CN116421782A - Polyamino acid composite material with nerve repair promoting function, and preparation method and application thereof - Google Patents
Polyamino acid composite material with nerve repair promoting function, and preparation method and application thereof Download PDFInfo
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
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- A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction
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Abstract
A polyamino acid composite material with nerve repair promoting function is prepared from polylactic acid-glycolic acid copolymer and poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) amine. The preparation method creatively uses PEG-MELG, adopts PLGA and PEG-MELG as raw materials, blends the raw materials into a blend fiber membrane through an electrostatic spinning technology, and prepares the polyamino acid composite material with the function of promoting nerve repair through vacuum drying, so that the polyamino acid composite material has better mechanical support, and can promote the repair of nerve cell axons and guide the climbing growth of growth cones.
Description
Technical Field
The invention relates to a polyamino acid composite material with a nerve repair promoting function, and a preparation method and application thereof, belonging to the technical field of medical biological materials.
Background
Nerve injury is a common and frequent type of clinical injury, which is caused by partial or total nerve cell injury caused by various traumas and other conditions, often causes sensory and motor dysfunction or loss of patients, and seriously affects the daily life quality and work efficiency of the patients. It is urgent to find therapeutic measures effective in treating nerve damage; the key point of the research hot trend of nerve tissue regeneration engineering for repairing the damage of the nervous system is that materials are used for supporting the growth of nerve cells, and the better materials can be used as an adhesion matrix of implanted cells or host nerve stem cells, and can promote the growth of the nerve cells by themselves or degradation products thereof; for example, the use of nerve conduits (Nerve Guide Conduit, NGC) can provide a number of beneficial conditions for regeneration after nerve breaks or long cuts. In nerve regeneration, the two ends of defective nerve can be temporarily fixed and supported, the axon of neuron is guided to axially grow, so that the exogenous nerve is avoided, neuroma is formed, a relatively isolated microenvironment is provided for nerve regeneration, the neurotrophic factors required by nerve regeneration are enriched, the invasion of exogenous miscellaneous cells is reduced, and the formation of glial scars is prevented.
Peripheral nerve injury is generally caused by trauma, ischemia, etc., and excessive fiber deposition and scarring of distal damaged nerves affect nerve signaling, loss of function, and even nerve disuse. Because of the complex structure and function of the peripheral nerve tissue, once the peripheral nerve tissue is damaged, the peripheral nerve tissue has high disability rate, poor prognosis and difficult treatment, and becomes an important problem in the current clinic. Peripheral nerve injury can cause sensory and motor dysfunction and irreversible tissue atrophy, resulting in reduced quality of life for the patient and also a heavy burden on society. Thus, peripheral nerve damage has been the most challenging problem of trauma surgery due to the structural function of the peripheral nerve and the specificity of the pathological process.
Clinically, at present, the two ends of the broken nerve are generally directly anastomosed for the nerve fracture injury (< 5 mm) with shorter distance; for more serious long-distance nerve defects (> 10 mm), the tension is too high when the direct suturing is performed, and an autologous nerve is transplanted or other nerve repair materials are implanted to reduce the suturing tension so as to assist the nerve growth. However, the autologous nerve transplantation has the problems of limited quantity, poor biocompatibility and difficult control of tissue similarity, and can cause the problems of sensory and functional disturbance of the innervation area of the donor, thereby greatly limiting the wide application of the autologous nerve transplantation. Thus, there is a great clinical need to find new nerve repair materials.
Nerve conduit products currently marketed at home and abroad, for example: 1. patent application CN202210377724.2 discloses a double-layer tubular product for promoting defective nerve regeneration, which is composed of a tubular outer layer formed by a human-compatible biodegradable material and a tubular inner layer adhered to the inner wall thereof, the tubular inner layer being a hydrogel layer formed of polyethylene glycol. The biocompatible biodegradable material is selected from the group consisting of: polylactic acid or its derivative, gelatin and its derivative, chitosan, polyurethane, polycaprolactone, water insoluble cellulose and its derivative, etc. The polylactic acid derivative is selected from the group consisting of: polylactic acid-polyethylene glycol copolymer, lactic acid-glycolic acid copolymer (PLGA), PLGA-polyethylene glycol copolymer, lactic acid-caprolactone copolymer (PCLA), PCLA-polyethylene glycol copolymer. The weight ratio of the tubular outer layer to the tubular inner layer is 100:20-100, and the thickness of the tube wall of the double-layer tubular product is 50-500 mu m. 2. Patent application CN202110504965.4 discloses a preparation method of an active oxygen response hydrogen sulfide release nerve conduit, the conduit comprises a fiber conduit, the interior of the fiber conduit is filled with temperature-sensitive polyamino acid hydrogel, the interior of the temperature-sensitive polyamino acid hydrogel is loaded with polyamino acid nano-micelles, the polyamino acid nano-micelles are loaded with active oxygen response hydrogen sulfide donors, and the active oxygen response hydrogen sulfide donors comprise arylborate functionalized peroxythiocarbamate and derivatives thereof. The method utilizes the active oxygen environment excessively generated by peripheral nerve injury to activate the hydrogen sulfide donor loaded in the nerve conduit, eliminates active oxygen and simultaneously realizes hydrogen sulfide response release, inhibits the expression of active oxygen and inflammatory factors based on the anti-inflammatory, antioxidant and macrophage polarization regulation effects of the hydrogen sulfide, and further performs feedback regulation to avoid the excessive generation of the hydrogen sulfide, thereby achieving the purpose of dosing according to the requirement and creating a good regeneration microenvironment for injured nerves. 3. Patent application CN200910177405.1 discloses a repair material for promoting defective nerve regeneration, which comprises a biodegradable material compatible with human body and a macromolecule material pore-forming agent, and application of the pore-forming agent in preparing the repair material for promoting defective nerve regeneration.
