Functionalized nerve repair system and uses thereof

Abstract

The application relates to the technical field of biomedical materials, and provides a functionalized nerve repair system and application thereof, the functionalized nerve repair system comprising a functionalized nerve repair composite conduit and an adhesive; the application innovatively provides a "glue-conduit-nerve tissue" triple chemical crosslinking system; the aldehyde group functionalized PEG adhesive is combined with the functionalized nerve conduit for the first time, the conduit is fixed through Schiff base reaction between the aldehyde group of the adhesive and the amino group of the natural material of the conduit (first crosslinking network, second crosslinking network), meanwhile, the tissue adhesion is realized through Schiff base reaction between the aldehyde group of the adhesive and the amino group on the surface of the nerve tissue (third crosslinking network), a one-time crosslinking and double-anchored chemical bonding fixing mechanism is formed, and the method is significantly superior to the physical wrapping or single adhesion mode in the prior art.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a functionalized nerve repair system and its application. Background Technology

[0002] Peripheral nerve injury (PNI) is a common traumatic condition that can lead to sensory loss, motor dysfunction, and even limb paralysis. Current clinical repair methods mainly include direct end-to-end anastomosis (only applicable to short-range defects), autologous nerve transplantation (which has limitations such as donor site damage and limited donor sources), and nerve conduit bridging. Nerve conduits are considered the most promising alternative to autologous nerve transplantation, but existing conduits still have fundamental limitations.

[0003] Existing nerve conduit materials are mainly divided into two categories: natural materials (acellular matrix, collagen, silk fibroin, chitosan, etc.) have excellent biocompatibility, which is conducive to Schwann cell adhesion and axonal growth, but their mechanical properties are insufficient, they are prone to collapse in vivo, and their degradation is often too rapid, making it difficult to provide continuous support; synthetic polymer materials (PCL, PLA, PLGA, etc.) have excellent mechanical properties and controllable degradation, but they lack bioactive sites and have strong surface hydrophobicity, which is not conducive to cell adhesion and active guidance of nerve regeneration. A single material system cannot simultaneously meet the dual requirements of nerve conduits for mechanical support and bioactive guidance.

[0004] In recent years, researchers have attempted to combine synthetic and natural materials through composite or layered structures to achieve synergistic performance. For example, CN113633430A uses a composite assembly structure of outer tube + orientation membrane + hollow tube + hydrogel, but it has many components, complex processes, and a long outer shell degradation time of 12-24 months, posing a risk of chronic foreign body reaction; CN110420355A is a bilayer structure (inner decellularized matrix / polyester orientation membrane, outer polyester layer), but it lacks a transition layer between synthetic and natural materials, resulting in weak interlayer bonding and abrupt performance changes; CN101579246B is a silk fibroin bilayer vessel with an inner layer of oriented fibers and an outer layer of vertical fibers, but it uses only a single natural material, resulting in limited mechanical properties.

[0005] Meanwhile, polyethylene glycol (PEG) has shown unique advantages in the field of nerve repair. Multiple preclinical and clinical studies have demonstrated that PEG-mediated axonal fusion can promote the repair of severed axonal membranes and accelerate sensory recovery. Existing technology also discloses a photoactivated polymer system for non-invasive, sutureless repair of peripheral nerves (TISSIUM's COAPTIUM® Connect with TISSIUM® LIGHT system DEN240066 / K251957), consisting of a photocurable polymer and a protective anastomosis cavity, used for non-invasive, sutureless repair of peripheral nerve injuries with gap closure ≤1cm. However, this product has the following shortcomings: ① It requires a dedicated external photocurable platform (TISSIUM LIGHT) for activation, increasing equipment requirements and operational steps; ② The 3D-printed cannula is a purely synthetic polymer material, providing only a physical channel and lacking the bioactivity to guide the directional growth of axons; ③ Its applicability is limited (only applicable to nerve gap closures ≤1cm).

[0006] Furthermore, aldehyde-functionalized PEG derivatives (such as CHO-PEG-CHO and multi-arm PEG-aldehyde) have been widely used in hydrogel construction, biomolecular cross-linking, and tissue engineering. These materials can form reversible imine bonds with amine-containing compounds via Schiff base reactions under mild conditions, exhibiting characteristics such as rapid reaction, no toxic byproducts, and excellent biocompatibility. However, current technologies lack a systematic design for combining aldehyde-based PEG adhesives with functionalized nerve conduits to achieve non-invasive, sutureless nerve repair through a triple chemical cross-linking process of "glue-conduit-tissue."

[0007] In summary, existing technologies have the following common shortcomings: ① Individual adhesives or catheters are developed independently, lacking a systematic combination scheme; ② Existing catheters are mostly simple double-layer structures, lacking a continuous material gradient transition from synthetic to natural materials; ③ Existing non-invasive sutureless techniques require external light sources, and the catheters lack bioactive guiding functions; ④ No dual anchoring design utilizing aldehyde-amino Schiff base reaction to simultaneously achieve catheter fixation and tissue adhesion has been found.

[0008] Therefore, developing a non-invasive, sutureless, functional nerve repair system that combines the advantages of chemical cross-linking without sutures, active guidance from functional catheters, and systematic integration is of significant clinical importance and market demand. Summary of the Invention

[0009] The technical problem to be solved by this invention is that existing systems have low fixation strength and low cell compatibility, and require complex suturing processes. To address this, this invention provides a functionalized nerve repair system and its application, which achieves the goals of simple operation, high mechanical effect, and significant repair effect.

[0010] To address the aforementioned technical problems, in a first aspect, the present invention provides a functionalized nerve repair system, the functionalized nerve repair system comprising a functionalized nerve repair composite conduit and an adhesive;

[0011] The functional nerve repair composite conduit has a tubular structure, comprising an inner layer, a middle layer, and an outer layer. The outer layer is a fiber layer formed by electrospinning synthetic polymer materials and / or natural materials. The middle layer is a composite fiber layer formed by electrospinning synthetic polymer materials and natural materials. The inner layer is a natural fiber layer formed by electrospinning natural materials. The degradation rates of the outer, middle, and inner layers form a gradient, and the outer, middle, and inner layers are fixed by cross-linking.

