CN108159493B - Preparation method of alginate-hydrogel nanofiber scaffold - Google Patents
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- A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
Abstract
The invention relates to a preparation method of an alginate-hydrogel nanofiber scaffold, which comprises the following steps: preparing a polycaprolactone solution; preparing a sodium alginate solution; adopting a double-nozzle electrostatic spinning system to carry out electrostatic spinning to obtain a polycaprolactone-sodium alginate nanofiber membrane; completely soaking the polycaprolactone-sodium alginate nanofiber membrane in a calcium chloride solution, fully washing with an ethanol water solution, freeze-drying, soaking and washing with chloroform, sequentially washing with ethanol and distilled water, and freeze-drying to obtain the alginate-hydrogel nanofiber scaffold. The alginate-hydrogel nanofiber scaffold has a stable three-dimensional structure and good biocompatibility, can be applied to 3D cell culture, and can optimize the penetration depth of cells.
Description
Technical Field
The invention belongs to the field of nanofiber scaffolds, and particularly relates to a preparation method of an alginate-hydrogel nanofiber scaffold.
Background
Electrospun nanofibers are a common scaffold material for modeling extracellular matrix structures in tissue engineering. It is not clear whether the cell culture environment of the nanoweb can represent the true in vivo environment. Because of the intermingling of the cells themselves and the porous nature of electrospun nanofibers, the cells are allowed to migrate horizontally on the lattice, interfering with the three-dimensional displacement of the cells on the nanofiber scaffold. Since the difference between two-dimensional and three-dimensional cell culture environments has a significant impact on cell metabolism, three-dimensional cell culture has become a hot spot for tumor tissue modeling and stem cell culture applications. For example, chinese patent "method for preparing collagen sponge-nanocellulose-based three-dimensional cell scaffolds" (201710515612.8) reported that three-dimensional cell scaffolds were prepared by preparing collagen sponge and nanocellulose separately and then mixing them. The method has the advantages of reliable result, simple and convenient operation and strong repeatability, can effectively promote the three-dimensional growth and proliferation of cells for a long time, has a certain effect on the generation of various human three-dimensional micro tissues, particularly the formation of liver cancer tissue functions, but has obvious defects, does not overcome the problem of cell surface culture, and needs to arrange the cells on a bracket by a biological printing technology instead of automatically diffusing the cells on the bracket; the chinese patent "biphasic porous three-dimensional cell culture scaffold" (CN102719391A) reports a three-dimensional culture system with all conveniences of a two-dimensional cell culture system, and this ideal three-dimensional cell culture system will need to have a first step of making it possible for a user to easily observe the growth of cells in the three-dimensional cell culture scaffold as in the case of using a planar cell culture plate, and the preparation method thereof is the first step: firstly, preparing a semi-finished porous three-dimensional scaffold composed of a crude fiber phase. The second step is that: and spraying fine fibers on the outer surface of the semi-finished porous three-dimensional scaffold by adopting an electrostatic spinning technology. The spraying of the electrostatic spinning technology on the semi-finished porous three-dimensional scaffold is influenced by the depth of the three-dimensional scaffold, the spinning density of the semi-finished porous three-dimensional scaffold is reduced along with the depth, and the outer surface of the porous three-dimensional scaffold is easily covered, so that the electrostatic spinning in the inner part is rarely carried out. The thickness of the semi-finished porous three-dimensional scaffold composed of the crude fiber phase in the first step is limited in the second step, and the electrostatic spinning density in the inside of the formed scaffold is extremely unstable.
As a latest research hotspot in tissue engineering simulation, compared with the traditional two-dimensional cell culture, the three-dimensional cell culture brought by the three-dimensional cell scaffold has the advantages that: 1) closer to the in vivo growth pattern of cells 2) may enhance understanding of the structure-function relationship of normal and pathological tissues; 3) the research direction is wider; 4) the research result is more reliable. In recent years, the three-dimensional cell scaffold has good application potential in the field of tissue engineering simulation, can provide a growth environment closer to cells in vivo, and realizes more ideas of tissue engineering cell simulation on the basis of the growth environment, including interaction between cells and growth factors and between cells and medicines, deepening understanding of structure-function relationship of normal and pathological tissues and the like. The electrostatic spinning technology is also a popular method for preparing the three-dimensional nano cell scaffold, the prepared cell scaffold has high specific surface area, a three-dimensional net structure, continuous gaps, high porosity and the like, perfectly meets the requirements of people, and can be accompanied with antibacterial property. An electrospun material with biocompatibility, degradability and good mechanical properties, such as: synthetic polymer compounds such as polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), Polycaprolactone (PCL), and Polyurethane (PU).
