Neural restoration guiding catheter, preparation method and application

Abstract

The invention relates to a nerve repair guide catheter, a preparation method and application, an electrostatic spinning technology is adopted to construct a nanofiber membrane which is directionally and orderly arranged so as to guide nerve axons to directionally grow, and cell energy metabolism is promoted and tissue repair and regeneration are accelerated through metabolism and conversion of degradation products. Therefore, the nerve injury repairing effect better than that of traditional matrix materials without biological activity, such as PCL, is achieved. According to the invention, a novel solution is provided for solving the problem of insufficient biological activity of a traditional nerve conduit material by constructing a degradable high polymer material with biological activity. Therefore, the regeneration efficiency of peripheral nerve injury repair is improved, and the application prospect of the bioactive material in the fields of tissue engineering and regenerative medicine is expanded.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nerve conduit, in particular to a nerve repair guide conduit, a preparation method and application. BACKGROUND

[0002] Peripheral nerve injury (PNI) is a common disabling disease, which is usually caused by trauma, surgery, infection or other chronic diseases. PNI has high incidence, limited treatment methods and poor recovery effect. The main methods for treating peripheral nerve injury include autologous nerve transplantation or artificial material transplantation. At present, autologous nerve transplantation is the gold standard for clinical treatment, but the shortage of donor nerve tissue and complications such as scar tissue formation limit its therapeutic effect. Therefore, nerve guidance conduits (NGCs) prepared from biomaterials are widely used in the repair of PNI. Biomaterials can be divided into natural materials and synthetic materials, natural materials include collagen, silk fibroin, chitosan, etc., synthetic materials include polycaprolactone (PCL), polylactic acid (PLA) and polyurethane (PU) and the like. In addition, enhancing the metabolic state of cells is conducive to the anabolism and mitosis of cells, thereby accelerating tissue repair and regeneration. In the process of nerve tissue regeneration and repair, the growth of residual nerve, the migration and proliferation of SCs will all lead to the increase of energy consumption and metabolic level of cells. Studies have shown that cellular bioenergetics (CBE) can be used to regulate the metabolic state of cells, and by controlling energy substrates, it can regulate metabolism-related biosynthesis and mitotic behavior, thereby accelerating tissue repair and regeneration. The tricarboxylic acid cycle (TCA cycle) is a key pathway of cellular energy metabolism, as a core link of organic matter oxidative metabolism, it can significantly affect the process of cellular energy metabolism, and further affect the repair and regeneration of damaged tissues. The key intermediates in the process of TCA cycle, such as succinic acid, citric acid and α-ketoglutaric acid, are the core components of TCA cycle, which can significantly affect the process and efficiency of energy metabolism. Among them, succinic acid (also known as succinate, SuA) is an essential circulating metabolite in the TCA cycle, which serves as a substrate for succinate dehydrogenase (SDH) and helps to produce energy in the basic mitochondrial metabolic pathway. In addition, some metabolic regulators and exogenous small molecules, such as itaconic acid, can form competitive inhibition with specific enzymes of the TCA cycle, thereby affecting energy metabolism and immune regulation processes. Itaconic acid is one of the highest degree of induction metabolites in activated macrophages, which can participate in energy supply and inflammatory signal transduction through double regulation as an endogenous metabolic intermediate, and has antimicrobial and immunomodulatory activity. Studies have shown that itaconic acid can inhibit the production of SDH, regulate the level of succinic acid in cells, inhibit aerobic glycolysis, and reduce the production of ROS and IL-1β. However, there is still a lack of research on using NGCs with biological activity as a substrate material to treat large segment nerve tissue injury in the field of nerve repair. Therefore, it is of great significance to prepare nerve repair materials with biological activity, non-immunogenicity and degradability.

[0003] Therefore, the present application is proposed. SUMMARY

[0004] The application aims to provide a nerve repair guide catheter, a preparation method and an application, so as to solve the problem that bioactivity, non-immunogenicity and degradability cannot be combined in an artificial nerve catheter in the prior art.

