CN113101413B - Ordered hydrogel fiber scaffold, preparation method and application thereof - Google Patents

Ordered hydrogel fiber scaffold, preparation method and application thereof Download PDF

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CN113101413B
CN113101413B CN202010030224.2A CN202010030224A CN113101413B CN 113101413 B CN113101413 B CN 113101413B CN 202010030224 A CN202010030224 A CN 202010030224A CN 113101413 B CN113101413 B CN 113101413B
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modified
collagen
fibrin
spinning solution
solution
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CN113101413A (en
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戴建武
李晓然
陈珍妮
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Suzhou Institute of Nano Tech and Nano Bionics of CAS
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Suzhou Institute of Nano Tech and Nano Bionics of CAS
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Abstract

The invention discloses an ordered hydrogel fiber scaffold, a preparation method and application thereof. The ordered hydrogel scaffold is prepared by modifying collagen and fibrin and combining the Michael addition reaction and the liquid electrostatic spinning technology, has the advantages of simple process, safety, no toxicity, no organic solvent discharge and residue, can better simulate the structure and components of a natural extracellular matrix, has high water content and orientation, can create a more appropriate microenvironment for tissue regeneration and damage function recovery after carrying substances such as bioactive molecules and the like, can induce directional differentiation of stem cells, and can be applied to nerve repair materials and products with spinal cord damage repair function.

Description

Ordered hydrogel fiber scaffold, preparation method and application thereof
Technical Field
The invention relates to a biomedical material, in particular to an ordered hydrogel fiber scaffold, a preparation method and application thereof, belonging to the technical field of bioengineering.
Background
Spinal cord injury is a central nervous disease in which cells are lost, dead, and nerve fibers are broken in spinal cord tissue caused by various pathogenic factors, functionally resulting in various degrees of motor, sensory, and neurological disorders, and even paralysis in severe cases.
In recent years, many researches are carried out on the treatment of spinal cord injury by using tissue engineering scaffold materials, and corresponding material preparation methods are various, wherein a more common method is realized by using an electrostatic spinning technology,
the electrostatic spinning technology is a simple way for obtaining the tissue engineering scaffold, and the obtained electrospun fiber has the characteristics of large specific surface area, controllable diameter, various topological structures and the like. However, most of the conventional electrospinning uses an organic agent as a solvent, which affects the properties of the natural polymer material during the dissolution process, and the organic agent volatilizes during the electrospinning process, and the organic agent remained in the scaffold material has a certain toxic effect on cells.
Therefore, the development of a safe and environment-friendly tissue engineering scaffold preparation process with good biocompatibility has become a problem to be solved in the field.
Disclosure of Invention
The invention mainly aims to provide an ordered hydrogel fiber scaffold, a preparation method and application thereof, so that the defects in the prior art are overcome.
In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:
the embodiment of the invention provides a preparation method of ordered hydrogel fibers, which comprises the following steps:
respectively modifying collagen and fibrin, and then respectively dissolving the modified collagen and the fibrin in a water phase system to form modified collagen spinning solution and modified fibrin spinning solution, wherein the modified collagen and the modified fibrin respectively have a first reaction group and a second reaction group, and a Michael addition reaction can be carried out between the first reaction group and the second reaction group;
and (2) converging the modified collagen spinning solution and the modified fibrin spinning solution into a spinning nozzle for outputting by adopting a liquid electrostatic spinning method, and combining the modified collagen and the modified fibrin through the Michael addition reaction to prepare and form the ordered hydrogel fiber.
The embodiment of the invention also provides a preparation method of the ordered hydrogel scaffold, which comprises the following steps:
respectively modifying collagen and fibrin, and then respectively dissolving the modified collagen and the fibrin in a water phase system to form modified collagen spinning solution and modified fibrin spinning solution, wherein the modified collagen and the modified fibrin respectively have a first reaction group and a second reaction group, and a Michael addition reaction can be carried out between the first reaction group and the second reaction group;
dissolving bioactive factors and/or medicinal compounds in at least one of the modified collagen spinning solution and the modified fibrin spinning solution, then respectively adding the modified collagen spinning solution and the modified fibrin spinning solution into one spinning solution channel by using a liquid electrostatic spinning method, converging the modified collagen spinning solution and the modified fibrin spinning solution into one spinning nozzle through the two spinning solution channels for outputting, and combining the modified collagen and the modified fibrin therein through the Michael addition reaction, thereby preparing the ordered hydrogel scaffold loaded with the bioactive factors and/or the medicinal compounds.
The embodiment of the invention also provides the ordered hydrogel fiber and the ordered hydrogel scaffold prepared by any one of the methods.
The embodiment of the invention also provides application of the ordered hydrogel fiber or the ordered hydrogel scaffold in cell culture.