However, the current technology for preparing three-dimensional cell scaffolds and providing approximate cell growth environment is not mature, and it is not easy to realize displacement in three dimensions because the cells are horizontally displaced due to the unfixed cell positions and the porosity of the fibers, which facilitates the three-dimensional displacement. The results, which are intuitive from the perspective, are: cells on the surface of the three-dimensional cell scaffold are not easy to permeate into the depth of the scaffold. Therefore, it is urgently needed to prepare a three-dimensional nanofiber cell scaffold which can optimize the penetration depth of cells.
Disclosure of Invention
In order to solve the problems, the invention provides a preparation method of an alginate-hydrogel nanofiber scaffold, which is characterized in that the alginate-hydrogel nanofiber scaffold is prepared by adopting a coaxial co-spinning and removing technology, wherein sodium alginate and polycaprolactone are injected together through an inner needle and an outer needle, and the polycaprolactone can support the fiber form of the sodium alginate until the sodium alginate is crosslinked in a calcium chloride solution. The polycaprolactone layer was then peeled off by repeated soaking in chloroform, while the cross-linked alginate-hydrogel nanofiber scaffold remained intact inside. Therefore, the alginate-hydrogel nano-scaffold produced by utilizing the polycaprolactone nano-fiber can be used for 3D cell culture due to better biocompatibility.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of an alginate-hydrogel nanofiber scaffold comprises the following steps:
1) dissolving polycaprolactone in chloroform/methanol solvent to obtain polycaprolactone solution;
2) dissolving sodium alginate and Triton X-100 in a solvent to prepare a sodium alginate solution;
3) adopting a double-nozzle electrostatic spinning system, weighing 18g of the polycaprolactone solution prepared in the step 1) and placing the polycaprolactone solution in an outer nozzle of the double-nozzle electrostatic spinning system, weighing 20g of the sodium alginate solution prepared in the step 2) and adding the sodium alginate solution into an inner nozzle of the double-nozzle electrostatic spinning system, and carrying out electrostatic spinning to obtain a polycaprolactone-sodium alginate nanofiber membrane;
4) dissolving calcium chloride in an ethanol water solvent to obtain a calcium chloride solution, completely soaking the polycaprolactone-sodium alginate nanofiber membrane obtained in the step 3) in the prepared calcium chloride solution, soaking at room temperature for 6 hours, fully washing with the ethanol water solution, and freeze-drying;
5) soaking and washing the nanofiber membrane subjected to freeze drying in the step 4) with chloroform, then sequentially washing with ethanol and distilled water until the chloroform is completely removed, and carrying out freeze drying to obtain the alginate-hydrogel nanofiber scaffold.
Preferably, in the polycaprolactone solution prepared in the step 1), the volume ratio of the chloroform to the methanol is 3:1, and the content of the polycaprolactone in the solution is 16 wt%.
Preferably, in the sodium alginate solution prepared in step 2), the solvent is a mixture of distilled water and dimethyl sulfoxide, and the volume ratio of distilled water to dimethyl sulfoxide is 19: 1.
Preferably, in the sodium alginate solution prepared in step 2), the content of the sodium alginate in the solution is 2 wt%, and the content of the Triton X-100 in the solution is 0.5 wt%.
Preferably, the spinning conditions in the step 3) are that the flow rate of the solution in the outer nozzle is controlled to be 0.5ml/h, the flow rate of the solution in the inner nozzle is controlled to be 2.5ml/h, the diameter of the collecting roller is 100mm, the length of the collecting roller is 200mm, the rotating speed is 100rpm, the distance between the nozzle and the roller is 12cm, and the spinning voltage is 20 kV.
Preferably, in the step 3), the temperature is controlled to be 23-26 ℃ and the humidity is controlled to be 25-35% in the electrostatic spinning process.
