TWI458827B - Method for preparing nerve graft - Google Patents

Description

Method for preparing nerve graft

The invention relates to a preparation method of a nerve graft, in particular to a preparation method of a nerve graft for transplantation of an organism.

The nervous system consists mainly of neurons and neuron glial cells, which form a complex and specific communication domain for linking with other tissues or organs for functional coordination. In the nervous system, neurons receive neurons to receive stimuli, conduct and output neurotransmitters for tissue or organ communication, and glial cells perform neuronal physical support, nutrient supply, and regulatory communication. Features such as message speed. Each neuron consists of a cell body and a neurite depending on the type. The neurites extend from the cell body and grow toward other neurons or other cells (eg, muscle cells), where the neurites Divided into axon (axon) and dendrites (dendrite). In general, the stimulus is received by the dendrites and transmits impulses to the cell body, impulses are transmitted through the axons to the ends of the axons, and conductive material is released to trigger other cells.

Since the nervous system plays a coordinating role between tissues and organs in the living body, its importance is self-evident. Previously, neurological deficits due to impaired nervous system were clinically common disabling diseases. Among them, the nerve system is implanted to repair the damaged nervous system, and the neurosurgery is used to repair the nervous system damage caused by various conditions. An important means of injury. Previous nerve grafts are typically "branched" neural tubes at the ends of the damaged portion of the nervous system, which are tubular structures made of biodegradable materials. Neurons at one end of the damaged portion of the nervous system grow neurites along the inner wall of the neural tube to reach the other end of the damaged portion of the nervous system.

In general, the length of the damaged site that needs to be repaired by implanting the neural tube is long, and the growth process of the neurite is very slow, so the neural tube is used to repair the damaged nervous system. The repair time required is longer.

In view of this, it is necessary to provide a preparation method of a nerve graft, and the nerve graft prepared by the preparation method can reduce the repair time of the damaged nervous system.

A method for preparing a nerve graft, comprising the steps of: providing a cultivating layer comprising a carbon nanotube membrane structure and a protein layer disposed on a surface of the carbon nanotube membrane structure; Planting a plurality of nerve cells on the surface; and cultivating the plurality of nerve cells until the plurality of nerve cells grow a plurality of neurites and the neurites between the plurality of nerve cells are connected to each other to form a neural network.

Compared with the prior art, the preparation method of the nerve graft forms a growth layer by providing a protein layer on the surface of the carbon nanotube membrane structure, and forms the neural network on the surface of the cultivation layer. The carbon nanotube membrane structure has the advantages of good elasticity, good ductility and low density, so the nerve graft can be cut, stretched and implanted according to the shape and size of the damaged part of the damaged nervous system. Damaged part. The neural network has biological activity and signal transmission capabilities such that the neural graft including the neural network also has biological activity and signal transmission capabilities. When the nerve graft is implanted in a damaged part of the organism, due to neurons in the nerve implant The distance from the neurons at both ends or edges of the damaged portion is relatively close, so that the damaged portion can be made by directly suturing the neurons in the nerve implant and the neurons at the edge of the damaged portion. The two ends establish a signal transmission capability to complete the nerve repair of the damaged part, thereby saving the growth time of the neurite and reducing the repair time of the damaged nervous system.

100‧‧‧Neural graft

10‧‧‧cultivation

12‧‧‧Nano Carbon Membrane Structure

14‧‧‧protein layer

20‧‧‧Neural Network

22‧‧‧ nerve cells

24‧‧‧Nervous

FIG. 1 is a schematic flow chart of a method for preparing a nerve graft according to an embodiment of the present invention.

Figure 2 is a scanning electron micrograph of a carbon nanotube film.

Figure 3 is a scanning electron micrograph of a carbon nanotube rolled film.

Figure 4 is a scanning electron micrograph of a carbon nanotube film.

FIG. 5 is a side view of a nerve graft according to an embodiment of the present invention.

FIG. 6 is a schematic top view of a nerve graft according to an embodiment of the present invention.

FIG. 7 is a scanning electron micrograph of a carbon nanotube film structure provided by an embodiment of the present invention.

FIG. 8 is a transmission electron micrograph of a carbon nanotube film structure provided by an embodiment of the present invention.

Figure 9 is a transmission electron micrograph of a cultivating layer provided in an embodiment of the present invention.

FIG. 10 is a scanning electron micrograph of a neural cell implanted on the growth layer differentiated into a plurality of neurites according to an embodiment of the present invention.

Figure 11 is a scanning electron micrograph of an unstained nerve graft provided in an embodiment of the present invention.