However, the main effects of the nerve conduit products still stay in providing basic physical support for damaged nerve regeneration, connecting stumps, preventing surrounding tissue from being pressed, and the like, most of the products do not effectively integrate novel biomedical materials for deep and targeted optimization, and the currently marketed nerve conduit products still lack the effects of guiding nerve regeneration and extension.
Biomedical materials have been developed through three major stages. The first generation biological materials mainly comprise various biological inert materials, mainly comprise bone nails, bone plates, artificial joints and the like. The second generation biological material is represented by bioactive glass, biological ceramic, absorbable suture and other bioactive materials. The third generation biological material is represented by bionic materials such as tissue engineering scaffold materials, in-situ tissue regeneration materials and the like, and the generation is focused on optimizing the physical properties such as micro-nano structure, biomechanics and the like of the fine control material or combining various chemical or biological factors so as to stimulate tissue repair.
The electrostatic spinning technology is a technology for spinning a product by using a polymer solution under high-voltage static electricity, in particular to a technology for carrying the polymer solution or a melt to thousands to tens of thousands of volts of high-voltage static electricity, and charged polymer liquid drops are accelerated at the Taylor cone vertex of a capillary under the action of an electric field force. When the electric field force is large enough, the polymer droplets overcome the surface tension to form a jet of thin fluid. The solvent evaporates or solidifies during the trickle jet process and eventually falls onto the receiving means to form a fibrous mat resembling a nonwoven fabric. The electrostatic spinning can prepare fibers with diameters of tens to hundreds of nanometers, and the product has higher porosity and larger specific surface area, diversified components and uniform diameter distribution, and has high application value in the fields of biomedicine, environmental engineering, textile and the like.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, an object of the present invention is to provide a composite material of polyamino acids having a function of promoting nerve repair.
According to a first embodiment of the present invention, there is provided a polyamino acid composite material having a nerve repair promoting function, made of polylactic acid-glycolic acid copolymer (PLGA) and poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) amine (PEG-MELG), wherein the mass ratio of the polylactic acid-glycolic acid copolymer and the poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) amine is 20 to 24:76 to 80, preferably 21 to 23:77 to 79, preferably 23:77.
The poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) amine is a polyethylene glycol modified poly methionine material, is one of a plurality of polyethylene glycol-polyamino acid block copolymers, is a high molecular organic matter, is widely used in the fields of chemical industry and medicine, has many excellent properties such as water solubility, non-volatility, physiological inertia and the like, and utilizes the amphiphilic copolymer of the polyethylene glycol to adsorb on the surface of the material to further improve the biocompatibility of the nerve sleeve and facilitate the purification of products.
According to a second embodiment of the present invention, there is provided a method for preparing a polyamino acid composite material having a function of promoting nerve repair, comprising the steps of:
(1) Dissolving polylactic acid-glycolic acid copolymer and poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) amine in an organic solvent, and stirring and mixing uniformly to obtain an electrospinning stock solution;
(2) Injecting the electrospinning stock solution into an injector, then carrying out electrostatic spinning treatment under the action of a thrust pump, and receiving the product through a cylindrical metal rotary receiving disc to obtain blended spinning;
(3) After the electrostatic spinning treatment is finished and all the blended spinning is received by a cylindrical metal rotary receiving disc, the receiving speed of the receiving disc is adjusted to obtain a blended fiber membrane;
(4) And (3) sending the obtained blend fiber membrane into a vacuum drying oven for vacuum drying, and removing residual solvent to obtain the polyamino acid composite material with the function of promoting nerve repair.
Further, as a more preferred embodiment of the present invention, in the step (1), the stirring is performed at room temperature for 12 to 16 hours; the organic solvent is selected from hexafluoroisopropanol or dichloromethane; preferably 13 to 15 hours; preferably 12 to 15 hours; preferably 14 to 15 hours.
