Magnetic response biological adhesive tape as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of peripheral nerve injury repair, and particularly relates to a magnetic response biological adhesive tape (BCFO-GM) and a preparation method and application thereof. The adhesive tape comprises a group of methacrylic acid gelatin (N3-GelMA) modified by a functional monomer azide and a group of methacrylic acid gelatin (DBCO-GelMA) modified by dibenzyl cyclooctene, wherein the group of methacrylic acid gelatin (N3-GelMA) can be clicked. The suture-free self-adhesive biological adhesive tape prepared by the preparation method disclosed by the invention is self-adhered through a chemical click reaction to form tight wrapping, and provides protection for injured nerves, so that the nerves are quickly connected. The result of the embodiment shows that the BCFO-GM prepared by the invention has good biocompatibility, not only can promote motor function and neurosensory recovery and promote in-vivo nerve or myelin sheath regeneration, but also can inhibit gastrocnemius amyotrophy after PNI operation.

Description

Technical Field

[0001] The invention belongs to the technical field of peripheral nerve injury repair, and specifically relates to a magnetic responsive biological tape and a preparation method and application thereof. Background Art

[0002] Peripheral nerve injury (PNI) is a common clinical disease usually caused by trauma, surgical complications or neural tumors, often leading to long-term disability and loss of motor and sensory functions. Unlike the central nervous system, the peripheral nervous system has the ability to self-regenerate after injury. However, the nerve regeneration process after peripheral nerve injury is complicated, and the repaired peripheral nerves usually show functional limitations or incomplete functions, which can easily cause post-traumatic limb defects. Therefore, it is imperative to develop effective treatments for peripheral nerve injury.

[0003] At present, microsurgery is usually used clinically to directly repair peripheral nerves, connect the proximal and distal ends of the nerves, and shorten the axon reconnection distance through extra-neural or peri-neural sutures in a tension-free manner. However, this surgical intervention treatment method has certain limitations. First, the effective connection of nerves cannot be guaranteed, which may lead to partially reversible neuronal atrophy, reduce the production of neurotrophic factors, and hinder the accelerated regeneration of nerves; second, the presence of foreign suture materials can lead to intraneural inflammation, increased fibrosis, and scar tissue formation, which further leads to axonal dislocation, delayed remyelination, and failure of nerve recovery; in addition, the operation requires sutures to accurately connect the two sides of the nerve, which takes a long time and requires high suturing skills from the doctor. Therefore, establishing a more convenient and effective nerve rapid connection solution is of greater clinical significance for grassroots hospitals.

[0004] In addition to the need for effective and rapid connection of nerves, the microenvironment of the damaged nerve site also needs to be improved. As the substance of nerve pulses, electrical signals are highly involved in the high-speed and real-time transmission of biological information. At present, research on the neuromodulation of nerve cells or tissues by electrical stimulation and the promotion of nerve regeneration continues to emerge. Studies have shown that electrical stimulation can enhance axonal growth during nerve repair and accelerate sensory and motor recovery. However, traditional electrical stimulation is performed by implanting electrodes, which are invasive and may cause secondary damage and inflammatory responses.

[0005] To address the above issues, there is an urgent need to develop a new way to effectively and quickly connect nerves. Summary of the invention

[0006] The purpose of the present invention is to provide a magnetic responsive biological tape. The magnetic responsive biological tape provided by the present invention can not only effectively and quickly connect damaged peripheral nerves, but also improve postoperative motor ability and nerve sensation recovery.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The invention provides a magnetically responsive biological adhesive tape, which comprises a group of clickable functional monomers; the functional monomers are methacrylate gelatin modified with azide and methacrylate gelatin modified with dibenzylcyclooctene.

[0009] Preferably, the azide-modified methacrylate gelatin and dibenzylcyclooctene-modified methacrylate gelatin are prepared by reacting dibenzocyclooctyne-N-hydroxysuccinimide ester and azide-N-hydroxysuccinimide ester with methacrylate gelatin.

[0010] Preferably, the inner layer of the biotape is loaded with magnetoelectric nanomaterials; the magnetoelectric nanomaterials are composed of piezoelectric materials and magnetic response materials; the piezoelectric material is barium titanate; and the magnetic response material is cobalt oxide iron.

