CN111569158B

CN111569158B - Preparation method of bionic tissue engineering scaffold with photo-thermal responsiveness and controllable drug release

Info

Publication number: CN111569158B
Application number: CN202010380785.5A
Authority: CN
Inventors: 李贵才; 刘逸凡; 慕函朔; 刘毅恒; 韩琦; 梁佳琦; 张林辉; 刘恒全; 张鲁中; 杨宇民
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2020-05-08
Filing date: 2020-05-08
Publication date: 2022-05-03
Anticipated expiration: 2040-05-08
Also published as: CN111569158A

Abstract

The invention discloses a preparation method of a bionic tissue engineering scaffold with photo-thermal responsiveness and controllable drug release. The method comprises the following specific steps: preparing bioactive nanoparticles; preparing an electrospinning receiving substrate; preparing a high molecular biological material solution, and blending and electrospinning the bioactive nano-particles and the high molecular biological material solution; and stripping the stent to obtain the stent. The stent has excellent biocompatibility, mechanical property, intelligent responsiveness and drug controlled release performance, can accelerate nerve regeneration, and solves the problems of unsatisfactory long-distance nerve defect repair effect and deficient function recovery. Provides ideal material and preparation method for nerve recovery of nerve injury patients.

Description

Preparation method of bionic tissue engineering scaffold with photo-thermal responsiveness and controllable drug release

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to a preparation method of a bionic tissue engineering scaffold with photo-thermal responsiveness and controllable drug release.

Background

Peripheral nerve injury is a common disease in clinic, and for the patient with short defect gap, precision suture or small gap sleeve can be used for directly repairing the defect, but for long-distance nerve injury, direct suture is not feasible. At present, with the development of microsurgical devices and techniques, the clinical treatment effect of peripheral nerve injury is continuously improved, and the adoption of more methods is to use a tissue engineering graft for bridging, which is proved to be an effective method for repairing peripheral nerve injury, but the repairing effect is still different from that of an autologous nerve graft, so that the development of a novel tissue engineering nerve graft is urgently needed.

Researches find that the surface topology of the graft plays an important role in regulating cell growth and tissue regeneration. The surface of the bracket has the advantages that the bionic topological structure can change the cell form and arrangement characteristics and can influence the growth and functions of the cells. The biomaterial with the topological structure is more beneficial to the growth of cells, and the cells grown on the scaffold according to the topological orientation formed by the topological structure have better orientation. The micro topological structure is formed on the nano fiber scaffold prepared by the electrostatic spinning method, and the adhesion, development, growth and differentiation of various cells on the tissue engineering scaffold can be obviously shown. The surface topological scaffold has related research reports in nerve regeneration, but the independent topological structure has limited effects of regulating and promoting nerve regeneration.

The bioactive molecule is a substance with different biological functions of promoting cell adhesion and growth, and can be protein, polypeptide, growth factor, etc. The loading of bioactive molecules into the graft has a significant promoting effect on cell and tissue regeneration. At present, the variety of bioactive molecules for peripheral nerve regeneration is more, including YIGSR, IKVAV, NGF, bFGF and the like, which can better promote the nerve regeneration process. However, after being implanted into the body, the bioactive molecules in the implant have the problems of poor stability and easy loss, and the nutrient supply is insufficient in the later period, so that the long-term implantation fails and the nerve function is difficult to recover. Thus, achieving a controlled release of bioactive molecules has a critical role in the success or failure of a nerve regeneration graft.

Smart responsive materials are a class of functional or smart polymers that respond by a variety of stimuli (electrical, thermal, optical, acoustical, magnetic) such as bending, contracting, folding, relaxing, or creeping. The research on the improvement of tumor treatment efficiency, drug controlled release and in vitro diagnosis of the intelligent responsive material is quite extensive, but the research on the controlled release of the drug in the implant by combining the intelligent responsive material with the tissue engineering implant is rarely reported. For the implanted artificial implant, the intelligent responsive material can carry out remote controlled drug release treatment on a specific injury part, and can deform under the action of external stimulation, so that the form and the function of the implant can be remotely and automatically regulated, and the implant with a complex three-dimensional form structure can be conveniently constructed. Thereby bringing convenience to patients and reducing the risk brought by secondary operation. Therefore, the intelligent responsive material has great potential application value in the field of tissue engineering.