Figure 12 is a scanning electron micrograph of a nerve graft after staining according to an embodiment of the present invention.

Referring to FIG. 1 , the present invention provides a method for preparing a nerve graft, comprising: S10, providing a cultivating layer, wherein the cultivating layer comprises a carbon nanotube membrane structure and disposed on a surface of the carbon nanotube membrane structure. a protein layer; S20, implanting a plurality of nerve cells on the surface of the protein layer; and S30, cultivating the plurality of nerve cells until the plurality of nerve cells grow a plurality of neurites and the neurites between the plurality of nerve cells are connected to each other to form a neural network road.

In the step S10, the carbon nanotube film structure is composed of a plurality of carbon nanotubes. The plurality of carbon nanotubes may extend substantially parallel to a surface of the carbon nanotube membrane structure. Preferably, the plurality of carbon nanotubes are connected by a Van der Waals attractive force to form a self-supporting structure. The so-called "self-supporting" means that the carbon nanotube film structure can maintain its own specific shape without being disposed on a surface of a substrate. Since a large number of carbon nanotubes in the self-supporting carbon nanotube membrane structure are attracted to each other by van der Waals force, the carbon nanotube membrane structure has a specific shape to form a self-supporting structure. When the carbon nanotube membrane structure is a self-supporting structure, the carbon nanotube membrane structure may be a membrane-like structure formed by at least one carbon nanotube membrane, and when the carbon nanotube membrane structure comprises a plurality of nanometer membrane structures In the case of the carbon nanotube film, the plurality of carbon nanotube films are stacked, and the adjacent carbon nanotube films are combined by van der Waals force. Since the carbon nanotube film is basically composed of a carbon nanotube and a carbon nanotube The carbon nanotube film structure has the advantages of good elasticity, good ductility and low density, and is convenient for cutting and stretching.

Referring to FIG. 2, the carbon nanotube film may be a carbon nanotube flocculation membrane, and the carbon nanotube membrane is a self-supporting nanometer obtained by flocculation of a carbon nanotube raw material. Carbon tube membrane. The carbon nanotube flocculation membrane comprises carbon nanotubes which are intertwined and uniformly distributed. The carbon nanotubes have a length greater than 10 microns, preferably from 200 microns to 900 microns, such that the carbon nanotubes are intertwined with each other. The carbon nanotubes are attracted to each other by van der Waals forces to form a network structure. Since the large number of carbon nanotubes in the self-supporting carbon nanotube flocculation membrane are attracted to each other and entangled by van der Waals force, the carbon nanotube flocculation membrane has a specific shape to form a self-supporting structure. . The carbon nanotube flocculation membrane is isotropic. The carbon nanotubes in the carbon nanotube flocculation membrane are uniformly distributed and randomly arranged to form a plurality of gaps or micropores having a size ranging from 1 nm to 500 nm. The gap or micropores can increase the specific surface area of the carbon nanotube membrane and infiltrate more protein.

The carbon nanotube film may be a carbon nanotube rolled film, which is a self-supporting carbon nanotube film obtained by rolling a carbon nanotube array. The carbon nanotube rolled film comprises uniformly distributed carbon nanotubes, and the carbon nanotubes are arranged in the same direction or in different directions. The carbon nanotubes in the carbon nanotube rolled film partially overlap each other and are attracted to each other by van der Waals force, and the carbon nanotube film has good flexibility and can be flexibly folded. In any shape without breaking. Moreover, since the carbon nanotubes in the carbon nanotube rolled film are attracted to each other by the van der Waals force, the carbon nanotube film is a self-supporting structure. The surface shape of the carbon nanotubes in the carbon nanotube rolled film and the growth substrate forming the carbon nanotube array Forming an angle β, wherein β is greater than or equal to 0 degrees and less than or equal to 15 degrees, the angle β is related to the pressure applied to the carbon nanotube array, and the larger the pressure, the smaller the angle, preferably, the carbon nanotube The carbon nanotubes in the rolled film are aligned parallel to the growth substrate. The carbon nanotube rolled film is obtained by rolling a carbon nanotube array, and the carbon nanotubes in the carbon nanotube rolled film have different arrangement forms according to different rolling methods. Specifically, the carbon nanotubes can be arranged in disorder; referring to FIG. 3, when rolling in different directions, the carbon nanotubes are arranged in different orientations; when crushed in the same direction, the carbon nanotubes are along a The orientation is preferred and the orientation is preferred. The length of the carbon nanotubes in the carbon nanotube rolled film is greater than 50 microns.