[0011] The present invention also provides a method for preparing the magnetic responsive biological tape, comprising the following steps:

[0012] S1. Preparation of aqueous cobalt oxide iron: heating triacetylacetonate iron, cobalt diacetylacetonate, 1,2-hexadecanediol, oleic acid, oleylamine and benzyl ether under nitrogen protection to reflux, purifying and centrifuging the purified product to obtain a purified product; dispersing the purified product and mixing it with water, anhydrous ethanol and polyvinyl pyrrolidone through ultrasonic vibration, drying it, dispersing it with a dispersant, and filtering it to obtain aqueous cobalt oxide iron;

[0013] S2, preparing magnetoelectric nanomaterials: removing the supernatant from the aqueous phase cobalt oxide iron in step S1, adding a barium titanate precursor solution, and obtaining magnetoelectric nanomaterials after ultrasonic drying and calcination;

[0014] S3, adding dibenzocyclooctyne-N-hydroxysuccinimide ester and azide-N-hydroxysuccinimide ester to trimethylamine and methacrylate gelatin solution respectively, stirring, dialyzing and then freeze-drying to obtain a white solid product, dissolving in a buffer solution to obtain dibenzylcyclooctene-modified methacrylate gelatin and azide-modified methacrylate gelatin solutions, adding the magnetoelectric nanomaterial and photoinitiator of step S2 respectively and stirring evenly; spreading the dibenzylcyclooctene-modified methacrylate gelatin solution evenly in a mold, exposing it to ultraviolet light to polymerize into a hydrogel, and then spreading the azide-modified methacrylate gelatin solution evenly on the upper layer of the dibenzylcyclooctene-modified methacrylate gelatin hydrogel, exposing it to ultraviolet light for 30-40s to polymerize into a hydrogel, and finally preparing a magnetic responsive biotape;

[0015] The purified product is chloroform and acetone in a mass ratio of 1:3.

[0016] Preferably, in step S1, the molar ratio of triacetylacetonate iron, cobalt diacetylacetonate, 1,2-hexadecanediol, oleic acid, oleylamine and benzyl ether is 2:1:0:6:6:20; the heating and reflux process is: heating to 200° C. for 2 h, and heating and reflux at 300° C. for 1 h; and the dispersant is hexane.

[0017] Preferably, the barium titanate precursor solution in step S2 is prepared from citric acid, isopropyl titanium, ethanol, barium carbonate and water.

[0018] Preferably, in step S3, the concentration of the dibenzylcyclooctene-modified methacrylate gelatin and the azide-modified methacrylate gelatin solution is 10% w / v; the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate; the thickness of the hydrogel is 250 μm; and the ultraviolet light is 405 nm.

[0019] The present invention also provides the use of the magnetic responsive biological tape or the magnetic responsive biological tape prepared by the above preparation method in promoting the recovery of motor function and nerve sensation after peripheral nerve injury surgery.

[0020] The present invention also provides the use of the magnetic responsive biological tape or the magnetic responsive biological tape prepared by the above preparation method in promoting nerve regeneration and myelin regeneration after peripheral nerve injury surgery.

[0021] The present invention also provides the use of the magnetic responsive biological tape or the magnetic responsive biological tape prepared by the above preparation method in promoting the high expression of NF 200 protein and S100β protein in nerve cells.

[0022] Beneficial effects of the present invention:

[0023] The BCFO-GM prepared by the present invention adheres by itself through a chemical click reaction to form a tight package, provides protection for injured nerves, and enables nerves to quickly establish connections, which reduces the difficulty of surgical operations, shortens the regeneration time, and eliminates secondary puncture site damage and suture-related inflammation.

[0024] The magnetically responsive biotape (BCFO-GM) prepared by the present invention can quickly establish neural connections without suturing, realize wireless, non-invasive electrical stimulation to accelerate the healing of peripheral nerve injuries, and has good biocompatibility. It can not only promote the recovery of motor function and nerve sensation, but also promote the high expression of NF 200 protein and S100β protein in nerve cells to promote nerve or myelin regeneration in vivo, and can also inhibit gastrocnemius muscle atrophy after PNI surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0026] Figure 1 HE staining of rat heart, liver, spleen, lung and kidney;

[0027] Figure 2 Schematic diagram of animal surgery and staining;

[0028] Figure 3 This is a diagram of rat footprints and SFI index analysis;

[0029] Figure 4 This is a data chart of the drowning time and number of drowning times in rats’ forced swimming;