The embodiment of the invention also provides application of the ordered hydrogel fiber or the ordered hydrogel scaffold in preparation of a product with a spinal cord injury repair function.
Compared with the prior art, the ordered hydrogel fiber is prepared by modifying collagen and fibrin and then combining the Michael addition reaction and the liquid electrostatic spinning technology, has the advantages of simple process, safety, environmental protection and no organic solvent discharge and residue, can better simulate the structure and components of natural extracellular matrix, has high water content and orientation, and further can create a more appropriate microenvironment for tissue regeneration and damage function recovery and induce directional differentiation of stem cells after a corresponding ordered hydrogel support carries bioactive molecules, so that the ordered hydrogel fiber can be applied to nerve repair materials and can be applied to repair spinal cord injury.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic view of a liquid electrostatic spinning apparatus used in an exemplary embodiment of the present invention;
FIGS. 2A-2D illustrate fiber bundle macro-topography at receive times of 30s, 90s, 5min, and 10min, respectively, in an embodiment of the invention;
FIGS. 3 a-3 b, 3 c-3 d show SEM images of fiber bundles at a turntable speed of 160rpm/min and 200rpm/min, respectively, in accordance with an embodiment of the present invention;
FIG. 4 shows Tuj-1 after culturing for 7d in one embodiment of the present invention+And Olig-2+Fluorescent microscope photograph (scale: 50 μm);
FIG. 5 shows Tuj-1 after culturing for 7 days in one embodiment of the present invention+And GFAP+The fluorescence microscopic photograph of (1) shows (on the scale of 50 μm).
Detailed Description
In view of the deficiencies in the prior art, the inventors of the present invention have made extensive studies and extensive practices to provide technical solutions of the present invention. The technical solution, its implementation and principles, etc. will be further explained as follows.
One aspect of the embodiments of the present invention provides a method for preparing an ordered hydrogel fiber, including:
respectively modifying collagen and fibrin, and then respectively dissolving the modified collagen and the fibrin in a water phase system to form modified collagen spinning solution and modified fibrin spinning solution, wherein the modified collagen and the modified fibrin respectively have a first reaction group and a second reaction group, and a Michael addition reaction can be carried out between the first reaction group and the second reaction group;
and (2) converging the modified collagen spinning solution and the modified fibrin spinning solution into a spinning nozzle for outputting by adopting a liquid electrostatic spinning method, and combining the modified collagen and the modified fibrin through the Michael addition reaction to prepare and form the ordered hydrogel fiber.
In some embodiments, the preparation method specifically comprises: dissolving collagen in 0.3-1.0mol/L acetic acid aqueous solution to form collagen solution with concentration of 3-10mg/ml, adjusting pH value of the obtained collagen solution to 5.5-6, adding 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) and N-hydroxysuccinimide (NHS) with mass ratio of 2:1-4:1, adding N-2-aminoethylmaleimide (Mal) with molar ratio of collagen and N-2-aminoethylmaleimide of 1:5-1:1, stirring overnight at room temperature, and post-treating to obtain modified collagen.
In some embodiments, the preparation method specifically comprises: dissolving fibrinogen (Cys) in 5-8mol/L urea-containing phosphate buffered saline, and adding tris (dicarbonylethyl) phosphate (TCEP) so that the molar ratio of fibrin to tris (dicarbonylethyl) phosphate is 1:20-1: and 10, carrying out light-resistant reaction at room temperature, and carrying out post-treatment to obtain the modified fibrin.
In some embodiments, the preparation method specifically comprises: dissolving the modified collagen in deionized water to form the modified collagen spinning solution with the concentration of 1.5-2 wt%.
In some embodiments, the preparation method specifically comprises: dissolving modified fibrin in physiological saline containing 50-100mg/ml urea to form modified fibrin spinning solution with the concentration of 2-4 wt%.
In some embodiments, the preparation method specifically comprises: and respectively adding the modified collagen spinning solution and the modified fibrin spinning solution into a spinning solution channel, converging the spinning solution into a spinning nozzle through the two spinning solution channels, outputting the spinning nozzle, and preparing the ordered hydrogel fiber by using a liquid electrostatic spinning method.
Preferably, the liquid electrospinning method adopts process conditions including: the flow velocity of the modified collagen spinning solution and the modified fibrin spinning solution in the spinning solution channel is 0.6-1.0ml/h, the voltage is set to be 3-4kV, the rotating speed of the liquid receiving device is 100-plus-material 200rpm/min, and the receiving distance is set to be that the spinning nozzle is contacted with the receiving solution.
The diameter, the length and the like of the ordered hydrogel fiber can be regulated and controlled by adjusting the flow speed and the voltage of each spinning solution, the rotating speed of a liquid receiving device and the like.