Preferably, in the calcium chloride solution prepared in the step 4), the ethanol-water solvent is a mixed solution of ethanol and distilled water in a volume ratio of 3:7, and the mass fraction of the calcium chloride is 0.002-2%.
Preferably, in the step 5), the nanofiber membrane freeze-dried in the step 4) is soaked and washed with chloroform for 10 times, and each soaking time is 1 h. The polycaprolactone in the nanofiber membrane can be gradually removed by soaking and washing with chloroform for multiple times, so that the polycaprolactone can be completely removed.
In order to further crosslink sodium alginate in the electrospun nanofibers, a polycaprolactone-sodium alginate nanofiber membrane is soaked in calcium chloride solutions with different concentrations to enable the polycaprolactone-sodium alginate nanofiber membrane to spontaneously generate crosslinking behavior, calcium chloride is dissolved in an ethanol water solution with the volume ratio of ethanol to distilled water being 3:7, the mass fraction of the calcium chloride solution is controlled within the range of 0.002-2%, the polycaprolactone-sodium alginate nanofiber membrane is completely soaked in the calcium chloride solution and incubated at room temperature for 6 hours, the ethanol water solution is used for fully washing after the incubation is finished, the calcium chloride is removed from the nanofiber membrane, and then the polycaprolactone-sodium alginate nanofiber membrane is freeze-dried to remove ethanol, water and other components in the nanofiber membrane and maintain the original shape. And soaking the freeze-dried nanofiber membrane in chloroform for 10 times, wherein the soaking time is 1 hour each time, so that polycaprolactone is gradually peeled off. Then washed with ethanol and distilled water in order to remove chloroform, and finally freeze-dried to maintain the shape of the nanofiber membrane.
An alginate-hydrogel nanofiber scaffold prepared by the preparation method of the alginate-hydrogel nanofiber scaffold.
Further, the fiber diameter of the alginate-hydrogel nanofiber scaffold is 88 +/-47 nm
After the polycaprolactone is peeled from the crosslinked alginate-hydrogel nanofiber scaffold, the nanofiber scaffold maintains good fiber shape, and the fiber density of the nanofiber scaffold is gradually reduced along with the gradual reduction of the concentration of calcium chloride. It was found that the average diameter of the fiber of the nanofiber scaffold before the polycaprolactone was peeled off was 555. + -. 173nm, however, whatever the concentration of calcium ion reached, the fiber diameter of the alginate-hydrogel nanofiber scaffold was reduced to 88. + -.47 nm after the polycaprolactone was peeled off. The reduction in fiber diameter of the alginate-hydrogel nanofiber scaffold confirmed that the polycaprolactone covering the sodium alginate was completely removed by the chloroform, and the crosslinked alginate-hydrogel nanofiber scaffold maintained the morphology of the fibers due to the encapsulation of the polycaprolactone. However, after washing, the fibers in the alginate-hydrogel nanofiber scaffold crosslinked under the condition of medium and low concentration of calcium ions have low mechanical properties, most of the fibers are washed away, and only the fibers with better mechanical properties are remained.
The invention has the beneficial effects that:
the invention adopts a coaxial electrostatic spinning technology to form a sodium alginate nanofiber scaffold wrapped by polycaprolactone, then the sodium alginate is further crosslinked in a calcium chloride solution, and the method for removing the polycaprolactone is adopted to finally prepare the alginate-hydrogel nanofiber scaffold.
(1) This application spins and gets rid of the technique through coaxial altogether, sodium alginate and polycaprolactone are injected into jointly through interior outer needle and are carried out electrostatic spinning, collect the non-woven fabrics that forms three-dimensional structure receiving, detach the polycaprolactone afterwards, the polycaprolactone can support the three-dimensional structure's of sodium alginate fiber form until sodium alginate cross-linking in calcium chloride, soak repeatedly in chloroform and get rid of behind the polycaprolactone, the cross-linked alginate-aquogel nanofiber support can also keep intact, and not change three-dimensional structure's fiber form because of the getting rid of polycaprolactone, and this alginate-aquogel nanofiber support has better biocompatibility, can be applied to 3D cell culture.
(2) The alginate-hydrogel nanofiber scaffold optimizes the penetration depth of cells, particularly the nanofiber scaffold taking hydrogel as a raw material, so that research needing taking hydrogel as a scaffold material can be cultured in the scaffold closer to the real living conditions of the cells to obtain a more real and reliable research result.