[0030] Figure 5 This is a rat nerve conduction map;

[0031] Figure 6 This is a graph showing the experimental results of the von Frey and Hargreaves tests in rats;

[0032] Figure 7 The figure shows the immunofluorescence staining results of S100β protein and NF200 protein in rat sciatic nerve tissue;

[0033] Figure 8 The immunofluorescence staining images of β-III-tubulin protein and MBP protein in rat sciatic nerve tissue;

[0034] Fig. 9 This is the HE staining result of rat sciatic nerve cross section;

[0035] Fig.10 This is the result of rat LFB myelin staining;

[0036] Fig.11 This is the TEM result of the cross section of the rat sciatic nerve;

[0037] Fig.12 Quantitative data graphs of the number of myelinated axons, diameter of myelinated axons, thickness of myelin sheath and G ratio (%) of rats;

[0038] Fig.13 This is a picture of the rat gastrocnemius muscle and the gastrocnemius muscle wet weight ratio result diagram;

[0039] Fig.14 HE staining of rat gastrocnemius muscle;

[0040] Fig.15 This is a Masson staining image of rat gastrocnemius muscle. DETAILED DESCRIPTION

[0041] The present invention provides a self-adhesive biological tape that responds to magnetic field stimulation and does not require sutures, and is used for peripheral nerve injury repair. The tape is made of a group of clickable functionalized monomers (azide-modified methacrylate gelatin (N3-GelMA) and dibenzylcyclooctene-modified methacrylate gelatin (DBCO-GelMA)), which self-adhere through chemical click reactions to form a tight package, provide protection for injured nerves, and enable nerves to quickly establish connections, which reduces the difficulty of surgical operations, shortens the regeneration time, and eliminates secondary puncture site damage and suture-related inflammation. In addition, this study uses a magnetoelectric nanomaterial BCFO (CFO@BTO) composed of a piezoelectric material (barium titanate BTO) and a magnetic response material (cobalt oxide iron CFO) to load on the inner layer of the biological tape. When the self-adhesive biological tape wraps around the damaged nerve tissue, under the action of an external magnetic field, wireless and non-invasive electrical stimulation for peripheral nerve injury repair is achieved, accelerating nerve regeneration growth.

[0042] "Clickable functionalized monomers" are monomers designed with specific functional groups that can participate in click chemistry reactions. Click chemistry is a synthetic strategy that uses a class of highly specific, efficient and reliable chemical reactions to build complex molecular structures. These reactions are usually carried out under mild conditions, with high yields, few by-products, and easy operation and purification.

[0043] Hematoxylin-eosin staining, referred to as HE staining, is one of the commonly used staining methods in paraffin section technology. Hematoxylin staining solution is alkaline, which mainly makes the chromatin in the cell nucleus and the ribosomes in the cytoplasm purple-blue; eosin is an acidic dye, which mainly makes the components in the cytoplasm and extracellular matrix red. When cells age or undergo degeneration, they will show strong eosinophilic staining. When collagen fibers age and undergo transparent degeneration, the eosin color changes from light to dark. When cells evolve into malignant tumor cells, the cells usually grow larger, the chromatin in the cell nucleus is very dark, and the arrangement is abnormal.

[0044] SFI is the abbreviation of "Sciatic Function Index", which can be translated into Chinese as sciatic nerve function index. This term is widely known in the medical community, especially in the British medical community, and its abbreviation is among the most popular terms among 3,598 terms. SFI is mainly used to evaluate the health status and functional recovery of the sciatic nerve, and is commonly used in rehabilitation assessments after nerve injury, such as after surgery or during the treatment of spinal cord injury. For example, in studies on postoperative rats, SFI is used to detect the recovery of nerve function, including pain threshold, conduction velocity, and muscle status.

[0045] The glial cells in the peripheral nervous system are called Schwann cells, which are distributed along the processes of neurons. Schwann cells are wrapped around nerve fibers, which are called myelinated nerve fibers. The morphology and function of Schwann cells in myelinated nerve fibers and unmyelinated nerve fibers are different. The outer surface of Schwann cells has a basement membrane, which can secrete neurotrophic factors, promote the survival of damaged neurons and the regeneration of their axons, and participate in the formation of nerve fibers in the peripheral nervous system.

