High-conductivity peripheral nerve socketed pipe and preparation method thereof

Abstract

The invention relates to the technical field of biomedical materials, in particular to a high-conductivity peripheral nerve socketed pipe and a preparation method thereof. The nerve socketed pipe is a hollow cylindrical socketed pipe made of a material containing poly (octylene-citrate-co-octanol) POCO, and a regeneration promoting material is loaded in the hollow cylindrical socketed pipe; the regeneration promoting material comprises fetal calf serum and a DMEM cell culture medium. The POCO has the advantages of conductive activity, nerve regeneration induction, free radical scavenging ability, oxidation resistance, deformation resistance and the like, and has good degradability and biocompatibility, so that the nerve socketed pipe is prepared from poly (octylene-citrate-co-octanol), and the growth of axons is accelerated through electrical stimulation. Furthermore, at least one conductive monomer of pyrrole, aniline, p-phenylenediamine, o-methoxyaniline, thiophene, 3-hexylthiophene, 3-thiopheneacetic acid and 3, 4-ethylenedioxythiophene is adopted for modifying the POCO hollow cylindrical socketed pipe through in-situ oxidative polymerization, and the performance of the POCO hollow cylindrical socketed pipe serving as the neural restoration material is further improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a highly conductive peripheral nerve sheath and its preparation method. Background Technology

[0002] A peripheral nerve conduit is a medical device or biomaterial used to repair peripheral nerve injuries. It primarily guides nerve regeneration, serving as an alternative to traditional nerve transplantation. When peripheral nerves (such as those in the arm or leg) are severed due to injury or surgery, the nerve fibers need to regrow and reconnect. The conduit acts as a "bridge," employing a small-gap connection method. A 2mm gap is left between the severed nerve ends within the conduit, which is filled with regenerative materials (such as growth factors, stem cells, or collagen matrix) to provide physical guidance and biochemical support for axonal regeneration. Unlike traditional methods that rely on autologous nerve transplantation (such as harvesting the sural nerve from the lower leg), which can cause donor site dysfunction, the conduit reduces secondary surgical trauma, lowers the rate of nerve fiber misconnection, improves the success rate of nerve repair, shortens suture time, eliminates neuroma formation, reduces pain, and avoids the problem of donor nerve shortage. Peripheral nerve sheathing tubes are suitable for short-distance peripheral nerve defects (usually <3cm, but some materials can be extended to 5cm), and for the repair of small-diameter nerves such as digital nerves and facial nerves. They have the advantages of being minimally invasive and reducing donor site complications.

[0003] Peripheral nerve cannulas typically require good mechanical properties and biodegradability, such as high-stiffness materials (e.g., PCL) and flexible materials (e.g., collagen). Currently, mass-produced cannulas include synthetic materials, natural biomaterials, and composite materials. Synthetic materials mainly include absorbable materials such as polylactic acid (PLA) and polycaprolactone (PCL), which degrade over time, avoiding secondary removal; and non-absorbable materials, such as silicone tubes, which require subsequent surgical removal and are now rarely used. Natural biomaterials include decellularized neural matrix (e.g., decellularized nerves derived from animals) and collagen tubes, which are closer to the natural neural microenvironment. Composite materials combine material scaffolds with bioactive substances (e.g., neurotrophic factors, Schwann cells) to enhance regeneration. Neuronal cells possess electrical activity, and electrical signal transmission is one of the basic functions of nerve fibers; the transmission of bioelectrical signals is crucial for the functional recovery of peripheral nerves. However, current materials do not possess high conductivity and cannot accelerate axonal growth through electrical stimulation. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a highly conductive peripheral nerve sheath and its preparation method. The nerve sheath has good conductivity, biocompatibility, free radical scavenging ability, antioxidant properties, sufficient elasticity and biocompatibility, solving the technical problem that existing sheath materials cannot simultaneously achieve free radical scavenging ability, biocompatibility, mechanical properties and high electrical conductivity.

[0006] (II) Technical Solution

[0007] In a first aspect, the present invention provides a highly conductive peripheral nerve sheath, which is a hollow cylindrical sheath made of a material comprising poly(octylmethylene-citrate-cooctanol)POCO, wherein a regeneration-promoting material is loaded in the hollow cylindrical sheath; the regeneration-promoting material comprises fetal bovine serum and DMEM cell culture medium.

[0008] Secondly, the present invention provides a method for preparing a highly conductive peripheral nerve sheath, comprising:

[0009] S1. Preparation of poly(octylmethylene-citrate-cooctanol) prepolymer uPOCO

[0010] A mixture of citric acid, 1,8-octanediol, and octanol is melted at 160-175℃ and reacted at 140-150℃ for 2.5-5 hours to obtain a crude product containing the prepolymer uPOCO. The crude product is dissolved in 15%-35% ethanol for precipitation purification. The precipitate is diluted with ethanol to a mass concentration of 30-55% to obtain an ethanol solution of the prepolymer uPOCO.

[0011] S2. Fabricate a hollow cylindrical sleeve.

[0012] A molding solution is prepared by mixing a PVA solution, a chitosan solution, and an ethanol solution of the prepolymer uPOCO obtained in S1. The molding solution is then transferred to a molding mold consisting of an inner core rod and an outer casing, or a hollow cylindrical structure is formed by electrospinning. The structure is cured at 50-80℃ for 1.5-5 hours until dry, and then left at room temperature for 12-24 hours to allow excess solvent to evaporate. Afterward, polycondensation and curing are carried out at 50-80℃ for 4-8 days to obtain a semi-finished product. The semi-finished product is then acetylated in an acetylation reagent for 1-24 hours. After acetylation, the outer casing is removed, and the remaining part is soaked in deionized water to dissolve the PVA. The formed hollow cylindrical casing is then peeled off from the inner core rod or the cylindrical spinneret and placed in DMEM medium containing ethanol to remove unreacted monomers.