(3) In the process of crosslinking sodium alginate and removing polycaprolactone, the regularity of the influence of the concentration of calcium ions in the calcium chloride solution on the stent can control the parameters of the stent, so that the controllability of the stent is ensured, and the corresponding alginate-hydrogel nanofiber stent meeting the requirements can be manufactured according to different requirements of experiments.
Drawings
Figure 1 is a TEM image of alginate-hydrogel nanofiber scaffolds in example 1.
FIG. 2 is a picture of alginate-hydrogel nanofiber scaffolds prepared in examples 1-3.
FIG. 3 is a photograph of cell migration under the tracer technique.
Fig. 4 is cell migration data for the alginate-hydrogel nanofiber scaffolds prepared in example 1.
Fig. 5 is cell migration data for the alginate-hydrogel nanofiber scaffolds prepared in example 2.
FIG. 6 shows the cell migration data in Gel 2.
Detailed Description
In order to better illustrate the present invention and facilitate the understanding of the technical solutions of the present invention, the present invention is further described in detail by the following examples. The following examples are merely illustrative of the present invention and do not represent or limit the scope of the claims, which are defined by the claims.
Example 1
A preparation method of an alginate-hydrogel nanofiber scaffold comprises the following steps:
1) mixing chloroform and methanol according to a volume ratio of 3:1, dissolving polycaprolactone in a chloroform/methanol mixed solution, and preparing a polycaprolactone solution with the polycaprolactone content of 16 wt%;
2) mixing distilled water and dimethyl sulfoxide according to a volume ratio of 19:1, dissolving sodium alginate and Triton X-100 in a mixed solution of distilled water and dimethyl sulfoxide, and preparing to obtain a sodium alginate solution, wherein the content of sodium alginate in the solution is 2 wt%, and the content of Triton X-100 in the solution is 0.5 wt%;
3) adopting a double-nozzle electrostatic spinning system, weighing 18g of polycaprolactone solution prepared in the step 1) and placing the polycaprolactone solution in an outer nozzle of the double-nozzle electrostatic spinning system, weighing 20g of sodium alginate solution prepared in the step 2) and adding the sodium alginate solution into an inner nozzle of the double-nozzle electrostatic spinning system, and carrying out electrostatic spinning to obtain a polycaprolactone-sodium alginate nanofiber membrane, wherein the spinning conditions are as follows: controlling the flow rate of the solution in the outer nozzle to be 0.5ml/h, the flow rate of the solution in the inner nozzle to be 2.5ml/h, the diameter of the collecting roller to be 100mm, the length to be 200mm, the rotating speed to be 100rpm, the distance between the nozzle and the roller to be 12cm, the spinning voltage to be 20kV, the temperature to be controlled to be 23-26 ℃, and the humidity to be 25-35%;
4) dissolving calcium chloride in an ethanol water solvent to obtain a calcium chloride solution, wherein the ethanol water solvent is a mixed solution of ethanol and distilled water in a volume ratio of 3:7, the content of the calcium chloride is 2 wt%, completely soaking the polycaprolactone-sodium alginate nanofiber membrane obtained in the step 3) in the prepared calcium chloride solution at room temperature for 6 hours, fully washing with the ethanol water solution, and freeze-drying;
5) soaking and washing the nanofiber membrane subjected to freeze drying in the step 4) with chloroform for 10 times, soaking for 1h each time, then sequentially washing with ethanol and distilled water until chloroform is completely removed, and freeze drying to obtain the alginate-hydrogel nanofiber scaffold.
An alginate-hydrogel nanofiber scaffold prepared by the preparation method.
The fiber diameter of the alginate-hydrogel nanofiber scaffold is 88 +/-47 nm.
As shown in fig. 1, fig. 1a is a scanning electron microscope picture of the polycaprolactone-sodium alginate nanofiber membrane soaked in the calcium chloride solution for 6 hours in the step 4), and fig. 1b is a scanning electron microscope picture of the alginate-hydrogel nanofiber membrane fully washed by the ethanol aqueous solution in the step 4), so that the alginate-hydrogel nanofiber membrane can keep good fiber shape after the polycaprolactone is stripped from the calcium chloride crosslinked alginate-hydrogel nanofiber membrane.
