CN112250805B - Preparation method of artificial nervous tissue engineering material, material and application thereof - Google Patents
Preparation method of artificial nervous tissue engineering material, material and application thereof Download PDFInfo
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- CN112250805B CN112250805B CN202011162014.5A CN202011162014A CN112250805B CN 112250805 B CN112250805 B CN 112250805B CN 202011162014 A CN202011162014 A CN 202011162014A CN 112250805 B CN112250805 B CN 112250805B
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Abstract
The invention provides a preparation method of an artificial neural tissue engineering material, which comprises the following steps: (1) preparation of a pre-polymerization solution: acrylamide is used as a monomer, diethoxyacetophenone is used as an initiator, and acrylamide, polylysine, N-N, methylene acrylamide, diethoxyacetophenone and N, N, N ', N' -tetramethyl ethylenediamine are sequentially added into a container to form a pre-polymerization liquid; (2) preparation of hydrogel Material: and adding the pre-polymerization solution into the template, and reacting for 3-4 hours under ultraviolet illumination to form the hydrogel material. The invention solves the technical problem that the prior art lacks materials which can be effectively applied to the repair of nervous system injury, in particular materials which can reconstruct the nerve electrical signal conduction in the early stage.
Description
Technical Field
The invention belongs to the technical field of biological materials, and particularly relates to a preparation method of an artificial neural tissue engineering material, the material and application thereof.
Background
The nervous system is a functional regulatory system which plays a leading role in the human body and is composed of a central part and peripheral parts thereof, wherein the central part comprises the brain and the spinal cord, and the peripheral parts constitute the peripheral nervous system. At present, millions of people all over the world suffer from nervous system injury, the latest global epidemiological survey in 2015 shows that the incidence rate of peripheral nerve injury is as high as 13-23/100000, and about 100 ten thousand cases of peripheral nerve injury are newly increased every year in China, wherein about 30 ten thousand cases of nerve defect are caused.
There are great difficulties in the repair of neurological damage in the clinic. In the case of peripheral nerves, when acute axonal injury is formed early after injury, dystrophic vesicles begin to form at the ends, and the stump axons disintegrate in the 24-72 hours later after injury, schwann cells proliferate, and macrophages begin the process of clearing. In addition, the tissues innervated by the damaged peripheral nerves lose the trophism of nerves, the muscles lose the contractile function, the muscle tone disappears, and the muscles lose the nerve-suppressing action to cause fibrillation, which accelerates muscle atrophy. After the muscle atrophy, fibrous tissues around the muscle are deposited, and the connection of regenerated nerves and the reconstruction end plate of the muscle fiber is influenced.
Unlike peripheral nerves, the central nervous system is damaged by very limited spontaneous regenerative functions through apoptosis and necrosis, with the consequences of spinal cord injury being mainly hemiplegia, paraplegia, and dysuria, as well as a decline in cognitive, motor, and sensory functions (e.g., parkinson's disease, alzheimer's disease, and multiple sclerosis). Loss of vision from retinal diseases also falls into this category (e.g., retinitis pigmentosa and age-related macular degeneration). At present, only drug intervention treatment can be carried out, and the efficacy is limited to delaying the progress of the disease. In addition, because of the unique anatomical structure of the central nervous system, transplanted cells and other components are not favored at the injured site, drugs and other bioactive components are difficult to penetrate the blood-brain barrier, further increasing the difficulty of central nervous repair.
In view of the above, there is a need to find a better way to repair damaged nerves, whether in the central or peripheral nervous system. The current nerve tissue engineering provides a good regeneration environment for the damage repair of peripheral nerves.