Further, as a more preferred embodiment of the present invention, in step (1), the electrospinning dope: the mass percentage of the polylactic acid-glycolic acid copolymer (PLGA) is 8.6-12.3%; preferably 8.8 to 12.0 percent; preferably 9 to 12%; preferably 9.5 to 11.5%; preferably 10 to 11%.
Further, as a more preferred embodiment of the present invention, in step (1), the poly (ethylene glycol) block poly (γ -benzyl-L-methionine) is 18.2 to 23.1% by mass; preferably 18.5 to 23.0%; preferably 19 to 22%; preferably 20 to 21%.
Further, as a more preferable embodiment of the invention, in the step (2), the injection speed of fibrinogen in the electrospinning process is 1-3 ml/h, the loading voltage is 10-20 kV, and the receiving distance is 5-25 cm; preferably, the speed is 1.5-2.5 ml/h, the loading voltage is 12-18 kV, and the receiving distance is 10-20 cm; preferably, the speed is 1-2 ml/h, the loading voltage is 10-15 kV, and the receiving distance is 5-15 cm; preferably at a speed of 2-3 ml/h, a loading voltage of 15-20 kV and a receiving distance of 15-25 cm.
Further, as a more preferred embodiment of the present invention, in the step (2), the diameter of the blend spinning is 1.5 to 2.5 μm; preferably 1.6 to 2.2 μm; preferably 2.0 to 2.5 μm; preferably 1.5 to 2.0. Mu.m.
Further, as a more preferred embodiment of the present invention, in the step (2), the receiving speed of the cylindrical metal rotary receiving pan is adjusted to 700 to 2000r/h; preferably 800-1800 r/h; preferably 1000 to 1500r/h; preferably 1500 to 2000r/h.
Further, as a more preferable embodiment of the present invention, in the step (4), the vacuum degree of the vacuum drying is 0.03Mpa to 0.06Mpa, the temperature is 18 ℃ to 23 ℃ and the time is 36 to 48 hours; preferably, the vacuum degree is 0.04 Mpa-0.05 Mpa, the temperature is 19 ℃ to 22 ℃ and the time is 40 h to 45h; preferably, the vacuum degree is 0.04 Mpa-0.06 Mpa, the temperature is 20 ℃ to 23 ℃ and the time is 38 h to 48h; preferably, the vacuum degree is 0.03 Mpa-0.05 Mpa, the temperature is 18 ℃ to 20 ℃ and the time is 39 h to 45h.
According to a third embodiment of the present invention, there is provided an application of the polyamino acid composite material having a nerve repair promoting function prepared by the preparation method described above in the field of biomedical materials for preparing biomedical nerve repair cannulas.
Further, as a more preferable embodiment of the invention, the biomedical nerve repair sleeve has a wall thickness of 0.1-0.3 mm; preferably 0.15 to 0.25mm; preferably 0.2 to 0.3mm; preferably 0.1 to 0.2mm.
The sleeve is a bioabsorbable and white sleeve pipe, can effectively play a role in blocking a closed environment, and provides a sufficient closed growth space for injured nerves; meanwhile, the device can simulate in-vivo growth environment to the greatest extent, provides good space and mechanical properties for nerve repair, and accelerates nerve repair efficiency.
According to a fourth embodiment of the present invention, there is provided a cannula having a nerve repair promoting function, the cannula being prepared by crimping a polyamino acid composite material prepared by the above-mentioned preparation method into a shape having an inner diameter of 2mm to 5mm and a length of 3cm to 5cm by a tube rolling cutting technique, the cannula being suitable for clinical treatment of peripheral nerve injury of not more than 20 mm.
The principle of the present application: in recent years, there has been increasing evidence of the relative support that the micro-nano structure and biomechanical regulation of implantable materials can affect, and even remodel, the microenvironment of peripheral nerve injury. Because the occurrence and growth of neurons is always dependent on interactions with extracellular components, intercellular communication in the dynamic microenvironment. The mechanical properties of extracellular substrates can regulate signal transduction of axon biological activity, such as electrical conduction and protein transport, and soft materials close to nerve tissue or elastic modulus < 1kPa can promote neuron viability and neurite elongation 【1】 . The cells have very sensitive reaction to the local microenvironment in which they are locatedThe invention adopts PLGA and PEG-MELG as raw materials, adopts an electrostatic spinning treatment technology to prepare the composite material, utilizes the prepared composite material to prepare the nerve repair sleeve, and achieves the aims of safely and efficiently promoting nerve repair by accurately regulating and controlling interface structures and biomechanical characteristics, reconstructing local microenvironment of peripheral nerves, providing guiding effect for directional growth of neuron axons and matching a degradation period with a repair period through fine material proportion while providing basic physical support, connecting stubs, defending surrounding tissue compression and other basic functions.