In conclusion, the surface topology structure of the implant and the internal loading of the controllably released bioactive molecules have a significant effect on promoting nerve regeneration, but there are few reports on the related research of applying the bionic topological implant for realizing the controlled release of the bioactive molecules by adopting an intelligent responsive material to the peripheral nerve injury repair.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the existing problems and the defects of the prior art, the invention aims to solve the technical problem of constructing the artificial nerve graft which internally contains the intelligent response controlled release bioactive molecules and has the bionic micro-nano topological structure on the surface so as to better realize the function of accelerating nerve regeneration and provide beneficial choices for patients with peripheral nerve injury clinically. Therefore, the invention provides a preparation method of the bionic tissue engineering scaffold with photo-thermal responsiveness and controllable drug release. Preparing a surface micro-nano topological electrostatic spinning receiving substrate by adopting a micro-molding technology, modifying photo-thermal nanoparticles to obtain photo-thermal nanoparticles which have better dispersibility and load bioactive biomolecules, uniformly blending the modified photo-thermal nanoparticles and a high molecular biological material solution, and electro-spinning the mixed solution onto the electrostatic spinning receiving substrate with the surface having the micro-nano topological structure by utilizing an electrostatic spinning technology to obtain the artificial nerve graft internally containing the bioactive molecules with intelligent responsiveness and controlled release and with the bionic micro-nano topological structure on the surface. The surface of the graft has an anisotropic micro-nano topological structure, so that the distribution form and arrangement characteristics of nerve cells on the stent can be changed, and the functions of the nerve cells can be influenced. The implant with the topological structure is more beneficial to the migration of cells, and leads nerve cells to grow along the existing topological structure, so that the migration and growth of tissues are accelerated. Meanwhile, under the irradiation of near infrared light, the response of the internal photo-thermal nanoparticles realizes the controllable release of the loaded bioactive molecules, thereby being beneficial to the long-term function exertion and the improvement of the treatment effect of the nerve regeneration graft implanted into the body. The invention can accelerate nerve regeneration by controlling the concentration of bioactive factors released by photo-thermal stimulation of nano particles and the synergistic effect of the anisotropic topological structure on the surface of the bracket to induce the growth of nerve cells, and solves the problems of unsatisfactory long-distance nerve defect repair effect and deficient function recovery at present.

The technical scheme is as follows: in order to achieve the purpose, the invention adopts the following technical scheme: the invention provides a preparation method of a bionic tissue engineering scaffold with photo-thermal responsiveness and controllable drug release, aiming at better regulating and controlling the migration and growth of nerve tissues, improving the long-term effect of a nerve regeneration graft on repairing peripheral nerve injury and providing important reference for the treatment and rehabilitation of patients with peripheral nerve injury clinically.

The bionic tissue engineering scaffold with photo-thermal responsiveness and controllable drug release is characterized in that the scaffold body material is a high molecular biological material with excellent biocompatibility; the scaffold contains controllable-release bioactive molecules with nerve regeneration promoting function, and the bioactive molecules are loaded by nanoparticles with photothermal effect; the surface of the scaffold is provided with an anisotropic topological structure capable of regulating and controlling the oriented growth and migration of cells; the support can better simulate the microenvironment for nerve regeneration.

As an optimization: the polymer biomaterial with excellent biocompatibility is chitin, chitosan, alginate, collagen, Polycaprolactone (PCL) or Polylactide (PLA).

As an optimization: the bioactive molecule capable of promoting the nerve regeneration function and controllable releasing is polysaccharide, nucleic acid, protein, lipid, polypeptide or growth factor.

As an optimization: the nano particles with the photo-thermal effect are carbon nano tubes, ferroferric oxide or gold nano particles.

As an optimization: the anisotropic topological structure capable of regulating and controlling the oriented growth and migration of cells comprises a microscopic nano topological structure and a regular micro-nano groove.

As an optimization: the support can better simulate the microenvironment for nerve regeneration, including glial cells, nerve growth factors and extracellular matrix.

The preparation method of the bionic tissue engineering scaffold with photo-thermal responsiveness and controllable drug release comprises the following steps:

(1) preparing bioactive nanoparticles loaded with bioactive molecules with nerve regeneration promoting function;

(2) preparing a high molecular biological material solution, blending the bioactive nano-particles and the high molecular biological material solution, and then electrospinning

(3) Preparing an electrostatic spinning receiving substrate with an anisotropic topological structure on the surface by using a micro-molding method; (ii) a

(4) The bionic topological tissue engineering scaffold with the nerve regeneration promoting function is prepared by adopting an electrostatic spinning method.

As an optimization: the method for modifying the photo-thermal nanoparticles in the step (1) comprises the following steps: the photo-thermal nanoparticles can be coated and modified by dopamine hydrochloride (DOPA), genipin or aminopropyl ethoxysilane coupling Agent (APTE).

As an optimization: in the step (2), the blending mass-volume ratio of the bioactive nano-particles to the polymer biological material solution is 1:100 to 1: 10000.

As an optimization: the mould used in the micro-moulding method in the step (3) comprises a PDMS mould or a PMMA mould; the electrospinning receiving substrate can be a natural biological material substrate or a synthetic biological material substrate, and the substrate is formed by stamping on a cover glass through a micro-molding method.