[0012] The adhesive contains aldehyde groups. The adhesive forms a first cross-linking network with the inner layer of the functional nerve repair composite catheter through the aldehyde groups, forms a second cross-linking network with the middle layer of the functional nerve repair composite catheter through the aldehyde groups, and forms a third cross-linking network with the surface of the nerve tissue through the aldehyde groups. The first cross-linking network, the second cross-linking network, and the third cross-linking network fix the functional nerve repair composite catheter to the nerve tissue.

[0013] In the first and second cross-linking networks of this invention, the aldehyde active groups of the adhesive can react with the amino groups of the natural materials in the inner and middle layers of the conduit to form a three-dimensional cross-linking network. In the third cross-linking network, the free aldehyde groups of the adhesive can target and bind to active sites such as amino groups in nerve tissue cell membrane proteins and extracellular matrix to generate a cross-linking system in situ.

[0014] The adhesive used in this invention acts similarly to glue, bonding ducts and nerve tissue. Generally, there is no specific ratio limit; any amount that satisfies the cross-linking effect can be used.

[0015] The functional nerve repair system provided by this invention features a first, second, and third cross-linking network that interconnects and works synergistically to firmly lock the functional nerve repair composite conduit and nerve tissue into an integrated structure. This significantly improves fixation strength compared to physical fixation methods, eliminating the need for additional sutures post-operatively and reducing surgical trauma and suture complications. The dual cross-linking network possesses controllable slow degradation characteristics, with a degradation cycle perfectly matching the nerve regeneration cycle. It gradually degrades after nerve regeneration and formation, leaving no residue or foreign body stimulation. Simultaneously, the cross-linking network can slowly release functional components such as neurotrophic factors, achieving targeted and long-lasting nerve repair regulation, greatly improving the success rate of peripheral nerve injury repair and shortening the patient's functional recovery period.

[0016] The degradation rate of the present invention is calculated based on the time required for the structure to completely degrade and disappear when placed in PBS buffer containing 1 μg / mL collagenase.

[0017] This invention features a three-layer gradient composite structure consisting of an outer layer, a middle layer, and an inner layer. Each layer is prepared using an electrospinning process, resulting in a uniform fiber structure, good pore connectivity, and precise control over pore size, porosity, fiber orientation, and degradation rate.

[0018] The degradation rate gradient can be formed by increasing the degradation rate from the outer layer to the inner layer, matching the physiological law of peripheral nerve regeneration: the outer layer degrades slowly to maintain the overall structural stability of the duct in the long term; the middle layer degrades moderately to adapt to the initial proliferation and growth of nerve tissue; the inner layer degrades rapidly to reserve sufficient space for the growth and remodeling of new nerve tissue, avoiding the failure of the microenvironment due to excessively rapid degradation of the duct, or the compression of new nerve tissue due to excessively slow degradation.

[0019] Preferably, the synthetic polymer material includes any one or a combination of at least two of polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), polylactic acid-glycolic acid copolymer (PLGA), polylactide-caprolactone copolymer (PLCL), or polylactic acid-polyethylene glycol copolymer (PLA-PEG).

[0020] Preferably, the natural material includes any one or a combination of at least two of the following: decellularized matrix, collagen, chitosan, or silk fibroin.

[0021] Preferably, the average pore size of the outer layer is 50~500nm, for example, it can be 0nm, 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, etc., and the porosity is 60%~80%, for example, it can be 60%, 62%, 65%, 68%, 70%, 72%, 75%, 78%, 80%, etc.

[0022] Preferably, the axial orientation of the inner layer of the fibrous functional nerve repair composite conduit is oriented, with a fiber orientation degree higher than 75%. In this invention, the highly axially oriented fiber structure can form a physical guiding channel, precisely guiding the nerve axons to extend orderly along the conduit axis, significantly improving the nerve regeneration rate and regularity.

[0023] Preferably, the adhesive comprises a multi-arm polyethylene glycol containing aldehyde groups, with a molecular weight of 5000~40000 Da, for example, 5000 Da, 8000 Da, 10000 Da, 15000 Da, 20000 Da, 25000 Da, 30000 Da, 35000 Da, 40000 Da, etc., and the degree of substitution of the aldehyde groups is 8%~15%, for example, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%; the multi-arm polyethylene glycol containing aldehyde groups exists in the form of being dissolved in phosphate buffer, and the concentration of the multi-arm polyethylene glycol containing aldehyde groups in the phosphate buffer is 8%~15%, for example, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, etc.

[0024] The adhesive used in this invention is different from existing adhesives, which are mostly general-purpose adhesives that rely on single physical bonding, instant curing, and lack biological specific bonding capabilities. The aldehyde-based adhesive of this invention uses multi-arm polyethylene glycol as the skeleton and active aldehyde groups as the crosslinking core, which can match the physiological structure and regeneration needs of nerve tissue and work with composite catheters to construct an integrated repair system.

[0025] The degree of aldehyde substitution in this invention refers to the percentage of terminal hydroxyl groups in the polyethylene glycol molecular chain that are successfully replaced by aldehyde groups.

[0026] The multi-arm polyethylene glycol of this invention refers to a star-shaped branched structure in which multiple independent polyethylene glycol molecular chains are radiating outward from a central molecule. Each outwardly extending molecular chain is considered an arm, and the structure can be two-armed, four-armed, or eight-armed. The more arms, the more terminal active reaction sites a single molecule can provide, and the stronger the ability to construct a molecular spatial network. Based on this, aldehyde functionalization modification is performed, that is, aldehyde active functional groups are grafted to the end of each molecular chain of the multi-arm polyethylene glycol, ultimately forming an aldehyde-modified multi-arm polyethylene glycol containing highly active crosslinking sites. This is the key to the high-density, three-dimensional double crosslinked network that this invention can construct.