Another aspect of an embodiment of the present invention provides a method for preparing an ordered hydrogel scaffold, including:
respectively modifying collagen and fibrin, and then respectively dissolving the modified collagen and the fibrin in a water phase system to form modified collagen spinning solution and modified fibrin spinning solution, wherein the modified collagen and the modified fibrin respectively have a first reaction group and a second reaction group, and a Michael addition reaction can be carried out between the first reaction group and the second reaction group;
dissolving bioactive factors and/or medicinal compounds in at least one of the modified collagen spinning solution and the modified fibrin spinning solution, then respectively adding the modified collagen spinning solution and the modified fibrin spinning solution into one spinning solution channel by using a liquid electrostatic spinning method, converging the modified collagen spinning solution and the modified fibrin spinning solution into one spinning nozzle through the two spinning solution channels for outputting, and combining the modified collagen and the modified fibrin therein through the Michael addition reaction, thereby preparing the ordered hydrogel scaffold loaded with the bioactive factors and/or the medicinal compounds.
In some embodiments, the preparation method specifically comprises: dissolving collagen in 0.3-1.0mol/L acetic acid water solution to form collagen solution with concentration of 3-10mg/ml, adjusting pH of the obtained collagen solution to 5.5-6, adding 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in a mass ratio of 2:1-4:1, adding N-2-aminoethylmaleimide (Mal), the mol ratio of the collagen to the N-2-aminoethylmaleimide is 1:5-1:1, the mixture is stirred at room temperature overnight, and then modified collagen is obtained after post-treatment, and then dissolving the modified collagen in deionized water to form the modified collagen spinning solution with the concentration of 1.5-2 wt%.
In some embodiments, the preparation method specifically comprises: dissolving fibrinogen (Cys) in phosphate buffer solution containing 5-8mol/L urea, adding tris (dicarbonylethyl) phosphate (TCEP) at a molar ratio of fibrin to tris (dicarbonylethyl) phosphate of 1:20-1:10, carrying out dark reaction at room temperature, carrying out post-treatment to obtain modified fibrin, and dissolving the modified fibrin in physiological saline containing 50-100mg/ml urea to form modified fibrin spinning solution with the concentration of 2-4 wt%.
Preferably, the liquid electrospinning method adopts process conditions including: the flow velocity of the modified collagen spinning solution and the modified fibrin spinning solution in the spinning solution channel is 0.6-1.0ml/h, the voltage is set to be 3-4kV, the rotating speed of the liquid receiving device is 100-plus-material 200rpm/min, and the receiving distance is set to be that the spinning nozzle is contacted with the receiving solution. Preferably, the spinning time used therein is within 10 min.
Preferably, the receiving liquid in the liquid receiving device is a solution containing 2-10mg/ml EDC and 1.2-5mg/ml NHS.
The diameter, the length, the structure and the like of the ordered hydrogel stent can be regulated and controlled by adjusting the flow rate and the voltage of each spinning solution, the rotating speed of the liquid receiving device and the like.
In some embodiments, the bioactive factor (0-7ng/ml) and/or the pharmaceutical compound (0-50ng/ml) are homogeneously mixed in the water-soluble spinning solution.
In some embodiments, the biologically active factor includes, but is not limited to, neurotrophic factor 3(Neurotrophin-3, NT-3).
In some embodiments, the pharmaceutical compound includes, but is not limited to, Paclitaxel (PTX).
Another aspect of an embodiment of the present invention provides an ordered hydrogel fiber made by any of the methods described above.
Another aspect of an embodiment of the present invention provides an ordered hydrogel scaffold prepared by any of the methods described above.
Further, the ordered hydrogel scaffold may be formed by aggregation of a plurality of ordered hydrogel fibers, and may be, for example, a bundle of ordered hydrogel fibers.
In some embodiments, the ordered hydrogel scaffold may further comprise ordered hydrogel fibers (defined as first fibers) carrying bioactive factors and ordered hydrogel fibers (defined as second fibers) carrying pharmaceutical compounds, and the first fibers and the second fibers may be distributed in different regions of the ordered hydrogel scaffold, for example, may be distributed at the top end and the bottom end of the ordered hydrogel scaffold, respectively, or may be distributed at the inner periphery, the outer periphery, and the like of the ordered hydrogel scaffold, respectively, without being limited thereto.
Another aspect of an embodiment of the present invention provides a cell culture device comprising the ordered hydrogel fiber or the ordered hydrogel scaffold, a cell wall and a substrate, the ordered hydrogel fiber or the ordered hydrogel scaffold being fixed on the substrate, the cell wall being disposed around the ordered hydrogel fiber or the ordered hydrogel scaffold, thereby forming a cell.
In some embodiments, an ordered hydrogel fiber scaffold supporting NT-3 and paclitaxel may be immobilized on a substrate such as a glass slide, and a fluid cell wall is immobilized around the ordered hydrogel fiber scaffold to form a cell. Then, the neural stem cells can be inoculated on the surface of the ordered hydrogel fiber scaffold, and the research on the directional induced differentiation of the neural stem cells in vitro can be carried out.
