CN111671979A - Nerve repair material - Google Patents

Abstract

The invention discloses a nerve repairing material, which is treated by adopting natural strong cations (such as protamine); on one hand, the material can be firmly combined with the CSPG (exogenous) in the material, and on the other hand, the material can be combined with the CSPG (endogenous) at the injury part of the nerve tissue, so that the inhibition effect of the exogenous and endogenous CSPG on nerve regeneration can be effectively slowed down and prevented; meanwhile, the ECM adopts a plant source cell removal reagent, so that on one hand, the three-dimensional structure of the ECM can be maintained relatively well, on the other hand, more growth factors and beneficial active ingredients (such as HA) in the ECM can be reserved, and by using the nerve repair material (such as a nerve conduit), the nerve repair material not only can promote the growth cone of the neuron to break through scar tissues and is beneficial to nerve regeneration, but also HAs good mechanical property and enough support strength to guide the axial growth of nerve cells; meanwhile, the production is simple, only one-step treatment is added in the prior art, the used raw materials are safe, and the product belongs to a new product for nerve repair.

Description

Nerve repair material

Technical Field

The invention relates to the technical field of nerve repair materials, in particular to a nerve conduit which is used for repairing damaged or broken nerves.

Background

Nerve injury, which is often caused by trauma and other conditions, is a type of injury that is common and frequent in clinic, and often causes sensory and motor dysfunction or loss, which seriously affects the quality of daily life and efficiency of work of patients. The search for effective therapeutic measures for treating nerve damage is urgent; nerve tissue regeneration engineering has become a research heat tide for repairing nervous system injury, the key point is that materials are adopted to support the growth of nerve cells, better materials can serve as an attachment matrix of implanted cells or host nerve stem cells, and the growth of the nerve cells can be promoted by the materials or degradation products of the materials; for example, the use of Nerve conduits (NGCs) can provide a number of advantageous conditions for Nerve regeneration after Nerve disruption or long-distance truncation. During nerve regeneration, the nerve regeneration device can temporarily fix and support two ends of a defective nerve, guide axon axial growth of a neuron, avoid outgrowth and neuroma formation, provide a relatively isolated microenvironment for nerve regeneration, enrich neurotrophic factors required by nerve regeneration, reduce invasion of exogenous impurity cells and prevent formation of glial scars.

The nerve repair material should have the following basic characteristics: (1) the biodegradable polyester film has biocompatibility and biodegradable characteristics, the degradation rate is suitable and controllable, and degradation products are nontoxic; (2) supporting and promoting axon growth, having suitable mechanical properties (strength, flexibility) to facilitate surgical operations; (3) the material is convenient to synthesize and easy to process and form. The ideal nerve repair material firstly has good biocompatibility of tissues and cells; secondly, the appropriate mechanical property is needed, and the growth and extension of axons are facilitated, and meanwhile, harmful factors and various unfavorable components do not exist, so that the regeneration of neurons is inhibited or destroyed; finally has good guiding function to the regeneration of the axon of the neuron.

The existing nerve repair materials are divided into degradable materials and non-degradable materials according to whether the materials are degradable or not, wherein the degradable materials mainly comprise: natural sources of materials and synthetic materials. Materials of natural origin, such as extracellular matrix (ECM), gelatin, chitosan, alginate, cellulose, hyaluronic acid, liposomes, fibroin, etc., have good histocompatibility and low antigenicity, like ECM materials also have a natural pore structure system, which is beneficial for cell adhesion, but generally have poor biomechanical properties and lack sufficient physical strength to protect damaged parts; synthetic materials, usually polymeric materials, such as PGA, PLA, PLGA, etc., which have good physical properties, including mechanical strength, flexibility, degradability, permeability; however, the artificially synthesized material has certain cytotoxicity and poor biocompatibility, so that a host has stronger inflammatory reaction. Some synthetic materials, although immune tolerant to the host, are poorly compatible with cells and are not conducive to cell adhesion and tissue repair. The mixed material compounded by natural materials and artificial materials can make up the defects of single materials, has certain advantages but also has no complete advantages, and is difficult to achieve the perfect degree in the aspects of various indexes of the micro three-dimensional space structure, the biomechanical property, the biocompatibility, the content of beneficial active ingredients, the material degradation speed, the activity of degradation products and the like of the repair material.

The acellular nerve repair material is the product which is most researched and is the best seen at present and can really and effectively promote the repair of nerve injury.

The prior patent technology is as follows:

d1: the invention patent 201711282138.5 of Shandong meaningful science and technology Co., Ltd, relates to an extracellular matrix nerve repair membrane and a preparation method thereof; the disclosed neuroprosthesis membrane is a porous membrane obtained by treating peripheral nerves of a xenogeneic animal. The invention also discloses a preparation method of the extracellular matrix nerve repair membrane, which can reserve beneficial components for promoting nerve regeneration to the maximum extent after the peripheral nerves of the heterogeneous animals are subjected to cell removal treatment, and the prepared extracellular matrix nerve repair membrane has good safety and can specifically promote the regeneration of damaged nerves.

D2: the invention patent 201911040953.X of Chinese medical university, a nerve repair catheter prepared from a novel composite material and a preparation method thereof; the preparation method disclosed by the method comprises the steps of obtaining the neural adventitia catheter, and carrying out decellularization treatment on the neural adventitia catheter to obtain a decellularized neural adventitia catheter; the outer layer of the acellular nerve adventitia catheter is wrapped by a high polymer material. The method separates the adventitia from the nerve fiber, so that the nerve adventitia can form a good lumen structure, and the outside of the nerve adventitia is wrapped by a layer of high polymer material, so that the catheter is tougher and is not easy to collapse; the donor nerve source is wider, the preparation is carried out according to the nerve materials with different diameter requirements, and the method is suitable for repairing large-gap nerve defects.

D3: the invention patent 201710124327.3 of Beijing Bopfurri applied to Biotechnology Limited, a acellular nerve repair material, a preparation method and application. The decellularized nerve repairing material disclosed therein comprises collagen, a polysaccharide substance, an active factor and a nerve regeneration promoting factor, has a three-dimensional network porous structure, is non-immunogenic, is degradable in vivo, and may be in the form of a sheet or a hollow tube. And the decellularized raw material is porcine small intestine submucosa tissue.

D4: the invention patent 201510679135.X applied by Wenzhou medical university discloses a acellular nerve repair material applied by combining with acellular nerves, which consists of nerves of acellular allogeneic or acellular xenogeneic organisms, a repair material, a tubulin inhibitor, a neurotrophic factor and stem cells, has good biocompatibility, provides nutrient supply required by nerve regeneration repair, can maintain the optimal physicochemical and biological microenvironment for nerve regeneration for a long time, and has double functions of preventing neuroma formation and promoting defective nerve repair.