Taking preparation of a PCL/CS scaffold modified by YR \ DFO-Dopa @ MWCNT as an example, the bionic topological tissue engineering scaffold prepared by the method comprises bioactive molecules dopamine Dopa and polypeptide YR which have nerve regeneration promoting function and can be controllably released, and the bioactive molecules (dopamine Dopa and polypeptide YR) are loaded by nano-particle carbon nano-tubes with photo-thermal effect; the electrostatic spinning receiving plate prepared by the micro-molding method is obtained by stamping the chitosan artemisia seed mixed glue, and the stamped stamp surface has a bionic topological structure, so that the surface of the bracket can have an anisotropic topological structure after an electrostatic spinning film of the chitosan artemisia seed stamp is stripped from the chitosan artemisia seed stamp by electrospinning; meanwhile, the bionic topological tissue engineering scaffold prepared by the method is prepared from a macromolecular chemical material polycaprolactone PCL and a natural material chitosan CS, and has the advantages of good biocompatibility and the like by means of the condition that the macromolecular chemical material polycaprolactone material has excellent fiber forming capability under the electrostatic spinning technology, uniform fiber thickness and strong mechanical property, and the conditions that the chitosan material is difficult to maintain effective electrostatic spinning operation when being subjected to electrostatic spinning independently are overcome, so that the conditions that the chitosan spinning has poor fiber forming capability, nonuniform fiber thickness and insufficient mechanical property are caused. Meanwhile, the excellent biocompatibility of the chitosan, the cell recognition signal on the surface of the chitosan and the high affinity with nerve cells are utilized, so that the PCL chitosan mixed scaffold is stable in performance and is more beneficial to the regeneration research of peripheral nerve cells.

The method specifically comprises the following steps:

1. preparing a nano particle loaded with bioactive molecules and having a photothermal effect, comprising the following steps:

(1) putting 0.24g of Tris (hydroxymethyl) aminoethane into 200ml of Milli-Q, fully stirring to prepare a Tris solution with the pH value of 8.5, and titrating the Tris solution to 8.5 by using a method of titrating NaOH if the pH value is lower than 8.5;

(2) adding dopamine Dopa powder into a Tris solution to prepare a polydopamine solution, fully stirring, adding nanoparticles into the polydopamine solution after the solution is fully stirred to become reddish brown, adding 1ml of polydopamine solution into every 10mg of nanoparticles, carrying out light-shielding oscillation reaction for 72 hours or more, centrifuging at 10000r/min, and then drying the rest precipitate in a drying box to obtain Dopa @ MWCNT; wherein, the ratio of the Dopa to the Tris solution is that 2mg of Dopa is added into 1ml of Tris solution to prepare polydopamine solution;

(3) wrapping the container filled with the mixed solution with tinfoil for shading treatment, poking a plurality of holes at the opening of the container by using sharp objects to ensure that oxygen in the air can be contacted with the solution, placing the container wrapped with the tinfoil and filled with the mixed solution into a mechanical shaking table for oscillation, removing supernatant from the container of the shaking table after oscillation, taking out the solution, precipitating and drying;

(4) Washing the dried Dopa @ MWCNT once by using Milli-Q, putting the washed solution into a centrifuge for centrifugation, discarding the centrifuged supernatant, and drying and precipitating to obtain Dopa @ MWCNT powder;

(5) adding the Dopa @ MWCNT powder into a mixed solution of a DFO solution and a YR solution, placing the solution into a mechanical shaking table to vibrate, taking out the solution from the shaking table after vibration, placing the solution into a centrifugal machine to centrifuge, wherein the centrifugation speed is 10000r/min, and the time is 3 min; wherein the concentration of the prepared DFO solution is 200 mug/ml, the concentration of the YR solution is 200 mug/ml, and the volume ratio of the prepared DFO solution of 200 mug/ml to the mixed solution of 200 mug/ml YR solution is 1: 1; adding maximum 10mg Dopa @ MWCNT powder into a mixed solution of 1ml of DFO solution and 1ml of YR solution, wherein the mixed solution needs to be vibrated in a mechanical shaker for 6 hours or more to ensure that oxygen in the air can contact the solution;

(6) and discarding the centrifuged supernatant, and drying the remaining precipitate in a drying oven to obtain YR \ DFO-Dopa @ MWCNT.

2. Preparing a chitosan and artemisia seed solution, which comprises the following steps:

(1) dissolving glacial acetic acid in water, wherein the concentration of the glacial acetic acid solution is 3%; wherein, the glacial acetic acid solvent is water, and the volume ratio of the glacial acetic acid to the water is 3: 100, respectively;

(2) Preparing a chitosan solution: the 3% glacial acetic acid solution is a solvent of chitosan, the concentration of the chitosan solution is 3%, and in the preparation process, the just prepared chitosan solution needs to be stirred for 24 hours; wherein the mass-volume ratio of the chitosan to the glacial acetic acid solution is 3: 100, respectively;

(3) adding 0.1g of artemisia desertorum seeds into every 5ml of chitosan solution, stirring the prepared chitosan and artemisia desertorum seed solution for 24 hours, wherein the mass-volume ratio of the artemisia desertorum seeds to the chitosan solution is 1: 500, a step of;

(4) and (3) putting the prepared chitosan and artemisia seed solution into a 4-degree refrigerator for sealing and storing.