Another aspect of an embodiment of the invention provides the use of the ordered hydrogel fiber, the ordered hydrogel scaffold, or the cell culture device in cell culture.
Another aspect of the embodiments of the present invention provides a use of the ordered hydrogel fiber or the ordered hydrogel scaffold in preparing a product having a spinal cord injury repair function.
Another aspect of the embodiments of the present invention provides a functional product comprising the ordered hydrogel scaffold loaded with bioactive factors including Neurotrophin-3 (NT-3); and, the product has at least the following functions: inducing differentiation of neural stem cells into neurons and oligodendrocytes, promoting myelination of damaged areas, and, inducing axonal regeneration.
Another aspect of an embodiment of the present invention provides a functional product comprising the ordered hydrogel scaffold loaded with a pharmaceutical compound, the pharmaceutical compound comprising Paclitaxel (PTX); and, the product has at least a function of inducing differentiation of neural stem cells into neurons.
Another aspect of an embodiment of the invention provides a functional product comprising the ordered hydrogel scaffold of claim loaded with neurotrophin 3 and paclitaxel; and, the product has at least a function of promoting the formation of a neural circuit.
It is well known in the art that spinal cord is composed of millions of ordered nerve fibers, and after spinal cord injury, the ordered scaffold not only can bridge two broken ends of the injury region, but also can be used as a carrier of cells and bioactive factors. Neural Stem Cells (NSCs) are Cells in the central nervous system that have the ability to self-renew and the potential for multipotentiality. But self-functional repair is difficult to achieve due to the limited number of NSCs in the lesion area and the inhibitory microenvironment.
The foregoing embodiment of the present invention combines the michael addition reaction and the liquid electrospinning technology to prepare the collagen/fibrin ordered hydrogel fiber, which can better simulate the structure and components of the natural extracellular matrix, and has high water content and orientation. Furthermore, after the ordered hydrogel scaffold prepared by the embodiment of the invention carries some medicinal compounds and bioactive molecules, a more suitable microenvironment can be created for tissue regeneration and recovery of the injury function, and the ordered hydrogel scaffold can also induce the directional differentiation of stem cells and is suitable for being used as a scaffold material for spinal cord injury repair.
In the previous embodiment of the present invention, the michael addition reaction and the water-soluble liquid electrospinning technology are used to prepare the collagen/fibrin ordered hydrogel fiber scaffold, wherein the michael addition reaction conditions are mild, and the gel curing time is fast, while the water-soluble electrospinning is non-toxic to bioactive factors and cells because the solvent used in the water-soluble electrospinning is water, and the biological activities of the loaded NT-3, paclitaxel, cells, etc. can be better maintained.
The ordered hydrogel fiber scaffold provided by the previous embodiment of the invention can provide a guiding direction for cell growth.
The NT-3-loaded ordered hydrogel scaffold induces neural stem cells to differentiate towards neurons and oligodendroglia, promotes myelination of a damaged area, and induces axon regeneration.
Wherein, the biological function bracket loaded with the paclitaxel induces the neural stem cells to differentiate towards the neurons.
Wherein, the composite scaffold material carrying NT-3 and paclitaxel can promote the formation of a neural loop, thereby being beneficial to the regeneration of tissues and the recovery of the function of a damaged area.
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention are described in further detail below with reference to the accompanying drawings and several preferred embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. The test methods in the following examples are carried out under conventional conditions without specifying the specific conditions. 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.
Example 1 preparation of ordered hydrogel fibers
1) Preparation of modified collagen
Collagen (500mg) was completely dissolved in 0.5mol/L acetic acid solution to form a collagen solution having a concentration of 10mg/ml, the pH of the collagen solution was adjusted to 5.5 to 6 with 1M sodium hydroxide, and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC)/N-hydroxysuccinimide (NHS) (both having a mass of 400mg/200mg) and N-2-aminoethylmaleimide (100mg) were sequentially added to the solution, followed by stirring at room temperature overnight. Dialyzed against 1M aqueous sodium chloride for 1 day (5000-. Freezing at-80 deg.C overnight, freeze-drying in freeze-drying machine to obtain modified collagen, and storing at 4 deg.C.
2) Preparation of modified fibrin
Fibrinogen (500mg) was dissolved in 150mM PBS containing 8mol/L of urea to form a solution having a concentration of 5mg/ml, and after complete dissolution, Tris (dicarbonylethyl) phosphate (Tris (2-carbonylethyl), TCEP. HCL, 40mg) was added and stirred at room temperature in the dark for 30min to allow the TCEP. HCL to sufficiently open the fibrinogen disulfide bond to introduce more thiol groups. After completion of the reaction, the reaction mixture was dialyzed against light with 50mM PBS containing 0.1 wt% acetic acid (all wt% unless otherwise specified) for 3 days (12000-14000 molecular weight cut-off dialysis bag). Freezing at-80 deg.C overnight, freeze-drying in freeze-drying machine to obtain modified fibrin, and storing at-20 deg.C.