D5: the invention patent of the applicant's Lushi application No. CN201010147529.8 discloses a preparation method and application of a neural tissue matrix derived tissue engineering scaffold material. Nerve tissues are taken as raw materials, matrix components (including nano-scale collagen microfilaments, fibronectin microfilaments and laminin microfilaments) which are beneficial to nerve regeneration are extracted by means of drug expansion, mechanical crushing, enzymolysis treatment, dialysis collection and the like, and immunogenic components (including Schwann cells, phospholipid and axons) which are not beneficial to nerve regeneration are removed. The prepared nerve tissue matrix source material can be further prepared into a three-dimensional porous oriented scaffold through directional crystallization, freeze drying and crosslinking independently or by being matched with other high polymer materials, or a nano-level film is prepared by utilizing an electrospinning technology and then wound to form a nerve regeneration conduit.

D6: the invention patent of the applicant's Lushi application No. CN201010033726.7, a tissue engineering nerve scaffold and a preparation method and application thereof; discloses a method for removing Chondroitin Sulfate Proteoglycan (CSPGs) in allogenic nerves by adopting chondroitinase ABC to obtain a basal membrane tube of an extracellular matrix as a tissue engineering nerve scaffold; is suitable for repairing peripheral nerve defects, has very obvious effect of promoting axon regeneration, and is a better medical material for nerve transplantation.

D7: the invention discloses an invention patent of Shandong province eye research application No. CN201810845538.0, a acellular nerve matrix material and a preparation method and application thereof, and discloses a method for preparing the acellular nerve matrix material. The material can be used for nerve regeneration and repair.

D1, D2, D3, D4 and D5 are acellular nerve repair materials, but Chondroitin Sulfate Proteoglycan (CSPGs) which is substances for inhibiting neuron regeneration in the materials are not removed, so that the speed of nerve tissue regeneration and the quality of recovery are necessarily affected to different degrees, and particularly, the difficulty is great when the nerve repair materials are used for achieving nerve tissue regeneration and function recovery under the condition that old nerve damaged tissues, particularly large-area glial scars exist at nerve damaged positions and contain more CSPGs. The nerve repair material of the invention as invented by D3, paragraph [0009] introduces that it contains a polysaccharide substance, which is a composition comprising chondroitin sulfate and hyaluronic acid; meanwhile, as shown in example 1 of paragraph [0068], the content of chondroitin sulfate was 4512. mu.g/g on the average; it is clear that the inventors consider chondroitin sulfate to be beneficial for nerve repair and regeneration; it is not realized at all, and in the field of nerve tissue repair, a great number of domestic and foreign research articles and reports exist, which show that chondroitin sulfate has obvious inhibition effect on the regeneration of neurons and the extension of neurite growth cones; for example, CSPGs inhibit the Regeneration of neurites and dendrites (Silver and Miller, 2004, Regeneration after and the neurological scar, Nature reviews Neuroscience, 5, 146-); Jian-Long Zou et al found that when DRG distal end of newborn axon was covered with 10ug/ml of CSPGs, it was found to significantly inhibit axon regeneration and lateral sprouting, the density of newborn axon was also significantly reduced, and CSPGs covered group, and there was significant collapse of newborn axon (Spatial distribution of CSPGs in the new regeneration of the tissue-treated path, Experimental Neurology, 2018, 307, 37-44). In fact, a large number of studies have been carried out to show that at the site of injury following injury to the nervous tissue, glial scars are produced, which are composed mainly of astrocytes and Chondroitin Sulfate Proteoglycans (CSPGs) and act as barriers to axonal extension and regeneration. The growth cone in the axon of the neuron can not pass through the glial scar, thereby directly influencing the continuous growth and extension of the glial scar, and further causing the serious obstruction of the regeneration of nerve tissues.

At present, it is believed that the mechanical barrier formed by the glial scar block is not enough to completely block axon regeneration, and the more important blocking effect is due to a plurality of chemical substances for inhibiting axon growth, such as Chondroitin Sulfate Proteoglycan (CSPGs), Myelin Associated Glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp), involution protein (Nogo), growth cone collapse inhibitory factor membrane damage glycan (IMP), secreted by various nerve cells, and these axon regeneration inhibiting substances activate related signal pathways by binding different receptors, thereby playing a role in inhibiting nerve regeneration, and forming a chemical barrier for axon regeneration, and the chemical barrier formed by these substances blocks the regeneration effect of nerve injury, which is far stronger than the mechanical barrier. The main factor playing a role in inhibition is CSPGs, which not only have the property of inhibiting the growth of axons, but also can block the differentiation of neural stem cells into neurons.

Obviously, in the nerve repair materials, if Chondroitin Sulfate (CS) or Chondroitin Sulfate Proteoglycan (CSPGs) are contained, the regeneration of nerve tissues is generally influenced more or less directly and adversely; in view of the inhibitory effect of chondroitin sulfate components such as CSPGs on nerve tissue regeneration, researchers have treated nerve scaffolds with chondroitinase ABC in order to obtain positive effects on nerve tissue regeneration.

Cafferty et al observed that transgenic mice expressing chondroitinase ABC (Chondrotinase ABC, Ch-ABC or ChABC for short) still have strong axon regeneration capacity after dorsal root cutting of spinal nerves and faster recovery of motor function (Functional axonal regeneration negative allergic tissue modifier two gel fracture surface proteins J Neurosci, 2007, 27, 2176-loop 2185). There have also been attempts by schacs to promote regrowth of CNS axons, used in conjunction with chebc and other methods. Fouad et al used peripheral nerve material, olfactory ensheathing cells and ChABC injection in combination to treat nerve transection injury, which combination therapy enhanced axonal regeneration and promoted restoration of motor function (Combining Schwann cell bridges and muscle-inducing neuron recruitment of the spinal cord. J Neurosci, 2005,25, 1169-. Studies such as the huangyu flute show that ChABC can inhibit the apoptosis of photoreceptor cells by degrading CSPGs abnormally deposited on rat degenerative retina, thereby promoting the repair of damaged retina (chondroitinase relieves the apoptosis of the photoreceptor cells of the rat degenerative retina, the university of Yangzhou, 2012, 4, 465 and 466). Zhang Yu et al have proved that the combined application of the glial cell line-derived neurotrophic factor sustained-release microspheres and the NogoA and ChABC sustained-release microspheres can effectively promote the repair of the nerve regeneration function of the injured nerves of rats (Chinese tissue engineering research, 2012, 16, 5401-; wangying research and the like finds that the acellular rat sciatic nerve material can repair the defected sciatic nerve of the rat after being injected into Mesenchymal Stem Cells (MSCs) through ChABC treatment (abstracts of 2011 annual meeting articles of Chinese society of anatomy); kangan et al found that ChABC combined with MSCs can promote the expression of Vascular Endothelial Growth Factor (VEGF) in repairing nerve defects (abstracts of the 2012 annual meeting of the Chinese society of anatomy). Following transection injury of nerve tissue, nerve regeneration and functional recovery requires not only the growth of damaged axons, but also the formation of effective synaptic connections so that nerve impulses are properly conducted.