The area of the carbon nanotube rolled film is substantially the same as the size of the carbon nanotube array. The thickness of the carbon nanotube film is related to the height of the carbon nanotube array and the pressure of the rolling, and may be between 0.5 nm and 100 μm. It can be understood that the larger the height of the carbon nanotube array and the smaller the applied pressure, the larger the thickness of the prepared carbon nanotube rolled film; on the contrary, the smaller the height of the carbon nanotube array, the more the applied pressure Large, the smaller the thickness of the prepared carbon nanotube rolled film. There is a gap between adjacent carbon nanotubes in the carbon nanotube film, thereby forming a gap between 1 nm and 500 nm in the carbon nanotube film. Or micropores. The gap or micropores can increase the specific surface area of the carbon nanotube membrane and infiltrate more protein.

The carbon nanotube film may be a carbon nanotube film, and the carbon nanotube film is a self-supporting structure composed of a plurality of carbon nanotubes. Referring to FIG. 4, the plurality of carbon nanotubes are arranged in a preferred orientation along the length direction of the carbon nanotube film. The preferred orientation means that the overall extension direction of most of the carbon nanotubes in the carbon nanotube film is substantially in the same direction. Moreover, the overall extension direction of the majority of the carbon nanotubes is substantially parallel to the nai The surface of the carbon tube is pulled.

Further, most of the carbon nanotubes in the carbon nanotube film are connected end to end by van der Waals force. Specifically, each of the carbon nanotubes of the majority of the carbon nanotubes extending in the same direction in the carbon nanotube film is connected end to end with the carbon nanotubes adjacent in the extending direction by van der Waals force . Of course, there are a few carbon nanotubes in the carbon nanotube film that deviate from the extending direction. These carbon nanotubes do not constitute an obvious alignment of the majority of the carbon nanotubes in the carbon nanotube film. influences. The self-supporting carbon nanotube film does not require a large-area carrier support, but only provides support force on both sides, and can be suspended as a whole to maintain its own film state, that is, the carbon nanotube film is placed (or When fixed on two supports arranged at a certain distance, the carbon nanotube film located between the two supports can be suspended to maintain its own film state. The self-supporting is mainly achieved by the presence of continuous carbon nanotubes extending through the end-to-end extension of the van der Waals force in the carbon nanotube film. Specifically, the plurality of carbon nanotubes extending substantially in the same direction in the carbon nanotube film are not absolutely linear and may be appropriately bent; or are not completely aligned in the extending direction, and may be appropriately deviated from the extending direction. . Therefore, it is not possible to exclude partial contact between the carbon nanotubes juxtaposed in the majority of the carbon nanotubes extending substantially in the same direction. Specifically, the carbon nanotube film comprises a plurality of continuous and aligned carbon nanotube segments. The plurality of carbon nanotube segments are connected end to end by van der Waals force. Each carbon nanotube segment consists of a plurality of carbon nanotubes that are parallel to each other. The carbon nanotube segments have any length, thickness, uniformity, and shape. The carbon nanotube film has good light transmittance and the visible light transmittance can reach more than 75%.

When the carbon nanotube membrane structure comprises a plurality of carbon nanotube membranes, the plurality of nanocarbons The tube-pull film is laminated to form a layered structure. The thickness of the layered structure is not limited, and the adjacent carbon nanotube film is bonded by van der Waals force. Preferably, the layered structure comprises a carbon nanotube film having a layer number of less than or equal to 10 layers, so that the number of carbon nanotubes per unit area is small, and the Raman light intensity of the carbon nanotube itself is made strong. It is kept in a small range, thereby reducing the Raman peak intensity of the carbon nanotubes in the Raman spectrum. The carbon nanotubes in the adjacent carbon nanotube film in the layered structure have an intersection angle α between the α and the α is greater than 0 degrees and less than or equal to 90 degrees. When the carbon nanotubes in the adjacent carbon nanotube film have an intersection angle α, the carbon nanotubes in the composite carbon nanotube film are intertwined to form a nerve graft. The mechanical properties of the carbon nanotube membrane structure are increased. In this embodiment, the carbon nanotube film structure comprises a plurality of layers of carbon nanotube film laminated, and the intersection angle α between the carbon nanotubes in the adjacent carbon nanotube film is substantially equal to 90 degrees. That is, the extending directions of the carbon nanotubes in the adjacent carbon nanotube film are substantially parallel.