Infrared spectrum characterization of modified collagen and modified fibrin: the collagen modification process is to dehydrate and condense the carboxyl of the collagen with the amino of N-2-aminoethylmaleimide, so that the collagen is modified with maleimide groups. The amido bonds of the modified collagen are increased, the stretching vibration of the collagen relative to the procollagen amide I and amide II is enhanced, and the successful modification of the maleimide groups on the collagen is proved. The fibrin is modified by reducing disulfide bond of fibrin into sulfhydryl by TCEP.HCL at 2600cm-1Characteristic peaks of mercapto group appear on the left and right. The successful modification of fibrin into thiolated fibrin is proved.
3) Preparation of modified collagen/fibrin ordered hydrogel fiber
The modified collagen was dissolved in an aqueous solution to form a modified collagen solution having a concentration of 2 wt%. The modified fibrin was dissolved in normal saline containing urea (100mg/ml) to form a modified fibrin solution having a concentration of 4 wt%. The ordered hydrogel fiber is prepared by combining the Michael addition reaction and the liquid electrostatic spinning technology.
Specifically, referring to fig. 1, the modified collagen solution and the modified fibrin solution are respectively added into a 1ml syringe, and the two syringes are finally collected to an outlet of the syringe by a three-way device. The injection speed of the two syringes is 0.8ml/h, the high-voltage power supply voltage is set to be 3kV, the rotating speed of the liquid receiving device is 160rpm/min, and the receiving distance is that the needle head (with the specification of 23G or 25G) just contacts with the receiving solution (the solution containing 2-10mg/ml EDC/1.2-5mg/ml NHS). As the electrospinning time increased (30s, 90s, 5min, 10min), the macroscopic diameter of the hydrogel micelles increased, as shown in FIGS. 2A-2D. While keeping the pushing speed, voltage and receiving distance of the refraction pump unchanged, the diameter of the single fiber bundle can be regulated and controlled by changing the rotating speed of the rotating disc, as shown in figures 3A-3B (rotating speed 160rpm/min) and figures 3C-3D (rotating speed 200 rpm/min).
Example 2: this example is essentially the same as example 1, except that: respectively dissolving collagen with different masses in acetic acid aqueous solutions with the concentrations of 0.3mol/L, 0.5mol/L and 1.0mol/L to form collagen solutions with the concentrations of 3mg/ml, 8mg/ml and 10mg/ml, adjusting the pH value of the obtained collagen solution to 5.5-6, and respectively adding the collagen solutions with the mass ratios of 2:1 and 3: 1. 4:1 of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide and N-hydroxysuccinimide, then adding different molar amounts of N-2-aminoethylmaleimide respectively, and enabling the molar ratio of collagen to N-2-aminoethylmaleimide to be 1:5, 1: 3. 1:1, stirring at room temperature overnight, performing post-treatment to obtain a series of modified collagen products, and dissolving the modified collagen products in water to form a series of modified collagen spinning solutions with the concentration of 1.5-2 wt%.
Example 3: this example is essentially the same as example 1, except that: respectively dissolving fibrinogen with different masses in phosphate buffer salt solutions containing 5mol/L, 7mol/L and 8mol/L of urea, then adding tris (dicarbonylethyl) phosphate with different masses, and enabling the molar ratio of the fibrinogen to the tris (dicarbonylethyl) phosphate to be 1:20, 1: 15. 1:10, reacting at room temperature in a dark place, performing post-treatment to obtain a series of modified fibrin products, and dissolving the modified fibrin products in a series of normal saline containing urea with the concentration of 50-100mg/ml to form a series of modified fibrin spinning solutions with the concentration of 2-4 wt%.