In the field of regeneration and repair after nerve injury (especially in the peripheral nervous system), exciting results have been achieved with the common goal of directing regenerated nerve fibers directly into the distal nerve sheath and achieving point-to-point precise docking to promote axonal regeneration and functional recovery. After extensive research, surgeons have now been able to obtain some interesting synthetic materials to bridge the neurological deficit. Jafar Ai et al found that use of Poly-D, L-lactic-co-glycolic acid (PLGA) as a Neural scaffolding material can improve cell adhesion, neural cell division and outgrowth of Neural synapses (Ai, J., kiasat-Dolatabadi, A., ebrahis-Barough, S., ai, A., lotfibakhsuish, N., norouzi-Javidan, A., saberi, H., arjmand, B.and Aghayan, H. (2013), polymeric Scaffolds in Neural Tissue Engineering: A review. Archives of Neuroscience,1 (1), pp.15-20). Serivisoot et al studied the characteristics of PEDOT scaffolds and found that PEDOT can increase axonal nerve conduit growth. PANI and PEDOT were chemically synthesized and then added to the collagen solution to make a cell suspension. This three-dimensional conductive collagen gel exhibits good cellular compatibility while increasing the growth of nerve axons compared to non-conductive collagen gels (sirivisot, s., pareta, r. And Harrison, b. (2013). Protocol and cell responses in a three-dimensional conductive collagen gel scans with conductive polymers for tissue regeneration. Interface Focus,4 (1), pp. 20130050-20130050). Ghasemii-mobarake et al prepared an electrostatic Conductive nanofiber scaffold Using PANI and PCL/Gelatin, on which Neural Stem Cells (NSCs) were seeded and electrically stimulated, and showed enhanced adhesion, proliferation and neurite outgrowth of Neural Stem Cells (NSCs) (ghasemii-mobarake, l., prabhakaran, m., morshend, m., nasr-eshahani, m. And Ramakrishna, s. (2009) electric Stimulation of new Cells Using Conductive Nanofibrous scans for new Tissue Engineering Part a,15 (11), pp.3605-3619). Pires et al found that Carbon Nanotubes (CNTs) have excellent electrical conductivity, large stiffness and high aspect ratio, have strong ability to absorb strain and induce electrical conduction, while maintaining the stability of the scaffold structure (Pires, F., ferreira, Q., rodrigues, C., morgado, J.and Ferreira, F. (2015). Neural stem cell differentiation by electrical stimulation using a cross-linked PEDOT substrate: expanding the user of biological modulated coupled diffusion for Neural tissue engineering. Biological analysis Acta (BBA) -General substrates, 1850 (6), 1858-1158).
However, there is little research interest on how to restore the conduction of the neuroelectric signal as soon as possible in the early stage of injury repair. The earlier the conduction of nerve electrical signals at the nerve injury position is established, the earlier the pathway from the spinal cord to peripheral nerves is opened, the nerve function can be more timely and effectively promoted to be recovered, and the muscular atrophy of the injured nerve branch and join in marriage area is prevented.
Disclosure of Invention
Therefore, the invention provides a preparation method of an artificial neural tissue engineering material, the material and application thereof, and solves the technical problem that the prior art lacks a material which can be effectively applied to repairing nervous system injury, in particular a material which can reconstruct nerve electrical signal conduction in the early stage.
In order to solve the technical problems, the invention provides a preparation method of an artificial neural tissue engineering material, which comprises the following steps:
(1) Preparing a pre-polymerization liquid: acrylamide is used as a monomer, diethoxyacetophenone is used as an initiator, and acrylamide, polylysine, N-N, methylene acrylamide, diethoxyacetophenone and N, N, N ', N' -tetramethyl ethylenediamine are sequentially added into a container to form a pre-polymerization liquid;
(2) Preparation of hydrogel material: and adding the pre-polymerization solution into the template, and reacting for 3-4 hours under ultraviolet illumination to form the hydrogel material.
Preferably, the step (1) is: adding a PBS solution into a container, then adding acrylamide while stirring until the solution is clear; adding polylysine, stirring for 3-5 min, adding N-N, methylene acrylamide and diethoxyacetophenone, and stirring for 8-12 min; then adding N, N, N ', N' -tetramethyl ethylenediamine, and stirring for 15-20 min to form a pre-polymerization solution.
Preferably, in the step (1), 40ml of PBS solution, 6.14g of acrylamide, 0.414g of polylysine, 0.005g of N-N, methylene acrylamide and 300 μ L of diethoxyacetophenone and 0.05ml of N, N, N ', N' -tetramethylethylenediamine are added.
Preferably, the step (2) is: and dropping the prepolymer into a polytetrafluoroethylene template, and reacting for 3-4 hours under 365nm ultraviolet light to form the hydrogel material.
Preferably, the step (2) further comprises: after ultraviolet irradiation, the template is taken out, soaked in PBS solution, washed for 45min by a shaking table and washed for 3 to 5 times.
The invention also provides an artificial nervous tissue engineering material prepared by the preparation method.
The invention also provides application of the artificial neural tissue engineering material for repairing nerve injury, preparing a drug slow-release device and constructing a three-dimensional structure by 3D printing.
Preferably, in the early stage of nerve injury repair, nerve electrical signal conduction is reconstructed, support and stimulation are provided for the injured nerve, and the formation of tissue scars is reduced.
Preferably, the material is used as a scaffold material for nerve tissue engineering in the later period of nerve injury repair to promote the regeneration of the axon of the distal nerve.
According to the preparation method of the artificial nervous tissue engineering material, acrylamide is selected as a monomer, diethoxyacetophenone is selected as an initiator, the monomer dosage and the proportion of the monomer to the initiator, the monomer to a cross-linking agent N-N, and methylene acrylamide are set, so that the mechanical property of the artificial nervous tissue engineering material is improved, the material has better toughness and lower viscoelasticity, and the mechanical property of the material is further improved.
