CN114525629B

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Publication number: CN114525629B
Application number: CN202210163894.0A
Authority: CN
Inventors: 魏强; 孙骞
Original assignee: Chengdu Weiwo Technology Co ltd
Current assignee: Hunan Meibai Biomedical Co ltd
Priority date: 2022-02-22
Filing date: 2022-02-22
Publication date: 2023-05-12
Anticipated expiration: 2042-02-22
Also published as: CN114525629A

Abstract

The invention discloses a periodic cycle low-temperature preparation method and application of an electrostatic spinning film containing curled nanofibers, wherein the method comprises the following steps: dissolving raw materials to obtain an electrostatic spinning prefabricated solution; precooling the electrostatic spinning solution, which is different from the room temperature and higher temperature range set by the existing electrostatic spinning technology, stabilizing an electric field by utilizing an aluminum foil sheet under the control of periodic low temperature, and adjusting parameters of an electrostatic spinning machine, so that curled nanofibers can be obtained and can be prepared into films; the nanofiber membrane prepared at low temperature in the periodic cycle is used for cell culture after being subjected to sterilization treatment, and can effectively promote cell adhesion and induce stem cell osteogenic differentiation; the preparation method of the curled nanofiber prepared at low temperature in a cyclic cycle is efficient and simple, the preparation process is good in stability, and the material is good in biocompatibility.

Description

Periodic cycle low-temperature preparation method and application of electrostatic spinning film containing curled nanofibers

Technical Field

The invention relates to the technical field of medical biological electrostatic spinning materials, in particular to a cyclic low-temperature preparation method and application of an electrostatic spinning film containing curled nanofibers.

Background

The electrospinning technology is one of the most commonly used methods for preparing nanofibers, and has been widely used in various fields of medicine, biology, environment, energy, etc. The traditional electrostatic spinning technology is limited by a preparation method, can only be used for manufacturing nanofibers in a straightened state under the double effects of a strong electric field and a high-speed roller, and is difficult to manufacture nanofibers with a bending form, so that popularization and application of the electrostatic spinning nanofiber technology are hindered.

At present, few reports on the electrostatic spinning preparation effect are made by independently controlling temperature parameters, and few researches are based on the purpose of reducing the viscosity of an electrostatic spinning solution to improve the processing temperature. The low-temperature environment increases the solution viscosity to a certain extent, so that the electrostatic spinning processing is prevented from subconsciously excluding the low-temperature control from the conventional electrostatic spinning preparation method, and the development of related researches is influenced.

The topology of the extracellular matrix has an important influence on the cell behavior, and the microscopic topology of the extracellular matrix is complex and diverse so as to adapt and regulate different physiological microenvironments. For example, collagen fibers of the tenocyte extracellular matrix have a crimp structure to promote nonlinear hardening of the tissue under tension, and fibrous membranes having a crimped morphology are capable of absorbing more tension than fibrous membranes in a straightened state, thereby cushioning the mechanical load created by the attached muscle or bone. Furthermore, the spring-like behavior of the coiled structure may protect the muscle from tearing when contracting. It has been found that cell edge bending behavior can promote accumulation of mechanical strength of cells and promote osteogenic differentiation of cells. The traditional electrospun nanofiber surface with only linear type is difficult to adapt to the complex life activity requirement. Therefore, changing the topological structure limitation of the electrostatic spinning film, the preparation of nanofiber materials with curled morphology is the focus of current research.

Disclosure of Invention

Accordingly, it is an object of the present invention to provide a cyclic low temperature preparation method of an electrospun film containing crimped nanofibers; the second object of the invention is to provide the application of the electrostatic spinning film of the nanofiber prepared by the method in promoting cell adhesion; the invention also aims to provide the application of the electrostatic spinning membrane of the nanofiber prepared by the method in promoting the osteogenic differentiation of stem cells.