[0013] S3, Intermediate Processing

[0014] Soak the hollow cylindrical sleeve in an ethanol PBS solution containing antibiotics for 20-30 hours, then replace it with an antibiotic PBS solution and soak for another 20-30 hours; then transfer it to a low-glucose DMEM medium containing antibiotics and soak for 1-4 hours, then rinse with deionized water, and then soak in low-glucose DMEM for 12-24 hours.

[0015] S4, loaded with regeneration-promoting materials

[0016] Next, it is soaked overnight in high-glucose DMEM containing fetal bovine serum and antibiotics, then soaked in 70-75% ethanol for 15-25 minutes, and then soaked again overnight in high-glucose DEME medium containing fetal bovine serum and antibiotics to obtain POCO sleeves for nerve repair.

[0017] Preferably, in step S1, the mixture is prepared by mixing citric acid, 1,8-octanediol, and octanol in a molar ratio of 9:10:2. Preferably, in step S1, the crude product is dissolved in 15%-35% ethanol for precipitation purification at least twice.

[0018] Preferably, in S2, the PVA solution concentration is 50 mg / mL, the chitosan solution mass concentration is 2-10%, the ethanol solution of the prepolymer uPOCO mass concentration is 30-55%, and the PVA solution, chitosan solution, and uPOCO ethanol solution are mixed in a mass ratio of (1-5):(1-10):(10-1) to obtain the molding liquid.

[0019] Preferably, in S2, the acetylation reagent is a combination of acetic anhydride and methanol, wherein the volume ratio of acetic anhydride to methanol is 1:10-10:1. After acetylation, the outer shell is removed, and the remaining part is soaked in deionized water overnight to dissolve PVA. The formed hollow cylindrical sleeve is peeled off from the inner core rod or the cylindrical spinneret and placed in DMEM medium containing 20 v / v% ethanol to remove unreacted monomers.

[0020] Preferably, in S3, the hollow cylindrical sleeve is soaked in a 20v / v% ethanol PBS solution containing 1% penicillin / streptomycin for 24h, and then the solution is replaced with a PBS solution containing 1% penicillin / streptomycin and soaked for another 24h; then, it is transferred to a low-glucose DMEM medium containing 1% penicillin / streptomycin and soaked for 2h, then rinsed with deionized water, and then soaked in low-glucose DMEM for 12-24h.

[0021] Preferably, in step S4, the tube is soaked overnight in high-glucose DMEM containing 10% fetal bovine serum and 1% penicillin / streptomycin, then soaked in 70-75% ethanol for 20 minutes, and then soaked again overnight in high-glucose DMEM containing 10% fetal bovine serum and 1% penicillin / streptomycin to obtain the POCO sleeve tube for nerve repair; the soaking temperature is 36.5-37.5℃.

[0022] The solution contains a 1% penicillin / streptomycin (P / S) antibacterial combination, with 1 mL of penicillin / streptomycin bispecific antibiotic stock solution added to every 100 mL of solution. The penicillin / streptomycin bispecific antibiotic stock solution is typically a commercially available ready-to-use formulation. Common stock solution concentrations are: penicillin (10,000 U / mL) + streptomycin (10,000 μg / mL), resulting in a final concentration of: penicillin: 100 U / mL (1% × 10,000 U / mL); streptomycin: 100 μg / mL (1% × 10,000 μg / mL).

[0023] Thirdly, the present invention also provides a functionalized modified highly conductive peripheral nerve sheath, comprising a hollow sheath and a regenerative material loaded in the hollow sheath; the regenerative material comprises fetal bovine serum and DMEM cell culture medium.

[0024] The hollow sleeve is made of poly(octylmethylene-citrate-cooctanol) as an elastic base material and modified with a conductive polymer on the surface of the elastic base material. The conductive polymer contains a conductive monomer selected from at least one of pyrrole, aniline, p-phenylenediamine, o-methoxyaniline, thiophene, 3-hexylthiophene, 3-thiopheneacetic acid, and 3,4-ethylenedioxythiophene. The regeneration-promoting material also contains an antibiotic; the conductive polymer and the elastic base material are combined in the form of an interpenetrating network structure.

[0025] Fourthly, the present invention provides a method for preparing a functionalized, highly conductive peripheral nerve sheath, comprising:

[0026] Step 1: Preparation of poly(octylmethylene-citrate-cooctanol) prepolymer uPOCO

[0027] A mixture of citric acid, 1,8-octanediol, and octanol (molar ratio (5-9):(6-10):(1-4)) was melted at 160-175℃ and reacted at 140-150℃ for 2.5-5 h to obtain a crude product containing the prepolymer uPOCO. The crude product was dissolved in 15%-35% ethanol for precipitation purification. The precipitate was diluted with ethanol to a mass concentration of 30-55% to obtain an ethanol solution of the prepolymer uPOCO.

[0028] Step 2: Make a hollow cylindrical sleeve.