[0027] The aldehyde-containing multi-arm polyethylene glycol includes any one or a combination of at least two of the following: aldehyde-containing two-arm polyethylene glycol, aldehyde-containing four-arm polyethylene glycol, or aldehyde-containing eight-arm polyethylene glycol.

[0028] Preferably, the adhesive further includes a polyamine compound.

[0029] The polyamino compound includes a mixture of polylysine and polyethyleneimine, or any one or a combination of at least two of polylysine or polyethyleneimine.

[0030] In this invention, an adhesive is preferably composed of a polyamino compound and a multi-arm polyethylene glycol containing aldehyde groups. The two components can be mixed in equal volumes. The amino groups can react with the aldehyde groups to form a Schiff base reaction to form a cross-linked hydrogel, which helps to strengthen the density of the cross-linked network, improve the curing speed and bonding strength of the adhesive, and at the same time improve the bioadhesion of the adhesive.

[0031] Preferably, the adhesive further includes neurotrophic factors, which include any one or a combination of at least two of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and glial cell-derived neurotrophic factor (GDNF).

[0032] The concentration of the neurotrophic factor in this invention is 10~200 ng / mL, for example, it can be 10 ng / mL, 30 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, etc.

[0033] The adhesive also includes functional additives, including butylated hydroxytoluene and / or brilliant blue.

[0034] Secondly, the present invention provides the application of the functionalized nerve repair system as described in the first aspect in the preparation of medical devices for treating peripheral nerve injuries.

[0035] The functionalized nerve repair system provided by this invention is suitable for non-invasive, sutureless repair of peripheral nerve injuries. Compared with existing technologies, this invention has the following advantages: 1. Simple operation: The chemical cross-linking system does not require an external light curing platform, simplifying the surgical procedure, reducing equipment requirements, and making it more suitable for medical institutions at different levels, which is conducive to product promotion and popularization. 2. Wider range of indications: The functionalized catheter can bridge longer segmental nerve defects (more than 10 mm), breaking through the limitation of existing non-invasive, sutureless products that are only suitable for closure of gaps ≤1 cm. 3. Excellent repair effect: Animal experiments have confirmed that the repair effect of this system reaches or approaches the gold standard of autologous nerve transplantation. 4. Flexible catheter structure: The catheter contains at least two layers, and a double-layer or triple-layer gradient structure can be selected according to clinical needs, taking into account both mechanical support and bioactive guidance. 5. Controllable process: The electrospinning and chemical cross-linking processes are mature, highly controllable, and easy to scale up production. 6. Combination product strategy: The catheter and adhesive can be sold separately or in combination, forming a multi-level commercialization strategy.

[0036] Implementing this invention has at least the following beneficial effects:

[0037] 1. This invention innovatively provides a triple chemical crosslinking system of "adhesive-catheter-nerve tissue": This invention is the first to combine aldehyde-functionalized PEG adhesive with functionalized nerve conduits. The conduit is fixed by the Schiff base reaction between the aldehyde group of the adhesive and the amino group of the natural material of the conduit (first crosslinking network, second crosslinking network). At the same time, the tissue is adhered by the Schiff base reaction between the aldehyde group of the adhesive and the amino group on the surface of the nerve tissue (third crosslinking network). This forms a chemical bonding and fixation mechanism of one crosslinking and double anchoring, which is significantly better than the physical encapsulation or single adhesion method of the prior art.

[0038] 2. Chemical cross-linking without light source curing: Unlike the photo-activated physical encapsulation of existing systems, this invention uses an aldehyde-amino Schiff base chemical cross-linking reaction, which can be rapidly cured in situ at room temperature without the need for an external light curing platform. This simplifies the surgical procedure, reduces the requirements for equipment, and is more suitable for medical institutions at different levels.

[0039] 3. Synergistic design of functionalized catheters and adhesives: The natural material (containing amino groups) of the inner layer of the catheter in this invention has three functions: providing bioactive guidance for axonal growth, serving as a cross-linking reaction site for adhesive to achieve catheter fixation, and loading neurotrophic factors to promote nerve regeneration, thus realizing an integrated design of structure and function.

[0040] 4. Flexibility and innovation of catheter structure: The catheter of this invention has a three-layer structure. The inner layer is an amino-containing natural material oriented fiber layer, which provides axon growth guidance and adhesive cross-linking sites. The outer layer is a synthetic polymer material or natural material, which provides mechanical support and anti-adhesion function. The middle blend layer can realize a gradient structure in which the material composition gradually transitions from the outside to the inside, further optimizing the synergy between mechanical properties and biological activity.

[0041] The three-layer fiber is formed by sequential electrospinning, continuous deposition, and interweaving. The middle layer is a composite layer of polymer and natural materials. According to the principle of like dissolves like, the middle layer has the same similar material as the inner and outer layers, which can achieve a firm bonding between different layers through electrospinning.

[0042] 5. Stepped degradation time design: The degradation time of each layer of the conduit is matched with the nerve regeneration cycle (inner layer 4-10 weeks, middle layer 8-12 weeks, outer layer 12-24 weeks). The outer layer degrades slowly to maintain mechanical support during the critical window period of nerve regeneration, while the inner layer degrades rapidly to provide growth space for new axons.

[0043] 6. Expanded indications: The functional catheters of this system can bridge longer segmental nerve defects (more than 10 mm), compared with existing methods that are only suitable for closing gaps of ≤1 cm, thus broadening the indications.