In order to achieve the above purpose, the present invention provides the following technical solutions:

1. the low-temperature preparation method of the periodic cycle of the electrostatic spinning film containing the curled nanofiber comprises the following steps:

(1) Weighing raw materials, adding a solvent, and stirring to completely dissolve the raw materials to prepare an electrostatic spinning prefabricated solution;

(2) Carrying out low-temperature pretreatment on the electrostatic spinning prefabricated solution prepared in the step 1 to prepare an electrostatic spinning working solution;

(3) And (3) pouring the electrostatic spinning working solution prepared in the step (2) into an electrostatic spinning needle tube, assembling the electrostatic spinning needle tube onto an electrostatic spinning machine, maintaining the cyclic low temperature, installing an anode and a cathode, and adjusting electrostatic spinning parameters to prepare the electrostatic spinning nanofiber membrane.

In the preferred embodiment of the present invention, in the step (1), the raw materials include, but are not limited to, one or more of polycaprolactone, polyvinyl alcohol, polyvinylpyrrolidone, polylactic acid, polyethersulfone, chitosan and gelatin, and the molecular weight of the raw materials is 5000-1000000.

In the step (2), the temperature of the low-temperature pretreatment is between-40 and 10 ℃ and the time of the low-temperature pretreatment is between 30 and 60 minutes.

In the step (3), the temperature of the cyclic low temperature is-10 ℃ and the cyclic interval is 0-120 min.

2. The electrostatic spinning film containing the curled nanofiber, which is prepared by the method, is applied to promotion of cell adhesion.

Preferably, the cells are adipose-derived mesenchymal stem cells hASCs, or bone marrow mesenchymal stem cells BMSCs, or mouse embryonic fibroblasts NIH3T3, or mouse fibroblasts L929, or human umbilical vein endothelial cells HUVEC.

3. The electrostatic spinning membrane containing the curled nanofibers, which is prepared by the method, is applied to promoting the osteogenic differentiation of stem cells.

Preferably, the stem cells are adipose-derived mesenchymal stem cells hASCs, or/and bone marrow mesenchymal stem cells BMSCs.

The preferred fiber curvature of the electrospun film containing crimped nanofibers is 0.15-0.5 μm^-1 。

Preferably, the electrostatic spinning film is subjected to aseptic treatment before use.

The invention has the beneficial effects that: the invention discloses a low-temperature cyclic preparation method of an electrostatic spinning film containing curled nanofibers, which comprises the steps of preparing an electrostatic spinning solution by using a solvent to dissolve raw materials, adopting low-temperature pre-cooling the electrostatic spinning solution, accurately controlling the cyclic temperature of a spinning environment, and preparing an electrostatic spinning film material containing curled nanofibers by adjusting electrostatic spinning parameters.

The invention provides a method for controlling electrostatic spinning at a low temperature in a cyclic cycle for the first time, which not only avoids the obstruction of solution viscosity increase to spinning, but also searches for the influence of low-temperature environment on electrostatic spinning, prepares the electrostatic spinning film with a curled structure, and makes up the technical blank in the field.

Compared with the existing electrostatic spinning technology, the invention makes up the defect that the existing electrostatic spinning equipment can only prepare linear materials, greatly widens the application range of the electrostatic spinning technology, enriches the types of biomedical materials simulating extracellular matrixes, and can be used for exploring the influence of material interfaces on cell behaviors. The nanofiber material prepared by the invention has good biocompatibility, chemical stability and degradability, low cost and simple and adjustable process, and has good research and application prospects as a novel biomedical material.