Reference is made to: 【1】 Stukel, J.M.; willits, R.K. mechanical transmission of Neural Cells Through Cell-Substrate interactions. Tissue engineering. Part B, reviews 2016,22,173-182, doi:10.1089/ten.TEB.2015.0380.
Compared with the prior art, the technical scheme provided by the application has the following advantages:
1. the poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) amine (PEG-MELG) is creatively used, the poly lactic-co-glycolic acid (PLGA) and the poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) amine (PEG-MELG) are adopted as raw materials, the blend fiber membrane is prepared by blending through an electrostatic spinning technology, and then the polyamino acid composite material with the function of promoting nerve repair is prepared through vacuum drying, so that the poly amino acid composite material has better mechanical support, and can promote the repair of nerve cell axons and guide the climbing growth of growth cones.
2. The coiled nerve tissue repair sleeve can be manufactured by a coiled tube cutting technology, the polyamino acid composite material provides a relatively closed space which is not interfered by other tissues for nerve repair, guiding and protecting the repair of nerve axons, and simultaneously, the regeneration and extension of nerves are promoted by utilizing the special structural advantages of the material.
3. The preparation of the polyamino acid composite material by adopting the electrostatic spinning technology is convenient for constructing the microstructure surface structure of the polyamino acid composite material in a later stage, defines the pushing speed of fibrinogen to be 1-3 ml/h, the loading voltage to be 10-20 kV, the receiving distance to be 5-25 cm, and the diameter of the blended yarn to be 1.5-2.5 mu m, and adjusts the rotating receiving speed to be 700-2000 r/h, so that the polyamino acid composite material not only has better mechanical support, but also can promote the restoration of nerve cell axons and can guide the climbing growth of growth cones.
Drawings
FIG. 1 is a flow chart of a process for preparing a polyamino acid composite material having a function of promoting nerve repair;
FIG. 2 is a graph showing SFI scores of sciatic nerve function after surgery of the sleeve laboratory mice prepared in example 1 and comparative example 6 of the present application;
FIG. 3 is a plot of CD3 lymphocyte staining (20X) of the surgical field tissue sections of the sleeve experimental animals prepared in example 1 and comparative example 6 of the present application;
FIG. 4 is a schematic structural diagram of a nerve repair sleeve with nerve repair promoting function prepared by the method.
Detailed Description
In order to better understand the technical solutions in the present application, the following description will clearly and completely describe the technical solutions in the embodiments of the present application in conjunction with the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only some embodiments of the present application, not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the present disclosure, are within the scope of the present disclosure.
It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. In the description of the present application, the meaning of "a plurality" or "a number" is two or more, unless explicitly defined otherwise.
It should be understood that the structures, proportions, sizes, etc. shown in the drawings are for illustration purposes only and should not be construed as limiting the scope of the present disclosure, since any structural modifications, proportional changes, or dimensional adjustments made by those skilled in the art should not be made in the present disclosure without affecting the efficacy or achievement of the present disclosure.
In recent years, polyamino acid materials have demonstrated great potential application values in biological applications such as antibacterial, antifouling, antitumor, gene delivery, tissue engineering and immunoregulation.
According to a first embodiment of the present invention, there is provided a polyamino acid composite material having a nerve repair promoting function, made of polylactic acid-glycolic acid copolymer (PLGA) and poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) amine (PEG-MELG), wherein the mass ratio of the polylactic acid-glycolic acid copolymer and the poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) amine is 20 to 24:76 to 80, preferably 21 to 23:77 to 79, preferably 23:77.
The poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) amine used in the present application is a polymer organic substance, and is widely used in the fields of chemical industry and medicine, and has many excellent properties: the amphiphilic copolymer of poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) amine is adsorbed on the surface of the material to further improve the biocompatibility of the nerve sleeve and facilitate the purification of the product.
According to a second embodiment of the present invention, there is provided a method for preparing a polyamino acid composite material having a function of promoting nerve repair, comprising the steps of:
(1) Dissolving polylactic acid-glycolic acid copolymer and poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) amine in an organic solvent, and stirring and mixing uniformly to obtain an electrospinning stock solution;
(2) Injecting the electrospinning stock solution into an injector, then carrying out electrostatic spinning treatment under the action of a thrust pump, and receiving the product through a cylindrical metal rotary receiving disc to obtain blended spinning;
(3) After the electrostatic spinning treatment is finished and all the blended spinning is received by a cylindrical metal rotary receiving disc, the receiving speed of the receiving disc is adjusted to obtain a blended fiber membrane;
(4) And (3) sending the obtained blend fiber membrane into a vacuum drying oven for vacuum drying, and removing residual solvent to obtain the polyamino acid composite material with the function of promoting nerve repair.