[0029] Mix the PVA solution, chitosan solution, and ethanol solution of the prepolymer uPOCO obtained in step 1 to obtain a molding solution. Transfer the molding solution to a molding mold consisting of an inner core rod and an outer shell, or use electrospinning to form a hollow cylindrical structure. Cure at 50-80℃ for 1.5-5 hours until dry, and place at room temperature for 12-24 hours to allow excess solvent to evaporate. Then continue polycondensation and polymerization at 50-80℃ for 4-8 days to obtain a semi-finished product. Acetylate the semi-finished product in an acetylation reagent for 1-24 hours. After acetylation, remove the outer shell, and soak the remaining part in deionized water to dissolve the PVA. Peel the formed hollow cylindrical sleeve from the inner core rod or the cylindrical spinneret and place it in DMEM medium containing ethanol to remove unreacted monomers.

[0030] Step 3, Intermediate Processing

[0031] Soak the hollow cylindrical sleeve in an ethanol PBS solution containing antibiotics for 20-30 hours, then replace it with an antibiotic PBS solution and soak for another 20-30 hours; then transfer it to a low-glucose DMEM medium containing antibiotics and soak for 1-4 hours, then rinse with deionized water, and then soak in low-glucose DMEM for 12-24 hours.

[0032] Step 4: In-situ polymerization for functionalization modification

[0033] The prepolymer uPOCO is diluted 50-200 times with ethanol; the hollow cylindrical sleeve from step 3, the diluted uPOCO, and the conductive monomer are incubated at room temperature for 1-2 days; then the hollow cylindrical sleeve is transferred to a polymerization solution containing the conductive monomer, an oxidation catalyst (APS, KPS, enzyme catalysis, or H2O2 system), and phytic acid, and incubated with shaking during incubation. The mixture is then vigorously mixed overnight at 0-5°C (preferably 4°C) to allow in-situ polymerization of uPOCO and the conductive monomer on the hollow cylindrical sleeve; the conductive monomer is selected from at least one of pyrrole, aniline, p-phenylenediamine, o-methoxyaniline, thiophene, 3-hexylthiophene, 3-thiopheneacetic acid, and 3,4-ethylenedioxythiophene.

[0034] Step 5: Loading regeneration materials

[0035] Incubate in 70-75% ethanol for 10-20 min, and rinse with high glucose DMEM containing FBS and antibiotics until the rinsing solution no longer changes color (to remove excess polymerization solution); then, incubate the hollow cylindrical sleeve in high glucose DMEM containing FBS and antibiotics at room temperature for 20-30 h, change the incubation solution the next day, and continue incubation for 20-30 h.

[0036] Preferably, the antibiotics in each solution are a combination of penicillin and streptomycin, and 1 mL of penicillin / streptomycin bispecific antibiotic stock solution is added to every 100 mL of solution; the soaking temperature is 36.5-37.5℃.

[0037] In step 4, phytic acid plays a crucial role in the in-situ polymerization of uPOCO and conductive monomers on the hollow cylindrical sleeve. Phytic acid functions as a dopant, crosslinking agent, and structure regulator. As a dopant, it enhances conductivity, optimizes stability, constructs three-dimensional networks, induces porous structures, and aids in neural repair. Phytic acid is a natural polyphosphate compound containing six phosphate groups. It can bind to the polymer chains of conductive monomers through protonic acid doping, removing electrons from the polymer to form holes (p-type doping), significantly enhancing conductivity. During polymerization, phytic acid may promote the formation of porous structures (pore size 1-10 μm) through phase separation or a template effect, facilitating cell migration and nutrient diffusion (suitable for neural regeneration applications).

[0038] High-glucose DMEM contains a glucose concentration of 1000-4500 mg / L, which is suitable for cells with high metabolic demands (such as neurons); low-glucose DMEM contains a glucose concentration of <1000 mg / L, which is close to physiological blood glucose levels.

[0039] In this application, the size design of the nerve conduit must match the anatomical structure of the target nerve and optimize the regenerative microenvironment. Its inner diameter, outer diameter, and thickness (wall thickness) are typically adjusted according to the application scenario (e.g., peripheral nerve defect length, nerve type) and material properties. Clinically, the inner diameter of the conduit is generally 10-20% larger than the diameter of the damaged nerve to allow space for edema and avoid compression. The length is usually 1.5-2 times the length of the defective nerve (e.g., a 10mm defect requires a 15-20mm conduit). Commonly, the inner diameter (ID) of peripheral nerve conduits is 1.0-6.0mm, for rat sciatic nerves (~1.3mm) and human digital nerves (~2-3mm); the outer diameter (OD) is 1.5-6.5mm, and the wall thickness is 0.2-1.0mm. A wall that is too thin is prone to collapse, while a wall that is too thick may hinder nutrient diffusion.

[0040] (III) Beneficial Effects

[0041] This invention uses poly(octylmethylene-citrate-cooctanol) to make a nerve sheath. Poly(octylmethylene-citrate-cooctanol) has advantages such as electrical conductivity, nerve regeneration induction, free radical scavenging ability, antioxidant properties, deformation resistance, and high tensile strength. It also has good degradability and biocompatibility, which is beneficial for accelerating the growth of nerve axons through subsequent electrical stimulation.

[0042] Furthermore, this invention also provides a functionalized, highly conductive peripheral nerve sheath, which is made from a poly(octylmethylene-citrate-cooctanol) elastic substrate material and a conductive polymer modified on the surface of the elastic substrate material. The conductive polymer is formed by polymerization of uPOCO and conductive monomers, and the conductive monomers are selected from at least one of pyrrole, aniline, p-phenylenediamine, o-methoxyaniline, thiophene, 3-hexylthiophene, 3-thiopheneacetic acid, and 3,4-ethylenedioxythiophene (conductivity order: EDOT > pyrrole ≈ thiophene > aniline). The nerve sheath obtained by functionalizing the conductive polymer has higher conductivity, resistance to deformation, cell activity, free radical scavenging ability (antioxidant capacity), and nerve repair effect compared with the unfunctionalized version, which is beneficial for inducing nerve regeneration. Attached Figure Description

[0043] Figure 1 SEM images of the highly conductive peripheral nerve sheaths prepared in Examples 1 and 2 at magnification of 30k. Detailed Implementation

[0044] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to specific embodiments.