[0044] 7. Excellent safety and operability: Schiff base reaction conditions are mild and there are no toxic byproducts. The imine bonds of the reaction products are reversible under physiological conditions, which is beneficial to material degradation and tissue remodeling. The two-component system is easy to mix before use and operate. The addition of staining agents to the adhesive can achieve intraoperative visualization, and the addition of neurotrophic factors can further promote nerve regeneration. Attached Figure Description

[0045] Figure 1 This is a physical image of the nerve repair composite catheter of Embodiment 1 of the present invention.

[0046] Figure 2 This is a SEM image of the outer layer of the nerve repair composite conduit in Embodiment 1 of the present invention.

[0047] Figure 3 This is a SEM image of the inner layer of the nerve repair composite conduit in Embodiment 1 of the present invention. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0049] Example 1

[0050] PLGA / PLGA and collagen / collagen triple-layer conduit system with four-arm PEG-aldehyde adhesive

[0051] 1. Material Preparation

[0052] (1) Preparation of the three-layer nerve repair conduit, specifically as follows: Figure 1 As shown, the SEM images of the outer and middle layers after spinning are as follows: Figure 2 As shown, the inner layer SEM image is as follows: Figure 3 As shown.

[0053] Outer spinning solution: Dissolve PLGA (LA:GA=75:25, molecular weight 100 kDa) in a mixed solvent of dichloromethane and dimethylformamide (volume ratio 4:1) to prepare a 12% mass fraction solution. Add 8% PEG (molecular weight 2000) by mass of PLGA and stir at room temperature until completely dissolved.

[0054] Intermediate layer spinning solution: PLGA and type I collagen in a mass ratio of 7:3 are dissolved in hexafluoroisopropanol (HFIP) to prepare a 10% mass fraction solution, and stirred at room temperature until completely dissolved.

[0055] Inner spinning solution: Type I collagen is dissolved in hexafluoroisopropanol (HFIP) to prepare a solution with a mass fraction of 8%, and stirred at room temperature until completely dissolved.

[0056] Multi-needle sequential electrospinning technology was employed, with the following spinning parameters: voltage 18 kV, receiving distance 15 cm, and feed rate 1.0 mL / h. The rotating receiving device rotated at 2000 rpm. The inner, middle, and outer spinning solutions were sequentially deposited onto a rotating drum to form a three-layer composite fiber membrane. After drying, the membrane was wound into a tubular shape (inner diameter 1.5 mm, wall thickness 0.5 mm) and cross-linked for later use.

[0057] (2) Preparation of aldehyde-based PEG adhesives

[0058] Component A (PEG-aldehyde solution): Dissolve four-armed PEG-aldehyde (4-PEG-CHO, molecular weight 20,000 Da, degree of aldehyde substitution 12%) in phosphate buffer (PBS, pH 7.4) to prepare a 10% (w / v) solution, filter to sterilize and use for later use.

[0059] Component B (polyamine solution): Polylysine (molecular weight 3,000 Da) and polyethyleneimine (PEI, branched structure, molecular weight 1,800 Da) are mixed at a mass ratio of 3:1, dissolved in PBS (pH 8.0) to prepare a solution with a total concentration of 8% (w / v), filtered and sterilized for later use.

[0060] Instructions for use: Wrap the severed nerve tissue inside the three-layer nerve repair catheter. Apply an appropriate amount of two-component adhesive to both ends of the severed nerve (place adhesive components A and B in a two-component syringe and inject simultaneously). Ensure that the adhesive is in full contact with the nerve tissue and the catheter to achieve a firm adhesion between the catheter and the nerve tissue.

[0061] The aldehyde-based PEG adhesive forms a first cross-linking network with the inner layer of the functional nerve repair composite catheter through the aldehyde groups, forms a second cross-linking network with the middle layer of the functional nerve repair composite catheter through the aldehyde groups, and forms a third cross-linking network with the surface of the nerve tissue through the aldehyde groups. The first cross-linking network, the second cross-linking network, and the third cross-linking network fix the functional nerve repair composite catheter to the nerve tissue.

[0062] Mechanism of action: The inner and middle layers of the nerve repair conduit contain a large number of amino groups in type I collagen, and the adhesive on the four walls contains aldehyde groups. The amino and aldehyde groups undergo a Schiff base cross-linking reaction, and the adhesive forms a strong adhesion with the inner layer of the functional nerve repair composite conduit. The surface components of the nerve tissue also contain amino groups, and the adhesive forms a strong adhesion with the surface of the nerve tissue, ultimately achieving a strong adhesion between the conduit and the nerve tissue.

[0063] 2. Adhesive performance characterization

[0064] (1) Gel time test: The gel time was determined using the inverted test tube method. Component A and component B were mixed in equal volumes (0.5 mL each) and shaken rapidly. The time from mixing to gel formation was recorded. The results showed that the gel time was 45 ± 10 seconds at 25°C. By changing the degree of aldehyde substitution in component A (8%–15%), the gel time could be adjusted between 30 seconds and 3 minutes to meet the needs of different surgical procedures.

[0065] (2) Adhesion strength test: The adhesion performance of the adhesive was evaluated by the overlap shear test. Using isolated rat sciatic nerve tissue as the adhesion substrate, component A and component B were mixed in equal volumes and then coated onto the surface of the nerve tissue, with an adhesion area of ​​approximately 1 cm². 2 After curing at room temperature for 5 minutes, the material was tested using a universal testing machine at a tensile speed of 10 mm / min.

[0066] The results showed that the overlap shear adhesion strength between the adhesive and the nerve tissue was 35.2 ± 4.8 kPa. Further testing using the inner collagen fiber membrane of the duct as the adhesion substrate yielded an overlap shear adhesion strength of 28.5 ± 3.9 kPa between the adhesive and the duct. Using a two-interface adhesion model of nerve tissue and duct, the overall adhesion strength of the adhesive simultaneously bonding the nerve tissue and duct interface was 32.8 ± 5.2 kPa.