Example 2
A preparation method of an alginate-hydrogel nanofiber scaffold comprises the following steps:
1) mixing chloroform and methanol according to a volume ratio of 3:1, dissolving polycaprolactone in a chloroform/methanol mixed solution, and preparing a polycaprolactone solution with the polycaprolactone content of 16 wt%;
2) mixing distilled water and dimethyl sulfoxide according to a volume ratio of 19:1, dissolving sodium alginate and Triton X-100 in a mixed solution of distilled water and dimethyl sulfoxide, and preparing to obtain a sodium alginate solution, wherein the content of sodium alginate in the solution is 2 wt%, and the content of Triton X-100 in the solution is 0.5 wt%;
3) adopting a double-nozzle electrostatic spinning system, weighing 18g of polycaprolactone solution prepared in the step 1) and placing the polycaprolactone solution in an outer nozzle of the double-nozzle electrostatic spinning system, weighing 20g of sodium alginate solution prepared in the step 2) and adding the sodium alginate solution into an inner nozzle of the double-nozzle electrostatic spinning system, and carrying out electrostatic spinning to obtain a polycaprolactone-sodium alginate nanofiber membrane, wherein the spinning conditions are as follows: controlling the flow rate of the solution in the outer nozzle to be 0.5ml/h, the flow rate of the solution in the inner nozzle to be 2.5ml/h, the diameter of the collecting roller to be 100mm, the length to be 200mm, the rotating speed to be 100rpm, the distance between the nozzle and the roller to be 12cm, the spinning voltage to be 20kV, the temperature to be controlled to be 23-26 ℃, and the humidity to be 25-35%;
4) dissolving calcium chloride in an ethanol water solvent to obtain a calcium chloride solution, wherein the ethanol water solvent is a mixed solution of ethanol and distilled water in a volume ratio of 3:7, the content of the calcium chloride is 0.02 wt%, completely soaking the polycaprolactone-sodium alginate nanofiber membrane obtained in the step 3) in the prepared calcium chloride solution, soaking for 6 hours at room temperature, fully washing with the ethanol water solution, and then freeze-drying;
5) soaking and washing the nanofiber membrane subjected to freeze drying in the step 4) with chloroform for 10 times, soaking for 1h each time, then sequentially washing with ethanol and distilled water until chloroform is completely removed, and freeze drying to obtain the alginate-hydrogel nanofiber scaffold.
An alginate-hydrogel nanofiber scaffold prepared by the preparation method.
The fiber diameter of the alginate-hydrogel nanofiber scaffold is 88 +/-47 nm.
Example 3
A preparation method of an alginate-hydrogel nanofiber scaffold comprises the following steps:
1) mixing chloroform and methanol according to a volume ratio of 3:1, dissolving polycaprolactone in a chloroform/methanol mixed solution, and preparing a polycaprolactone solution with the polycaprolactone content of 16 wt%;
2) mixing distilled water and dimethyl sulfoxide according to a volume ratio of 19:1, dissolving sodium alginate and Triton X-100 in a mixed solution of distilled water and dimethyl sulfoxide, and preparing to obtain a sodium alginate solution, wherein the content of sodium alginate in the solution is 2 wt%, and the content of Triton X-100 in the solution is 0.5 wt%;
3) adopting a double-nozzle electrostatic spinning system, weighing 18g of polycaprolactone solution prepared in the step 1) and placing the polycaprolactone solution in an outer nozzle of the double-nozzle electrostatic spinning system, weighing 20g of sodium alginate solution prepared in the step 2) and adding the sodium alginate solution into an inner nozzle of the double-nozzle electrostatic spinning system, and carrying out electrostatic spinning to obtain a polycaprolactone-sodium alginate nanofiber membrane, wherein the spinning conditions are as follows: controlling the flow rate of the solution in the outer nozzle to be 0.5ml/h, the flow rate of the solution in the inner nozzle to be 2.5ml/h, the diameter of the collecting roller to be 100mm, the length to be 200mm, the rotating speed to be 100rpm, the distance between the nozzle and the roller to be 12cm, the spinning voltage to be 20kV, the temperature to be controlled to be 23-26 ℃, and the humidity to be 25-35%;
4) dissolving calcium chloride in an ethanol water solvent to obtain a calcium chloride solution, wherein the ethanol water solvent is a mixed solution of ethanol and distilled water in a volume ratio of 3:7, the content of the calcium chloride is 0.006 wt%, completely soaking the polycaprolactone-sodium alginate nanofiber membrane obtained in the step 3) in the prepared calcium chloride solution, soaking at room temperature for 6 hours, fully washing with the ethanol water solution, and freeze-drying;
5) soaking and washing the nanofiber membrane subjected to freeze drying in the step 4) with chloroform for 10 times, soaking for 1h each time, then sequentially washing with ethanol and distilled water until chloroform is completely removed, and freeze drying to obtain the alginate-hydrogel nanofiber scaffold.