Example 4 neural Stem cell culture Using NT-3 and paclitaxel-Supported modified collagen/fibrin ordered hydrogel scaffolds as carriers
1) Preparation of NT-3 and paclitaxel-loaded collagen/fibrin ordered hydrogel scaffold
Preparing a collagen/fibrin ordered hydrogel scaffold carrying NT-3: a modified collagen solution having a concentration of 2 wt% and a modified fibrin solution having a concentration of 4 wt% were prepared in the same manner as in example 1. Then NT-3 is dissolved in deionized water, NT-3 and modified collagen solution/fibrin solution are mixed evenly in a vortex mode to prepare electrostatic spinning solution, and then the collagen/fibrin ordered hydrogel scaffold carrying NT-3 is prepared by referring to the operation of the step 3) in the embodiment 1 (wherein the flow rate of the modified collagen spinning solution and the modified fibrin spinning solution in the spinning solution channel can be set to be 0.6ml/h-1.0ml/h, the voltage can be set to be 3-4kV, the rotation speed of the liquid receiving device can be set to be 100-160rpm/min, and the receiving solution can be a solution containing 2-10mg/ml EDC and 1.2-5mg/ml NHS), which is a hydrogel fiber bundle. The ordered hydrogel scaffolds were mounted on a slide using dow corning 3140 silicone adhesive, and the cell walls were mounted around the ordered hydrogel scaffolds to form a cell pool. Using EDC/NHS, Ca2+Thrombin as cross-linker, the pH of the cross-linker solution was adjusted to 7.4 with 58% beta-disodium glycerophosphate pentahydrate (beta-GP) and cross-linked at 37 ℃ for 3 h. After crosslinking was complete, sterilization was carried out with 75% ethanol at 37 ℃ for 4 h. Washing with PBS for about 20min, and drying the water around the ordered hydrogel scaffold with sterile filter paperFor use.
② preparing the collagen/fibrin ordered hydrogel bracket which carries paclitaxel: modified collagen solution and modified fibrin solution were prepared according to the method of example 1. Then, paclitaxel is dissolved in dimethyl sulfoxide (DMSO), and then the paclitaxel and the modified collagen solution/fibrin solution are mixed evenly by vortex to prepare an electrostatic spinning solution, and then the collagen/fibrin ordered hydrogel scaffold carrying paclitaxel is prepared by the operation of the step 3) of the reference example 1. Referring to the operation of the first part, the operations of fixing, crosslinking, sterilizing and the like of the ordered hydrogel scaffold are optimized. After sterilization, the hydrogel scaffold was washed with PBS for about 20min, and the water around the ordered hydrogel scaffold was blotted with sterile filter paper for use.
2) NT-3 and paclitaxel loaded collagen/fibrin ordered hydrogel scaffold for stem cell culture
The hippocampus was isolated from ICR neonatal mice within 24h of birth, minced, digested with pancreatin in at 37 ℃ in a cell culture chamber, blown evenly with a pipette at 5min intervals, and digested with PBS. The incompletely digested tissue was filtered through a 400 μm filter at 300rpm/2min, the supernatant was collected and 1000rpm/5min, the supernatant was discarded, and the cells were resuspended in proliferation medium. Then transferred to a culture flask for balling culture. Digesting neurosphere into single cells after passage for 2 times, respectively inoculating on the NT-3 and paclitaxel-loaded collagen/fibrin ordered hydrogel fibrous scaffold, wherein the cell inoculation density is 1 × 105-2×105。
3) NT-3-loaded collagen/fibrin ordered hydrogel fibrous scaffold for promoting differentiation of neural stem cells towards oligodendroglia and neurons
Preparing the collagen/fibrin ordered hydrogel fiber scaffold carrying NT-3 with the concentrations of 0, 1ng/ml, 10ng/ml and 50ng/ml according to the method of the part I. Neural stem cells were seeded at the same seeding density on the surface of the 4 NT-3 concentrations of scaffold, 3 replicates per concentration set. Then culturing the cells in a cell culture box for about 6 hours in an adherent manner, and gently washing the non-adherent cells by using PBS. 400 μ l of differentiation medium was added. And replacing the differentiation culture medium every two days, and identifying the differentiation result after culturing for 7 days. The specific operation is as follows: fixing 4% paraformaldehyde at room temperature for 30min, and washing with PBS for 3 times; incubating 0.08% Triton X-100 at room temperature for 10min, and washing with PBS for 3 times; 5% BSA was blocked at room temperature for 30min, washed 3 times with PBS; TUj-1, Olig-2 primary antibody and PBS are incubated overnight at 4 ℃ for 3 times; incubating the second antibody at 37 ℃ for 40min, and washing the second antibody with PBS for 3 times; nuclei were stained with 1. mu.l/mL DAPI for 20min, washed 3 times with PBS, and pictures were taken by scanning under a fluorescence microscope at the same exposure intensity. As shown in figure 4, the NSCs on the ordered hydrogel fiber scaffold carrying NT-3 with the concentration of 1ng/ml have the best effect of promoting the neural stem cells to differentiate towards oligodendroglia and neurons, and are beneficial to forming a marrow sheath in the damaged area to promote the axon growth.