The nerve tissue material is decellularized, and immunogenic cell components are removed through decellularization treatment, so that immunological rejection can be effectively avoided, and meanwhile, most of the original space frameworks and important ECM proteins such as laminin and fibronectin and the like of nerves can be reserved in the nerves, are similar to the growth environment of neurons, and can well guide the extension growth of new axons. However, the products of the two inventions of D6 and D7 use ChABC to degrade CSPGs so as to eliminate the inhibitory effect of Chondroitin Sulfate (CS) and/or CSPGs on the regeneration of neurons; however, in this patent, the decellularized tissue, particularly the decellularized porcine nerve tissue (e.g., in the tibial nerve), itself contains relatively low concentrations of chondroitin sulfate compared to hyaluronic acid; but the chondroitinase ABC with higher concentration is used, and the action time is longer, and the 37 ℃ reaches 6 hours. Because chondrosulphatase ABC is a mixed enzyme, it can digest not only chondroitin sulphate but also hyaluronic acid therein, degrading it into di-oligosaccharide; because chondroitin sulfate is adopted, the acellular nerve tissues are treated at high concentration for a long time, hyaluronic acid and cross-linking thereof are inevitably damaged, and the mechanical properties such as tensile strength of the acellular materials are directly influenced; because the natural hyaluronic acid macromolecules in the decellularized material have excellent natural crosslinking effect (crosslinking between collagen and hyaluronic acid), the mechanical property of the ECM can be enhanced.

Therefore, the method for removing the chondroitin sulfate can be simplified, and the mechanical property of the material is not damaged, which is the main purpose of the invention.

Disclosure of Invention

The first object of the present invention is to provide a novel nerve repair material which does not contain chondroitin sulfate proteoglycan (exogenous CSPGs from the patient's perspective) having an inhibitory effect on neuronal regeneration.

The second purpose is that: the provided nerve repair material HAs good biomechanical properties (such as tensile strength) and more beneficial active ingredients (such as HA) for promoting healing.

The third purpose is that: the components of the nerve repair material can be combined with the CSPGs at the wound of the nerve tissue (from the perspective of a patient, the nerve repair material belongs to endogenesis), and the inhibition effect of the endogenous CSPGs at the wound on nerve regeneration can be effectively slowed and stopped.

The fourth purpose is that: the provided nerve repair material has good antibacterial and anti-inflammatory effects on the basis of good biocompatibility and low immunogenicity.

In order to realize the purpose of the invention, the technical scheme adopted by the invention is as follows:

the nerve repairing material is treated by a CSPGs antagonist, the CSPGs antagonist is a strong positive ion macromolecule, the molecular weight of the strong positive ion macromolecule is 2-3 ten thousand, and the strong positive ion macromolecule is one of natural polymers, derivatives thereof or analogues thereof or a compound thereof.

Furthermore, the CSPGs antagonist is one of protamine, protamine derivatives, protamine structural mimics or the combination thereof, so as to achieve the purpose of firmly combining the protamine strong cations with the CSPGs.

Further, the treatment in the treatment with the CSPGs antagonist means one or a combination of soaking, painting and spraying the nerve repair material with a solution containing 0.1 to 10% of one or a combination of protamine, a protamine derivative and a protamine structure mimic.

Further, the protamine is one or a mixture of salmon, herring, red trout and mammals, including monoprotamine, biprotamine and tripprotamine.

Further, the treatment refers to soaking the acellular nerve repair material by using a solution containing 0.5-2% of protamine, wherein the soaking is performed for 30 minutes to 5 hours each time, then the soaking is performed for 10 to 50 minutes by using a buffer solution, the soaking and cleaning steps can be repeated for one to three times, and after the last time of the protamine soaking, the material is directly dried and shaped without being washed by the buffer solution.

Further, the nerve repair material may include a degradable material, which is one of a synthetic polymer, a natural polysaccharide polymer, a collagen polymer, or a composite thereof, and is combined with the acellular material by electrospinning, bonding, or suturing, mainly using an acellular material (ECM).

Further, the synthetic polymer is one of polyamino acid, polycaprolactone, polylactic acid, polyglycolic acid, polycaprolactone, polyethylene glycol and polyamino acid or a composition thereof.

Further, the natural polysaccharide polymer material is one or a combination of cellulose, chitosan and alginic acid.

Furthermore, the cell-removed material refers to a material for removing cells from a tissue of a mammal, wherein the mammal is a pig, a cow, a sheep, a horse, a donkey, a camel or a rabbit, and the tissue refers to one or a combination of peritoneum, dermis, small intestine submucosa, gastric membrane, periosteum, pericardium, fascia, nerve and blood vessel.

The further optimization scheme is that the acellular nerve repairing material is acellular porcine small intestine submucosa, sciatic nerve and femoral artery blood vessels.

The further optimization scheme is that the pig is an adult pig with the feeding age of more than 10 months, and the cell removal material is jejunum submucosa.

In a further optimization scheme, the decellularization is carried out by using one of a physical method, a chemical method and a biological method or a combination of the physical method, the physical method is an osmotic pressure method, the chemical method is surfactant treatment, and the biological method is protease digestion; the protease is trypsin.

The further optimization scheme is that the cells are removed by a chemical method, and the used surfactant is one of SDS, TritonX-100, TritonX-200, sodium deoxycholate and plant source surfactant or the composition thereof.

The further optimization scheme is that the cell removing reagent is a plant source surfactant, and the optimized reagent is as follows: one or a combination of triterpene saponin and steroid saponin; the effective working concentration for decellularization is 0.05-3% by weight (W/W).

Further, the nerve repairing material is in a hollow tubular shape or a sheet shape, the length is 1-5cm, the diameter is 0.1-0.9cm, and the thickness of the tube or the sheet is 0.1-1.5 mm.

Further, specifically, a solution containing 0.5-2% of protamine is used for soaking a semi-finished product or a finished product of the nerve repair material at 37 ℃ for 1-2 hours, then the semi-finished product or the finished product is washed by PBS buffer solution for 10-15 minutes, the soaking and washing steps are repeated for one to three times, and after the last soaking, the washing by the buffer solution is not needed.

Further, the protamine is protamine derived from salmon, trout and herring; preferably, the molecular weight is in the range of 8000 daltons or less, preferably 4000-6000 daltons.

Further, the protamine can also comprise pharmaceutically acceptable inorganic salts such as sulfate, acetate, phosphate, hydrochloride, carbonate and the like, and organic salt forms such as maleate, citrate, tartrate, sulfonate, salicylate, malate and the like; preferably protamine sulfate.