It should be noted that the polyamino acid composite material prepared by the method has the same primary structure (amino acid residue and peptide bond) and similar secondary structure (alpha-helix and beta-sheet) as the natural protein and polypeptide materials, and the characteristics are the inherent advantages of the polyamino acid composite material as a biological material.
Specifically describing, in the embodiment of the present invention, in the step (1), stirring is performed at room temperature for 12 to 16 hours; the organic solvent is selected from hexafluoroisopropanol or dichloromethane.
Specifically describing, in the embodiment of the present invention, in step (1), the electrospinning dope: the mass percentage of the polylactic acid-glycolic acid copolymer (PLGA) is 8.6-12.3%, and the mass percentage of the poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) is 18.2-23.1%.
Specifically describing, in the embodiment of the invention, in the step (2), the injection speed of fibrinogen in the electrostatic spinning treatment process is 1-3 ml/h, the loading voltage is 10-20 kV, and the receiving distance is 5-25 cm.
Specifically, in the embodiment of the present invention, in the step (2), the diameter of the blend spinning is 1.5 to 2.5 μm.
Specifically describing, in the embodiment of the invention, in the step (2), the receiving speed of the cylindrical metal rotary receiving disc is adjusted to 700-2000 r/h.
Specifically describing, in the embodiment of the invention, in the step (4), the vacuum degree of vacuum drying is 0.03 Mpa-0.06 Mpa, the temperature is 18-23 ℃ and the time is 36-48 h.
According to a third embodiment of the present invention, there is provided an application of the polyamino acid composite material having a function of promoting nerve repair prepared by the preparation method as described above for preparing biomedical nerve repair cannulas.
Specifically describing, in the embodiment of the invention, the wall thickness of the biomedical nerve repair sleeve is 0.1-0.3 mm.
Example 1
As shown in fig. 1, 2, 3 and 4, a polyamino acid composite material with nerve repair promoting function is prepared from polylactic acid-glycolic acid copolymer (PLGA) and poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) amine (PEG-MELG), wherein the mass ratio of the polylactic acid-glycolic acid copolymer to the poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) amine is 23:77.
A preparation method of a polyamino acid composite material with a nerve repair promoting function comprises the following steps:
(1) Dissolving polylactic acid-glycolic acid copolymer and poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) amine in hexafluoroisopropanol, stirring at room temperature for 13h, and uniformly mixing to obtain an electrospinning stock solution; the electrospinning stock solution comprises the following components: the mass percentage of the polylactic acid-glycolic acid copolymer (PLGA) is 10 percent, and the mass percentage of the poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) is 20 percent;
(2) Injecting the electrospinning stock solution into an injector, and then carrying out electrostatic spinning treatment under the action of a propelling pump, wherein the pushing speed of fibrinogen in the electrostatic spinning treatment process is 1ml/h, the loading voltage is 10kV, the receiving distance is 5cm, the product is received by a cylindrical metal rotary receiving disc, and the receiving speed of the receiving disc is adjusted to 700r/h, so that the blending spinning with the diameter of 1.5 mu m is obtained;
(3) After the electrostatic spinning treatment is finished, when all the blended spinning is received by a cylindrical metal rotary receiving disc, a blended fiber membrane is obtained;
(4) And (3) sending the obtained blend fiber membrane into a vacuum drying oven for vacuum drying, wherein the vacuum degree of the vacuum drying is 0.03Mpa, the temperature is 18 ℃ and the time is 36 hours, and removing the residual solvent to obtain the polyamino acid composite material with the function of promoting nerve repair.
The application of the polyamino acid composite material with the nerve repair promoting function prepared by the preparation method is used for preparing the biomedical nerve repair sleeve, and the wall thickness of the biomedical nerve repair sleeve is 0.1mm.
Example 2
As shown in fig. 1 and 4, a polyamino acid composite material with nerve repair promoting function is prepared from polylactic acid-glycolic acid copolymer (PLGA) and poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) amine (PEG-MELG), wherein the mass ratio of the polylactic acid-glycolic acid copolymer to the poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) amine is 20:80.