The protein layer is disposed on one surface or two opposite surfaces of the carbon nanotube membrane structure for imparting hydrophilicity and biocompatibility to the carbon nanotube layer, thereby enabling the growth layer to Provide a suitable environment for the cultivation and growth of the nerve cells. Preferably, the protein in the protein layer is a soluble protein. The protein is selected from the group consisting of a fibrous protein, a plasma protein, an enzyme protein, and the like. In this embodiment, the protein is a mammalian serum such as porcine serum, bovine serum or human serum. The serum not only provides a suitable environment for the growth and growth of the nerve cells, but also provides a growth factor for the nerve cells as they grow.

The preparation method of the cultivation layer is not limited, and only the protein layer and the carbon nanotube can be made The membrane structure can be mixed together. For example, the protein solution may be infiltrated into the carbon nanotube membrane structure by immersing the carbon nanotube membrane structure in a protein solution, thereby allowing proteins in the protein solution to adhere to the nanometer. One surface or two oppositely disposed surfaces of the carbon tube membrane structure form a protein layer. The protein solution may also be sprayed on one surface or two oppositely disposed surfaces of the carbon nanotube membrane structure such that the protein layer is disposed on one surface or two oppositely disposed surfaces of the carbon nanotube membrane structure. The protein solution may also be dropped on one surface or two oppositely disposed surfaces of the carbon nanotube membrane structure, and the protein layer may be disposed on one surface or two opposite sides of the carbon nanotube membrane structure by means of a ruthenium membrane. Set the surface. Generally, when the thickness of the carbon nanotube film structure is large, the protein can be controlled to be disposed only on one surface of the carbon nanotube film structure. Whereas when the thickness of the carbon nanotube membrane structure is small, the protein is usually disposed on opposite surfaces of the carbon nanotube membrane structure. The protein solution may include, in addition to the protein, a biological medium that dissolves the protein, and the type of the biological medium is not limited and may be modulated depending on the type of the protein. Usually, the concentration of the protein in the protein solution is 50% or more and 100% or less. In this embodiment, the protein concentration in the protein solution is 100%, that is, the protein solution is a pure protein and does not require solvent dissolution.

Since the carbon nanotube membrane structure comprises a plurality of carbon nanotubes and a gap exists between the plurality of carbon nanotubes to form a plurality of micropores, the protein solution is infiltrated when the protein solution infiltrates the carbon nanotube membrane structure The surface of the plurality of carbon nanotubes can be infiltrated into the inner surface of the carbon nanotube membrane structure. Of course, in the cultivating layer, not all the surface of the carbon nanotubes can be infiltrated with a protein layer, but only need to be located in the nerve A part of the surface of the carbon nanotube membrane structure of the cell is infiltrated with a protein solution to form a protein layer on the surface of the carbon nanotube membrane structure, so that the carbon nanotube membrane structure is hydrophilic and Biocompatibility enables the cultivating layer to function as a carrier for the growth of nerve cells. This is because, because of the hydrophobicity of the carbon nanotubes, the structure of the carbon nanotube membrane composed of carbon nanotubes does not provide a suitable hydrophilic environment for nerve cell growth. When the surface of the carbon nanotube is covered with a hydrophilic and non-toxic protein layer, the structure composed of the carbon nanotubes covered with the protein can provide a suitable hydrophilic environment for cell growth. In this embodiment, the method for preparing the incubation layer may further include the following steps: S11, providing the carbon nanotube membrane structure; S12, infiltrating the carbon nanotube membrane structure with a protein solution; and S13, The culture layer is formed by sterilizing a carbon nanotube membrane structure infiltrated with a protein solution.

In step S12, the manner in which the carbon nanotube membrane structure is impregnated with the protein solution is not limited, and only the protein in the protein solution adheres to the surface of the carbon nanotube membrane structure to form a protein layer. For example, infiltration of the protein solution can be achieved by immersing the carbon nanotube membrane structure in the protein solution. Infiltration of the protein solution can also be achieved by spraying the protein solution on the carbon nanotube membrane structure. In this embodiment, in order to achieve infiltration of the protein solution, the carbon nanotube membrane structure is selected to be immersed in pure protein. The soaking time of the carbon nanotube membrane structure depends on the specific structure of the nanotube membrane structure and the specific composition of the protein solution, and can only make most of the carbon nanotubes in the carbon nanotube membrane structure. Infiltrate with a protein solution. Typically, the carbon nanotube membrane structure has a soaking time of more than 2 hours. The environment in which the carbon nanotube membrane structure is immersed is not limited, and only the protein is not deteriorated. Generally, the soaking process can be carried out under normal temperature and normal pressure environments.