4) Paclitaxel-loaded collagen/fibrin ordered hydrogel fibrous scaffold for promoting differentiation of neural stem cells towards neurons
Preparing collagen/fibrin ordered hydrogel fiber scaffold carrying paclitaxel with concentration of 0, 0.85ng/ml, 1.7ng/ml and 7ng/ml according to the method of the second part. Neural stem cells were seeded at the same seeding density on the surface of the 4 paclitaxel concentrations of scaffolds, with 3 replicates per concentration set. Then culturing the cells in a cell culture box for about 6 hours in an adherent manner, and gently washing the non-adherent cells by using PBS. 400 μ l of differentiation medium was added. And replacing the differentiation culture medium every two days, and carrying out differentiation result identification 7 days after the neural stem cells are cultured. The subsequent operations were the same as those in step 3) of example 2, except that the primary antibody used was different (TUj-1 was used here, GFAP was used as the primary antibody). As shown in FIG. 5, NSCs on the ordered hydrogel fiber scaffold loaded with paclitaxel at a concentration of 1.7ng/ml showed the best effect of promoting differentiation of neural stem cells toward neurons.
According to the embodiment of the invention, the ordered hydrogel fiber scaffold carrying NT-3/paclitaxel is successfully prepared by combining the Michael addition reaction and the water-soluble electrostatic spinning technology, and the scaffold can be rapidly molded and has high water content. The paclitaxel-loaded collagen/fibrin ordered hydrogel fiber scaffold promotes the differentiation of neural stem cells towards neurons; the NT-3-loaded ordered hydrogel fibrous scaffold promotes differentiation of neural stem cells towards oligodendroglia and neurons, induces myelination, promotes axon extension, and is favorable for formation of neural circuits and repair of spinal cord injury functions.
The aspects, embodiments, features and examples of the present invention should be considered as illustrative in all respects and not intended to be limiting of the invention, the scope of which is defined only by the claims. Other embodiments, modifications, and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed invention.
It should be understood that the order of steps or the order in which particular actions are performed is not critical to the invention, so long as the teachings of the invention remain operable. Further, two or more steps or actions may be performed simultaneously.
While the invention has been described with reference to illustrative embodiments, it will be understood by those skilled in the art that various other changes, omissions and/or additions may be made and substantial equivalents may be substituted for elements thereof without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from its scope. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims.

Claims (20)

1. A method for preparing ordered hydrogel fibers, comprising:
respectively modifying collagen and fibrin, and then respectively dissolving the modified collagen and the fibrin in a water phase system to form modified collagen spinning solution and modified fibrin spinning solution, wherein the modified collagen and the modified fibrin respectively have a first reaction group and a second reaction group, and a Michael addition reaction can be carried out between the first reaction group and the second reaction group;
and (2) converging the modified collagen spinning solution and the modified fibrin spinning solution into a spinning nozzle for outputting by adopting a liquid electrostatic spinning method, and combining the modified collagen and the modified fibrin through the Michael addition reaction to prepare and form the ordered hydrogel fiber.
2. The preparation method according to claim 1, which specifically comprises:
dissolving collagen in 0.3-1.0mol/L acetic acid aqueous solution to form a collagen solution with the concentration of 3-10mg/mL, adjusting the pH value of the obtained collagen solution to 5.5-6, adding 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide and N-hydroxysuccinimide in a mass ratio of 2:1-4:1, then adding N-2-aminoethylmaleimide, enabling the molar ratio of the collagen to the N-2-aminoethylmaleimide to be 1:5-1:1, stirring at room temperature overnight, and carrying out post-treatment to obtain modified collagen; and
dissolving fibrinogen into phosphate buffer solution containing 5-8mol/L urea, adding tris (dicarbonylethyl) phosphate, enabling the molar ratio of fibrinogen to tris (dicarbonylethyl) phosphate to be 1:20-1:10, carrying out dark reaction at room temperature, and carrying out post-treatment to obtain the modified fibrin.
3. The preparation method according to claim 1, which specifically comprises:
dissolving modified collagen in water to form 1.5-2 wt% modified collagen spinning solution; and
dissolving the modified fibrin in physiological saline containing 50-100mg/mL of urea to form modified fibrin spinning solution with the concentration of 2-4 wt%.
4. The preparation method according to claim 1, which specifically comprises: respectively adding the modified collagen spinning solution and the modified fibrin spinning solution into a spinning solution channel, converging the spinning solution into a spinning nozzle through the two spinning solution channels, outputting the spinning nozzle, and preparing and forming the ordered hydrogel fiber by using a liquid electrostatic spinning method;
wherein, the liquid electrostatic spinning method adopts the following process conditions: the flow rate of the modified collagen spinning solution and the modified fibrin spinning solution in the spinning solution channel is 0.6-1.0mL/h, the voltage is set to be 3-4kV, the rotating speed of the liquid receiving device is 100 plus materials and 200rpm/min, and the receiving distance is set to be that the spinning nozzle is contacted with the receiving solution.
5. The method according to claim 4, wherein the receiving solution in the liquid receiver is a solution containing 2-10mg/mL EDC and 1.2-5mg/mL NHS.