[0046] β-III-tubulin protein (β-tubulin class III, TUBB3) is a subtype of tubulin, mainly expressed in neurons, involved in neurogenesis, axon guidance and maintaining the structural integrity of neurons. β-III-tubulin protein plays a key role in the development and regeneration of the nervous system, especially in the differentiation and migration of neurons.

[0047] MBP protein Myelin basic protein (MBP) is the main protein of myelin in the central nervous system (CNS) and peripheral nervous system (PNS). It is located on the inner side of the myelin sheath and helps maintain the stability of myelin structure and function. MBP plays a vital role in the myelination of nerve cells. It is one of the main components of the myelin sheath surrounding the axon. The expression level of MBP can be used as an indicator to judge the degree of damage to the central nervous system, which is of great significance for the diagnosis and treatment of central nervous system diseases.

[0048] VonFrey, also known as the ciliary mechanical stimulation needle, is an experimental method commonly used in pain research to assess the pain sensitivity of animals under pressure pain stimulation. The scoring standard is based on the pain threshold to judge the animal's response to different degrees of stimulation, aiming to provide objective, repeatable and quantifiable results.

[0049] The Hargreaves test is a method used to assess the response of mice and rats to thermal stimulation and is primarily used to quantify the sensitivity of these animals to thermal pain. The test assesses pain sensitivity by measuring the time it takes an animal to react to infrared heat stimulation, known as the thermal response latency.

[0050] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0051] The production processes, experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technical personnel in the field can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0052] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in terms of their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art.

[0053] Example 1 Preparation of magnetically responsive biotape (BCFO-GM)

[0054] 1. Preparation of magnetoelectric nanomaterials (BCFO)

[0055] (1) Preparation of barium titanate (BTO) precursor solution: The BTO precursor solution is prepared by mixing solution A and solution B. Solution A: 0.5 g citric acid, 24 μl titanium isopropoxide, 15 ml ethanol; Solution B: 0.1 g citric acid, 0.0158 g barium carbonate (BaCO3), 15 ml water.

[0056] (2) Preparation of aqueous cobalt oxide iron (CFO): 2mmol of triacetylacetonate iron [Fe(acac)3], 1mmol of acetylacetonate cobalt di[Co(acac)2], 10mmol of 1,2-hexadecanediol, 6mmol of oleic acid, 6mmol of oleylamine, and 20mL of benzyl ether were heated to 200°C in a three-necked flask under nitrogen protection for 2h, heated to reflux at 300°C for 1h, purified with chloroform and acetone in a ratio of 1:3, 7500rpm for 10min, and the product was redispersed in 15ml of hexane. 10ml of the oil phase solution was mixed with 30ml of water, 6ml of anhydrous ethanol, and 1g of PVP, and subjected to vigorous ultrasonic vibration for 50min to obtain an oil-in-water microemulsion. A small amount of insoluble oleic acid and insoluble matter were removed to obtain a stable microemulsion. Dry in an oven at 60°C to obtain a colloid, add 20 ml of deionized water to redisperse, filter to obtain an aqueous phase CFO, centrifuge at 5000 rpm for 5 min, obtain a precipitate, and dissolve it in water again or in anhydrous ethanol.

[0057] (3) The aqueous CFO was added to the BTO precursor solution and ultrasonicated for 20 minutes. The above mixture was placed in an oven at 80°C overnight to obtain a dry gel. The temperature was increased to 600°C at 5°C / min and calcined for 6 hours. The temperature was decreased at 1°C / min to finally obtain a BCFO nanomaterial with an average particle size of 20 nm.

[0058] 2. Preparation of Magnetically Responsive Biotape (BCFO-GM)

[0059] Methacrylate gelatin (NH2-GelMA) was dissolved in water, stirred at 60°C, and then cooled to 30°C for use. Dibenzocyclooctyne-N-hydroxysuccinimide ester (DBCO-NHS) and azide-N-hydroxysuccinimide ester (N3-NHS) were added to trimethylamine, and then methacrylate gelatin NH2-GelMA solution was added, respectively, and stirred at 30°C for 5 hours. The reaction mixture was then dialyzed with deionized water at 30°C for 2 days.