In step S12, since the carbon nanotube membrane structure is a self-supporting structure, the carbon nanotube membrane structure is directly immersed in the protein solution. Of course, in order to reduce the probability of damage caused by the surface tension of the liquid when the carbon nanotube film structure is immersed in the protein solution, the step S12 may further include the following steps: S121, the carbon nanotube film The structure is pre-set on a hydrophobic substrate surface. In order to bind the carbon nanotube film structure to the hydrophobic substrate, the carbon nanotube film structure disposed on the surface of the hydrophobic substrate may be subjected to an organic solvent treatment. The hydrophobic substrate must also be non-toxic to the organism so as not to disrupt the activity of the protein. Preferably, the hydrophobic substrate may also have good ductility, such as a silicone substrate.

In step S13, the manner in which the carbon nanotube membrane structure infiltrated with the protein solution is sterilized is not limited, and only most of the bacteria in the protein solution can be killed. For example, the protein solution can be sterilized by high temperature sterilization or ultraviolet light sterilization. In this embodiment, the protein solution is sterilized by high temperature sterilization. Of course, in order to prevent the protein in the protein solution from being destroyed, the temperature at the time of high temperature sterilization must not exceed 220 degrees. In the present embodiment, the temperature at the time of high temperature sterilization is approximately 120 degrees. It can be understood that this step S13 can be omitted when there are few bacteria in the protein solution. When the carbon nanotube membrane structure infiltrated with the protein solution is sterilized in the step S13, the solvent or moisture in the protein solution is reduced. Typically, the protein infiltrated in the structure of the carbon nanotube membrane The qualitative solution solidifies as the solvent or moisture decreases, thereby forming the protein layer on the surface of the carbon nanotube film structure.

In step S10, in order to increase the adhesion of the cultivation layer to the nerve cells and provide a growth environment more suitable for the nerve cells, after the protein layer is formed, the step S10 may further include the following steps: S14, in the cultivation layer. A poly-D-lysine (PDL) layer is formed on the surface of the protein layer. Specifically, the incubation layer may be immersed in a polylysine solution having a polylysine concentration of approximately 20 micrograms per milliliter.

In step S20, the nerve cells include nerve cells of a mammal, and preferably, the nerve cells are hippocampal neurons. The method of implanting a plurality of nerve cells on the surface of the layer is not limited, and a solution containing the nerve cells may be sprayed or coated on the surface of the layer, or the layer may be immersed in the nerve cell containing nerve cells. In the above, only the nerve cell fluid may be covered by the culture layer. In order for the nerve cell fluid to cover the incubation layer, the nerve cell fluid may be contained in a culture dish. The incubation layer can be suspended in the culture dish. It may also be disposed on a bottom surface of the culture dish, and only the culture liquid may cover the nerve cells. When the incubation layer is disposed on the bottom surface of the culture dish, a hydrophobic substrate must be disposed between the cultivation layer and the bottom surface of the culture dish to prevent the nerve cells from growing on the bottom surface of the culture dish.

In step S30, the culture environment of the nerve cells is not limited, and only neurites can be grown. Generally, the nerve cells can grow in a normal temperature and a normal pressure environment. That is, by placing the nerve cells in an indoor environment, the nerve cells can grow, and proteins in the culture layer such as bovine serum can provide a growth factor and promote the nerves. Cell growth. Of course, the incubation environment may also be close to the in vivo growth environment of the organism providing the nerve cells. For example, when the nerve cell is a hippocampal nerve cell taken from a mouse, the growth environment in the mouse can be simulated.

The nerve cells are capable of growing a plurality of neurites when incubated. The neurites include dendrites and axons (Axon). When there is only one nerve cell on the surface of the growth layer, the nerve cells of the nerve cell randomly grow in various directions along the surface of the growth layer. However, since the nerve cells themselves release a factor that induces directional growth of the neurites, when the plurality of nerve cells are disposed on the surface of the growth layer, the nerve cells of the nerve cells have a tendency to grow along adjacent nerve cells. Thereby the adjacent nerve cells can be connected and communicated. Therefore, by controlling the distribution of nerve cells in the growth layer, the growth direction of the neurites can be controlled. For example, if the neural cell line is randomly and evenly distributed on the surface of the incubation layer, the nerve cells will each grow a neurite out to connect with adjacent cells, when all or most of the nerve cells of the plurality of nerve cells are When a neurite is connected between adjacent nerve cells, the plurality of nerve cells form the neural network by the neurites, so that the plurality of nerve cells can communicate with each other. If the neurites of adjacent nerve cells meet, they will merge into the same neurite. For example, when the nerve cells are arranged in a line or array on the surface of the layer, and the nerve cells in the longitudinal direction are close to each other, and the nerve cells in the lateral direction are far apart, at this time, The nerve cells grown by the nerve cells may extend substantially in the longitudinal direction. In order to enable the nerve cells to be arranged in a line or array on the surface of the incubation layer, the carbon nanotubes in the carbon nanotube film may be selected to extend substantially in the same direction. Through cultivation, most of the nerve cells adjacent to each other pass through The neurites establish a connection to form the neural network. The neural network forms the nerve graft together with the incubation layer.

