A novel double-layer nerve guide conduit for inducing nerve regeneration and a preparation method thereof

Abstract

The application discloses a novel double-layer nerve guide conduit for inducing nerve regeneration and a preparation method thereof. The conduit is composed of an outer layer of polyvinyl alcohol (PVA) hydrogel conduit and an inner layer of conductive hydrogel. The conductive hydrogel is prepared by compounding a gelatin polymer (PAMB-G) modified by 3-amino-4-methoxybenzoic acid (AMB) with decellularized extracellular matrix (dECM) extracted from pig tendons, and then cross-linking by 1-ethyl-(3-dimethylaminopropyl) carbodiimide / N-hydroxysuccinimide (EDC / NHS). The novel double-layer nerve guide conduit prepared by the application has the ability to induce nerve regeneration around defects, can realize the recovery of 10-30 mm peripheral nerve structure and function, and the final recovery effect is equivalent to that of the "gold standard" autologous nerve graft.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a novel nerve guiding conduit for inducing nerve regeneration and its preparation method. Background Technology

[0002] Peripheral nerve injury is typically caused by trauma, surgery, infection, or disease. These factors can lead to nerve fiber rupture, contusion, or compression, interfering with nerve signal transmission and causing adverse effects such as sensory disturbances, muscle weakness, and loss of function. Currently, treatment methods for peripheral nerve injury include non-surgical treatment and surgical reconstruction. Surgical reconstruction includes nerve anastomosis, autologous nerve transplantation, and allogeneic nerve transplantation; however, these treatment methods all have certain limitations. In recent years, nerve conduits fabricated using tissue engineering techniques have been widely used in the field of peripheral nerve regeneration. Nerve conduit scaffold materials possess good biocompatibility, biodegradability, and structural support to maintain the biological microenvironment required for nerve repair.

[0003] Current research on nerve conduits mainly suffers from problems such as poor bioactivity, slow nerve regeneration, and poor functional recovery due to nerve scarring. Therefore, developing a novel double-layered nerve-guiding conduit that induces nerve regeneration and applying it to the repair of peripheral nerve tissue defects is of great significance. Summary of the Invention

[0004] To address the current technological shortcomings in the research of nerve conduit materials and to solve the problems of poor bioactivity and poor function in existing nerve conduit materials, the first objective of this invention is to propose a novel double-layered nerve guiding conduit that induces nerve regeneration. The second objective of this invention is to develop a method for preparing the conduit.

[0005] To achieve the objectives of the invention described above, the present invention is implemented through the following technical solution: A novel double-layered nerve guiding conduit for inducing nerve regeneration, the double-layered nerve guiding conduit consisting of an outer PVA hydrogel conduit and an inner layer filled with conductive composite hydrogel.

[0006] As a further preferred embodiment of the present invention, the diameter of the PVA hydrogel conduit is 2-5 mm and the length of the PVA hydrogel conduit is 15-40 mm.

[0007] As a further preferred embodiment of the present invention, the conductive composite hydrogel is a dECM@PAMB-G composite hydrogel.

[0008] The second objective of this invention discloses a method for preparing the novel double-layered nerve guiding conduit, comprising the following steps: S1: PVA is dissolved and dispersed in deionized water at 95 ℃ to prepare a PVA solution of a certain concentration. The PVA solution is poured into a mold and frozen and thawed 5 times at -20 ℃ and 37 ℃ respectively. The PVA hydrogel conduit is obtained by demolding. S2: A gelatin solution with a mass fraction of 40% was prepared by dissolving gelatin powder in deionized water. A certain amount of AMB powder was added at 55 °C. After complete stirring and dispersion, ammonium persulfate was added to catalyze the oxidative polymerization reaction for 36 h. Then, the reaction solution was dialyzed in deionized water and freeze-dried to prepare PAMB-G polymer. S3: Dissolve the prepared PAMB-G polymer in MES buffer at 37 °C and stir to obtain a 10% mass fraction solution. Then add an appropriate amount of dECM and stir at 37 °C for 16 h to obtain a homogeneous solution. Then add EDC and NHS crosslinking agent in sequence to obtain dECM@PAMB-G composite hydrogel, and freeze dry for later use. S4: The freeze-dried dECM@PAMB-G composite hydrogel was coated with paraffin and cut into rectangular prisms with a side length of 0.5 mm. Then, it was cut to a suitable length and inserted into the PVA hydrogel conduit prepared in S1. It was placed in PBS (pH=7.4) buffer solution at 37 ℃ for 2 h. After freeze-drying, a double-layer nerve guiding conduit was obtained.