[0005] To achieve one of the above-mentioned purposes, the application provides the following technical scheme:

[0006] A preparation method of a nerve repair guide catheter comprises the following steps:

[0007] S100, 1,3-propanediol, 1,4-butanediol, itaconic acid, succinic acid and sebacic acid are mixed according to a molar ratio of 0.25:0.25:0.05:0.315:0.135; after mixing, esterification is carried out under a nitrogen atmosphere to obtain an esterification liquid;

[0008] S200, under the protection of a nitrogen gas flow, tetrabutyl titanate is added to the esterification liquid as a catalyst, then the esterification liquid is heated and subjected to a polycondensation reaction under vacuum until a climbing rod effect appears, and a product PPBISS is obtained;

[0009] S300, PCL and PPBISS are dissolved in an organic solvent trifluoroethanol to obtain a PCL / PPBISS mixed solution;

[0010] S400, the PCL / PPBISS mixed solution is prepared into an orderly arranged PCL / PPBISS fiber membrane through an electrospinning process;

[0011] S500, after the PCL / PPBISS fiber membrane is treated by a plasma method to be hydrophilic, ultraviolet disinfection is carried out, then the PCL / PPBISS fiber membrane is wrapped into a tube to form a nerve repair guide catheter.

[0012] Preferably, in the step S100:

[0013] The esterification reaction is carried out at a temperature of 180°C.

[0014] Preferably, in the step S200:

[0015] The amount of tetrabutyl titanate added to the esterification liquid is 0.1% by mass fraction;

[0016] The polycondensation reaction is carried out at a temperature of 220°C, and after vacuumizing, a pressure of <300 Pa needs to be reached.

[0017] Preferably, in the step S300:

[0018] The mass concentration of the PCL / PPBISS mixed solution is 10%.

[0019] Preferably, in the step S400:

[0020] The parameters of the electrospinning process are: solution propulsion speed 1 mL / h, positive electrode voltage +16 kV, negative electrode voltage -2 kV, receiving distance 14 cm, and drum rotation speed 1000 r / min.

[0021] Preferably, in the step S500:

[0022] The time for hydrophilization by the plasma method is 15 s, and the time for ultraviolet disinfection is 15 min.

[0023] 8. A nerve repair guiding conduit prepared by the method of any one of claims 1-6, or the use of a nerve repair guiding conduit of claim 7 in the treatment of peripheral nerve injury.

[0024] To achieve the above-mentioned purpose two, the present application provides the following technical solutions:

[0025] A nerve repair guiding conduit prepared by the method as described above.

[0026] To achieve the above-mentioned purpose three, the present application provides the following technical solutions:

[0027] A nerve repair guiding conduit prepared by the method as described above, or the use of a nerve repair guiding conduit as described above in the treatment of peripheral nerve injury.

[0028] Compared with the prior art, the present application has important significance. Based on the mechanism of intracellular endogenous metabolites (succinic acid and itaconic acid) driving mitochondrial energy metabolism to regulate cell function, the present application designs and synthesizes degradable bioactive polyester materials containing succinic acid and itaconic acid functional monomers. Further, the present application uses electrospinning technology to construct a nanofiber membrane with directional and ordered arrangement to guide the directional growth of nerve axons, and promotes cell energy metabolism through the metabolic conversion of degradation products to accelerate tissue repair and regeneration, thereby achieving a better effect of nerve injury repair than traditional PCL and other non-bioactive substrate materials. The present application provides a new solution to the problem of insufficient bioactivity of traditional nerve conduit materials by constructing biologically active degradable polymer materials. Thus, the regeneration efficiency of peripheral nerve injury repair is improved, and the application prospect of bioactive materials in the field of tissue engineering and regenerative medicine is expanded.

[0029] In order to make the above-mentioned purpose, features and advantages of the present application more obvious and easy to understand, the following preferred embodiments are specifically described below with reference to the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0030] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 The results are shown in the 1H-NMR spectrum of the PPBISS prepared in this invention.

[0032] Figure 2 The results are from the permeation gel chromatography (GPC) test of the PPBISS prepared in this invention.

[0033] Figure 3 The images show the SEM images and diameter distributions of PCL / PPBISS fiber membranes prepared under different mixing ratios of PCL / PPBISS in this invention.

[0034] Figure 4 The effect of PCL / PPBISS fiber membranes prepared under different mixing ratios of PCL / PPBISS in this invention on DRG axon extension;

[0035] Among them, (A) the growth morphology of DRG on different fibrous membranes; (B) statistics on the average length of nerve axon growth; and (C) statistics on the longest length of nerve axon growth.

[0036] Figure 5 The images show the surface morphology of PCL / PPBISS fiber membranes prepared under different mixing ratios in this invention after being embedded in vivo for 2, 4, and 8 weeks. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0038] Example 1 (PB55 group)

[0039] 1,3-propanediol, 1,4-butanediol, itaconic acid, succinic acid, sebacic acid were mixed together according to the molar ratio of 0.25:0.25:0.05:0.315:0.135; after mixing, esterification reaction was carried out at 180°C under nitrogen atmosphere to obtain an esterification liquid;

[0040] Under the protection of nitrogen gas flow, 0.1% of tetrabutyl titanate by mass fraction was added to the esterification liquid as a catalyst, then the esterification liquid was heated to 220°C and subjected to polycondensation reaction under vacuum condition (<300 Pa) by using melt polycondensation method, until the climbing rod effect appeared, to obtain the product PPBISS.