3. The preparation method of the electrostatic spinning receiving plate comprises the following steps:

(1) dripping a drop of chitosan and artemisia seed solution on a glass wafer by using an injector, imprinting the topological anisotropic topological structure hydrogel on the glass wafer on which the solution is dripped, ensuring that no bubbles are generated in the solution between the anisotropic topological structure hydrogel and the glass wafer, extruding and discharging the generated bubbles, and standing the imprinted anisotropic topological structure hydrogel and the glass wafer for 24 hours at room temperature; wherein, the topological structure units of the anisotropic topological structure hydrogel are respectively 10 μm, 30 μm or 50 μm;

(2) The anisotropic topology hydrogel was separated from the glass wafer. Putting the stripped glass wafer into a big dish, and then carrying out alkali treatment by using a NaOH solution with the solution concentration of 4%;

(3) carefully cleaning the glass wafer for more than three times by using Milli-Q until the cleaning solution is neutral by pH test paper, airing the cleaned glass wafer in a vacuum drying oven, observing under a light mirror after airing, and drying and storing after confirming no error; the topological structure observed under the optical lens has no large amount of small bubbles, if a large amount of small bubbles appear, the alkali treatment step is not completely sufficient, and the alkali treatment is carried out;

(4) the aluminum foil is covered on a hard board, so that the surface of the receiving plate has electric conductivity, the conductive substance of the receiving plate is the aluminum foil, the aluminum foil is used for connecting electrodes on the back of the receiving plate, and the front of the receiving plate is stuck with a glass wafer which is air-dried, stored, complete, clear and free of a large number of small bubbles and has an anisotropic topological structure, and the receiving plate is the receiving end of an electrospinning machine.

4. The bionic tissue engineering scaffold with photo-thermal responsiveness and controllable drug release is prepared by utilizing an electrostatic spinning technology, and comprises the following steps:

(1) preparing 10 wt% PCL solution, shaking and stirring 1g PCL and 10ml hexafluoroisopropanol at room temperature for 1d, and taking out for storage; wherein the mass volume ratio of the PCL to the hexafluoroisopropanol solution is 1: 10;

(2) Preparing 2 wt% CS solution, shaking and stirring 0.2gCS and 10ml hexafluoroisopropanol for 1d, taking out and storing; wherein the mass volume ratio of CS to hexafluoroisopropanol is 1: 50;

(3) uniformly mixing 2 wt% of CS solution and 10 wt% of PCL solution, and mixing 5ml of PCL solution and 200 mu L of CS solution, wherein the volume ratio of the PCL solution to the CS solution is 25:1, and the solution is uniformly mixed at room temperature for 1 d;

(4) uniformly mixing the prepared PCL/CS solution with YR \ DFO-Dopa @ MWCNT powder, wherein the mass-volume ratio of the YR \ DFO-Dopa @ MWCNT powder to the PCL/CS mixed solution is controlled within the range of 0.01-5 mg/mL;

(5) putting the mixed solvent into an electrostatic spinning transmitting end injector to serve as a transmitting end; taking a glass wafer with an anisotropic topological structure on the surface as a receiving end of electrostatic spinning, adjusting parameters and then carrying out electrospinning, wherein the distance between a needle head and a receiving plate during electrospinning is 15cm, the voltage during electrospinning is 20.0kv, the speed during electrospinning is 0.160ml/h, the needle head selected during electrospinning is a No. 19 needle head, the electrospinning time is 5h, and the temperature during electrospinning is set to be room temperature;

(6) the spun receiving plate with the support is taken down and placed in a glass vessel with distilled water to strip the spinning membrane support, the process of stripping spinning is carried out in water, the topological structure is easy to be damaged by dragging in the air, the stripping spinning is to separate the support from a glass wafer of the receiving plate, the side with the topological structure turned out is placed on a new transparent glass sheet upwards, the stripped support is dried and stored in a drying box, the stripped glass wafer with the topological structure is dried and stored in the drying box, and the glass wafer with the topological structure can be recycled for many times if the topological structure is not damaged after being dried.

The technical effects are as follows: compared with the prior art, the invention has the following advantages.

1. The intelligent responsive biological material is developed for the first time and is used for constructing the nerve regeneration graft so as to realize the treatment and repair of the nerve tissue injury and obviously improve the success rate of treating the nerve injury patient by the tissue engineering nerve graft.

2. The photo-thermal nano-particles loaded with bioactive molecules and the bionic function topology are combined to construct the bionic tissue engineering nerve graft with controllable release of the bioactive molecules for the first time, the photo-thermal nano-particles have quick thermal responsiveness and can realize real-time remote control on release of the bioactive molecules, the micro-nano topological structure can regulate and accelerate migration and growth of tissues and cells, the biological functionality of the nerve graft is greatly improved, and the nerve graft is more beneficial to long-term treatment and rehabilitation of patients with nerve tissue injury in clinic.