[0009] As a further preferred embodiment of the present invention, in step S1, the mass concentration of the PVA solution is 8-12%.

[0010] As a further preferred embodiment of the present invention, in step S2, the mass ratio of AMB powder to gelatin powder is (0.075~0.15):1.

[0011] As a further preferred embodiment of the present invention, in step S3, the mass ratio of the amount of dECM added to the amount of PAMB-G polymer is (0.2~0.6):1.

[0012] As a further preferred embodiment of the present invention, in step S3, the dECM is prepared by the following method: fresh porcine Achilles tendon slices are washed with PBS, the washed slices are soaked in a mixture containing 0.5 wt% Triton X-100 and 0.5 wt% SDS for 24 h, washed again with PBS, and then soaked in HAc solution, thoroughly rinsed with distilled water, freeze-dried to obtain dECM hydrogel, and stored in a refrigerator at 4°C for later use.

[0013] Compared with the prior art, the advantages of the present invention are: The internal scaffold of the double-layer nerve guiding conduit of this invention is a composite hydrogel material of dECM and conductive gelatin. By filling the PVA hydrogel conduit with conductive hydrogel composited with dECM, a biomimetic microenvironment very similar to natural nerve tissue is obtained. This conduit can effectively promote Schwann cell proliferation, axonal growth, and myelin formation, which is beneficial to nerve regeneration. In vitro cytotoxicity experiments and rat sciatic nerve transection model experiments confirmed its biocompatibility and nerve regeneration capacity. Compared with the "gold standard" autologous nerve grafts, the dECM@PAMB-G / PVA nerve conduit showed comparable performance in promoting nerve regeneration and functional recovery. Attached Figure Description

[0014] Figure 1 These are the tensile stress-strain curves of the nerve guiding conduits in Examples 1, 2, and 3; Figure 2 The conductivity of the nerve guiding conduits in Examples 1, 4, and 5; Figure 3 The biocompatibility of the nerve guiding conduits in Examples 1, 6, and 7; Figure 4 These are photographs of the autologous nerve graft and PVA catheter at 4 and 8 weeks after the repair in Example 1. Figure 5 This is Example 1, a functional recovery test 8 weeks after repair of autologous nerve grafts and PVA catheters. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0016] Example 1 This embodiment discloses a novel double-layered nerve guiding conduit for inducing nerve regeneration and its preparation method, comprising the following steps: S1: Dissolve and disperse PVA in deionized water at 95 ℃ to prepare a 10% PVA solution by mass. Then pour the solution into a mold and freeze and thaw it 5 times at -20 ℃ and 37 ℃ respectively. Demold to obtain PVA hydrogel conduit. S2: Dissolve 1.0 g of gelatin powder in deionized water to prepare a gelatin solution with a mass fraction of 40%. Add 100 mg of AMB powder at 55℃, stir and disperse completely, then add ammonium persulfate to catalyze the oxidative polymerization reaction for 36 h. Then, dialyze the reaction solution in deionized water and freeze-dry to prepare PAMB-G polymer. S3: Dissolve 1.0 g of the prepared PAMB-G polymer in MES buffer at 37 °C and stir to obtain a 10% mass fraction solution. Then add 0.4 g of dECM and stir at 37 °C for 16 h to obtain a homogeneous solution. Then add EDC and NHS crosslinking agent in sequence to obtain dECM@PAMB-G composite hydrogel, and freeze dry for later use. S4: The freeze-dried dECM@PAMB-G composite hydrogel was coated with paraffin and cut into rectangular prisms with a side length of 0.5 mm. Then, it was cut to a suitable length and inserted into the PVA hydrogel conduit prepared in S1. It was placed in PBS (pH=7.4) buffer solution at 37 ℃ for 2 h. After freeze-drying, a double-layer nerve guiding conduit was obtained.