An alginate-hydrogel nanofiber scaffold prepared by the preparation method.
The fiber diameter of the alginate-hydrogel nanofiber scaffold is 88 +/-47 nm.
As shown in fig. 2, fig. 2a is a scanning electron microscope picture of the polycaprolactone-sodium alginate nanofiber membrane obtained after electrostatic spinning, fig. 2b, fig. 2c, and fig. 2d are scanning electron microscope pictures of the alginate-hydrogel nanofiber scaffolds prepared in examples 1 to 3, respectively, it can be known that after the polycaprolactone is peeled off from the alginate-hydrogel nanofiber scaffold crosslinked by calcium chloride, the nanofiber scaffold maintains a good fiber form, and the fiber density in the alginate-hydrogel nanofiber scaffold gradually decreases as the concentration of calcium chloride gradually decreases. Due to the wrapping of polycaprolactone, the alginate-hydrogel nanofiber scaffold maintains the shape of fibers, however, after washing, the fibers in the alginate-hydrogel nanofiber scaffold after crosslinking under the condition of medium and low concentration of calcium ions have low mechanical properties, most of the fibers are washed away, and only the fibers with better mechanical properties are reserved.
Example 4
A preparation method of an alginate-hydrogel nanofiber scaffold comprises the following steps:
1) mixing chloroform and methanol according to a volume ratio of 3:1, dissolving polycaprolactone in a chloroform/methanol mixed solution, and preparing a polycaprolactone solution with the polycaprolactone content of 16 wt%;
2) mixing distilled water and dimethyl sulfoxide according to a volume ratio of 19:1, dissolving sodium alginate and Triton X-100 in a mixed solution of distilled water and dimethyl sulfoxide, and preparing to obtain a sodium alginate solution, wherein the content of sodium alginate in the solution is 2 wt%, and the content of Triton X-100 in the solution is 0.5 wt%;
3) adopting a double-nozzle electrostatic spinning system, weighing 18g of polycaprolactone solution prepared in the step 1) and placing the polycaprolactone solution in an outer nozzle of the double-nozzle electrostatic spinning system, weighing 20g of sodium alginate solution prepared in the step 2) and adding the sodium alginate solution into an inner nozzle of the double-nozzle electrostatic spinning system, and carrying out electrostatic spinning to obtain a polycaprolactone-sodium alginate nanofiber membrane, wherein the spinning conditions are as follows: controlling the flow rate of the solution in the outer nozzle to be 0.5ml/h, the flow rate of the solution in the inner nozzle to be 2.5ml/h, the diameter of the collecting roller to be 100mm, the length to be 200mm, the rotating speed to be 100rpm, the distance between the nozzle and the roller to be 12cm, the spinning voltage to be 20kV, the temperature to be controlled to be 23-26 ℃, and the humidity to be 25-35%;
4) dissolving calcium chloride in an ethanol water solvent to obtain a calcium chloride solution, wherein the ethanol water solvent is a mixed solution of ethanol and distilled water in a volume ratio of 3:7, the content of the calcium chloride is 0.002 wt%, completely soaking the polycaprolactone-sodium alginate nanofiber membrane obtained in the step 3) in the prepared calcium chloride solution, soaking for 6 hours at room temperature, fully washing with the ethanol water solution, and then freeze-drying;
5) soaking and washing the nanofiber membrane subjected to freeze drying in the step 4) with chloroform for 10 times, soaking for 1h each time, then sequentially washing with ethanol and distilled water until chloroform is completely removed, and freeze drying to obtain the alginate-hydrogel nanofiber scaffold.