The artificial nerve tissue engineering material prepared by the invention is very similar to human nerve signal conduction, can sense nerve electrochemical signals, is very suitable for nerve bridging, and can respond nerve signals and guide nerve axons to grow to the correct direction. The artificial nerve formed by the ionic gel is connected into the broken nerve, so that the conduction of nerve electrical signals can be quickly reestablished in the early repair stage, the far end is continuously stimulated to prevent atrophy, certain support and stimulation can be given to tissues around the damaged nerve, the formation of tissue scars is slowed down, and a good biological microenvironment is provided for further nerve regeneration and repair. Meanwhile, the repair later stage can also be used as a scaffold material for nerve tissue engineering to promote the regeneration of the axon of the distal nerve. Therefore, the research of the nervous system injury repair technology based on the novel conductive ion hydrogel material has great significance for human life engineering.
The artificial nerve tissue engineering material prepared by the invention has adjustable mechanical property and good biocompatibility, thus being used as a biological scaffold for cell culture and cell and gene delivery, being assembled with biological agents to guide fusion, migration and division among cells, and simultaneously having binding sites of growth factors, peptides or cytokines. The material can identify chemical substances on certain specific pathogenic cells, further cause gel expansion or degradation to release a drug, realize targeted therapy and promote nerve repair.
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.
FIG. 1 is a diagram showing the results of cell safety evaluation experiments of the artificial neural tissue engineering material prepared in example 1 of the present invention;
FIG. 2 is a graph showing the results of an electrochemical analysis experiment on a hydrogel material prepared in example 1 of the present invention;
FIG. 3 is a graph showing the experimental results of the conductivity of electrical stimulation of the hydrogel material prepared in example 1 of the present invention;
FIG. 4 is a schematic view of a hydrogel material prepared in example 1 of the present invention.
Detailed Description
In order to explain the technical content, the objects and the effects of the present invention in detail, the following description will be given with reference to the embodiments.
The reagents and raw materials used in the present specification are all commercially available products except for specific instructions.
Example 1
The embodiment provides a preparation method of an artificial neural tissue engineering material, which comprises the following steps:
(1) Adding 40mL of PBS solution into a cleaning beaker, and then adding 6.14g of acrylamide while stirring until the solution is clear;
(2) Adding polylysine 0.414g, stirring for 3min, adding N-N0.005 g, methacrylamide, and diethoxyacetophenone 300 μ L, and stirring for 10min; adding 0.05mL of N, N' -tetramethylethylenediamine, and stirring for 20min to form a uniform prepolymerization solution;
(3) And (3) dripping the prepolymer obtained in the step (2) into a polytetrafluoroethylene mold, irradiating for 3 hours under an ultraviolet lamp, taking out, soaking in a PBS solution, and washing for 45min in a shaking table for 5 times in total.
The artificial neural tissue engineering material is obtained, as shown in figure 4. The following tests were carried out on this material.
Cell safety evaluation experiment:
(1) The detection was carried out by the CCK-8 method, using mouse fibroblasts (L929 cells) as model cells, and incubating L929 cells (5X 10 cells) in a 6-well plate 4 Cells/ml medium, total 3 ml medium containing 10% fetal bovine serum, 1% double antibody);
(2) After culturing for 6 hours in an incubator (37 ℃), adding a sample plate after cells are attached to the wall successfully, and adding 10 groups in total as parallel control;
(3) After incubating the sample for 24 hours, washing the sample with PBS for three times, adding 10% CCK-8 solution (CCK-8 is dissolved in a culture medium) to incubate in an incubator for 2 hours, and testing the absorbance (480 nm) in a pore plate by using an enzyme-labeling instrument to calculate the safety;
(4) The cell viability of the sample pieces after treatment was calculated by using the plate containing the sample pieces as a positive control and the absorbance value defined as 100% and the plate containing no cells as a negative control and the absorbance value defined as 0%. Based on the above experiment, the experimental results obtained in the experimental step are shown in fig. 1, and the cell survival rate of the sample slice after treatment is 89.762%.
Characterization of the materials:
by fixing the sheet hydrogel material at two ends of a tensile machine to perform a mechanical tensile test, when the tensile deformation is 12mm, the stress can reach over 36 kPa.