[0045] This invention discloses a method for preparing a highly conductive peripheral nerve sheath, comprising the following steps:

[0046] S1. Preparation of poly(octylmethylene-citrate-cooctanol) prepolymer uPOCO

[0047] Citric acid, 1,8-octanediol, and octanol were mixed in a molar ratio of (5-9):(6-10):(1-4), melted at 160-175℃, and reacted at 140-150℃ for 2.5-5 hours to obtain a crude product containing the prepolymer uPOCO. The crude product was dissolved in 15%-35% ethanol for precipitation purification 2-3 times. The precipitate was diluted with ethanol to a mass concentration of 30-55% to obtain an ethanol solution of the prepolymer uPOCO.

[0048] S2. Fabricate a hollow cylindrical sleeve.

[0049] The PVA solution (50 mg / mL), chitosan solution (prepared with acetic acid solution as solvent to form 2%-10% chitosan solution), and ethanol solution of the prepolymer uPOCO obtained in S1 were mixed at a mass ratio of (1-5):(1-10):(10-1) to obtain the molding solution.

[0050] The molding liquid is transferred to a molding mold consisting of an inner core and an outer shell, or a hollow cylindrical structure is formed using electrospinning. It is cured at 50-80℃ for 1.5-5 hours until dry, and then left at room temperature for 12-24 hours to allow excess solvent to evaporate. Afterward, it undergoes further polycondensation and curing at 50-80℃ for 4-8 days to obtain a semi-finished product. The semi-finished product is then acetylated in an acetylation reagent for 1-24 hours. The acetylation reagent is a composition of acetic anhydride and methanol, in which the volume ratio of acetic anhydride to methanol is 1:10-10:1.

[0051] After acetylation, the outer shell is removed, and the remaining part is soaked in deionized water at 36.5-37.5℃ overnight to dissolve PVA. The formed hollow cylindrical sleeve is peeled off from the inner core or cylindrical spinneret and soaked in DMEM medium containing ethanol at 36.5-37.5℃ to remove unreacted monomers.

[0052] S3, Intermediate Processing

[0053] Hollow cylindrical sleeves were immersed in a 20 v / v ethanol PBS solution containing 1% penicillin / streptomycin at 36.5-37.5°C for 24 h. Then, the PBS solution was replaced with another 1% penicillin / streptomycin solution, and the immersion continued at 36.5-37.5°C for another 24 h. Afterward, the sleeves were transferred to a low-glucose DMEM medium containing 1% penicillin / streptomycin and immersed at 36.5-37.5°C for 2 h. They were then rinsed with deionized water and immersed in low-glucose DMEM at 36.5-37.5°C for 12-24 h. The prepared sleeves were then ready for functionalization modification with poly(3,4-ethylenedioxythiophene) (PEDOT), aniline, etc.

[0054] S4, Load-bearing regeneration material

[0055] The POCO sleeve was soaked overnight at 36.5-37.5°C in high-glucose DMEM containing 10 wt% fetal bovine serum (FBS) and 1% penicillin / streptomycin. Finally, it was soaked in 70-75% ethanol for 15-25 minutes, and then soaked again overnight at 36.5-37.5°C in high-glucose DEME medium containing 10 wt% fetal bovine serum (FBS) and 1% penicillin / streptomycin. This yielded the POCO sleeve for nerve repair.

[0056] The reaction mechanisms and functions of each step in the above preparation method are as follows:

[0057] S1 is the synthesis of uPOCO prepolymer. Citric acid, as a tricarboxylic acid, provides crosslinking sites, and the crosslinking density can be controlled slightly below the theoretical value (1.0). 1,8-Octanediol is the main chain extender, constructing the polymer backbone, and octanol is the end-capping agent. Melting at 160-175℃ disrupts the citric acid crystal structure, promoting a homogeneous reaction. Polycondensation at 140-150℃ forms the poly(octylmethylene citrate) backbone. Ethanol purification improves the purity of the prepolymer (>85%). The conjugated structure of citric acid in POCO provides π-electron transport channels, giving the material conductivity, while the octanediol segments impart elasticity, resulting in a superior Young's modulus.

[0058] S2 is a sleeve molding process: constructing a ternary composite system of PVA, chitosan, and uPOCO. PVA serves as a sacrificial template or porogen, forming a porous structure; chitosan promotes cell adhesion and has antibacterial properties; and uPOCO acts as a conductive matrix, providing structural support. The molding die allows for precise control of the inner diameter (±0.1 mm). The fiber diameter produced by electrospinning is 300-800 nm, which is beneficial for increasing the specific surface area. When using electrospinning, less or no PVA can be used during molding. Acetylation treatment improves the lipophilicity of the reinforcing material.

[0059] S3 is an intermediate treatment (biocompatibility treatment): ethanol-PBS solution: gradually replaces the organic solvent (residual ethanol <0.1%), antibiotic treatment: reduces the risk of infection, and low-glucose DMEM (1 g / L glucose). This avoids high-glucose-induced oxidative stress and is closer to the neuronal microenvironment.

