Preparation method of nervous tissue scaffold and nervous tissue scaffold

Abstract

The invention provides a preparation method of a nervous tissue scaffold and the nervous tissue scaffold, the method comprises the following steps: adding lysine diisocyanate and a catalyst into molten polyethylene glycol and polycaprolactone, carrying out a heating reaction in a nitrogen atmosphere, then adding 1, 3-propylene glycol, and carrying out primary chain extension modification to obtain a first polymer; putting the first polymer into deionized water, stirring and emulsifying, then adding an alginate aqueous solution, and mixing to obtain a first mixed solution; fully mixing the first mixed solution and the chitosan solution to obtain a second mixed solution; and preparing the nervous tissue scaffold through an electrostatic spinning instrument. Polyethylene glycol and polycaprolactone are used as polymer matrixes and are mixed with an alginate aqueous solution, and polyethylene glycol and the like on the surface of a first polymer can physically interact with groups on an alginate molecular chain, so that alginate macromolecules are wound and combined on the surfaces of particles, the average particle size is increased, and the porosity is improved.

Description

Technical Field

[0001] The invention relates to the technical field of neural tissue scaffolds, and in particular to a neural tissue scaffold preparation method. Background Art

[0002] Neural tissue defects bring great distress and pain to patients. Currently, autologous nerve transplantation, gene therapy, growth factor induction and other technologies can be used to treat and repair nerve defects. The self-repair ability of large sections of damaged peripheral nerve bundles and damaged central nervous tissue is quite limited. Tissue engineering technology can simulate the structure and characteristics of natural nerves, provide a suitable microenvironment for the regeneration and repair of nerve cells, and use biomaterials, cell culture technology and bioengineering technology to construct neural tissue substitutes with specific structures and functions to restore the function of damaged nerves.

[0003] In the existing technology, natural polymers such as collagen, hyaluronic acid, chitosan, sodium alginate, and silk fibroin are often used in neural tissue scaffolds due to their good biocompatibility, but they have poor stability and certain immunogenicity to the human body. Summary of the invention

[0004] In view of the deficiencies of the prior art, the purpose of the present invention is to provide a method for preparing a neural tissue scaffold, aiming to solve the technical problem of poor stability of natural polymer material scaffolds in the prior art.

[0005] In order to achieve the above object, the present invention is implemented by the following technical scheme: a method for preparing a neural tissue scaffold, comprising the following steps:

[0006] Lysine diisocyanate and a catalyst are added to molten polyethylene glycol and polycaprolactone, and the mixture is heated and reacted in a nitrogen atmosphere, and then 1,3-propylene glycol is added to perform a chain extension modification to obtain a first polymer;

[0007] The first polymer is placed in deionized water for stirring and emulsification, and a sodium hydroxide solution is added to adjust the pH value to 7-8, and then an alginate aqueous solution with a concentration of 7wt%-10wt% is added to mix to obtain a first mixed solution;

[0008] The first mixed solution and the chitosan solution are fully mixed in a ratio of 1:1 to obtain a second mixed solution;

[0009] Concentrating the second mixed solution by wind speed method until the concentration of polyethylene glycol in the second mixed solution reaches 20wt%-30wt%;

[0010] The concentrated second mixed solution is injected into a syringe, and a liquid stream is ejected from the syringe under preset parameters by an electrospinning apparatus, and a nerve tissue scaffold is prepared after peeling and drying.

[0011] According to one aspect of the above technical solution, the molar ratio of polyethylene glycol to polycaprolactone in the first mixed solution is 0.1:0.8-0.2:0.9.

[0012] According to one aspect of the above technical solution, the molar ratio of lysine diisocyanate to 1,3-propylene glycol in the first mixed solution is 3:0.5-4:1.5.

[0013] According to one aspect of the above technical solution, before the step of placing the first polymer into deionized water for stirring and emulsification, the method further comprises:

[0014] The first polymer is cooled in a cold water bath, and then L-lysine solution is added dropwise to perform secondary chain extension modification.

[0015] According to one aspect of the above technical solution, before the step of cooling the first polymer using a cold water bath, the method further comprises:

[0016] After the primary chain extension modification is completed, acetone is added to the first polymer to reduce the viscosity.

[0017] According to one aspect of the above technical solution, during the heating reaction in a nitrogen atmosphere, the reaction temperature is 90° C. and the reaction time is 2 hours.

[0018] According to one aspect of the above technical solution, the catalyst is organic bismuth with a concentration of 0.1 wt%.