Drawings

In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention provides the following drawings for description:

FIG. 1 is a schematic diagram of a loop type cyclic low temperature electrostatic spinning device;

FIG. 2 is a scanning electron microscope image of an electrospun film (stress: surface morphology of a conventional straightened state nanofiber film; curved: surface morphology of a crimped state nanofiber film);

FIG. 3 is a graph of fiber curvature statistics (stress: conventional straightened state nanofiber curvature statistics; curved: curled state nanofiber curvature statistics);

FIG. 4 is a graph showing the results of 7-day osteogenic differentiation of mesenchymal stem cells measured with alkaline phosphatase (ALP) (A: the result of cell differentiation induced by a conventional straightened state nanofiber membrane; B: the result of cell differentiation induced by a curled state nanofiber membrane);

FIG. 5 cell adhesion spread graph (A: conventional straightened state cell spread; B: coiled state cell spread);

FIG. 6 cell edge curvature statistics (stress: cell edge curvature statistics for conventional straightened state nanofiber membrane adhesion; curved: cell edge curvature statistics for crimped state nanofiber membrane adhesion);

FIG. 7 cell vertex count (stress: cell vertex count for conventional straightened state nanofiber membrane adhesion; curved: cell vertex count for crimped state nanofiber membrane adhesion);

FIG. 8 shows alkaline phosphatase (ALP) expression levels seven days after culturing mesenchymal stem cells with electrospun membranes at different cycling temperatures;

FIG. 9 shows the alkaline phosphatase (ALP) expression levels seven days after the mesenchymal stem cells were cultured in the electrospun films obtained in different cycle periods.

Reference numerals illustrate:

1-a spinning solution storage injection tube; 2-electrostatic spinning solution; 3-stabilizing an electric field aluminum foil layer; 4-positive electrode clamps; 5-spinning a needle; 6-electrospinning the film; 7-receiving roller; 8-a base; 9-a negative electrode; 10-a temperature controller.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to limit the invention, so that those skilled in the art may better understand the invention and practice it.

The loop type periodic cycle low-temperature electrostatic spinning device adopted by the embodiment of the invention is shown in figure 1, and comprises the following components: a spinning solution storage injection tube 1; an electrostatic spinning solution 2; a stable electric fieldaluminum foil layer 3; apositive electrode clip 4; aspinning needle 5; an electrospun film 6; areceiving roller 7; a base 8; anegative electrode 9; atemperature controller 10.

Example 1 periodic cyclic Low temperature preparation of electrospun films containing crimped nanofibers

The periodic low-temperature preparation method of the electrostatic spinning film containing the curled nanofiber is realized by the following steps:

(1) Weighing polycaprolactone or other polymer materials, adding the weighed polycaprolactone or other polymer materials into solvents such as hexafluoroisopropanol and the like, and stirring for 3-24 hours to completely dissolve the polycaprolactone or other polymer materials to obtain an electrostatic spinning prefabricated solution; other polymer materials include but are not limited to polycaprolactone, polyvinyl alcohol, polyvinylpyrrolidone, polylactic acid, polyethersulfone, chitosan, gelatin and the like, and the molecular weight is between 5000 and 1000000.

(2) Pre-cooling the electrostatic spinning prefabricated solution in a refrigerator at the temperature of minus 20 ℃ for 30-60 min to obtain electrostatic spinning working solution;

(3) Adding the precooled electrostatic spinning working solution into an electrostatic spinning needle tube, fixing the electrostatic spinning needle tube on an electrostatic spinning machine, accurately controlling the ambient temperature (-10 ℃) through cyclic low temperature, installing an anode and a cathode, and adjusting electrostatic spinning parameters as follows: the voltage is 10 KV to 30KV, the injection speed is 0.008 mL/min to 0.1mL/min, the rotation speed of a receiving roller is 100 rpm to 1000rpm, the needle head is 21 # to 28 # and the distance from the positive needle head to the receiving roller is 5 cm to 30cm, the spinning time is 1 h to 10h, the curled nanofiber can be obtained, the thickness of the electrostatic spinning film is 1 mu m to 5000 mu m, and the fiber diameters are 50 nm to 900 nm; the curvature of the fiber is 0.001-200 mu m^-1 Between them.

The coiled nanofiber membrane prepared by adopting a cyclic low-temperature method is subjected to sterilization treatment by 75% alcohol, is washed for 5 times by using sterile PBS, is inoculated with mesenchymal stem cells (hASC, BMSC), or fibroblasts (NIH 3T3, L929), or endothelial cells (HUVEC), is bent to have a morphology which can promote cell adhesion, induces osteogenic differentiation of the mesenchymal stem cells (hASC, BMSC), and detects the results related to analysis.