[0060] S4 is a regenerative loading material: Serum proteins are modified using 10% fetal bovine serum to form a protein adsorption layer on the POCO sheath, containing laminin (promoting axonal growth). A second ethanol treatment with 70-75% ethanol is used for sterilization (sterilization at 121℃ will damage the structure) to maintain pore size stability and avoid significant shrinkage. A high-glucose DMEM final treatment simulates the acute microenvironment after nerve injury, promoting Schwann cell migration. The treatment process is carried out at a constant temperature of 36.5-37.5℃ to maintain protein conformational stability and match the optimal temperature for nerve regeneration.

[0061] The nerve sleeve tube of the present invention has electrical conductivity (to meet the requirements of nerve electrical signal conduction), porosity (to facilitate nutrient diffusion and vascularization), and degradability (to match the nerve regeneration time window).

[0062] The method for preparing functionalized, highly conductive peripheral nerve sheaths is as follows:

[0063] (1) Functional modification through in-situ polymerization

[0064] The prepolymer uPOCO was diluted 50-200 times with ethanol. The hollow cylindrical sleeve prepared in S3 above, the diluted uPOCO, and the conductive monomer were incubated at room temperature for 1-2 days. Then, the hollow cylindrical sleeve was transferred to a polymerization solution containing the conductive monomer, APS (ammonium persulfate), and phytic acid and incubated with shaking during incubation. The mixture was vigorously mixed overnight at 0-5°C (preferably 4°C) to allow in-situ polymerization of uPOCO and the conductive monomer on the hollow cylindrical sleeve. The conductive monomer was selected from at least one of pyrrole, aniline, p-phenylenediamine, o-methoxyaniline, thiophene, 3-hexylthiophene, 3-thiopheneacetic acid, and 3,4-ethylenedioxythiophene (EDOT).

[0065] (2) Load-loaded regeneration materials

[0066] The device was incubated in 70% ethanol for 15 min and then rinsed with high-glucose DMEM containing 10 wt% FBS and 1% penicillin / streptomycin until the rinsing solution no longer changed color (complete removal of excess polymerization solution). After that, the hollow cylindrical sleeve was incubated at room temperature in 10 wt% FBS and high-glucose DMEM containing 1% penicillin / streptomycin for 24 h. The incubation solution was changed the next day, and the device was incubated at 36.5-37.5℃ for another 24 h to obtain the conductive polymer functionalized neural sleeve.

[0067] In this process, hollow cylindrical sleeves, diluted uPOCO, and conductive monomers are incubated at room temperature for 1-2 days for pre-wetting and adsorption. This allows the diluted uPOCO to permeate into the micropores of the sleeves and bind to the chitosan amino groups via hydrogen bonds. The conductive monomers are adsorbed onto the citric acid conjugated backbone of uPOCO through π-π stacking. During the polymerization stage, ammonium persulfate (APS) provides free radicals, while phytic acid acts as both a dopant and a crosslinking agent. Low-temperature control (0-5℃, preferably 4℃) suppresses side reactions and improves polymer regularity. APS can also be derived from low-toxicity KPS, biocompatible enzyme catalysis, or an H2O2 system.

[0068] Diluted uPOCO enters the pores of the casing via permeation (prepolymer uPOCO is diluted 50-200 times with ethanol to avoid clogging the adsorption pores). The carboxyl groups of its citric acid units pre-assemble with conductive monomers (such as EDOT) through hydrogen bonding or electrostatic interactions, providing anchoring sites for subsequent polymerization. Under the action of an oxidant (ammonium persulfate), the enol structure of uPOCO (derived from citric acid) can co-oxidize with conductive monomers (such as pyrrole) to form an interpenetrating polymer network (IPN), enhancing mechanical strength. The conjugated ester bonds of uPOCO (C=O and ortho-double bonds) act as bridges for electron hopping, compensating for the interstitial conduction defects of pure conductive polymers (such as PEDOT). Furthermore, the octanol segments of uPOCO remain hydrophobic, forming a microphase-separated structure with the FBS protein adsorption layer, balancing conductivity and cellular compatibility. uPOCO's participation in polymerization prevents excessive self-polymerization of conductive monomers (such as EDOT) that could lead to increased brittleness. Phytic acid coordinates with both the hydroxyl groups of uPOCO and the π-system of the conductive monomer to form a ternary composite conductive phase.

[0069] During the loading and regeneration process, treatment with 70-75% ethanol sterilizes the material and simultaneously opens its pores to promote FBS protein penetration and adsorption. High-sugar DMEM is used in a two-stage loading process to form a serum protein coating on the sleeve surface. Penicillin / streptomycin inhibits microbial contamination caused by residual oxidants and promotes the anchoring of neurotrophic factors (such as NGF and BDNF) to the conductive polymer via electrostatic interactions.

[0070] The neural tube sheath prepared by functionalizing conductive polymers exhibits higher conductivity, resistance to deformation (increased Young's modulus), cell activity, free radical scavenging ability (antioxidant capacity), and nerve repair effect compared to the original functionalized version, which is beneficial for inducing nerve regeneration.

[0071] The following description is based on preferred embodiments of the present invention.

[0072] Example 1

[0073] This embodiment provides a method for preparing poly(octylmethylene-citrate-cooctanol) (POCO) nerve sheaths, the specific steps of which are as follows: (1) A mixture of citric acid, 1,8-octanediol and octanol (molar ratio 9:10:2) is melted at 160°C and then reacted at 145°C for 3 hours to synthesize the prepolymer uPOCO. Afterwards, it is purified three times with 25% ethanol. After purification, the prepolymer uPOCO is diluted to 45% (weight percentage) in ethanol for use in the synthesis of peripheral nerve sheaths.