3. The preparation method disclosed by the invention is non-toxic, harmless, green and environment-friendly, simple, convenient and feasible, is easy to operate, has adjustable near-infrared illumination time, and does not influence the activity of cells and tissues and the scientificity of experimental results in the illumination process. The dosage of the photo-thermal nano particles can be flexibly changed according to actual needs so as to achieve the regulation and control of different release rates of bioactive molecules, the biomaterial scaffold with different mechanical properties can be obtained through the electro-spinning technology and the regulation and control of biomaterial component parameters, and the personalized tissue engineering nerve graft with photo-thermal responsiveness and topological functionalization is obtained so as to match the clinical requirements of different patients, thereby realizing the personalized treatment and application of the nerve tissue engineering and regenerative medicine.

Drawings

FIG. 1 is a schematic view of the modification of photothermal nanoparticles of the present invention;

FIG. 2 is a schematic view of the topological preparation of the present invention having a surface with an anisotropic topological structure;

FIG. 3 is a schematic diagram of a bionic nerve scaffold with biological activity prepared by electrostatic spinning according to the invention;

FIG. 4 is a schematic representation of a scanning electron microscope topography of a modified MWCNT of the present invention;

FIG. 5 is a schematic view of the optical microscope observation of the surface topography of the electrospun neural stent of the present invention;

FIG. 6 is a schematic view of fluorescence observation of the growth morphology of Schwann cells on the surface of a nerve scaffold.

Detailed Description

In order to make the technical solution of the present invention more fully understood by those skilled in the art, the present invention will be further described with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the teachings of the present invention, and such equivalents also fall within the scope of the appended claims.

Detailed description of the preferred embodiment 1

Preparing nanoparticles loaded with bioactive molecules with a photothermal effect in a first step:

Tris solution with PH of 8.5 is prepared by Tris of Tris of nanometer of 50mg of poly of nanometer of the poly of at last of nanometer of 10ml of nanometer of 10ml of nanometer of at last. Packaging the container with the mixed solution with tinfoil, protecting from light, ventilating, and placing into a mechanical shaker for oscillation. And after shaking, removing the solution from the shaking table container, taking out the supernatant, taking out the precipitate and drying the precipitate. And then the dried powder is washed by Milli-Q. Centrifuging again, removing the supernatant, and drying the precipitate to obtain the Dopa @ MWCNT powder. Dopa @ MWCNT powder was added to 20ml of DFO/YR mixed solution and shaken in a mechanical shaker. After shaking, the solution is taken out of the shaking table and put into a centrifuge for centrifugation. And finally, discarding the supernatant, and drying the precipitate to obtain YR \ DFO-Dopa @ MWCNT.

The second step is to prepare a chitosan and artemisia seed solution:

dissolving glacial acetic acid in water to prepare a glacial acetic acid solution with the concentration of 3%. Adding chitosan powder into 3% glacial acetic acid solution as chitosan solvent, adding 7g chitosan into 100ml 3% glacial acetic acid solution, sealing the beaker with preservative film, and stirring on a magnetic stirrer for 24 h. Then 2g sargassum pallidum is added into the chitosan solution, and the mixture is stirred for 24 hours. And (3) putting the prepared chitosan and artemisia seed solution into a refrigerator at 4 ℃ for sealed storage.

Thirdly, preparing an electrostatic spinning receiving plate with an anisotropic topological structure:

dripping a drop of chitosan and artemisia seed solution on a glass wafer by using a syringe, impressing the hydrogel with the anisotropic topological structure and the topological structure width of 30 mu m on the glass wafer on which the solution is dripped, and standing for 24h at room temperature. After 24h the anisotropic topology hydrogel and the glass disc were separated. The removed glass disc was placed in a petri dish and subjected to alkali treatment with 4% NaOH solution for 15 min. And carefully cleaning the glass wafer for more than three times by using Milli-Q till the glass wafer is neutral, airing the glass wafer, observing the glass wafer under a light mirror, and drying and storing the glass wafer after no error is confirmed. The aluminum foil is coated on a cardboard, which is used on the back for connecting the electrodes. And adhering a glass wafer with an anisotropic topological structure on the front surface of the aluminum foil plate. The receiving plate is the receiving end of the electrospinning machine.

Fourthly, preparing the bionic tissue engineering scaffold with photo-thermal responsiveness and controllable drug release by utilizing an electrostatic spinning technology:

firstly, connecting a chitosan artemisia desertorum electrospinning receiving plate with a topological structure distance of 10 mu m to an electrode, then injecting YR \ DFO-Dopa @ MWCNT-PCL/CS mixed solution (the volume ratio of 10 wt% PCL to 2 wt% CS is 50:1) into a special electrostatic spinning injector, and carrying out electrospinning parameters: the voltage is 20kV, the distance from the needle head to the receiving plate is 15cm, the pushing speed is 0.160ml/h, and the time is 5 h. And (3) peeling the YR/DFO-Dopa @ MWCNT-PCL/CS scaffold from the glass slide in distilled water after electrospinning, and drying in a drying box after peeling to obtain the bionic topological tissue engineering scaffold with the nerve regeneration promoting function on the surface.