Further, the nerve repair material is one of natural biodegradable materials and artificially synthesized biodegradable materials or a combination thereof.

Further, the natural biodegradable material is one of collagen material, chitosan and alginic acid or a combination thereof.

Furthermore, the mammal is pig, cattle, sheep, horse, donkey, camel, rabbit, and the tissue refers to one or the combination of peritoneum, dermis, small intestine submucosa, periosteum, pericardium, fascia, nerve and blood vessel.

Further, the mammalian tissue is porcine small intestinal submucosa, sciatic nerve, and femoral artery blood vessel.

Further, the decellularization is carried out by using one of a physical method, a chemical method, a biological method or a combination thereof, wherein the physical method is an osmotic pressure method, the chemical method is a surfactant treatment, and the biological method is protease digestion; the protease is trypsin, and the surfactant is one or a combination of SDS, Triton X-100, Triton X-200, sodium deoxycholate, plant-derived pentacyclic triterpene saponin and steroid saponin; the effective working concentration of the surfactant is 0.05-3% by weight.

In addition, some cytokines, such as growth factors and adhesion factors, such as Nerve Growth Factor (NGF) and brain-derived neurotrophic factor (BDNF), are added into the nerve repair material, and the cytokines are controlled or slowly released, so that the extension and the regeneration of damaged neuron axons can be promoted, and the regeneration and the functional rehabilitation of nerve tissues, such as nerve conduction velocity recovery and Compound Muscle Action Potential (CMAP) normal, are facilitated.

In addition, the technical scheme can also comprise the steps of crushing the decellularized tissue (after removing impurities, degreasing, decellularizing, DNA and alpha-galactoside), adding a protease solution, and stirring until the protease solution is dissolved to obtain a decellularized solution; adding 0.1-2% (w/w) protamine into the cell matrix removing solution, mixing, injecting into self-made moulds with different shapes and sizes, and freeze-drying and shaping to obtain the nerve repair material.

The invention has the beneficial effects that:

1. compared with the prior art, the nerve repair material provided by the invention is not neutralized by digestive enzymes, immunological antibodies or conventional gliosis resisting agents, but is treated by components with obvious antagonism with CSPGs, such as protamine, so that the components for inhibiting the growth of neuron axons contained in the nerve repair material are firmly combined, the treatment method is simple and convenient, the treatment time is greatly shortened, and the mechanical property of the material is not damaged (in the prior art, CSPGs are not removed, the mechanical property is reserved, or CSPGs are removed, but the mechanical property is reduced). The method has the advantages of convenient operation, low preparation cost, strong clinical practicability and good effect; easy operation and control and wide application prospect.

2. The nerve repair material provided by the invention is moderately overloaded with the CSPGs antagonist, can not only shield the inhibitory action of the CSPGs contained in the material on nerve regeneration, but also be combined with the original and residual components for inhibiting axon growth in the glial scar of the operative part of a patient, overcomes the double inhibitory action of exogenous and endogenous CSPGs on nerve regeneration, and is obviously beneficial to the repair of neurons and the regeneration and functional rehabilitation of nerve tissues.

3. The nerve repairing material provided by the invention has good mechanical property, sufficient support strength and durability, difficult deformation and better rebound resilience so as to guide axial growth of nerve cells; even for the repair of long-distance nerve injury, the nerve repair material not only has better strength and hardness, but also has slower degradation speed, and can also repair the injury of the long nerve.

4. The nerve repair material provided by the invention has good biocompatibility and reasonable natural three-dimensional porosity, is favorable for cell adhesion and efficient transportation and utilization of nutrients; the material contains more beneficial components, and has obvious guiding and promoting effects on nerve tissue regeneration.

5. The nerve repair material provided by the invention is nontoxic and harmless in material selection, can be completely biodegradable, and does not cause inflammation by main degradation products; the main material is a acellular matrix, has no immunogenicity or low immunoreactivity, and has relatively controllable degradation speed; is suitable for repairing different types and different thicknesses of nerve defects (such as the nerve repair of old people is slow, and the nerve repair of children and teenagers is fast, and the nerve defects of large nerves and small nerves).

6. The protamine loaded by the nerve repair material provided by the invention also has an antibacterial effect, and is degraded in a human body to become arginine, so that the nerve repair material is beneficial to the health of the human body; protamine also has effects of anti-tumor, relieving fatigue, enhancing liver function, stimulating pituitary gland to release gonadotropin, etc.

The invention mechanism is as follows:

1. a nerve repair material which is treated by adopting natural strong cations (such as protamine); on one hand, the composite can firmly combine with the CSPGs (exogenous) in the material, and on the other hand, the composite can also combine with the CSPGss (endogenous) at the nerve tissue injury, so that the inhibition effect of the exogenous and endogenous CSPGs on nerve regeneration can be effectively slowed down and prevented.

2. The nerve repair material is mainly a decellularization material, and a plant source decellularization reagent is adopted, so that on one hand, the three-dimensional structure of the ECM can be well maintained, and on the other hand, more growth factors and beneficial active ingredients (such as HA and heparin) in the ECM can be reserved.

3. The nerve repairing material (such as nerve conduit) of the invention can not only promote the growth cone of the neuron to break through scar tissue and be beneficial to nerve regeneration, but also has good mechanical property and enough supporting strength to guide the axial growth of nerve cells.

4. The inspiration or intelligence contribution of the invention firstly comes from the deep analysis of the reasons of nerve regeneration disorder, in particular to the deep understanding of the action mechanism of CSPGs, so that the RPTP state on the surface of nerve cells is changed; secondly, the accurate understanding of the structural composition, the component characteristics and the functions of various glycosaminoglycans (GAGs) and Proteoglycans (PG) in the acellular matrix (ECM), and finally, the accurate understanding of strong cationic natural polymers and the scientific positioning of active components represented by natural arginine polymers (represented by various protamine) are realized.

5. Protamine can bind to CSPGs tightly, reducing or eliminating the axon growth inhibitory activity of CSPGs, thereby allowing Oligodendrocyte Precursor Cells (OPCs) at nerve wounds (glial scars) to migrate to demyelinating sites and promote their differentiation into mature Oligodendrocytes (ODCs), thereby remyelinating nerve tissue; protamine can also inhibit the activity of other substances of axonal growth, such as Myelin Associated Glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp), Nogo protein (involution protein), growth cone collapse inhibitor membrane damaged glycan (IMP), thereby significantly improving repair and regeneration of nerve myelin; is beneficial to promoting the growth and proliferation of Schwann cells, guiding the regeneration of neuron axons and recovering the nerve function.

6. The invention needs to skillfully combine the frontier knowledge of the multidomain interdisciplinary disciplines of pharmacy (protamine), pathology (neuron regeneration, growth cone extension, sciatic nerve function index SFI), biochemistry (hyaluronic acid), tissue engineering (cell removal process), pig raising knowledge (piglets, slaughtered commercial pork pigs, adult pigs and boars), natural phytochemicals (such as triterpene saponin) and the like, is absolutely unobvious, and can not easily obtain the technical scheme of the invention through common knowledge and simple non-intellectual labor, and the invention has certain difficulty and height.