A preparation method of a polyamino acid composite material with a nerve repair promoting function comprises the following steps:
(1) Dissolving polylactic acid-glycolic acid copolymer and poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) amine in methylene dichloride, stirring at room temperature for 12 hours, and uniformly mixing to obtain an electrospinning stock solution; the electrospinning stock solution comprises the following components: the mass percent of the polylactic acid-glycolic acid copolymer (PLGA) is 8.6, and the mass percent of the poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) is 18.2;
(2) Injecting the electrospinning stock solution into an injector, and then carrying out electrostatic spinning treatment under the action of a propelling pump, wherein the pushing speed of fibrinogen in the electrostatic spinning treatment process is 3ml/h, the loading voltage is 20kV, the receiving distance is 25cm, the product is received by a cylindrical metal rotary receiving disc, and the receiving speed of the receiving disc is adjusted to 2000r/h, so that the blending spinning with the diameter of 2.5 mu m is obtained;
(3) After the electrostatic spinning treatment is finished, when all the blended spinning is received by a cylindrical metal rotary receiving disc, a blended fiber membrane is obtained;
(4) And (3) sending the obtained blend fiber membrane into a vacuum drying oven for vacuum drying, wherein the vacuum degree of the vacuum drying is 0.04Mpa, the temperature is 20 ℃, the time is 48 hours, and the residual solvent is removed, so that the polyamino acid composite material with the function of promoting nerve repair is obtained.
The application of the polyamino acid composite material with the nerve repair promoting function prepared by the preparation method is used for preparing the biomedical nerve repair sleeve, and the wall thickness of the biomedical nerve repair sleeve is 0.3mm.
Example 3
As shown in fig. 1 and 4, a polyamino acid composite material with nerve repair promoting function is prepared from polylactic acid-glycolic acid copolymer (PLGA) and poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) amine (PEG-MELG), wherein the mass ratio of the polylactic acid-glycolic acid copolymer to the poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) amine is 23:77.
A preparation method of a polyamino acid composite material with a nerve repair promoting function comprises the following steps:
(1) Dissolving polylactic acid-glycolic acid copolymer and poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) amine in hexafluoroisopropanol, stirring at room temperature for 16h, and uniformly mixing to obtain an electrospinning stock solution; the electrospinning stock solution comprises the following components: 12.3% by mass of polylactic-co-glycolic acid (PLGA) and 23.1% by mass of poly (ethylene glycol) block poly (gamma-benzyl-L-methionine);
(2) Injecting the electrospinning stock solution into an injector, and then carrying out electrostatic spinning treatment under the action of a propelling pump, wherein the pushing speed of fibrinogen in the electrostatic spinning treatment process is 2ml/h, the loading voltage is 15kV, the receiving distance is 15cm, the product is received by a cylindrical metal rotary receiving disc, and the receiving speed of the receiving disc is adjusted to 1400r/h, so that the blending spinning with the diameter of 2.0 mu m is obtained;
(3) After the electrostatic spinning treatment is finished, when all the blended spinning is received by a cylindrical metal rotary receiving disc, a blended fiber membrane is obtained;
(4) And (3) sending the obtained blend fiber membrane into a vacuum drying oven for vacuum drying, wherein the vacuum degree of the vacuum drying is 0.06Mpa, the temperature is 23 ℃, the time is 42h, and the residual solvent is removed, so that the polyamino acid composite material with the function of promoting nerve repair is obtained.
The application of the polyamino acid composite material with the nerve repair promoting function prepared by the preparation method is used for preparing the biomedical nerve repair sleeve, and the wall thickness of the biomedical nerve repair sleeve is 0.2mm.
Comparative example 1
The difference from example 1 is that: the receiving speed of the cylindrical metal rotary receiving disc is adjusted to 500r/h, and other conditions are unchanged.
Comparative example 2
The difference from example 1 is that: a nerve repair composite material is prepared by adding poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) amine (PEG-MELG), and replacing PEG-MELG with PGA under the same conditions.
The sleeve manufactured in the comparative example 2 is tested by adopting a catheter implantation local immunohistochemical staining experiment method, and the test result shows that the blended spinning fiber membrane manufactured in the comparative example 2 cannot adjust the degradation period, can cause serious inflammatory reaction and cannot promote nerve repair.
Comparative example 3
The difference from example 1 is that: a nerve repair composite material made from polylactic acid-glycolic acid copolymer (PLGA) and poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) amine (PEG-MELG), wherein the mass ratio of the polylactic acid-glycolic acid copolymer to the poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) amine is 15:85, with the other conditions unchanged.
Referring to the relevant detection step of mechanical test method 4.3 of standard JIS T0401-2013 holder, the blended yarn fiber membrane product obtained in comparative example 3 was placed intact on a parallel plate jig so that the instrument could measure the radial force of the product, and the sleeve was compressed at a uniform speed of 2mm/min at (37.+ -. 2) ℃ until the outer diameter was reduced by 50%, and the load required to deform the sleeve by 50% of the outer diameter was measured.
Through testing, the blended yarn fiber membrane prepared by the method of the comparative example 3 has no stress, the extrusion resistance is reduced, and the space required during nerve regeneration cannot be maintained.
Comparative example 4
The difference from example 3 is that: the loading voltage of the injected fibrinogen is 30kV, and other conditions are unchanged.
Comparative example 5
The difference from example 3 is that: the loading voltage of the injected fibrinogen is 7kV, and other conditions are unchanged.