Specific example 2

Preparing nanoparticles loaded with bioactive molecules and having a photothermal effect:

tris (Tris-hydroxymethyl-aminoethane) is taken to prepare a Tris solution with the pH value of 8.5, then Dopa powder is taken to be added into the Tris solution to prepare a polydopamine solution, and finally 100mg of nanoparticles are added into 10ml of the polydopamine solution. Packaging the container with the mixed solution with tinfoil, protecting from light, ventilating, and placing into a mechanical shaker for oscillation. And after shaking, removing the solution from the shaking table container, taking out the supernatant, taking out the precipitate and drying the precipitate. And then the dried powder is washed by Milli-Q. Centrifuging again, removing the supernatant, and drying the precipitate to obtain the Dopa @ MWCNT powder. Dopa @ MWCNT powder was added to 20ml of DFO/YR mixed solution and shaken in a mechanical shaker. After shaking, the solution is taken out of the shaking table and put into a centrifuge for centrifugation. And finally, discarding the supernatant, and drying the precipitate to obtain YR \ DFO-Dopa @ MWCNT.

The second step is to prepare a chitosan and artemisia seed solution:

dissolving glacial acetic acid in water to prepare a glacial acetic acid solution with the concentration of 3%. Adding chitosan powder into 3% glacial acetic acid solution as chitosan solvent, adding 3g chitosan into 100ml 3% glacial acetic acid solution, sealing the beaker with preservative film, and stirring on a magnetic stirrer for 24 h. Then 2g sargassum pallidum is added into the chitosan solution, and the mixture is stirred for 24 hours. And (3) putting the prepared chitosan and artemisia seed solution into a refrigerator at 4 ℃ for sealed storage.

dripping a drop of chitosan and artemisia seed solution on a glass wafer by using a syringe, impressing hydrogel with an anisotropic topological structure and a topological structure width of 10 mu m on the glass wafer on which the solution is dripped, and standing for 24h at room temperature. After 24h the anisotropic topology hydrogel and the glass disc were separated. The removed glass disc was placed in a petri dish and subjected to alkali treatment with 4% NaOH solution for 15 min. And carefully cleaning the glass wafer for more than three times by using Milli-Q till the glass wafer is neutral, airing the glass wafer, observing the glass wafer under a light mirror, and drying and storing the glass wafer after no error is confirmed. The aluminum foil is coated on a cardboard, which is used on the back for connecting the electrodes. And adhering a glass wafer with an anisotropic topological structure on the front surface of the aluminum foil plate. The receiving plate is the receiving end of the electrospinning machine.

firstly, connecting a chitosan artemisia desertorum electrospinning receiving plate with a topological structure distance of 30 mu m to an electrode, then injecting YR \ DFO-Dopa @ MWCNT-PCL/CS mixed solution (the volume ratio of 10 wt% PCL to 2 wt% CS is 25:1) into a special electrostatic spinning injector, and carrying out electrospinning parameters: the voltage is 20kV, the distance from the needle head to the receiving plate is 15cm, the pushing speed is 0.160ml/h, and the time is 5 h. And (3) peeling the YR/DFO-Dopa @ MWCNT-PCL/CS scaffold from the glass slide in distilled water after electrospinning, and drying in a drying box after peeling to obtain the bionic topological tissue engineering scaffold with the nerve regeneration promoting function on the surface.

Specific example 3

tris (Tris-hydroxymethyl-aminoethane) is taken to prepare a Tris solution with the pH value of 8.5, then Dopa powder is taken to be added into the Tris solution to prepare a polydopamine solution, and finally 150mg of nano particles are added into 10ml of the polydopamine solution. Packaging the container with the mixed solution with tinfoil, protecting from light, ventilating, and placing into a mechanical shaker for oscillation. And after shaking, removing the solution from the shaking table container, taking out the supernatant, taking out the precipitate and drying the precipitate. And then the dried powder is washed by Milli-Q. Centrifuging again, removing the supernatant, and drying the precipitate to obtain the Dopa @ MWCNT powder. Dopa @ MWCNT powder was added to 20ml of DFO/YR mixed solution and shaken in a mechanical shaker. After shaking, the solution is taken out of the shaking table and put into a centrifuge for centrifugation. And finally, discarding the supernatant, and drying the precipitate to obtain YR \ DFO-Dopa @ MWCNT.

The second step is to prepare a chitosan and artemisia seed solution:

dissolving glacial acetic acid in water to prepare a glacial acetic acid solution with the concentration of 3%. Adding chitosan powder into 3% glacial acetic acid solution as chitosan solvent, adding 5g chitosan into 100ml 3% glacial acetic acid solution, sealing the beaker with preservative film, and stirring on a magnetic stirrer for 24 h. Then 2g sargassum pallidum is added into the chitosan solution, and the mixture is stirred for 24 hours. And (3) putting the prepared chitosan and artemisia seed solution into a refrigerator at 4 ℃ for sealed storage.