[0041] PCL and PPBISS were dissolved together in the organic solvent trifluoroethanol to obtain a PCL and PPBISS mixed solution, with a mass concentration of 10% and a weight ratio of PCL to PPBISS of 5:5.

[0042] The sample bottle containing the polymer solution was placed on a magnetic stirrer for stirring for more than 24 h until the polymer sample was completely dissolved and uniformly mixed. The electrospinning parameters were as follows: solution propulsion speed 1 mL / h, positive electrode voltage +16 kV, negative electrode voltage -2 kV, receiving distance 14 cm, and drum rotation speed 1000 r / min.

[0043] Using a high-speed rotating drum collector (1000 r / min), the coupling effect of electric field and mechanical drawing was adjusted to induce the fibers to arrange in the circumferential direction of the drum, and a uniaxial ordered topological structure was obtained. According to the process parameters under this condition, after electrospinning for 20 hours, an ordered PCL / PPBISS fiber membrane was obtained.

[0044] The above PCL / PPBISS fiber membrane was cut into a long strip with a width of 12 mm. After 15 s of plasma hydrophilic treatment, the front and back surfaces of the material were each subjected to 15 min of ultraviolet sterilization. Then the material was wound into a tube in a super-clean bench to prepare a nerve repair guide catheter with an inner diameter of 1.5 mm and a length of 12 mm.

[0045] Example 2 (PB28 group)

[0046] In this example, the weight ratio of PCL to PPBISS was 2:8, and the rest was the same as in Example 1.

[0047] Example 3 (PB82 group)

[0048] In this example, the weight ratio of PCL to PPBISS was 8:2, and the rest was the same as in Example 1.

[0049] Example 4 (PCL group)

[0050] In this embodiment, the weight ratio of PCL to PPBISS is 10:0, and the rest is the same as example 1.

[0051] Example 5 (PPBISS group)

[0052] In this embodiment, the weight ratio of PCL to PPBISS is 0:10, and the rest is the same as example 1.

[0053] In the above examples, the PPBISS group cannot form a fiber membrane during the electrospinning process due to the absence of PCL. Therefore, the remaining experimental data are for the nerve repair guide conduit prepared by the remaining several groups of examples.

[0054] First, the prepared PPBISS in the example process was analyzed by nuclear magnetic resonance hydrogen spectrum.

[0055] Ensure that the material composition structure is consistent with the expected results, Figure 1 The proton chemical shift at 1.30 ppm (l, k), 1.61 ppm (j) and 2.29 ppm (i) is the characteristic absorption peak of the sebacic acid unit (-(CH2)4-CH2-CH2-COO-). The proton chemical shift at 1.97 ppm (c) and 4.12 ppm (d) is the (-(CH2)-CH2-O-) group in the 1,3-propanediol unit, and the proton chemical shift at 2.63 ppm (h) is the characteristic absorption peak of the (-COO-(CH2)2-) group in the succinic acid unit. The proton chemical shift at 6.76 ppm (g), 5.63 ppm (f) and 3.30 ppm (e) belongs to the (-CO-C(=CH2)-CH2-) group in the itaconic acid unit. The proton chemical shifts at 4.17 ppm (a) and 1.71 ppm (b) belong to the characteristic absorption peaks of the (-O-CH2-CH2-) group in the 1,4-butanediol unit, which indicates that the PPBISS material has been successfully prepared.

[0056] It tests the GPC of the PPBISS prepared in the example process.

[0057] The prepared PPBISS material is tested for molecular weight using permeation gel chromatography. Table 1 and Figure 2For the GPC test results, it can be seen that the number average molecular weight Mn in the PPBISS is 2.97 x 104 Dal; the weight average molecular weight Mw is 16.79 x 104 Dal, and the main peak molecular weight Mp is 6.05 x 104 g / mol. In addition, the Mn of the secondary peak 2 is 0.079 x 104 Dal, the Mw is 0.13 x 104 Dal, and the Mp is 850 g / mol. It shows that the molecular weight distribution of the PPBISS material is relatively wide (PDI = 5.66), and there is a certain amount of oligomer, which may be related to the random polycondensation process of the polyester synthesis. In combination with the above nuclear magnetic structure analysis, it can be shown that the PPBISS has been successfully synthesized.