As shown in fig. 2, the surface morphology of the nanofiber membrane prepared at the temperature of 10-40 ℃ in the prior art is in a straightened state (stress) as shown in a scanning electron microscope image of an electrostatic spinning membrane, while the surface morphology of the nanofiber membrane prepared at the low temperature in the periodic cycle of the invention is in a curled state (curved);

as shown in FIG. 3, the curvature of the nanofiber in the conventional straightened state ranges from 0 to 0.16 μm as can be seen by counting the curvature of the fiber^-1 (stress); the curvature range of the nano fiber in the curled state prepared under the low temperature condition of the periodic cycle of the invention is 0.02-0.78 mu m^-1 ；

Alkaline phosphatase (ALP) is a marker enzyme that reflects catabolic levels in bone tissue and plays a key role in calcification. Calcium ions are deposited on collagen under the action of ALP to complete the matrix mineralization process, bone tissues are formed by calcification of bone matrix, and the bone matrix is synthesized and secreted by osteoblasts; ALP activity of osteoblasts is highest when the matrix starts calcification, and lowest when the calcification is near the end, and the activity reflects the differentiation degree and the functional state of osteoblasts to a certain extent. As shown in fig. 4, the mesenchymal stem cells were assayed for 7-day osteogenic differentiation by alkaline phosphatase (ALP), and it was found that the effect of inducing cell differentiation by the nanofiber membrane in a curled state was better than that by the conventional nanofiber membrane in a straightened state (fig. 4, a).

As shown in fig. 5, the cell adhesion spreading effect indicates that the cells adhere only to the straight fibers (fig. 5, a), but can span multiple fibers on the crimped fibers (fig. 5, b). It is generally believed that the cell adhesion is only sufficiently good that it can be forced to accomplish cross-adhesion, a necessary condition for the cell to develop its own function and behavior. Cells on straight fibers are usually confined to the framework of the straight fibers and more can only stretch against the edges of the fibers, effectively being spatially limited to some extent by the fibers. We therefore say that the cells spread better on the crimped fibre membrane.

As shown in FIG. 6, the cell edge curvature of the nanofiber membrane adhered to the conventional straightened state was 0.06-0.64 μm as shown by the cell edge curvature statistics^-1 (Straight) compared with the crimped nanofiber membrane, the cell edge curvature of the adhesion is 0.14-0.72 μm^-1 (cured) is larger;

as shown in FIG. 7, it is clear from the cell apex count statistics that the nanofiber membrane in a curled state is adhered to cell apexes 2-21 (curved) compared to cell apexes 0-11 (stress) adhered to the nanofiber membrane in a conventional straightened state.

It is apparent that the edge bending effect exhibited by cells on crimped fibers is not limited by the morphological spatial constraints of the crimped fibers, which are fashioned by the cells forming a cross-over type of adhesion on the different fibers. These dangling arcuate edges of the cells are key information on the effect of cell adhesion. It is found that the suspended circular arc structure formed by cells needs to maintain the self cytoskeleton, high-expression cytoskeletal proteins are gathered at the circular arcs, unique cell behaviors are formed, related signal paths are activated, cell adhesion is facilitated, and cell lineage differentiation is promoted. The increase in the number of cell vertices is related to the increase in cell arcs, wherein the cell edge curvature is defined to be 1 vertex area, a crossing arc-shaped structure is arranged between the two vertices, and on the other hand, the increase in the number of the vertices also means that the cell has stronger capability of actively sensing surrounding extracellular matrix and more adhesion sites. Whereas the edges of straight fibers typically exhibit spreading attached to the fibers, there is no need to aggregate more cytoskeleton in these areas to maintain the structure, unlike the above-described cell-to-fiber non-contact spanning arc structure, which does not form an aggregation of cytoskeletal proteins, which does not form a specific pathway that stimulates cell behavior and function.