[0019] According to one aspect of the above technical solution, the ratio of polyethylene glycol to acetic acid solution in the polyethylene glycol solution is 1:50.

[0020] According to one aspect of the above technical solution, the molar ratio of 1,3-propylene glycol to L-lysine in the first mixed solution is 1:1.

[0021] According to one aspect of the above technical solution, the preset parameters include the electrospinning voltage parameter of 12kV-22kV and the syringe propulsion speed of 1.5mL / h-2mL / h.

[0022] On the other hand, the present invention provides a neural tissue scaffold, which is prepared by the neural tissue scaffold preparation method in the above technical solution.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: by using polyethylene glycol (PEG) and polycaprolactone (PCL) as polymer matrices, the basic structure and physical properties of the scaffold are provided, and they are mixed with an alginate aqueous solution. The hydrophilic carboxylate ions, polyethylene glycol, etc. on the surface of the first polymer can physically interact with carboxyl groups, hydroxyl groups, etc. on the alginate molecular chain, so that the alginate macromolecules are entangled and bound to the particle surface, resulting in an increase in the average particle size, thereby increasing the porosity. At the same time, lysine diisocyanate (LDI) and a catalyst can be used to improve the crosslinking degree and stability of the polymer. 1,3-propylene glycol and L-lysine solution are used as chain extenders to help adjust the chain length of the polymer and the intermolecular interaction. Mixing the first mixed solution with the chitosan solution can improve the biocompatibility and promote the proliferation and differentiation of nerve cells on the basis of ensuring the mechanical properties of the scaffold. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a flow chart of the method for preparing a neural tissue scaffold in the first embodiment of the present invention;

[0025] Figure 2 It is a comparison diagram of the cell adhesion rates of the neural tissue scaffolds prepared in various embodiments of the present invention and the comparative examples;

[0026] The following specific implementation manner will further illustrate the present invention in conjunction with the above-mentioned drawings. DETAILED DESCRIPTION

[0027] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. Several embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0028] It should be noted that when an element is referred to as being "fixed to" another element, it may be directly on the other element or there may be a central element. When an element is considered to be "connected to" another element, it may be directly connected to the other element or there may be a central element at the same time. The terms "vertical", "horizontal", "left", "right" and similar expressions used herein are for illustrative purposes only.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0030] Embodiment 1

[0031] See also Figure 1 , which is a flow chart of a method for preparing a neural tissue scaffold in a first embodiment of the present invention. As shown in the figure, the method comprises the following steps:

[0032] Step S100, lysine diisocyanate and a catalyst are added to molten polyethylene glycol and polycaprolactone, and the mixture is heated to react in a nitrogen atmosphere, and then 1,3-propylene glycol is added to perform a chain extension modification to obtain a first polymer.

[0033] Specifically, in this embodiment, during the heating reaction in the nitrogen atmosphere, the reaction temperature is 90° C. and the reaction time is 2 hours; the catalyst is organic bismuth with a concentration of 0.1 wt %.

[0034] Specifically, in some application scenarios of the present embodiment, lysine diisocyanate and 0.1 wt% of organic bismuth are added to molten polyethylene glycol and polycaprolactone in a three-necked flask equipped with a thermometer and a mechanical stirrer, and the mixture is reacted at 90° C. for 2 hours in a nitrogen atmosphere. The temperature is then lowered to 70° C., and 1,3-propylene glycol is added for the first chain extension modification, and the reaction time is 1 hour.

[0035] Step S200, placing the first polymer into deionized water for stirring and emulsification, adding sodium hydroxide solution to adjust the pH value to 7-8, and then adding alginate aqueous solution with a concentration of 7wt%-10wt% to mix to obtain a first mixed solution.

[0036] Specifically, in some application scenarios of this embodiment, the stirring and emulsifying time is 1.5 hours, and the concentration of the alginate aqueous solution is preferably 8wt%. In addition, in this embodiment, before the emulsified emulsion is mixed with the alginate aqueous solution, the emulsion needs to be adjusted to a solid content of 15wt% by rotary evaporation.

[0037] The molar ratio of polyethylene glycol to polycaprolactone in the first mixed solution is 0.1:0.8-0.2:0.9. In this embodiment, it is preferably 0.16:0.85. The molar ratio of lysine diisocyanate to 1,3-propylene glycol in the first mixed solution is 3:0.5-4:1.5. In this embodiment, it is preferably 3.6:1.