[0067] (3) Swelling rate test: The cured adhesive gel (10 mm in diameter and 2 mm in thickness) was immersed in PBS at 37°C, and weighed at regular intervals to calculate the swelling rate. The results showed that the equilibrium swelling rate after 24 hours was 18±3%, which was significantly lower than that of most reported PEG hydrogel systems (usually >50%). The low swelling rate helps to avoid compression of nerve tissue.

[0068] (4) Cell compatibility test: In accordance with ISO 10993-5 standard, L929 fibroblasts and primary Schwann cells were used to perform CCK-8 cytotoxicity test on the adhesive extract. The results showed that the cell survival rate was >92%, indicating that the adhesive has good cell compatibility.

[0069] (5) Characterization of the performance of the conduit: the outer fiber diameter is about 420±90 nm, the average pore size is about 180 nm, and the porosity is about 68%; the inner fiber diameter is about 280±60 nm, and the fibers are highly oriented along the axial direction of the conduit (orientation degree 85%); the tensile strength is 4.8±0.5 MPa, and the elongation at break is 120±15%.

[0070] 3. Conclusion

[0071] Example 1 verified the basic feasibility of combining a double-layered catheter with a four-armed PEG-aldehyde adhesive. The adhesive has a suitable gel time, excellent adhesion strength, low swelling rate and good cell compatibility, and the catheter has good mechanical properties and orientation guidance structure.

[0072] Example 2

[0073] Eight-arm PEG-aldehyde adhesive + PCL / silk fibroin three-layer gradient catheter system (loaded with NGF)

[0074] 1. Material Preparation

[0075] (1) Preparation of a three-layer gradient nerve repair conduit

[0076] Outer spinning solution: Dissolve PCL (molecular weight 80 kDa) in HFIP to prepare a 10% mass fraction solution, add 10% PCL mass of PEG (molecular weight 2000), and stir at room temperature until completely dissolved.

[0077] Intermediate layer spinning solution: PCL and silk fibroin are mixed at a mass ratio of 7:3 and dissolved in HFIP to prepare a solution with a total mass fraction of 12%. Stir at room temperature until completely dissolved.

[0078] Inner spinning solution: Dissolve regenerated silk fibroin in formic acid to prepare a 10% (w / w) solution, and stir at room temperature until completely dissolved.

[0079] Spinning parameters: voltage 20 kV, receiving distance 12 cm, feed speed 1.2 mL / h, roller speed 2500 rpm. Three-layer composite fiber membranes are spun sequentially, dried, and wound into tubes (inner diameter 2.0 mm, wall thickness 0.6 mm), and then subjected to gradient dehydration treatment with ethanol.

[0080] (2) Preparation of aldehyde-based PEG adhesives (containing NGF)

[0081] Component A (PEG-aldehyde solution): Dissolve octagonal PEG-aldehyde (8-PEG-CHO, molecular weight 40,000 Da, degree of aldehyde substitution 10%) in PBS (pH 7.4) to prepare a 12% (w / v) solution. Add nerve growth factor (NGF, final concentration 100 ng / mL) and brilliant blue (0.02% w / v), filter to sterilize, and use for later use.

[0082] Component B (Polyamino solution): Polylysine (molecular weight 5,000 Da) and polyethyleneimine (PEI, molecular weight 1,800 Da) are mixed at a mass ratio of 2:1 and dissolved in PBS (pH 8.0) to prepare a solution with a total concentration of 10% (w / v). Antioxidant BHT (0.05% w / v) is added, and the solution is filtered and sterilized before use.

[0083] Usage method: Same as in Example 1.

[0084] The aldehyde-based PEG adhesive forms a first cross-linking network with the inner layer of the functional nerve repair composite catheter through the aldehyde groups, forms a second cross-linking network with the middle layer of the functional nerve repair composite catheter through the aldehyde groups, and forms a third cross-linking network with the surface of the nerve tissue through the aldehyde groups. The first cross-linking network, the second cross-linking network, and the third cross-linking network fix the functional nerve repair composite catheter to the nerve tissue.

[0085] Mechanism of action: The silk fibroin material in the inner and middle layers of the nerve repair conduit contains a large number of amino groups, and the octagonal PEG adhesive contains a large number of aldehyde groups. The amino and aldehyde groups undergo a Schiff base cross-linking reaction, and the adhesive forms a strong adhesion with the inner layer of the functional nerve repair composite conduit. The components on the surface of the nerve tissue also contain amino groups, and the adhesive forms a strong adhesion with the surface of the nerve tissue, ultimately achieving a strong adhesion between the conduit and the nerve tissue.

[0086] 2. Adhesive performance characterization

[0087] (1) Gel time test: gel time is 60±15 seconds (25℃).

[0088] (2) Adhesion strength test: The overlap shear test showed that the adhesion strength of the adhesive to nerve tissue was 38.6±5.1 kPa, and the adhesion strength to the duct was 31.2±4.3 kPa. Compared with Example 1, the increased number of crosslinking sites in the eight-arm PEG slightly improved the adhesion strength.

[0089] (3) NGF release curve: ELISA detection showed that NGF in the adhesive could be released in vitro for more than 6 weeks, with the release concentration maintained within the biological activity window of 15~45 ng / mL, and the release behavior conformed to the first-order kinetic model.

[0090] (4) Cell compatibility test: CCK-8 assay showed cell viability >90%.

[0091] (5) Characterization of catheter performance: The outer layer of the catheter has a fiber diameter of about 380±80 nm, an average pore size of about 150 nm, and a porosity of about 70%; the inner layer has a fiber diameter of about 300±70 nm and an orientation degree of 82%; the tensile strength is 5.0±0.6 MPa and the elongation at break is 115±12%; the in vitro degradation experiment showed that the inner layer was basically degraded in 7 weeks, the middle layer was significantly degraded in 11 weeks, and the outer layer was completely degraded in 18 weeks, showing a step-degradation characteristic.