The terms described in the specification of the present invention

A first part: technical terms related to nervous tissue

1. Schwann cell (Schwanncell, SC): is a glial cell of the peripheral nervous system and plays a key role in axonal myelination, organization of peripheral neurons, maintenance of normal neuronal function, formation of synapses, and responses to nerve injury and neuropathic pain.

2. Wallerian Degeneration (WD), which is an antegrade degeneration of axons and myelin sheaths due to injury of upper motor neurons; in 1850, Waller first described that when the glossopharyngeal and sublingual nerves of frogs were severed, it was found that, in addition to the damaged nerve fibers themselves, the distal portions including axons and myelin sheaths were also denatured, cleaved, and phagocytosed. This phenomenon is widely present in the peripheral and central nervous systems; histology of the severed distal peripheral nerve alters Wallerian degeneration; central nervous system Wallerian degeneration can occur in the neurofibrillary tracts such as the corticospinal tract, and the corticocephalic tract, and common causes are cerebral infarction, cerebral hemorrhage, and tumor and demyelinating disease.

3. Glial cells (Glial cells) are a large group of cells in nervous tissues, except neurons, which also have processes, but no dendrites or axons, and are widely distributed in the central and peripheral nervous systems. In mammals, the ratio of glial cell number to neuronal cell number is about 10: 1; glial cells in the nervous system: there are mainly astrocytes, oligodendrocytes (which are collectively called macroglial cells) and microglia. Glial cells are traditionally considered connective tissues, which serve only to connect and support various neural components, and in fact, glia also serve to distribute nutrients, participate in repair and phagocytosis, and differ from normal connective tissues in morphology, chemical characteristics, and embryonic origin. The characteristics of histology are as follows: glial cells have complex and diverse structures and express abundant secretory products, which contain most of neurotransmitters, neuropeptides, hormones and neurotrophic factor receptors, ion channels, neuroactive amino acid affinity vectors, cell recognition molecules, and can secrete a variety of neuroactive substances (growth factors, neurotrophic factors, cytokines, etc.).

4. Oligodendrocyte (ODC), a type of glial cell, originates in the ectoderm, and has a morphology: the dark ovoid nucleus is surrounded by a thin, deep layer of cytoplasm, with the chromatin clustering, with a few radial branching processes, often in the form of beads. The cell body is smaller than the astrocyte, about 6-8 microns wide and round. Cytoplasmic organelles are immersed in a dark matrix filled with moderately dense colloidal particles.

5. Glial scar: after the nerve tissue is damaged, a large number of astrocytes rapidly enter an activation proliferation stage, and then, the astrocytes, macrophages and extracellular matrixes secreted by the astrocytes and the macrophages form a glial scar, wherein the glial scar mainly contains CSPGs; histologically, glial scars consist of astrocytes and connective tissue, and are the primary barriers to inhibit neuronal axonal regeneration. Glial scars are not merely physical and mechanical barriers to nerve regeneration, and many studies have now shown that chemical barriers formed by biochemical components in scars (e.g., CSPGs, etc.) should be the major barriers to the extension of nerve processes or growth cones, and to the regeneration of nerve tissue.

6. The Tyrosine Phosphatase Protein Receptor sigma (RPTP sigma) is a specific Receptor that affects the action of neurite outgrowth. RPTP σ is a member of the leukocyte antigen family, and is composed of immunoglobulin-like (Ig) and fibronectin iii (FN iii) repetitive fragments, and the like. The receptor is widely distributed in glands and nervous systems (highly expressed in hippocampus, cerebral cortex, olfactory bulb, retina and inferior ventricular tunica), and is involved in the development of nerves and the regeneration of nervous tissues: mainly manifested by inhibition of axonal growth and involvement in the formation of glial scars. RPTP σ has a conserved, positively charged extracellular domain, and studies have shown that Chondroitin Sulfate (CS), a negatively charged polysaccharide chain in CSPGs, is the binding site for RPTP σ.

A second part: introduction of active ingredients related to nerve repair materials.

1. Chondroitin Sulfate Proteoglycans (CSPGs)

The structural composition is a group of proteins which are covalently bonded with Chondroitin Sulfate (CS), and the proteins are composed of one or more linear CSs formed by covalently connecting Core Protein (CP) and glycosaminoglycan (GAGs) chains and widely distributed in various parts of organisms such as nervous tissues, connective tissues and the like. In this patent, unless otherwise specified, the CSPGs generally include CSPGs alone and or CS; the Core Protein (CP) is composed of a hyaluronic acid domain, an immunoglobulin-like domain, an endothelial cell growth factor-like domain, a CS chain-linked domain, a lectin-like domain and an intact regulatory protein domain, and is classified into agrececan family (including Aggrecan, neuroproteoglycan, short proteoglycan, etc.), NG2, phacan, etc. more than 30, such as Tenascon, agrecen, Versican, Brevican, Neurocan, Phosphacan and 2, etc., according to the different CSPGs of CP and GAGs chains. Studies have shown that several CSPGs are significantly up-regulated in glial scar formation after nervous system injury, such as phosphoproteoglycan, neuroproteoglycan, brevican, versican and NG2, which are closely related to the role of glial scar in inhibiting axonal regeneration. Studies have shown that CSPGs are a chemical barrier that hinders regeneration after nerve injury, the part of which that plays a major role is its CS chain, and is related to the location and extent of sulfation; by means of preventing synthesis of CSPGs, blocking the way of action of the CSPGs, degrading CS chains and the like, the nerve regeneration inhibition effect of the CSPGs can be eliminated, and nerve injury repair can be promoted.

2. Chondroitin Sulfate (CS): is a glycosaminoglycan (GAG) which is extracted from animal cartilage tissue and has a relative molecular mass (Mr) of 10-50 kDa. CS can be subdivided into different types, CS-A, CS-B (dermatan), CS-C, CS-D, CS-E, etc., depending on the position and amount of sulfation.

3. Anti-gliosis agents: refers to an agent capable of reducing gliosis; common, the first is anti-nogoA also known as endoplasmic reticulum protein 4(Reticulon4), neuroendocrine specific protein, neurite outgrowth inhibitors, NOGO, neurite outgrowth inhibitors 220; the second type is an enzyme preparation such as chondroitinase ABC (E.C. No. 4.2.2.4), β -D-xyloside (E.C. No. 217.897.1), type I collagenase (E.C. No. 232-582-9), mitomycin-C (CASNO)50-07-7), MMP-3-matrix metalloproteinase (E.C. No: 3.4.24, angiotensin converting enzyme (ACEa, e.c. No. 3.4.15.1); the third class is antibodies: such as anti-NogoA, anti-TGFP 1, 2 and 3 anti-NG-2 domains, others such as: decorin (Decorin) (e.g., human Decorin, e.g., Uniprot accession No. P07585, PAPN-. beta.aminopropionyl, mannose-6-phosphate (CASNO: 3672-15-9), oxidized recombinant human galectin-1, Copaxone (Copaxone) (glatiramer acetate), and tripeptide (ser-gly-gly).