[0060] The solution was then freeze-dried to obtain white solid products, dibenzylcyclooctene-modified methacrylate gelatin (DBCO-GelMA) and azide-modified methacrylate gelatin (N3-GelMA). Prepare DBCO-GelMA and N3-GelMA solutions (10% w / v, PBS), then add 500 μg / mL BCFO nanomaterials and photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) (1% w / v) respectively and stir thoroughly, spread the DBCO-GelMA solution evenly in a mold with a length of 1 cm, a width of 500 μm, and a height of 500 μm, with a required thickness of 250 μm, and DBCO-GelMA polymerizes into a hydrogel after being exposed to 405 nm ultraviolet light (UV) for 30 s, and then spread the N3-GelMA solution evenly on the upper layer of the DBCO-GelMA hydrogel with a thickness of 250 μm, and N3-GelMA polymerizes into a hydrogel after being exposed to 405 nm ultraviolet light (UV) for 30 s, and finally prepare the BCFO-GM tape.

[0061] Example 2 Biocompatibility of BCFO-GM

[0062] Hematoxylin-eosin staining, referred to as HE staining, is one of the commonly used staining methods in paraffin section technology. Hematoxylin staining solution is alkaline, which mainly makes the chromatin in the cell nucleus and the ribosomes in the cytoplasm purple-blue; eosin is an acidic dye, which mainly makes the components in the cytoplasm and extracellular matrix red. When cells age or undergo degeneration, they will show strong eosinophilic staining. When collagen fibers age and undergo transparent degeneration, the eosin color changes from light to dark. When cells evolve into malignant tumor cells, the cells usually grow larger, the chromatin in the cell nucleus is very dark, and the arrangement is abnormal.

[0063] The biosafety of BCFO-GM tape in vivo was evaluated. Tissue toxicity was further monitored by HE staining of major organs of rats, including heart, liver, spleen, lung, and kidney. Figure 1 As shown, the chromatin color in the cell nucleus is normal, and the cell growth state and arrangement are normal, indicating that the BCFO-GM tape has good biocompatibility and its degradation in the body does not cause any obvious systemic toxicity.

[0064] Example 3 BCFO-GM promotes motor function and nerve sensation recovery

[0065] 1. Motor function experiment

[0066] Four groups of rats with the same breeding conditions underwent the same artificial mechanical peripheral nerve injury. The four groups of rats were divided into nerve exposure (Sham group), nerve rupture (PNI group), simple end-to-end suture (ETE group) and BCFO-GM tape treatment group (BCFO-GM group) for animal surgery.

[0067] Schematic diagram of animal surgery results Figure 2 shown.

[0068] Compared with nerve exposure (Sham group), nerve rupture (PNI group) and simple end-to-end suture (ETE group), BCFO-GM tape could easily wrap the ruptured nerve (BCFO-GM group).

[0069] Three months after surgery, the regenerated sciatic nerve was exposed and evaluated. It showed reconnection of the distal and proximal sites, no neuroma formation at the injury site in all groups, and complete degradation of the material visible to the naked eye.

[0070] The recovery of motor function is closely related to the structural and pathophysiological aspects of peripheral nerves. HE staining was performed on the cells of postoperative rats. Results Compared with the ETE group, the axonal fibers in the BCFO-GM group were well organized and continuous at the injury site. The nerves in the PNI group showed intermittent nerve fiber distribution and poor reconnection with many connective tissues. It was concluded that the BCFO-GM tape provided a suitable microenvironment for axon regeneration.

[0071] Motor function recovery was assessed using walking trajectory (SFI) analysis. SFI analysis was used to record the rats' footprints and gait at 4, 8, and 12 weeks after surgery.

[0072] The results are as follows Figure 3As shown, SD rats in the Sham group and the BCFO-GM group treated with a magnetic field (referred to as the BCFO-GM+ME group) showed better walking ability compared with the PNI group, based on shorter footprints and identifiable toes. In addition, the SFI values ​​showed that the rats in the BCFO-GM+ME group had a faster recovery of motor function than the rats in the ETE group at 8 and 12 weeks after surgery.

[0073] The rat forced swim test was used to evaluate the repair of nerve damage by strenuous exercise of the rat hind limbs. Figure 4 The experimental results showed that within 15 minutes, the PNI group had hind limb movement disorders due to sciatic nerve injury, and the number of times and total time of submergence were significantly higher than those of the Sham group, while the number of times and total time of submergence of rats in the BCFO-GM+ME group were significantly lower than those in the PNI group and the ETE group, and were closest to the Sham group. The above results show that BCFO-GM+ME can accelerate the recovery of the exercise ability of PNI rats, and even enable intense exercise.