[0092] 3. Conclusion

[0093] Example 2, based on Example 1, uses an eight-arm PEG-aldehyde adhesive loaded with NGF, and upgrades the catheter to a three-layer gradient structure. The adhesive strength and sustained-release performance of the adhesive are further improved, and the stepped degradation design of the catheter is better matched with the nerve regeneration cycle.

[0094] Example 3

[0095] Four-arm PEG-OPA adhesive + PCL / PLGA / chitosan / dECM / BDNF four-component gradient catheter system

[0096] 1. Material Preparation

[0097] (1) Preparation of a three-layer gradient nerve repair conduit (four-component system)

[0098] Outer spinning solution: PCL and PLGA (LA:GA=85:15) are mixed at a mass ratio of 1:1 and dissolved in HFIP to prepare a solution with a mass fraction of 11%. PEG is added at a total mass of 8% of PCL / PLGA and stirred at room temperature until completely dissolved.

[0099] Intermediate layer spinning solution: PCL, PLGA and chitosan are mixed in a mass ratio of 4:3:3. Chitosan is pre-dissolved in trifluoroacetic acid (TFA) and then mixed with the PCL / PLGA HFIP solution to prepare a solution with a total mass fraction of 13%.

[0100] Inner spinning solution: Dissolve porcine decellularized matrix (dECM) powder in HFIP to prepare a 7% (w / w) solution, add chitosan (dECM:chitosan = 8:2), and add BDNF (final concentration 100 ng / mL).

[0101] Spinning parameters: voltage 22 kV, receiving distance 18 cm, feed speed 0.8 mL / h, roller speed 3000 rpm. Three-layer composite fiber membranes were spun sequentially. The resulting fiber membranes were wound into tubes (inner diameter 1.8 mm, wall thickness 0.55 mm) and then thermally crosslinked at 60℃ for 2 hours.

[0102] (2) Preparation of aldehyde-based PEG adhesives (OPA system)

[0103] Component A (PEG-OPA solution): Four-arm PEG-o-phthalaldehyde (4-PEG-OPA, molecular weight 20,000 Da, o-phthalaldehyde substitution degree 8%) was dissolved in PBS (pH 7.4) to prepare a 6% (w / v) solution. BDNF (final concentration 50 ng / mL) and Brilliant Blue (0.02% w / v) were added, and the solution was filtered and sterilized before use. The aldehyde reactivity of OPA-terminated groups is significantly higher than that of conventional benzaldehyde, enabling rapid cross-linking at lower concentrations.

[0104] Component B (polyamine solution): Polylysine (molecular weight 5,000 Da) and polyethyleneimine are mixed at a mass ratio of 3:1, dissolved in PBS (pH 8.0) to prepare a solution with a total concentration of 8% (w / v), filtered and sterilized for later use.

[0105] Usage method: Same as in Example 1.

[0106] The aldehyde-based PEG adhesive forms a first cross-linking network with the inner layer of the functional nerve repair composite catheter through the aldehyde groups, forms a second cross-linking network with the middle layer of the functional nerve repair composite catheter through the aldehyde groups, and forms a third cross-linking network with the surface of the nerve tissue through the aldehyde groups. The first cross-linking network, the second cross-linking network, and the third cross-linking network fix the functional nerve repair composite catheter to the nerve tissue.

[0107] Mechanism of action: The decellularized matrix (dECM) of the inner layer of the nerve repair conduit and the chitosan of the middle layer both contain a large number of amino groups, and the four-arm PEG-phthalaldehyde adhesive contains a large number of aldehyde groups. The amino groups and aldehyde groups undergo Schiff base cross-linking reaction, and the adhesive forms a strong adhesion with the inner and middle layers of the functional nerve repair composite conduit. The components on the surface of the nerve tissue also contain amino groups, and the adhesive forms a strong adhesion with the surface of the nerve tissue, ultimately achieving a strong adhesion between the conduit and the nerve tissue.

[0108] 2. Adhesive performance characterization

[0109] (1) Gel time test: The gel time was 35±10 seconds (25℃). Due to the high reactivity of OPA, the gel speed was significantly faster than in Example 1 and Example 2, and the critical gel concentration was reduced to about 1% (w / v).

[0110] (2) Adhesion strength test: The overlap shear test showed that the adhesion strength of the adhesive to the nerve tissue was 42.3±5.8 kPa and the adhesion strength to the duct was 35.6±4.5 kPa, both of which were significantly better than those of Example 1 and Example 2 (P<0.05), confirming the advantages of the OPA system in terms of adhesion performance.

[0111] (3) Cell compatibility test: CCK-8 assay showed cell viability >93%. Due to the lower aldehyde concentration required by the OPA system (6% vs 10%-12%), cell compatibility was slightly improved.

[0112] (4) Characterization of duct performance: The outer layer of the duct has a fiber diameter of about 380±75 nm, an average pore size of about 150 nm, and a porosity of about 70%; the inner layer has a fiber diameter of about 250±50 nm and an orientation degree of 88%; the tensile strength is 5.5±0.7 MPa and the elongation at break is 110±10%; in vitro degradation experiment: about 18 weeks for the outer layer, about 12 weeks for the middle layer, and about 7 weeks for the inner layer; retention of dECM active ingredients: ELISA detection showed that the collagen content retained in the inner layer was about 65%, the glycosaminoglycan (GAG) content was about 12%, and the growth factor retention rate was about 40%.

[0113] 3. Animal experiments

[0114] (1) Animal model: 24 adult male SD rats (weighing 200-250 g) were randomly divided into 3 groups: the composite system group of this invention (n=8), the TISSIUM COAPTIUM® system control (positive control, n=8), and the blank control group (resection without repair, n=8). After anesthesia, the right sciatic nerve was exposed, and the middle 10 mm of the nerve was removed to create a 10 mm segmental defect.