[0074] (2) Prepare a 50 mg / mL polyvinyl alcohol (PVA) solution. Prepare an 8 wt% chitosan solution using acetic acid solution. Mix the PVA solution, chitosan solution, and uPOCO solution in a ratio of 3:8:5 to obtain a molding solution. Transfer the molding solution to a molding mold (consisting of an inner core rod and a mold outer sleeve) and cure at 70°C for 3.5 h until dry. Then place it at room temperature for 12 h to allow excess solvent to evaporate. After evaporation, the sleeve is cured by polycondensation at 70°C for 5 days. The cured semi-finished product is acetylated for 5 h in an acetylation reagent. The acetylation reagent is composed of acetic anhydride and methanol in a volume ratio of 1:1. Afterward, remove the molding mold outer sleeve, soak the POCO sleeve in deionized water overnight to dissolve the polyvinyl alcohol (PVA), and peel it off from the inner core rod. Then place the POCO sleeve in DMEM medium containing 20% ​​ethanol to remove unreacted monomers.

[0075] (3) The POCO sheath was soaked in 20% ethanol phosphate buffer (PBS) containing 1% penicillin / streptomycin for 24 h (37°C), followed by soaking in PBS containing 1% penicillin / streptomycin for 24 h (37°C). After that, the POCO sheath was soaked in low-glucose DMEM medium (glucose concentration less than 1 g / L) containing 1% penicillin / streptomycin for 2 h, then rinsed with deionized water, and then low-glucose DMEM was added to the peripheral nerve sheath and soaked for 12 h.

[0076] (4) Finally, the POCO sheath was soaked overnight in high-glucose DMEM (4 g / L glucose concentration) containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. Then it was soaked in 70% ethanol for 20 minutes, and then soaked again overnight (37°C) in high-glucose DMEM (4 g / L) containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin to remove excess ethanol residue on the membrane, thus obtaining the POCO peripheral nerve sheath.

[0077] Example 2

[0078] This embodiment is based on embodiment 1, and the hollow cylindrical sleeve obtained in step (3) is functionalized and modified as follows:

[0079] The prepolymer uPOCO was diluted 100 times with ethanol. The hollow cylindrical sleeve obtained in step (3), the diluted uPOCO, and EDOT were incubated at room temperature for 2 days. The sleeve was then transferred to a polymerization solution containing 28.5% EDOT, 14.2% ammonium persulfate solution (concentration 1.24M), and 57.3% phytic acid (stabilizer). The mixture was incubated in centrifuge tubes placed on a shaker and vigorously mixed overnight at 4°C.

[0080] After polymerization, the sheath was incubated in 70% ethanol for 15 minutes and then rinsed in high-glucose DMEM containing 10% FBS (fetal bovine serum) and 1% penicillin / streptomycin until the rinsing solution stopped discoloring to remove excess polymerization solution. The sheath was then incubated at room temperature in high-glucose DMEM (4 g / L) containing 10% FBS and 1% penicillin / streptomycin for 24 hours. The incubation solution was changed the next day, and the incubation was continued at 37°C for another 24 hours to obtain a conductive polymer-functionalized neural sheath.

[0081] like Figure 1 The image shows SEM images of the highly conductive peripheral nerve sheaths prepared in Examples 1 and 2 at magnification of 30k. As can be seen from the comparison between the right and left images, the nerve sheath prepared in Example 2 forms several sheet-like coverings on the surface of the elastic substrate material, which are conductive polymers.

[0082] The physical properties of the peripheral nerve sheath tubes with an inner diameter of 6 mm and an outer diameter of 6.5 mm prepared by Examples 1 and 2 and poly(octyl methylene maleate (anhydride) citrate) POMaC were tested, including conductivity, resistance to deformation, and mechanical strength. The test results are shown in Table 1.

[0083] Table 1:

[0084] Types of materials Electrical conductivity (S / m) Young's modulus Fracture strength POMaC casing 0.2 312 kPa 6N Example 1 0.5 467kPa 32N Example 2 2.9 723kPa 31N

[0085] Note: The POMaC sleeve is a POMaC replacement for the POCO in Example 1.

[0086] Biological properties such as cell activity and free radical scavenging ability (antioxidant) of peripheral nerve sheaths with an inner diameter of 6 mm and an outer diameter of 6.5 mm, prepared by Examples 1 and 2 and poly(octyl methylene maleate (anhydride) citrate) POMaC, were tested.

[0087] The test method is as follows, and the test results are shown in Table 2.

[0088] (1) Cell viability test

[0089] RSC96 rat Schwann cell line was incubated in high-glucose DMEM medium supplemented with 5% (v / v) fetal bovine serum. The medium was placed in a cell culture incubator. The cultured cells were divided into three groups: the first group was a blank control group, the second group was an experimental group, and the second group was cultured with POMaC tubing, tubing from Example 1, and tubing from Example 2, respectively. During the culture period, electrical stimulation was applied at a current intensity of 10 mV, a frequency of 20 Hz, and a stimulation time of 0.5 h / d for 3 consecutive days.

[0090] The three groups of cells were cultured under the same conditions for 3 days, and cell viability in each group was detected using CCK8 assay at the same time each day. It should be noted that cell viability represents cell growth rate and division activity, and thus reflects the promoting effect on peripheral nerve regeneration.

[0091] The cell activity of the blank control group on the first day was recorded as 1 (reference value). After testing, the cell activity of the blank control group on the second day was 1.83, and the cell activity of the blank control group on the third day was 2.30.