Electrochemical analysis of materials
Selecting 1/3 of rat sciatic nerve middle section to be fully dissociated and removing nerve bundle envelopes, cutting a sciatic nerve section with the length of 1cm, placing the sciatic nerve section on an insulating plate, dripping 37 ℃ and 0.9% of normal saline to be fully wetted, keeping nerves complete in a sham operation group, cutting a control group in the middle of the nerves and removing 5mm of nerve fibers, carefully sucking the normal saline to keep nerve amputation ends on two sides in a non-contact manner, bridging the hydrogel group on the basis of the control group, bridging the hydrogel ends on the two sides with the 5mm of hydrogel to ensure that the hydrogel is fully contacted with the nerve amputation ends, carefully sucking the normal saline, and preparing to carry out limb motion function determination. And (3) observing the motion function of the lower limbs: the amplitude of lower limb muscular movement was evaluated as the rate of change of tibia-knee-femur angulation, and knee joint angulation rate = (electrical stimulation posterior angulation-electrical stimulation anterior angulation)/electrical stimulation anterior angulation was calculated. The experimental results are shown in fig. 2, and the knee joint angulation change rate control group is similar to the hydrogel bridging group, and is obviously larger than the knee joint angulation change rate separation group. The results of the conductivity test of the electrical stimulation are shown in fig. 3, and the current passing through the proximal end, the distal end and the broken end (material) of the nerve end, i.e. the electrical conductivity, is respectively detected by using probes. According to the constant voltage, the larger the current passing through, the better the conductivity; 2 measurements per site, so there are two peaks per site; the results show that the material is electrically conductive in accordance with the original nerve (proximal, distal).
The artificial nerve tissue engineering material has excellent mechanical property and good biocompatibility, can be used as a nerve scaffold to guide and support nerve regeneration at the broken end of nerve injury, can realize the conduction of bioelectric signals of nerves at early stage, provides a cell transmission carrier and a drug release reservoir, can sense the change of environmental temperature or substance component concentration, realizes the controllable slow release of bioactive factors, and provides a self-sensing, self-adjusting and self-adapting multiple microenvironment for nerve injury repair. According to the invention, the specific contents of N, N ' -methylene bisacrylamide and diethoxyacetophenone are adjusted, so that the material has excellent toughness which can be matched with the mechanical property of a damaged tissue, and the prepared material can achieve good matching of the mechanical property of the damaged tissue from the aspects of strength, toughness, crosslinking density, water content and porosity by using the combination of acrylamide, polylysine, N-N, methylene acrylamide, diethoxyacetophenone and N, N, N ', N ' -tetramethyl ethylenediamine and exploring, researching and developing the using amount of the combination.
It should be understood that the above embodiments are only examples for clarity of description, and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. This list is neither intended to be exhaustive nor exhaustive. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (7)
1. A preparation method of an artificial neural tissue engineering material is characterized by comprising the following steps:
(1) Preparing a pre-polymerization liquid: acrylamide is used as a monomer, diethoxyacetophenone is used as an initiator, and acrylamide, polylysine, N-N, methylene acrylamide, diethoxyacetophenone and N, N, N ', N' -tetramethyl ethylenediamine are sequentially added into a container to form a pre-polymerization liquid, wherein the molar concentration ratio of the acrylamide to the polylysine to the N-N, methylene acrylamide to the diethoxyacetophenone to the N, N, N ', N' -tetramethyl ethylenediamine is (1700-2600): (65-100): (0.6-1.0): (30-45): (6-10);
(2) Preparation of hydrogel material: adding the prepolymerization solution into a template, and reacting for 3-4 hours under ultraviolet illumination to form a hydrogel material;
wherein the step (1) is as follows: adding a PBS solution into a container, adding acrylamide while stirring until the solution is clear; adding polylysine, stirring for 3-5 min, adding N-N, methylene acrylamide and diethoxy acetophenone, and stirring for 8-12 min; then adding N, N, N ', N' -tetramethyl ethylenediamine, and stirring for 15-20 min to form a pre-polymerization solution.
2. The method for preparing the artificial neural tissue engineering material according to claim 1, wherein the step (2) is: and dropping the prepolymer into a polytetrafluoroethylene template, and reacting for 3-4 hours under 365nm ultraviolet light to form the hydrogel material.
3. The method for preparing the artificial neural tissue engineering material according to claim 2, wherein the step (2) further comprises: after ultraviolet irradiation, the template is taken out, soaked in PBS solution, washed for 45min by a shaking table and washed for 3 to 5 times.
4. An artificial neural tissue engineering material characterized by being produced by the production method according to any one of claims 1 to 3.
5. The use of the artificial neural tissue engineering material according to claim 4, for nerve injury repair, for the preparation of drug delivery devices and for 3D printing to construct three-dimensional structures.
6. The use of the artificial neural tissue engineering material according to claim 5, wherein in the early stage of nerve injury repair, nerve electrical signal conduction is reconstructed, support and stimulation are provided for the injured nerve, and the formation of tissue scar is reduced.
7. The use of the artificial neural tissue engineering material according to claim 5, which is used as a scaffold material for neural tissue engineering to promote the regeneration of distal nerve axons in the later stage of nerve injury repair.
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