According to the general rule of a scanning electron microscope measuring method of standard GB/T16594-2008 micron-scale length, a scanning electron microscope is used for photographing the fiber surfaces of the inner side pipe wall and the outer side pipe wall of the blended yarn samples prepared in comparative examples 1, 4 and 5 respectively, at least 10 points (except fiber intersections) are randomly selected on a photo to measure the fiber diameters, and an average value is obtained.
The blended yarn prepared by the method of comparative example 1 was tested to have a diameter of 5 μm and lost the designed micro-pore structure. The blended yarn prepared by the method of comparative example 4 had a diameter of 0.9 to 1.2. Mu.m, and the yarn was too fine and the mechanical supporting effect was deteriorated. The blended yarn prepared by the method of comparative example 5 has a diameter of 3.3-4.0 μm, and the fiber is too thick, loses the designed micro-pore structure, cannot promote the restoration of nerve cell axons, and cannot guide the climbing growth of growth cones.
Comparative example 6
Commercial products
Nerve Guide, registration certificate number: NGC2020082111, manufacturer: polyganics Innovations B.V. (Rozenburglaan), netherlands.
1. Assessment of nerve repair effect
1. Sciatic nerve function index (SFI) detection
The detection method and the standard are as follows: the sciatic nerve function index (SFI) detection is used, and the specific method is as follows: a wood groove with two ends being opened and 60cm long, 10cm wide and 10cm high is made, 70g of white paper is cut into the same width as the wood groove, and then the white paper is paved at the bottom of the groove. After the two hind limbs of the rat are immersed in the pigment for coloring the ankle joints, the rat is placed at one end of the groove, so that the rat can walk to the other side of the groove by itself, and 5-6 footprints are left on each hind limb. Selection of well-blotted footprints 3 indicators of normal foot (N) and injured side foot (E) were measured separately: A. PL (footprint length); B. TS (toe width); C. IT (medial toe width). Substituting the index into the Bain formula to calculate the sciatic nerve function index. The Bain formula is: SFI=109.5 (ETS-NTS)/NTS-38.3 (EPL-NPL)/NPL+13.3 (EIT-NIT)/NIT-8.8. Sciatic nerve function index sfi=0 is normal mice, and sciatic nerve function index sfi= -100 is fully injured mice.
The composite materials prepared by the methods of example 1 and comparative example 6 were used to prepare cannulas, and sciatic nerve repair detection was performed using each set of cannulas, and each set of treatment conditions were as follows:
group of sham operations: only dissecting epidermis, and not performing sciatic nerve dissociation operation of the mice;
negative control group: after the sciatic nerve separation operation is carried out on the mice, no repair operation is carried out, and the mice are directly sutured;
example 1 group (abbreviated as "example" in the drawings): after the sciatic nerve separation operation is performed on the mice, a repair operation is performed by using the sleeve made of the polyamino acid composite material of example 1;
comparative example 6 group (abbreviated as "comparative example" in the drawings): after the sciatic nerve disruption operation was performed on the mice, a repair operation was performed using a cannula made of the nerve repair composite material of comparative example 6.
The experimental results are shown in FIG. 2.
As shown in fig. 2, the SFI score was significantly lower 15 days after surgery in all groups of experimental mice relative to the normal control mice, and the sham operated group had recovered to near normal mouse level by the time of re-evaluation 30 days after surgery, suggesting that the sham operated group had no nerve damage, and the score at 15 days after surgery was lower than normal possibly caused by factors such as local edema, wound pain, etc. in the operative area. The remaining groups of rats with sciatic nerve disruption, with or without cannula repair, continued decline in score 30 to 45 days post-surgery, suggesting sustained nerve function impairment, with the negative control group with no catheter repair declining most significantly. After 45 days, the negative control group score continued to decrease, but none of the catheter group scores continued to decrease and began to rise slightly, suggesting that neurological function and lower limb muscle strength recovered as the proximal regeneration from the severed nerve extended. The scores for each conduit group were significantly different from the negative control group (P < 0.05) with no statistical differences between the conduit groups. 90 days after operation, the nerve function score of each group starts to be stable, and compared with the comparative example 6, the catheter control score prepared by the polyamino acid composite material of the example 1 of the application is higher in positive and better in nerve repair effect.
2. Local immunohistochemical staining experiment for catheter implantation
The composite materials prepared in example 1 and comparative example 6 were prepared into cannulas by the same method, and the prepared cannulas were used to perform a catheter implantation local immunohistochemical staining experiment on laboratory mice, and the treatment conditions of each group were as follows:
normal group: the experimental mice are not subjected to nerve-separation operation and are not implanted with materials;
negative control group: the experimental mice are subjected to nerve-separation operation without implanting materials;
example 1 group (abbreviated as "example" in the drawings): performing nerve dissociation operation on the experimental mice, and implanting the sleeve manufactured in the embodiment 1 of the application to perform repair operation;
comparative example 6 group (abbreviated as "comparative example" in the drawings): the experimental mice were subjected to nerve-separation surgery, and the cannulas prepared in comparative example 6 were implanted for repair surgery.