dripping a drop of chitosan and artemisia seed solution on a glass wafer by using a syringe, impressing the hydrogel with the anisotropic topological structure and the topological structure width of 50 mu m on the glass wafer on which the solution is dripped, and standing for 24h at room temperature. After 24h the anisotropic topology hydrogel and the glass disc were separated. The removed glass disc was placed in a petri dish and subjected to alkali treatment with 4% NaOH solution for 15 min. And carefully cleaning the glass wafer for more than three times by using Milli-Q till the glass wafer is neutral, airing the glass wafer, observing the glass wafer under a light mirror, and drying and storing the glass wafer after no error is confirmed. The aluminum foil is coated on a cardboard, which is used on the back for connecting the electrodes. And adhering a glass wafer with an anisotropic topological structure on the front surface of the aluminum foil plate. The receiving plate is the receiving end of the electrospinning machine.

firstly connecting a chitosan artemisia desertorum electrospinning receiving plate with a topological structure distance of 50 mu m to an electrode, then injecting YR \ DFO-Dopa @ MWCNT-PCL/CS mixed solution (the volume ratio of 10 wt% PCL to 2 wt% CS is 25:1) into a special electrostatic spinning injector, and carrying out electrospinning parameters: the voltage is 20kV, the distance from the needle head to the receiving plate is 15cm, the pushing speed is 0.160ml/h, and the time is 5 h. And (3) peeling the YR/DFO-Dopa @ MWCNT-PCL/CS scaffold from the glass slide in distilled water after electrospinning, and drying in a drying box after peeling to obtain the bionic topological tissue engineering scaffold with the nerve regeneration promoting function on the surface.

Specific example 4

(1) preparing Tris solution with pH of 8.5 with Tris (hydroxymethyl) aminoethane, adding Dopa powder into Tris solution to prepare polydopamine solution, and adding nano particles into polydopamine solution

(2) And then wrapping the container of the mixed solution with tinfoil for light-shielding and ventilation treatment, placing the container into a mechanical shaking table for oscillation, and removing the supernatant from the shaking table container after oscillation, taking out the supernatant, precipitating and drying. Then the dried powder was washed once with Milli-Q. Centrifuging again, removing supernatant, and drying and precipitating to obtain Dopa @ MWCNT powder

(3) Adding the Dopa @ MWCNT powder into a mixed solution of the DFO solution and the YR solution, placing the mixed solution into a mechanical shaking table to shake, taking the solution out of the shaking table after shaking, and placing the solution into a centrifugal machine for centrifugation. Finally, discarding the supernatant, and drying the precipitate to obtain YR \ DFO-Dopa @ MWCNT

The second step is to prepare a chitosan and artemisia seed solution:

(1) the glacial acetic acid is prepared into a 3% glacial acetic acid solution.

(2) Taking out chitosan powder, putting chitosan as solute and 3% glacial acetic acid solution as solvent into a beaker according to the proportion, putting a rotor, and sealing the opening of the beaker by using a preservative film to prevent acetic acid from volatilizing. And (3) placing the beaker into a magnetic stirrer to stir for 24 hours, and preparing a 3% chitosan solution.

(3) And taking out the stirred solution after 24 hours, and taking out the artemisia desertorum seeds. Adding Artemisia desertorum seed into the solution. And continuously stirring for 24 hours by using a magnetic stirrer, and sealing the mouth of the beaker by using a preservative film. And preparing the required CS + SA solution. And the prepared solution is stored in a 4-degree refrigerator.

(1) the prepared CS + SA solution was drawn into a 1ml syringe. A drop of the solution was applied to the glass wafer using a syringe. And then covering the anisotropic topological structure hydrogel on a glass wafer to imprint a CS + SA solution, and simultaneously ensuring that no bubbles are generated in the solution between the anisotropic topological structure hydrogel and the glass wafer. Then, the mixture was left to stand in the air at room temperature for 24 hours.

(2) After 24h, the anisotropic topology hydrogel and the glass disc were separated. At this point, the CS + SA solution has solidified to have an anisotropic topology and adhered to the glass wafer.

(3) And (3) putting the taken glass wafer into a large dish, carefully immersing the glass wafer in a 4% NaOH solution for treatment, soaking the glass wafer in the NaOH solution for half an hour, carefully cleaning the glass wafer for more than three times by using triple distilled water until the cleaning solution is neutral through pH test paper, and airing and storing for later use to obtain the electrostatic spinning receiving plate with the anisotropic topological structure.

(1) preparing an aluminum foil and a hard board, covering and wrapping the hard board with the aluminum foil, wherein the aluminum foil is a receiving board, taking out the stored glass wafer with the anisotropic topological structure, enabling the side with the anisotropic topological structure to face outwards, and adhering the back of the glass sheet to the surface of the receiving board, namely the electrostatic spinning receiving board is manufactured and can be directly used as a receiving end of an electric spinning machine.

(2) Preparing 10 wt% of PCL, wherein the mass volume ratio of the PCL to hexafluoroisopropanol is 1:10, and stirring for 1d by shaking. Preparing 2 wt% of CS, wherein the mass volume ratio of CS to hexafluoroisopropanol is 1:50, shaking and stirring for 1d, and taking out.

(3) Uniformly mixing 2 wt% of CS and 10 wt% of PCL at room temperature, placing 5ml of 10 wt% of PCL and 200 mu L of 2% of wtCS in a shaking table, shaking and stirring for 1d, and taking out.