[0017] Example 2 The preparation method of a novel double-layered nerve guiding conduit for inducing nerve regeneration according to this embodiment includes the following steps: S1: Dissolve and disperse PVA in deionized water at 95 ℃ to prepare a 12% PVA solution by mass. Then pour the solution into a mold and freeze and thaw it 5 times at -20 ℃ and 37 ℃ respectively. Demold to obtain PVA hydrogel conduit. S2: Dissolve 1.0 g of gelatin powder in deionized water to prepare a gelatin solution with a mass fraction of 40%. Add 100 mg of AMB powder at 55℃, stir and disperse completely, then add ammonium persulfate to catalyze the oxidative polymerization reaction for 36 h. Then, dialyze the reaction solution in deionized water and freeze-dry to prepare PAMB-G polymer. S3: Dissolve 1.0 g of the prepared PAMB-G polymer in MES buffer at 37 °C and stir to obtain a 10% mass fraction solution. Then add 0.4 g of dECM and stir at 37 °C for 16 h to obtain a homogeneous solution. Then add EDC and NHS crosslinking agent in sequence to obtain dECM@PAMB-G composite hydrogel, and freeze dry for later use. S4: The freeze-dried dECM@PAMB-G composite hydrogel was coated with paraffin and cut into rectangular prisms with a side length of 0.5 mm. Then, it was cut to a suitable length and inserted into the PVA hydrogel conduit prepared in S1. It was placed in PBS (pH=7.4) buffer solution at 37 ℃ for 2 h. After freeze-drying, a double-layer nerve guiding conduit was obtained.

[0018] Example 3 The preparation method of a novel double-layered nerve guiding conduit for inducing nerve regeneration according to this embodiment includes the following steps: S1: Dissolve and disperse PVA in deionized water at 95 ℃ to prepare a PVA solution with a mass fraction of 8%. Then pour the solution into a mold and freeze and thaw it 5 times at -20 ℃ and 37 ℃ respectively. Demold to obtain PVA hydrogel conduit.

[0019] S2: Dissolve 1.0 g of gelatin powder in deionized water to prepare a gelatin solution with a mass fraction of 40%. Add 100 mg of AMB powder at 55℃, stir and disperse completely, then add ammonium persulfate to catalyze the oxidative polymerization reaction for 36 h. Then, dialyze the reaction solution in deionized water and freeze-dry to prepare PAMB-G polymer. S3: Dissolve 1.0 g of the prepared PAMB-G polymer in MES buffer at 37 °C and stir to obtain a 10% (w / w) solution. Then add 0.4 g of dECM and stir at 37 °C for 16 h to obtain a homogeneous solution. Then add EDC and NHS crosslinking agent in sequence to obtain dECM@PAMB-G composite hydrogel, and freeze-dry for later use.

[0020] S4: The freeze-dried dECM@PAMB-G composite hydrogel was coated with paraffin and cut into rectangular prisms with a side length of 0.5 mm. Then, it was cut to a suitable length and inserted into the PVA hydrogel conduit prepared in S1. It was placed in PBS (pH=7.4) buffer solution at 37 ℃ for 2 h. After freeze-drying, a double-layer nerve guiding conduit was obtained.

[0021] Example 4 The preparation method of a novel double-layered nerve guiding conduit for inducing nerve regeneration according to this embodiment includes the following steps: S1: Dissolve and disperse PVA in deionized water at 95 ℃ to prepare a 10% PVA solution. Then pour the solution into a mold and freeze and thaw it 5 times at -20 ℃ and 37 ℃ respectively. Demold to obtain PVA hydrogel conduit.