An alginate-hydrogel nanofiber scaffold prepared by the preparation method.
The fiber diameter of the alginate-hydrogel nanofiber scaffold is 88 +/-47 nm.
Example 5
A preparation method of an alginate-hydrogel nanofiber scaffold comprises the following steps:
1) mixing chloroform and methanol according to a volume ratio of 3:1, dissolving polycaprolactone in a chloroform/methanol mixed solution, and preparing a polycaprolactone solution with the polycaprolactone content of 16 wt%;
2) mixing distilled water and dimethyl sulfoxide according to a volume ratio of 19:1, dissolving sodium alginate and Triton X-100 in a mixed solution of distilled water and dimethyl sulfoxide, and preparing to obtain a sodium alginate solution, wherein the content of sodium alginate in the solution is 2 wt%, and the content of Triton X-100 in the solution is 0.5 wt%;
3) adopting a double-nozzle electrostatic spinning system, weighing 18g of polycaprolactone solution prepared in the step 1) and placing the polycaprolactone solution in an outer nozzle of the double-nozzle electrostatic spinning system, weighing 20g of sodium alginate solution prepared in the step 2) and adding the sodium alginate solution into an inner nozzle of the double-nozzle electrostatic spinning system, and carrying out electrostatic spinning to obtain a polycaprolactone-sodium alginate nanofiber membrane, wherein the spinning conditions are as follows: controlling the flow rate of the solution in the outer nozzle to be 0.5ml/h, the flow rate of the solution in the inner nozzle to be 2.5ml/h, the diameter of the collecting roller to be 100mm, the length to be 200mm, the rotating speed to be 100rpm, the distance between the nozzle and the roller to be 12cm, the spinning voltage to be 20kV, the temperature to be controlled to be 23-26 ℃, and the humidity to be 25-35%;
4) dissolving calcium chloride in an ethanol water solvent to obtain a calcium chloride solution, wherein the ethanol water solvent is a mixed solution of ethanol and distilled water in a volume ratio of 3:7, the content of the calcium chloride is 1.00 wt%, completely soaking the polycaprolactone-sodium alginate nanofiber membrane obtained in the step 3) in the prepared calcium chloride solution, soaking for 6 hours at room temperature, fully washing with the ethanol water solution, and then freeze-drying;
5) soaking and washing the nanofiber membrane subjected to freeze drying in the step 4) with chloroform for 10 times, soaking for 1h each time, then sequentially washing with ethanol and distilled water until chloroform is completely removed, and freeze drying to obtain the alginate-hydrogel nanofiber scaffold.
An alginate-hydrogel nanofiber scaffold prepared by the preparation method.
The fiber diameter of the alginate-hydrogel nanofiber scaffold is 88 +/-47 nm.
To determine the effect of calcium ion concentration on cell penetration depth, cells were fluorescently labeled in the alginate-hydrogel nanofiber scaffolds prepared in examples 1 and 2, and then the length of the cells vertically crossing the alginate-hydrogel nanofiber scaffold was measured under a microscope. The specific operation is as follows: placing 3mg alginate-hydrogel nanofiber scaffold in a container containing 0.1mL 50 μ g mL in an environment at 4 deg.C-1The protein solution was in a round PDMS (polydimethylsiloxane) grid with a depth of 2mm and a diameter of 8 mm. After 12h, the reaction solution is obtainedThe well plate filled with alginate-hydrogel nanofiber scaffold was used for cell culture, while a set of control was set, a substance (Gel2) was generated by crosslinking 100 microliters of 2% mass fraction sodium alginate Gel with 100 microliters of calcium chloride. 1 x 10 of5NIH3T3 cells were seeded into the zonulin-coated culture plate, and then placed in DMEM medium containing 10% FBS for 12 h. Then, a 25 μm deep red cell tracking solution was added to the medium to stain the cells with fluorescence. The cell-covered culture plate was inverted and placed on top of the alginate-hydrogel nanofiber scaffold. Migration of fluorescently labeled cells was monitored by confocal laser scanning microscopy every 3h for the first 12h and every 24h for the next 5 days. And (3) exciting a 633nm laser, an emission filter and a 640-700 nm band-pass filter by using a helium-neon laser to vertically scan the alginate-hydrogel nanofiber support respectively. As shown in fig. 3-6, it is observed that the alginate-hydrogel nanofiber scaffold prepared by the method of the present invention has good penetration depth, and especially the alginate-hydrogel nanofiber scaffold crosslinked at high calcium ion concentration shows very superior cell penetration ability, and the comparison with the cell migration in Gel2 can be used to draw this conclusion very intuitively. And the permeability of the alginate-hydrogel nanofiber scaffold treated under different calcium concentrations is different, and because the concentration of calcium ions in a certain range directly influences the crosslinking degree of the alginate-hydrogel nanofiber scaffold, the permeability and the permeability speed are in direct proportion to the calcium ion concentration in the certain range.