[0115] The composite system of the present invention is as follows: the three-layer gradient conduit prepared in Example 3 is used to bridge the defect, and then component A and component B are mixed in equal volumes and sprayed onto the connection between the conduit and the nerve end using a dual-syringe. The mixture is then left to stand at room temperature for 2 minutes to complete in-situ curing.

[0116] TISSIUM control (positive control) group: Repair was performed according to the TISSIUM system operating instructions.

[0117] Blank control group: The nerve was removed but not repaired, and was directly sutured.

[0118] (2) Postoperative assessment (12 weeks postoperatively):

[0119] Gross observation: No obvious adhesions were observed in the composite system group of this invention. The catheter was easily separated from the surrounding tissue, and regenerated nerve tissue was visible continuously passing through the entire length of the catheter within the lumen. No obvious adhesions were also observed in the TISSIUM control group.

[0120] Sciatic nerve function index (SFI): The SFI of the composite system group of this invention was -60.5±4.2, the SFI of the TISSIUM control group was -66.2±4.9 (P<0.01), and the SFI of the autologous nerve transplantation group was -58.3±5.2.

[0121] Nerve conduction velocity: 42.8±4.0 m / s for the composite system group of this invention, and 36.8±3.8 m / s for the TISSIUM control group.

[0122] Composite muscle action potential (CMAP) amplitude: 91% ± 6% of the autologous transplantation group in the composite system group of this invention.

[0123] Histological analysis: Regenerated nerve tissue was harvested 12 weeks postoperatively and stained with toluidine blue. The myelinated nerve fiber count in the composite system group of this invention was 2,150 ± 180 / mm. 2 Compared with the autologous transplantation group (2,120±180 / mm), 2 The results were comparable to, and significantly better than, the TISSIUM control group (1,850 ± 195 / mm). 2 (P<0.01).

[0124] Myelin thickness (G-ratio): 0.67±0.04 for the composite system group and 0.65±0.04 for the autologous transplantation group.

[0125] Immunofluorescence staining: Immunofluorescence staining at 4 weeks post-surgery showed that S-100-positive Schwann cells formed continuous cell chains along the oriented fiber direction; NF-200 staining at 8 weeks post-surgery showed that the regenerated axons had filled the entire length of the duct and were arranged in an orderly manner.

[0126] The wet weight ratio of gastrocnemius muscle was 0.68±0.05 in the composite system group and 0.71±0.05 in the autologous transplantation group. There was no significant difference between the two groups (P>0.05).

[0127] 4. Conclusion

[0128] The four-arm PEG-OPA adhesive (containing BDNF) + PCL / PLGA / chitosan / dECM / BDNF four-component gradient catheter system in Example 3 achieved or approached the performance level of autologous nerve transplantation in all performance indicators, significantly outperforming the TISSIUMCOAPTIUM® system. The OPA system, by reducing aldehyde concentration while increasing adhesion strength, balances safety and efficacy, making it a preferred adhesive for nerve repair. Animal experiments confirmed that this system can effectively repair a 10 mm sciatic nerve defect in rats, with repair effects comparable to autologous nerve transplantation.

[0129] Comparative Example 1 (The catheter material does not contain amino groups and cannot cross-link with the adhesive)

[0130] Eight-arm PEG-aldehyde adhesive + PCL / PLGA double-layer conduit (free of natural materials)

[0131] 1. Material Preparation

[0132] (1) Double-layer nerve repair conduit (containing no natural materials):

[0133] Outer spinning solution: Same as the outer layer in Example 2, PCL (Mw 80 kDa) dissolved in HFIP, mass fraction 10%, with 10% PEG 2000 added.

[0134] Inner spinning solution: PCL and PLGA (LA:GA=75:25, Mw 100 kDa) are mixed at a mass ratio of 1:1 and dissolved in HFIP, with a total mass fraction of 12%, and contain no natural materials (no collagen, no silk fibroin, no chitosan, no dECM).

[0135] Spinning parameters: same as in Example 2 (voltage 20 kV, distance 12 cm, feed rate 1.2 mL / h, roller 2500 rpm). A double-layer fiber membrane was sequentially spun and wound into a tube (inner diameter 2.0 mm, wall thickness 0.6 mm).

[0136] (2) Aldehyde-based PEG adhesive: exactly the same as component A (octagonal PEG-aldehyde, 40 kDa, degree of substitution 10%, concentration 12% w / v, containing NGF 100 ng / mL) and component B (polylysine:PEI=2:1, total concentration 10% w / v) in Example 2.

[0137] 2. Adhesive performance characterization

[0138] (1) Gel time test: The gel time was determined by inverted test tube method. Component A (octagonal PEG-aldehyde group) and component B (polyamino solution) were mixed in equal volumes and then shaken quickly. The results showed that the gel time was 60±12 seconds at 25℃, which was not significantly different from that in Example 2 (60±15 seconds), indicating that the Schiff base crosslinking reaction of the adhesive itself was not affected by the catheter material.

[0139] (2) Adhesion strength test: The adhesion performance of the adhesive was evaluated by the overlap shear test. When using isolated rat sciatic nerve tissue as the adhesion substrate, the adhesion strength between the adhesive and the nerve tissue was 37.9±4.8 kPa, which was comparable to that in Example 2 (38.6±5.1 kPa), indicating that the second cross-linking network (aldehyde-amino Schiff base reaction) between the adhesive and the nerve tissue could be formed normally. However, when using the inner layer of the conduit (PCL / PLGA synthetic fiber membrane) as the adhesion substrate, the adhesion strength between the adhesive and the conduit was only 3.2±1.1 kPa, which was much lower than that in Example 2 (31.2±4.3 kPa). This is because the inner layer of the conduit is composed entirely of synthetic polymers and lacks active groups such as amino groups on its surface, so it cannot form the first cross-linking network (Schiff base reaction) with the aldehyde groups of the adhesive, and only has very weak physical adsorption. Further testing was conducted using a two-interface adhesion model of nerve tissue and conduit (adhering the nerve stump to the inner wall of the conduit at the same time), and the overall adhesion strength was only 8.5±2.3 kPa, and slight disturbance could cause the conduit to detach from the nerve.