[0022] S2: Dissolve 1.0 g of gelatin powder in deionized water to prepare a gelatin solution with a mass fraction of 40%. Add 150 mg of AMB powder at 55℃, stir and disperse completely, then add ammonium persulfate to catalyze the oxidative polymerization reaction for 36 h. Then, dialyze the reaction solution in deionized water and freeze-dry to prepare PAMB-G polymer. S3: Dissolve 1.0 g of the prepared PAMB-G polymer in MES buffer at 37 °C and stir to obtain a 10% (w / w) solution. Then add 0.4 g of dECM and stir at 37 °C for 16 h to obtain a homogeneous solution. Then add EDC and NHS crosslinking agent in sequence to obtain dECM@PAMB-G composite hydrogel, and freeze-dry for later use.

[0023] S4: The freeze-dried dECM@PAMB-G composite hydrogel was coated with paraffin and cut into rectangular prisms with a side length of 0.5 mm. Then, it was cut to a suitable length and inserted into the PVA hydrogel conduit prepared in S1. It was placed in PBS (pH=7.4) buffer solution at 37 ℃ for 2 h. After freeze-drying, a double-layer nerve guiding conduit was obtained.

[0024] Example 5 The preparation method of a novel double-layered nerve guiding conduit for inducing nerve regeneration according to this embodiment includes the following steps: S1: Dissolve and disperse PVA in deionized water at 95 ℃ to prepare a 10% PVA solution. Then pour the solution into a mold and freeze and thaw it 5 times at -20 ℃ and 37 ℃ respectively. Demold to obtain PVA hydrogel conduit.

[0025] S2: Dissolve 1.0 g of gelatin powder in deionized water to prepare a gelatin solution with a mass fraction of 40%. Add 75 mg of AMB powder at 55°C, stir and disperse completely, then add ammonium persulfate to catalyze the oxidative polymerization reaction for 36 h. Then, dialyze the reaction solution in deionized water and freeze-dry to prepare PAMB-G polymer. S3: Dissolve 1.0 g of the prepared PAMB-G polymer in MES buffer at 37 °C and stir to obtain a 10% (w / w) solution. Then add 0.4 g of dECM and stir at 37 °C for 16 h to obtain a homogeneous solution. Then add EDC and NHS crosslinking agent in sequence to obtain dECM@PAMB-G composite hydrogel, and freeze-dry for later use.

[0026] S4: The freeze-dried dECM@PAMB-G composite hydrogel was coated with paraffin and cut into rectangular prisms with a side length of 0.5 mm. Then, it was cut to a suitable length and inserted into the PVA hydrogel conduit prepared in S1. It was placed in PBS (pH=7.4) buffer solution at 37 ℃ for 2 h. After freeze-drying, a double-layer nerve guiding conduit was obtained.

[0027] Example 6 The preparation method of a novel double-layered nerve guiding conduit for inducing nerve regeneration according to this embodiment includes the following steps: S1: Dissolve and disperse PVA in deionized water at 95 ℃ to prepare a 10% PVA solution. Then pour the solution into a mold and freeze and thaw it 5 times at -20 ℃ and 37 ℃ respectively. Demold to obtain PVA hydrogel conduit.

[0028] S2: Dissolve 1.0 g of gelatin powder in deionized water to prepare a gelatin solution with a mass fraction of 40%. Add 100 mg of AMB powder at 55℃, stir and disperse completely, then add ammonium persulfate to catalyze the oxidative polymerization reaction for 36 h. Then, dialyze the reaction solution in deionized water and freeze-dry to prepare PAMB-G polymer. S3: Dissolve 1.0 g of the prepared PAMB-G polymer in MES buffer at 37 °C and stir to obtain a 10% (w / w) solution. Then add 0.2 g of dECM and stir at 37 °C for 16 h to obtain a homogeneous solution. Then add EDC and NHS crosslinking agent in sequence to obtain dECM@PAMB-G composite hydrogel, and freeze-dry for later use.

[0029] S4: The freeze-dried dECM@PAMB-G composite hydrogel was coated with paraffin and cut into rectangular prisms with a side length of 0.5 mm. Then, it was cut to a suitable length and inserted into the PVA hydrogel conduit prepared in S1. It was placed in PBS (pH=7.4) buffer solution at 37 ℃ for 2 h. After freeze-drying, a double-layer nerve guiding conduit was obtained.