[0092] (2) Free radical scavenging rate test

[0093] Free radical scavenging rate was measured to assess antioxidant activity. After cutting open the tubing, the samples were cut into circular pieces using a 6 mm biopsy punch. The samples were incubated at 37°C in 200 μM DPPH dissolved in ethanol for 24 hours. PTFE was used as a negative control, and ascorbic acid as a positive control. The positive control measurements were subtracted from all samples to obtain a blank. Absorbance was measured at 320 nm.

[0094] Table 2:

[0095]

[0096] The test results above show that, compared with the POMaC sheath, the peripheral nerve sheaths prepared in Examples 1-2 all exhibit better electrical conductivity, resistance to deformation, and mechanical strength, resulting in superior nerve repair effects and antioxidant properties. Compared with Example 1, the peripheral nerve sheath prepared in Example 2 has higher electrical conductivity and Young's modulus, exhibiting good mechanical and elastomer behavior; it also has a higher free radical scavenging rate, increasing from 16% to 25%, enhancing antioxidant activity. Furthermore, prolonged cell activity suggests that the peripheral nerve sheath prepared in Example 2 improves nerve repair effects.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. These modifications or substitutions, or combinations of technical features in the above embodiments that do not conflict with each other, can be made in accordance with the manner described in the embodiments. These modifications, substitutions or combinations do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A highly conductive peripheral nerve sheath, characterized in that, A hollow cylindrical sleeve pipe is made of a material containing poly(octamethylene-limonite-co-octanol) POCO, and a regenerative material is loaded in the hollow cylindrical sleeve pipe; the regenerative material contains fetal bovine serum and DMEM cell culture medium.

2. A method for preparing a highly conductive peripheral nerve sheath, characterized in that, The application comprises the following steps: S1, preparing a poly(octamethylene-limonite-co-octanol) prepolymer uPOCO A mixture of citric acid, 1,8-octanediol and octanol is melted at 160-175°C, and reacted at 140-150°C for 2.5-5h to obtain a crude product containing the prepolymer uPOCO; the crude product is dissolved in 15%-35% ethanol for precipitation purification, and the precipitate is diluted with ethanol to a mass concentration of 30-55% to obtain an ethanol solution of the prepolymer uPOCO; S2, making a hollow cylindrical sleeve pipe The PVA solution, the chitosan solution and the ethanol solution of the prepolymer uPOCO obtained in S1 are mixed to obtain a molding liquid, the molding liquid is moved to a molding mold composed of an inner core rod and an outer sleeve or is molded into a hollow cylindrical structure by using an electrospinning process, and is cured at 50-80°C for 1.5-5h until dry, and is placed at room temperature for 12-24h to evaporate the excess solvent; then, the semi-finished product is subjected to post-polymerization condensation curing at 50-80°C for 4-8 days, and is acetylated in an acetylation reagent for 1-24h; after the acetylation is completed, the outer sleeve is removed, the remaining part is soaked in deionized water to dissolve the PVA, the molded hollow cylindrical sleeve pipe is peeled off from the inner core rod or the round rod-shaped jet receiving body, and is placed in a DMEM medium containing ethanol to discharge the unreacted monomers; S3, intermediate treatment The hollow cylindrical sleeve pipe is soaked in an ethanol PBS solution containing antibiotics for 20-30h, and then is replaced with a PBS solution containing antibiotics for continuous soaking for 20-30h; then, the pipe is transferred into a low-sugar DMEM culture medium containing antibiotics for soaking for 1-4h, and then is washed with deionized water, and is soaked in a low-sugar DMEM for 12-24h after the washing; S4, loaded with a regenerative material The pipe is soaked in a high-sugar DMEM containing fetal bovine serum and antibiotics overnight, is soaked in 70-75% ethanol for 15-25min, and is soaked in a high-sugar DMEM containing fetal bovine serum and antibiotics overnight again, thereby obtaining a POCO sleeve pipe for nerve repair.

3. The method of claim 2, wherein, In S1, the mixture is prepared by using citric acid, 1,8-octanediol and octanol in a molar ratio of (5-9):(6-10):(1-4); the crude product is dissolved in 15%-35% ethanol for precipitation purification for more than twice.

4. The preparation method according to claim 2, characterized in that, In S2, the PVA solution has a concentration of 50mg / mL, the chitosan solution has a mass concentration of 2-10%, the ethanol solution of the prepolymer uPOCO has a mass concentration of 30-55%, and the PVA solution, the chitosan solution and the ethanol solution of uPOCO are mixed in a mass ratio of (1-5):(1-10):(10-1) to obtain the molding liquid.

5. The preparation method according to claim 2, characterized in that, In S2, the acetylating agent is a mixture of acetic anhydride and methanol, the volume ratio of acetic anhydride to methanol in the mixture is 1:10-10:1; after the acetylation is completed, the outer sheath is removed, the remaining part is soaked in deionized water overnight to dissolve PVA, the formed hollow cylindrical sleeve is peeled off from the inner core rod or the round rod-shaped jet receiving body, and is placed in a DMEM medium containing 20v / v% ethanol to discharge unreacted monomers.