[0140] Comparative Example 2 (The adhesive does not contain aldehydes and cannot adhere to moist nerve tissue)

[0141] Fibrin Glue + Four-Component Gradient Catheter System of Example 3

[0142] 1. Material Preparation

[0143] (1) Nerve repair catheter: The four-component gradient catheter (PCL / PLGA / chitosan / dECM, with BDNF in the inner layer) is exactly the same as that in Example 3.

[0144] (2) Adhesive: Clinically commonly used fibrin glue (containing no aldehydes) is selected. Typical composition:

[0145] Component A: Human fibrinogen (80 mg / mL), aprotinin (3000 KIU / mL), dissolved in sodium chloride solution.

[0146] Component B: Human thrombin (500 IU / mL), calcium chloride (40 mmol / L).

[0147] Before use, mix equal volumes using a dual syringe. After application, a fibrin clot will form within seconds.

[0148] 2. Adhesive performance characterization

[0149] (1) Gel time test: The gel time was determined by inverting the test tube. After mixing equal volumes of component A (fibrinogen solution) and component B (thrombin solution) of fibrin glue, the gel time was approximately 10 ± 3 seconds (25℃). This curing speed is extremely fast, which is not conducive to adjusting the catheter position during the operation, and clots are easily formed when the coating is not uniform.

[0150] (2) Adhesion strength test: The adhesion performance of the adhesive was evaluated by the overlap shear test. When using isolated rat sciatic nerve tissue as the adhesion substrate, the adhesion strength of fibrin glue to wet nerve tissue was only 5.6±2.1 kPa, which was much lower than 42.3±5.8 kPa in Example 3. This is because fibrin glue mainly relies on the physical entanglement of fibrin with the tissue surface and a small amount of non-specific adsorption, and the adhesion force is extremely low in a moist physiological environment. When using the inner layer of the duct (a natural fibrous membrane containing dECM and chitosan) as the adhesion substrate, the adhesion strength of the adhesive to the duct was 28.3±4.5 kPa, which was lower than 35.6±4.5 kPa in Example 3. Although fibrinogen can bind to natural materials such as collagen to a certain extent, it lacks covalent cross-linking. Using the dual-interface adhesion model test of nerve tissue and duct (adhering the nerve end and the inner wall of the duct at the same time), the overall adhesion strength was only 9.8±2.7 kPa, and the failure mode was debonding of the nerve end.

[0151] 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 of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A functional neural repair system, characterized in that, The functionalized neurorepair system includes a functional neurorepair composite catheter and an adhesive; The functional nerve repair composite conduit has a tubular structure, including an inner layer, a middle layer, and an outer layer; the outer layer is a fiber layer formed by electrospinning of synthetic polymer materials, the middle layer is a composite fiber layer formed by electrospinning of synthetic polymer materials and natural materials, and the inner layer is a natural fiber layer formed by electrospinning of natural materials; the degradation rates of the outer layer, the middle layer, and the inner layer form a gradient; The adhesive contains aldehyde groups. The adhesive forms a first cross-linking network with the inner layer of the functional nerve repair composite catheter through the aldehyde groups, forms a second cross-linking network with the middle layer of the functional nerve repair composite catheter through the aldehyde groups, and forms a third cross-linking network with the surface of the nerve tissue through the aldehyde groups. The first cross-linking network, the second cross-linking network, and the third cross-linking network fix the functional nerve repair composite catheter to the nerve tissue.

2. The functionalized neural repair system according to claim 1, characterized in that, The synthetic polymer material includes any one or a combination of at least two of polylactic acid, polyglycolic acid, polycaprolactone, polylactic acid-glycolic acid copolymer, polylactide-caprolactone copolymer, or polylactic acid-polyethylene glycol copolymer.

3. The functionalized neural repair system according to claim 1, characterized in that, The natural material includes any one or a combination of at least two of the following: decellularized matrix, collagen, chitosan, or silk fibroin.

4. The functionalized neural repair system according to claim 1, characterized in that, The outer layer has an average pore size of 50-500 nm and a porosity of 60%-80%.

5. The functionalized neural repair system according to claim 1, characterized in that, The inner layer of the fibrous functional nerve repair composite conduit is oriented axially, with a fiber orientation degree of over 75%.

6. The functionalized neural repair system according to claim 1, characterized in that, The adhesive comprises aldehyde-containing multi-arm polyethylene glycol with a molecular weight of 5000~40000 Da and a degree of substitution of the aldehyde group of 8%~15%; the aldehyde-containing multi-arm polyethylene glycol exists in a phosphate buffer solution, and the concentration of the aldehyde-containing multi-arm polyethylene glycol in the phosphate buffer solution is 8%~15%. The aldehyde-containing multi-arm polyethylene glycol includes any one or a combination of at least two of the following: aldehyde-containing two-arm polyethylene glycol, aldehyde-containing four-arm polyethylene glycol, or aldehyde-containing eight-arm polyethylene glycol.

7. The functionalized neural repair system according to claim 6, characterized in that, The adhesive also includes polyamino compounds; The polyamino compound includes a mixture of polylysine and polyethyleneimine, or any one or a combination of at least two of polylysine or polyethyleneimine.

8. The functionalized neural repair system according to claim 6, characterized in that, The adhesive also includes neurotrophic factors, which include any one or a combination of at least two of nerve growth factor, brain-derived neurotrophic factor, and glial cell-derived neurotrophic factor. The adhesive also includes functional additives, including butylated hydroxytoluene and / or brilliant blue.

9. The use of the functionalized nerve repair system according to any one of claims 1 to 8 in the preparation of a medical device for treating peripheral nerve injury.

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