[0030] Example 7 The preparation method of a novel double-layered nerve guiding conduit for inducing nerve regeneration according to this embodiment includes the following steps: S1: Dissolve and disperse PVA in deionized water at 95 ℃ to prepare a 10% PVA solution. Then pour the solution into a mold and freeze and thaw it 5 times at -20 ℃ and 37 ℃ respectively. Demold to obtain PVA hydrogel conduit.

[0031] S2: Dissolve 1.0 g of gelatin powder in deionized water to prepare a gelatin solution with a mass fraction of 40%. Add 100 mg of AMB powder at 55℃, stir and disperse completely, then add ammonium persulfate to catalyze the oxidative polymerization reaction for 36 h. Then, dialyze the reaction solution in deionized water and freeze-dry to prepare PAMB-G polymer. S3: Dissolve 1.0 g of the prepared PAMB-G polymer in MES buffer at 37 °C and stir to obtain a 10% (w / w) solution. Then add 0.6 g of dECM and stir at 37 °C for 16 h to obtain a homogeneous solution. Then add EDC and NHS crosslinking agent in sequence to obtain dECM@PAMB-G composite hydrogel, and freeze-dry for later use.

[0032] S4: The freeze-dried dECM@PAMB-G composite hydrogel was coated with paraffin and cut into rectangular prisms with a side length of 0.5 mm. Then, it was cut to a suitable length and inserted into the PVA hydrogel conduit prepared in S1. It was placed in PBS (pH=7.4) buffer solution at 37 ℃ for 2 h. After freeze-drying, a double-layer nerve guiding conduit was obtained.

[0033] Performance testing experiment (1) Tensile property test: The nerve guiding conduits from Examples 1, 2, and 3 were selected for tensile testing at a stretching rate of 2 mm / min. Figure 1 The figure shows the tensile stress-strain curve of the nerve guiding catheter. Analysis of the figure shows that the catheter has the best tensile performance when the mass fraction is 10%. Decreasing the concentration reduces the cross-linking density, while increasing the concentration will lead to poor solution dispersion uniformity, both of which will result in poor performance.

[0034] (2) Conductivity test: Conductivity tests were performed on the nerve guidance catheters selected from Examples 1, 3, and 4. Figure 2 The data shows that the conductivity of the nerve-guided catheter increases with the increase of AMB addition.

[0035] (3) In vitro cell culture test: The neural guiding conduits from Examples 1, 6, and 7 were co-cultured with PC12 cells. A 5mm span scratch was created between the cells, and the intercellular spacing was observed at 8h and 24h. Figure 3 The images show scratch-scratch microscopy images of the cells. The results indicate that, compared to the control group, the cells in all the nerve guiding conduit groups showed good activity.

[0036] (4) In vivo animal experiments: Eighteen rats aged 7-8 weeks and weighing 200-230g were selected. The right side of the posterior spine of the rats was first disinfected and shaved. The sciatic nerve was then used as a nerve damage model. A 15-20mm section of the rat sciatic nerve was surgically removed. In the positive control group, autologous nerve transplantation was used, and the removed nerve was reversed 180° and sutured. In the experimental group, the nerve-inducing repair catheter scaffold prepared in Example 1 was used for suturing. In the negative control group, a hollow PVA catheter prepared in step S1 of Example 1 was used. Each group consisted of 15 rats. At weeks 4 and 8, the nerve repair sites in any three rats were dissected to observe the effect of nerve repair.

[0037] Figure 4These are photographs of the nerve tissues in the positive control group, experimental group, and negative control group of the animal experiment at weeks 4 and 8. In this experimental group, the nerves had fully recovered by week 8. Figure 4 It can be seen that the nerves had already achieved basic bridging by week 4, and the nerve structure in the experimental group was repaired after week 8, with the outer tube resembling an autologous nerve graft.

[0038] Further testing was conducted on the induction of peripheral nerve function recovery using a nerve-guided catheter, and the results were as follows: Figure 5 As shown, analysis of electrical signal transmission and walking trajectory reveals that the PVA catheter in the negative control group had a poor effect on the repair of peripheral nerves, while the autologous nerve graft and the experimental group had good effects on the recovery of nerve function, and the treatment effects of the two were comparable.