4. Myelin-associated glycoprotein (MAG): is a minor glycoprotein component in the PNS and Central Nervous System (CNS).

5. A perineuronal network (PNN), a highly organized glycoprotein lattice structure, is a network of extracellular matrix that surrounds the cell bodies and proximal neurites of specific types of neurons; the main components of the composition are Chondroitin Sulfate Proteoglycan (CSPGs) and Heparan Sulfate Proteoglycan (HSPG).

6. Chondroitinase ABC: is an intracellular enzyme derived from various bacteriA, which catalyzes the action of 4-chondroitin sulfate (CS-A), 6-chondroitin sulfate (CS-C), dermatan sulfate (CS-B) and Hyaluronic Acid (HA) in glycosaminoglycan chains in extracellular matrix components, and the products are unsaturated disaccharides and oligosaccharides. The chondrosulphatase ABC carries out enzymolysis on glycosaminoglycan side chains of chondroitin sulphate proteoglycan, thereby eliminating the inhibition effect of CSPGs on the growth of nerve regeneration axons and indirectly promoting the regeneration and repair of nerve tissues. Topical application of chondrosulphatase ABC to nerve damage is considered to be one of the most promising approaches to promote regenerative repair of damaged nerves; however, under the action of various physical and chemical factors in vivo, the chondroitinase ABC is inactivated quickly and is difficult to carry out continuous enzymolysis on Chondroitin Sulfate Proteoglycan (CSPGs) at nerve injury positions.

7. Protamine (PTM): is an alkaline protein extracted and separated from the fish spermary; generally consists of about 30 to 50 amino acids, is rich in arginine, is alkaline, can be dissolved in water and dilute acid, is not easy to be dissolved in organic solvents such as ethanol, acetone and the like, has good stability, and is not solidified by heating. Protamine can be classified into 3 kinds of monoprotamine, biprotamine and tripprotamine according to the difference of the kinds and the amount of the basic amino acid components. Wherein the monoprotamine contains only one component arginine, such as salmon, herring and rainbow trout protamine; the bis-protamine contains arginine, histidine or lysine, such as carp protamine; the three protamine contains 3 basic amino acids, such as silver carp protamine and sturgeon protamine.

8. Heparin is a sulfate radical-rich linear chain biomacromolecule compound with negative charges, is a mucopolysaccharide with an abnormal and complex structure, and has the functions of anticoagulation, antithrombotic generation, smooth muscle cell proliferation resistance, inflammation resistance and the like. The precise structure of heparin is unknown and is believed to be composed of α -L-iduronic acid-2-sulfate, N-sulfo- α -D-glucosamine-6-sulfate, β -D-glucuronic acid and N-sulfo- α -D-glucosamine-6-sulfate joined by glycosidic linkages to form a "tetrasaccharide" as a building block from which the polysaccharide is polymerized.

And a third part: technical terms related to nerve repair materials

1) Tissue composite patches, tissue regeneration materials, biological membranes, biological repair membranes, biological scaffolds, degradable membranes, absorbable membranes, Bio-Mesh, Bio-Patch, bioscafold, ECM membranes, which are different in surface terminology but basically the same in purpose and use; the meaning of the terms above, unless otherwise specified, is intended to be equivalent in nature in the present invention.

2) Nerve repair materials, Nerve scaffolds, Nerve conduits, artificial Nerve materials and corresponding english terms such as Nerve Guide Conduit (NGC), are equivalent in meaning to these terms or terms in the present invention unless otherwise specified.

3) Extracellular matrix ecm (extracellular matrix): is a non-cellular component present in all tissues and organs, which not only provides necessary physical support for cellular tissues, but also provides a suitable place and microenvironment for the normal physiological activities of various cells; but also plays an important role in lever regulation on the aspects of morphogenesis of tissues, chemotaxis and differentiation of cells, important physiological biochemistry, biomechanics and the like, thereby influencing or regulating the functions of tissues and organs. 50% of the cell's function is determined by the external microenvironment created by the extracellular matrix.

4) Tissue-derived extracellular matrix (ECM) is obtained by treating allogeneic or xenogeneic tissues through cytotechnological processes, and its basic and application studies have become research hot in the research fields of tissue engineering and regenerative medicine. The extracellular matrix scaffold material without cell tissues is characterized in that on the basis of effectively removing cell components with immunogenicity in natural tissues, a naturally occurring internal three-dimensional scaffold structure and structural protein components (usually Collagen Collagen is taken as a main component and can contain elastin, Elastins and the like), special protein components (including Fibronectin (FN), fibronectin (fibronectin), Laminin (LN), fibrillin and the like) in the structure are retained to the maximum extent, various cell growth factors (such as fibroblast FGF, transforming growth factor TGF, vascular endothelial growth factor VEGF) are carried in the scaffold material, and a small amount of but very important epidermal growth factor EGF and insulin-like growth factor-1 (IGF-1) are possibly also contained in the scaffold material, and various beneficial components and growth factors such as glycosaminoglycan (GAGs), proteoglycan (including HSPG, CSPGs and the like) are also contained in the extracellular matrix, the unique internal structure and natural composition of these tissues is not perfectly reproducible and accurately modeled by synthetic materials.

5) Unless otherwise specified or explained in detail, the terms or terms as used herein are understood to be substantially the same or equivalent, and the main component is collagen (mainly, the content thereof is greater than 50%).

6) The nerve repair material is a sheet-shaped or tubular material used for temporarily or permanently repairing nerve tissue defects. The material includes non-absorbable synthetic materials such as polytetrafluoroethylene/polyurethanes, etc., synthetic biodegradable materials (such as absorbable synthetic materials such as polylactic acid/polycaprolactone), and natural biodegradable materials, including purified collagen materials, decellularized (animal-derived tissue) ECM materials, and further ECM materials including different sources such as allogenic, xenogenic animals, etc.