[0074] 2. Nerve conduction experiment

[0075] Electrophysiological methods were used to detect the functional recovery of injured peripheral nerves in rats 12 weeks after surgery. The biological signal acquisition system displayed the action potential signals of the sciatic nerve trunks in different groups. When the signal amplitude was larger, the conductivity of the nerve was more optimized, the number of axons with conduction function was more, the latency was shorter, the nerve conduction velocity was lower, and the degree of nerve myelin regeneration was higher.

[0076] The results are as follows Figure 5 As shown in the figure, the BCFO-GM group and the Sham group had similar peak shapes and higher average CMAP peak amplitudes; the BCFO-GM group was closest to the control group, with no significant difference, while the PNI group and the ETE group were significantly different from both the control group and the BCFO-GM group. This indicates that the BCFO-GM group can promote the conduction of nerve signals.

[0077] 3. Sensory function experiment

[0078] Sensory function recovery is also an important aspect of nerve regeneration after transection. The mechanical and thermal sensory functions of rats were tested by von Frey and Hargreaves tests, respectively.

[0079] The results are as follows Figure 6As shown in the figure, all animals began to recover some mechanical sensation 4 weeks after surgery. Over time, the mechanical sensation function of rats in the ETE and BCFO-GM groups gradually recovered, which was significantly different from the PNI group. It was also found that rats in the ETE and BCFO-GM groups began to gradually recover thermal sensation over time 4 weeks after surgery, and the withdrawal latency of the BCFO-GM group was significantly lower than that of the other experimental groups at the 8th and 12th weeks after surgery, indicating that BCFO-GM accelerated the recovery of thermal function in PNI rats.

[0080] Example 4 In vivo nerve regeneration and myelin regeneration experiments

[0081] Nerve exposure (Sham group), nerve rupture (PNI group), and simple end-to-end suture (ETE group), BCFO-GM tape could easily wrap the ruptured nerve (BCFO-GM group).

[0082] The effect of BCFO-GM tape on promoting nerve regeneration in vivo was evaluated 12 weeks after surgery. Immunofluorescence staining was performed on the cross-sections and longitudinal sections of the obtained nerve segments to specifically evaluate the axonal reconnection and myelination after nerve transection and repair. NF200 protein is a marker protein for axonal regeneration. When axons grow, myelinated axons accumulate more neurofilaments, which promotes the increase of axon diameter.

[0083] The results are as follows Figure 7 As shown, the fluorescence intensity of Schwann cell-specific marker protein (S100β protein) and neurofilament protein (NF200) in the BCFO-GM group was significantly increased. The high expression of NF200 and S100β protein indicated that BCFO-GM can promote the regeneration of axons and Schwann cells.

[0084] Immunofluorescence staining of β-III-tubulin protein and MBP protein was selected to further verify the role of BCFO-GM in nerve regeneration. In neural cells, Tuj-1 protein is mainly expressed in neurons and may be involved in neurogenesis.

[0085] The experimental results are as follows Figure 8 As shown, the expression of Tuj-1 protein in the BCFO-GM group was high, which was significantly different from that in the PNI group and the ETE group. At the same time, the MBP in the BCFO-GM group showed a stronger fluorescence intensity than that in the other groups. The above results indicate that the BCFO-GM group more significantly promotes nerve regeneration and myelination.

[0086] To further investigate the remyelination of regenerated nerves, the nerves were subjected to H&E staining, LFB staining and transmission electron microscopy (TEM) imaging.

[0087] The results of H&E staining at 4 weeks after surgery were as follows Fig. 9As shown in the figure, the nerve structure of the PNI group was sparse, and at 8 and 12 weeks after surgery, the nerve structure of the PNI group was even sparser and more disordered. Compared with the PNI group, the regenerated nerve bundles in the BCFO-GM group were dense and orderly. At 8 and 12 weeks after surgery, the morphological order of the BCFO-GM group was further improved, and the nerve morphology was closest to that of the Sham group, which was better than the other groups.

[0088] LFB myelin staining is a commonly used staining method to show the morphological structure and pathological changes of nerve myelin. The results of LFB experiment are consistent with those of H&E staining. Fig.10 As shown, compared with the Sham group, the myelin sheath structure in the PNI group was sparser and more disordered, the myelin sheath density in the BCFO-GM group was denser than that in the ETE group, and over time, the nerve fiber density in the BCFO-GM group was higher and more tightly arranged, indicating that nerve regeneration and remyelination were better in the BCFO-GM group.