[0038] Preferably, in this embodiment, before the step of placing the first polymer into deionized water for stirring and emulsification, the method further comprises:

[0039] Step S101, cooling the first polymer using a cold water bath, and then dropping an L-lysine solution for secondary chain extension modification. Specifically, in this step, the temperature is lowered to below 40° C. in a cold water bath by removing the heating device, and then adding an L-lysine solution for secondary modification. Preferably, the molar ratio of 1,3-propylene glycol to L-lysine in the first mixed solution is 1:1.

[0040] Furthermore, before the step of cooling the first polymer using a cold water bath, the method further comprises:

[0041] Step S102, after the primary chain extension modification is completed, acetone is added to the first polymer to reduce the viscosity. Specifically, by adding acetone to the reaction system, the viscosity can be reduced. In addition, after the secondary chain extension modification is completed, the acetone needs to be precipitated by dialysis after emulsification is completed.

[0042] Step S300, the first mixed solution and the chitosan solution are fully mixed in a ratio of 1:1 to obtain a second mixed solution. It is easy to understand that chitosan has excellent biocompatibility and can promote the proliferation and differentiation of nerve cells, but its flexibility and elasticity are poor. By mixing the chitosan solution with the first mixed solution to prepare a composite scaffold, its mechanical properties can be improved and it is easier to shape.

[0043] Step S400, concentrating the second mixed solution by wind speed method until the concentration of polyethylene glycol in the second mixed solution reaches 20wt%-30wt%. In this embodiment, the concentration of polyethylene glycol in the second mixed solution is 25wt%.

[0044] Step S500, injecting the concentrated second mixed solution into a syringe, ejecting a liquid stream from the syringe under preset parameters through an electrospinning apparatus, and preparing a neural tissue scaffold after peeling and drying.

[0045] Specifically, the preset parameters include an electrospinning voltage parameter of 12kV-22kV and a syringe propulsion speed of 1.5mL / h-2mL / h. In some application scenarios of this embodiment, the second mixed solution is electrospun at a relative humidity of 50% and a temperature of 25°C, with a spinning voltage of 20kV, a distance between the needle and the receiving electrode of 10cm, and an extrusion rate of 0.8mL / h; after drying, it is peeled off from the aluminum foil and placed in a constant temperature and humidity chamber at a temperature of 40°C and a humidity of 90%RH for 36 hours to obtain a neural tissue scaffold.

[0046] In summary, the method for preparing a neural tissue scaffold in the above-mentioned embodiment of the present invention provides the basic structure and physical properties of the scaffold by using polyethylene glycol (PEG) and polycaprolactone (PCL) as polymer matrices, and mixes them with an aqueous alginate solution. The hydrophilic carboxylate ions, polyethylene glycol, etc. on the surface of the first polymer can physically interact with carboxyl groups, hydroxyl groups, etc. on the alginate molecular chain, so that the alginate macromolecules are entangled and bound to the particle surface, resulting in an increase in the average particle size, thereby increasing the porosity. At the same time, lysine diisocyanate (LDI) and a catalyst can be used to improve the crosslinking degree and stability of the polymer. 1,3-propylene glycol and L-lysine solution are used as chain extenders to help regulate the chain length and intermolecular interactions of the polymer. Mixing the first mixed solution with the chitosan solution can improve the biocompatibility and promote the proliferation and differentiation of nerve cells on the basis of ensuring the mechanical properties of the scaffold.

[0047] Embodiment 2

[0048] The second embodiment of the present invention provides a method for preparing a neural tissue scaffold. The preparation method in this embodiment is basically the same as that in the first embodiment, except that:

[0049] In this embodiment, the molar ratio of lysine diisocyanate to 1,3-propylene glycol in the first mixed solution is 3:0.5; the molar ratio of polyethylene glycol to polycaprolactone in the first mixed solution is 0.1:0.8.

[0050] Embodiment 3

[0051] The third embodiment of the present invention provides a method for preparing a neural tissue scaffold. The preparation method in this embodiment is basically the same as that in the first embodiment, except that:

[0052] In this embodiment, the molar ratio of lysine diisocyanate to 1,3-propylene glycol in the first mixed solution is 4:1.5; the molar ratio of polyethylene glycol to polycaprolactone in the first mixed solution is 0.2:0.9.

[0053] Embodiment 4

[0054] The fourth embodiment of the present invention provides a method for preparing a neural tissue scaffold. The preparation method in this embodiment is basically the same as that in the first embodiment, except that:

[0055] In this embodiment, the concentration of the alginate aqueous solution is 7 wt %.