(4) Uniformly mixing and stirring the prepared PCL/CS solution and YR \ DFO-Dopa @ MWCNT powder to ensure that the PCL/CS solution is fully stirred

(5) In a super clean bench, a receiving plate with anisotropic topological structure electrostatic spinning is placed at the receiving end of an electrostatic spinning machine, and the receiving plate is connected with an electrode; and meanwhile, injecting the mixed solvent into an injector special for electrostatic spinning, namely an emitting end of the electrostatic spinning machine, adjusting the propelling speed of the emitting end and pulling down a super clean bench protective cover after confirming that the whole machine is assembled without errors when the emitting end and the receiving end of the electrostatic spinning machine are assembled, and switching on a power supply to start the electrostatic spinning process.

(6) Closing the instrument after the electrostatic spinning lasts for 5 hours, taking down the spun receiving plate with the bionic topological tissue engineering bracket, peeling the bracket and the receiving plate in a glass dish filled with distilled water, and peeling without rough pulling so as to avoid destroying the topological structure on the bracket or causing deformation of the topological structure in the peeling process. The stripped stent was placed on a transparent glass disc placed in a glass dish in advance. Taking the scaffold out of water, airing and storing at room temperature to obtain the tissue engineering scaffold which has the bioactive molecules with the function of promoting nerve regeneration in the scaffold and the bionic topological structure on the surface of the scaffold.

Claims

1. A preparation method of a bionic tissue engineering scaffold with photo-thermal responsiveness and controllable drug release is provided, wherein a scaffold body material is a high molecular biological material with excellent biocompatibility; the method is characterized in that: the scaffold contains controllable-release bioactive molecules with the function of promoting nerve regeneration, and the bioactive molecules are loaded by nanoparticles with a photothermal effect; the surface of the scaffold is provided with an anisotropic topological structure capable of regulating and controlling the oriented growth and migration of cells; the support can better simulate the microenvironment for nerve regeneration;

(1) preparing bioactive nano particles loaded with bioactive molecules with the function of promoting nerve regeneration;

(2) preparing a high molecular biological material solution;

(3) preparing an electrostatic spinning receiving substrate with an anisotropic topological structure on the surface by adopting a micro-molding method;

(4) and (3) mixing the (1) and the (2) and then electrospinning the mixture on a receiving substrate to prepare the bionic tissue engineering scaffold with photo-thermal response and controllable drug release.

2. The method of claim 1 for preparing a biomimetic tissue engineering scaffold with photo-thermal response and controlled drug release, characterized in that: the polymer biomaterial with excellent biocompatibility is chitin, chitosan, alginate, collagen, Polycaprolactone (PCL) or Polylactide (PLA).

3. The method of claim 1 for preparing a biomimetic tissue engineering scaffold with photo-thermal response and controlled drug release, characterized in that: the bioactive molecule capable of promoting the nerve regeneration function and controllable releasing is polysaccharide, nucleic acid, protein, lipid, polypeptide or growth factor.

4. The method of claim 1 for preparing a biomimetic tissue engineering scaffold with photo-thermal response and controlled drug release, characterized in that: the nano particles with the photo-thermal effect are carbon nano tubes, ferroferric oxide or gold nano particles.

5. The method of claim 1 for preparing a biomimetic tissue engineering scaffold with photo-thermal response and controlled drug release, characterized in that: the anisotropic topological structure capable of regulating and controlling the directional growth and migration of cells comprises a directional nano topological structure, a regularly arranged micro-groove structure or a combination of the directional nano topological structure and the regularly arranged micro-groove structure.

6. The method of claim 1 for preparing a biomimetic tissue engineering scaffold with photo-thermal response and controlled drug release, characterized in that: the support can better simulate the microenvironment for nerve regeneration, including a physical microenvironment, a glial microenvironment, a nerve growth factor microenvironment and an extracellular matrix microenvironment.

7. The method of claim 1 for preparing a biomimetic tissue engineering scaffold with photo-thermal response and controlled drug release, characterized in that: the method for modifying the photo-thermal nanoparticles in the step (1) comprises the following steps: the photo-thermal nano-particles can be coated and modified by dopamine hydrochloride (DOPA), genipin or aminopropyl ethoxysilane coupling Agent (APTE); the polymer solution in step (2) may be a natural or synthetic polymer solution having good biocompatibility.

8. The method of claim 1 for preparing a biomimetic tissue engineering scaffold with photo-thermal response and controlled drug release, characterized in that: the mould used in the micro-moulding method in the step (3) comprises a PDMS mould or a PMMA mould; the electrostatic spinning receiving substrate can be a natural biomaterial or synthetic biomaterial substrate, and the substrate is formed by stamping on a cover glass by a micro-molding method.

9. The method of claim 1 for preparing a biomimetic tissue engineering scaffold with photo-thermal response and controlled drug release, characterized in that: in the step (4), the blending mass-volume ratio of the bioactive nano particles to the polymer biological material solution is 1:100 to 1: 10000.