the fiber surface is a balsam pear-like skin rough structure with a plurality of micro-nano-scale pores.
Cells were cultured using the 3D porous aerogel prepared in this example, and were subjected to hematoxylin and eosin staining (HE), and as a result of observation under a microscope, many cells smoothly migrated to the edge in the middle of the scaffold along the boundary, and finally exhibited a crypt-like structure, as shown in fig. 3. Due to the ideal pore size, the stents have guidance, can provide more growth space for cells, and are favorable for cell adhesion and migration.
Example 2
1. Ultrasonically dispersing PHB and chitosan in isopropanol solution to prepare 10% (m/v) spinning solution, transferring the spinning solution into a 10mL injector, spinning under the conditions that the ambient temperature is 25 +/-3 ℃, the humidity is 50% +/-5%, the spinning voltage is 15kV, the spinning speed is 6mL/min and the receiving distance is 15cm, and collecting the nano-fibers by adopting a coagulation bath which is a mixture of tert-butyl alcohol and water (v/v) = 1.
2. The collected nanofibers were transferred to a beaker and sonicated for 1h in an ice bath.
3. And freezing the nanofiber mixed solution obtained by ultrasonic dispersion by using liquid nitrogen, putting the frozen nanofiber mixed solution into a freeze dryer, and freeze-drying for 48 hours to prepare the 3D porous aerogel.
4. And (3) soaking the obtained aerogel by using 1% genipin, and crosslinking for 2 hours under the heating condition of 30 ℃ to obtain the nanofiber 3D porous aerogel.
The aerogel prepared by the embodiment is of a nanofiber 3D three-dimensional structure, a porous net structure is formed by fibers in a staggered mode, the surface of each fiber is rough, and the aerogel is rich in pores. The aerogel is used for culturing cells, shows excellent biocompatibility and no toxicity to the cells, and can promote cell adhesion, migration and growth.
Example 3
1. Dissolving 80% deacetylated chitosan in 90% acetic acid water solution, heating and stirring at 50 deg.C for 4 hr to obtain 8% (m/v) chitosan solution, and adding appropriate amount of gelatin to regulate viscosity of spinning solution. After being mixed uniformly, the mixture is transferred into a 10mL injector, the ambient temperature is 25 ℃ plus or minus 3 ℃, the humidity is 50 percent plus or minus 5 percent, the spinning voltage is 20kV, the spinning speed is 6mL/min, the spinning is carried out under the condition that the receiving distance is 13cm, and the nano-fibers are collected by adopting a coagulation bath from tert-butyl alcohol to water (v/v) = 9.
2. And (3) filtering and cleaning the collected coagulation bath mixed liquid for multiple times by using the tert-butyl alcohol aqueous solution in the same proportion, and then carrying out ultrasonic dispersion for 30min.
3. And freezing the nanofiber mixed solution obtained by ultrasonic dispersion by using liquid nitrogen, putting the frozen nanofiber mixed solution into a freeze dryer, and freeze-drying for 48 hours to prepare the 3D porous aerogel.
4. And (3) placing the obtained aerogel into a 1% genipin aqueous solution, soaking and crosslinking for 2 hours at 30 ℃, taking out, and performing vacuum drying to obtain the nanofiber 3D porous aerogel.
The aerogel prepared by the embodiment is of a nanofiber 3D three-dimensional structure, a porous net structure is formed by fibers in a staggered mode, the surface of each fiber is rough, and the aerogel is rich in pores. The aerogel is used for culturing cells, shows excellent biocompatibility and no toxicity to the cells, and can promote cell adhesion, migration and growth.
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 (9)
1. The preparation method of the nanofiber 3D porous aerogel is characterized by comprising the following steps:
s1, dissolving a degradable biological piezoelectric material in a solvent to prepare a spinning solution;
s2, spraying the spinning solution obtained in the step S1 into a coagulating bath under the action of an electric field, carrying out ultrasonic dispersion in an ice bath for 0.5-1 h, and then carrying out freeze drying to obtain the nanofiber aerogel;
and S3, carrying out cross-linking treatment on the nanofiber aerogel obtained in the step S2 to obtain the nanofiber 3D porous aerogel.
2. The method for preparing nanofiber 3D porous aerogel according to claim 1, characterized in that: in the step S1, the degradable biological piezoelectric material is one or more of poly-beta-hydroxybutyric acid, chitosan, chitin and cellulose; the solvent is one or more of dimethylformamide, isopropanol, trifluoroethanol, tetrahydrofuran and glacial acetic acid.
3. The preparation method of nanofiber 3D porous aerogel according to claim 1, characterized in that: in step S2, the coagulation bath is an aqueous solution of tert-butanol.
4. The method for preparing nanofiber 3D porous aerogel according to claim 3, characterized in that: the volume ratio of the tertiary butanol to the water in the tertiary butanol aqueous solution is (1-9): 1.
5. The method for preparing nanofiber 3D porous aerogel according to claim 1, characterized in that: in the step S2, the freeze drying time is 24-48 h.
6. The preparation method of nanofiber 3D porous aerogel according to claim 1, characterized in that: in step S3, the crosslinking is chemical crosslinking.
7. The method for preparing nanofiber 3D porous aerogel according to claim 6, characterized in that: the crosslinking agent used for the chemical crosslinking is genipin.
8. A nano-fibrous 3D porous aerogel produced according to the method of any of claims 1 to 7, characterized in that: the fibers in the nanofiber 3D porous aerogel are staggered to form a net structure, and the fiber surface is rough and is provided with a plurality of through holes.
9. Use of the nanofiber 3D porous aerogel of claim 8 in a tissue engineering scaffold material.
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| CN106390197A (en) * | 2016-11-02 | 2017-02-15 | 武汉纺织大学 | Stress active tissue engineering scaffold material and preparation method thereof |
| CN106390208A (en) * | 2016-10-09 | 2017-02-15 | 华南理工大学 | Three-dimensional support material containing hierarchical porous structures and preparation and application |
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| CN106390208A (en) * | 2016-10-09 | 2017-02-15 | 华南理工大学 | Three-dimensional support material containing hierarchical porous structures and preparation and application |
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