[0058] Table 1 GPC results

[0059] Peak M n (×10 4 Dal M w (×10 4 Dal M P ( x 10 4 g / mol)]]> PDI 1 2.97 16.79 6.05 5.66 2 0.079 0.13 0.09 1.60

[0060] The micro-morphology of the PCL / PPBISS fiber membrane is studied and analyzed.

[0061] The oriented nanofiber prepared by electrospinning can guide the growth of neuron processes and arrange them along the fiber direction, thereby improving the efficiency of nerve regeneration. Therefore, the electrospinning process of the PPBISS material needs to be explored to prepare oriented nanofiber, and its process feasibility is evaluated in combination with macroscopic observation and SEM test. Since the solution of the single biobased polyester elastomer PPBISS and trifluoroethanol is spun on the tin paper surface, a relatively viscous film is formed, which is difficult to form a unidirectional ordered arrangement of fiber structure. This may be because the PPBISS multi-component copolyester molecular chain has high flexibility, and the fiber cannot be quickly solidified after forming during electrospinning, resulting in adhesion and fusion between the fibers. In view of the above defects of insufficient processability of PPBISS spinning, PCL commonly used in nerve conduit preparation is introduced for blending modification. PCL and PPBISS are co-dissolved in trifluoroethanol solvent in different mass ratios (10:0, 8:2, 5:5, 2:8), and a series of oriented composite fiber membranes (labeled as PCL, PB82, PB55 and PB28, respectively) are prepared by electrospinning technology. The fiber morphology is characterized by scanning electron microscopy, and the diameter distribution is counted by ImageJ software. As shown in FIG. 1, each group of fiber membranes shows a significant oriented arrangement feature, confirming that the optimized process can obtain fiber materials with unidirectional ordered arrangement structure. Among them, the average fiber diameter of the PCL group is 0.50 ± 0.20 μm, the average fiber diameter of the PB82 group is 0.83 ± 0.40 μm, the average fiber diameter of the PB55 group is 0.94 ± 0.38 μm, and the average fiber diameter of the PB28 group is 0.99 ± 0.44 μm. Figure 3

[0062] The differentiation of DRG axons is studied and analyzed. ​

[0063] The cultured DRG can be used for the study of axon growth and development. The DRG of fetal rats is used as an in vitro neuron model for differentiation experiments to explore the growth and differentiation of DRG axons on different material polyester composite fiber membranes. After 5 days of differentiation culture, the length of the axons is quantitatively counted, and the results are shown in Figure 4 .

[0064] After 5 days of differentiation culture, the nerve axons of DRG in each group are arranged along the fiber orientation direction, indicating that the unidirectional and ordered nanofiber has a guiding effect on the nerve axons. With the increase of the content of PPBISS in the material, the growth length of the nerve axons of DRG is longer, indicating that the introduction of PPBISS material has a promoting effect on the growth of DGR axons. As shown in Figure 4 (B), after 5 days of differentiation culture, the average growth length of DRG in PCL group, PB82 group, PB55 group and PB28 group is 578.6±49.41 μm, 631.2±132.9 μm, 967.9±339.1 μm and 1145.0±361.6 μm respectively. There is no significant difference between PCL group and PB82 group (ns), and there is significant difference between PB55 group and PB28 group (P < 0.1), and the average length of nerve axons in these two groups is longer, and the significant difference with other groups is P < 0.0001, and the average growth length of DRG nerve axons on PB28 group material is the highest, which is 2.0 times of PCL group. As shown in Figure 4 (C), after 5 days of culture in the cell differentiation culture solution, the longest growth length of DRG in PCL group is the shortest (657.1±10.1 μm), PB82 group is slightly longer (882.2±29.3 μm), PB55 group (1460.0±5.5 μm) is longer, and PB28 group is the longest, which is 1675.0±129.4 μm, about 2.5 times of pure PCL group, and there is significant difference between each group. In summary, with the increase of PPBISS component in the material, the average growth length and the average longest growth length of nerve axons will increase. Compared with pure PCL material, the increase of PPBISS component can help to enhance the differentiation promoting effect of the material on nerve cells.

[0065] The in vivo degradation of the material is tested and analyzed.