The carbon nanotube membrane structure is a macroscopic membrane-like structure, and the area thereof can generally reach 15 mm×15 mm or more. Specifically, the length of the carbon nanotube membrane structure can reach 300 m or more and the width can reach More than 0.5 meters. The carbon nanotube membrane structure has the advantages of good elasticity, good ductility, no metal and low density, and can be directly implanted into the living body. Therefore, the nerve graft body whose main carrier is the carbon nanotube membrane structure can be cut, stretched, and implanted into the damaged portion according to the shape and size of the damaged portion of the damaged nervous system. The neural network has biological activity and signal transmission capability, so that the nerve graft including the neural network also has biological activity and signal transmission capability. When the nerve graft is implanted into a damaged part of the living body, since the distance between the neuron in the nerve implant and the neurons at both ends or edges of the damaged part is short, it can be directly The manner in which the neurons in the nerve implant and the neurons at the edge of the damaged site are sutured establishes a signal transmission capability at both ends of the damaged portion, and nerve repair of the damaged portion is completed, thereby saving the neurite The growth time reduces the repair time of the damaged nervous system. It can be understood that even when the nerve implant is implanted into the damaged site, no direct suturing is performed, because the distance of the neurons at the edge of the damaged portion of the neuron in the nerve implant is smaller than the received The distance between the neurons at both ends of the lesion is reduced. Therefore, by implanting the nerve implant, the growth time of the neurites can also be reduced, thereby reducing the repair time of the damaged nervous system.

It is to be noted that, in general, the carbon nanotubes in the structure of the carbon nanotube membrane refer to a carbon nanotube that has not been subjected to chemical or physical treatment, such as a carbon nanotube that has not been subjected to surface hydrophilic treatment, That is, the carbon nanotubes are pure carbon nanotubes. Of course, nano carbon If the carbon nanotubes in the membrane structure are modified carbon nanotubes, they are not only toxic to nerve cells, and should be within the scope of the present invention, only the carbon nanotubes. Modification does not have any substantial contribution to the achievement of the present invention because, after the protein layer covers the carbon nanotube, the nerve cells are not in direct contact with the carbon nanotubes, the nanocarbon The surface structure of the tube is in fact negligible.

The nerve graft provided by the present invention may include a product obtained by culturing a neural network formed by a plurality of nerve cells and neurites on the surface of the growth layer including the carbon nanotube membrane structure and the protein layer by the above-described method for preparing a nerve graft.

Referring to FIG. 5, the nerve graft 100 includes a seed layer 10 and a neural network 20 distributed on the surface of the layer 10.

The incubation layer 10 includes a carbon nanotube membrane structure 12 and a protein layer 14 disposed on one surface or opposite surfaces of the carbon nanotube membrane structure 12. In the present embodiment, the protein layer 14 is disposed only on one surface of the carbon nanotube film structure 12.

The carbon nanotube membrane structure 12 includes a plurality of carbon nanotubes substantially parallel to the surface of the carbon nanotube membrane structure, and adjacent carbon nanotubes are connected to each other by van der Waals to form a self-supporting structure. The carbon nanotube film structure 12 includes at least one carbon nanotube film, which may be a carbon nanotube film as shown in FIG. 2, and a carbon nanotube film in FIG. And the carbon nanotube film in Figure 4. In the present embodiment, the carbon nanotube film structure 12 includes a plurality of laminated films disposed in a plurality of layers, and the adjacent drawn films are bonded to each other by a vantage force. In the adjacent drawn film, the extending direction of the carbon nanotubes may have an intersection angle, and preferably, the crossing angle is 90 degrees. The carbon nanotube film junction The thickness of the structure 12 can be set according to specific needs. Typically, the carbon nanotube membrane structure 12 has a thickness greater than 0.3 microns and less than 60 microns. In this embodiment, the carbon nanotube film structure 12 has a thickness of approximately 0.6 microns.