6. The preparation method according to claim 2, characterized in that, In S3, the hollow cylindrical sleeve is soaked in a 20v / v% ethanol PBS solution containing 1% penicillin / streptomycin for 24h, and then is replaced with a PBS solution containing 1% penicillin / streptomycin for continuous soaking for 24h; thereafter, the hollow cylindrical sleeve is transferred into a low-sugar DMEM medium containing 1% penicillin / streptomycin for soaking for 2h, and then is rinsed with deionized water and is placed in a low-sugar DMEM for soaking for 12-24h; In S4, the hollow cylindrical sleeve is soaked in a high-sugar DMEM containing 10% fetal bovine serum and 1% penicillin / streptomycin overnight, is soaked in 70-75% ethanol for 20min, and is again soaked in a high-sugar DMEM containing 10% fetal bovine serum and 1% penicillin / streptomycin overnight, thereby obtaining the POCO sleeve for nerve repair; The soaking temperature is 36.5-37.5℃.

7. A functionalized, highly conductive peripheral nerve sheath, characterized in that, The hollow sleeve and the regenerative material loaded in the hollow sleeve; the regenerative material comprises fetal bovine serum and DMEM cell culture medium; The hollow sleeve is prepared by using poly(octamethylene-limonite-co-octanol) as an elastic base material and modifying a conductive polymer on the surface of the elastic base material, the conductive polymer comprises a conductive monomer selected from at least one of pyrrole, aniline, p-phenylenediamine, o-methylaniline, thiophene, 3-hexylthiophene, 3-thiophene acetic acid and 3,4-ethylenedioxythiophene; and the conductive polymer is combined with the elastic base material in the form of an interpenetrating network structure.

8. A method of making a functionally modified, highly conductive peripheral nerve cuff, comprising: providing a cuff having a plurality of electrodes; providing a plurality of nerve fibers; and functionally modifying the plurality of nerve fibers to increase the conduction of action potentials through the plurality of nerve fibers. The method comprises the following steps: Step 1, preparing a poly(octamethylene-limonite-co-octanol) prepolymer uPOCO A mixture of citric acid, 1,8-octanediol and octanol is melted at 160-175℃, and is reacted at 140-150℃ for 2.5-5h to obtain a crude product containing the prepolymer uPOCO; the crude product is dissolved in 15%-35% ethanol for precipitation purification, and the precipitate is diluted with ethanol to a mass concentration of 30-55% to obtain an ethanol solution of the prepolymer uPOCO; Step 2, making a hollow cylindrical sleeve In S2, the acetylating agent is a mixture of acetic anhydride and methanol, the volume ratio of acetic anhydride to methanol in the mixture is 1:10-10:1; after the acetylation is completed, the outer sheath is removed, the remaining part is soaked in deionized water overnight to dissolve PVA, the formed hollow cylindrical sleeve is peeled off from the inner core rod or the round rod-shaped jet receiving body, and is placed in a DMEM medium containing 20v / v% ethanol to discharge unreacted monomers. Mixing the PVA solution, the chitosan solution and the ethanol solution of the prepolymer uPOCO obtained in step 1 to obtain a molding liquid, moving the molding liquid into a molding mold composed of an inner mandrel and an outer sleeve or using an electrospinning process to mold a hollow cylindrical structure, curing at 50-80℃ for 1.5-5h until dry, and placing at room temperature for 12-24h to evaporate the excess solvent; then continuing the post-polymerization condensation curing at 50-80℃ for 4-8 days to obtain a semi-finished product; acetylating the semi-finished product in an acetylating reagent for 1-24h, after the acetylation is completed, removing the outer sleeve, and placing the rest in deionized water to dissolve the PVA, peeling the molded hollow cylindrical sleeve from the inner mandrel or the round rod-shaped jet receiving body, and placing in a DMEM medium containing ethanol to discharge the unreacted monomers; Step 3, intermediate processing Soaking the hollow cylindrical sleeve in an ethanol PBS solution containing antibiotics for 20-30h, and then replacing it with a PBS solution containing antibiotics for continued soaking for 20-30h; then transferring it into a low-sugar DMEM medium containing antibiotics for soaking for 1-4h, and then rinsing with deionized water, and placing in a low-sugar DMEM for soaking for 12-24h after rinsing; Step 4: functional modification by in-situ polymerization Diluting the prepolymer uPOCO with ethanol by 50-200 times; incubating the hollow cylindrical sleeve of step 3, the diluted uPOCO and the conductive monomer at room temperature for 1-2 days; then transferring the hollow cylindrical sleeve into a polymerization solution containing a conductive monomer, an oxidation catalyst and phytic acid, and incubating, during which shaking and vigorous mixing at 0-5℃ overnight are performed, so that the uPOCO and the conductive monomer are in-situ polymerized on the hollow cylindrical sleeve; the conductive monomer is selected from at least one of pyrrole, aniline, p-phenylenediamine, o-methoxyaniline, thiophene, 3-hexylthiophene, 3-thiophene acetic acid and 3,4-ethylenedioxythiophene; Step 5: loading the pro-regenerative material Then incubating in 70-75% ethanol for 10-20min, and rinsing with a high-glucose DMEM containing FBS and antibiotics until the rinsing liquid no longer changes color; then incubating the hollow cylindrical sleeve in a high-glucose DMEM containing FBS and antibiotics at room temperature for 20-30h, and replacing the incubation liquid the next day for continued incubation for 20-30h.

9. The production method according to claim 8, characterized by, The antibiotic in each solution is a bacteriostatic combination of penicillin / streptomycin, and 1mL of a penicillin / streptomycin double-antibiotic stock solution is added per 100mL of the solution; the soaking temperature is 36.5-37.5℃.

10. The method of claim 8, wherein, In step 1, the mixture is prepared according to the molar ratio of (5-9) citric acid:(6-10) 1,8-octanediol:(1-4) octanol. In step 1, the mixture is prepared according to the molar ratio of (5-9) citric acid:(6-10) 1,8-octanediol:(1-4) octanol.

Images