7) The collagen membrane products for nerve tissue repair are divided into three types according to components or processes: firstly, a decellularized matrix membrane (ECM membrane), a non-decellularized matrix membrane (non-ECM membrane), and a mixed membrane (in structural composition, both ECM and non-ECM membranes exist); non-ECM membranes can be divided into two types, one type is a purified collagen membrane (mainly containing type I collagen), which is mostly taken from connective tissues such as tendon, dermis, peritoneum, small intestine submucosa and the like of animals, pure type I and/or type III collagen is extracted by a specific treatment technology, and then the membrane with a certain structure is prepared by freeze drying and the like; another class is non-purified collagen membranes, which may be combined with other substances, including, but not limited to, degradable polymers or polymeric substances, such as polylactic acids, chitosan, etc., and prepared by other methods (e.g., electrospinning, etc.), and may use other additives (including cross-linking agents, modifiers, protective agents, antimicrobial agents, etc., to impart or enhance certain properties to the membrane material; typically, collagen is mainly type I collagen, type III collagen, or a mixture thereof; and collagen may also include a certain proportion of type II, type IV, type VI, or type VIII collagen, or any combination thereof, or any type of collagen.

8) Decellularized (ECM) material: including, but not limited to, fascia from Small Intestinal Submucosa (SIS), peritoneum, pericardium, amnion, dermis, ligament, tendon, SIS, diaphragm (diaphragm), omentum, muscle, or organ from cultured mammals such as pigs, cattle, horses, sheep, donkeys, camels, dogs, rabbits, etc.; biomaterials obtained by mature processes of degreasing, decellularization, DNA removal, removal of other antigens (such as galactosides) contain mainly type I collagen, while ECM from parts of the tissue (such as the dermis) may also contain less than 50% elastin.

9) Small intestine submucosa sis (smallintestinalsubmucosa), small intestine tissue including jejunum and ileum part, the rest part after removing small intestine mucosa layer, muscular layer and serosa layer, using collagen as main component, accounting for more than 80%.

10) Glycosaminoglycans gags (glycoaminoglycans): also called as mucopolysaccharide, is one of heteropolysaccharide, mainly exists in animal connective tissue, and is an important raw material participating in normal physiological activities of tissue, soft tissue and the like; glycosaminoglycans can be classified into 5 major classes, Hyaluronic Acid (HA), Chondroitin Sulfate (CS), Dermatan Sulfate (DS), Keratan Sulfate (KS), heparan sulfate and Heparin (HP), according to the type of monosaccharide residues, the linkages between residues and the number and position of sulfate groups. Heparin (Heparin) among glycosaminoglycans inhibits platelet aggregation and platelet growth factor release, inhibiting the excessive formation of thrombus and scar tissue.

11) Hyaluronic Acid (Hyaluronic Acid): also known as hyaluronic acid (Hyaluronan), also known as uronic acid, a natural linear polysaccharide, is a linear homoglycan formed by repeated alternating linkage of disaccharide units of glucuronic acid and N-acetylphthaleinglucosamine, is an anionic polymer, unlike other mucopolysaccharides, which does not contain sulfur. Hyaluronic acid shows various important physiological functions in a body by unique molecular structure and physicochemical properties, such as regulating permeability of a blood vessel wall, regulating protein, water electrolyte diffusion and running, promoting cell proliferation, promoting tissue regeneration and wound healing; hyaluronic acid is contained in all tissues and body fluids throughout the body and is a very important active ingredient in the extracellular matrix (ECM).

12) Acellular matrix actm (acellular tissue matrix) or acellular tissue matrix: the method is characterized in that specific reagents and treatment modes are adopted to fully remove or inactivate components which can generate immunological rejection reaction, such as cells, viruses, DNA and the like in animal organs or tissues, preserve the integrity of the original natural three-dimensional structure to the maximum extent, and preserve cell growth factors and active functional components in the original matrix to the greatest extent; the decellularized ECM has the characteristics of natural three-dimensional (3D) structure, containing bioactive factors, being capable of being degraded by a host receptor, being easy to induce the receptor stem cells to move (be easy) and differentiate and the like, is widely applied to clinic and is used for repairing and regenerating tissues (congenital defect and acquired wound); the acellular matrix is a novel tissue regeneration scaffold, has good biocompatibility, degradability and harmlessness, and the ECM has large plasticity in form.

13) Sciatic nerve function index (SFI) test: a wooden groove with the length of 60cm, the width of 10cm and the height of 10cm and two open ends is manufactured, 70g of white paper is cut into the same length and width as the wooden groove, and then the white paper is laid at the bottom of the groove. After coloring the double hind limbs of the rat by soaking the pigment in the double ankle joints, the rat is placed at one end of the groove and walks to the other side of the groove by itself, and 5-6 footprints are left on each hind limb. The footprints with clear footprints were selected to measure 3 indices of normal (N) and injured lateral (E) feet, respectively: A. PL (footprint length); B. TS (toe width); C. IT (medial toe width). Substituting the indexes into Bain formula to calculate sciatic nerve function index. Bain formula: 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, -100 is complete injury.

As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods, and/or steps, which are of the type described herein and/or which will be apparent to those skilled in the art upon reading this disclosure. The term "about" or "near" refers to a range of values in a statistical sense, and ranges can be within an order of magnitude, typically within 50%, further within 20%, still more typically within 10%, and even more typically within 5% of the specified value or range. The permissible variation encompassed by the term "about" or "approximately" depends on the particular system under study and can be readily recognized by one skilled in the art.

The term "effective amount" or "therapeutically effective amount" refers to an amount of an active agent sufficient to induce a desired biological result, e.g., to promote and/or restore neuronal regeneration and/or neurite outgrowth. The result can be a reduction in the signs, symptoms, or causes of the disease, or any other desired alteration of the biological system.

Drawings

Fig. 1 is a schematic view of a hollow tubular nerve conduit according to one embodiment.

Fig. 2 is a schematic diagram of a coiled sheet-like nerve conduit according to one embodiment.

The principles and aspects of the present invention will be further described with reference to specific embodiments; it is to be understood that these examples are for the purpose of illustration only and are not intended to limit the scope of the present invention; the examples are not intended to limit the scope of the invention in any way, and various processes and methods not described in detail in the following examples are conventional methods well known in the art.

Detailed Description

The first embodiment is as follows:

1. material taking and pretreatment: in a slaughterhouse, after commercial pork pigs (with the weight of about 100 kg) are slaughtered, taking small intestines of the commercial pork pigs, and removing a mucous layer, a muscular layer, a serosal layer and lymph nodes of the small intestines and jejunum by using a mechanical scraping method; leaving Small Intestinal Submucosa (SIS), soaking in 1.0% acetic acid solution at a ratio of SIS to acetic acid solution of 1:10 for 45 min, and soaking in purified water;

2. and (3) disinfection: soaking the mixture of SIS and 20% ethanol solution at a ratio of 1:10 at room temperature for 100 min under ultrasonic condition, sterilizing, and ultrasonic cleaning with purified water for 3 times;

3. degreasing: soaking SIS in 95% ethanol at a ratio of SIS to ethanol (W/W) of 1:10 under ultrasonic condition for 2 hr; then washing for 3 times by using deionized water;