[0089] TEM images of regenerated nerves were used to further demonstrate the degree of myelination and axonal maturation of nerve fibers. Fig.11 As shown. The myelin sheath in the Sham group was round, dense, uniform and orderly, while in the PNI group, the myelin sheath was loosely arranged and the myelin edge was thin and disordered. The BCFO-GM group was closest to the Sham group, with abundant myelinated nerve fibers, and the myelin sheath was in a typical layered structure with clear and orderly edges. At the same time, the ETE group and the BCFO-GM group also showed more small and thin myelin tissue aggregations. This is the period of axon regeneration, when the myelin sheath is too thin to adapt to the diameter of the axon. The larger and thicker myelin sheath tissue contained in the BCFO-GM group also reflects that the BCFO-GM group has the ability to accelerate axon regeneration and myelination compared with the ETE group.

[0090] The number of myelinated axons, diameter of myelinated axons, thickness of myelin sheath and G ratio (%) of rats were quantified 12 weeks after surgery. Fig.12 shown.

[0091] The BCFO-GM and ETE groups had more myelinated axons than the Sham group, which may be due to the larger myelin sheath in the control group. At the same time, the average diameter of myelinated axons in the BCFO-GM group was greater than that in the PNI and ETE groups, but did not reach the level of the Sham group. There was no significant difference in diameter between the BCFO-GM and Sham groups, indicating that the axons tended to mature. In addition, in terms of myelin sheath thickness, the axons in the BCFO-GM group also showed greater remyelination than those in the PNI and ETE groups.

[0092] The average G ratio is an important indicator of axonal myelination, which is defined as the ratio of axon area to myelin area, which indicates the optimal function and structure of nerve myelination. The results showed that the G ratio distribution trend of the BCFO-GM group was comparable to that of the Sham group, and had a lower G ratio than the PNI group and the ETE group. These results indicate that the BCFO-GM group exhibited a more active effect on myelin regeneration in regenerating nerves than the ETE group, with satisfactory nerve recovery function.

[0093] Example 5 BCFO-GM inhibits gastrocnemius muscle atrophy after PNI surgery

[0094] Nerve exposure (Sham group), nerve rupture (PNI group), and simple end-to-end suture (ETE group), BCFO-GM tape could easily wrap the ruptured nerve (BCFO-GM group).

[0095] Delayed axonal regeneration after peripheral nerve injury usually leads to progressive muscle atrophy and poor functional recovery. The results of the analysis of the gastrocnemius muscle innervated by the tibial branch of the sciatic nerve are as follows Fig.13 shown.

[0096] Gastrocnemius muscles were collected at 4, 8, and 12 weeks after surgery, and the wet weight ratio (injured side / healthy side) was calculated. After injury and repair, the muscle weight of the PNI group was significantly reduced compared with the Sham group at week 4, but the ETE group and BCFO-GM group were statistically different from the PNI group. Muscle atrophy was more obvious in the PNI group at weeks 8 and 12, and the BCFO-GM group had a more significant effect in preventing gastrocnemius muscle atrophy than the ETE group.

[0097] Masson staining refers to the use of two or three anionic dyes mixed together, with collagen fibers appearing blue and muscle fibers appearing red. It is one of the staining methods used to display fibers and inflammatory factors in tissues.

[0098] H&E staining of gastrocnemius muscle Fig.14 As shown and Masson staining as Fig.15 As shown. The results also reflected the results of the wet weight ratio. Due to muscle atrophy, the muscle fiber area of ​​the PNI group was significantly lower than that of the Sham group over time, while the muscle fiber area of ​​the BCFO-GM group was larger than that of the PNI group and the ETE group. Correspondingly, the muscle fiber diameter of the BCFO-GM group was also significantly larger than that of the PNI group and the ETE group. In addition, the percentage of collagen area in the Sham group and the BCFO-GM group was significantly lower than that in the PNI group and the ETE group, while the collagen deposition in the ETE group gradually decreased over time, and there was no significant difference between the Sham group and the BCFO-GM group at week 12. These results show that the BCFO-GM group has a significant effect in inhibiting muscle atrophy and promoting motor function recovery after PNI.