6. A method for preparing an ordered hydrogel scaffold, comprising:
respectively modifying collagen and fibrin, and then respectively dissolving the modified collagen and the fibrin in a water phase system to form modified collagen spinning solution and modified fibrin spinning solution, wherein the modified collagen and the modified fibrin respectively have a first reaction group and a second reaction group, and a Michael addition reaction can be carried out between the first reaction group and the second reaction group;
dissolving bioactive factors and/or medicinal compounds in at least one of the modified collagen spinning solution and the modified fibrin spinning solution, then respectively adding the modified collagen spinning solution and the modified fibrin spinning solution into one spinning solution channel by using a liquid electrostatic spinning method, converging the modified collagen spinning solution and the modified fibrin spinning solution into one spinning nozzle through the two spinning solution channels for outputting, and combining the modified collagen and the modified fibrin therein through the Michael addition reaction, thereby preparing the ordered hydrogel scaffold loaded with the bioactive factors and/or the medicinal compounds.
7. The preparation method according to claim 6, which specifically comprises:
dissolving collagen in 0.3-1.0mol/L acetic acid aqueous solution to form a collagen solution with the concentration of 3-10mg/mL, adjusting the pH value of the obtained collagen solution to 5.5-6, adding 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide and N-hydroxysuccinimide in a mass ratio of 2:1-4:1, then adding N-2-aminoethylmaleimide, enabling the molar ratio of the collagen to the N-2-aminoethylmaleimide to be 1:5-1:1, stirring at room temperature overnight, carrying out post-treatment to obtain modified collagen, and then dissolving the modified collagen in water to form a modified collagen spinning solution with the concentration of 1.5-2 wt%;
dissolving fibrinogen into phosphate buffer solution containing 5-8mol/L urea, adding tris (dicarbonylethyl) phosphate, enabling the molar ratio of fibrinogen to tris (dicarbonylethyl) phosphate to be 1:20-1:10, carrying out light-shielding reaction at room temperature, carrying out post-treatment to obtain modified fibrin, and dissolving the modified fibrin in physiological saline containing 50-100mg/mL urea to form modified fibrin spinning solution with the concentration of 2-4 wt%.
8. The preparation method according to claim 7, wherein the liquid electrospinning method adopts process conditions comprising: the flow rate of the modified collagen spinning solution and the modified fibrin spinning solution in the spinning solution channel is 0.6-1.0mL/h, the voltage is set to be 3-4kV, the rotating speed of the liquid receiving device is 100-160rpm/min, and the receiving distance is set to be that the spinning nozzle is contacted with the receiving solution.
9. The method according to claim 8, wherein the receiving solution in the liquid receiver is a solution containing 2-10mg/mL EDC and 1.2-5mg/mL NHS.
10. The method of claim 6, comprising: and dissolving the bioactive factor and/or the medicinal compound in the modified collagen spinning solution and/or the modified fibrin spinning solution, wherein the spinning solution output from the spinning nozzle contains the bioactive factor with the concentration of 0-7ng/mL and/or the medicinal compound with the concentration of 0-50 ng/mL.
11. The method of claim 10, wherein the bioactive factor comprises neurotrophic factor 3.
12. The method of claim 10, wherein the pharmaceutical compound comprises paclitaxel.
13. An ordered hydrogel fiber produced by the method of any one of claims 1-5.
14. An ordered hydrogel scaffold prepared by the method of any one of claims 6-12.
15. A cell culture device comprising the ordered hydrogel fiber of claim 13 or the ordered hydrogel scaffold of claim 14, a cell wall, and a substrate, the ordered hydrogel fiber or the ordered hydrogel scaffold being affixed to the substrate, the cell wall being disposed around the ordered hydrogel fiber or the ordered hydrogel scaffold to form a cell.
16. Use of the ordered hydrogel fiber of claim 13, the ordered hydrogel scaffold of claim 14, or the cell culture device of claim 15 in cell culture.
17. Use of the ordered hydrogel fiber of claim 13 or the ordered hydrogel scaffold of claim 14 in the preparation of a product having a spinal cord injury repair function.
18. A functional product comprising the ordered hydrogel scaffold of claim 13, wherein the ordered hydrogel scaffold is loaded with a bioactive factor, wherein the bioactive factor comprises neurotrophic factor 3; and, the product has at least the following functions: inducing differentiation of neural stem cells into neurons and oligodendrocytes, promoting myelination of damaged areas, and, inducing axonal regeneration.
19. A functional product comprising the ordered hydrogel scaffold of claim 13, loaded with a pharmaceutical compound comprising paclitaxel; and, the product has at least a function of inducing differentiation of neural stem cells into neurons.
20. A functional product comprising the ordered hydrogel scaffold of claim 13, loaded with neurotrophic factor 3 and paclitaxel; and, the product has at least a function of promoting the formation of a neural circuit.
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