[0056] Embodiment 5

[0057] The fifth embodiment of the present invention provides a method for preparing a neural tissue scaffold. The preparation method in this embodiment is basically the same as that in the first embodiment, except that:

[0058] In this embodiment, the concentration of the alginate aqueous solution is 10 wt %.

[0059] Embodiment 6

[0060] The sixth embodiment of the present invention provides a nerve tissue scaffold, which is prepared by the nerve tissue scaffold preparation method in the above embodiment.

[0061] Comparative Example 1

[0062] In this comparative example, the steps of the above preparation method are substantially the same as those of Example 1, except that:

[0063] The concentration of the alginate aqueous solution is 4 wt %.

[0064] The scaffolds prepared in Example 1, Example 4, Example 5 and Comparative Example 1 were placed in culture medium and cultured for 12 hours to evaluate the adhesion rate of the scaffolds. Then 0.1 mL of PC12 cell suspension was inoculated on each scaffold at a seeding density of 1×105 cells / mL. After 4 hours of culture, the scaffolds were removed, the number of remaining cells was recorded, and the cell adhesion rate was calculated according to the following calculation formula:

[0065]

[0066] Where C is the seeding density, V is the scaffold volume, and R is the number of remaining cells.

[0067] The comparison results are as follows Figure 2 As shown. Figure 2 It can be seen that the cell adhesion rate increases significantly with the increase in the concentration of the alginate aqueous solution, indicating that the hydrophilic carboxylate ions, polyethylene glycol, etc. on the surface of the first polymer interact with the hydrogen bonding force of the alginate molecules, thereby increasing the porosity and facilitating the transport of liquids and nutrients and subsequent cell growth.

[0068] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0069] The above-described embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a neural tissue scaffold, characterized in that: The following steps are involved: Lysine diisocyanate and a catalyst are added to molten polyethylene glycol and polycaprolactone, and the mixture is heated and reacted in a nitrogen atmosphere, and then 1,3-propylene glycol is added to perform a chain extension modification to obtain a first polymer; The first polymer is placed in deionized water for stirring and emulsification, and a sodium hydroxide solution is added to adjust the pH value to 7-8, and then an alginate aqueous solution with a concentration of 7wt%-10wt% is added to mix to obtain a first mixed solution; The first mixed solution and the chitosan solution are fully mixed in a ratio of 1:1 to obtain a second mixed solution; Concentrating the second mixed solution by wind speed method until the concentration of polyethylene glycol in the second mixed solution reaches 20wt%-30wt%; The concentrated second mixed solution is injected into a syringe, and a liquid stream is ejected from the syringe under preset parameters by an electrospinning apparatus, and a nerve tissue scaffold is prepared after peeling and drying.

2. The method for preparing a neural tissue scaffold according to claim 1, characterized in that: The molar ratio of polyethylene glycol to polycaprolactone in the first mixed solution is 0.1:0.8-0.2:0.

9.

3. The method for preparing a neural tissue scaffold according to claim 1, characterized in that: The molar ratio of lysine diisocyanate to 1,3-propylene glycol in the first mixed solution is 3:0.5-4:1.

5.

4. The method for preparing a neural tissue scaffold according to claim 1, characterized in that: Before the step of placing the first polymer into deionized water for stirring and emulsification, the method further comprises: The first polymer is cooled in a cold water bath, and then L-lysine solution is added dropwise to perform secondary chain extension modification.

5. The method for preparing a neural tissue scaffold according to claim 4, characterized in that: Before the step of cooling the first polymer using a cold water bath, the method further comprises: After the primary chain extension modification is completed, acetone is added to the first polymer to reduce the viscosity.

6. The method for preparing a neural tissue scaffold according to claim 1, characterized in that: During the heating reaction in a nitrogen atmosphere, the reaction temperature was 90° C. and the reaction time was 2 hours.

7. The method for preparing a neural tissue scaffold according to claim 1, characterized in that: The catalyst is organic bismuth with a concentration of 0.1 wt%.

8. The method for preparing a neural tissue scaffold according to claim 4, characterized in that: The molar ratio of 1,3-propylene glycol to L-lysine in the first mixed solution is 1:

1.

9. The method for preparing a neural tissue scaffold according to claim 1, characterized in that: The preset parameters include an electrospinning voltage parameter of 12 kV-22 kV and a syringe propulsion speed of 1.5 mL / h-2 mL / h.

10. A neural tissue scaffold, characterized in that: The neural tissue scaffold is prepared by the neural tissue scaffold preparation method described in any one of claims 1 to 9.

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