[0099] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A magnetically responsive biotape, characterized in that: The biological adhesive tape comprises a group of clickable functional monomers; the functional monomers are methacrylate gelatin modified with azide and methacrylate gelatin modified with dibenzylcyclooctene.

2. The bio-tape according to claim 1, characterized in that: The azide-modified methacrylate gelatin and dibenzylcyclooctene-modified methacrylate gelatin are prepared by reacting dibenzocyclooctyne-N-hydroxysuccinimide ester and azide-N-hydroxysuccinimide ester with methacrylate gelatin.

3. The magnetically responsive biotape according to claim 1, characterized in that: The inner layer of the biological tape is loaded with magnetoelectric nanomaterials; the magnetoelectric nanomaterials are composed of piezoelectric materials and magnetic response materials; the piezoelectric material is barium titanate; and the magnetic response material is cobalt oxide iron.

4. The method for preparing the magnetic responsive biological tape according to claim 1, characterized in that: The following steps are involved: S1. preparing aqueous cobalt oxide iron: heating triacetylacetonate iron, cobalt diacetylacetonate, 1,2-hexadecanediol, oleic acid, oleylamine and benzyl ether under nitrogen protection to reflux, purifying and centrifuging the purified product to obtain a purified product; The dispersed purified product is mixed with water, anhydrous ethanol and polyvinyl pyrrolidone by ultrasonic oscillation, and then dried, dispersed with a dispersant, and filtered to obtain aqueous cobalt oxide iron; S2, preparing magnetoelectric nanomaterials: removing the supernatant from the aqueous phase cobalt oxide iron in step S1, adding a barium titanate precursor solution, and obtaining magnetoelectric nanomaterials after ultrasonic drying and calcination; S3, adding dibenzocyclooctyne-N-hydroxysuccinimide ester and azide-N-hydroxysuccinimide ester to trimethylamine and methacrylate gelatin solution respectively, adding trimethylamine and methacrylate gelatin solution respectively, stirring, dialyzing and then freeze-drying to obtain a white solid product, dissolving it in a buffer solution to obtain dibenzylcyclooctene-modified methacrylate gelatin and azide-modified methacrylate gelatin solution, adding the magnetoelectric nanomaterial and photoinitiator of step S2 respectively and stirring evenly; spreading the dibenzylcyclooctene-modified methacrylate gelatin solution evenly in a mold, exposing it to ultraviolet light to polymerize into a hydrogel, and then spreading the azide-modified methacrylate gelatin solution evenly on the upper layer of the dibenzylcyclooctene-modified methacrylate gelatin hydrogel, exposing it to ultraviolet light for 30-40s to polymerize into a hydrogel, and finally preparing a magnetic responsive biotape; The purified product is chloroform and acetone in a mass ratio of 1:

3.

5. The preparation method according to claim 4, characterized in that: In the step S1, the molar ratio of triacetylacetonate iron, cobalt diacetylacetonate, 1,2-hexadecanediol, oleic acid, oleylamine and benzyl ether is 2:1:0:6:6:20; the heating and reflux process is: heating to 200° C. for 2 hours, and heating and reflux at 300° C. for 1 hour; the dispersant is hexane.

6. The preparation method according to claim 4, characterized in that: In step S2, the barium titanate precursor solution is prepared from citric acid, isopropyl titanium, ethanol, barium carbonate and water.

7. The preparation method according to claim 4, characterized in that: The concentration of the dibenzylcyclooctene-modified methacrylate gelatin and the azide-modified methacrylate gelatin solution in step S3 is 10% w / v; the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate; the thickness of the hydrogel is 250 μm; and the ultraviolet light is 405 nm.

8. Use of the magnetic responsive biotape described in any one of claims 1 to 3 or the magnetic responsive biotape prepared by the preparation method described in any one of claims 4 to 7 in promoting motor function and nerve sensation recovery after peripheral nerve injury surgery.

9. Use of the magnetic responsive biotape described in any one of claims 1 to 3 or the magnetic responsive biotape prepared by the preparation method described in any one of claims 4 to 7 in promoting nerve regeneration and myelin regeneration after peripheral nerve injury surgery.

10. Use of the magnetic responsive biotape according to any one of claims 1 to 3 or the magnetic responsive biotape prepared by the preparation method according to any one of claims 4 to 7 in promoting high expression of NF 200 protein and S100β protein in nerve cells.

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