It should be understood by those skilled in the art that equivalent substitutions of raw materials and additions of auxiliary components, selection of specific modes, or combination of specific features described in the above embodiments may be made without departing from the principle of the present invention, and the equivalents, the additions of auxiliary components, the combination of specific features, and the like are also considered to be within the scope of the present invention.
Claims (6)
1. The preparation method of the alginate-hydrogel nanofiber scaffold is characterized by comprising the following steps of:
1) dissolving polycaprolactone in chloroform/methanol solvent to obtain polycaprolactone solution; the volume ratio of chloroform to methanol is 3:1, and the content of polycaprolactone in the solution is 16 wt%;
2) dissolving sodium alginate and Triton X-100 in a solvent to prepare a sodium alginate solution; the solvent is a mixture of distilled water and dimethyl sulfoxide, and the volume ratio of the distilled water to the dimethyl sulfoxide is 19: 1; the content of the sodium alginate in the solution is 2 wt%, and the content of the Triton X-100 in the solution is 0.5 wt%;
3) adopting a double-nozzle electrostatic spinning system, weighing 18g of the polycaprolactone solution prepared in the step 1) and placing the polycaprolactone solution in an outer nozzle of the double-nozzle electrostatic spinning system, weighing 20g of the sodium alginate solution prepared in the step 2) and adding the sodium alginate solution into an inner nozzle of the double-nozzle electrostatic spinning system, and carrying out electrostatic spinning to obtain a polycaprolactone-sodium alginate nanofiber membrane; controlling the flow rate of the solution in the outer nozzle to be 0.5mL/h and the flow rate of the solution in the inner nozzle to be 2.5 mL/h;
4) dissolving calcium chloride in an ethanol water solvent to obtain a calcium chloride solution, completely soaking the polycaprolactone-sodium alginate nanofiber membrane obtained in the step 3) in the prepared calcium chloride solution, soaking at room temperature for 6 hours, fully washing with the ethanol water solution, and freeze-drying; the ethanol water solvent is a mixed solution of ethanol and distilled water in a volume ratio of 3:7, and the mass fraction of calcium chloride is 0.002-2%;
5) soaking and washing the nanofiber membrane subjected to freeze drying in the step 4) with chloroform, then sequentially washing with ethanol and distilled water until the chloroform is completely removed, and carrying out freeze drying to obtain the alginate-hydrogel nanofiber scaffold.
2. The method for preparing the alginate-hydrogel nanofiber scaffold as claimed in claim 1, wherein the spinning conditions in the step 3) are that the diameter of the collecting roller is 100mm, the length of the collecting roller is 200mm, the rotating speed is 100rpm, the distance between the nozzle and the roller is 12cm, and the spinning voltage is 20 kV.
3. The method for preparing the alginate-hydrogel nanofiber scaffold according to claim 1, wherein in the step 3), the temperature is controlled to be 23-26 ℃ and the humidity is controlled to be 25-35% in the electrospinning process.
4. The method for preparing the alginate-hydrogel nanofiber scaffold according to claim 1, wherein in the step 5), the nanofiber membrane freeze-dried in the step 4) is soaked and washed with chloroform for 10 times, and each soaking time is 1 h.
5. An alginate-hydrogel nanofiber scaffold prepared by the method for preparing an alginate-hydrogel nanofiber scaffold as claimed in any one of claims 1 to 4.
6. The alginate-hydrogel nanofiber scaffold of claim 5, wherein the fiber diameter of said alginate-hydrogel nanofiber scaffold is 88 ± 47 nm.
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