Example 2 determination of optimal cycle temperature and time

In order to obtain the optimal crimping electrospinning parameters for inducing the osteogenic differentiation of cells, the temperature and time of the cycle were analyzed, respectively, and the optimal cycle temperature and cycle time were determined from the 7-day osteogenic differentiation results of mesenchymal stem cells measured by alkaline phosphatase (ALP). Other parameters were kept within the range of the optimal electrospinning preparation settings shown in example 1.

1. The cycle time was determined to be 10min and the cycle temperature range was set as shown in table 1:

TABLE 1

By culturing mesenchymal stem cells with electrospun membranes obtained by setting different circulation temperatures, and detecting after seven days, the expression level of alkaline phosphatase (ALP) can be determined to be 5 ℃ to-5 ℃ and 0 ℃ to-10 ℃ as shown in the following figure 8.

2. Maintaining the optimal circulation temperature range from 5 ℃ to-5 ℃ and from 0 ℃ to-10 ℃, and determining the circulation period range as shown in table 2:

TABLE 2

Grouping	Cycling temperature range (. Degree. C.)	Cycle period (min)
			1	5～-5	10、20、30、60
2	0～-10	10、20、30、60

Mesenchymal stem cells were cultured by setting the electrospun membranes obtained in different cycle periods, and after seven days, the measurement was performed, and the alkaline phosphatase (ALP) expression level was as shown in FIG. 9 below, and finally, the optimal cycle temperature and cycle time were determined as shown in FIG. 3:

TABLE 3 Table 3

Grouping	Cycling temperature range (. Degree. C.)	Cycle period (min)
			1	5～-5	20
2	0～-10	10

The curvature of the curled electrostatic spinning optimal fiber obtained by the two groups of optimal circulation temperature and circulation period is about 0.15 mu m^-1 ～0.5μm^-1 。

The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.

Claims

1. A cyclic low temperature preparation method of an electrostatic spinning film containing curled nanofibers, comprising the steps of:

(2) Carrying out low-temperature pretreatment on the electrostatic spinning prefabricated solution prepared in the step 1 to prepare an electrostatic spinning working solution, wherein the temperature of the low-temperature pretreatment is-20 ℃, and the time of the low-temperature pretreatment is 30-60 min;

(3) Filling the electrostatic spinning working solution prepared in the step 2 into an electrostatic spinning needle tube, assembling the electrostatic spinning needle tube onto an electrostatic spinning machine, maintaining the cyclic low temperature, mounting a positive electrode and a negative electrode, and adjusting electrostatic spinning parameters to prepare an electrostatic spinning nanofiber membrane; the cyclic low temperature is 0 ℃ to-10 ℃ and the cyclic period is 10min.

2. The method according to claim 1, wherein in the step (1), the raw material comprises one or more of polycaprolactone, polyvinyl alcohol, polyvinylpyrrolidone, polylactic acid, polyethersulfone, chitosan and gelatin, and the molecular weight of the raw material is 5000-1000000.

3. Use of an electrospun membrane comprising crimped nanofibers prepared according to any one of claims 1-2 to promote cell adhesion.

4. The use according to claim 3, wherein the cells are adipose-derived mesenchymal stem cells, hASCs, or bone marrow mesenchymal stem cells, BMSCs, or mouse embryonic fibroblasts, NIH3T3, or mouse fibroblasts, L929, or human umbilical vein endothelial cells, HUVECs.

5. Use of an electrospun membrane comprising crimped nanofibers prepared according to any one of claims 1-2 to promote osteoblast differentiation of stem cells.

6. The use according to claim 5, wherein the stem cells are adipose-derived mesenchymal stem cells, hASCs, or/and bone marrow mesenchymal stem cells, BMSCs.

7. The use according to claim 3 or 5, characterized in that the electrospun film comprising crimped nanofibers has an optimal fiber curvature of 0.15-0.5 μm^-1 。

8. Use according to claim 3 or 5, characterized in that the electrospun film is subjected to a sterile treatment before use.