[0066] To evaluate the in vivo degradation characteristics of PCL / PPBISS composite materials, the morphology evolution of the material at different time points (2, 4, 8 weeks) is observed by subcutaneous implantation model system Figure 5). The results show that the fiber materials in PCL group, PB82 group, PB55 group and PB28 group gradually begin to degrade in the animal body, and with the extension of time, the material degradation is more obvious at the 4th and 8th week. At the 2nd week, the surface of PCL group is smooth, without obvious degradation signs, the surface of PB82, PB55 and PB28 groups is rougher, and the surface of PB28 group appears local defects, indicating that the PPBISS component accelerates the initial degradation of the material. At the 4th week, the surface of PCL group is slightly rough, the surface of PB82 group is rougher, the surface of PB55 group is significantly roughened, and the fiber of PB28 group is obviously broken and damaged. At the 8th week, the surface of PCL group continues to be roughened, but still maintains the structural integrity, the fiber breakage and damage of PB82, PB55 and PB28 groups are intensified, and the phenomenon of PB28 group is more obvious. In addition, at the 4th and 8th week, the fiber orientation degree of each group is obviously poor, which is due to the synergistic effect of mechanical stress (muscle activity) and chemical degradation in the body. The above results show that the materials in each group will obviously degrade after being implanted into the animal body, and with the passage of time, the degradation phenomenon will be more obvious. Among them, the degradation performance of PCL material is the worst, and with the increase of PPBISS component in the material, the material degradation is more obvious with time, which shows that the increase of PPBISS proportion can significantly enhance the hydrophilicity of the material and increase the content of degradable ester functional groups, thereby promoting the penetration of body fluid and enzymatic hydrolysis, and finally showing the degradation of the material from the surface to the inside.

[0067] In summary, the nerve repair guide catheter prepared by the present application is designed and synthesized by using the degradable bioactive polyester material (PPBISS) containing succinic acid and itaconic acid functional monomers. Further, the electrospinning technology is used to construct the directional and ordered nanofiber membrane to guide the directional growth of nerve axons, and the metabolic transformation of degradation products is used to promote cell energy metabolism and accelerate tissue repair and regeneration, so that the nerve injury repair effect is better than that of traditional substrate materials such as PCL which do not have biological activity

[0068] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application. It should be noted that similar reference numbers and letters represent similar items in the following drawings, therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

Claims

1. A method of making a nerve repair guiding catheter, characterized by, It comprises the following steps: S100, 1, 3-propanediol, 1, 4-butanediol, itaconic acid, butanedioic acid, sebacic acid are mixed according to the molar ratio of 0.25:0.25:0.05:0.315:0.135; after mixing, esterification reaction is carried out under nitrogen atmosphere to obtain esterification liquid; S200, under the protection of nitrogen flow, tetrabutyl titanate is added as catalyst in the esterification liquid, then the esterification liquid is heated and polycondensation reaction is carried out under vacuum condition by using melt polycondensation method, until the climbing rod effect appears, the product PPBISS is obtained; S300, PCL and PPBISS are dissolved in organic solvent trifluoroethanol to obtain PCL and PPBISS mixed solution; S400, the PCL and PPBISS mixed solution is prepared into ordered PCL / PPBISS fiber membrane by electrospinning process; S500, after the PCL / PPBISS fiber membrane is treated by plasma method and then sterilized by ultraviolet, the PCL / PPBISS fiber membrane is wrapped into a tube to form a nerve repair guide catheter.

2. The method of claim 1, wherein the nerve repair guiding catheter is prepared by the steps of: In the step S100: The temperature for esterification reaction is 180℃.

3. The method of claim 1, wherein the nerve repair guiding catheter is prepared by the steps of: In the step S200: The amount of tetrabutyl titanate added in the esterification liquid is 0.1% by mass fraction; The temperature for polycondensation reaction is 220℃, and after vacuumizing, it needs to reach <300 Pa.

4. The method of claim 1, wherein the neurorestoration guide catheter is prepared by the steps of: In the step S300: The mass concentration of PCL and PPBISS mixed solution is 10%.

5. The method of claim 1, wherein the neurorestoration guide catheter is prepared by the steps of: In the step S400: The parameters of electrospinning process are: solution propelling speed 1 mL / h, positive electrode voltage +16 kV, negative electrode voltage-2 kV, receiving distance 14 cm, and drum rotating speed 1000 r / min.

6. The method of claim 1, wherein the neurorestoration guide catheter is prepared by the steps of: In the step S500: The time for plasma method hydrophilic is 15s, and the time for ultraviolet sterilization is 15min.

7. A neurorestoration guide catheter, characterized by, Prepared by the method of any one of claims 1-6.

8. A nerve repair guide catheter prepared by the method of any one of claims 1-6, or the use of the nerve repair guide catheter of claim 7 in the treatment of peripheral nerve injury.

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