[0039] Replacing the double-layered nerve guiding catheter scaffold used in the experimental group of the animal experiment with the one prepared in Examples 2-7 resulted in a final repair effect comparable to that of autologous nerve repair.

[0040] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A novel double-layered nerve guiding conduit for inducing nerve regeneration, characterized in that, The double-layered nerve guiding conduit consists of an outer PVA hydrogel conduit and an inner conductive composite hydrogel filling layer. The conductive composite hydrogel is a dECM@PAMB-G composite hydrogel, which is prepared by the following method: gelatin powder is dissolved in deionized water to prepare a 40% gelatin solution. A certain amount of AMB powder is added at 55 °C, and after complete stirring and dispersion, ammonium persulfate is added to catalyze an oxidative polymerization reaction for 36 h. The reaction solution is then dialyzed in deionized water and freeze-dried to prepare PAMB-G polymer. The prepared PAMB-G polymer is dissolved in MES buffer at 37 °C and stirred to obtain a 10% solution. Then, an appropriate amount of dECM is added, and the solution is stirred at 37 °C for 16 h to obtain a homogeneous solution. Finally, EDC and NHS crosslinking agents are added sequentially to obtain the dECM@PAMB-G composite hydrogel. The AMB powder is 3-amino-4-methoxybenzoic acid powder; the mass ratio of the AMB powder to the gelatin powder is (0.075~0.15):1; The mass ratio of the amount of dECM added to the amount of PAMB-G polymer is (0.2~0.6):

1.

2. The novel double-layered nerve guiding conduit for inducing nerve regeneration according to claim 1, characterized in that, The diameter of the PVA hydrogel conduit is 2-5 mm, and the length of the PVA hydrogel conduit is 15-40 mm.

3. The method for preparing the novel double-layered nerve guiding conduit according to any one of claims 1-2, characterized in that, Includes the following steps: S1: PVA is dissolved and dispersed in deionized water at 95 ℃ to prepare a PVA solution of a certain concentration. The PVA solution is poured into a mold and frozen and thawed 5 times at -20 ℃ and 37 ℃ respectively. The PVA hydrogel conduit is obtained by demolding. S2: A gelatin solution with a mass fraction of 40% was prepared by dissolving gelatin powder in deionized water. A certain amount of AMB powder was added at 55 °C. After complete stirring and dispersion, ammonium persulfate was added to catalyze the oxidative polymerization reaction for 36 h. Then, the reaction solution was dialyzed in deionized water and freeze-dried to prepare PAMB-G polymer. S3: Dissolve the prepared PAMB-G polymer in MES buffer at 37 °C and stir to obtain a 10% mass fraction solution. Then add an appropriate amount of dECM and stir at 37 °C for 16 h to obtain a homogeneous solution. Then add EDC and NHS crosslinking agent in sequence to obtain dECM@PAMB-G composite hydrogel, and freeze dry for later use. S4: The freeze-dried dECM@PAMB-G composite hydrogel was coated with paraffin and cut into rectangular prisms with a side length of 0.5 mm. Then, it was cut to a suitable length and inserted into the PVA hydrogel conduit prepared in S1. It was placed in PBS (pH=7.4) buffer solution at 37 ℃ for 2 h. After freeze-drying, a double-layer nerve guiding conduit was obtained.

4. The method for preparing a double-layered nerve guiding conduit according to claim 3, characterized in that, In step S1, the mass concentration of the PVA solution is 8-12%.

5. The method for preparing a double-layered nerve guiding conduit according to claim 3, characterized in that, In step S3, the dECM is prepared by the following method: fresh porcine Achilles tendon slices are washed with PBS, the washed slices are soaked in a mixture containing 0.5 wt% Triton X-100 and 0.5 wt% SDS for 24 h, washed again with PBS, and then soaked in HAc solution. After thorough rinsing with distilled water, the dECM hydrogel is obtained by freeze drying and stored in a refrigerator at 4°C for later use.

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