The protein layer 14 is composed of a soluble protein. The so-called soluble protein, that is, the protein has good hydrophilicity. The thickness of the protein layer 14 is not limited, and only a hydrophilic environment can be provided. Typically, the protein layer 14 has a thickness of from 0.3 microns to 2 microns. In this embodiment, the protein layer 14 has a thickness of approximately 0.5 microns. Macroscopically, the protein layer 14 may optionally be disposed on only one surface or opposite surfaces of the carbon nanotube membrane structure 12. Microscopically, the protein in the protein layer 14 easily penetrates into the interior of the carbon nanotube membrane structure 12 and coats some or all of the carbon nanotubes in the carbon nanotube membrane structure 12, There is no distinct interface between the protein layer 14 and the carbon nanotube membrane structure 12. Generally, when the thickness of the carbon nanotube film structure 12 is thin, for example, when the thickness of the carbon nanotube film structure 12 is less than or equal to 3 μm, the protein in the protein layer 14 easily penetrates into the The interior of the carbon nanotube membrane structure 12 substantially covers all of the carbon nanotubes in the carbon nanotube membrane structure 12. When the thickness of the carbon nanotube film structure 12 is relatively thick, for example, when the thickness of the carbon nanotube film structure 12 is greater than or equal to 3 micrometers, the protein in the protein layer 14 can penetrate into the tissue. The inside of the carbon nanotube membrane structure 12 is generally only coated with the carbon nanotube membrane structure 12 near the carbon nanotubes of the neural network 20. In this embodiment, the protein in the protein layer 14 substantially coats all of the carbon nanotubes in the carbon nanotube membrane structure 12.

The neural network 20 is disposed on the protein layer 14 away from the carbon nanotube membrane structure A surface of 12. When the nerve graft 100 includes only one protein 14 disposed on one surface of the carbon nanotube membrane structure 12, the nerve graft 100 includes only one neural network 20. When the protein layer 14 includes two protein layers 14 respectively disposed on opposite surfaces of the carbon nanotube membrane structure 12, the nerve graft 100 may include two neural networks 20 respectively disposed therein. The two protein layers 14 are remote from the surface of the carbon nanotube membrane structure 12, and may also include only one neural network 20 disposed on the surface of one of the protein layers 14. In the present embodiment, the nerve graft 100 includes only one neural network 20.

It can be understood that, in order to improve the bacteriostasis of the biological graft 100 and increase the life of the nerve graft 100, the nerve graft 100 may further include a poly-lysine layer disposed on the neural network 20 Between the protein layer 14 and the protein layer 14.

Referring to FIG. 6, the neural network 20 includes a plurality of neural cells 22 and a plurality of neurites 24 extending from the plurality of nerve cells 22. The number of neurites 24 extending from each of the nerve cells 22 is not limited, and only the biological connection between the plurality of nerve cells 22 can be established to enable the plurality of nerve cells 22 to communicate with each other. For example, one of the nerve cells 22 may extend out of the plurality of neurites 24 or may not extend out of any of the neurites 24.

The nerve graft 100 of the present invention has a neural network 20 in the nervous system in the repairing organism disposed on the surface of the incubation layer 10. The carbon nanotube membrane structure 12 in the incubation layer 10 is a self-supporting structure consisting essentially of carbon nanotubes, and has the advantages of no metal, good elasticity, non-corrosion, good ductility and low density. Therefore, the carbon nanotube membrane structure 12 can be implanted into the organism along with the plurality of nerve cells 12 connected by the plurality of neurites 14 for repairing the damaged nervous system in the organism, and can be rooted The nerve graft 100 is cut or stretched according to the wound area of the nervous system in the living body.

Hereinafter, a method for preparing a nerve graft of the present invention and a nerve graft will be described in detail with reference to the accompanying drawings and specific embodiments.

The invention provides a preparation method of a nerve graft, which comprises the following steps:

S210, immersing a carbon nanotube membrane structure in a protein solution.

In step S210, the carbon nanotube film structure comprises a plurality of layers of carbon nanotube film, and the direction of extension of the carbon nanotubes between the adjacent carbon nanotube films has an intersection angle. Referring to Figures 7 and 8, preferably, the angle of intersection is substantially equal to 90 degrees. The protein solution is pure bovine serum solution. Referring to FIG. 9, when the carbon nanotube membrane structure is immersed from the protein solution for about 1.5 hours, most of the surface of the carbon nanotube membrane may be infiltrated with a protein solution.

S220, taking out the carbon nanotube membrane structure from the protein solution, and performing high temperature sterilization treatment at 120 degrees Celsius.