The detection method and the standard are as follows: 1 month after the modeling operation, the local stimulation condition of each group of implant materials is judged by performing operation area dissection on a normal group, a negative control group, an example 1 group and a comparative example 6 group and performing CD3 lymphocyte staining on an operation part sample, and the experimental result is shown in figure 3.
As can be seen from fig. 3, after implantation of the nerve conduit material in each group, no significant increase in lymphocyte staining occurred in both the example 1 group and the allograft group relative to the sham-operated group and the negative control. The comparative example 6 group is a conventional chitosan nerve repair catheter experiment, and the positive staining of the lymphocytes of the comparative example 6 group without the polyamino acid composite material is obviously increased. From the experimental results, the sleeve made of the polyamino acid composite material can reduce potential stimulation to local immune cells.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A polyamino acid composite material with nerve repair promoting function, which is characterized in that: is prepared from polylactic acid-glycolic acid copolymer and poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) amine;
the mass ratio of the polylactic acid-glycolic acid copolymer to the poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) amine is 20-24:76-80.
2. A preparation method of a polyamino acid composite material with a nerve repair promoting function comprises the following steps:
(1) Dissolving polylactic acid-glycolic acid copolymer and poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) amine in an organic solvent, and stirring and mixing uniformly to obtain an electrospinning stock solution;
(2) Injecting the electrospinning stock solution into an injector, then carrying out electrostatic spinning treatment under the action of a thrust pump, and receiving the product through a cylindrical metal rotary receiving disc to obtain blended spinning;
(3) After the electrostatic spinning treatment is finished and all the blended spinning is received by a cylindrical metal rotary receiving disc, the receiving speed of the receiving disc is adjusted to obtain a blended fiber membrane;
(4) And (3) sending the obtained blend fiber membrane into a vacuum drying oven for vacuum drying, and removing residual solvent to obtain the polyamino acid composite material with the function of promoting nerve repair.
3. The method for preparing a polyamino acid composite having a nerve repair promoting function according to claim 2, characterized by: in the step (1), stirring is carried out for 12-16 h at room temperature; the organic solvent is selected from hexafluoroisopropanol or dichloromethane.
4. The method for preparing a polyamino acid composite having a nerve repair promoting function according to claim 2, characterized by: in step (1), in the electrospinning dope: the mass percentage of the polylactic acid-glycolic acid copolymer is 8.6-12.3%.
5. The method for preparing a polyamino acid composite having a nerve repair promoting function according to claim 2, characterized by: in the step (1), the mass percentage of the poly (ethylene glycol) block poly (gamma-benzyl-L-methionine) is 18.2-23.1%.
6. The method for preparing a polyamino acid composite having a nerve repair promoting function according to claim 2, characterized by: in the step (2), the pushing speed of fibrinogen in the electrostatic spinning treatment process is 1-3 ml/h, the loading voltage is 10-20 kV, and the receiving distance is 5-25 cm.
7. The method for preparing a polyamino acid composite having a nerve repair promoting function according to claim 2, characterized by: in the step (2), the diameter of the blend spinning is 1.5-2.5 μm.
8. The method for preparing a polyamino acid composite having a nerve repair promoting function according to claim 2, characterized by: in the step (2), the receiving speed of the cylindrical metal rotary receiving disc is adjusted to 700-2000 r/h.
9. The method for preparing a polyamino acid composite having a nerve repair promoting function according to claim 2, characterized by: in the step (4), the vacuum degree of the vacuum drying is 0.03-0.06 Mpa, the temperature is 18-23 ℃ and the time is 36-48 h.
10. Use of the polyamino acid composite material having function of promoting nerve repair prepared by the preparation method of any one of claims 1 to 9 for preparing biomedical nerve repair cannulas.
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| KR101541999B1 (en) * | 2014-03-28 | 2015-08-05 | 동국대학교 산학협력단 | A nerve conduit and the method of using the same |
| US20170021056A1 (en) * | 2015-07-20 | 2017-01-26 | Northwestern University | Peptide amphiphile biomaterials for nerve repair |
| CN109331225A (en) * | 2018-12-07 | 2019-02-15 | 广州贝奥吉因生物科技有限公司 | A kind of degradable composite nerve conduit and preparation method thereof |
| CN109758617A (en) * | 2019-03-11 | 2019-05-17 | 宁波光远致信生物科技有限公司 | A kind of neural restoration film and its preparation method and application |
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