4. and (3) cell removal: soaking SIS30 min in 0.8% saponin solution at 4 deg.C under ultrasonic condition; then washing the SIS by using a 0.5 percent saponin solution with the same concentration for 15 minutes; then soaking the SIS for 20 minutes by using a PBS-EDTA solution; the above-mentioned decellularization step can be repeated once;

5. DNA and α -galactoside removal: respectively treating the SIS with a DNA enzyme solution and an alpha-galactosidase solution, and flushing the SIS with a PBS solution in a flowing water mode for three times in the middle and later, wherein each time lasts for ten minutes;

6. SIS was placed in a freshly prepared 1.5% w/v protamine solution, acted (binding reaction) at 37 degrees for 30 minutes, followed by rinsing with PBS buffer; soaking for 30 minutes again, taking out, and rinsing with buffer solution;

7. shaping: stacking the SIS semi-finished product obtained in the step 6 in three layers, placing the three layers in a tubular or U-shaped mold, freezing and drying in vacuum, and finally cutting, sterilizing and packaging; the obtained final product has length of 1-5cm, diameter of 0.1-0.9cm, thickness of 0.1-1mm, and shape of nerve repairing material as shown in figure 1 and figure 2.

Example two: control preparation, decellularized SIS (not treated with protamine solution)

The steps are basically the same as the first embodiment, and the difference is that the sixth step is not operated.

Example three: acellular nerve repair material (culling sow SIS)

The steps are basically the same as the first embodiment, and the difference is only that the pigs are different in month age; the parts of the materials to be taken are the same; for example, commercial meat sows are only fed for 5-6 months, and for example, culled sows (the feeding months are more than 40 months old).

Example four: detection of mechanical properties of acellular nerve repair material

Detecting the nerve conduit samples prepared in examples 1, 2 and 3; the specific methods and results are given in the following table:

as can be seen from the comparison between the first and second examples in the above table, the tensile strength of the protamine-treated SIS is not affected, but the mechanical properties of the material are slightly enhanced, which is significantly improved compared with the prior art (the mechanical properties of the material are reduced). By comparing the first and third examples, it can be seen that with dam-derived SIS, the mechanical properties of the material are further improved.

Example five: detection of chondroitin sulfate content in acellular nerve repair material

After drying, the nerve conduit samples prepared in the examples 1, 2 and 3 are subjected to detection of bioactive factors; detecting the content of the chondroitin sulfate by adopting a commercial ELISA kit; the sample pretreatment method is to adopt a low-temperature grinding method for treatment, and the detection results are as follows:

from the above table, it can be seen that the residual chondroitin sulfate content in the SIS material after protamine treatment has been greatly reduced compared to that without protamine treatment. The treatment time is also greatly reduced compared to the method using enzyme treatment.

Example six: animal nerve defect simulation experiment

Experiments were performed using the artificial nerve conduits prepared in examples 1, 2, and 3; the number of experimental rats was 24. After anesthesia of the abdominal recumbent position, the right lower thigh was opened longitudinally, the lateral femoral muscle group was separated with a small forceps, the surgical microscope was adjusted, the sciatic nerve was separated with a surgical hook, the nerve 3.5 cm long was exposed, and a nerve defect model was created manually by cutting 2 cm. Selecting an artificial nerve conduit with the length of 3.5 centimeters, inserting a needle at the tail end of the conduit, penetrating the nerve at the far end of the nerve, inserting the needle from the inner end of the conduit, withdrawing the needle from the outer side of the conduit, sewing in a U shape, fixing the catheter and knotting; the sarcolemma and skin layers were then sutured together. Eight weeks after surgery, sciatic nerve function index (SFI) was measured for each test group, with the results as follows:

it can be seen from the above table that after protamine was used, sciatic nerve function index could more rapidly trend towards 0 (normal state) and the effect of the dams was more pronounced.

Numerous simple variations or adaptations or combinations will occur to those skilled in the art in light of the foregoing description; therefore, the invention is not to be limited to the details of the examples and the substitution of common general knowledge without departing from the spirit of the claims.

Claims (10)

1. The nerve repairing material is characterized in that the nerve repairing material is treated by a strong cationic natural polymer to achieve the purpose of combining chondroitin sulfate proteoglycan, and the strong cationic natural polymer is one of protamine, protamine derivatives, protamine structure mimics or the treatment of a composition of the protamine, the protamine derivatives and the protamine structure mimics.

2. The nerve repair material according to claim 1, wherein the treatment is one or a combination of soaking, smearing and spraying the nerve repair material with a solution containing 0.1-10% of protamine, protamine derivatives, protamine structure mimics or a combination thereof.

3. The nerve repair material according to claim 1, wherein the protamine is one or a mixture of protamine from salmon, herring, red trout and mammals, including monoprotamine, biprotamine and tripprotamine.

4. The nerve repairing material according to claim 1, wherein the treatment is soaking the acellular nerve repairing material in a solution containing 0.5-2% of protamine, wherein the soaking is performed for 30 minutes to 3 hours each time, then washing with a buffer solution for 10 to 50 minutes, and the soaking and washing steps can be repeated one to three times, and after the last soaking in the protamine solution, the buffer solution is not used for washing.

5. The nerve repairing material according to claim 1, wherein the nerve repairing material is mainly decellularized material, and has a hollow tubular shape or a sheet shape, a length of 1-5cm, a diameter of 0.1-1cm, and a thickness of 0.1-1 mm.

6. The decellularized material of claim 5, wherein the decellularized material is a decellularized mammalian tissue, the mammal is a pig, a cow, a sheep, a horse, a donkey, a camel, a rabbit, and the tissue is one of peritoneum, dermis, gastric membrane, small intestine submucosa, periosteum, pericardium, fascia, nerve, blood vessel, or a combination thereof.

7. The decellularized material of claim 5, wherein said decellularized material is one or a combination of decellularized porcine small intestine submucosa, sciatic nerve and femoral artery blood vessel; the pigs are adult pigs with the age of more than 10 months.

8. The decellularized material of claim 5, wherein the decellularization is carried out by using one of a physical method, a chemical method, a biological method or a combination thereof, the physical method is an osmotic method, the chemical method is a surfactant treatment, and the biological method is a protease digestion; the protease is trypsin.

9. The acellular nerve repair material according to claim 5, wherein the acellular nerve repair material is an acellular mammalian tissue, and the acellular method is to use one of SDS, Triton X-100, Triton X-200, sodium deoxycholate, plant-derived pentacyclic triterpene saponin, steroid saponin or a combination thereof as an acellular surfactant at an effective working concentration of 0.05-3% by weight.

10. The material for repairing a denervated nerve according to claim 5, wherein said material for repairing a denervated nerve comprises a degradable material, and said degradable material is one of a synthetic polymer, a natural polysaccharide polymer, a collagen material, or a composite thereof; the degradable material can be fixedly combined with the decellularized material by one or a combination of electrostatic spinning, adhesion and suturing.

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