In step S220, after the carbon nanotube membrane structure is taken out from the protein solution, it can be subjected to heat sterilization treatment in a dry box. The sterilization temperature of the drying oven is approximately 120 degrees. After the sterilization treatment, the protein in the protein solution is substantially solidified, and a protein layer is formed on the surface of the carbon nanotube membrane structure to form a seed layer.

S230, immersing in the poly-lysine solution in the incubation layer.

In step S230, the concentration of polylysine in the polylysine solution is approximately 50%. By soaking, the surface of the layer is adhered with polylysine and provides an aqueous environment.

S240, adding a nerve cell liquid to the soaked culture layer until the nerve cell liquid covers the cultivation layer.

In step S240, the incubation layer may be suspended in a petri dish or placed on a silicone substrate.

S250, cultivating the plurality of nerve cells attached to the culturing layer, causing the plurality of nerve cells to grow a plurality of neurites connected between the plurality of nerve cells, thereby forming a neural network in the cultivating layer.

The cultivation environment of the nerve cells is an ordinary indoor environment, and the cultivation time can be determined according to actual needs. Therefore, in the environment of step S240, referring to FIG. 10, the various conditions are maintained, and the nerve cells are differentiated into a plurality of neurites by culturing in an indoor environment for about 15 days. When the nerve cells are grown, the protein, such as bovine serum, is capable of providing a growth factor for growth of the neural cells. After the plurality of nerve cells on the plurality of nerve cells are connected to each other, the neural network and the transplant body are formed. Referring to FIG. 11 and FIG. 12, the nerve graft is not stained by scanning electron micrograph and stained. After the transmission electron micrograph. It can be clearly seen from the above-mentioned projection electron micrograph that a plurality of nerve cells in the nerve graft are connected by neurites. Meanwhile, as shown in FIG. 11, although some nerve cells extend out of a plurality of neurites, they are not connected to other nerve cells through the plurality of neurites, but this does not affect the nerve graft as a whole. The nature of the activity.

In summary, the present invention has indeed met the requirements of the invention patent, and has filed a patent application according to law. please. However, the above description is only a preferred embodiment of the present invention, and it is not possible to limit the scope of the patent application of the present invention. Equivalent modifications or variations made by persons skilled in the art in light of the spirit of the invention are intended to be included within the scope of the following claims.

20‧‧‧Neural Network

22‧‧‧ nerve cells

24‧‧‧Nervous

Claims (9)

A method for preparing a nerve graft, comprising the steps of: providing a carbon nanotube membrane structure, the carbon nanotube membrane structure comprising a plurality of carbon nanotubes, the plurality of carbon nanotubes extending in a direction parallel to the a surface of the carbon nanotube membrane structure, and the plurality of carbon nanotubes are connected by van der Waals to form a self-supporting structure; the self-supporting structure of the carbon nanotube membrane structure is immersed in a protein solution and Curing, forming a protein layer on opposite surfaces of the carbon nanotube film structure; forming a poly-lysine layer on the surface of the protein layer; implanting a plurality of nerve cells on the surface of the poly-lysine layer; And cultivating the plurality of nerve cells until the plurality of nerve cells grow a plurality of neurites and the neurites between the plurality of nerve cells are connected to each other to form a neural network. The method of preparing a nerve graft according to claim 1, wherein each nerve cell in the neural network comprises at least one neurite protruding from an adjacent nerve cell. The method of preparing a nerve graft according to claim 1, wherein the neurites include dendrites and axons. The method for producing a nerve graft according to claim 1, wherein the protein in the protein layer is a soluble protein. The method for producing a nerve graft according to the above aspect, wherein the protein concentration in the protein solution is 50% or more and 100% or less. The method for preparing a nerve graft according to claim 1, wherein the carbon nanotube membrane structure comprises at least one carbon nanotube membrane, and the carbon nanotube membrane comprises a carbon nanotube flocculation membrane. , carbon nanotube film or nano carbon tube film. The method for preparing a nerve graft according to claim 6, wherein the carbon nanotube membrane structure comprises a plurality of laminated layers of carbon nanotube membranes, and the adjacent carbon nanotube membranes are passed between Wall connection. The method for preparing a nerve graft according to claim 1, wherein the carbon nanotube membrane structure is composed of the plurality of carbon nanotubes, and the plurality of carbon nanotubes pass through a van der Waals force End-to-end and axially extending in the same direction. The method of producing a nerve graft according to claim 8, wherein the nerve protrusions in the nerve cells extend in the same direction.